FRONT COVER

The owlish look of an owl is due to the facial ruff which layers of stiff feathers form around the face. The ruff is a sound collecting device (see abstract 184). Photo taken by Lois ."'1.acBird.

BACK COVER

Spread of the fluorescent tracer 6-COOH fluorescein amoo.g hepatocytes in rat liver. The dye has been micronjected into a single hepatocyte; intercellular transfer of dye is by ~ay of the channels of the gap junctions (see abstract 138). Photo by D. J. Meyer, S. B. Yancey and J.-P. Revel. A REPORT FOR THE YEAR 1979-80

ON THE RESEARCH AND OTHER ACTIVITIES

OF THE

DIVISION OF BIOLOGY

AT THE

CALIFORNIA INSTITUTE OF TECHNOLOGY

PASADENA, CALIFORNIA ii

STAFF OF filOLOGY 1980 Constance R. Katz: Compilation, typing and layout.

Elizabeth T. Hanson: Copy editing.

The helpful assistance of JoAnn Chikahiro, Lody Kempees and Isabella Lubomirski is greatly appreciated. A special thanks to Bernita Larsh for her guidance, assistance and support.

RESEARCH REPORTS

Much of the research work summarized here has not yet been reported in print, in many instances because it is not yet complete. For that reason this report is not intended as a publication and should not be cited as such. Individual projects should be referred to only if specific permission to do so is obtained from the investigator responsible for the material. References are made here to published papers bearing on the projects reported. Publications by members of the Division, covering the period July 1979-June 1980, are listed separately, at the end of the research reports of each group. iii

TABLE OF CONTENTS

Page

Introduction. . • . . . • . . . 1 Staff of Instruction and Research 9

Research Reports

MOLECULAR BIOLOGY AND BIOCHEMISTRY

Giuseppe Attardi - Summary. . . . • • • • . . • • 17 1. Transcription of the HeLa cell mitochondrial genome . 17 2. 5'-End sequence analysis of mitochondrial RNA species 18 3. 3'-End sequence analysis of mitochondrial RNA species 18 4. Cloning of human mitochondrial DNA • • • • • • • • 19 5. Amino acid sequence determination of the amino terminus of cytochrome oxidase subunit I 19 6. Cross-linking HeLa cell mitochondrial DNA in situ with·psoralen ...... 19 7. In vitro synthesis of human cytochrome c ...... • . • ...... 20 8. Studies on the stability of amplified DHFR activity in Mtx-resistant HeLa and V A -B cell lines. 20 9. Characterization of DHFR mRNA • • • . . . . • • • . . • • • • • . . • .2 . . . . • • 21 10. Double minute chromosomes in methotrexate-resistant human cell lines . 21 11. Purification of human dihydrofolic acid reductase and determination of its kinetic and physical parameters • ...... 21 12. Cloning of cDNA from purified DHFR mRNA •••••••••••• 22 13. DNA from double minute chromosomes in human methotrexate-resistant cells 22

James Bonner - Summary • • • • • • • • • • • • • • • • • 23 14. The role of his tone acetylation in gene expression ...... 24 15. The fine structure and evolution of the rat serum albumin gene. 25 16. Preparation of cloned cDNA probes for the a-fetoprotein gene . 25 17. The DNA sequence of rat a-fetoprotein gene. 25 18. Analysis of rat a-fetoprotein gene . • . 25 19. Nucleosome assembly in vitro . . . • . 26 20. Unfolding of the nucleosome in high salt 27 21. Active chromatin"' . . . . . 27

Eric H. Davidson - Summary • 28 22. Single copy DNA polymorphism of sea urchins in the genus Stronglyocentrotus 29 23. Analysis of sequence polymorphism in regions surrounding structural genes. 29 24. Human single copy DNA polymorphism • • • • • • • • . • • • • • • • • 29 25. Sequence organization around a structural gene in sea urchin DNA . . . . . 30 26. Isolation of insulin-like DNA sequences from the sea urchin ...••... 30 27. DNA rearrangements around homologous structural genes in two sea urchins . 30 28. Evolution of sea urchin DNA coding sequences ...... • . . . 31 29. Repetitive sequences of the sea urchin genome •.....•.•. 31 30. Nucleotide sequence analysis of the firi.e structure of repetitive DNA 31 31. Isolation and characterization of a gene battery . • . . . • . . . 32 32. Cycles of oogenesis in wild and laboratory-maintained sea urchins . • . . . 32 33. Sea urchin egg poly(A}+ RN As contain transcripts of repeated DNA sequences 32 34. Nature and fate of repetitive sequence transcripts in the sea urchin egg. 33 35. Repetitive sequence transcripts in Xenopus egg RN A ...... 33 36. Complexity of RNA in developing oocytes of Drosophila. • • • • • • • 33 37. Electron microscope visualization of transcription in Echinoderm oocytes 33 38. RNA synthesis in the fertilized mouse egg. • • • • • • • • • • • • • 34 39. Synthesis of vitelline layer proteins of sea urchin eggs. . . • . . . . . 34 40. Limited complexity of RNA in micromeres of 16-cell sea urchin embryos 34 41. Messenger RNA prevalence in sea urchin embryos measured with cloned cDNAs 35 42. Regulation of mRNA prevalence in the sea urchin embryo .... 35 43. Patterns of expression of mRNA sequences of sea urchin embryos 35 44. Rates of accumulation and turnover of individual messenger RNAs 36 45. Turnover rates of structural gene transcripts in nuclear RN As • • 36 iv

William J. Dreyer - Summary • • • • • • . • • . • . • . 37 46. Heritable molecular diversity in tumor cell"."'surface antigens . 38 4 7. Molecular characterization of human tumor markers 38 48. The immune response to solubilized tumor antigens . . . . . 39 49. Immunomicrospheres for cell detection and sorting . . . . . 39 50. Amino acid sequence analysis using gas-phase sequence technology 40 51. Development of a new, ultrasensitive mass spectrometer along research and clinical lines 40

Leroy E. Hood - Summary . • • . • • . • • • • • • • • • • • • • • • • • • • • • 41 52. Generation of diversity and regulation of expression in immunoglobulin heavy chain genes 43 53. Sequence and organization of immunoglobulin heavy chain variable region genes . . . . 43 54. Sequence studies of a-1,3 dextran binding antibodies ...... 44 55. Heavy chain variable region sequences from phosphorylcholine-binding myeloma proteins 45 56. A family of V region genes: The a-1,3 dextran system • . • . . . 45 57. Mechanisms of class switching in immunoglobulin heavy chain genes 45 58. Heavy chain switching • • • • • • • • • • • • . • . . • • . 46 59. Studies of the mouse immunoglobulin-D constant region gene 47 60. Isolation of immunoglobulin E gene • • . • • . . • ...... 47 61. The analysis of human immunoglobulin genes in health and disease 48 62. Two clones of a B-cell lymphoma synthesize three species of immunoglobulin mu chains . 48 63. The immunoglobulin mu chains of secreted and membrane-bound IgM differ in their c-terminal segments • . • • • • • • • • • 49 64. Do T cells express B-cell immunoglobulin genes? • • • • . • • • • 49 65. Structural studies of the genes coding for transplantation antigens • 50 66. Analyses of RTl products using two-dimensional polyacrylamide gels 51 67. Structure of murine Ia antigens: Two-dimensional electrophoretic analyses and HPLC tryptic peptide maps • ...... • • . 51 68. Development and application of protein microsequencing technology 52 69. A DNA sequenator .•••••.••••.•.•.•.•••• 52

Norman H. Horowitz - Summary ...... 54 70. Cellular siderophores of Aspergillus nidulans and Penicillium chrysogenum and the effect of low water activity. • • • • . • . • • • • • • • 54 71. Extracellular siderophores of Aspergillus nidulans and Penicillium chrysogenum. 54 72. Experiments with Neurospora crassa 55

Tom Maniatis - Summary • . • . • 55 73. Molecular cloning of the human S-like globin gene cluster . 56 74. The nucleotide sequence of the human S-globin gene • • • 57 75. The structure and evolution of the human S-globin gene family. 57 76. Nucleotide sequence analysis of rabbit S-globin genes. • • • • 57 77. The nucleotide sequence of a rabbit S-globin pseudogene • . • 58 78. Tissue-specific DNA methylation in a cluster of rabbit S-like globin genes. 59 79. Isolation and structural characterization of the human a-like globin gene cluster ••• 59 80. The structure of a human a-globin pseudogene (Wal) and its relationship to a-globin gene duplication . . • • • • • • • • • • • • • • • • • • 59 81. Characterization of intergenic repeat sequences of the human a-like globin gene cluster. 60 82. The nucleotide sequence of the human embryonic 7,;-globin genes •••••.• 60 83. Transformation of mouse erythroleukemia cells with cloned human globin genes . . 61 84. In vitro transcription of globin genes • • • • • • • • • . • . • • • . • • • • . 61 85. Identification of the human S-globin gene promoter • . . • . . • . . . . • • . 62 86. Isolation and partial characterization of the Drosophila alcohol dehydrogenase gene. 62

Elliot M. Meyerowitz - Summary . 63

Herschel K. Mitchell - Summary . 64 87. Hair phenocopies and developmental regulation in Drosophila 64 88. Rapid changes in gene expression in differentiating wing tissue in Drosophila. 64 89. Morphogenesis of wing hairs as observed by electron microscopy • • . • • • 65 90. Translational regulation of gene expression following heat shock • • . • • • 65 91. Heat shock at 37° induces synthesis of more than 50 proteins in larval salivary glands 66 92. The aromatic crosslink of insect cuticle. 66

James H. Strauss Jr. - Summary . . . . 67 93. Biochemical studies of large plaque and small plaque Sindbis virus 67 94. Genetic exchange between Sindbis virus and Western equine encephalitis virus 67 95. Comparative studies of the 3'-terrninal sequences of several alphavirus RN As 68 96. Cloning of DNA sequences complementary to the Sindbis virus 26S RNA. 68 97. Analysis of cDN A to Sindbis 26S RN A. . • • . • • • • • • • • • 68 98. Locating the panhandle structure on Semliki Forest virus virion RNA •• 69 v

99. Structural studies of Sindbis virus glycoproteins . • • • • • • . • 69 100. Carboxy-terminal sequencing of the Sindbis structural proteins. • • 69 101. N-Terminal sequences of blocked proteins of Sindbis virus . . . • . 70 102. Purification and characterization of E3 in the Sindbis virus infection 70 103. Electron microscopic studies of Sindbis virus maturation 70 104. Reversibility of envelope glycoprotein mutants of Sindbis virus. 71 105. Monoclonal antibodies to Sindbis proteins . • • • • • • • • • 71

CELLULAR BIOLOGY AND BIOPHYSICS

Howard C. Berg - Summary • • • • • . . . 75 106. Adaptation in E. coli chemotaxis • • • • . • 75 107. Signal-processing times in E. coli chemotaxis 75 108. Energetics of flagellar rotation ...... 76 109. Selection of non-chemotactic mutants of Streptococcus V4051 76 110. Search for steps in flagellar rotation 77 111. Mechanism of gliding motility . . • 77 112. Light antennas in phototactic algae. 77

Charles J. Brokaw - Summary • . . 78 113. Further work with anti-tubulins . . 78 114. The dynein cross-bridge cycle • • • 78 115. Asymmetrical beating of demembranated sea urchin sperm flagella. 79 116. Movement of spermatozoa in viscous environments ...... 79 117. Computer simulation of cross-bridge behavior in rigor muscle and flagella . 79

Max Delbriick . . • • . • • • • • • • . . . . . • • • • • • 80 118. Light-induced carotene synthesis in mad mutants of Phycomyces . 80 119. Identification of proteins involved in carotenoid biosynthesis by two-dimensionafgel electrophoresis ...... 81 120. Protein phosphorylation in Phycomyces and its behavioral mutants 81 121. Role of cAMP in the sensory transduction process in Phycomyces. 81 122. Search for photoreceptor .mutants . . . . . 82 123. Studies with riboflavin analogues...... 82 124. Stable and unstable dar alleles of Phycomyces • • • • • • • 83 125. Nuclear behaviors in Phycomyces zygospores . . . • . . . 84 126. Some new aspects of the avoidance response of Phycomyces • 84

Jolm J. Hopfield - Summary • • • • • 85 127. Proofreading in biochemical processes 85

Elias Lazarides - Summary • . . . • 85 128. Desmin and vimentin coexist at the periphery of the myofibril Z disc 86 129. The synthesis and distribution of desmin and vimentin during myogenesis in vitro 86 130. Association of a high molecular weight protein with avian muscle desmin 87 131. The synthesis and deployment of filamin in chicken skeletal muscle. . . . . 87 132. In vitro translation of intermediate filament subunit proteins ...... 88 133. Characterization of phoshorylation sites of desmin and vimentin ...... 88 134. Identification of endogenous substrates for chicken muscle transglutaminase . 89 135. The 68,000 dalton neurofilament-associated polypeptide is a component of non-neuronal cells 89

Jean-Paul Revel - Summary • . . • • • . • • • • • • • • • • • • • • . . . • • • • 90 136. An analysis of the protein components of rat liver gap junctions ...... • • . 90 137. A comparison of the gap junctional proteins of liver and lens...... • • • . 91 138. A quantitative analysis of intercellular communication in normal and regenerating rat liver 92 139. An estimate of the conductance of a single connexon in rat liver ...... 92 140. In vivo incorporation of [355] methionine into gap junctional protein in rat liver . 92 141. Thioacetamide-induced changes in gap junctional morphology ...... 93 142. Formation of gap junctions during embryonic development of the chick lens 93

Anthonie Ven Harreveld. • • . • • . • . • • • • • • • • • • • • • • 94 143. Mechanism of transparency changes in the retina...... 94 144. A histological method for the electron microscopic localization of chloride 95

CELLULAR NEUROBIOLOGY

Jeremy P. Broekes - Summary • . . . . . • • • • • • • • • • • • • • • . . . • 99 145. Purification of glial growth factor (GGF), a new component of the brain and pituitary . 99 vi

146. Glial growth factor in brain ...... • 100 147. Preliminary studies of myelination in vitro ...... 100 148. Generation of monoclonal antibodies to specific neural cells • 100 149. Effect of diphtheria toxin and mutant proteins on cultured rat Schwann cells. 101 150. Studies on the effects of gonadal hormones upon the differentiation of dissociated neurons in vitro. 102

A. James Hudspeth - Summary • • 102 151. Analysis of the microphonic response recorded from the sacculus in vitro 103 152. Adaptation in the response of hair cells to sustained stimuli . • . . 103 153. Electrical responses from solitary hair cells . . . . • . . . • . . 104 154. Gating kinetics of the transduction element in vertebrate hair cells. 105 155. An in vitro study of ototoxicity due to aminoglycoside antibiotics. 105

Henry A. Lester - Summary • • • • • • • • • • • . • . • • • 106 156. Perfusion of cells containing acetylcholine receptors • • . • • • 106 157. Numerical reconstruction of relaxation and fluctuation experiments at nicotinic synapses 107 158. Photoisomerization of azobenzenes. • • • . • . . . • . 107 159. Photochemical probes of the agonist-receptor complex • . • • • • • • • • • • . • • 107 160. The muscarinic acetylcholine receptor of frog atrial fibers • • • • • • • • • • . • • 108 161. How photoisomerizable azobenzene compounds affect acetylcholine receptors of fish muscle • 108 162. Photochemically labile precursors of calcium, protons, and cyclic nucleotides 109 163. Studies of nerve and muscle in cell cultures 109

Felix Strumwasser - Summary • • • • • • 110 164. Localization of ELH-like immunoreactivity in the Aplysia nervous system 111 165. Microinjection of protein kinase into cultured bag cell neurons • 112 166. Protein phosphorylation in bag cell neurons • • • • • • • 112 167. Atrial gland extracts from Aplysia brasiliana . • • • • • 113 168. L-proline blocks hippocampal excitatory synaptic activity . 113

NEUROBIOLOGY AND BEHAVIORAL BIOLOGY

John M. Allman - Summary • • • • • • • • . . . . • . . • . . • . . • • • . • . . • 117 169. Neurons selective for stimulus dimension in the dorsolateral area of the owl monkey • • • • 118 170. Orientation selectivity and responsiveness to moving random dot arrays in MT, DL, DM and M 119 171. Innervation and control of the facial muscles 121

Derek H. Fender - Summary • • • • • • • • 122 172. Binocular hysteresis and fusion with voluntary eye motions 122 173. Eye movements in reading. • • • • • • • • • • • • • • 123 174. Rapid adaptation in the retina • • . . . • • • • • • • • 123 175. Localization and depth perception processes in the monkey 123 176. LGN and deep-source localization through evoked potentials • 124 177. Auditory scalp potentials evoked by monaural/binaural coherent/incoherent noise stimulation 124 178. Superposition of responses to partial-field visual stimulation • 124 179. Localization of VESP for a nonspherical head • • • • • • • 125 180. Combined spatial and temporal localization of VESP sources . 125

Masakazu Konishi • • • • • . • • • . • . . • . . • • . 126 181. Androgen increases protein synthesis within the avian brain song control system 126 182. The cellular nature of the critical period for sexual differentiation in the zebra finch 126 183. Neurophysiological studies of singing birds. 127 184. Use of phase by the owl auditory system 127

Marianne E. Olds - Summary. • • • • • 128 185. The role of the locus coeruleus in stimulation reward 129 186. Anatomical basis for the reinforcing effects of opioids 129 187. Opiate sensitivity profiles of neurons in regions supporting rewarding brai~ stimulation 130 188. Ontogeny of the stimulant effects of morphine and methionine-enkephalin in rats. 130 189. Plasticity during auditory conditioning in the deep layers of the superior colliculus but not in the inferior colliculus...... • ...... 131 190. Afferents to MGB and deep layers of the superior colliculus studied with the horseradish peroxidase method 131

John D. Pettigrew - Summary • • • 132 191. The two foveae of the hawk's retina subserve two visual pathways 133 192. Co-evolution of nocturnal vision and frontally-placed eyes? 133 193. Avian geomagnetic field perception . • • • • • • • • • • • • 134 vii

194. Prolonging the "critical period" for plasticity in the visual cortex. • • . • . • • 134 195. Brief visual experience can modify ocular dominance of cortical neurons in kittens 135 196. Binocular neural mechanisms in goats and sheep . • . • • . • • • • • • • • 135 197. Ocular dominance columns in the visual cortex of monocularly deprived owls . . 136 198. Further anatomical features in the visual system of owls • • • • • • • • • • 137 199. Binocular facilitation and inhibition in the lateral geniculate nucleus of the cat. 137 200. Functional classes of auditory cortical neurons distinguished by sensitivity to sound location 138 201. Cell shrinkage in geniculate neurons following brief unilateral blockade of retinal ganglion cell activity • • • • • • . • • • • • • • • . • • • . • 138 202. Cyclic nucleotides in the synaptic plasticity of visual cortex . • • • • • . • . . 139 203. Single cell responses in cat visual cortex: Their modulation by iontophoresis of NE 139 204. Brief perfusion of norepinephrine increased the neural plasticity in kitten visual cortex 139 205. Morphology of catecholaminergic terminals in kitten visual cortex • • . • . • • • 140 206. Coexistence of catecholaminergic terminals and acetylcholinesterase-sensitive neurons in cat visual cortex . • . • • • • • • • • • . . . . . • . • • . 140 207. Regrowth of catecholamine terminals in cat visual cortex following localized lesions 140 208. Total deprivation of visual afferents from one eye: A comparison with conventional monocular lid suture • 141

R. W. Sperry - Summary. • • • • • • • 142 209. Somatognostic abilities of the right and left cerebral hemispheres 142 210. Unilateral spatial neglect and spatial disorientation ••••••• 143 211. Goal-conflict in split brains . . • • • • • • • • • • • • • • • 143 212. Identification and cross-comparison of faces by human commissurotomy patients . 144 213. Dichotic ear difference is a poor measure of hemispheric dominance 144 214. Further anatomical features of interhemispheric connections • • 145 215. Behavioral functions of commissural connections • • • . • . • • . 145 216. Visual preferences of the two hemispheres of split-brain monkeys. • 145 217. Hemispheric differences in processing sequential comparisons in monkeys 145 218. Recognition of individuals and facial expression by the two hemispheres of split-brain monkeys • 146

David c. Van llssen - Summary. • • • • • • • • • • • • • • • • • • • • • 147 219. Areal boundaries and topographic organization of the ventral posterior area (VP) 147 220. Architectonic identification of the boundaries of V3 in the macaque monkey • 148 221. Patchy and non-patchy connections between Vl and V2 in the macaque • • 148 222. The representation of the visual field in the macaque middle temporal area 149 223. Two-dimensional maps of the lateral geniculate nucleus • • • • • • • • 149 224. Effect of impulse blockade on the elimination of neuromuscular synapses • 150

NEUROGENETICS

Seymour Benzer - Summary • • • • • • • • • • • • • • • • • • • • • • • 153 225. Profiles of the giant fiber of Drosophila as revealed by Lucifer yellow injection 153 226. The physiology of Shaker (Sh) mutants • • • . • . • . . . • • 153 227. Genetic organization of the Shaker (Sh) gene. • • • • • • • • • • • 154 228. Polypeptides of Drosophila specific to the central nervous system 154 229. Electrophysiological studies of flightless mutants of Drosophila 154 230. A myofibrillar defect in indirect flight muscle caused by deficiency of chromosomal bands 11A6-7 . . . . • • • • • • • • 155 231. The dunce mutant and cAMP phosphodiesterase • • . • • 155 232. Immunological studies of PDE in Drosophila melanogaster • 156

Ronald J. Konopka - Summary • • • • • • • • • • • • • 157 233. A membrane model for the Drosophila circadian oscillator. 157 234. Longevity of clock mutants in artificial environmental cycles 157 235. Pharmacological studies on the Drosophila nervous system • . 158 236. Mapping and characterization of the andante clock mutant 158 237. Isolation of new clock mutants...... • . . . . . • • 158 238. Innervation of antennae and legs in wild-type and homeotic mutant flies. 158

DEVELOPMENTAL GENETICS AND IMMUNOGENETICS

Edward B. Lewis - Summary • • • • • • • • • • • • • • 163 239. Developmental effects of deleting the Polycomb gene. • • 163 240. Clonal analysis of Polycomb in the abdomen of Drosophila . 164 241. Identification of two new genes that appear to act as bithorax complex repressors 164 242. An improved four-winged fly • • • • • • 165 243. Molecular analysis of the bithorax complex • • • • • • • • • • • • • • • • • 165 viii

BIOLOGY/CHEMISTRY GRADUATE STUDENTS

244. Nature of integrated FeL-V provirus...... 167 245. Characterization of the actin genes of Drosophila 167 246. Cuticle genes • • . • • • • • • • • • • • • • 168 247. Milliseconds time resolution of cation transport by reconstituted purified acetylcholine receptor. 168

Visiting Lecturers ...... 171 Graduates ...... 173 Financial Support ...... 174 Author lndex (by page number). . • 178 Financial Support lndex (by page number) 180 INTRODUCTION 2

Berg

Hopfield

Meyerowitz 3

New Faeulty

Our faculty has been strengthened by the addition of Drs. Howard C. Berg, John J. Hopfield, and Elliot M. Meyerowitz.

Dr. Howard Berg's appointment as Professor of Biology began October 1, 1979. Dr. Berg received his B.S. degree from Caltech in 1956, and, more recently, was at the University of Colorado where he was a Prdfessor of Molecular, Cellular, and Developmental Biology. He is well known for his research on the rotary motor responsible for bacterial motility and its regulation during characteristic behavior.

Dr. John Hopfield received a joint appointment as the Roscoe G. Dickinson Professor of Chemistry and Biology and took residence at Caltech in January 1980. Dr. Hopfield came from Princeton University where he was a Professor of Physics. He is also a member of the technical staff at Bell Laboratories.

Dr. Elliot Meyerowitz became an Assistant Professor of Biology effective January 1, 1980. He came from Stanford University and will continue the analysis of the glue-protein gene region and will also pursue the problems of eye-brain developmental interaction in Drosophila. We are happy to announce that Dr. Mary Kennedy, presently a postdoctoral fellow at Yale University, will join the faculty in January 1981 as Assistant Professor of Biology.

Konishi

Endowed Professorship

Professor Mark Konishi was appointed the Bing Professor of Behavioral Biology effective January 1, 1980. Professor Konishi is well known for his studies of song and hearing in birds. There are interesting parallels between the development of speech. in man and that of song in birds, and Professor Konishi's studies of vocal development in songbirds have given useful insights into the ontogeny of human speech. 4

Sperry

Honors

Professor Roger Sperry was honored this year with many awards recognizing his contributions to psychobiology: The Wolf Prize in Medicine, the Ralph Gerard Award of the Society for Neuroscience, the International Visual Literacy Association Special Award for 1979, the Albert Lasker Medical Research Award, and an honorary D.Sc. degree from The Rockefeller University.

Honorary Degrees, Appointments, Lectureships

Professor James Bonner has been appointed an Honorary Professor of the Sinkiang University, Urumchi, Sinkiang Autonomous Region and Professor of the Fudan University, Shanghai, People1s Republic of China.

These appointments mark a resurgence of Caltech contacts with Mainland China which also included visits by Professor Eric Davidson to lecture at Shanghai Institute of Biochemistry of the Academia Sini<~a and the appointment of Boning Gao as a graduate student from China in Professor Davidson's laboratory.

Professor Leroy Hood gave the Howard Taylor Ricketts Lecture at the University of Chicago.

Professor John Pettigrew is spending his leave of absence at the National Vision Research Institute in Carlton, Victoria, Australia.

Professor Jean-Paul Revel was apointed a Visiting Professor of Cell Biology at New York University.

Professor Felix Strumwasser gave the Lang Lecture at the Marine Biological Laboratory, Woods Hole, Massachusetts, on July 25, 1980. 5

Birthday Celebration

In celebration of Professor Ray Owen's 65th birthday, a symposium entitled 11 Frontiers in Imrnunogenetics" was held at Baxter Hall on June 2-3, 1980. The prime mover for the symposium was William Hildemann, a former graduate student of Ray Owen and now a Professor at UCLA. The speakers on the program were all former students or immediate colleagues of Ray Owen. Many of the participants are shown clustered around Ray in the above photograph. The banquet held in connection with the symposium featured a i;>erformance by the Caltech Women's Glee Club, a skit depicting life in Ray's coffee room, and a slide presentation showing Ray and his co-workers over the years. The proceedings of the symposium will be published as a monograph by Elsevier-North Holland.

Braun Laboratories of Cell Biology and Chemistry

Construction of this new building began December 1979. Completion is expected in the Spring of 1982. This building will provide 75,000 sq. ft. of facilities for a new program which will emphasize basic research in biology and chemistry that is relevant to modern medicine, and in particular, will permit expansion of the work of our Division in the field of immunology. Three occupants of the Braun Laboratories have been identified: Professor Leroy Hood, whose program is centered around immunology; Professor Michael Raftery, whose primary interest now is the nature of receptor molecules and ion channels on cell surfaces; and Professor John Richards, who works on the structure and function of proteins, including immunoglobins. Five additional investigators will be recruited. The picture above is a rendering of the proposed completed facility and on the next page is a photograph of the construction in progress. 6

BraWl Laboratories - progress to date

Cranmer Retirement

On December 26, 1979 an era came to an end. After 25 years of serving as the Division Chairman's Secretary, Geraldine Cranmer retired. She had been on hand throughout the reigns of four division heads-seven years with George Beadle (one year was with James Bonner while Beadle was at Oxford), seven years with Ray Owen, nine years with Robert Sinsheimer (one year was with Norman Horowitz while Sinsheimer was in Switzerland), and two years with Norman Horowitz. With appreciation and thanks for her devoted service, the members of the Division of Biology wish Geraldine well in her retirement.

Faculty

With the conclusion of Professor Horowitz' term of office as Chairman of the Division, the following appointments took effect this Fall, 1980: Chairman: Leroy E. Hood Associate Chairman: Charles J. Brokaw Executive Officer: A. James Hudspeth Executive Officer: James H. Strauss BIOLOGY DIVISION STAFF

Instruction

Research

Administrative

9

STAFF OF INSTRUCTION AND RESEARCH DIVISION OF moLOGY Norman H. Horowitz, Chairman Charles J. Brokaw, Executive Officer Masakazu Konishi, Executive Officer

Board of Trustees Professor of Biology Emeritus

Max.Delbriick, Ph.D., Sc.D., Nobel Laureate ••• Biology

Professors Emeriti

Henry Borsook, Ph.D. M.D. • • • • • Biochemistry Sterling Emerson, Ph.D. • • • • • • • Genetics George E. MacGinitie, M.A. • Biology Anthonie Van Harreveld, Ph.D., M.D. Physiology

Professors

Giuseppe Attardi, M.D •••• • • • • • • • • • • • • • • • • Biology Seymour Benzer, Ph.D., D.Sc. James G. Boswell Professor of Neuroscience Howard c. Berg, Ph.D. . Biology James Bonner, Ph.D. • • Biology Charles J. Brokaw, Ph.D. Biology Eric H. Davidson, Ph.D. Biology William J. Dreyer, Ph.D. Biology Derek H. Fender, Ph.D. Biology and Applied Science Leroy E. Hood, M.D., Ph.D. Ethel Wilson Bowles and Robert Bowles Professor of Biology John J. Hopfield, Ph.D. • • Roscoe G. Dickinson Professor of Chemistry and Biology Norman H. Horowitz, Ph.D. • • • • • • • • • • • • • • • Biology Masakazu Konishi, Ph.D. , Bing Professor of Behavioral Biology Edward B. Lewis, Ph.D. Thomas Hunt Morgan Professor of Biology Tom Maniatis, Ph.D. • • • • • • • • • • • • • • • • • • Biology Herschel K. Mitchell, Ph.D. • • • • • • • • • • • • • • • Biology Ray D. Owen, Ph.D., Sc.D. • • • • • • • • • • • • • • • Biology Dean of Students and Vice President for Student Affairs Jean-Paul Revel, Ph.D. . . Albert Billings Ruddock Professor of Biology Roger W. Sperry, Ph.D., Sc.D. Hixon Professor of Psychobiology Felix Strumwasser, Ph.D. • • • • • • . • . • • • • • • • Biology

Sherman Fairchild Distinguished Seholars

William Hayes, Ph.D. • • • • • Biology William R. Levick, M.B., B.S. . Biology Donald M. MacKay, Ph.D. Biology Francois-Xavier Wilhelm, Ph.D. Biology

Senior Research Associate

Roy J. Britten, Ph.D. • • • • • • • • • • • • • • • • • Biology

Research Associates

Charles R. Hamilton, Ph.D. . • Biology Barbara R. Hough-Evans, Ph.D. Biology Peter H. Lowy, Doctorandum Biology Marianne E. Olds, Ph.D. Biology

Del Webb Visiting Associates

Sudarshan Malhotra, Ph.D. • • . • • • • • • • • University of Alberta Evelyn L. Teng, Ph.D. University of Southern California School of Medicine

Gosney Visiting Associates

Yoshiki Hatta, M.D. • . • • University of Tokyo Carlo Morandi, Ph.D. • Universita di Pavia, Italy 10

Visiting Associates

James F. Baker, Ph.D••• • • . • . • • Pitzer College Welcome W. Bender, Ph.D. • • • Stanford Medical School Hermes Bravo, D.D.S. • Universidad Catolica de Chile Raymond P. Briggs, Ph.D. • • • • • • Chaffey College Joel Buxbaum, M.D. • • • Manhattan Veterans Administration Hospital Kathryn L. Calame, Ph.D. University of California, Los Angeles Patrice L. French, Ph.D. . • . . University of California, Los Angeles NestOr F. Gonzfilez-Cadavid, Ph.D. • . Universidad Central de Venezuela Theodore A. Goodman, M.D. • University of California, San Diego Rodney M. Hewick, Ph.D. Imperial Cancer Research Fund, London Dennis J. Hocevar, Ph.D. • • • University of Southern California Jeffrey J. Hubert, Ph.D. Scripps Clinic and Research Foundation Stanley Klein, Ph.D. . • • • • • • Claremont College I. Richard Lapidus, Ph.D. • • • Stevens Institute of Technology Keh-Chi Ling, Ph.D. • • • • • • • International Rice Institute EveLynn McGuinness, Ph.D. University of California, Los Angeles Ronald L. Meyer, Ph.D. • . • University of C.alifornia, Irvine Josette M. Michelot, M.D. . .. French National Institute for Medical Research Oscar L. Miller, Ph.D. . • • • • • University of Virginia Julio Montoya, Ph.D. Universidad Complutense de Madrid Joel Myerson, Ph.D. University of California, Riverside Kunio Nakai, M.D. • . Wakayama Medical College Joel Nargeot, Ph.D. University of Tours, France Tamotsu Ootaki, Ph.D. Yamagata University Edward F. Pate, Ph.D. • • • Aerospace Corporation Ruggero Pierantoni, Ph.D. • Consiglio Nazionale del Ricerca Lajos Piko, D. v.M. • . • • Veterans Administration Medical Center Jose M. Sala-Trepat, Ph.D. • Laboratoire d'Enzymologie, CNRS Theo B. Sonderegger, Ph.D. • • • • • University of Nebraska Mamoru Umemoto, Ph.D. • • • • • • Osaka City University R. Bruce Wallace, Ph.D. • • City of Hope Research Institute Norma P. Williams, Ph.D. • • • • • • • • Howard University Ji-hou Xin, B.S. • Shanghai Institute of Biochemistry Eran Zaidel, Ph.D. • • • University of California, Los Angeles

Associate Professors

John M. Allman, Ph.D. • • • • Biology A. James Hudspeth, Ph.D., M.D. Biology Henry A. Lester, Ph.D. • • • Biology John D. Pettigrew, M.B., B.S. Biology James H. Strauss Jr., Ph.D. Biology David C. Van Essen, Ph.D. Biology Assistant Professors

Jeremy P. Brockes, Ph.D •• Biology Ronald J. Konopka, Ph.D •• Biology Elias Lazarides, Ph.D. • • Biology Elliot M. Meyerowitz, Ph.D •• Biology Gosney Senior Research Fellows

Peter F. R. Little, Ph.D. Biology Nancy S. Petersen, Ph.D. Biology 11

Senior Research Fellows

David M. Anderson, Ph.D. Biology Dorwin L. Birt, Ph.D. Biology Sandra J. Ewald, Ph.D. Biology Eri Heller, Ph.D. Biology Michael W. Hunkapiller, Ph.D. Biology Leonard K. Kaczmarek, Ph.D. Biology Takuji Kasamatsu, M.D., Ph.D. Biology Minnie McMillan, Ph.D. Biology Michael D. Manson, Ph.D. Biology Menasche M. Nass, Ph.D. . Biology Makoto Okuno, Ph.D. Biology Nicholas J. Proudfoot, Ph.D. Biology M. Viswana th Reddy, D.Sc. Biology John W. Roberts, Ph.D. Biology Janet M. Roman, Ph.D. Biology Ellen G. Strauss, Ph.D. Biology Terry L. Thomas, Ph.D. Biology S. Barbara Yancey, Ph.D. Biology Jung-Rung Wu, Ph.D. Biology

Del Webb Research Fellow

Ma.'rk Gurney, Ph.D.

Gosney Research Fellows

Carlos V. Cabrera, Ph.D. Alberto Ferrus, Ph.D. Ze 'ev Lev, Ph.D.

Weizmann Fellows

Pamela L. Mellon, Ph.D. David E. Presti, Ph.D.

Research Fellows

Marylouise Ary, Ph.D. John W. Grula, Ph.D. Monica Mottes, Ph.D. David J. Asai, Ph.D. Leah T. Haimo, Ph.D. Jeanne M. Nerbonne, Ph.D. Martha W. Bond, Ph.D. Terrence J. Hall, Ph.D. William T. Newsome III, Ph.D. Eugene T. Butler III, Ph.D. Ross C. Hardison, Ph.D. Clare M. O'Connor, Ph.D. Ricardo Castro-Gonzalez, Ph.D. Eliot M. Herman, Ph.D. Manfred K. Otto, Ph.D. Anne Chomyn, Ph.D. Henry V. Huang, Ph.D. R. Gilles Plourde, M.D. Toni R. Claudio, Ph.D. Michael T. Hyson, Ph.D. Vilayanur S. Ramachandran, Ph.D. Loring G. Craymer III, Ph.D. Linda L. Jagodzinski, Ph.D. Elizabeth A. Repasky, Ph.D. Laura De Francesco, Ph.D. Makkuni Jayaram, Ph.D. Winfield S. Sale, Ph.D. Richard H. Douglas, Ph.D. Lawrence M. Kauvar, Ph.D. Richard H. Scheller, Ph.D. Jan W. Duncan, Ph.D. Laurence A. Lasky, Ph.D. Monica H. M. Shander, Ph.D. Philip w. Early, Ph.D. Richard M. Lawn, Ph.D. C.-K. James Shen, Ph.D. Susan G. Ernst, Ph.D. Leslie L. Leutwiler, Ph.D. Robert E. Sheridan Jr., Ph.D. James L. Fobes, Ph.D. Maria-Luisa Maccecchini, Ph.D. Michael Steinmetz, Ph.D. Costantin Flytzanis, Dr.rer.nat. Gordon C. Machray, Ph.D. Katherine A. Stygall, Ph.D. John Frelinger, Ph.D. Christian G. Merkel, Ph.D. Mark A. Tanouye, Ph.D. Edward F. Fritsch, Ph.D. David J. Meyer, Ph.D. Murray F. Teitell, Ph.D. Sinobu c. Fujita, Ph.D. Andrew Moiseff, Ph.D. Martin M. Weinstock, Ph.D. Paul A. Galland, Ph.D. Gordon P. Moore, Ph.D. John E. Wiktorowicz, Ph.D. Robert S. Goodenow, Ph.D. Kevin W. Moore, Ph.D. Kendrick N. Williams, Ph.D.

Graduate Fellows and Assistants

Gary Aston-Jones, B.A. Steven H. Green, B.S. William T. Newsome III, B.S. John R. Bell, B.S. Mark E. Gurney, B.A. Bruce J. Nicholson, B.Sc. John L. Bixby, B.A. Tim Hunkapiller, B.S. Deanna K. Ojala, B.Sc. Beverley J. Bond, A.B. Kent R. Jennings, B.Sc. Dominic Orr, B.Sc. Arlene Y. Chiu, M.Sc. Larry E. Johnson, B.S., M.D. Jing-hsiung James Ou, B.S. David P. Corey, B.A. Nelson D. Johnson, B.S. Vann P. Parker, B.S. Frank Costantini, B.S. Lawrence c. Katz, B.A. Steven E. Petersen, M.A. 12

Stephen T. Crews, B.A. Marilyn R. Kehry, B.S. James W. Posakony, B.S. Alice M. Cronin, B.A. Stuart K. Kim, B.A. Antonio A. Reyes, B.S. Madeline A. Crosby, B.S. Michael W. Klymkowsky, B.S. Charles M. Rice III, B.S. Thomas E. Crowley, B.S. Mitchell Kronenberg, B.A. Arthur H. Roach, B.Sc. Mark M. Davis, B.A. Mauri E. Krouse, S.S. Thomas D. Sargent, A.B. Philip W. Early, B.S. Baruch Kuppermann, A.B. Loveriza A. Sarmiento, B.S. Ruth A. Eatock, M.Sc. Elizabeth H. Lacy, B.A. James W. Schilling, A.B. Jay W. Ellison, B.S. Joyce E. Lauer, A.B. Brian S. Seed, B.S. Douglas A. Fisher, A.B. Greg Erwin Lemke, S.B. Jeffrey E. Segall, B.A. John Frelinger, B.S. David E. Levy, B.A. Beverly Taylor Sher, B.A. Karl J. Fryxell, B.A., B.S. Richard s. Lewis, B.S. Sandra L. Shotwell, A.B. Boning Gao, B.S. Donna L. Livant, B.A. David W. Sivertsen, B.S. David L. Gard, B.S. James S. Mccasland, B.S., B.A. Randall E. Smith, B.S. Karen E. Gaston, M.A. Kenneth L. Marton, B.S. Michael P. Snyder, B.A. Robert A. Gelfand, M.A. Jeffrey N. Masters, B.S. Betty A. Vermeire, B.S. David A. Goldberg, B.S. John H. R. Maunsell, B.S. Chung Wang, M.S. Richard H. Gomer, B.A. Jeffrey T. Mayne, S.B. Wilson C.-s. Wu, B.A. Herman J. Gordon, B.A. Katherine S. Mixter, B.A. Joanne M. Yeakley, B.S. Bruce L. Granger, B.A. Jay J. Myers, B.A., M.A.

Members of the Professional staff Gisela W. Charlang, Ph.D. Francis M. Miezin, MSEE

Research Staff Cynthia Akutagawa Margaret M. Griffith Stella Olive Eugene Akutagawa, B.S. Les B. Grim, B.A. Susan Ker-hwa Ou, M.S. Maria Alonso, S.S. Nancy I. Harris Wanda L. Owens Fargo Balliett, B.S. Suzanna J. Horvath, Ph.D. Jeanne P. Patalano, B.S. David R. Balzer Jr., B.A. Richard A. Jacobs, B.S., M.S. A. George Pauls Rosalie A. Beer, B.S. Jeannette Johnstone Tony B. Ramey, B.S. Celeste A. Berg, B.A. Bertha E. Jones Jane Rigg, B.A. Alison E. Blake Gertrude Jordan Joan Roach, B.A. Ellen B. Blau, S.S. Benneta T. Keeley Ronald M. Satchell Rebecca F. Bodor Chin Sook Kim, M.S. Janet Sauer, B.S. Ingelore S. Bonner Rosina Kinzel Floyd R. Schlechte, B.S. Paul K. Cartier III, A.B. Phyllis F. Knudsen, M.S. John M. Scotese, M.S. Margaret E. Chamberlin, B.S. Laura E. Kochevar, B.S. Carol L. Shotwell John Wen-Kiang Chu, B.S. Patrick F. Koen Mark E. Silver, B.A. Maria A. Clancy Susan Shu-Ai Tsai Lai, B.S. Thomas F. Simonick, B.S. Sheila Collins, B.S. Patrick S. Leahy, B.S. Nelson L. Smith, B.S. M. Patricia Conley, B.S. Shian Yun Lee, M.A. Robert D. Smyth, Ph.D. Adriana Cortenbach Thomas E. Lee, B.S. Devra C. Spurr Maria A. DeBruyn Teresita U. Legaspi, A.B. Delilah A. Stephens, B.A. B. Lynn DeOgny, B.S. Edith M. Lenches, B.S. Teresa M. Stevens Arger L. Drew Jamie P. Leverette, B.S. Marjorie Sturgis Martin M. Echols, B.A. Ilga Lielausis Robert W. Tajima Jean E. Edens Lois MacBird Jane T. Traub, B.S. Eveline Eichenberger Josephine Macenka, B.S. Joseph F. Venti Leah Ellenberg, Ph.D. Douglas S. Mechaber, M.A. Caroline Vermaes Vincent R. Farnsworth, M.A. Mathilda Meulemans Daniel P. Viele, B.S., M.S. Doris T. Finch, B.A. Cecilia Mong, B.S. Jessie Walker Helen Gay, B.S. Catherine Davis Moser, A.B. Steven M. Wells Ana L. Gharzeddine Jean Mueller, B.A. Eva Westmorland Anna Gil, B.S. Jeanette Navest Gayle-Linda Westrate Terrence Giugni Bradford Ng, B.S. Donna R. Williams Carlotta A. Glackin, B.A. Margaret S. Norton Nancy K. Wischhmen, B.A. Eef Goedemans Mary M. Nousek, B.S. Maria Yu-Li Yang, B.S. Maria R. Gomez Catherine D. O'Connell Annette S. Yuen, A.A. Geoffrey J. Graham, M.S. Edward Ogawa 13

Student Assistants

Jeffrey R. Binkley Eric Hanson Richard C. Penrock Bonnie Blam ick Teri A. Hermsen Grace Pilpa Roberta J. Brandenburg David Kaleung Ho John B. Reinitz Kenneth H. Britten Joseph Man-Hei Ho Constance S. Royden Julie Brook James J. Host Miriam L. Rus-ch Carolyn A. Burhenn David S. Kamins Anne W. Schmidt Kenneth L. Campos Solomon Kebede Leon E. Schwartz Jeffrey D. Carpenter Hedy L. Kindler Eric Sinn Francisca A. Castorena Jimmy Kwok Ching Lam A. Gregory Sorensen Peggy Chang David D. Lo Michael D. Stein Thomas A. Cross Donald C. Lo Ann H. Tamashiro Paul Cummings Bruce D. Malamud John D. Terrell Bryan C. Dunkeld Linda B. McAllister John C. Whitehead Susan M. Ebersole Gary C. Mockli William H. Wright III Steven T. Eckmann Richard o. Nepi! Fonda Bai-Yueh Wu Joseph A. Garcia Jr. John J. Ngai Lui Kwong Yu Sarkis J. Ghazarian Carl Nicholson John R. Yuen Hilary L. Glazer Stephan G. Nicholson Kenneth M. Gray Phillip A. Patten

ADMINlSTRATIVESTAFF

Michael Miranda, Administrator Geraldine S. Cranmer, Division Secretary Bernita Larsh, Division Secretary

Accounting Kerckhoff Marine Laboratory

Lody Kempees, Supervisor Robert K. Blue, Superiittendent Sandra L. Carta Peggy R. Bierer Ruth M. Erickson James P. Currie Joe R. Deem Animal Rooms Glen Epstein Jara Lewin Milton Grooms, Supervisor William Smith Sergio Bretado Peter L. Vignaroli Terrance L. Jones Julie L. Mangan Machine Shop Gildardo Vega Roberto Vega Frank L. Ostrander, Supervisor Richard J. Broderick Beekman Laboratories John Klemic

JoAnn Chikahiro - Accounting Secretarial Dale D. Linder - Animal Rooms Michael P. Walsh - Electronics Shop Jane Chacon, Supervisor David C. Hodge Christine R. Bartel John N. Power Stephanie A. Canada Nancy M. Gill - Grants Joanne H. Dugdale Elizabeth T. Hanson - Secretarial Constance R. Katz Elizabeth F. Koster Grants J. Beth McGrath Vickie Oldenburg Barbara B. Cathey Renee Thorf Isabella Lubomirski Elizabeth A. Vagner Stockroom

Instrument Fabrication Shop William F. Lease, SUpervisor Giao K. Do James J. Gilliam Jane c. Keasberry Linda McCasland Instrument Repair Shop

William B. Bowman, Supervisor Gilbert N. Granillo

MOLECULAR BIOLOGY AND BIOCHEMISTRY

Giuseppe Attardi

James Bonner

Roy J. Britten

Eric H. Davidson

William J. Dreyer

Leroy E. Hood

Norman H. Horowitz

Tom Maniatis

Elliot M. Meyerowitz

Herschel K. Mitchell

James H. Strauss Jr.

17

Professor: Giuseppe A tta.rdi mRNAs start directly at, or very near to an AUG or AUA Gosney Visiting Associate: Carlo Morandi triplet (which is a methionine codon in human mito­ Visiting Associates: Nest6r F. GonzB.lez-Cadavid, Julio Montoya chondria), with the exception of one which starts at an Research Fellows: Anne Chomyn, Laura De Francesco, AUU. The available evidence indicates that the terminal Christian G. Merkel, Monica Mottes Graduate Students: Robert A. Gelfand, Jeffrey N. or subterminal AUGs and AUAs, and possibly also the Masters, Deanna K. Ojala, terminal AUU, are initiator codons for the corre"sponding Research Staff: Doris T. Finch, Helen Gay, Susan Shu-Ai Tsai Lai; Benneta T. Keeley polypeptides. Laboratory Staff: Arger L. Drew, Maria R. Gomez, A correlation of the 5'-end proximal sequences of the Rosina K. T. Kinzel, Wanda L. Owens human mitochondrial mRNAs with the NH -terminal 2 Support: The work described in the following research sequence of subunit I of human cytochrome ·c oxidase and reports has been supported by: American Cancer Society with genetically characterized yeast gene sequences has American Heart Association allowed the identification of the mRNA, and therefore of European Molecular Biology Organization E. S. Gosney Fund the coding stretch in mit-DNA, for subunits I and III of National Institutes of Health, USPHS cytochrome c oxidase and for cytochrome b. National Science Foundation Spanish Ministerio de Universidad e Investigacion Another area of interest in our laboratory in the past Universidad Central de Venezuela year has been the phenomenon of dihydrofolic acid reductase (DHFR) gene amplification, in particular its Summary: In the past year, our laboratory has continued mechanism, dynamics and control, in methotrexate-resis­ the analysis of the human mitochondrial genetic system at tant human cell lines which we have recently isolated. the level of the organization of genes and transcr-ipts, Several of these lines have been extensively analyzed to while developing a new line of investigation on the determine the level of their DHFR activity, the stability phenomenon of selective, drug-induced nuclear gene of their phenotype and their chromosomal constitution. In amplification in human cells. all these lines, a greatly increased DHFR activity has The conclusion of extensive investigations on the been found; this increased activity is unstable, that is, structural, metabolic and mapping properties of the declines, though with different kinetics, upon removal of transcription products of HeLa cell mitochondrial DNA the selective agent. Furthermore, all variants analyzed (mit-DN A) has allowed us to develop a coherent picture of exhibit the presence of "double minute chromosomes" gene organization in human mit-DNA and a plausible which are now under investigation. model of expression of this genome. Thus, transcription The human DHFR has been purified from the parental mapping and DNA and RN A sequencing data have revealed cell lines and from a methotrexate-resistant variant, and that the gene arrangement in. human mit-DNA is characterized in its physical and enzymatic properties. extremely compact, with the heavy strand sequences Experiments are in progress to clone the cDNA synthe­ which code for the rRNAs, poly(A)-containing RNAs and sized from DHFR-specific mRNA, with the aim of using it tRNAs forming a continuum for the quasitotality of its as a probe for the molecular dissection of gene length. Furthermore, tRNA genes separate from each amplification. other nearly all the mitochondrial DNA segments coding for the individual rRNAs and poly(A)-containing RNAs, 1. TRANSCRIPTION OF THE HeLll CELL thus providing the punctuation in the reading of mito­ MITOCHONDRIAL GENOME chondrial DNA information. The mapping and sequencing Investigators: Deanna K. Ojala, Christian G. Merkel, data are thus consistent with a model of transcription of Robert A. Gelfand, Julio Montoya the H-strand in the form of a single molecule which is The mitochondrial genome has been shown to encode, processed by precise endonucleolytic cleavages before and in addition to two rRNAs and a number of tRNAs, at least after each tRNA sequence to yield the mature products 18 discrete poly(A)-containing RNA species. A variety of or, in some cases, processing intermediates. techniques, including solution hybridization, Southern One striking result of the sequencing analysis of HeLa blots, Northern blots and Berk and Sharp analyses, were cell mitochondrial RNAs has been that, in contrast to all utilized to localize these polyadenylated species with known eukaryotic mRNAs, the putative mitochondrial respect to a detailed restriction map. Seventeen species 18 were thus successfully localized with an accuracy of about polyacrylamide/urea gels. These analyses were greatly 30-40 nucleotides, allowing a number of interesting facilitated by the development of a scaled-up version of observations to be made concerning the process of the micrococcal nuclease procedure for the preparation of transcription of this genome. First, as was found for the mitochondrial RNA. Thus, it has become possible to rRNA transcripts (Biology 1979, No. 4; Ojala and Attardi, prepare microgram amounts of individual RNA species. 1980), the polyadenylated components are transcribed The first of the species to be sequenced was that of colinearly; no intervening sequences exist. As with the RNA 16, which encodes subunit II of cytochrome c rRNA genes, this is in contrast to the situation observed oxidase. The 5'-end was found to begin directly with the in mitochondrial DNA from yeast, where a complicated AUG initiator codon and in addition, by comparison with processing system has been demonstrated (Van Ommen, the DNA sequence (Barrell, Bankier and Drouin, 1979), Groot and Grivell, 1979). Second, there appears to exist was shown to be immediately contiguous to the 31-end of a no overlapping of the mature polyadenylated species, the tRNA aspartate gene (Ojala, Montoya and Attardi, 1980). rRNAs and the tRNAs (within the resolution described); Therefore, this species lacks a typical ribosome binding furthermore, with the exception of the D-loop region and site. another small region near the origin of replication, the To determine if the lack of a 51-end noncoding stretch mitochondrial DNA sequences appear to be completely is a general characteristic of human mitochondrial utilized for the synthesis of the transcripts. Third, a poly(A)-containing RN~s, nine additional species were comparison of the positions of the DNA sequences which subsequently sequenced. Results thus obtained revealed encode the polyadenylated transcripts with the positions that, with one exception, these RN As begin with AUG or of the tRNA genes, as derived from the DNA sequence (F. AUA (which is a methionine codon in human mito­ Sanger, personal communication), shows that the majority chondria), or have 1 to 8 nucleotides preceding these of these transcripts are flanked at both 5'- and 3'-ends by codons. Moreover, all of the species which have been a tRNA gene. shown to be flanked by a tRNA at their 5'-end (Attardi et A model has .been proposed whereby the tRNA al., 1980) were found, by comparison with DNA sequence sequence or secondary structure may play a role in the data (F. Sanger, personal communication), to begin processing events which form the mature species. immediately following the tRNA 3'-end terminal Specifically, the tRNA sequences would provide signals nucleotide. for precise endonucleolytic cleavage events which would References: generate the mature species from a larger precursor. To Attardi, G., Cantatore, P., Ching, E., Crews, S., Gelfand, R., Merkel, c., Montoya, J. and Ojala, D. (1980) In: provide support for this model, the precise positions of the The Organization and Expression of the Mitochondrial 3'- and 51-ends of the DNA sequences coding for the Genome, A. M. Kroon and C. Saccone (Eds.). North­ Holland, Amsterdam. In press. polyadenylated species with respect to the tRNA genes Barrell, B. G., Bankier, A. T. and Drouin, J. (1979) Nature are at present being determined (see Biology 1980, Nos. 2 282: 189-194. Ojala, D. K., Montoya, J. and Attardi, G. (1980) Nature. and 3). In press. References: Ojala, D. and Attardi, G. (1980) J. Mo!. Biol. 138: 411-420. Van Ommen, G.-J. B., Groot, G. S. P. and Grivell, L. A. (1979) Cell 18: 511-523. 3. 3'-END SEQUENCE ANALYSIS OF MITOCHONDRIAL RNA SPECIES Investigators: Deanna K. Ojala, Julio Montoya

2. 5'-END SEQUENCE ANALYSIS OF Work has begun to sequence the 3'-end proximal MITOCHONDRIAL RNA SPECIES segments of several mitochondrial poly(A)-containing Investigators: Julio Montoya, Deanna K. Ojala RN A species. Briefly, the approach utilizes phased The 5'-end proximal segments of several mitochondrial oligo(dT) primers and cDNA synthesis with reverse tran­ poly(A)-containing RN A species have been sequenced. scriptase., Preliminary results obtained for four species The procedure which was used involves base-specific which are\known to be flanked by a tRNA at their 3'-ends enzymatic cleavage of RNA labeled at the 5'-end, and comparison of these results with DNA sequence data followed by electrophoresis of the digested products in (F. Sanger, personal communication) are consistent with 19

an immediate juxtaposition of the 31 terminus of the rearrangements are systematically found in the DNA of poly(A)-containing RNA with the tRNA 5'-terminal the established recombinant plasmids. nucleotide. A fifth RNA species, which lacks a tRNA at its 31-end 5. AMINO ACID SEQUENCE DllTERMINATION OF THE AMINO TERMINUS OF CYTOCHROME has also been analyzed. In this case, the 3'-end terminal OXIDASE SUBUNIT I nucleotide was found to be immediately contiguous to the Investigators: Anne Chomyn, Michael W. Hunkapiller· 5'-end of an adjacent mRNA. The mitochondrial DNA of human cells codes for the three largest subunits of cytochrome c oxidase (Hare, 4. CLONING OF HUMAN MITOCHONDRIAL DNA Ching and Attardi, 1980). We have succeeded in purifying Investigator: Monica Mottes the largest subunit (COi), of apparent molecular weight The cloning of the whole mitochondrial DNA from 44,600, as estimated from the migration in SDS-urea gels, HeLa cells in Escherichia coli has been attempted at first and in deriving a partial amino acid sequence of an amino using lambda Charon 4A as a vector. Negative results in terminal segment. This has allowed us to identify its these initial experiments suggested a switch to the putative messenger RNA from among those mitochondrial plasmid vector pBR322, which could be used for cloning DNA-coded RNAs for which the sequence of a 5'-end both the entire mitochondrial genome and fragments of it, proximal segment has been determined (Biology 1980, No. obtainable by restriction endonuclease digestion. Mit­ 2). This RNA, RNA species number 9, according to the DN A was cut with several enzymes: Barn HI (1 single numbering system of Amalric et al. (1978) is 1620 cut), Barn Hl-EcoRI (2 cuts), Hind III (3 cuts) and ligated nucleotides long, long enough to encode a protein of 540 amino acids or 64,800 daltons. Identifying the mRNA for with similarly cut pBR322 DNA. Putative recombinant 8 this protein also allows us to map indirectly the gene for colonies, selected according to the phenotype (ApRTc ), this protein subunit in the DNA, since a transcription map were subsequently recognized by colony hybridization. Colonies scored after transformation of xl 776 strain with is available (Ojala et al., 1980). The map position of the gene for COi then corresponds to coordinates 55/100 to mit-DNA cut at its unique Barn site and ligated with Bam­ cut pBR322 gave, in a proportion of 90%, positive signals 66/100, relative to the origin taken as 0/100, with the direction of heavy strand transcription proceeding in the in the hybridization assay with a mit-DNA probe, indi­ cating that they contain mit-DNA. On the other hand, counterclockwise direction. 8 ApRTc colonies produced by pBR322 DNA ligated with References: Amalric, F., Merkel, Gelfand, R. and Attardi, G. Hind Ill or Barn-RI fragments of the rnit-genorne failed to c., (1978) J. Mo!. Biol. 118: 1-25. hybridize with the specific probe. Hare, J., Ching, E. and Attardi, G. (1980) Biochemistry 19: Further restriction analysis of 18 positive clones 2023-2030. Ojala, D., Merkel, C., Gelfand, R. and Attardi, G. (1980) revealed that, in all cases, the rnit-DNA, once integrated Cell. In press. into the plasmid, had undergone some deletion processes which also involved part of the vector DNA and produced 6. CROSS-LINKING HeLa CELL MITOCHONDRIAL heavily rearranged molecules. Surprisingly enough, 15 out DNA IN SITU WITH PSORALEN Investigator: Laura De Francesco of 18 plasmids examined by detailed mapping and Southern blotting experiments appear so far to be identical, the In previous investigations, the association of a protein remaining 3 being slightly smaller. In all cases, the structure of probable membrane derivation with HeLa cell mit-DNA sequence retained has a size of 2.3 kb (that is mitochondrial DNA, at or near its origin of replication, about 1/7 of the entire molecule), while the plasmid DNA has been demonstrated (Albring, Griffith and Attardi, is missing a 200 bp sequence adjacent to the Barn HI site. 1977). The site specificity of this protein-DNA complex Although the causes of the observed phenomena are tended to exclude a random adsorption of the protein not known, it seems from the available data that the structure to the DNA during extraction. However, the in deletion processes-which lead to stable molecules more vivo occurrence of the mitochondrial DNA-protein com­ compatible with the bacterial host-are subjected to plexes could not be demonstrated in the above mentioned strong constraints, since identical or very similar investigations. As an approach to the question of the in 20

vivo existence of these complexes, in the present work the immunoprecipitation with fixed S. aureus cells and the pattern of photochemical inter-strand cross-linking of immunoprecipitates analyzed by polyacrylamide gel HeLa cell mitochondrial DNA by psoralen derivatives has electrophoresis and fluorography, only one band was 125 been investigated. These compounds are known to observed and this ran as [ I]cytochrome c; no larger intercalate between adjacent base pairs of the DNA precursor was detected under these conditions. The same double helix and to covalently cross-link pyrimidines on result was obtained when poly(A)+ RN A from HeLa cell opposite DNA strands upon irradiation with long wave UV cytoplasmic free polysomes was translated in a light (320-380 nmeters). The cross-linking sites can be Micrococcus nuclease-treated reticulocyte lysate. identified by electron microscopic examination of the DNA under denaturing conditions. Since the cell mem­ branes are permeable to the psoralens, it is possible to 8. STUDIES ON THE STABILITY OF AMPLIFIED DHFR ACTIVITY IN Mtx-RESJSTANT HeLa AND study their cross-linking effect in vivo, and to use them as VA -B CELL LINES 2 probes for determining the association with DNA of Investigators: Jeffrey N. Masters, Benneta Keeley proteins or other molecules interfering with the cross­ Eight clones derived from HeLa cell and two clones linking of the DNA strands. The presence of a structure derived from VA -B cells which were previously made around the origin of replication of HeLa cell mit-DNA has 2 resistant to 2 x 10-4 M methotrexate (Mtx} (see Biology been confirmed by this study, which shows that in the 1979, No. 15) were assayed to determine if the amplified majority of mit-DNA molecules there is a region around DHFR activity was stable or unstable when the selection the origin which does not become cross-linked when the pressure of Mtx was removed. The assay is adapted from mit-DNA is cross-linked in situ, while the remainder of Frearson, Kit and Dubbs (1966) and measures the the molecule becomes densely cross-linked. In contrast, reduction of dihydrofolate to tetrahydrofolate in the the entire molecule can be cross-linked in vitro, a result presence of NADPH. which eliminates the possibility that the sequence itself in These results showed that, at the same level of Mtx that region is refractory to cross-linking. resistance, the DHFR activity of the resistant cells varied Reference: from 30 to 300 times the activity of the parental cells. Albring, M., Griffith, J. and Attardi, G. (1977) Proc. Nat. in Acad. Sci. USA 74, 1348-1352. When grown the absence of Mtx, all of the resistant cell lines lost DHFR activity (i.e., were unstable}, but at 7. IN VITRO SYNTHF.SJS OF HUMAN different rates. One cell line reached the parental DHFR CYTOCHROME C activity in 31 days while another was still losing activity Investigators: NestOr Gonz8Iez-Cadavid, Giuseppe Attardi after 241 days. Another may be stabilizing at a DHFR activity of about 26% of its amplified level (or 33 times In a project parallel to the study of the mechanism of the parental level). synthesis of nuclear-coded subunits of human cyto­ Mtx was added back to a portion of one of the HeLa chrome c oxidase, we are investigating the in vitro derived cell lines (10B-3R) when its DHFR activity had synthesis of human cytochrome c in order to obtain a dropped by 78%. 0.3% of these cells (10B-3R+R) survived comparative picture of their intracellular transport and and had the same amplified level of DHFR activity as processing and to develop an assay for future cloning 10B-3R cells. 10B-3R+R cells were also unstable with experiments. Liver free and membrane-bound polysomes respect to DHFR activity and lost it with the same and a Neurospora mitochondrial supernatant have been kinetics as 10B-3R. shown to synthesize cytochrome c, although there is no When portions of cell extracts from these cell lines are agreement as to whether the prosthetic group is linked to subjected to SDS-PAGE, a band corresponding to human the apoprotein in the cytoplasm or the mitochondria. DHFR (see Biology 1980, No. 11) can be seen in resistant We have prepared elee-trophoretically homogeneous cells but not in the pa.rental cells, and this band has the cytochrome c from human heart and liver and used it to same mobility in each cell line. raise specific antibodies in rabbits. When post-mito­ chondrial supernatants from HeLa cells labeled in vivo for Reference: Frearson, P. M., Kit, S. and Dubbs, D. R. (1966) Cancer 5 2 hr with f s]methionine were subjected to indirect Res. 26: 1653-1660. 21

9. CHARACTERIZATION OF DHFR mRNA correlation with degree of resistance to methotrexate, Investigator: Jeffrey N. Masters increase in level of DHFR activity or degree of instability

RNA has been extracted from six Mtx-resistant (MtxR) of the methotrexate resistance after removal of the drug. Thus, the variant 10B3 (a derivative of HeLa cell Bu25) cell lines, four derived from HeLa cell and two derived 8 which has only a few double minutes per cell (less than 10 from VA -B cell l~nes, and the Mtx-sensitive (Mtx ) 2 in the majority of cells) is more unstable in its drug parental cells. Poly(A)-containing RNA was isolated on resistance than the variant 6A3 (a derivative of the oligo(dT)-cellulose columns, treated with DNase I and VA -B cell line) which has a similar increase in DHFR electrophoresed on agarose-methylmercuric hydroxide 2 gels. An RN A species of 4 kb was seen in the MtxR cells activity, but a much larger number of double minutes per but not in the Mtxs cells. cell (the majority of cells having more than 100 up to This RN A species from one of the MtxR cell lines was several hundreds). However, an analysis of the quantitative behavior of the double minute chromosomes extracted from a gel (Wieslander, 1979) together with a corresponding region of the RNA electrophoretic pattern in the variant 6A3 in the absence of selective pressure has from the parental cell line. When the 4 kb RNA was used revealed that the loss of double minutes does parallel the decrease in DHFR activity after subtracting a basal level in an in vitro rabbit reticulocyte translation system, and the products were subjected to SDS-PAGE, a pronounced of stable increased activity (.!'25%). This result is band was observed with the same mobility as human consistent with the idea that the number of double DHFR (see Biology 1980, No. 11); no band of this intensity minutes in this variant, 6A3, reflects the degree of was visible in the control. unstable DHFR gene amplification, and that these genes may actually be carried, at least in part, by the double Reference: Wieslander, L. (1979) Anal. Biochem. 98: 305-309. minute chromosomes themselves. References: Kaufman, R. J., Brown, P. C. and Schimke, R. (1979) Proc. 10. DOUBLE MINUTE CHROMOSOMES IN Nat. Acad. Sci. USA 76: 5669-5673. METHOTRRXATE-RESISTANT HUMAN Levan, A. and Levan, G. (1978) Hereditas 88: 81-92. CELL LINES Investigators: Helen Gay, Jeffrey N. Masters, 11. PURIFICATION OF HUMAN DlliYDROFOLIC Giuseppe Attardi ACID REDUCTASE AND DETERMINATION OF Several variants of human cell lines resistant to high ITS KINETIC AND PHYSICAL PARAMETERS Investigator: Csrlo Morandi levels of methotrexate, a folic acid analog, have been isolated in this laboratory (Biology 1979, No. 15). These Dihydrofolic acid reductase (DHFR) has been purified lines all exhibit greatly increased levels of activity of from the HeLa cell derivative Bu25 and from a variant of dihydrofolic acid reductase (DHFR), the target enzyme of this cell line resistant to methotrexate (MTXR) (see the drug, presumably due to a drug-induced DHFR gene Biology 1979, No. 15) by ammonium sulfate precipitation amplification. In order to gain an insight into the possible followed by affinity chromatography (Kaufman and genetic changes leading to this increased DHFR activity, Kemerer, 1976; Alt, Kellems and Schimke, 1976). an analysis of the chromosomal constitution of these The two enzymes were estimated to have the same variants is being carried out. molecular weight-23,000 daltons-by using different All variants analyzed so far exhibit the presence of methods of polyacrylamide gel electrophoresis. small, usually paired, acentromeric chromosomal ele­ The Michaelis constants were determined for both ments, designated "double minute chromosomes," which enzymes and found to be identical: 5.7 x 10-S M for folic are substantially absent in the parental cell lines. The acid and 2 x 10-4 M for NADPH. Titration curves with presence of such chromosomal elements had been pre­ methotrexate are also identical for the two enzymes. viously reported in a variety of experimental and human The calculated specific activities correspond to 44,000 tumors and in methotrexate-resistant murine cell lines. U/mg, where one unit of folate reductase activity is the The average number and range of these double minute amount necessary to reduce 1 nmol of folate in 15 min chromosomes vary considerably among the methotrexate­ (Alt, Kellems and Schimke, 1976). The profiles of pH resistant human cell variants, without any obvious direct dependence of the DHFR activity are the same for the 22

two enzymes, with the maximum activity at pH 4. 7 5. minutes) in 6A3 cells, a methotrexate-resistant cell line Antiserum produced in rabbits immunized against pure derived from VA2 cells. From 4 x 108 cells, 70% of which DHFRase from MTXR cells, reacts equally well with pure are in arrested metaphase, we can purify 5-10 µg of enzyme prepared from the parental cells (MTXs). minutes DNA. This DNA will be tested first for the

References: presence of the DHFR gene using a cDNA probe at Alt, F. W., Kellems, R. E. and Schimke, R. T. (1976) J. present being prepared (see Biology 1980, No. 12). The Biol. Chem. 251: 3063-3074. DNA will be further characterized as to its sequence Kaufman, B. T. and Kemerer, V. F. (1976) Arch. Biochem. Biophys. 172: 289-300. complexity, the types of sequences present, and their arrangement. This type of information should give us 12. CLONING OF cDNA FROM some clues as to how gene amplification occurs. PURlFIED DHFR mRNA References: Investigators: Carlo Morandi, Jeffrey N. Masters Alt, F. W., Kellems, R. E., Bertino, J. R. and Schimke, R. At present we are synthesizing cDNA from the 4 kb T. (1978) J. Biol. Chem. 253: 1357-1370. Nunberg, J. H., Kaufman, R. J., Schimke, R. T., Urlaub, mRNA which has been purified from methotrexate­ G. and Chasin, L. A. (1978) Proc. Nat. Acad. Sci. USA 75: 5553-5556. resistant human cell lines and positively identified by in vitro translation as DHFR mRNA. DHFR cDNA will be inserted into pBR322 using poly dA/dT tailing and cloned in E. coli 2282 as bacterial host, which allows x PUBLICATIONS trimethoprim R selection of the clones which express Attardi, G. (1980) Organization and expression of the dihydrofolic acid reductase activity (Chang et al., 1978). mammalian mitochondrial genome: a lesson in Alternatively to phenotypic selection, Ap8 clones will be economy. Trends Biochem. Sci. In press. Attardi, G., Cantatore, P., Ching, E., Crews, S., Gelfand, screened by colony hybridization using the method devised R., Merkel, C., Montoya, J. and Ojala, D. (1980) The by St. John and Davis (1979). The cloned DHFR cDNA remarkable features of gene organization and will be used as a probe to study DHFR gene organization expression of human mitochondrial DNA. In: The Organization and Expression- of the Mitochondrial 8 in MTX as well as MTXR cell line DNAs. Genome, A. M. Kroon and C. Saccone (Eds.). North- Holland, Amsterdam. In press. , References: Attardi, G., Cantatore, P ., Ching, E., Crews, S., Gelfand, Chang, A. C. Y., Nunberg, J. H., Kaufman, R. J., Erlich, R., Merkel, c. and Ojala, D. (1979) The organization of H. A., Schimke, R. T. and Cohen, S. N. (1978) Nature the genes in the human mitochondrial genome and 275: 617-624. their mode of transcription. In: Extrachromosomal St. John, T. P. and Davis, R. W. (1979) Cell 16: 443-452. DNA, ICN-UCLA Symposium on Extrachromosomal DNA, Vol. 15, pp. 443-469. Attardi, G., Crews, S. T., Nishiguchi, J., Ojala, D. K. and 13. DNA FROM DOUBLE MINUTE CHROMOSOMES Posakony, J. W. (1979) Nucleotide sequence of a IN HUMAN METHOTREXATE-RESISTANT CELLS fragment of HeLa cell mitochondrial DNA containing Investigator: Anne Chomyri the precisely localized origin of replication. Cold Spring Harbor Symp. Quant. Biol. 43: 179-192. Double minute chromosomes occur in some human cell Cantatore, P. and Attardi, G. (1980) Mapping of nascent light and heavy strand transcripts on the physical map lines which have been selected for resistance to high of HeLa cell mitochondrial DNA. Nucleic Acids. Res. 8: 2605-2624. levels of methotrexate (Biology 1979, No. 15). These Crews, S. and Attardi, G. (1980) The sequences of the resistant cell lines have increased levels of the target small ribosomal RNA gene and the phenylalanine tRNA gene are joined end-to-end in human mitochondrial enzyme, dihydrofolate reductase (DHFR) (see Biology DNA. Cell 19: 775-784. 1980, No. 8). These increased levels are probably due to De Francesco, L. and Attardi, G. (1980) Analysis of amplification of the DHFR gene, as is the case with the sequence homology between human and mouse mito­ chondrial DNA. J. Mo!. Biol. 139: 85-93. mouse and Chinese hamster ovary cell lines (Alt et al., De Francesco, L., Attardi, G. and Croce, C. (1980) 1978; Nunberg et al., 1978). The double minute chromo­ Uniparental propagation of mitochondrial DNA in mouse-human cell hybrids. Proc. Nat. Acad. Sci. USA somes putatively carry at least some of the extra copies 77: 4079-4083. of this gene. Hare, J. F., Ching, E. and Attardi, G. (1980) Isolation, subunit composition and site of synthesis of human We have separated the double minute chromosomes cytochrome c oxidase. Biochemistry 19: 2023-2030. from the normal complement of human chromosomes (less Ojala, D. and Attardi, G. (1980) Fine mapping of the ribosomal RN A genes of HeLa cell mitochondrial DNA. than one contaminating normal chromosome per 800 J. Mo!. BioL 138: 411-420. 23

Ojala, D., Merkel, C., Gelfand, R. and Attardi, G. (1980) Ojala, D., Montoya, J. and Attardi, G. (1980) The putative The tRNA genes punctuate the reading of genetic mRNA for subunit II of human cytochrome c oxidase information in human mitochondrial DNA. Cell. In starts directly at the translation initiator codon. press. Nature. In press.

Professor: James Bonner introns, that is, the noncoding sequences which interrupt Sherman Pairehild Distinguished Scholar: Francois-Xavier Wilhelm the coding sequences of this gene. The a-fetoprotein g~ne Visiting Associates: Theodore A. Goodman, Josette M. is even more complicated, containing as it does about Michelot, Jose M. Sala-Trepat, R. Bruce Wallace, Marcelle L. Wilhelm 20,500 base pairs of DNA. Senior Research Pellow: Jung-Rung Wu The description of these genes and of their base Research Fellows: Ricardo Castro-Gonzalez, Linda L. Jagodzinski, Gordon C. Machray, Murray F. Teitell, composition of their coding sequences is detailed below. John E. Wiktorowicz The purpose of the exercise of isolating these genes, Graduate Student: Thomas D. Sargent Research staff: Ingelore S. Bonner, Carlotta A. Glackin, however, is to discover in what manner they are con­ Maria Yu-Li Yang trolled, that is, caused to be expressed or nonexpressed in Laboratory Staff: Stella Olive liver or in fetuses, respectively. To this end, we have SUpport: The work described in the following research conducted a massive program of trying to discover how to reports has been supported by: American Cancer Society reconstitute DNA into mini chromosomes, let us say a Biomedical Research Support Grant (NIH) 20,000-base-pair-long piece of DNA into a mini chromo­ Norman W. Church Foundation City of Hope Medical Center some containing 100 nucleosomes each consisting of eight Fairchild Foundation core histones and one histone 1 (Hl). French National Institute for Medical Research National Institutes of Health, USPHS The reconstitution of chromatin into nucleosomes Damon Runyon-Walter Winchell Cancer Fund occurs in life at a physiological concentration of about University of California, San Diego 0.25 M salt and in the absence of any other obvious Summary: Our group is dedicated to finding out how physical chemical factor. It is known that chromosomes eukaryote genes are transformed from nontranscribable to can be reconstituted, although imperfectly, by dissolving transcribable-that is, from inactive to active in the histones and DNA in 2 M salt and dialyzing away _the salt eukaryotic genome. To this end we have done a vast bit by bit to physiological ionic strength. However, such variety of experiments. During the past two years we reconstituted chromosomes are not authentic-the have devoted ourselves principally to the isolation of two nucleosome spacing is not authentic, the nucleosome individual genes: that for serum albumin which is composition is not as it is in native chromatin. We have expressed (in fact the most expressed gene) in the liver of therefore carried out a program to try to find out how to adult mammal, and that for a-fetoprotein which is the reconstitute chromatin at physiological ionic strength. In most expressed gene in the fetuses of mammals. The a­ this effort, we have been joined by Professor Douglas fetoprotein gene is turned off when the fetus is born. The Brutlag of Stanford University who is doing the same thing serum albumin gene is turned on at that time. The cx-­ with Drosophila embryo chromatin. fetoprotein gene is turned on again in cases of liver Mammalian DNA can be reconstituted with carcinogenesis, etc. mammalian histones, that is, the four core histones H2a, In the course of our isolation of these two genes, it has H2b, H3 and H4 in 0.2 M salt, provided that the turned out that the genetic material is complicated chromosomal protein, HMG 1, is simultaneously present. beyond anything that we had expected. For example, the The HMG 1 must be first incubated with histones and then serum albumin gene is about 14,500 base pairs long, this mixture incubated with DNA. It is possible also that con~ists of 14 coding sequences interrupted by 13 non­ a DNA nicking-closing enzyme may contribute to the coding sequences and ended by two ending sequences, each efficiency of chromatin reconstitution. 'Ye have not yet of which contains a repetitive sequence. The serum accomplished more than nucleosome reconstitution. albumin gene also contains two repetitive sequences in the Proper spacing between nucleosomes has not been 24

accomplished. It is possible that Hl is required for such surprising and significant result: the enzyme operates spacing. We do not know. under an ordered bireactant mechanism, i.e., as an initial When we can reconstitute fragments of DNA into mini step to the enzymatic reaction, histones must bind to the chromosomes, we will then be in a position to determine enzyme active site. This may induce the correct what nuclear components from rat liver or from fetal rat configuration in the active site required for acetyl CoA liver are required to make the serum albumin coding (the acetate donor in vivo) binding and for the reaction to fragments transcribable and the a-fetoprotein fragments proceed to completion. not transcribable and vice versa. We hope to accomplish We have observed that purified nucleosomes are, at this during the coming few months. best, slightly acetylated by exogenous enzyme. Since, in this state, the hydrophobic inner core of the nucleosome is

14. THE ROLE OF IDSTONE ACETYLATION IN buried and likely to be inaccessible to external macro­ GENE EXPRESSION molecules, we have reasoned that nucleosome acetylation Investigators: John E. Wiktorowiez, may require that the inner core be exposed, i.e., the Kenneth L. Campos* nucleosomes be in an "open" configuration (at present, this Histones are organized in the nucleus in a complex can be achieved in moderately high salt). It is possible structure called the nucleosome (Hewish and Burgoyne, that the enzyme may then attach to the "open" nucleo­ 1973; Kornberg and Thomas, 1974). The ~our histones, some (and only those nucleosomes) by virtue of mutual H2a, H2b, H3 and H4, interact in pairs with 140 base pairs hydrophobic attraction. The enzyme is thus brought into of DNA to form a core particle. An additional 60 bp of proximity of a localized saturating histone concentration DNA are bound to histone Hl and form a linker region and thus satisfies the first requirement for enzyme between core particles to complete the nucleosome activity, the binding of histone to the enzyme active site. structure. Since the histones interact with the DNA by In the presence of acetyl CoA, histones in the nucleosome virtue of ionic attractions, modification of the histone will then be acetylated and the loosening of the DNA­ side chains may cause alterations in the positive charge histone interaction will be achieved allowing, presumably, density and lead to a loosening of the histone-DNA transcription to occur. attraction, as well as conformational changes in the core We are at present investigating the validity of this particle (Wallace et al., 1977). Indeed, chemical acetyl­ hypothesis, as well as the possible role of histone ation of chromatin with acetic anhydride leads to an deacetylase and its inhibitors. We believe this approach increase in DNA-dependent RNA synthesis (Marushige, will allow us to answer the large question of why some 1976). In addition, highly acetylated histones are prefer­ nucleosomes are acetylated and others are not, and entially associated with template-active chromatin (Davie possibly therefore the larger question of why some genes and Candido, 1978). In order to understand nucleosomal are turned on and others not. modification, specifically acetylation, and its relationship References: to gene derepression, we have investigated the kinetic and Davie, J. A. and Candido, E. P. M. (1978) Proc. Nat. Acad. Sci. USA 75: 3574-3577. structural properties of histone acetyltransferase, the Hewish, D. and Burgoyne, L. (1973) Biochim. Biophys. nuclear enzyme responsible for nucleosome acetylation. Acta 474: 141-153. Recently, we have reported the purification of histone Kornberg, R. and Thomas, J. (1974) Science 184: 865-868. Marushige, K. (1976) Proc. Nat. Acad. Sci. USA 73: acetyltransferase (Wiktorowicz, Campos and Bonner, 3937-3941: 1980a) and in the course of this purification have observed Wallace, R. B.,. Sargent, T., Murphy, R. and Bonner, J. (1977) Proc. Nat. Acad. Sci. USA 74: 3244-3248. a marked tendency for the enzyme to aggregate with Wiktorowicz, J. E., Campos, K. L. and Bonner, J. (1980a) other proteins (primarily histones) into higher molecular Manuscript in preparation. Wiktorowicz, J. E., Campos, K. L. and Bonner, J. (1980b) weight aggregates. This has become significant since we Manuscript in preparation. have determined that the enzyme contains regions of *Undergraduate, California Institute of Technology. hydrophobicity which are probably located on the surface of the molecule. In addition, a rigorous analysis of the complex kinetics involved in the enzyme reaction (Wiktorowicz, Campos and Bonner, 1980b) has yielded a 25

15. THE FINE STRUCTURE AND EVOLUTION OF Eco RI rat library and also to screen a new library THE RAT SERUM ALBUMIN GENE constructed from Hae III partials. Between the two Investigators: Thomas D. Sergent, Maria Yu-Li Yang, libraries, we have isolated the entire a-fetoprotein gene James Bonner which appears to be approximately 20 kb in length. The goal of this project is to determine the nucleotide sequence of the entire rat serum albumin messenger RNA 17. THE DNA SEQUENCE OF and the nucleotide sequence of all 14 exons in this gene. RAT a-FETOPROTEIN GENE This has been done by sequencing cloned cDNA and Investigators: Linda L. Jagodzinski, Thomas D. Sargent genomic sequences by the Maxam and Gilbert method. The rat a-fetoprotein (AFP) gene coding sequence has The nucleotide sequence data can be used to demonstrate been inserted into plasmid pBR322. Seven AFP cDNA the relationship between the various domains and sub­ clones have been isolated. The DNA sequence of these domains in the albumin protein (Brown, 1976). These cDNA clones is being determined by the sequencing relationships can also be correlated to the structure of the method of Maxam and Gilbert. The sequence data will be albumin gene. Our results indicate that the present employed in the analysis of the AFP rat genomic clones. mammalian albumin gene may have arisen via a complex It will also give us the amino acid sequence of the AFP sequence of intragenic duplications of a primordial proto­ protein. A comparison of two developmentally-related alburnin gene that may have contained no intervening genes, the tat AFP and serum albumin genes, can be sequences. accomplished through their DNA and amino acid Ancillary results include the inference of the complete sequences. rat serum albumin amino acid sequence fr

19. NUCLEOSOME ASSEMBLY IN VITRO important step in the process of nucleosome assembly. To test this hypothesis, mixtures of HMG 1 and histones were Investigators: Gordon C.. Machray, Carlotta A. Glackin, Francois-Xavier Wilhelm, treated with the protein cross-linking reagent dimethyl Marcelle L. Wilhelm suberimidate and it was found that, in the presence of The role played by the structure of chromatin in the HMG 1, specific high molecular weight complexes were control of gene transcription is not well understood as yet. formed. When histones alone were cross-linked under the Nuclease digestion and electron microscopy studies have same ionic strength conditions, ·only dimers of histones shown that the nucleosomes associated with transcrip­ were formed. The role of the HMG protein would thus be tionally-active chromatin have an altered conformation as to stabilize a tetrameric or octameric complex of histones compared to nucleosomes of the bulk inactive chromatin. which would then be transferred to the DNA.

In order to study the role of f~ctors involved in the regulation of gene transcription, model systems can be used in which the purified components of chromatin are reconstituted in a controlled fashion. Previous methods such as dialysis from high to low salt concentration have been successful only in the reconsti­ tution of nucleosome core particles. More recently, nucleosome assembly under physiological salt conditions Figw-e 1. Mini chromosomes assembled in vitro at has been achieved by the mediation of protein extracts physiological ionic strength. Col E, DNA. histones and from various sources. We have found that partially­ HMG 1 were mixed in 0.1 M NaCl, 10 mM Tris and 0.1 mM EDTA. purified protein extracts of rat liver chromatin are able to mediate the assembly of nucleosomes from isolated DNA HMG 1 is not the only protein we suspect of playing a and histone at physiological ionic strength. The nucleo­ part in the assembly of nucleosomes. Nicking-closing somes thus formed sediment at llS, contain 140 bp of enzyme has been reported to mediate the assembly of DNA, and are revealed as identical to control nucleosomes nucleosomes in vitro (Germond et al., 1979). We have by electron microscopy. We have not observed, however, purified nicking-closing enzyme from rat liver and are the typical repeating structure of nucleosomes seen in now investigating its role in nucleosome assembly by itself native chromatin. In order to remove possible sources of and also in combination with HMG 1. Thus, nucleosome interference in the assembly process, we are now investi­ assembly may involve several factors; we are just gating the role of individual proteins, purified from beginning to understand the complexity of the assembly chromatin extracts, in nucleosome assembly. process. It has been shown recently that acidic polypeptides such as polyglutamic acid greatly faei!itate the assembly References: Germond, J.-E., Yaniv, M., Rouviere-Yaniv, J. and of nucleosomes at physiological ionic strength (Stein, Brutlag, D. (1979) Proc. Nat. Acad. Sci. USA 76: Whitlock and Biiia, 1979). HMG 1, a nonhistone protein 3779-3783. Stein, A., Whitlock, J. P. Jr. and Bina, M. (1979) Proc. extracted from chromatin by 0.35 M NaCl, is very rich in Nat. Acad. Sci. USA 76: 5000-5004. 27

When 10 mM sodium butyrate was used during the 20. UNFOLDING OF THE NUCLEOSOME IN IDGHSALT isolation and DNAase II fractionation of rat liver nuclei, Investigators: Marcelle L. Wilhelm, we found the following: (1) About 60% more A260 Francois-Xavier Wilhelm absorbing material is liberated after 5 minutes of The structure of chromatin is not static, but undergoes digestion, when the limit of digestion is reached, than structural changes necessary for the functions in which it with nontreated nuclei. This value is similar even using participates in the cell. Electron microscopic obser­ five times more enzyme, that is, between 10-50 vations as well as nuclease digestion studies have shown units/ A unit of nuclei, and until 60 minutes at least of 260 that subtle changes occur at the nucleosomal level during digestion. This difference is principally due to DNA replication or in the transcriptionally-active genes. digestion. The amount of total RNA liberated is Interestingly, recent transcription studies of in vitro essentially the same in both cases. This finding is in assembled chromatin have revealed that the propagation agreement with previous work of the author that shows rate of RNA polymerase along the nucleoprotein template that rat liver nuclei treated with 10 mM sodium butyrate is increased at high salt concentration. This has prompted incorporate about 60% more of acetyl groups into us to study in more detail the conformation of chromatin histones. In addition, the presence of sodium butyrate in high salt. We have used electron microscopic, causes a 40% increase in the DN Aase activity on pure hydrodynamic and spectroscopic techniques to study the DNA. (2) Following the DNAase II fractionation method conformation of the nucleosome and we have been able to developed in this lab, both the solubilized and the show that the nucleosome assumes a very extended precipitated chromatin fractions show a pattern of sizes conformation when the salt concentration is increased. which are multiples of approximately 200 base pairs (bp). The most direct evidence for the alteration of nucleosome The soluble fractions are mostly of 200-400 bp, in treated structure in high salt is obtained by electron microscopy as in nontreated nuclei. Two interesting bands of where the nucleosomes are no longer observed as beads approximately 70 and 100 bp are clearly identifiable only but have a rod-like shape. The unfolding of the in the digested and 2 mM MgC1 soluble fraction from 2 nucleosome could explain the mechanism by which the treated nuclei. (3) The DNAs from the soluble and passage of RNA polymerase is facilitated on chromatin precipitated fractions from sodium butyrate-treated and templates in high salt. There is some evidence that nontreated nuclei were hybridized to cDNA made from rat specific nonhistone proteins (HMG 14 and 17) are bound to liver polysomal poly(A) + total mRNA. The comparison. o f active genes in vivo. The interaction of these proteins the Cot curves indicates an impoverishment in comple­ with histones and/or the acetylation of histones could play mentary sequences to the cDNA in both fractions of the a role similar to that of the ionic strength by decreasing treated nuclei. This fact could be due to a degradation of the number of positive charges of the histones available the active sequences, as a result of a greater DNAase II for the interaction with the phosphate groups of DNA. It accessibility in the treated nuclei. would thus be of interest to study the structure of These data .suggest the theory that chromatin hyper­ chromatin assembled in vitro with acetylated histones, acetylation is the cause of the greater DNAase II together with HMG 14 and 17, and see whether it is digestion, and that this digestion is extended in the possible to unfold the nucleosome under more physio­ presence of sodium butyrate to new chromatin zones that logical conditions. are recognized by the acetyltransferase and acetylated. Our intention is to discover if HMG proteins are involved 21. ACTIVE CHROMATIN in this recognition. Investigator: Ricardo castro-GonzaJ.ez

There is much accumulated evidence that indicates PUBLICATIONS that a greater DNA nuclease susceptibility, an enrichment Bakke, A. C. and Bonner, J. (1979) The purification and in HMG proteins, and an increased histone acetylation are the histones of Dictyostelium discoideum chromatin. characteristics of active chromatin. It is also known that Biochemistry 18: 4556-4562. Bonner J. (1979) The World's People and the World's Food sodium butyrate increases histone acetylation because of Sup~ly, J. J. Head (Ed.). Scientific Publ. Division, its inhibitory effect on deacetylase activity. Carolina Biological Readers. 28

Douvas, A., Reyes-Lopez, P., Tan, E. and Stumph, W. Sell, S., Sala-Trepat, J. M., Sargent, T. D., Thomas, K., (1979) Isolation and characterization of nuclear ribo­ Nahon, J. L., Goodman, T. A. and Bonner, J. (1980) nucleoprotein complexes using human anti-RNP anti­ Molecular mechanisms of control of albumin and a.­ bodies. J. Biol. Chem. 254: 3608-3619. fetoprotein production. A system to study the early Dube, S. K., Wallace, R. B. and Bonner, J. (1979) State of effects of chemical hepatocarcinogens. Cell Biol. globin gene in Friend cell. Cold Spring Harbor Symp. Internat. Reports 4: 235-254. Quant. Biol. In press. Sell, s., Thomas, K., Michaelson, M., Sala-Trepat, J. M. Murphy, R. F., Wallace, R. B. and Bonner, J. (1980) and Bonner, J. (1979) Control of albumin and a­ Isolation of newly replicated chromatin by using fetoprotein expression in rat liver and in some shallow meterizimide gradients. Proc. Nat. Acad. Sci. transplantable hepatocellular carcinomas. Biochim. USA 77: 3336-3340. Biophys. Acta 564: 173-178. Sargent, T., Sala-Trepat, J., Wallace, R. B., Reyes, A. and Thomas, K., Skelly, H., Michaelson, M., Sala-Trepat, J. Bonner, J. (1979) The rat serum albumin gene. M., Sell, S. and Bonner, J. (1979) Expression of Analysis of cloned sequences. J. Supra. Malec. Struct. albumin and AFP genes in fetal rat liver and yolk sac. 8: 64. ICN-UCLA Symposia Molecular and Cellular Biology Sargent, T. D., Wu, J.-R., Sala-Trepat, J. M., Wallace, R. (Abstract). B., Reyes, A. A. and Bonner, J. (1979) The rat serum Wallace, R. B., Shaffer, J., Murphy, R. F ., Bonner, J., albumin gene: Analysis of cloned sequences. Proc. Hirose, T. and Itakura, K. (1979) Hybridization of Nat. Acad. Sci. USA 76: 3256-3260. synthetic oligodeoxyribonucleotides to 0X174 DNA: The effect of single base pair mismatch. Nucleic Acids Res. 6: 3543-3557.

Professor: Eric H. Davidson Summary: The work of our laboratory can be described in Senior Research Associate: Roy J. Britten* Research Associate: Barbara R. Hough-Evans very general terms as concerned with the molecular Visiting Amociates: Oscar L. Miller, Lajos PikO**, Ji-hou biology of development. This has come to include basic Xin Senior Research Fellows: David M. Anderson, John W. studies of the organization of genomic DNA sequences, _ Roberts, Terry L. Thomas and of changes in single copy (nonrepetitive) and in Gosney Research Fellows: Carlos V. Cabrera, Ze'ev Lev Research Fellows: Susan G. Ernst, Constantin Flytzanis, repeated sequences both between individuals (DNA poly­ John W. Grula, Terrence J. Hall, Laurence A. Lasky, morphism) and in evolution. Much of the analysis of DNA Gordon P. Moore, Richard H. Scheller Graduate Students: Frank Costantini, Jay W. Ellison, relationships has been done with sea urchins, and in Boning Gao, James W. Posakony particular with the local Southern California purple sea Research Staff: Maria Alonso, Alison E. Blake, Margaret E. Chamberlin, Steven T. Eckmann, Geoffrey J. urchin, Strongylocentrotus purpuratus. We keep a large Graham, Patrick S. Leahy, Jara Lewin, Michael Lusby, stock of these sea urchins in running seawater tanks at the Jean Mueller, Jane Rigg, Nelson L. Smith, Steven W. Trabert Division of Biology Kerckhoff Marine Laboratory at Laboratory Staff: Fargo Balliett, Joseph A. Garcia Jr., Corona del Mar, where several members of our laboratory Terrence Glugni, Linda B. McAllister, George Pauls, Juanito Villanueva are conducting their research. Besides investigating genomic DNAs, we have long *Concurrently a member of the staff of the Carnegie Institution of Washington. been interested in their various RNA transcription **Veterans Administration Medical Center, Sepulveda, products. Our ultimate aim is to increase our under­ California. standing of how gene expression is regulated, and in particular of the processes of gene regulation which take Support: The work described in the following research reports has been supported by: place during the development of eggs and embryos. A American Cancer Society number of related projects are being carried out which Biomedical Research Support Grant (NIH) California Foundation for Biochemical Research involve the analysis of nuclear RN As and messenger RNAs Carnegie Institution of Washington of sea urchin eggs, embryos, and adult tissues. Norman W. Church Foundation Charles B. Corser Fund for Biological Research Both DNA and RNA studies have been enriched and Deutsches Krebsforschungszentrum expanded by the application of recombinant DNA tech­ E. S. Gosney Fund National Institutes of Health, USPHS nology. We have constructed, and are making extensive National Science Foundation use of, libraries of total sea urchin DNA cloned in A phage University of Virginia Veterans Administration (some of these libraries were prepared with the DNA of a 29

single individual). Also available are sets of randomly­ sequence ranging from less than 1 % to more than 10%. selected cloned sea urchin DNA fragments, and Restriction endonuclease digestion has been used to individually-cloned repetitive sequences. More recently, investigate the sequence polymorphism in regions sur- cDNA libraries derived from the poly(A)+ cytoplasmic rounding several structural genes expressed in S. RNA sequences of gastrula and pluteus stage embryos purpuratus gastrula cDNA. When a single copy labeled have been prepared, and the libraries and individual clones probe is incubated with a blot of a restriction digest of an selected from them are contributing to a wide variety of individual DNA, one or two bands usually appear on an works in progress. autoradiograph. The variation of the positions of the bands for several individuals allows an estimate of the 22. SINGLE COPY DNA POLYMORPHISM OF number of "alleles" or restriction site variants for each SEA URCIIlNS IN THE GENUS enzyme. One cDNA plasmid probe (SpG19) gave the same STRONGLYOCENTROTUS band for all eight individual DNA Eco RI digests. Plasmid Investigators: John W. Grula, Terrence J. Hell, John Hunt•, Terrence Giugni, SpG30 also gave only one allele with Eco RI and two Roy J. Britten alleles were detected with Hind III. Thus, these two genes Since the first observation of a large amount (4%) of appear to occur in regions with little polymorphism. single copy DNA polymorphism in the sea urchin, Plasmid SpG6 showed four alleles with Eco RI, four with Stronglyocentrotus purpuratus (Britten, Cetta and Barn HI and two with Hind III, and is in a somewhat more Davidson, 1978), we have been interested in determining polymorphic region. Another structural gene region (Sp88) the generality of this variation among other species. has been examined in considerable detail as described Therefore, the single copy DNA polymorphism of two elsewhere in this report (Biology 1980, No. 25). Among related sea urchins, S. drobachiensis and S. franciscanus, eight individual DNAs, it shows eight alleles with Eco RI, has been measured. Both exhibit a slightly smaller degree six with Hae III, three with Pst and eight with Hind III. of polymorphism than s. purpuratus (2% and 3% for S. -Thus, this region is the most polymorphic so far studied by drobachiensis and S. franciscanus, respectively). this technique. Calculation suggests that it may be in a Additional measurements indicate that there are no length region showing about 4% polymorphism while SpG6 is in a differences in the duplexes formed with DNA from the region showing about 2%. The restriction analyses are same or an unrelated individual. This suggests that the consistent with single copy DNA polymorphism measured observed polymorphism in these species is mainly attribut­ by thermal stability and show different degrees of able to randomly distributed base substitutions and not polymorphism adjacent to different genes. DNA rearrangements. Studies utilizing cloned DNA are now in progress to determine the relative importance of 24. HUMAN SINGLE COPY DNA POLYMORPHISM these alternative processes in generating intraspecific Investigators: Terrence J. Hall, Terrence Giugni, single copy DNA variation. Michael Cummings*, Roy J. Britten Human single copy DNA polymorphism has recently Reference: Britten, R. J., Cetta, A. and Davidson, E. H. (1978) Cell been examined. DNA was isolated from a single individ­ 15: 1175-1186. ual. Two different 3H-DNA tracers were prepared by in *University of Hawaii, Honolulu, Hawaii. vitro labeling of a human fibroblast culture or by nick translation of sperm DNA and isolation of the single copy 23. ANALYSIS OF SEQUENCE POLYMORPHISM DNA. The tracers were reacted with DNAs from the IN REGIONS SURROUNDING same individual, from an unrelated individual, and a STRUCTURAL GENES mixture of DNA obtained from more than 20 individuals. Investigators: John W. Grula, John W. Roberts, Terry L. Thomas, Laurence A. Lasky, The thermal stability reduction for the interindividual Eric H. Davidson, Roy J. Britten duplexes compared to controls indicates that there is Thermal stability measurements have demonstrated approximately 1 % base substitution in human single copy about 4% single copy DNA sequence polymorphism in the DNA. This observation for total single copy is consistent sea urchin Strongylocentrotus purpuratus. There is with the data of Jeffreys (1979) concerning the degree of evidence that this is the average of differences in base substitution within an intervening sequence of a- 30

globin DNA. Lev, z., Thomas, T. L., Lee, A. S., Angerer, R. C., Britten, R. J. and Davidson, E. H. (1980) Devel. BioL Reference: 76: 322-340. Jeffreys, A. J. (1979) Cell 18: 1-10.

*Illinois Institute for the Study of Developmental 26. ISOLATION OF INSULIN-LIKE DNA Disabilities, Chicago, Illinois. SEQUENCES FROM THE SEA URCIIlN Investigators: Terry L. Thomas, Argiris Efstratiadis*, Eric H. Davidson 25. SEQUENCE ORGANIZATION AROUND A STRUCTURAL GENE IN SEA URCIIlN DNA The importance of insulin and other peptide hormones Investigators: Terry L. Thomas, NeJson L. Smith, in vertebrate physiology is well documented. Eric H. Davidson Interestingly, there are a number of examples of insulin­ We have cloned a sea urchin genomic DNA fragment, like activities in invertebrates, including the starfish, an Sp88, that contains a single copy sequence which codes for echinoderm. We have isolated three different insulin-like rare mRNAs. The number of Sp88 mRNA transcripts per DNA sequences from sea urchin DNA by screening our embryo changes drastically during _sea urchin development Stronglyocentrotus purpuratus bacteriophage A libraries (Lev et al., 1980). In addition, four distinct size classes of with a cloned rat insulin cDNA probe. This clone was Sp88 RNA transcripts have been found in early sea urchin constructed by Villa-Komaroff et al. (1978). Filter embryos (Lee et al., 1980). At least three species are hybridization experiments suggest about 70% homology localized on polysomes of the 16-cell embryo; however, by between these sea urchin A recombinants and the rat the gastrula stage the predominant RNA species is preproinsulin gene. These homologies are being confirmed approximately 6 kilobases (kb) in length and is represented by direct DNA sequencing• . almost exclusively in the nuclear RNA. Our interest in these sea urchin DNA clones is directed Sp88 has a complex sequence organization with several at the genome organization surrouqding the DNA se­ nonrepetitive DNA sequenee elements interspersed with quences homologous to the insulin gene, and the expres­ different repetitive sequence elements. However, all of sion of these sequences both in nuclear RNA and mRNA in the Sp88 sequences are located 3' to the coding region. embryonic and adult tissues. Preliminary RNA/DNA We have recently isolated longer DNA sequences con­ hybridization experiments with one of the insulin-like taining the Sp88 structural gene. This was accomplished sequences show that this DNA sequence is represented at by screening bacteriophage A libraries, containing approxi­ a very low level in both pluteus and intestine total RNA. mately 15 kb sea urchin DNA inserts, with labeled DNA We will also compare the DNA sequence of these insulin­ probes made from the codogenic region of Sp88. One of like recombinants with the already determined DNA these A recombinants, A-16, contains approximately 12 kb sequences of insulin structural genes of other species. of genomic DNA 5' to the original Sp88 coding region. Reference: With A-16, we are examining the longer range sequence Villa-Komaroff, L., Efstratiadis, A., Broome, S., organization surrounding the Sp88 codogenic sequence, Lomedico, P., Tizard, R., Naber, S. P., Chick, W. L. and Gilbert, W. (1978) Proc. Nat. Acad. Sci. USA 75: i.e., we wish to define with respect to the coding region 3727-3731. the location of repetitive sequence elements and other *Harvard University School of Medicine, Boston, codogenic and noncodogenic single copy DNA sequences. Massachusetts. Additionally, A-16 is being used to investigate the interesting and probably complex relationship between the 27. DNA REARRANGEMENTS AROUND primary transcripts of Sp88 and its respective mRNAs. HOMOLOGOUS STRUCTURAL GENES IN TWO SEA URCIIlNS This will allow us to determine whether the multiple Investigators: John W. Roberts, John W. Grula, polyadenylated transcripts seen in the 16-cell embryo are Eric H. Davidson, Roy J. Britten, processing intermediates or whether they are different Laurence A. Lasky

mRNAs that overlap at their 3' termini. Little is known about the nature of DNA sequence References: changes which underlie evolutionary change on· the pheno­ Lee, A. s., Thomas, T. L. Lev, z., Britten, .R. J. and typic level. However, it is clear that the genome shows Davidson, E. H. (1980) Proc. Nat. Acad. Sci. USA. In press. great evolutionary plasticity. The number of members of 31 families of repeated sequences change rapidly in evolu­ was reacted with total DNA from S. purpuratus, S. tion. When probes representing particular repeat families drobachiensis, S. franciscanus, Lytechinus pictus, and are reassociated with "Southern blots11 of DNA restriction Tripneustes gratilla. The heteroduplexes were isolated digests, bands are observed representing multiple precise and melted in the chaotropic solvent, tetraethyl copies as well as wide distributions of fragment sizes ammonium chloride (2.4 M). 'The lengths of duplexes were representing interspersed short repeats. When the DNA determined after digestion with 81 nuclease and correc­ from the related sea urchins Strongylocentrotus tions were made for the effect of length on thermal purpuratus and S. franciscanus are compared, particular stability. The temperature at which 50% of the cDNA repeat families show several different bands in each tracer remained as duplex was compared to previous species. This implies that a number of different events of measurements for the same set of species with total multiplication have occurred in this repeat family in both single copy DNA tracers. The results indicate that the genomes. The interspersed repeats also show large evolutionary rate of change for these coding sequences is contrasts in quantity between the two species. Thus, between one-half and two-thirds of that for the average mafly events of multiplication, insertion and deletion have single copy DNA sequence. occurred and more than one mechanism is probably involved. 29. REPETITIVE SEQUENCES OF THE To explore these DNA rearrangements, we have SEA URCHIN GENOME started comparing large (15-20 kb) pieces of DNA con­ Investigators: David M. Anderson, Richard H. Scheller, taining homologous structural genes which have been Linda B. McAllister, Steven W. Trabert isolated from the recombinant A phage libraries of two sea Three repetitive sequence families from the sea urchin urchins, S. purpuratus and S. franciscanus. Two plasmid genome were studied, each defined by homology with a + cDNA clones made from s. purpuratus gastrula poly(A) specific cloned probe one to a few hundred nucleotide RNA have been used to screen the libraries of each pairs (nt) long. Recombinant >- sea urchin DNA libraries species. Lambda recombinants containing an insert which were screened with these probes, and individual recombi­ is homologous to a prevalent poly(Ai+ RNA have been nants were selected which include genomic members of purified from each library. The A recombinants from each these families. Restriction mapping, gel blot, and kinetic species sharing this structural gene region will be com­ analyses were carried out to determine the organization pared directly by electron microscope analysis of hetero- of each repeat family. Sequence elements belonging to duplexes and restriction enzyme analysis. We will the first of the three repeat families were found to be measure rearrangement events, reiteration frequency and embedded in longer repeat sequences. These repeat relatedness of regions near each structural gene in both sequences frequently occur in small clusters. Members of species. the second repeat family are also found in a long repetitive sequence environment, but these repeats

28. EVOLUTION OF SEA URCHIN DNA usually occur singly in any given region of the DNA. The CODING SEQUENCES sequences of the third repeat are only 200 to 300 nt long, Investigators: Terrence J. Hall, Laurence A. Lasky, and are generally terminated by single copy DNA, though Roy J. Britten a few examples were found associated with other repeats. The thermal stabilities were measured for hetero­ These three repeat sequence families constitute sets of duplexes formed by the reassociation of total DNA from homologous sequence elements which relate distant five different sea urchin species with cDNA prepared regions of the DNA. + from Strongylocentrotus purpuratus gastrula poly(A) cytoplasmic RNA. The results indicate that the sequences 30. NUCLEOTIDE SEQUENCE ANALYSIS OF THE FINE STRUCTURE OF REPETITIVE DNA represented in the S. purpuratus gastrula cDNA are Investigators: James Posakony, Eric IL Davidson conserved to a greater extent than that of total single w. copy DNA. (Estimates from this laboratory indicate As part of our investigations into the nature of gastrula cDNA sequences represent less than 1% of the repetitive sequence families in the sea urchin genome, we single copy DNA complexity.) The gastrula cDNA tracer have been studying the fine structure of repeated DNA by 32

nucleotide sequence analysis. We would like to answer We are currently engaged in DNA sequencing and RNA several important questions, among which are: What are blotting experiments directed at understanding the func­ the main features of the internal organization of repeti­ tion of this 3' repetitive sequence family. tive sequences? What, if any, sequence relationships exist between different repeat families at a fine level? What

are the structural relationships between individual mem­ 32. CYCLES OF OOGENESJS IN WILD AND bers of a repetitive sequence family? LABORATORY-MAINTAINED SEA URCHINS We have determined the nucleotide sequences of Investigators: Patrick S. Leahy, Eric H. Davidson

several cloned repeat elements, representing both dif­ The reproductive state of female sea urchins from ferent repetitive families and different members of the representative wild populations and in laboratory culture same family. Our analysis of these data (1) indicates that has been monitored every 2-3 months for several years. repeats are generally complex sequences belonging to Data are based on counts of the numbers of previtello­ distinct families, (2) reveals the existence of significant genic, vitellogenic, and mature oocytes in disaggregated internal structural organization in at least some repeat ovaries, and on average egg yields when the animals elements, and (3) demonstrates a striking non-colinearity spawned artificially after KCl injection. As reported in of different members of the same repetitive family, previous investigations, fecundity of intertidal sea urchins indicative of fine-scale sequence rearrangement. is sharply seasonal. Two peaks of fertility per year were noted, and in the summer months virtually none of the 31. ISOLATION AND CHARACTERIZATION animals contain any large vitellogenic oocytes or mature OF A GENE BAITERY eggs. In contrast, a certain number of the female sea Investigators: Richard H. Scheller, David M. Anderson, urchins living at 50-70-foot depths in a kelp bed were Linda B. McAllister, Eric H. Davidson found to contain mature oocytes all through the year. The gastrula cDNA clone SpG2 contains 700 nucleo­ These urchins can be collected and maintained under tides of sea urchin DNA. This clone was cleaved into laboratory conditions, and will continue to show a useful three 200-300 nucleotide fragments (band 1, band 2 and asynchrony in egg production. band 3), and these were used individually to screen a A library of the sea urchin genome. Band 1 was found complementary to six distinct regions of the genome. 33. SEA URCHIN EGG POLY(A)+ RNAs CONTAIN Band 3, located on the 3' end of the cDNA, was found to TRANSCRIPTS OF REPEATED DNA SEQUENCES be reiterated about 100 times per haploid genome. Electron microscope heteroduplexing and R-loop experi­ Investigators: Frank Costantini, Eric H. Davidson ments revealed that the band 1 clones contain an 1800 The sea urchin genome includes over 100,000 short nucleotide coding region and two small divergent inter­ repetitive sequence elements interspersed with the single vening sequences. Hybridization selection with band 1 copy DNA regions. Earlier experiments showed that the followed by translation in a rabbit reticulocyte lysate genomic repeats belong to several thousand distinct system resulted in the synthesis of a 40,000 dalton sequence families, which in general share little or no protein. homology. Though the biological role of these sequences The band 3 clones (i.e., those that hybridize to band 3 is unknown, almost all of the diverse repeat families are but not band 1) contain transcribed regions flanking the represented in unfertilized egg RNA. Using cloned

band 3 repetitive element. Blots of these clones with fragments of rep~ated and single copy sea urchin DNA to randomly and oligo(dT) initiated oocyte poly(Ai+ cDNA analyze egg poly(A) + RNA, we found that half the mass show that the repetitive element is usually on the 3' end and most of the maternal mRNA sequences are covalently of the transcript. We have, therefore, a set of diverse associated with transcripts of short repetitive sequences. messenger RN As with a common 3' repetitive element, Only a restricted group of the diverse genomic repeat and present this as a structural definition of a gene families is significantly represented. The maternal battery. While the sequence organization is quite striking, messages fall into several hundred sets, each containing the function of this repetitive family is to date unknown. transcripts from a different repeat family. 33

34. NATURE AND FATE OF REPETITIVE the Eco RI site of pBR325. These plasmids were used to SEQUENCE TRANSCRIPTS IN THE transform E.coli strain HB101. We will use the subclones SEA URCHIN EGG containing repeats to determine their expression, preva­ Investigators: James W. Posakony, Eric H. Davidson lence and other properties in the Xenopus oocyte. It will Previous studies in this laboratory demonstrated that be of interest to compare our findings with what is the majority of the repetitive sequence families in the sea already known about repeated DNA transcripts in sea urchin genome are represented in the RNA of the urchin eggs. unfertilized egg, and that both strands of each repeat are Reference: represented approximately equally. Furthermore, it was Costantini, F. D., Scheller, R. H., Britten, R. J. and shown that most of the different single copy maternal Davidson, E. H. (1978) Cell 15: 173-187. mRNA sequences in the egg are covalently associated 36. COMPLEXITY OF RNA IN DEVELOPING with repetitive sequence transcripts. OOCYTES OF DROSOPHILA We are interested in the fate of the egg repeat­ Investigators: Barbara R. Hough-Evans, containing transcripts after fertilization. For example, Marcelo Jacobs-Lorena*, Eric IL Davidson how are they localized with respect to the nuclei and cytoplasm of the early cleavage-stage embryo? Are they Mass separation of Drosophila egg chambers into subjected to RNA processing steps? Do any of them several size classes was accomplished by means of a become associated immediately with ribosomes for trans­ column of nylon nets of graduated mesh size, and RNA lation? was extracted from each egg chamber of each size class. As a basis for approaching these questions, we have We measured the complexity of the RNAs by hybridizing investigated the nature of the repetitive sequence tran­ them with labeled single copy Drosophila DNA. Egg scripts in the polyadenylated fraction of egg RNA. Using chamber RN As from all stages of oogenesis, including cloned DNA probes, we have found that the four repeat early pre-vitel!ogenesis, contained single copy DNA tran­ families studied so far are represented by multiple large scripts equal in complexity to the RN A of the mature (3-10 kilobase) transcripts, and that the transcripts Drosophila egg. complementary to the individual strands of each repeat *Case Western Reserve University School of Medicine, are of distinct sizes. Cleveland, Ohio.

37. ELECTRON MICROSCOPE VISUALIZATION OF 35. REPETITIVE SEQUENCE TRANSCRIPTS IN TRANSCRIPTION IN ECHINODERM OOCYTES XENOPUS EGG RNA Investigators: Barbara R. Hough-Evans, Investigators: Margaret E .. Chamberlin, Eric H. Davidson Oscar L. Miller, Eric H. Davidson

Repetitive sequences in mature oocytes of the sea We have used a special technique (Miller and Beatty, urchin have been extensively studied in this laboratory 1969) to spread out chromatin from sea urchin oocytes for (Costantini et aL, 1978). We wish to observe and compare electron microscope observations. We found closely­ various characteristics of repetitive sequence transcripts packed gradients of ribosomal RNA transcripts, s~parated in the .oocytes of another organism, Xenopus laevis. by typical nontranscribed spacer regions. Although In preliminary experiments we self-annealed poly(A)+ oocytes of several size classes were examined, from early RNA from Xenopus stage 6 (mature) oocytes to repetitive vitellogenic (40 mic.ron diameter) to late, nearly mature RNA Cots and visualized it in the electron microscope. A oocytes, we did not find nonribosomal transcripts in great deal of duplex is observed, indicating transcripts of similar arrays, although gradients of more widely spaced repetitive sequence in the poly(A)+ DNA. In order to transcripts were seen. Thus, typical lampbrush chromo­ further characterize these transcripts, we have con­ some arrangements of long transcripts such as have been structed clones of genomic repeats. We accomplished this observed in frog oocyte chromatin appear to be absent by subcloning Hae III digests of random clones from a from sea urchin oocytes. A comparative study using Charon 4 whole genome library of X. laevis. The Hae Ill oocytes of a starfish (in which typical lampbrush chromo­ fragments were annealed with an excess of the appro­ somes have been observed with light microscopy) has been priate decamer and ligated into the Barn site of pBR322 or initiated. 34

Reference: vitelline layer (VL) proteins during development of sea Miller, 0. L. and Beatty, B. R. (1969) Science 164: 955-957. urchin oocytes? What is the length of time needed to accumulate the steady state amount of a given VL prot-ein 38. RNA SYNTHESJS IN THE FERTILIZED in the mature egg? In order to try to answer these MOUSE EGG questions, we first isolated VL proteins from mature sea Investigators: Lajos Piko*, Kerry B. Clegg* urchin eggs, separated them on two-dimensional acryl­ Previous studies have shown that the mouse egg amide gels, and stained the second-dimension gel to inherits a substantial amount of stored RNA but that new characterize the VL proteins. About 30 different proteins embryo-derived RN As are synthesized at least from the have been identified in this way. 2-cell stage onward. Whether the pronuclei of the 1-cell We then dissected developing oocytes of different fertilized egg are active in RNA synthesis has not been sizes out of sea urchin ovaries, and separated 40 µ, 50 µ, determined, due in part to the low permeability of the egg 60 µ and 70 µ (diameter) oocytes (the mature eggs are to most labeled precursors. "'70-80 µ in diameter). The oocytes were incubated with 35 We have investigated RNA synthesis in the mouse egg [ s ]methionine to label newly-synthesized proteins. after labeling with r3H]adenosine which is taken up about Labeled samples were analyzed together with carrier lOOX more efficiently than uridine. Total RN A was vitelline layer proteins. If the VL proteins (and their extracted by an SDS-proteinase K-phenol procedure and mRNAs) are prevalent, some of the newly-labeled proteins the labeled products were analyzed by LiCI precipitation, will coincide with carrier VL proteins on two-dimensional binding to poly(U) sepharose, electrophoresis in polyacryl- gels. In experiments to date, these coincident spots have amide gels, and enzymatic digestion. The specific not been seen, and we conclude tentatively that the activity of the ATP pool was monitored. The following vitelline layer proteins are synthesized by a set of rare main results were obtained: (1) Transfer RN A is heavily mRNAs. labeled at the -CCA terminal, but some new synthesis of transfer RNA also takes place. (2) The synthesis of 40. LIMITED COMPLEXITY OF RNA IN ribosomal RNA is either absent or occurs at a rate of less MICROMERES OF 16-CELL SEA URCHIN EMBRYOS than 0.5% that in 816-cell embryos (on a per cell basis). Investigators: Susan G. Ernst, Erie H. Davidson (3) Heterogeneous nuclear RNA is synthesized at an equilibrium level of about 0.5 pg per egg. (4) A Sea urchin eggs divide into cells equal in size until the substantial amount (about 0.25 pg over 5 hr) of poly(A) fourth division. This unequal division produces a 16-cell tracts are added to previously nonpolyadenylated, stored embryo with three distinguishable cell types. Micromeres, RNA. (5) On the basis of internally localized rH ]adeno­ the smallest of the cells, give rise to the cells which form sine label, new synthesis of poly(Al-containing RNA also the primary mesenchyme and produce the embryo skele­ occurs at a low rate. (6) Newly polyadenylated RNA and ton, later in development. newly synthesized poly(A)-containing RNA are associated We examined the RNA of micromeres isolated from with polysomes. These data indicate that the fertilized 16-cell embryos, and found that, in contrast to the mouse egg synthesizes several types of RNA with the cytoplasm of the cells in the rest of the embryo, notable exception of ribosomal RNA. Cytoplasmic poly­ micromere cytoplasm contains few, if any, complex adenylation is significant and may represent recruitment maternal RN A species which are not represented in the of stored matemal messenger RNA. polysomes. Both polysomal and cytoplasmic RNAs contain about 75% of the sequence complexity of maternal *Veterans Administration Medical Center, Sepulveda, California. mRNA. Total 16-cell embryo cytoplasmic RNA includes all the sequence complexity of maternal mRNA, although 39. SYNTHESIS OF VITELLINE LAYER PROTEINS the polysomal mRNA complexity again amounts to only OF SEA URCHIN EGGS 75% of the maternal 37 million nucleotides of diverse Investigators: Boning Gao, Barbara R. Hough-Evans, sequence. Eric H. Davidson It appears that the differences which determine the What is the concentration of mRN A coding for micromere pattern of differentiation occur subsequent to 35

the 16-cell stage, and do not result from localization of We have isolated large genomic clones (10-20 kb in very complex sets of maternal RNA sequences. length), which contain the SpG30 and SpG6 cDNA sequences, from a A phage-sea urchin genome library. We 41. Ml!SSENGER RNA PREVALENCE IN SEA are at present isolating the genomic clones containing URCIIlN EMBRYOS MEASURED WITH SpGlO. These clones are being mapped using a variety of CLONED eDNAs restriction enzymes and the Southern blotting technique, Investigators: Laurence A. Lasky, Ze'ev Lev, 32 Ji4tou Xin, Eric H. Davidson with the appropriate cDNA clones as P-labeled probes. Future experiments will revolve around determining Measurements of mRNA prevalence during sea urchin the nature of the factors which establish the cytoplasmic development were made by reacting cDNA clone colonies concentrations of these three mRNA sequences. Detailed with labeled cDNA transcribed from unfertilized egg and + transcription mapping using both Sl nuclease protection embryo poly(A) RN As. The number of cytoplasmic and electron microscope technology will allow us to transcripts per embryo complementary to three cDNA measure the extent of the transcribed regions in these clones was determined independently by titration with clones. Subsequent experiments will deal with the poly(At RNA in solution, and the amount of cDNA bound genomic sequence organization surrounding these tran­ to these clones in colony hybridization was shown to be scribed regions. Finally, it is hoped that these clones will proportional to the concentration of the respective be amenable to transcription analysis using either oocyte poly(A)+ RNAs in the embryo cytoplasm. Using these injection, or in vitro transcription with sea urchin egg clones as standards for comparison, we found that at the lysates or more purified RNA polymerase II systems. gastrula stage, the most prevalent mRNA species occur at about 10 6 molecules per embryo. If all cells were equivalent, this would be a few hundred molecules per 43. PATTERNS OF EXPRESSION OF mRNA SEQUENCES OF SEA URCIIlN EMBRYOS cell. By pluteus stage, the prevalence of some sequences Investigators: Ji-hou Xin, Laurence A.. Lasky, has increased more than 10-fold. Most, though not all, Eric IL Davidson sequences prevalent in later embryos are also present in the maternal RNA of the unfertilized egg. For most Libraries of cDNA clones have been constructed by cloning of cDNA-cytoplasmic poly(A)+ RNA hybrids of sea poly{A) + RNA sequences, the prevalence levels deter- urchin gastrula and pluteus stage embryos. Hybridization mined during oogenesis are maintained throughout early of cDNA clone libraries with cDNA transcribed from the development, while a minority of sequences display sharp + poly(A) RNA of unfertilized eggs and of gastrula and stage-specific changes. pluteus stage embryos has been used to describe the distribution of mRNA prevalence classes in these 42. REGULATION OF mRNA PREVALENCE IN THE SEA URCIIlN EMBRYO embryos. The results show that for most abundant + Investigators: Ze'ev Lev, Laurence A. Lasky, poly{A) RNA sequences, the prevalence level is main- Gordon P. Moore, Erie H. Davidson tained throughout all embryo stages. In addition, some Three cDNA clones, SpGIO, SpG6 and SpG30, have genes are expressed in a stage-specific manner, although been selected from a gastrula cDNA clone library on the they are in the minority (see Biology 1980, No. 42). 32 basis of very different hybridization signals with P­ It would be interesting to know whether these preva­ labeled gastrula cDNA. The approximate cytoplasmic lent sequences are also expressed in adult tissues. We are prevalence of these clones has been determined using a therefore screening the cDN A libraries with intestine, variety of methods including colony hybridization (see coelomocyte and other adult tissue cDNAs. Thus, it may Biology 1980, No. 41), RNA-DNA titration, and labeling be possible to find some cDNA clones which are specific kinetics. These experiments have demonstrated the for the embryo. The embryo-specific clones will be following average cytoplasmic prevalence levels: SpGlO, 4 further characterized with respect to their presence and copies/cell; SpG6, 20 copies/cell; SpG30, 200 copies/eel!. state in adult tissue hnRNA. It is hoped that in this way Thus, these clones represent poly(A) mRN A sequences the mechanisms which control the expression of these which vary in cytoplasmic concentration by almost two sequences specifically in the early embryo will be orders of magnitude. elucidated. 36

44. RATES OF ACCUMULATION AND TURNOVER RNA (Ellison, Britten and Davidson, 1980; Biology 1980, OF INDIVIDUAL MESSENGER RNAs No. 44). Therefore a comparative study should provide Investigators: Jay Ellison, James Moore, w. evidence as to which one of either the rate of transcrip­ F.ric H. Davidson tion or the fraction of precursor RN As that are processed In order to learn about the relative contribution of and exported to the cytoplasm is the primary factor in factors which affect the prevalence of mRN A species, we determining the prevalence of cytoplasmic RN As. have measured both the rates of transport of transcripts References: to the cytoplasm and the half-lives of individual mRN As. Davidson, E. H. and Britten, R. J. (1979) Science 204: Sea urchin embryos were labeled with [3H]guanosine 1052-1059. Ellison, J. W., Britten, R. J. and Davidson, E. H. (1980). In during the blastula-gastrula stages of development (26-44 preparation. hours after fertilization). Cytoplasmic poly(A)+ RNA was Lasky, L. A., Lev, Z., Xin, J.-H., Britten, R. J. and Davidson, E. H. (1980) Proc. Nat. Acad. Sci. USA. In prepared at various times after addition of label. These press. 3 H-RNA samples were incubated with filters containing PUBLICATIONS an excess of a given cDNA plasmid. The rate of incorporation of radioactivity into an individual poly(A)+ Anderson, D. M., Scheller, R. H., Posakony, J. W., McAllister, L. B., Trabert, S. W., Britten, R. J. and RN A species could be obtained from the cpm bound to the Davidson, E. H. (1980) Repetitive sequences of the sea filters. By measuring the 3H-GTP specific activity we urchin genome. 1. Distribution of members of specific repetitive families. Submitted for publication. can convert the hybridization data to units of mass, and Britten, R. J., Hall, T. J., Lev, Z., Moore, G. P., Lee, A. we thus derive: (1) molecules per minute per cell S., Thomas, T. L. and Davidson, E. H. (1979) Examples of evolution and expression in the sea urchin genome. transported to the cytoplasm, and (2) half-life of the In: Eucaryotic Gene Regulation, ICN-UCLA Sym­ mRNA species. A number of cDNA clones representing posium on Molecular and Cellular Biology, Vol. 14, R. Axel, T. Maniatis and C. F. Fox (Eds), pp. 219-227. RNAs of different prevalence in gastrula embryos have Academic Press, New York. been analyzed in this way. The data obtained thus far Costantini, F. D., Britten, R. J. and Davidson, E. H. (1980) Message sequences and short repetitive sequences are indicate that more prevalent RNAs have both a greater interspersed in sea urchin egg poly(A)+ RN As. Nature. rate of transport and a longer half-life than the rarer In press. Ernst, S. G., Hough-Evans, B. R., Britten, R. J. and RNAs. Transcripts present at 1-5 copies per cell have Davidson, E. H. (1980) Limited complexity of the RNA half-lives similar to that of the total poly( At RN A in micromeres of 16-cell sea urchin embryos. Devel. Biol. In press. (2-3 hr), while the more abundant species are more stable Hall, T. J., Grula, J. W., Davidson, E. H. and Britten, R. J. (t, >12 hr). (1980) Evolution of sea urchin non-repetitive DNA. J. ·- Malee. Evol. In press. 45. TURNOVER RATES OF STRUCTURAL GENE Hough-Evans, B. R., Jacobs-Lorena, M., Cummings, M. R., TRANSCRIPTS IN NUCLEAR RNAs Britten, R. J. and Davidson, E. H. (1980) Complexity of RNA in eggs of Drosophila melanogaster and Musca Investigators: Carlos Cabrera, F.ric H. Davidson v. domestica. Genetics. In press. Jacobs-Lorena, M., Hough-Evans, B. R., Britten, R. J. and The purpose of this study is to test a proposition Davidson, E. H. (1980) Complexity of RNA in (Davidson and Britten, 1979) which was developed upon developing oocytes of Drosophila melanogaster. Devel. Biol. 76: 509-513. recent findings of the ubiquitous representation of nuclear Lasky, L. A., Lev, Z., Xin, J.-H., Britten, R. J, and RNAs (nRNAs) in distinct tissues of the adult sea urchin Davidson, E. H. (1980) Messenger RNA prevalence in sea urchin embryos measured with cloned cDNAs. and in embryonic stages. Submitted for publication. Our experimental approach makes use of a cDN A Lee, A. S., Thomas, T. L., Lev, Z., Britten, R. J, and Davidson, E. H. (1980) Four sizes of transcript pro­ library made from poly( RN A at the gastrula stage At duced by a single sea urchin gene expressed in early (Lasky et al., 1980). An excess of individual cDNA clones embryos. Proc. Nat. Acad. Sci. USA. In press. Lev, Z., Thomas, T. L., Lee, A. S., Angerer, R. C., differing in prevalence in the cytoplasm is being hybrid­ Britten, R. J. and Davidson, E. H. (1980) Develop­ 3 ized to nRNAs labeled with [ H]guanosine at different mental expression of two cloned sequences coding for rare sea urchin embryo messages. Devel. Biol. 76: times. The rate constants for synthesis and degradation 322-340. of the individual nRN A molecules are then calculated, Moore, G. P., Costantini, F. D., Posakony, J. W., Davidson, E. H. and Britten, R. J. (1980) Evolutionary taking into account the specific activity of the endo­ conservation of repetitive sequence expression in sea genous GTP pool, as a function of the labeling time. urchin egg RNAs. Science 208: 1046-1048. Similar data have been obtained for the cytoplasmic 37

Professor: William J. Dreyer which control their synthesis will certainly provide Visiting Associate: Rodney M. Hewick considerable insight into the basic mechanisms involved in Senior Research Fellow: Janet M. Roman Graduate Students: Douglas A. Fisher, David E. Levy their synthesis and function. Such molecules are scarce, Research Staff: Barbara L. DeOgny, Jeanne P. Patalano, since they are expressed predominantly on the cell Gayle-Linda Westrate Laboratory Staff: Jeffrey R. Brinkley, Carolyn A. surface; however, if detailed knowledge of the structure Burhenn, Barbara-Lynn Cass of these molecules is obtained, it is possible to devise Support: The work described in the following research appropriate probes and to clone the relevant genes. reports has been supported by: Molecules (antigens) which have certain properties Biomedical Research Support Grant (NIH) Norman W. Church Foundation expected of area code molecules have been detected on E. I. DuPont de Nemours & Company cultured cell lines derived from both rodent and human The Max C. Fleischmann Foundation National Institutes of Health, USPHS tumors. Some cell-surface antigens differ on each cell National Science Foundation line studied. In other words tumor A, derived from a given tissue type, differs in a stable and heritable fashion Summary: The genetic code and the basic structure of from tumors B, C, D, etc., also derived from the same DNA has been understood for so long that the subject is tissue. Tumor B displays a different "unique" antigen on now taught at the high school level. In spite of this, an its surface and so do tumors C and Do Other classes of article published last year expresses both the excitement antigens are shared to differing degrees among various and the complete mystery which continue to surround the tumor cell lines. Only in the case of tumors of the basic question of how genetic programs control the immune system is the phenomenon of unique tumor development of living organisms: antigenicity understood. In that case, the heritable and "For the real amazement, if you want to be unique antigenic properties of different B-cell lines are amazed, is the process. You start out as a single cell derived from the coupling of a due to spliced genes which code for the variable regions of sperm and an egg. this divides into two, then antibody molecules. In addition to the theoretical four, then eight, and so on, and at a certain stage there emerges a single cell which will questions raised by the existence of tumor antigens of have as all its progeny the human brain. The various types, they are also of interest in relation to mere existence of that cell should be one of the great astonishments of the earth ... No one possible immunological control of cancer and as diagnostic has the ghost of an idea how this works and aids (Seli, 1980). nothing else in life can ever be so puzzling." Cell-surface molecules are relatively scarce since they From "On Embryology" by Lewis Thomas (1979) are found in only a small fraction of the total c.ell volume; Humans have always been fascinated by this subject; however, new experimental approaches now greatly indeed numerous scientists at Caltech have concerned simplify the process of isolation of cell-surface molecules, themselves with developmental biology since the time the the study of their structure, the cloning of the genes Biology Division was founded over 50 years ago. Never­ which code for such molecules, and the analysis of cloned theless, it is only in the past few years that a combination genes. These include: (1) New immunological methods of new experimental methods and theoretical insights has including the technology for the production of monoclonal generated research programs which promise definitive antibodies and immunoabsorbants. (2) Methods for answers to questions which surround this subject. Many isolating useful quantities of cell-surface proteins from scientists now agree that families of cell-surface recog­ cultured cell lines. (3) Highly sensitive and effective nition molecules (we call them area code molecules) are instruments and methods for the study of the structure of differentially expressed on cell lineages as they unfold cell-surface proteins and glycoproteins. (4) Methods for (Dreyer, Gray and Hood, 1967; Hood, Huang and Dreyer, using the knowledge gained by the use of the previous 1977). Area code molecules are believed to play a central approaches to clone the relevant genes and to determine role in programmed cell migration and selective cell their base sequence. associations which develop during embryogenesis. Just as The abstracts which follow not only describe the has been true in studies of the generation of diversity in biological systems which we are studying at this time, but the immune system, studies of the amino acid sequence of also reflect the considerable investment which we have area code molecules and of the base sequence of genes made in developing the instruments and procedures which 38

now make it possible to carry out definitive studies of separate tumor lines vary in the amino acid sequence of interesting cell-surface proteins. their envelope proteins. The origin of this heterogeneity is unknown; however, it may reflect unique recombination References: Dreyer, W. J., Gray, W. R. and Hood, L. (1967) Cold Spring events among endogenous viral envelope genes or between Harbor Symp. Quant. Biol. 32: 353-367. viral and cellular genetic material. Hood, L., Huang, H. V. and Dreyer, W. J. (1977) J. Supramol. Struct. 7: 531-559. We are presently investigating whether the molecules Sell, S. (Ed.) (1980) Cancer Markers: Diagnostic and identified in this study function as tumor transplantation Developmental Significance. Humana Press, Clifton, New Jersey. antigens and/or as cell-surface proteins important in Thomas, L. (1979) On Embryology. In: The Medusa and normal development. the Snail, pp. 156-157. Viking Press.

47. MOLECULAR CHARACTERIZATION OP 46. HERITABLE MOLECULAR DIVERSITY IN HUMAN TUMOR MARKERS TUMOR CELL-SURFACE ANTIGENS Investigators: Janet M. Roman, Barbara L. DeOgny Investigators: Jeanne P. Patalano, David E. Levy, Barbara L. DeOgny, Janet M. Roman The recent widespread application of hybridoma tech­ A two-dimensional gel electrophoretic analysis of the nology to the study of human tumors has led to the cell-surface proteins of a series of syngeneic, UV light­ production of a large number of monoclonal antibodies induced murine fibrosarcomas has revealed heterogeneity directed against human tumor markers. The patterns of within a family of proteins antigenically related to the reactivity of these antibodies raise a number of envelope protein (gp70) of C-type retroviruses. This interesting questions concerning the nature of surface variability involves differences in the apparent molecular antigens found on tumor cell lines, the relationship of weights of molecules immunoprecipitated by syngeneic molecules on different tumor lines to each other, and the anti-tumor sera and by xenogeneic anti-viral sera. relationship of these molecules to molecules on normal Although not all distinct tumor lines can be differentiated tissues. by this technique, two classes of tumors are defined on We are using a series of immunological and protein the basis of these two-dimensional patterns. From each chemical methods to obtain definitive answers to these tumor can be precipitated two series of molecules with questions. separate molecular weights, each with extensive internal We have obtained cell lines of human fibrosarcomas, isoelectric point variability. The multiplicity of iso­ carcinomas, melanomas, neuroblastomas, gliomas, and electric points is presumably due to a heterogeneous sarcomas, as well as a variety of cell lines of normal number of terminal, acidic carbohydrate moieties on a tissues, from R. Saxton and D. Morton (UCLA, Division of single amino acid sequence. The source of the molecular Oncology, Department of Surgery), R. Seeger (UCLA, weight differences is presently unknown. These molecular Department of Pediatrics, Allergy, and Immunology), and patterns are stable, heritable characteristics of the tumor S. Rasheed and M. Gardner (USC, Department of lines whether passaged in vivo or in vitro. We have found Pathology). These groups have made available to us any that even early passage tumors are productively infected of the other cell lines in their libraries of frozen cells. with an endogenous retrovirus. However, a two- We have also obtained a series of monoclonal anti­ dimensional gel analysis of the viral proteins recovered bodies against these human tumor lines from our from the tumors does riot contain either the full collaborators in Southern California and elsewhere. The complexity of the cell-surface proteins or the between­ antibodies are being used on an analytical scale for class differences. immunoprecipitation and characterization of a series of Two-dimensional maps of tyrosine-containing tryptic antigens by two-dimensional gel electrophoresis and pep­ peptides have so far failed to reveal structural differences tide mapping. Immunoabsorbants will be used to isolate among the cell-surface proteins of varying molecular molecules for more detailed comparisons including amino weights. However, this technique has indicated that the acid sequence analysis when appropriate. cell-surface proteins are distinct from the envelope The combination of newly-available monoclonal proteins of the virus derived from that cell line. Pre­ antibodies and a series of advanced methods and unique liminary data indicate that the viruses isolated from instruments which have recently been developed at 39

Caltech makes this project fully feasible. Jn addition to suppression, subcutaneous injection of the same tumor increasing our basic understanding of the molecular and extract results in specific protection from tumor genetic nature of these molecules, this information should challenge. In addition to this tumor-unique immunogenic open up opportunities for the diagnosis and therapy of activity of the extracts, immunization with intact tumor -human cancers. cells has defined a cross-reacting transplantation antigen shared between tumor lines. We hope to utilize these 48. THE IMMUNE RESPONSE TO SOLUBllJZED responses to solubilized tumor material in a fractionation TUMOR ANTIGENS scheme to identify the molecules responsible for the Investigators: David E. Jeanne P. Patalano Levy, induction of specific and cross-reactive immune suppres­ The immune response of mice to syngeneic fibro­ sion and cytotoxicity. sarcomas is being investigated to provide a means of References; characterizing and isolating the cell-surface molecules Kripke, M. L. (1974) J. Nat. Cancer Inst. 53: 1333-1336. Spellman, C. W., Woodward, J. G. and Daynes, R. A. responsible for tumor antigenicity. Previous studies of a (1977) Transplantation 24: 112-119. series of ultraviolet light-induced tumors showed that each independently-derived tumor line has a unique tumor 49. IMMUNOMICROSPHERES FOR CELL DETECTION transplantation antigen (TSTA) as well as a serologically­ AND SORTING defined common tumor-associated antigen. These tumors Investigators: Alan Rembaum*, William J. Dreyer are normally rejected by a naive host due to a primary We have developed a series of immunomicrosphere cytotoxic response to the TSTA (Kripke, 1974), but they reagents for use in cell detection and sorting which can be will grow progressively in hosts made susceptible by the (1) highly radioactive for ultrasensitive immunoassays; (2) induction of suppressor T lymphocytes through moderate intensely fluorescent, giving increased definition to exposure to UV light (Spellman, Woodward and Daynes, fluorescence microscopy and better signal-to-noise 1977). In order to dissect these immune phenomena, we resolution in cell-sorting instruments; (3) ferromagnetic are characterizing the cell-mediated responses to for a novel approach to the physical separation of cells; solubilized tumor antigens. We have demonstrated that (4) highly colored or electron-dense as specific tags in pretreatment of mice with a hypertonic salt-solubilized light or electron microscopy; and (5) carrying cytotoxic extract of tumor cells specifically suppresses the host's compounds as a drug delivery system targeted against ability to reject a lethal challenge of that tumor line, specific undesirable cells (Molday et al., 1975; Yen et al., while allowing normal regression of another, distinct UV­ 1976; Gordon et al., 1977; Rembaum and Dreyer, 1980). induced tumor. However, the administration of multiple We are presently developing two new advanced types doses of tumor extract following tumor challenge renders of microsphere reagents. The first type of microsphere is the mice susceptible to both the tumor liile used for far more intensely fluorescent than any of the previous extraction as well as an antigenically-distinct line. reagents. Feasibility studies have indicated that cells Tumor-specific immune suppression has been demon­ labeled with the new immunomicrospheres emit so much strated using extracts of in vitro-derived tumor cells. fluorescent light that they will be detected by ordinary Thus, the molecules responsible for this effect are tumor­ light microscopy while monitoring unlabeled cells with associated and are not contributed by any infiltrating, conventional optics. non-tumor cells of the host. We are currently evaluating The second type of new reagent is similarly fluores­ the cell types and any possible soluble factors involved in cent but also incorporates magnetic particles so that cells this immune suppression response through adoptive can be both separated and gi.onitored without the need for transfer experiments with defined populations and a fluorescence-activated cell sorter. extracts of lymphoid cells. These new reagents are being tested for use in These tumor extracts have also been found to be detection and separation of cell subpopulations, for immunogenic, demonstrating the importance of adminis­ example, subpopulations of T or B lymphocytes or specific tration route of antigen dose in determining the immune antigen-binding cells. Using a murine tumor system as a response (presumably by affecting antigen presentation). model, we are analyzing whether the abiity to detect an While intravenous administration results in immune increase in antigen-binding lymphocytes may be used as a 40

diagnostic tool to detect the presence of very small, The high sensitivity and efficiency of this new instru­ hidden tumors. ment will facilitate studies of proteins which are of great interest but which can only be isolated in minute amounts. References: Gordon, I. L., Dreyer, w. J., Yen, S. P. S. and Rembaum, A. (1977) Cell. Immunol. 28: 307-324. 51. DEVELOPMENT OF A NEW, ULTRASENSITIVE Molday, R. S., Dreyer, W. J., Rembaum, A. and Yen, S. P. MASS SPECTROMETER ALONG RESEARCH S. (1975) J. Cell Biol. 64: 75-88. AND CLINICAL LINES Rembaum, A. and Dreyer, W. J. (1980) Science 208: Investigators: John w. Belliveau*, Charles Griffin**, 364-368. Heinz G. Boettger** Yen, S. P. S., Rembaum, A., Molday, A. and Dreyer, W. (1976) In: Emulsion Polymerization, I. Riirma and J .. L. Over the last few years, scientists from Caltech and Gardon (Eds.), pp. 236-257. American Chemical Society, Washington, D.C. Caltech's Jet Propulsion Lab have joined together to conceive and construct a new type of mass spectrometer *Jet Propulsion Laboratory, Pasadena, California. which was designed to be at least 1000 times more sensitive than other existing instruments (Dreyer et al., 50. AMINO ACID SEQUENCE ANALYSIS USING 1974). The central difference between this and other GAS-PHASE SEQUENCE TECHNOLOGY instruments is an electro-optical ion detection (EOID) Investigators: Rodney M. Hewick, Suzanna J .. Horvath, Michael W. Hunkapiller, Leroy E. Hood, array, which allows for simultaneous monitoring of all William J. Dreyer mass ranges. We have just finished building a new type of micro­ At this time, several ultrasensitive mass spec­ sequencer which we have demonstrated can completely trometers are available as developmental prototypes at sequence 500 pmoles (J'0.5 µg) of an octapeptide and is JPL. Desiring to exploit the capabilities of these capable of sequencing subnanomole quantities of proteins instruments along as many lines as possible, several at higher repetitive yields and efficiencies than previously investigations are under way and/or planned: (I) A possible in any instrument. In this new instrument, the universal need exists for improved means of analyzing protein or peptide is simply dried onto a polybrene-treated mixtures of amino acid derivatives produced by the Edman glass fiber disc held in a glass cartridge. The various degradation reaction in an amino acid sequencer. Several refinements to this instrument include miniaturized detection methods are available, but up until now, only delivery valves to further reduce chemical noise, high performance liquid chromatography on reverse phase improved cartridge and solid support design, and solid­ supports could provide the sensitivity and resolution state programming. required for quantitative analysis of the derivatives from The chemistry which we have developed to make this all the amino acids commonly present in proteins. Pre­ instrument possible employs gas-phase reagents at critical viously, several protocols for mass spectrometric analysis points in the Edman reaction. In this approach to of phenylthiohydantoin (PTH) amino acids have been automatic sequential degradation, the large spinning cup described, but the sensitivity of the mass spectrometer used in Edman-type instruments is avoided, yet, in has remained a limiting factor until now. Using the contrast to the commercially available solid phase instru­ prototype instrument with a 14-inch EOID, reference ments, proteins need not be chemically coupled to the spectra of 13 PTH-amino acids have been obtained. High­ supporting matrix. During degradation the material is sensitivity experiments are currently under way and we exposed only to gas-phase reagents or to washing with expect to improve upon the several picomole sensitivity nonpolar solvents so that it remains fixed on the support. we have already reached. (2) Experiments have started in The miniaturized reaction cartridge, with reduced surface which the prototype instrumentTs high sensitivity and area, offers the potential for reducing chemical noise in simultaneous ion monitoring capability will be applied to a the analytical system used to identify the amino acid clinical application. One in every 6000 infants suffers derivatives. from congenital hypothyroidism, a hormone deficiency This instrument takes less than half the time required resulting in severe mental retardation (Dussault et al., by a commercial spinning cup sequencer to complete each 1976). If the infant is treated before three months of age, cycle of degradation and has a greatly reduced rate of however, the prognosis for achieving a normal intelligence reagent consumption. quotient is greatly improved. Because the clinical 41

symptoms before three months are often obscure, the mass spectrometer provides will make possible a number American Thyroid Association recommends the establish­ of very exciting applications hitherto unfeasible. ment of neonatal screening programs for congenital References: hypothyroidism (Committee of the American Thyroid Committee of the American Thyroid Association (1976) J. Pediatr. 89: 692-694. Association, 1976). Currently, radioimmunoassays (RIA) Dreyer, W. J., Kupperman, A., Boettger, H. G., Giffin, C. and enzyme immunoassays (EIA) are used. However, these E., Norris, D. D., Grotch, S. L. and Theard, L. P. (1974) are time-consuming and lack the high specificity inherent Clin. Chem. 20: 998-1002. Dussault, J. H., Letarte, J., Guyda, H. and Laberge, C. to a mass spectrometer. We are developing a clinical (1976) J. Pediatr. 89: 541-544. procedure designed to be fast and highly sensitive *Undergraduate, California Institute of Technology. Sup­ employing mass spectrometer identification to test for ported under the California Institute of Technology Summer Undergraduate Research Fellowship (SURF) the presence of thyroxin in blood samples. Such a Program, funded from the Prize Fund and Samuel Krown procedure consists of the isolation of thyroxin from the Donation. **Jet Propulsion Laboratory, Pasadena, California. blood sample; the chemical derivatization of the thyroxin to allow for mass spectrometer analysis; and the optimi­ PUBLICATIONS zation of the experimental conditions to allow for highest sensitivity and highest thru-put rate. It is hoped that the Dreyer, W. J. (1979) A progress report on two highly sensitive, nonisotopic assay systems. Antibiotics knowledge gained from the development of this model Chemother. 26: 155-157. clinical test can be used to develop a family of clinical Dreyer, W. J. and Rembaum, A. (1980) A new family of immunological reagents: immunomicrospheres. C.R.C. screening tests which would be related through their use Handbook Clio. Chem. In press. of a mass spectrometer for fast, sensitive, and efficient Rembaum, A. and Dreyer, W. J. (1980) Immunomicro­ spheres: reagents for cell labeling and separation. detection of a wide variety of molecules. Science 208: 364-368. In addition to these two projects, we have several Yen, S. P. S., Rembaum, A., Molday, R. S. and Dreyer, W. J. (1979) Functional colloid particles for immuno­ others planned and it is already apparent that the 1000- research emulsion polymerization. ACS Symposium fold increase in sensitivity that the new ultrasensitive Series, Washington, D.C., No. 24, 236-257.

Professor: Leroy E. Hood Ethel Wilson Bowles and Robert Bowles Visiting Associates: Joel Buxbaum, Kathryn L. Calame, Professorship Jeffrey J. Hubert The B.W. Foundation Senior Research Fellows: Sandra J. Ewald, Michael W. Deutsche Forschungsgemeinschaft 1-Iunkapiller, Minnie McMillan E. I. DuPont de Nemours & Co. Research Fellows: Martha W. Bond, Richard H. Douglas, The Max C. Fleischmann Foundation Philip W. Early, John Frelinger, Robert S. Goodenow, Hoag Foundation Henry V. Huang*, Maria-Luisa Maccecchini, Kevin W. Louis B. Mayer Foundation Moore, Michael Steinmetz National lnstitutes of Health, USPHS Graduate Students: Stephen T. Crews, Mark M. Davis, National Science Foundation Douglas A. Fisher, Tim Hunkapiller, Nelson D. President's Venture Fund Johnson, Marilyn R. Kehry, Stuart K. Kim, Mitchell Prince Charitable Trusts Kronenberg, Donna L. Livant, James W. Schilling, Gordon Ross Medical Foundation Beverly Taylor Sher Damon Runyon-Walter Winchell Cancer Fund Research Staff: Paul K. Cartier III, Vincent R. Swiss National Agency Farnsworth, Suzanna J. Horvath, Chin Sook Kim Veterans Administration Laboratory Staff: Maria A. De Bruyn, Bertha E. Jones, Jessie Walker summary: ln 1965 Dreyer and Bennett made the *Division of Biology and Biomedical Engineering, Division prediction that antibody polypeptides would be encoded by of Engineering and Applied Science. two separate genes, a variable (V) and a constant (C), that Support: The work described in the following research were rearranged during the differer:itiation of antibody­ reports has been supported by: American Cancer Society, California Division producing (B) cells. This hypothesis had two unusual American Cancer Society, National features. First, this was the initial suggestion that Arthritis Foundation Biomedical Research Support Grant (NIH) eukaryotic genes might be. composed of multiple gene 42 segments ("split genes"). Second, the DNA rearrangement somatic mechanism for generating diversity in V gene was seen as a fundamental element of the molecular segments (Biology 1980, No. 55). machinery for antibody gene expression. Analyses in our (6) Antibody polypeptides are divided into homology laboratory and the laboratories of others using serological, units or discrete domains that include about 110 amino protein chemistry, genetical, and recombinant DNA tech­ acid residues. The coding regions for each of these niques have amply verified this insightful hypothesis and domains constitute distinct exons. The correlation of today it is a fundamental tenet of molecular immunology. functional domains with exons has important evolutionary We understand a great deal about the organization of implications for the antibody gene families and possibly antibody genes in the mouse and somewhat less about the for the evolution of other complex multigenic families various types of DNA rearrangements they undergo. Our (Calame et al., 1980). current state of knowledge can be summarized as follows. (7) Antibody molecules exist in two distinct environ­ (1) Antibodies are encoded by three unlinked families ments-as hydrophobic integral membrane proteins or of genes-two code for light (L) chains (A and K) and the receptors that trigger B-cell differentiation, and as third for heavy chains (H). hydrophilic effector molecules of humoral immunity in the (2) Light chains are encoded by several distinct gene serum. The µ chains of the membrane-bound and secreted elements-leader (L), variable (V), joining (J), and con­ lgM molecules have distinct C-termini (Biology 1980, Nos. stant ( C). During B-cell differentiation, the V and J gene 62 and 63). These two alternative forms of the µ chain segments are juxtaposed and together they encode the are generated by an elegant RNA splicing mechanism variable region of the light-chain polypeptide. This DNA from a single gene (Biology 1980, No. 52). rearrangement is termed variable-region formation or V-J The study of antibody genes and polypeptides affords joining. Germline DNA (DNA undifferentiated with unique opportunities to unravel the complexities of gene regard to antibody function) has been obtained from sperm organization and regulation of a fascinating and essential or embryos. Differentiated DNA usually is derived from eukaryotic system. Our future efforts in the antibody immunoglobulin-producing myeloma tumors or hybridomas. system will be directed along several genes line. (i) We (3) Heavy chains are encoded by the gene elements will analyze protein and DNA diversity in V regions to homologous to their light chain counterparts and, in elucidate the somatic mutational mechanism that appears addition, a third variable-region element termed D for to be operating (Biology 1980, Nos. 52-56). (ii) We will diversity (Biology 1980, No. 52). Thus in a heavy chain analyze in detail the molecular mechanism for the DNA gene, variable-region formation joins V, D and J gene rearrangements associated with class switching (Biology segments in contiguous alignment. 1980, Nos. 57 and 58). (iii) We will examine the (4) The heavy chain gene family has eight CH genes organization and rearrangement of antibody genes from (Cµ' co, cy3' cyl' cy2b' cy2a' CK, c,) (Biology 1980, normal B cells obtained using the fluorescence-activated Nos. 59 and 60). The developing B cell may shift from the cell sorter. (iv) We will examine special interesting expression of one CH gene to the expression of a second features concerning the organization or expression of CH gene. This shift changes the class or subclass of antibody genes (e.g., the mechanism for the expression of antibody expressed. This change requires a second type of o chains; the location of the E gene) (Biology 1980, DNA rearrangement termed a class switch or CH Nos. 59 and 60). (v) We will study the organization of switching (Biology 1980, Nos. 57 and 58). human antibody genes and their role, if any, in various (5) Antibody diversity or the diversity of the antigen­ human immunodeficiency diseases (Biology 1980, No. 61). binding V regions appears to be generated by several (vi) We will examine the evolution of a well-defined group distinct mechanisms: multiple germline V gene segments, of VH gene segment&-namely, those encoding the the combinatorial joining together of distinct V, D and J response to the simple antigen phosphorylcholine (Biology gene segments, alternative joining patterns for the V, D 1980, Nos. 53 and 56). and J elements that generate diversity at the corre­ We are just beginning the analysis of genes encoding sponding junctions, and the combinatorial association of two other interesting systems-T-cell receptors (Biology all light and heavy chains with one another (Biology 1980, 1980, No. 64) and gene products of the major histoeompat­ Nos. 53-56). There also appears to be an additional ibility complex (MHC) (Biology 1980, Nos. 65-67). Both T- 43 cell receptors and certain MHC gene products appear to I found that genes encoding heavy chain variable be encoded by families of genes. We are interested in regions are created somatically by joining three segments determining to what extent these systems share with the of DNA: VH' D, and JH. The JH gene segments are antibody genes common strategies for information closely linked to the lgM heavy chain constant region gene amplification and expression. in germline DNA, where they are widely separated from A major focus of our laboratory for the past five years VH gene segments. In an immunoglobulin-producing cell, has been the development of new instrumentation for one VH and one JH gene segment are joined, together with microchemical analysis (Biology 1980, Nos. 68 and 69). a D sequence which is probably also a germline gene This year we have employed our newly-designed spinning­ segment, to form the expressed heavy chain variable cup protein sequenator to analyze several different types region gene. Both combinatorial association of gene of human and mouse interferons (leukocyte, lympho­ segments and variations in the exact sites of DNA joining blastoid, and fibroblast) as well as several other molecules between gene segments can contribute to heavy chain available in very low quantities (Biology 1980, No. 68). variable region diversity. Based on observations of This instrument is capable of analyzing 10 picomole certain conserved nucleotides and spacer sequences samples. In collaboration with Drs. Rod Hewett and adjacent to unrearranged immunoglobulin gene segments, I William Dreyer, we have finished the construction of a have proposed a mechanism for variable region gene new gas-phase, solid state protein sequenator that rearrangement. during differentiation (Early et al., 1980a). ultimately may be capable of analyzing samples at even Secreted and membrane-bound forms of lgM heavy greater sensitivities and with more speed. In chains were found to be encoded by separate mRNAs collaboration with D. Caruthers at the University of transcribed from the same gene (Early et aL, 1980b). Colorado (Boulder), we now have under construction an These mRNAs differ only at their 3' ends, where one automatic DNA synthesiZer. We hope the combination of encodes a 20 amino acid secretory C-terminal segment, the ultrasensitive instrumentation for protein sequence and the other encodes a 41 amino acid transmembrane C­ analysis, and the ability to synthesize DNA probes terminal segment (Rogers et aL, 1980). Synthesis of the automatically, will enable us to approach the isolation of two forms of IgM heavy chain mRNA appears to be rare-messenger genes with greater speed and effective­ developmentally regulated by controlling the site of 3' ness than was heretofore possible. terminal polyadenylation. The site of polyadenylation defines the lengths of RN A transcripts and thereby References: Calame, K., Rogers, J., Early, P., Davis, M., Livant, D., determines which of two alternative RN A splicing Wall, R. and Hood, L. (1980) Nature 284: 452-455. Dreyer, W. J. and Bennett, J. C. (1965) Proc. Nat. Acad. patterns will be followed, leading to mRNAs encoding Sci. USA 54: 864-869. either the secreted or membrane-bound forms of IgM heavy chains. 52. GENERATION OF DIVERSITY AND REGULATION OF EXPRESSION IN IMMUNOGLOBULIN References: HEAVY CIIAIN GENES Early, P., Huang, H., Davis, M., Calame, K. and Hood, L. Investigator: Philip W. Early (1980a) Cell 19: 981-992. Early, P., Rogers, J., Davis, M., Calame, K., Bond, M., lmmunoglobulin heavy chains each display one of a Wall, R. and Hood, L. (1980b) Cell 20: 313-319. wide range of diverse antigen-binding variable regions. At Rogers, J., Early, P., Carter, C., Calame, K., Bond, M., Hood, L. and Wall, R. (1980) Cell 20: 303-312. least one class of immunoglobulin, IgM, contains heavy chains which exist as two forms, either bound to the 53. SEQUENCE AND ORGANIZATION OF outside of a cell membrane or linked by disulfide bonds in IMMUNOGLOBULIN HEAVY CHAIN secreted antibodies. I have used recombinant DNA VARIABLE REGION GENES techniques to isolate and determine the nucleotide Investigators: Stephen T. Crews, Henry V. Huang sequences of genes encoding mouse immunoglobulin heavy Remarkable advances have been made in recent years chains. This has enabled me to examine genetic bases for toward an understanding of the molecular basis of the diversity of heavy chain variable regions and for the antibody diversity and immunoglobulin gene structure. synthesis of membrane-bound and secreted forms of IgM Current unresolved problems of interest that concern us heavy chains. are: (1) the respective contributions of germline gene 44

sequences and somatic processes to antibody diversity; (102-117). One VH segment sequence has been found in 15 (2) linkage and genomic organization of variable region different proteins and undoubtedly represents a germline gene families; and (3) control of immunoglobulin gene gene segment. Other VH segments differ from this expression. Essential to an understanding of these prototype sequence by one to seven amino acid substitu­ problems is the isolation and sequence characterization of tions and may represent either other germline gene germline immunoglobulin heavy chain variable region segments or somatic variants of the known germline VH genes utilizing recombinant DNA techniques. Previously, gene segment. Nucleic acid sequence analysis should this laboratory had isolated a cloned cDNA copy of the resolve this point (Biology 1980, No. 56). mRNA coding for the 8107 myeloma heavy chain immuno­ Four JH segment sequences have been found multiple globulin (Early et al., 1979). The variable region times in dextran and other antibodies and are therefore sequences of this cDNA clone when hybridized to mouse germline gene segments. Two of these have been DNA by genomic blotting techniques reveal 10-20 puta­ identified directly in germline DNA (Biology 1980, No. tive germline variable region genes. Utilizing this probe, 52). One JH sequence has been identified which differs we have screened a library of mouse sperm DNA cloned in from a known germline JH gene segment only at its N­ the recombinant DNA vector, Charon 4A. Restriction terminus and may represent site-specific variation enzyme and DNA blotting analyses indicate that we have resulting from D-JH joining as previously postulated to isolated a minimum of 10 different variable region genes. occur in kappa light chains (Biology 1979, No. 58). Furthermore, we have been able to conclude that a The D segments are by far the most diverse part of the number of these genes are closely linked on the mouse heavy chain V region (Schilling et al., 1980a). Twelve chromosome. More specifically, there are two instances different amino acid sequences have been found for where three genes reside within 4 kilobases of each other residues 100 and 101 of dextran antibodies. The small size and another instance where two other genes are separated and high diversity of these segments make it difficult to by 14 kilobases. Presently, these genes are being assess the contribution of germline gene segments and sequenced either from the Charon 4A clones or plasmid somatic variational mechanisms to D segment diversity. subclones. The DNA sequences of these gerrnline variable Nucleic acid sequence analysis will be necessary to region genes, when compared to the large number of resolve this point. immunoglobulin protein sequences known, should, in Any dextran D segment appears to be able to join with principle, allow us to designate the corresponding germ­ any dextran VH and any dextran JH segment (Schilling et line and somatic mutational contributions to heavy chain al., 1980a). This combinatorial joining property appears to diversity. In addition, DNA sequencing and heteroduplex make a major contribution to total dextran antibody analysis of adjoining chromosomal regions may advance diversity. The 6 VH' 12 D, and 6 JH segments identified our understanding of the mechanisms of somatic mutation, could create 432 different dextran antibodies. How large of V-J joining, and of the evolution of these genes as well is the actual pool of dextran antibodies? Our failure to as reveal possible regulatory sites for gene expression. find a repeated VH sequence in 21 dextran antibodies examined suggests that it is in fact quite large. Referenee: Ear]y, P. W., Davis, M. M., Kaback, D. B., Davidson, N. This research was carried out in collaboration with and Hood, L. (1979) Proc. Nat. Acad. Sci. USA 76: Drs. Joe Davie and Brian Clevinger in the Department of 857-861. Microbiology at the Washington University Medical School 54. SEQUENCE STUDIES OF a-1,3 DEXTRAN in St. Louis. BINDING ANTIBODIES References; Investigators: James W. Schilling, Martha W. Bond Schilling, J., Clevinger, B., Davie, J. M. and Hood, L. We have expanded the total number of dextran-binding (1980a) Nature 283: 34-40. Schilling, J., Clevinger, B., Bond, M., Davie, J. and Hood, heavy-chain variable-region amino acid sequences deter­ L. (1980b) Manuscript in preparation. mined to 21 (Biology 1979, No. 59; Schilling et al., 1980b). We now have convincing evidence that the complete variable region is encoded by three separate germline gene segments: VH (residues 1-99), D (100-101) and JH 45

55. HEAVY CHAIN VARIABLE REGION SEQUENCES of VH gene segments encoding the murine immune FROM PHOSPHORYLCHOLINE-BINDING MYELOMA PROTEINS response to a-1,3 dextran, a polysaccharide found in Investigators: Nelson D. Johnson, Richard H. Douglas, bacterial cell walls. Recent protein sequence analyses Greg Sorensen* from this laboratory (Schilling et al., 1980) have suggested Individual vertebrates can make many thousands of that there are at least two to four dextran v H gene different antibodies. Immunologists have recently begun segments in BALB/c mice. The same study also has shown to discover, at a molecular level, what mechanisms that there are multiple D (diversity) segments for the

operate to generate this re~arkable diversity. Investi­ VDex genes. gations into this problem are currently proceeding in We want to address several immediate questions: How different though complementary directions: the study of many VH gene segments encoding the dextran response germline antibody genes (Early et al., 1980), somatic are there in BALB/c germ line DNA? Are there separate antibody genes (Early et aL, 1980) and protein sequences germline D gene segments (Brach et al., 1978) analogous (Johnson, Gearhart and Hood, 1980). An understanding of to the separate J gene segments already demonstrated at both germline and somatic antibody V coding regions will the DNA level for K (Seidman and Leder, 1979), ; (Brach help explain the mechanisms by which somatic antibody et aL, 1978), and the phosphorylcholine (PC)-binding v H variability is generated. (Early et al., 1980) genes? We are currently constructing We are currently analyzing the structures of variable a probe for the murine VDex gene segments using two regions from the heavy chains of hybridoma antibodies approaches. First, we are screening a genome library specific for phosphorylcholine. In the past year we have made from the DNA of myeloma tumor, M104E, which determined eight complete VH region sequences and have secretes an lgM antibody that binds dextran. Our purpose is to isolate the joined µ gene in order to subclone the V made a number of interesting observations about V region H diversity. These observations include the following: anti­ gene segment from it. Second, we have made 40 cDNA phosphorylcholine antibodies become considerably more clones from M104E mRNA and are currently searching diverse as they switch C regions from C (IgM) to C through them to find one which contains the V region. (IgG); the diversity that appears in IgG moiecules can, i~ The VDex probe will then be used to isolate VDex gene some cases, be correlated with acquisition of specific segments from a germline genomic library. functions; anti-phosphorylcholine light- and heavy-chain If the VDex genes are clustered in the BALB/c antibody polypeptides combinatorially associate to form genome, as appears to be true for at least some of the antibodies of varying specificity; and significant diversity V PC-like genes in the mouse (Biology 1980, No. 53), we intend to compare the organization of the v family in in this system seems to arise from unusual DNA joining Dex events (Johnson, Gearhart and Hood, 1980). various inbred strains of mice, particularly in those strains This research was carried out in collaboration with Dr. making a very poor response to a-1,3 dextran, and in wild Patricia Gearhart at the Carnegie Institution in mice. Our objectives in these studies are (1) to Baltimore, Maryland. investigate the evolution of the VDex gene family, and (2) to find out if in low-responder strains, there is a References: Early, P., Huang, H., Davis, M., Calame, K. and Hood L. structural difference in the VDex gene segment families. (1980) Cell 19: 981-992. ' Johnson, N. D., Gearhart, P. and Hood, L. (1980) Manu­ References: script in preparation. Brach, C., Hirama, M., Lenhard-Schuller, R. and Tonegawa, S. (1978) Cell 15: 1-14. *Undergraduate, California Institute of Technology. Early, P., Huang, H., Davis, M., Calame, K. and Hood, L. (1980) Cell 19: 981-992. Schilling, J., Clevinger, B., Davie, J. and Hood, L. (1980) Nature 283: 35-40. 56. AF AMILY OF V REGION GENES: Seidman, J. G. and Leder, P. (1979) Proc. Nat. Aead. Sci. THE a-1,3 DEXTRAN SYSTEM USA 76: 3450-3454. Investigator: Donna L. Livant 57. MECHANISMS OF CLASS SWITCHING IN How is the immune response to a particular antigen IMMUNOGLOBULIN HEAVY CHAIN GENES encoded in the genome? In order to answer this question, Investigators: Marie M. Davis, Stuart K. Kim we are currently investigating the structure of the group B lymphocytes initially express IgM molecules. After 46

antigenic stimulation of a particular B lymphocyte, a the heavy chain constant region gene locus occurs by different class of antibody can be produced in which the specific gene rearrangement or class-specific switching.

VH region remains the same but in which the C µ region References: has been replaced by another heavy chain constant region Davis, M., Calame, K., Early, P ., Livant, D., Joho, R., Weissman, J. and Hood, L. (1980a) Nature 283: (such as Cy' Ca or CE). The particular class of antibody 733-739. expressed by a B lymphocyte will determine its physio­ Davis, M., Kim, S. and Hood, L. (1980b) Science. In press. logical role, since each heavy chain constant region 58. HEAVY CHAIN SWITCHING mediates different effector functions such as complement fixation. We have shown that this "heavy-chain class Investigators: Kathryn L. caJame, Beverly Taylor Sher switchn occurs by DNA rearrangement (Davis et al., Our group has previously shown that formation of an 1980a). Specifically, we have cloned a rearranged, active immunoglobulin heavy chain gene requires two expressed a heavy chain gene, a6, from an IgA-producing DNA rearrangements: V-J joining which is common to myeloma, M603. We then compared a6 to the DNA both light and heavy chains, and CH switching which is surrounding the Cµ and C gene segments isolated from unique to heavy chains (Davis et aL, 1980). CH switching 0 germline DNA (mouse sperm). As shown by restriction allows B lymphocytes to express sequentially different mapping, heteroduplex analysis and sequencing data, there classes of immunoglobulin molecules which have identical is a "switch (S) site" in a6 located in the intervening antigen binding specificity. sequence between the J gene segment and the C gene. This project is designed to characterize further the The DNA in the intervening sequence upstream from" the mechanisms which mediate and regulate the CH switch site is derived from flanking sequence 5' to the switching. In collaboration with Dr. Patricia Gearhart germline Cµ gene whereas the DNA downstream from the (Carnegie Institution), we have three mouse tumors which switch site is derived from flanking sequence 51 to the secrete heavy chains of the IgM, IgA and IgG3 classes with germline Ca gene. In other words, a recombination has identical antigen-binding specificities. These are: hybrid­ occurred which, in effect, replaces the Cµ gene with the oma M2 (VHPCJHPCCµ), myeloma T15 (VHPCJHPCCa) C gene. 0 and hybridoma G3 (V HPCJHPCC y3). (V HPCJHPC In order to investigate further the mechanism of class encodes a specific phosphorylcholine binding variable switching, we have isolated the rearranged, expressed a region.) This unique system will allow us to compare CH heavy chain genes from two other myelomas, Y5236 and switching in two different heavy chain classes (a and y3) T15 (Davis, Kim and Hood, 1980b). These two clones were where identical VH and JH gene segments are used. compared to the germline C and C DNA configurations We have constructed genomic libraries in Charon 4A µ " and, as before, it was found that part of the intervening and Charon 28 from these three tumor DNAs. Heavy sequence in the rearranged gene was derived from chain joined clones containing V, J and C gene segments germline Cµ flanking sequence and part came from have been isolated from the M2 and Tl5 genomic libraries germline Ca flanking sequence. Each rearranged Ca gene (see Biology 1980, No. 57) libraries. We are currently allows us to identify unambiguously a switch site in the screening the G3 genomic library to isolate that joined germline C µ and Ca flanking sequences. In general, these clone. We also have germline VHPC' JH-Cµ' Ca and Cy3 switch sites occur in different places. However, the genomic clones. Comparison of the joined clones with germline Ca switch sites all occur within homologous their germline counterparts by Southern blotting and blocks of 30 nucleotides. This 11 switch11 sequence is heteroduplex analysis allows switch sites to be mapped repeated at least 17 times in over 1400 nucleotides of and subsequently sequenced. germ line Ca flanking sequences but has not, as yet, been The M2 joined clone, as predicted, contains only VHPC found in the germline Cµ or Cy flanking sequences. We and JH-Cµ germline sequences since CH switching is not hypothesize that this sequence is used as an enzyme involved in expression of IgM molecules. The Tl5 clone recognition site and that, since it is present next to the has been characterized by Davis and Kim (Biology 1980, Ca gene but not the Cy genes, such an enzyme could No. 57). We expect that analysis of the G3 clone will specifically replace a C µ gene with a Ca gene. In other demonstrate whether there are similar or different words, we believe that regulation of gene expression in switching sequences flanking C µ and C yJ for the y3 47 switch as compared to the a switch. Comparison of heavy-chain constant region genes, the expression of IgD switch sites in the y3 and a classes may reveal basic molecules does not involve DNA rearrangement. There­ similarities which may be required for all CH switching, fore, the simUltaneous expression of IgM and IgD as well as features unique to the two classes. molecules on the surface of B cells is presumably an RNA­ processing phenomenon. In collaboration with J. Rogers Reference: Davis, M. M., Calame, K., Early, P., Livant, D., Joho, R., and R. Wall in the Department of Microbiology at UCLA, Weissman, I. and Hood, L. (1980) Nature 283: 733-739. we are now examining this question. Preliminary results .indicate that in the rat myeloma tumor, the predominant 59. STUDIES OF THE MOUSE lMMUNOGWBULIN-D messenger species is a large (5-6 kb) molecule containing CONSTANT REGION GENE sequences homologous to all four of the plasmid subclones. Investigators: Kevin w. Moore, Tim Hunkapiller We also are attempting to demonstrate the existence of a Immunoglobulin D (IgD) is perhaps the least well primary transcript containing both c and C sequences. 11 0 characterized protein of the immunoglobulin heavy chain In the near future we plan to carry out nucleic acid family. This molecule is found predominantly on the sequence analyses of the above subclones to obtain information about the structure of the C gene, and to surface of B lymphocytes; only trace amounts can be 0 detected in normal serum. Moreover, both IgM and IgD examine the disposition of C 0 genes in other mouse molecules can be expressed simultaneously by a single B myeloma tumors, such as the a- and y3-producing tumors, cell, probably in association with the same variable region M603 and J606.

(Knapp et al., 1979). References: We have embarked upon a study of the C gene, which Bazin, H., Beckers, A., Urbain-Vansanten, G., Pauwels, R., 0 Bruyns, c., Tilkin, A. F., Platteau, B. and Urbain, J. codes for the constant region of IgD, in order to obtain (1978) J. Immunol. 121: 2077-2082. general structural information, to determine the mecha­ Calame, K., Rogers, J., Early, P., Davis, M., Livant, D., Wall, R. and Hood, L. (1980) Nature 284: 452-455. nisms operating at the nucleic acid level involved in Davis, M. M., Calame, K., Early, P. w., Livant, D. L., expression of IgD molecules, and in particular, to recon­ Joho, R., Weissman, I. L. and Hood, L. (1980) Nature 283: 733-739. cile the simultaneous expression of the C µ and C genes 0 Honjo, T. and Kataoka, T. (1978) Proc. Nat. Acad. Sci. with the deletion model of heavy chain gene expression USA 75: 2140-2144. Knapp, M. R., Jones, P. P., Black, S. J., Vitetta, E. s., (Honjo and Kataoka, 1978; Davis et al., 1980). Slavin, S. and Strober, s. (1979) J. Immunol. 123: We prepared poly(A)+ RNA from an IgD-secreting rat 992-999. McKeever, P. E., Kim, K. J., Nero, G. B., Laskov, R., myeloma tumor (Bazin et al., 1978). Using this RNA as a Merwin, R. M., Logan, W. J. and Asofsky, R. (1979) J. probe, we have identified and characterized two genomic Immunol. 122: 1261-1265. clones which contain the mouse C heavy-chain gene from 0 a library of mouse sperm DNA. One of these, µ 7 (Calame 60. ISOLATION OF lMMUNOGWBULIN E GENE Investigator: Maria-Luisa Maececchini et al., 1980), contains both the Cµ and c genes, while 0 the other, µ 37 (P. Early, unpublished), contains the C Hypersensitivity is mediated primarily by IgE anti­ 0 gene alone. Based on restriction map analysis, these bodies produced by allergic individuals in response to clones span 22 kilobases (kb) of germline DNA, and allergies. Augmented IgE biosynthesis is associated with overlap for about 11 kb. The C gene, as defined by various conditions representing ill health, and it does not + 0 hybridization to poly(A) RNA from the rat myeloma seem likely that IgE would have survived in evolution tumor, spans some 9-10 kb of DNA, beginning about 4 kb merely on the basis of its harmfuI properties. In fact, it to the 3' side of the cµ gene. These regions of seems that the IgE molecule plays an important role in hybridization are separated by about 3 kb of DNA and host defense against parasites and that it reacts with have been isolated as four subclones in the plasmid vector exogenous antigens entering through the respiratory and pBR322. gastrointestinal tract and the skin. The IgE molecuie has These subclones were used as probes for Southern blot a molecular weight of 190,000 and is composed of two analyses of DNA prepared from mouse liver, rat liver, two light and two heavy chains. The Fee: fragment is + + mouse µ 0 lymphomas (McKeever et al., 1979), and the composed of three domains and has about the same size as rat JgD myeloma. These data suggest that, unlike other the Feµ fragment. A comparison of e:, µ, O, y and a 48

chains suggests that the ancestral heavy chain consisted globulin heavy chain and fail to produce light chains of of four homology regions, which are still present in £ and either the K or A class. Our earlier studies have shown µ. Deletion of the primordial CH2 domain generated the that the cells synthesize a shortened heavy chain mRNA bridge or hinge region found in y, 0 and a chains. (15.5 S vs. 17 .5 S for a normal-sized chain) and no The development of IgE-secreting cells follows a translatable light-chain mRNA. Using the appropriate rather peculiar pathway. In fetal liver and spleen, only human probes, we are isolating the defective gene IgM-bearing cells are present. Within 24-48 hr after segments in order to carry out detailed structural birth, JgE-bearing cells appear in the spleen. More than analysis. This strategy has been extremely effective in 95% of these JgE-bearing cells carry surface JgM analyzing the genetic defects in a variety of thalassemias molecules. Upon stimulation, these lgM-JgE double for the globin genes and should be equally so in the studies producers become IgE secretors. The IgE-secreting cells of human immunodeficiency states. as well as the B memory cells committed for IgE do not

carry lgM on their surface. 62. TWO CWNES OF AB-CELL LYMPHOMA We are using recombinant DNA techniques to study the SYNTHESIZE THREE SPECIES OF IMMUNOGLOBULIN MU CHAINS molecular basis of the development of virgin B cells to Investigators: Sandra J. Ewald, Marilyn R. Kehry IgE-forming cells. We want to elucidate the mechanism involved in the production of JgM-lgE double bearing and B lymphocytes undergo a series of differentiation IgE-secreting cells. It is important to reconcile the steps, the first of which is characterized by the synthesis simultaneous expression of lgM and IgE in precursor cells of monomeric membrane IgM molecules that act as with the exclusive expression of IgE in secreting B cells. antigen receptors. Following antigenic stimulation, the B In order to answer these questions, we have isolated lymphocyte acquires the ability to secrete pentameric IgM DNA from IgE-producing cells. We are constructing a molecules. The mouse B-cell lymphoma, WEHI 279,

library in Charon 28 and screening it for the E gene using synthesizes both secreted and membrane-bound IgM a J-probe isolated from a piece of µ germline gene molecules. Since the heavy (µ) chains of secreted and containing the germline JH gene segment. Concomitant­ membrane-bound IgM differ in primary structure (see ly, we isolated mRNA and are synthesizing cDNA from Biology 1980, No. 63), we have been able to characterize

the poly(A)+ fraction. The E cDNA can be used as a probe three species of WEHI 279 µ chains, µ. (internal), µ I m for screening both germline and differentiated DNA. (membrane) and µs (secreted). These µ chains differ in their cellular location, their degree of glycosylation and 61. THE ANALYSIS OF HUMAN IMMUNOGWBULIN their polypeptide structure. GENES IN HEALTH AND DISEASE We have localized the µm chain in WEHI 279 cells to Investigator: Joel Buxbaum the plasma membrane and the µ. chain to the cytoplasm I Our work is directed toward applying the observations (see Biology 1979, No. 67). Pulse-chase experiments have made concerning the organization and structure of the demonstrated that the µ. pool contains precursors to both I genome controlling the production of murine immuno­ µm and µs chains. The intracellular µ chains differ from globulins to the analysis of human disease states in which their mature µ and µ chain counterparts in that the µ. m S I immunoglobulin synthesis is disturbed. To this end, we chains contain incompletely glycosylated complex carbo- have employed previously prepared plasmids containing hydrate structures. murine immunoglobulin gene fragments as hybridization Although the original WEHI 279 cell line has a high probes for the identification and isolation of the density of membrane IgM monomers and secretes negli­ homologous human genes from a library of human genomic gible amotints of IgM pentamers (Warner, Harris and fragments. We have isolated fragments which appear to Gutman, 1975), a recently isolated WEHI 279 clone, contain some of the desired human antibody genes. WEHI 279.1/12, was found to secrete 15-fold greater Our initial experiments are being carried out with a quantities of lgM molecules into the culture fluid. In cultured cell line obtained from patient with y heavy addition, the WEHI 279.1/12 cells also possess altered chain disease, a human lymphoproliferative disorder in concentrations of cell-surface differentiation markers which the malignant cells synthesize a deleted immuno- that are characteristic of more mature B cells (Sibley et 49 al., 1980). The WEHI 279 lymphoma therefore has in the structure of their C-terminal segments (Kehry et differentiated while in culture and represents a more al, 1980). The µm and µs chains have been distinguished advanced stage in the pathway of B-cell differentiation. as follows. (1) The µ chains are approximately 20 amino m Perhaps these cell lines will be useful in delineating the acids larger than µs chains. (2) The µm chains contain at regulatory mechanisms that mediate a developing B cell's least two phenylalanine residues not found in µ chains. s shift from synthesis of membrane-bound to secreted IgM. (3) The µm chains lack a methionine residue present in the These studies were carried out in collaboration with C-terminal segment of µs chains.. (4) The high mannose Dr. C. Sibley at the Department of Genetics, University carbohydrate moiety present in the C-terminal segment of of Washington, Seattle, Washington, and with Dr. W. µs chains is absent in µm chains. (5) The amino acids Raschke at the Salk Institute for Biological Studies, San present at the carboxyterminus of µm chains are different Diego, California. from those found in the carboxyterminal sequence of µ s chains. Sequence analysis of the µm mRNA predicts that References: Sibley, C. H., Ewald, S. J., Kehry, M. R., Douglas, R. H., µm chains contain a 41-residue C-terminal segment (Early Raschke, W. C. and Hood, L. E. (1980) J. Immunol. In et al, 1980; Rogers et al., 1980) .. The protein studies of press. Warner, N., Harris, A. and Gutman, G. (1975) In: µm chains are in agreement with the studies on the µm Membrane Receptors of Lymphocytes, M. Seligman, J. mRNA and the C µgene. L. Pred'homme and F. M. Kourilsky (Eds.), p. 203. American Elsevier, New York. These studies have been carried out in collaboration with Dr. W. Raschke at the Salk Institute for Biological 63. THE IMMUNOGLOBULIN MU CHAINS OF Studies, San Diego, California. SECRETED AND MEMBRANE-BOUND lgM DIFFER IN THEIR C-TERMINAL SEGMENTS References: Investigators: Marilyn R. Kehry, Sandra J. Ewald, Early, P., Rogers, J., Davis, M., Calame, K.. , Bond, M., Richard H. Douglas, Douglas Fambrough* Wall, R. and Hood, L. (1980) Cell 20: 313-319. Kehry, M., Ewald, S., Douglas, R., Sibley, C., Raschke, W., The lgM molecule is either a membrane-bound Fambrough, D. and Hood, L. (1980) Ce!L In press. Kehry, M., Sibley, Fuhrman, J., Schilling, J. and Hood, receptor on the surface of B lymphocytes or is secreted by c .. , L. E. (1979) Proc. Nat. Acad. Sci. USA 76: 2932-2936. plasma cells as a hydrophilic serum antibody. The Rogers, J., Early, P., Carter, c .. , Calame, K., Bond, M., Hood, L. and Wall, R. (1980) Cell 20: 303-312. secreted IgM molecules are pentamers that bind foreign antigens and fix complement in the bloodstream whereas *Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland. the membrane-bound lgM molecules are monomers that act as the antigen receptors that stimulate B-cell differ­ entiation. Previous studies on secreted IgM heavy ( µ ) 64. DO T CELLS EXPRESS B-CELL 8 IMMUNOGLOBULIN GENES? chains have revealed that the µ chain has five homology 8 domains, a variable and four constant (C) region domains Investigator: Mitchell Kronenberg (C 1, C 2, C 3 and C 4) followed by a nonhomologous µ µ µ µ Although T lymphocytes, like B lymphocytes, are hydrophilic C-terminal segment of 20 amino acid residues known to respond specifically to antigen, the molecules (Kehry et al., 1979). We have characterized the structural responsible for antigen recognition by T cells are com­ features at the carbohydrate and protein levels that pletely uncharacterized. Using antisera made to various distinguish secreted µ ( µs) chains and membrane-bound µ components or domains of B-cell immunoglobulin, much ( µm) chains. evidence has accumulated for expression of heavy chain Membrane-bound µ chains were purified from the variable regions by T lymphocytes.. The presence of heavy mouse B-cell lymphoma WEHI 279, and secreted WEHI 279 chain constant regions and light chains remains contro­ µ chains were isolated from the medium of hybrid versial. MPCllxWEHI 279 cells (see Biology 1979, No. 66). We Our approach to the T-cell receptor problem has been have compared the structures of the µm and µs chains by to utilize cloned B-cell,. DNA probes to test for immuno­ peptide mapping, microsequence, molecular weight, and globulin gene rearrangement and expression in T cells. carboxypeptidase analyses.. These studies demonstrate Our T cells have included: (1) whole thymus which is at that the µm and µs chains are very similar throughout least 95% T cells; (2) WEHl-22, a T-cell lymphoma; (3) their variable, C 1, C 2, C 3 and C 4 domains but differ HT-1, a monoclonal T cell which recognizes sheep red µ µ µ µ 50 blood cells and after stimulation with this antigen helps B L) coding for heavy chains of transplantation antigens cells to secrete immunoglobulin; (4) CTLLi6, a killer T have been identified in the H-2 complex. Surprisingly, cell which recognizes foreign histocompatibility antigens. amino acid sequence studies have shown that the alleles of We have used probes for the C , J and C, light chain the K and D locus are no more similar among each other K K A gene segments, and the JH' C and C heavy chain gene than they are to the alleles of the other locus, i.e., no µ " segments as well as a DNA sequence mediating B-cell "Kness" or 11 Dness" of the alleles have been found. heavy chain class switching. These B-cell probes have Despite their allotypic diversity, transplantation antigens been hybridized to T cell DNAs digested with various have been well conserved during evolution. About 75% restriction enzymes. We find no evidence for any B-cell homology has been observed between human and mouse immunoglobulin DNA rearrangement in T cells. We also transplantation antigens. have hybridized the C , C,, C and C probes to total Transplantation antigens have been found on the cell + K A µ CX cell poly(A) RNA from the T cells. Immunoglobulin RNA surface of every mammalian cell studied so far, although sequences are absent from the monoclonal cells and some cells seem to have extremely low amounts of H-2 present at about 0.6-16 copies per cell in thymus. We antigens (e.g., red blood cells). Cells of the immune attribute the RNA in thymus to slight B-cell contami­ system and also liver cells with a comparatively high nation of our preparations. The implication of these amount contain in the order of 0.01% H-2 antigen of total findings is that if T cells do express VH and VL regions to cellular protein. recognize antigen, they must employ their own J and C Our aim is to clone cDNA molecules derived from gene segments. mRNA molecules that code for polypeptide chains of We are currently testing whether T cells do in fact transplantation antigens. These cDNA clones will then be express B-cell V domains, using cloned T cells responsive used to study the structure and organization of the to antigens such as phosphorylcholine for which we have chromosomal genes which may help to clarify some of the obtained B-cell derived V region probes. questions indicated above. These experiments were carried out in collaboration As a starting materiai we use polysomal mRNA with Dr. James Watson from the Department of Micro­ isolated from lymphoma cell lines of different haplotypes biology at the University of California at Irvine. that have been shown to produce about 10 times more transplantation antigens than normal cells. Two such cell 65. STRUCTURAL STUDIES OF THE GENES lines were obtained from S. Nathenson and M. Mescher, CODING FOR TRANSPLANTATION ANTIGENS respectively. Messenger RNA from one of these cell lines Investigators: Miebael Steinmetz, Robert S. Goodenow has been fractionated by sucrose gradient centrifugation The major histocompatibility complex of the mouse and fractions containing message for 8 -microglobulin 2 (H-2 complex) codes for three groups of proteins which and the H-2 heavy chain have been identified by in vitro are involved in a variety of immunologic phenomena, e.g., translation and immunoprecipitation. Specific antisera allograft rej_ection, immune responsiveness, complement were obtained from H. Huang (rat anti mouse 8 - 2 activity. The proteins of two of these groups, the microglobulin) and S. Nathenson (rabbit anti H-2b). To transplantation antigens and the Ia molecules, are mem­ verify that the anti H-2b serum recognizes the iri vitro brane-bound glycoproteins. Both groups of proteins have translated H-2 heavy chain, we will compare peptide maps been shown to possess an exceptionally high degree of of immunoprecipitated protein labeled biosynthetically polymorphism. The reason for the presence of so many and by in vitro translation. The antisera will then be used alleles at each locus of this complex is not known nor is to screen cDNA libraries by in vitro translation of mRNA the molecular mechanism for the generation of this selected by hybridization. The cDNA libraries have been allotypic variability. synthesized from total poly(Al-containing mRNA as well

The transplantation antigens .~e composed of two as from mRNA enriched for -microglobulin and H-2 a2 noncovalently associated polypeptides, a heavy chain of heavy chain mRNA. 44,000-50,000 and a light chain of about 12,000 molecular weight. The light chain is identical to -microglobulin s2 and is not encoded by the H-2 complex. Three loci (K, D, 51

66. ANALYSES OF RTl PRODUCTS USING The I region controls a series of phenotypic traits which TWO-DIMENSIONAL POLYACRYLAMIDE GELS are intimately involved in the immune response. By Investigator: John G. Frelinger recombinatorial analysis, the I region has been divided A fundamental issue in the study of the major into five subregions, I-A, 1-B, I-J, 1-E and 1-C. histocompatibility complex (MHC) of mammals has been The only gene products from the I region which have the role of polymorphism. Studies on mice have suggested been directly identified are the la antigens. These that extensive polymorphism of MHC products is found in molecules are expressed,-predominantly on B lymphocytes both inbred and wild populations. There are extensive and are integral membrane proteins which appear highly differences between alleles, and wild mice exhibit many polymorphic by serological analysis. They are composed new alleles not represented in the inbred strains. In of two subunits of approximate molecular weights 35,000 contrast, studies on wild rats suggest that the poly­ (a) and 28,000 ( 8), which coimmunoprecipitate with a morphism of MHC products in rats (the RTl products) is third invariant polypeptide, n , of approximate molecular much more limited. Controversy exists. whether this weight 30,000. Distinct Ia molecules are encoded by the apparent lack of polymorphism is due to technical I-A and 1-E subregions. limitations in the typing procedures. We have therefore I have modified O'Farrell's two-dimensional electro­ utilized biochemical methods for analyzing the poly­ phoresis technique and analyzed Ia immunoprecipitates morphism of RTl products. We have used two-dimen­ and isolated Ia polypeptides. I find that the invariant sional polyacrylamide gels to analyze the RTl products of polypeptide, n , always specifically coimmunoprecipitates seven inbred strains of rats. We isolated the RTl products with Ia molecules and that the strength of its association using a heterologous rabbit anti-rat 8 -microglobulin is dependent upon haplotype. n is a methionine-rich 2 antiserum or crossreactive murine anti-1-Ak or anti-I-Ek protein of pl "' 8 and is not a precursor of known Ia serum. These studies indicate that all seven strains have polypeptides. Cell lysates contain at least 10 times more unique two-dimensional gel profiles except for the August n than can be immunoprecipitated with anti-Ia sera. and MNR strains. The class II (Ia-like) molecules of these Although the significance of the association of n with Ia strains are identical by this technique. molecules is not known at present, it may play a We feel this method of analysis will be extremely physiological role in the functioning of Ia molecules in the useful in the future. Since we can detect differences immune response. between the inbred strains examined, the next step will be I have also shown that the Ia polypeptides l have to characterize several inbred strains which are serologi­ previously characterized by partial N-terminal amino acid 1 cally identical, for example, Fischer and Lewis (RTl ). sequencing do not contain n . Using my high-pressure Further, we wish to extend this analysis to wild rats which liquid chromatography tryptic peptide map technique and

have been typed serologically. We will be able to recombinant and F 1 hybrid mice, I have genetically determine if the lack of polymorphism of RTl products in mapped the Aa' A and E la polypeptides to the I-A 8 8 wild rats is. real or apparent. We believe this will shed subregion. The observation that the polypeptide E , 8 light on the degree of polymorphism in rats and ultimately which is immunoprecipitated by anti-1-E subregion sera, on the requirements for polymorphism of MHCs in maps to the I-A subregion may provide a molecular general. mechanism to account for the gene complementation These studies are being carried out in collaboration controlling immune responsiveness. with Dr. Peter Wettstein at the Wistar Institute of These experiments are carried out in collaboration Anatomy and Biology, Philadelphia, Pennsylvania. with Dr. H. 0. McDevitt (Stanford Medical School, Stan­ ford, California), Dr. D. B. Murphy (Yale Medical School, New Haven, Connecticut) and Dr. J. A. Frelinger (USC 67. STRUCTURE OF MURINE Ia ANTIGENS: TWO-DIMENSIONAL ELECTROPHORETIC Medical School, Los Angeles, California). ANALYSES AND HPLC TRYPTIC PEPTIDE MAPS Investigator: Minnie McMillan

The Ia antigens are encoded in the I region of the major histocompatibility complex (H-2) of the mouse. 52

68. DEVELOPMENT AND APPLICATION OF The anticipated advantages are that the sequencing PROTEIN MICROSEQUENCING TECHNOLOGY process may be automated, that potentially longer Investigator: Michael W. Hunkapiller stretches of DNA may be sequenced, and that extreme Structural analysis of peptides and proteins of biologi­ sensitivities may be obtained in the next generation cal and medical importance is often inhibited by the machine which could use, for example, high-sensitivity scarcity of material available for study. For this reason, mass spectrometers (see Biology 1980, No. 51) to detect we have devoted considerable effort to improving the the DNA fragments. sensitivity of the automated Edman degradation technique At present we are characterizing various light source for peptide sequencing. By modifying a commercial and detector systems for incorporation into the peptide sequenator, we were able to decrease the amount sequenator. We are also investigating the resolving of peptide required for analysis to about 100 pmol, only power, run time, and stability of different gel compo­ 1 % that normally required (Hunkapiller and Hood, 1978). sitions. In parallel, we are establishing computer By building a new sequenator containing additional design algorithms which will be used to process the optical data modifications (Hunkapiller and Hood, 1980), by improving and to control the operations of the sequenator. the purity of the reagents used in the Edman chemistry, Reference: and by developing more sensitive and reliable analysis Sanger, F., Nicklen, S. and Carlson, A. R. (1977) Proc. Nat. Acad. Sci. USA 74: 5463-5467. methods for the phenylthiohydantoins produced by the Edman degradation (Johnson, Hunkapiller and Hood, 1979), *Bioinformation Systems, Division of Engineering and Applied Science, California Institute of Technology. we have reduced the peptide required for sequence analysis to 10 pmol. Using this new sequenator in collaboration with several other research groups at Caltech and elsewhere, we have PUBLICATIONS initiated sequence studies on a variety of proteins Blankenhorn, E. P., Cecka, J. M., Goetze, D. and Hood, L. (1980) Structure of la antigens of rats: IL Mouse previously obtainable in amounts too small for study. alloantisera demonstrate at least two distinct These include a series of human and mouse interferons, molecular species. Eur. J. Immunol. 10: 146-155. Calame, K., Rogers, J., Early, P., Davis, M., Livant, D., the subunits of the acetylcholine receptor from Torpedo Wall, R. and Hood, L. (1980) The mouse Cµ heavy chain californica, the gap junction proteins from rat liver and immunoglobulin segment contains three intervening lens tissues, large molecular weight forms of leucine­ sequences which appear to separate domains. Nature 284: 452-455. enkephalin (dynorphin) that are extremely potent Cecka, J. M., Blankenhorn, E. P., Goetze, D. and Hood, L. (1980) Structure of Ia antigens from rats: I. Micro­ analgesics, a platelet-derived growth factor important in sequence analyses of la antigens from three strains of tissue regeneration at wound sites, and diol dehydrase (a rats. Eur. J. lmmunol. 10: 140-145. Cecka, J. M., McMillan, M., Murphy, D. B., McDevitt, H. coenzyme B -dependent enzyme). 12 0. and Hood, L. (1979) Partial N-terminal amino acid sequence analyses and comparative tryptic peptide References: maps of murine Ia molecules encoded by the I-A Hunkapiller, M. W. and Hood, L. E. (1978) Biochemistry subregion. Eur. J. Immunol. 9: 955-963. 17: 2124-2133. Chapman, B. S., Tobin, A. J. and Hood, L. (1980) Early Hunkapiller, M. and Hood, L. E. (1980) Science 207: w. embryonic alpha-like globins: complete amino acid 523-525. Johnson, N. D., Hunkapiller, M. W. and Hood, L. E. (1979) sequences of II and II' globins from chicken. J. Biol. Chem. In press. Anal. Biochem. 100: 335-338. Chiu, A. Y., Hunkapiller, M. W., Heller, E., Stuart, D. K., Hood, L. E. and Strumwasser, F. (1979) Purification 69. A DNA SEQUENATOR and primary structure of the neuropeptide egg-laying hormone of Aplysia californica. Proc. Nat. Acad. Sci. Investigators: Henry Huang, Daniel Aranovieh*, USA 76: 6656-6660. Ben M. Ellert•, Gilbert D. Mccann• Choudhury, A. M., Kenner, G. W., Moore, S., Ramachan­ dran, L., Thorpe, W. D., Ramage, R., Docbray, G. We are constructing a DNA sequenator based upon K. J., Gregory, R. A., Hood, L. and Hunkapiller, M. (1980) polyacrylamide gel separation of DNA fragments synthe­ NH 2-terminal sequence of human big gastrin: sized by, for example, the dideoxy method (Sanger, sequence, synthetic and immunochemical studies. Hoppe Seyler. Submitted for publication. Nicklen and Carlson, 1977), and detecting the fragments Clevinger, B., Schilling, J., Hood, L. and Davie, J. (1980) Structural correlates of cross-reactive and individual by their UV absorption, instead of using the traditional idiotypic determinants on murine antibodies to cx(l-+ 3) radiolabeling method. dextran. J. Exptl. Med. 151: 1059-1070. 53

Davis, M., Calame, K., Early, P., Livant, D., Joho, R., Johnson, N. D., Hunkapiller, M. W. and Hood, L. E. (1979) Weissman, I. and Hood, L. (1980) An immunoglobulin Analysis of phenylthiohydantoin amino acids by high heavy chain gene is formed by at least two recombi­ performance liquid chromatography on DuPont Zorbax national events. Nature 283: 733-739. CN columns. Anal. Biochem. 100: 335-338. Davis, M., Early, P., Calame, K., Livant, D. and Hood, L. Joho, R., Weissman, I. L., Early, P., Cole, J. and Hood, L. (1979) The organization and rearrangement of heavy (1980) Organization of kappa light chain genes in chain immunoglobulin genes in mice. In: Eukaryotic germline and somatic tissue. Proc. Nat. Acad. Sci. Gene Regulation, ICN-UCLA Symposium, R. Axel, T. USA 77: 1106-1110. Maniatis and C. F. Fox (Eds.), pp. 393-406. Academic Kehry, M., Ewald, S., Douglas, R., Sibley, C., Raschke, W., Press, New York. Fambrough, D. and Hood, L. (1980) The membrane and Davis, M., Kim, S. and Hood, L. (1980) DNA sequences secreted mu chains differ in their C-terminal amino mediating heavy chain switching in alpha immuno­ acid sequence. Cell. In press. globulin genes. Science. In press. Knight, E. Jr., Hunkapiller, M. W., Korant, B. D., Hardy, Early, P., Huang, H., Davis, M., Calame, K. and Hood, L. R. w. and Hood, L. (1980) Human fibroblast interferon: (1980) An immunoglobulin heavy chain variable region amino acid analysis and N-terminal amino acid gene is generated from three segments of DNA: Vff, D sequence. Science 20?: 525-526. and JH. Cell 19: 981-992. Kronenberg, M., Watson, J. D., Davis, M. M., Early, P. W. Early, P., Rogers, J., Davis, M., Calame, K., Bond, M., and Hood, L. (1980) Helper and killer T cells do not Wall, R. and Hood, L. (1980) Two alternative mRNAs express B-cell immunoglobulin joining and constant can be produced from a single immunoglobulin mu gene region gene segments. J. Exptl. Med. Submitted for by developmentally regulated RNA processing. Cell publication. 20: 313-320. Lafay, F., Ewald, S. J., McMillan, M., Frelinger, J. A. and Elgin, S. C. R., Schilling, J. and Hood, L. E. (1979) Hood, L. E. (1980) Tryptic peptide map analysis of Sequence of histone 2B of Drosophila melanogaster. mouse transplantation antigens. lmmunogenetics. In Biochemistry 18: 5679-5685. press. Ewald, S. J., Klein, J. and Hood, L. E. (1979) Peptide map Okamura, H., Berthold, W., Hood, L., Hunkapiller, M., analysis of mutant transplantation antigens. Immuno­ Inoue, M., Smith-Johansen, H. and Tan, Y. H. (1980) genetics 8: 551-559. Human fibroblastoid interferon: immunosorbent Frelinger, J. G., Hood, L., Hill, S. and Frelinger, J. A. column chromatogrpahy and N-terminal amiito acid (1979) Epidermal la molecules of the mouse have a sequence. Biochemistry. In press. bone-marrow origin. Nature 282: 321-323. Raftery, M. A., Hunkapiller, M. W., Strader, C. D. and Goldstein, A., Tachibana, S., Cowney, L. I., Hunkapiller, Hood, L. (1980) The acetylcholine receptor: a complex M. and Hood, L. (1979) Dynorphin (1-13), an extra­ of homologous subunits. Science 208: 1454-1456. ordinary potent opioid peptide. Proc. Nat. Acad. Sci. Rogers, J., Early, P., Carter, C., Calame, K., Bond, M., USA 76: 6660-6670. Hood, L. and Wall, R. (1980) Two mRNAs with Hood, J. M., Huang, H. V. and Hood, L. (1980) A computer different 3' ends encode membrane and secreted forms simulation of evolution in a multigene family. J. of immunoglobulin mu chain. Cell 20: 303-312. Molec. Evol. In press. Schally, A. V., Huang, W. Y., Chang, R. C. C., Arimura, Hood, L. (1979) Antibodies: Diversity and genes. In: The A., Reddings, T. w., Millar, R. P., Hunkapiller, M. W. Chemistry and Physiology of the Human Plasma and Hood, L. E. (1980) Isolation and structure of pro­ Proteins, D. Bing (Ed.), pp. 49-58. Pergamon Press, somatostatin; a putative somatostatin precursor from New York. pig hypothalamus. Proc. Nat. Acad. Sci. USA. In Hood, L., Davis, M., Early, P., Calame, K., Kim, S. and press. Crews, S. (1980) Two types of DNA rearrangements in Schilling, J., Clevinger, B., Davie, J. M. and Hood, L. immunoglobulin genes. Cold Spring Harbor Symp. (1980) Amino-acid sequence analyses of homogeneous Quant. Biol. In press. antibodies to dextran: diversity patterns suggest that Hood, L. and Early, P. (1980) Organization and rearrange­ DNA rearrangements between V and J gene segments ments of heavy chain variable regions. In: Immuno­ occur in heavy chains. Nature 283: 35-40. globulin Genes and B-Cell Differentiation, J. R. Schwartz, B. D., McMillan, M., Shevach, E., Hahn, Y., Battisto and K. L. Knight (Eds.). Elsevier North Rose, S. M. and Hood, L. (1980) Partial N-terminal Holland, Inc. In press. amino acid sequences of guinea pig classic histocom­ Hood, L. and Schilling, J. (1980) Rearrangements of an patibility antigens. J. Immunol. In press. alpha immunoglobulin heavy chain gene. In: Mem­ Sibley, C., Ewald, S., Kehry, M., Douglas, R. and Hood, L. branes, Receptors, and the Immune Response, E. P. (1980) AB-cell lymphoma synthesizes three species of Cohen and H. Kohler (Eds.), pp. 371-380. Alan R. List, µ chain. J. Immunol. In press. Inc. Taira, H., Broeze, R. J., Jayaram, B. M., Lengyel, P ., Huang, H. V., Crews, S. T. and Hood, L. (1980) The Hunkapiller, M. W. and Hood, L. (1980) Mouse inter­ arrangement, rearrangement and diversification of ferons: NHi-terminal amino acid sequences of various antibody genes. In: Frontiers in Immunogenetics, W. species. Science 207: 528-530. Hindemann and J. Frelinger (Eds.). Elsevier North Weigert, M., Perry, R., Kelley, D., Hunkapiller, T., Holland, Inc., New York. In press. Schilling, J. and Hood, L. (1980) The joining of V and J Hunkapiller, M. W. and Hood, L. (1980) A new protein gene segments creates antibody diversity. Nature 283: sequenator with increased sensitivity. Science 20?: 497-499. 523-525. Weiner, S., Lowenstam, H. A., Taborek, B. and Hood, L. Hunkapiller, M. W., Strader, C. D., Hood, L. and Raftery, (1979) Fossil mollusk shell organic matrix components M. A. (1979) Amino terminal amino acid sequence of preserved for 80 million years. Paleobiology 5: 144-150. the major subunit of Torpedo californica acetylcholine Zoon, K. C., Smith, M. E., Bridgen, P. J., Anfinsen, C. B., receptor. Biochem. Biophys. Res. Comm. 91: 164-169. Hunkapiller, M. w. and Hood, L. E. (1980) Amino terminal sequence of the major component of human lymphoblastoid interferon. Science 207: 527-528. 54

Professor: Norman H. Horowitz phores were isolated from extracts and tentatively identi­ Visiting Associate: Norma P. Williams fied by thin-layer chromatography as ferricrocin in the Research Staff: Gisela Charlang, Bradford Ng case of A. nidulans and ferrichrome in the case of P. Support: The work described in the following research chrysogenum. Both of these compounds are cyclic reports has been supported by a Biomedical Research Support Grant (NIH). hexapeptides. Further identification tests are under way. Both species release cellular siderophores from conidia Summary: The acquisition of iron poses a special problem exposed to media of low water activity. Both species are for aerobic organisms because of the extreme insolubility more resistant to the effects of low water activity than is of ferric ion at physiological pHs. Microorganisms solve Neurospora. Whereas in N. crassa conidial germination is this problem by secreting specific compounds called strongly inhibited at aw ;;; 0.93, the equivalent inhibition in siderophores (formerly siderochromes) that solubilize iron A. nidulans and P. chrysogenum occurs at aw~ 0.87. In in the environment and transport it into the cell. The every case, the inhibition is relieved by addition of siderophores of filamentous fungi, of which about a dozen appropriate siderophores to the medium. are known, are trihydroxamates, usually cyclical, derived These results show that all three species are alike in from ornithine. producing distinct cellular and extracellular siderophores In the course of studying the water requirements of (see Biology 1980, No. 71), and also in their response to . Neurospora crassa, we observed that a germination factor low aw. The latter makes it possible to test the is lost from conidia exposed to solutions of low water effectiveness of different siderophores on each species. activity. The exposed conidia remained viable, but Some interesting results of such tests are summarized in required the unknown factor for germination. The the following abstract. germination response provided a bioassay that enabled us 71. EXTRACELLULAR SIDEROPHORES OF to isolate the factor, which we identified as ferricrocin, a ASPERGILLUS NIDULANS AND siderophore not previously known in Neurospora. This was PENICJLLIDM CHRYSOGENUM the first of a number of new findings concerning the Investigators: Gisela Charlang, Bradford Ng, siderophores of Neurospora that were made possible by Norman H. Horowitz the availability of a convenient and sensitive assay The siderophores secreted into the medium by cultures procedure for these substances. For example, we were of A. nidulans and P. chrysogenum growing under Fe­ able to show that ferricrocin and several other deficient conditions have been reported in the literature. siderophores are not secreted into the medium, but are They are, for A. nidulans, ferricrocin + ferrirhodin; and retained in the cells. A different siderophore, coprogen, for P. chrysogenum, coprogen. Our investigation of these is made for export. Until then, the accepted belief was species has confirmed the production of coprogen by P. that siderophores are exclusively extracellular agents. We chrysogenum as the principal extracellular siderophore, also could demonstrate the appearance of an active but only in cultures grown for six days or longer under uptake mechanism for siderophores shortly before the standard conditions. In younger cultures, the major onset of conidial germination. siderophores are two unidentified substances which we In order to assess the generality of these findings, we call P-a and P-8 . Of particular interest is the fact that have carried out experiments with Aspergillus nidulans P-a and P-S are at least an order of magnitude more and Penicillium chrysogenum. Some of the results are active for P. chrysogenum than is coprogen. They reported below. disappear from older cultures, however. In A. nidulans, we were unable to detect either of the siderophores reported for this species. Instead, we found 70. CELLULAR SIDEROPHORES OF ASPERGILLUS NIDULANS AND PENICILLIDM CHRYSOGENUM a compound (11 X") whose chromatographic properties dif­ AND THE EFFECT OF LOW WATER ACTIVITY fered clearly from those of either ferricrocin or ferri­ Investigators: Gisela Charlang, Bradford Ng rhodin. "X 11 has been isolated and identified as the cyclic Both of the species mentioned in the title produce triester N,N',N"-triacetylfusigen. The identification is different siderophores in (or on) the cells than they based on thin-layer chromatography and NMR spectro­ secrete into the medium.. The principal cellular sidero- scopy. The discrepancy between our results and those of 55

the earlier (European) authors is probably due to mis­ specifically defective in siderophore uptake and those classification of one of the strains. Ours was obtained with more general transport deficiencies. Five were from the Fungal Genetics Stock Center, Arcata, found to be normal in phenylalanine uptake and defective California. in ferricrocin uptake; six were defective in both uptakes. Biological tests showed that "X" is inactive for the An additional mutant was found that was slow to species that produces it, as well as for N. crassa and P. germinate, but was normal in ferricrocin transport. chrysogenum. In young (3-day) cultures of A. nidulans, The five specific mutants were crossed to wild type in however, we find two very active siderophores. These are order to isolate the mutant genes from the ornithineless similar to P-a and P-8 chromatographically and biologi­ background. So far, only one of the mutants has been cally. We refer to them as A-a and A-S. They, too, transferred to a wild-type background. Since the three disappear from older cultures. The!r identity is under ornithine genes are unlinked, only one progeny spore in 16 investigation. The biological significance of triacetyl­ will be of the desired "pure" type if it is also unlinked to fusigen, which is produced in large amounts by our A. them. nidulans, remains a mystery. In the course of this work, it was found that exogenous We are indebted to Dr. Robert Horowitz for the NMR arginine strongly represses ornithine synthesis by spectroscopy that identified 11xn. Neurospora. This opens up the possibility of using an

arginaseless mut~t repressed by arginine in future searches for siderophore-uptake mutants. 72. EXPERIMENTS WITH NEUROSPORA CRASSA Investigators: Gisela Charlllng, Norma P. Williams PUBLICATIONS As we reported last year (Biology 1979, No. 73), 11 Charlang, G., Ng, B. and Horowitz, N. H. (1980) Cellular mutants unable to utilize exogenous ferricrocin have been and extracellular siderophores of Aspergillus nidulans and Penicillium chrysogenum. In preparation. obtained in N. crassa. The mutants were induced in a Horowitz, N. H. (1979) Biological water requirements: In: triple-mutant that is blocked in the known pathways to Strategies of Microbial Life in Extreme Environments, M. Shilo (Ed.), pp. 15-27. Life Sciences Research omithine; this made it possible to start with cells depleted Report 13, Dahlem Konferenzen, Berlin. of siderophores. The mutant strains were tested for their Horowitz, N. H. (1979) Genetics and the synthesis of 14 proteins. In: Symposium on the Origins of ability to take up rHJferricrocin and [ cJphenylalanine Biochemistry: A Retrospect on Proteins. Ann. N.Y. in order to discriminate between those mutants Acad. Sci. 325: 253-266.

National Institutes of Health, USPHS Professor: Tom Maniatis National Science Foundation Gosney Senior Research Fellow: Peter F. R. Little Northwest Area Foundation Senior Research Fellow: Nicholas J~ Proudfoot Damon Runyon-Walter Winchell Cancer Fund Weizmann Research Fellow: Pamela L. Mellon Weizmann Fellowship Research Fellows: Eugene T. Butler In, Edward F. Fritsch, Ross C. Hardison, Richard M. Lawn, Monica summary: Our primary interest is in the molecular basis H. M. Shander, Che-Kun James Shen Graduate Students: David A. Goldberg, Elizabeth H. of differential gene activity in eukaryotes. Our approach Lacy, Joyce E. Lauer, Vann P. Parker, Brian S. Seed is to isolate and characterize segments of eukaryotic Research staff: Sheila Collins, Adriana Cortenbach, Anna Gil, Catherine E. Moser, Catherine D. O'Connell, chromosomal DNA which carry genes that are expressed Mark E. Silver during particular stages of development. To accomplish Laboratory Staff: Maria A. De Bruyn this we are using a combination of biochemical, genetic, SUpport: The work described in the following research and molecular cloning techniques. The availability of reports has been supported by: Rita Allen Foundation well-defined DNA fragments provides the opportunity for American Cancer Society studying the structure and function of individual tran­ Biomedical Research Support Grant (NIH) Norman w. Church Foundation scriptional units which comprise different developmental European Molecular Biology Organization pathways. Questions such as the precise location of E. s. Gosney Fund 56 transcription initiation and termination sites with respect the presence of two intervening sequences. to structural gene sequences, the timing of synthesis and turnover of specific gene transcripts, and the chromo­ 73. MOLECULAR CWNING OF THE HUMAN $-LIKE somal organization of coordinately or sequentially GWBIN GENE CLUSTER expressed genes are being addressed. Investigators: lldward F. Fritsch, Richard M. Lawn

During the past year we completed structural analyses The genes encoding human embryonic (e::), fetal (Gy, of the rabbit $-like and human e- and ex-like globin gene Ay) and adult (o, $) $-like globin polypeptides were clusters. These studies revealed an interesting and isolated as a set of overlapping cloned DNA fragments common pattern of gene organization and provided new from bacteriophage A libraries of high molecular weight information regarding the evolution of mammalian globin (15-20 kb) chromost9mal DNA. All five genes are tran­ genes. Most notable in this regard was the discovery of scribed from the same DNA strand and are arranged in the globin pseudogenes and an interesting family of repetitive order 5'-o-(13.3 kb)-8 y-(3.5 kb)-Ay-(13.9 kb)-o-(5.4 kb)-$- DNA sequences which are found in both the human a- and 3'. In addition to the five known 13-like globin genes, we e- and in the rabbit $-globin gene clusters. have detected two other 13-like globin sequences which do We have continued our efforts to establish in vivo not correspond to known polypeptides. assays of globin gene expression using DNA mediated gene A sequence which is repeated within the human 13-like transfer techniques in collaboration with Richard Axel and globin gene cluster (Fritsch, Lawn and Maniatis, 1980) is his laboratory at Columbia University. We have suc­ also interspersed with the human a-like globin genes (E. ceeded in introducing the human 13-globin gene into mouse Fritsch, J. Shen, R. Lawn and T. Maniatis, unpublished erythroleukemia cells. The transferred gene is expressed results), is highly repeated elsewhere in the genome at low levels, but its expression is not induced by DMSO. (Fritsch, Lawn and Maniatis, 1980), and is homologous to a Recently, we have established the feasibility of using sequence which is found in the rabbit $-like globin gene an in vitro transcription procedure to study the expression cluster (Shen and Maniatis, 1980; J. Shen, unpublished of cloned globin genes. Jn collaboration with Jim Manley results). Nucleotide sequence analysis of the copy of this and Malcolm Gefter at MIT, we have shown that the repeat located 5' to the Gy gene indicates that the repeat human 13-globin gene is accurately transcribed in vitro and is a member of a family of sequences which is reiterated the 5'-end of the 13-globin RNA transcript is identical to approximately 300,000 times in the human genome that of the $-globin mRNA isolated from erythroid cells. (Houck, Rinehart and Schmid, 1979; Jelinek et al., 1980). More recently, we have shown that all of the members of This family of repeated sequences is characterized by the the human globin gene family are accurately transcribed following set of diverse properties: (1) the repeats are in vitro and that genes which are transcribed relatively transcribed by RNA polymerase III in vitro (Duncan et al., less efficiently in vivo are expressed at correspondingly 1980; Fritsch et al., in preparation); (2) the repeats share low levels in vitro. The ability to faithfully transcribe sequence homology with an abundant small nuclear RNA globin genes in vitro has made it possible to identify molecule (Jelinek and Leinwand, 1978; Jelinek et al., globin gene promoters; this is being accomplished by 1980) and with double-stranded heterogeneous nuclear constructing deletion and point mutations near the human RNA (Jelinek et al., 1980; Fritsch, Lawn and Maniatis, 13-globin gene in vitro and then studying the effects on in 1980); (3) the repeats contain a sequence which is vitro transcription. homologous to a sequence found near the replication Another gene locus under study in the lab is the origins of SV40, polyoma and BK DNA tumor viruses alcohol dehydrogenase gene (ADH) of Drosophila melano­ (Jelinek et al., 1980). gaster. This locus is of interest because considerable References: genetic and biochemical data are available and the gene is Duncan, c., Biro, P. A., Chowdary, P. V., Elder, J. T., differentially expressed during development. The ADH Wang, R. R. C., Forget, B. G., De Riel, J. K. and Weissman, S. M. (1980) Proc. Nat. Acad. Sci. USA 76: gene was isolated from a library of Drosophila DNA using 5095-5099. a novel procedure which involves differential hybridi­ Fritsch, E. F., Lawn, R. M. and Maniatis, T. (1980) Cell 19: 959-972. zation using RNA from a wild type and an ADH deletion Houck, C. M., Rinehart, F. P. and Schmid, C. W. (1979) J. strain. Structural characterization of the gene revealed Mo!. Biol. 132: 389-306. 57

Jelinek, W.R. and Leinwand, L. (1978) Ce!l 15: 205-214. transcription and splicing. In particular, alignment of the Jelinek, w. R., Toomey, T. P., Leinwand, L., Duncan, C. sequences 5' to the human S-like globin genes revealed H., Biro, P. A., Choudary, P. V., Weissman, S. M., Rubin, c. M., Houck, c. M., Deininger, P. L. and two blocks of sequence homology which are present in Schmid, C. W. (1980) Proc. Nat. Acad. Sci. USA 77: analogous positions adjacent to most eukaryotic genes. 1398-1402. Shen, C.-K. J. and Maniatis, T. (1980) Cell 19: 379-391. The first homology block is an AT-rich sequence originally identified in the Drosophila histone gene cluster ("the 74. THE NUCLEOTIDE SEQUENCE OF THE HUMAN Hogness box"). A comparison of a number of different a­ B-GLOBIN GENE like globin genes has revealed that the AT-rich sequence Investigators: Richard M. Lawn, Catherine D. O'Connell, CATAAA is found 31 ±1 bp 5' to the mRNA capping sites, Argiris Efstratiadis* but that the sequence shared by all of the $-like genes is As a prerequisite for a detailed study of globin gene ATA. This sequence was therefore designated the ATA transcription and processing of mRNA precursors, we have box. The second homology block (designated the CCAAT determined the complete nucleotide sequence of the adult box) is located 77 ±10 bp 5' to each gene. B-globin gene. As predicted from earlier fine structure Previous comparisons of noncoding sequence in human, mapping studies (Lawn et al., 1978), the gene contains two mouse and rabbit S-like globin genes indicated that these introns of 130 bp and 848 bp located between the codons regions diverged by deletion and addition as well as by for amino acids 30 and 31 and 104 and 105, respectively. simple base substitution. Examination of the nucleotide Comparison of this sequence with the complete sequences surrounding putative deletion sites suggests nucleotide sequence of the other human B-like globin that short (2-8 nucleotides) direct repeats are involved in genes has led to interesting insights into the evolution of the generation of deletions. This pattern is remarkably S-globin genes and has revealed conserved 5' flanking similar to that observed for deletion hot spots in the lac i sequences which may be functionally significant. gene of E. coli (Farabaugh and Miller, 1978). On the basis Reference: of this information and an earlier model for the genera­ Lawn, R. M., Fritsch, E. F., Parker, R. C., Blake, G. and tion of frameshift mutation (Streisinger et al., 1966), we Maniatis, T. (1978) Cell 15: 1157-1174. proposed a model for the involvement of short direct *Department of Biological Chemistry, Harvard Medical repeat sequences in the generation of deletions in the School, Boston, Massachusetts. noncoding regions of S-like globin genes during evolution.

References: 75. THE STRUCTURE AND EVOLUTION OF THE Baralle, F. E., Shoulders, c. and Proudfoot, N. J. (1980) HUMAN B-GWBIN GENE FAMILY Cell. Jn press. Investigators: Argiris Efstratiadis*, James w. Posakony, Farabaugh, P. J. and Miller, J. H. (1978) J. Mo!. Biol. 126: Tom Maniatis, Richard M. Lawn, 847-863. Catherine D. O'Connell Lawn, R. M., Efstratiadis, A., O'Connell, C. and Maniatis, T. (1980) Cell. Jn press. The complete nucleotide sequences of the five human Slightom, J., Blechl, A. and Smithies, o. (1980) Cell. Jn press. S-like globin genes have been determined (Lawn et al., Spritz, R., De Riel, J., Forget, B. and Weissman, S. (1980) 1980; Spritz et al., 1980; Slightom, Blechl and Smithies, Cell. Jn press. Streisinger, G., Okada, Y., Emrich, J., Newton, J., 1980; Baralle, Shoulders and Proudfoot, 1980). We have Tsugita, A, Terzaghi, E. and Inouye, M. (1966) Cold collaborated with the research groups involved in these Spring Harbor Symp. Quant. Biol. 31: 77-84. sequencing studies to carry out a detailed analysis of the *Department of Biological Chemistry, Harvard Medical sequence information. The analysis provided the first School, Boston, Massachusetts~ description of the evolutionary relationship of a gene family based entirely on direct nucleotide sequence 76. NUCLEOTIDE SEQUENCE ANALYSIS OF comparison. In addition, the evolutionary relationship of RABBIT B-GWBIN GENES the embryonic e;-globin gene to other human S-like globin Investigators: Eugene T. Butler ID, Elizabeth H. Lacy, Ross C. Hardison genes _was defined for the first time.

The sequence comparison of the human S-like globin The polypeptide subunit composition of rabbit hem~ genes also revealed interesting sequence homologies in globin is developmentally ,regulated. Early in develop­ regions which are potentially involved in globin gene ment, t~o types of embryonic globin chains, designated X 58

(ex-like) and < ($-like), are synthesized in nucleated Lacy, E., Hardison, R. c., Quon, D. and Maniatis, T. (1979) Cell 18: 1273-1283. erythroid cells which originate in the blood islands of the Lacy, E. and Maniatis, T. (1980) Cell 21: 545-553. yolk sac (Melderis, Steinheider and Ostertag, 1974). Two Maniatis, T., Hardison, R. C., Lacy, E., Lauer, J., O'Connell, C., Quon, D., Sim, G. K. and Efstratiadis, types of nonallelic E chains appear to be present. The X A. (1978) Cell 15: 687-701. polypeptide is soon replaced by the adult cx-globin chain. Melderis, H., Steinheider, G. and Ostertag, W. (1974) Nature 250: 774-776. Later, as the site of erythropoiesis switches to the fetal liver, the adult $-globin polypeptide synthesis supplants that of the < chains (Kitchen and Brett, 197 4). The genes which encode the rabbit $-like globin 77. THE NUCLEOTIDE SEQUENCE OF A RABBIT $-GWBIN PSEUDOGENE polypeptides were obtained by screening a bacteriophage A Investigator: Elizabeth H. Lacy library of rabbit chromosomal DNA (Maniatis et al., 1978). Four 13-related globin gene sequences were shqwn to A cluster of four different $-like globin gene reside as a linked set in the rabbit genome in the sequences has been isolated from a bacteriophage A orientation 5'-$4-$3-$2-$ 1-3' (Lacy et al., 1979). library of rabbit chromosomal DNA in a set of overlapping Nucleotide sequence analysis of regions within and clones which together contain 44 kilobase pairs (kb) of flanking the genes was undertaken for gene identification contiguous DNA (Maniatis et al., 1978; Lacy et al., 1979). purposes and to determine sequences of DNA which may Approximately 5-8 kb of DNA separate each gene pair and serve for the regulation of globin gene expression. all four genes are transcribed from the same strand of Genes $4 and 133 are preferentially expressed in early DNA in the orientation 5'-B4-B3-B2-f31-3' (Lacy et al., rabbit embryos and appear to code for the < polypeptides 1979). Gene $2 hybridizes more efficiently to adult than (Hardison et al., 1979; E. Butler and R. Hardison, to embryonic B-globin mRNA sequences. However, no B2 unpublished results). Gene $ 1, which is expressed in adult mRNA transcripts have been detected in adult bone rabbit bone marrow, encodes the adult B-globin chain marrow and reticulocytes nor in embryonic erythroid cells (Hardison et al., 1979). Transcripts of the $2 region of (Hardison et al., 1979). One possible explanation for the the cluster do not accumulate in any tissue examined and apparent lack of e2 transcripts is that gene $2 is a globin nucleotide sequence analysis has shown that the locus pseudogene, a B-like sequence that does not code for a cannot code for a functional B-like globin polypeptide functional 13-globin polypeptide. A comparison of the (Lacy and Maniatis, 1980). nucleotide sequence of $2 with that of the rabbit adult e­ One strategy being employed to identify DNA globin gene, B1, reveals the presence of frameshift sequences which may be used for the temporal regulation mutations and premature termination codons in the of the expression of B-globin genes is to compare the protein coding sequence which render B2 unable to encode sequences of regions of DNA which flank the genes of a functional $-globin polypeptide. We therefore refer to distantly related species. B-globin genes which are this gene as ljJ e2 to in di ca te that it is a pseudogene. ljJ e2 expressed at ~alogous times in the differentiation of contains two intervening sequences at the same locations different organisms may be under the control of regula­ in the globin protein coding sequence as B1 and all other tory sites of evolutionarily conserved nucleotide sequence. sequenced B-globin genes. An examination of the DNA Preliminary results indicate that alignment of the 5' sequences at the intron/exon junctions suggests that a flanking regions of the rabbit $3 gene with that of a y­ putative 1jJ 132 precursor mRNA could not be normally globin gene which is expressed in fetal humans reveals a spliced. feature which the two genes share that distinguishes them Estimation of the time at which ljJ e2 diverged from its from the 5' flanking regions of the adult B-globin genes functional counterpart 131 indicates that the two genes (R. Hardison, unpublished observation). diverged well after the time at which adult and embryonic specific globin sequences began to appear (approximately References: 200 million years ago [Efstratiadis et al., 19801). Thus, Hardison, R. C., Butler, E. T. III, Lacy, E., Maniatis, T., Rosenthal, N. and Efstratiadis, A. (1979) Cell 18: the creation of the rabbit B pseudogene was not 1285-1297. coincident with the formation of the gene cluster. It Kitchen, H. and Brett, I. (1974) Ann. N.Y. Acad. Sci. 241: 653-670. seems likely that pseudogenes are simply a natural 59 consequence of the mechanisms by which multigene 79. ISOLATION AND STRUCTURAL CHARACTERIZATION OF THE HUMAN families evolve. <>-LIKE GLOBlN GENE CLUSTER References: Investigator: Joyce E. Lauer Efstratiadis, A., et al. (1980) Cell. In press. Hardison, R. c., Butler, E. T., Lacy, E., Maniatis, T., The adult (a) and embryonic (~) globin genes com­ Rosenthal, N. and Efstratiadis, A. (1979) Cell 18: prising the a-like globin gene cluster have been isolated 1285-1297. Lacy, E., Hardison, R. C., Quon, D. and Maniatis, T. (1979) (Lauer, Shen and Maniatis, 1980; Lauer, 1980). (1) Tu·e a­ Cell 18: 1273-1283. like genes are linked in the order 5'-r; 2-r; 1-i.lial-a2-al-3'. Maniatis, T., Hardison, R. c., Lacy, E., Lauer, J., O'Connell, C., Quon, D., Sim, G. K. and Efstratiadis, (2) jlal is a pseudogene for which no globin polypeptide A. (1978) Cell 15: 687-701. can be identified. (3) Comparison of the DNA regions flanking al and a2 indicates that each gene is located within an approximately 4 kb region of homology inter­ 78. TISSUE-SPECIFIC DNA METHYLATION 1N A CLUSTER OF RABBIT 6-LIKE GLOBlN GENES rupted by two short regions of nonhomology. The Investigator: Che-Kllll James Shen presence of these large blocks of homology in genes which are thought to have duplicated long ago suggests the The relationship between DNA methylation and existence of a mechanism for sequence matching. (4) The differential expression of rabbit 13-like globin genes was positions and the precise lengths of two types of deletions studied using restriction enzymes whose recognition which occur in cloned DNA indicate that deletion occurs sequences include the dinucleotide CpG (Bird and by homologous but unequal crossing-over between corre­ Southern, 1978). In particular, we made use of the sponding regions of al and a2. The lengths and positions restriction enzymes, Hpa II and Msp I, which cleave the of these deletions are indistinguishable from those of the sequence CCGG but are differentially inhibited by the two types of deletions which are associated with a­ presence of 5-methyl cytosine (Mann and Smith, 1977; thalassemia 2 (S. Embury, personal communication}, sug­ Waalwijk and Flavell, 1978; Singer, Robert-Ems and gesting that this common genetic disease results from Riggs, 1979). The methylation frequency of 13 CCGG sites which flank a set of four closely linked rabbit a -like homologous but unequal crossing-over between regions within and/or surrounding the adult cx-globin genes. (5) globin genes (Lacy et al., 1979; Hardison et al., 1979) was Identification of z; 2 and demonstration of the linkage of determined. This analysis revealed that certain sites z; surrounding embryonic and adult globin genes are 2 to the other a-like genes made it possible to locate the breakpoints of deletions associated with the a-thalassemia relatively undermethylated in DNA from embryonic and adult erythroid tissues, respectively. This pattern is most syndrome, hydrops fetalis. In one individual al, a2, i.lial and r; 1 are deleted, but z; 2 remains and z; polypeptide is pronounced for sites located near the 5'-ends of globin produced in large amount (Pressley et al., 1980). genes. Those sites are totally methylated in nonerythroid cells and partially methylated in erythroid cells. We Ordinarily c-globin is not detectable after 10 weeks gestation. The persistence of r;-globin production in cases conclude that the degree of CpG methylation in the rabbit 13-like globin gene cluster is correlated with gene activity, of hydrops fetalis appears to be the a-locus analogue of but the effect is confined to relatively small regions of persistent expression of y-globin caused by the a-locus disease HPFH. DNA. References: References: Lauer, J. (1980) Ph.D. Thesis, California Institute of Bird, A. P. and Southem, E. M. (1978) J. Mo!. Biol. 118: Technology. 27-47. Lauer, J., Shen, J. and Maniatis, T. (1980) Cell 20: Hardison, R. c., Butler, E. T., Lacy, E., Maniatis, T., 119-130. Rosenthal, N. and Efstratiadis, A. (1979) Cell 18: Pressley, L., Higgs, D. R., Clegg, J. B. and Weatherall, D. 1285-1297. J. (1980) Proc. Nat. Acad. Sci. USA 77: 3586-3589. Lacy, E., Hardison, R. C., Quon, D. and Maniatis, T. (1979) Cell 18: 1273-1283. 80. THE STRUCTURE OF A HUMAN <>-GLOBlN Mann, M. B. and Smith, H. o. (1977) Nucleic Acids Res. 4: 4211-4221. PSEUDOGENE (lJlal) AND ITS RELATIONSHIP TO <>-GLOBlN GENE DUPLICATION Singer, J., Robert-Ems, J. and Riggs, A. D. (1979) Science 203: 1019-1020. Investigator: Nicholas J. Proudfoot Waalwijk, C. and Flavell, R. A. (1978) Nucleic Acids Res. 5: 4631-4641. The human a-like globin gene cluster consists of five 60

closely-linked genes: two adult et-globin genes (al, a2), a Referenees: Lauer, J., Shen, C.-K. J. and Maniatis, T. (1980) Cell 20: gene which cannot be identified with a known globin 119-130. polypeptide (¢al), and two embryonic ~-globin genes (~l, Liebhaber, s. A., Goossens, M. J. and Kan, Y. W. (1980) Submitted for publication. ~ 2) (Lauer, Shen and Maniatis, 1980; see Biology 1980, No. 79). To determine whether 1jJcxl could encode an expressed 81. CHARACTERIZATION OF INTERGENIC REPEAT globin gene, its complete nucleotide sequence was deter­ SEQUENCES OF THE HUMAN 11-LIKE mined. Comparison of this sequence with a normal human GI.OBIN GENE CLUSTER et-globin gene (a2) (Liebhaber, Goossens and Kan, 1980) Investigator: Che-Km James Shen revealed that Wal contains both an initiator codon The sequence organization of intergenic regions within mutation and frameshift deletions which would prevent the human a-like globin gene cluster has been investi­ the production of an et-globin polypeptide. ¢al contains gated. Two types of repeated elements have been two intervening sequences with sizes and locations char­ identified within this gene cluster. Heteroduplex studies acteristic of mammalian a-globin genes. However, the and restriction mapping have demonstrated extensive alteration or absence of putative splicing sequences homology between the adult genes, al and a2. This region suggests that a primary transcript of Wal would not be of homology, which includes the coding sequence and 5' processed to produce a mature mRNA. Thus, Wal is an a­ flanking region, spans a region of 4 kb and is interrupted globin pseudogene, a gene which shares significant by two short regions of nonhomology. This homology has homology to a functional gene, but has acquired mutations been interpreted to be a result of the duplication event which prevent its expression. Although the presence and which gave rise to the two adult genes. similar location of pseudogenes in all of the mammalian The distribution of middle- or highly-repetitive globin gene clusters suggest that pseudogenes may have sequences within the a-globin gene cluster has been some as yet unidentified function (see Biology 1980, No. reve~led by probing Southern blots of restriction digests 77), the Simplest explanation for their existence is that of cloned a-cluster DNA with 32 P-labeled total genomic they are the natural consequence of the mechanisms of DNA. At least seven intergenic regions contain repetitive gene amplification and sequence divergence which act on DNA sequences. These repeated sequences cross­ multigene families during evolution. hybridize with the Alu family repeat which is also present The ,,Pal gene is located adjacent to two interrupted in the B-globin gene cluster and is highly repeated in the 4 kb duplication units, one containing the a2 gene and the human genome. Each of these seven copies of this repeat other the al gene. The leftward endpoints of this serves as a template for RNA polymerase III-dependent duplication were mapped by examination of hetero­ transcription in vitro. The transcripts produced range in duplexes which contain either the al or a.2 genes (Lauer, size from 500 nt to less than 40 nt. Shen and Maniatis, 1980). To establish the relationship Heteroduplex analysis is now being extended to the between the $al gene and the a-globin gene duplication regions flanking the embryonic a-like globin genes (' 1 and and to define the leftward endpoints of the duplication, '2) and the pseudogene (¢al) in order to determine the nucleotide sequence of the regions 3' to al, a2 and $al whether there are any major regions of sequence were determined. We found that the a2 gene duplication homology surrounding those genes. extends from the poly( A) addition site of $al to the poly(A) addition site of crl while the crl duplication 82. THE NUCLEOTIDE SEQUENCE OF THE extends from the poly(A) addition sites of crl and "2. The HUMAN EMBRYONIC ~-GI.OBIN GENES endpoint of each duplication unit consists of a repeated Investigators: Catherine D. O'Connell, pentanucleotide (GCCTG) separated by TGTGT. The Nicholas J. Proudfoot occurrence of this sequence in all three genes and its The complete nucleotide sequence analysis of one of location with respect to the endpoints of the n-globin gene the two embryonic r,;-globin genes (r,; 1) is in progress. duplication suggest that this sequence might be associated With approximately 75% of the sequence completed, the with the mechanism by which the genes were duplicated amino acid sequence predicted from the DNA sequence is or corrected. in excellent agreement with partially completed sequence r,;-globin polypeptide (J. Clegg, personal communication). 61

Although the r; 1 gene contains two intervening sequences 84. IN VITRO TRANSCRIPTION OF GLOBIN GENES at locations which are characteristic of other mammalian Investigators: Monica H. M. Sh.ander, Nicholas J. Proudfoot a-globin genes, the 3' introns deviate significantly from the normal pattern. Both introns are abnormally large for The human et- and S-globin gene clusters have been an a-like gene; 300-400 nucleotides rather than 100. isolated and a comparative DNA sequence analysis of Finally, intron 2 is a particularly remarkable sequence as most of the genes within these clusters has been com­ it closely resembles a simple repeated sequence (CG ) . pleted. The observation that certain regions of DNA 4 50 sequence are shared among the globin genes suggests that these sequences may be functionally important. However, 83. TRANSFORMATION OF MOUSE ERYTHRO­ actual identification of regulatory sequences such as LEUKEMIA CELLS WITH CLONED HUMAN GLOBIN GENES regulatory protein binding sites, RN A processing sites and Investigator: Pamela L. Mellon transcriptional initiation sites requires assays for gene expression. Cell-free extracts for RNA polymerase 11- A DNA fragment containing the human S-globin gene dependent transcription of cloned eukaryotic genes pro­ was introduced into mouse erythroleukemia cells (MEL vide an in vitro assay for globin gene expression (Manley cells) by thymidine kinase-mediated DNA transformation et al., 1980; Weil et al., 1979). These in vitro transcrip­ (Wold et al., 1979) in order to investigate the expression of this gene during MEL cell differentiation. MEL cells tion systems are capable of specific transcription of are a Friend virus transformed cell line which has the adenovirus genes and of the chicken ovalbumin and characteristics of an erythroid precursor cell. When conalbumin genes (Manley et al., 1980; Weil et al., 1979; Wasylyk et al., 1980). treated with any of a number of specific compounds, these cells differentiate along the erythroid pathway: hemo­ We have used the truncated template assay (Weil et globin synthesis is induced and cell division ceases. aJ., 1979) with HeLa whole cell extracts (Manley et al., 1980) to determine whether individual globin genes can A 7.6 kb DNA fragment containing the S-globin gene plus flanking sequences, and the herpes virus thymidine function as templates for specific in vitro transcription. kinase gene, were cloned together in pBR322. This All globin genes assayed appear to comprise independent recombinant plasmid was used to transform an MEL cell transcription units. Results of primer extension experi­ line which carried a mutation in the gene for thymidine ments suggest that the 5'-ends of the in vitro globin gene kinase (tk -). Selection for the tk +phenotype yielded five transcripts are coterminal with their respective mRNA capping sites. clones of transformed cells, which were shown to contain human $-globin DNA by blot hybridization experiments. The various globin genes are expressed at different Production of human S-globin RNA was demonstrated in levels in vivo, and during different developmental stages. two of these five cell lines by using a modified Berk-Sharp For example, the in vivo level of cS-globin nuclear mRNA precursor is 10-fold lower than that of the B-globin hybridization-Bl analysis (Berk and Sharp, 1977). This mRNA precursor. This difference could be due to a method, which involves an end-labeled DNA probe defect in 6-globin gene transcription. Consistent with (Weaver and Weissman, 1979) allows detection of human this possibility is the observation that the 8-globin gene is mRNA in the presence of a large amount of mouse e­ globin mRNA. We plan to study the effect of MEL cell transcribed less efficiently in vitro than the S-globin differentiation on the expression of the heterologous gene. We are also studying the .transcription in vitro of human S-globin gene. mutant S-globin genes and .of pseudogenes.

References: References: Berk, A. and Sharp, P. (1977) Cell 12: 721-732. Manley, J. L., Fire, A., Cano, A., Sharp, P.A. and Gefter, (1980) Weaver, R. and Weissman, (1979) Nucleic Acids Res. 'l: M. L. Proc. Nat. Acad. Sci. US,\. In press. c. Wasylyk, B., Kedinger, J., Corden, J., Brisen, and 1175-1193. o. Chambon, P. (1980) Nature 285: 367-373. Wold, B., Wigler, M., Lacy, E., Maniatis, T., Silverstein, s. Weil, P. A., Luse, D. S., Segall, J. and Roeder, R. G. and Axel, R. (1979) Proc. Nat. Acad. Sci. USA 76: (1979) Cell 18: 469-484. 5684-5688. 62

85. IDENTIFICATION OF THE HUMAN fl-GLOBIN criteria: in situ hybridization, in vitro translation of GENE PROMOTER mRNA selected by hybridization to the cloned DNA, and Investigators: Vann P. Parker, Monica H. M. Shander, comparison of the ADH protein sequence with a nucleo­ Nicholas J. Proudfoot tide sequence derived from the cloned DNA. We have begun studies to define the promoter Comparison of the restriction site maps from clones of sequences necessary for accurate RNA transcription of three different wild-type Drosophila strains revealed the the human S-globin gene. Comparative sequence analysis presence of a 200 nucleotide sequence in one strain which of the S-like globin genes has identified two regions 5' to is absent in the other two strains. The ADH mRNA the message sequence which are shared by all five S-like sequences were located within the cloned D_NA by genes. The first is the sequence CCAAT which is located hybridization-mapping experiments. Two intervening 80 bases 5' to the CAP site, and the second is an AT-rich sequences were identified within the ADH gene by limited region, the ATA box, which is located 30 bases 5' 'to the nucleotide sequence and Sl mapping experiments. CAP site. A series of deletion molecules was generated from a genomic clone of the human S-globin gene. The PUBLICATIONS parental clone was linearized with the enzyme Hpa I and Efstratiadis, A., Posakony, J. w., Maniatis, T., Lawn, R. shortened using the double-strand DNA exonuclease Bal M., O'Connell, C., Spritz, R. A., DeRiel, J. K., Forget, 31. New clones were isolated which contained the globin B., Weissman, S. M., Slightam, J. L., Blechl, A. E., Smithies, O., Baralle, F. E., Shoulders, and gene and various amounts of 5' flanking sequence. When c. c. Proudfoot, N. J. (1980) The structure and evolution of these clones were transcribed in vitro, it was shown that the human 8-globin gene family. Cell. In press. Fritsch, E. F., Lawn, R. M. and Maniatis, T. (1980) deletion of the CCAAT sequence did not abolish tran­ Molecular cloning and characterization of the human . scription of the gene, while deletion of the ATA sequence 8-like globin gene cluster. Cell 19: 959-972 • Goldberg, D. A. (1980) Isolation and partial characteri­ led to aberrant transcription. zation of the Drosophila alcohol dehydrogenase gene. We plan to extend these studies using additional Proc. Nat. Acad. Sci. USA. In press. Hardison, R. c., Butler, E. T., Lacy, E., Maniatis, T., deletion molecules and genes altered by point mutation of Rosenthal, N. and Efstratiadis, A. (1979) The struc­ specific base pairs. ture and transcription of four linked rabbit 13-like globin genes. Cell 18: 1285-12 97. Lacy, E., Hardison, R. c., Quon, D. and Maniatis, T. (1979) The linkage arrangement of four rabbit 8-like globin 86. ISOLATION AND PARTIAL CHARACTERIZATION genes. Cell 18: 1273-1283. OF THE DROSOPHILA ALCOHOL Lacy, E. and Maniatis, T. (1980) The nucleotide sequence DEHYDROGENASEGENE of a rabbit 8-globin pseudogene. Cell. In press. Lauer, J., Shen, C.-K. J. and Maniatis, T. (1980) The Investigators: David A. Gold>erg, Mark E. Silver chromosomal arrangement of human ex-like globin genes: Sequence homology and cx-globin gene The Drosophila alcohol dehydrogenase (ADH) gene deletions. Ce!l 20: 119-130. provides an interesting system for studying the molecular Lawn, R. M., Efstratiadis, A., O'Connell, c. and Maniatis, T. (1980) The nucleotide sequence of the human 8- basis of differential gene expression during development globin gene. Cell. In press. since it is a developmentally regulated gene for which Maniatis, T. (1980) Recombinant DNA procedures in the study of eukaryotic genes. In: Cell Biology: A considerable genetic and biochemical data are available. Comprehensive Treatise, L. Goldstein and D. Prescott For this reason, we have cloned the Drosophila ADH gene (Eds.), pp. 563-608. Academie Press, New York. Maniatis, T., Butler, E. T., Fritsch, E. F., Hardison, R. C., and partially characterized the physical structure of the Lacy, E., Lawn, R. M., Parker, R. C., Shen, C.-K. J. gene. (1979) The linkage arrangement of mammalian 8-like globin genes. In: Eukaryotic Gene Regulation, R. Adult RNA from wild-type flies was enriched in ADH Axel, T. Maniatis and C. F. Fox (Eds.), pp. 317-333. sequences by gel electrophoresis and then used to prepare Academic Press, New York. Maniatis, T., Fritsch, E. F., Lauer, J. and Lawn, R. M. labeled cDNA for screening a bacteriophage A library of (1980) The moleeular genetics of human hemoglobins. genomic Drosophila DNA. Of the clones which hybridized Ann. Rev. Genetics. In press. Proudfoot, N. J. and Maniatis, T. (1980) The structure of a in the initial screen, one clone was identified which human cx-globin pseudogene and its relationship to cx­ hybridized with labeled cDNA prepared from a wild-type globin gene duplication. Cell. In press. Proudfoot, N. J., Shander, M. H. M., Manley, J. L., Gefter, Drosophila strain but did not hybridize with cDNA M. L. and Maniatis, T. (1980) The structure, chromo­ prepared from an ADH deletion strain. This clone was somal arrangement and in vitro transcription of human globin genes. Science. In press. shown to contain ADH structural gene sequences by three 63

Shen, C.-K. J. and Maniatis, T. (1980) The organization of Wigler, M., Sweet, R., Sim, G. K., Wold, B., Pellicer, A., repetitive sequences in a cluster of rabbit a-like Lacy, E., Maniatis, T., Silverstein, s. and Axel, R. globin genes. Cell 19: 379-391. (1979) Transformation of mammalian cells with Shen, C.-K. J. and Maniatis, T. (1980) Tissue-specific DNA prokaryotic and eukaryotic genes. In: Eukaryotic methylation in a cluster of rabbit a-like globin genes. Gene Regulation, R. Axel, T. Maniatis and C. F. Fox Proc. Nat. Acad. Sci. USA. In press. (Eds.), pp. 457-475. Academic Press, New York. Sim, G. K., Kafatos, F. C., Jones, C. W., Koehler, M. D., Wold, B., Wigler, M., Lacy, E., Maniatis, T., Silverstein, S. Efstratiadis, A. and Maniatis, T. (1979) Use of a cDNA and Axel, R. (1979) Introduction and expression of a library for studies on evolution and developmental rabbit $-globin gene in mouse fibroblasts. Proc. Nat. expression of the Chorion gene families. Cell 18: Acad. Sci. USA 76: 5684-5688. 1303-1316.

Assistant Professor: Elliot M. Meyerowitz toward understanding the molecular mechanisms under- • Graduate Student: Madeline A. Crosby Research Staff: Celeste A. Berg lying the coordinate tissue-specific production of the three 68C RNA species and toward comprehending the Support: The work described in the following summary has been supported by: mechanism by which ecdysone represses gene activity at Biomedical Research Support Grant (NIH) the puff. Alfred P. Sloan Fund for Basic Research The second project involves the utilization of a new recombinant DNA vector that allows regular molecular Summary: The current research in this laboratory cloning of pieces of foreign DNA over 45,000 base pairs in involves two major projects directed toward the goal of length. A pool of random Drosophila chromosomal finding the biochemical mechanisms underlying fragments has been cloned using this vector; random differential gene expression and pattern formation in the members of this pool are being isolated and their locations development of the fruit fly, Drosophila melanogaster. The first project is a molecular study of the polytene of origin on the Drosophila chromosomes are being chromosome puff found at location 68C on the left arm of mapped, using the technique of in situ hybridization. the Drosophila third chromosome. This locus is Once a large catalogue of clone locations has been responsible for production of one of a group of poly­ obtained, a clone that maps near any desired Drosophila peptides secreted by the larval salivary gland just prior to could be obtained and used as a starting point -for a pupation, and used by the organism as a glue with which it process of successive isolation of overlapping cloned attaches itself to its substrate for the duration of the chromosome fragments, eventually leading to the isolation pupal period. The signal that starts RNA transcription at of a recombinant DNA moleeule containing the gene of the locus is unknown. Transcription is stopped by a direct interest. Thus, Drosophila loci whose genetic location is interaction of the steroid hormone ecdysone with the puff. known and whose function is important in development, Research to date has shown that the 68C puff contains the but whose biochemical products are unknown, could be DNA sequences that code for three different species of conveniently isolated. RNA which are produced simultaneously in the larval salivary glands but not in any other larval tissue. Detailed PUBLICATION maps of the DNA sequence topography at 68C have been prepared using recombinant DNA techniques; these maps Meyerowitz, E. M., Guild, G. M., Prestidge, L. S. and Rogness, D. S. (1980) A new high capacity cosmid permit experiments currently in progress that are directed vector and its use. Submitted for publication. 64

Professor: Herschel K. Mitchell all cases, there is an increased number of hairs from the Research Associate: Peter H. Lowy normal one per cell to four or five per cell and they are Gosney Senior Research Pellow: Nancy S. Petersen Graduate Students: Antonio A. Reyes, Loveriza A. separate from each other. In contrast, the multihair Sarmiento phenocopy induced by a heat shock (Mitchell and Lipps, Research Staff: Jean Edens, Joan Roach 1978) yields hairs which appear in bundles at one bundle Support: The work described in the following research per cell if the heat shock (40.8°C) is applied for 35 min or reports has been supported by: Earle C. Anthony Fellowship longer. However, a shorter treatment (25 min) yields Josephine V. Dumke Fund many separate but multiple hairs similar to the phenotype E. S. Gosney Fund National Institutes of Health, USPHS of mwh. Furthermore, if the mutant mwh is given a heat The Rockefeller Foundation shock of 35 min, then bundles of hairs appear as observed in the phenocopy. Thus, it appears that the differen­ Summary: A major center of attention of the past year tiating hair cells are affected in the same way by the has been concerned with the biochemical and molecular mutation and the heat shock. basis for the differentiation of Drosophila epithelial cells to give rise to hairs. Wing development in particular As noted in the foregoing paragraph, the mutant mwh provides an especially good model for studies of the affects wings, thorax and abdomen but the temporal production of complex morphology by individual cells. We aspects are not evident. On the other hand, the phenotype have made use of the mutants multiple wing hair (mwh) is dissected into component parts by looking at the and singed (sn) for examples of genetic modifications of phenocopies induced by heat shock. Hair bundles are hair morphology and heat shock phenocopies to determine induced on wings by heat shocks in the range of 37-40 hr temporal factors in the expression of genes which modify after puparium formation, on the thorax 4 hr later and on hair structure. the abdomen by treatment still 5 hr later. This is in spite of the fact that the wings and thorax come from the same We have eontinued also studies on the kineties of production and turnover of heat shock proteins and imaginal disc and the abdominal epithelium has a totally corresponding m RN As in Drosophila tissues and cell different origin. It is clear that each cell type has its own cultures. This is of special interest in relation to the temporal program of response (see Biology 1980, No. 88). protection of whole animals and cells against high References: temperature stresses by mild heat shock pretreatments. Garcia-Bellido, A. and Merriam, J.-R. (1971) Devel. Biol. 24: 61-87. As shown now in several laboratories, this phenomenon is Mitchell, H.K. and Lipps, L. S. (1978) Cell 15: 907-918. obtained with mammalian cells as well as with Drosophila cells. Furthermore, similar heat shock proteins are 88. RAPID CHANGES IN GENE EXPRESSION IN produced in a variety of eukaryotic cells from yeast to DIFFERENTIATING WING TISSUE IN DROSOPIIlLA humans. Investigators: Herschel K. Mitchell, Nancy S. Petersen We have continued work also on the biochemistry of the pupation process and the origins of the matrix proteins We observed earlier (Mitchell, Lipps and Tracy, 1977) involved in pupation and in the production of hairs and that patterns of protein synthesis change rapidly in bristles. Further work has also been carried out on the thoracic hypoderm of Drosophila during the time of chemical nature of crosslinking components in differentiation of bristles and hairs. We have now sclerotization. reexamined this phenomenon in differentiating wing tissue where nearly all of the cells are in synchrony in the production of hairs. With this more uniform system, the 87. HAIR PHENOCOPIES AND DEVELOPMENTAL changes in protein synthesis patterns are even more REGULATION IN DROSOPIIlLA extreme than observed for thoracic epithelium and Investigators: Herschel K. Mitchell, Nancy S. Petersen virtually all of the prominent components are turned on As reported many years ago (Garcia-Bellido and and off. We have shown furthermore that changes can Merriam, 1971), the mutant of Drosophila designated occur in 1 hr or less in vivo and we have demonstrated multiple wing hairs (mwh) is expressed not only on the that the protein changes are the direct result of transcrip­ wings but on the thorax and abdomen of the adult fly. In tional changes by extraction of mRNAs and translation in 65

vitro. This behavior now seems characteristic of non­ some rotation is required in order to move each hair away dividing but differentiating cells of Drosophila epithelium. from the edge of the cell which provides its structure. Besides the rapid turnover observed in a single cell Present information applies to the production of normal type, we have also noted that similar protein patterns hairs. Similar studies are in progress for the mutant, occur in different cell types but not at the same time. mwh, and for the multihair phenocopy which produces For example, a set of about 15 proteins appears in wing bundles of hairs. cells in the time range of 37 to 42 hr after puparium formation and what appears to be the same set appears in 90. TRANSLATIONAL REGULATION OF GENE EXPRESSION FOLLOWING HEAT SHOCK the thoracic epithelium about 4 hr later. The timing Investigators: Nancy S. Petersen, Herschel K. Mitehell correlates extremely well with the sensitive periods in the two tissues for the sensitive periods in which multihair Short exposures to high temperatures (40-42"C) shut phenocopies can be induced in each tissue by heat shock off both RN A and protein synthesis in Drosophila (Mitchell (see Biology 1980, No. 87). It is clear from these data and Lipps, 1978). The recovery of protein synthesis is that each cell type has its own programs of transcriptional much faster when a 35°C treatment (which turns on heat activities. shock genes) is given immediately prior to the 40°C treatment (Mitchell et al., 1979). The recovery of protein Reference: Mitchell, H. K., Lipps, L. S. and Tracy, U. M. (1977) synthesis always follows a definite pattern with the 70 K Biochem. Genetics 15: 575-587. heat shock protein being synthesized first followed by the smaller heat shock proteins and the 25° proteins in a specific sequence. We wished to see whether the recovery 89. MORPHOGENESIS OF WlNG HA1RS AS OBSERVED BY ELECTRON MICROSCOPY pattern of protein synthesis reflects the concentration of Investigators: Herschel K. Mitehell, Joan Roach, mRNA during the recovery period. We have isolated Nancy S. Petersen mRN A from larvae recovering from a 40° heat shock (both At the time in metamorphosis of Drosophila when each with and without a 37° pretreatment) and have translated wing cell produces a hair, there is no more cell division this RNA in the rabbit reticulocyte system. Under and each hair arises as an extrusion of cytoplasmic conditions where the amount of in vitro translation is material from each cell. The specific mechanisms by linearly dependent on mRNA concentration, we have which the morphogenesis occurs and its molecular basis found that messenger RNA concentrations of the major are unknown, but the system presents a good model of cell mRNAs remain constant throughout the recovery period differentiation and we now have a considerable amount of and that the only difference between pretreated and non­ information on morphogenetic variations (see Biology pretreated animals in the in vitro system is that heat 1980, No. 87) and on some of the biochemistry involved shock mRNA is present initially in the preheated animals. (Biology 1980, No. 88). We have therefore also looked at This means that the pattern of recovery of protein the normal process of hair formation by examining thin synthesis is due to selective translation of messenger sections from wing preparations at intervals through the RNAs. Further comparison of in vitro and in vivo protein stages of differentiation. We find that structures which synthesis patterns shows that pretreated animals translate become hairs appear about 32 hr after puparium formation message more efficiently than non-pretreated animals and that they lie fiat on a cell adjacent to the one from suggesting that the defect in initiation of protein which the extrusion originates. They are in fact covered synthesis caused by heat shock ~an be partially reversed up by a layer of proteinaceous material until about 45 hr. by pretreatment at 35°. Initially, the hair structures contain none of the normal References: cytoplasmic structures but only some amorphous granular Mitchell, H. K. and Lipps, L. S. (1978) Cell 15: 907-918. matter. Subsequently, at about 50 hr, microfibers appear, Mitchell, H. K., Moller, G., Petersen, N. S. and Sarmiento, L. S. (1979) DeveL Genetics 1: 181-192. the hairs stand up in the cell center and fibers are deposited at the periphery of the hair as well as in a circle around the base of the hair. During all these changes, there are very extensive modifications of cell shape and 66

91. HEAT SHOCK AT 3'l"INDUCES SYNTHESIS carbohydrate chitin. When larval cuticle hardens to pupal OF MORE THAN 50 PROTEINS IN cuticle, an aromatic crosslink is formed from N-acetyl­ LARVAL SALIVARY GLANDS dopamine that could involve protein segments only or Investigators: Carolyn Buzin*, Nancy S. Petersen protein as well as chitin. The crosslinked cuticle is We have used two-dimensional gels to compare pro­ resistant to proteolytic and chitinolytic enzymes, and teins synthesized at 25~ in Drosophila larval salivary while it can be solubilized by. mild acid hydrolysis, the glands with those synthesized immediately following a portion representing the crosslink is released as a free 37•c heat treatment. More than 50 new (heat shock) ketocatechol, leaving no clue to its original attachment proteins have been identified. About 30 of these are in (Andersen, 1976). Because the presence of chitin might the molecular weight (m.w.) range 68 K-70 K and we call shield cuticle from enzymatic attack, we tried to raise these the 70 K group. There are 12 heat shock proteins chitin-less Drosophila pupae from eggs on medium con­ between 45 K and 68 K; one 34 K protein and eight in the taining the chitin synthesis inhibitor diflubenzuron (Post, m.w. range 20-28 K. A vecy short exposure of these gels De Jong and Vincent, 1974). At 1.1 to 1.3 ppm, 30% shows the pattern previously reported (Mirault et al., survived to emerge as flies but none at 1.6 ppm. 1978) with seven spots in the 70 K region and seven other Inhibitor-treated pupal cases had 40% less glucosamine heat shock proteins. than controls, indicating a reduction but not the desired All of the new heat shock proteins we see with absence of chitin. The low-chitin pupal cases were still molecular weight less than 68 K probably represent resistant to proteolytic enzymes. distinct polypeptides since they are single spots on the References: gel. In the 70 K group, however, there are runs of Andersen, S. O. (1976) In: The Inse~t Integument, H. R. proteins of the same molecular weight that differ from Hepburn (Ed.), pp. 121-144. Elsevier, New York. Post, L. C., De Jong, B. J. and Vincent, W. R. (1974) each other only in charge. Some of these spots may be Pestic. Biochem. Physiol. 4: 473-483. the result of post-translational modification of a smaller number of polypeptides. We have compared the in vivo synthesized heat shock proteins with those synthesized from larval salivary gland heat shock RNA in vitro (in the rabbit reticulocyte system). Most of the 70 K group of PUBLICATIONS proteins are still present in the in vitro system. This suggests either that there is extensive post-translational Buzin C. and Petersen, N. S. (1980) Two-dimensional gel an~lysis of heat shock proteins synthesized in D. modification in the rabbit reticulocyte system or that melanogaster cell lines and salivary glands. Manu- there are many more heat shock proteins in this group script in preparation. . Chomyn, A. and Mitchell, H. K. (1980) Use of antibody to than had previously been identified. determine the distribution of the 83 K heat shock protein. Manuscript in preparation. We have identified four new heat shock proteins in the Mitchell, H. K. (1978) Ernst Hadorn. Ann. Rev. Genetics 20-28 K m.w. range, 12 new proteins in the range between 12: 1-3. 58 and 68 K and raised the possibility that there are more Mitchell, H. K., Moller, G., Petersen, N. S. and Lipps­ Sarmiento, L. (1979) Specific protection from P.heno­ in the 70 K group. This means that the task of identifying copy induction by heat shock. Devel. Genetics 1: 181-192. and mapping heat shock genes is not nearly complete. Mitchell H. K. and Petersen, N. S. (1980) Rapid changes in iene expression in differentiating tissues of Reference: Drosophila. Manuscript in preparation. Mirault, M. E., Goldschmi~t-Clermont, M., Moran, ~., Petersen, N. s., Moller, G. and Mitchell, H. K. (1979) Arrigo, A. P. and Tissieres, A. (1978) Cold Sprmg Genetic mapping of the coding regions for three heat Harbor Symp. Quant. Biol. 42: 819-827. shock proteins in D. melanogaster. Genetics 92: *City of Hope Medical Center, Duarte, California. 891-902. Petersen, N. s. and Mitchell, H. K. (1980) Regulation of protein synthesis following heat shock. Am. Chem. 92. THE AROMATIC CROSSLINK OF Soc. Abstracts. In press. Petersen, N. S. and Mitchell, H. K. (1980) Translational INSECT CUTICLE regulation during recovery from heat shock. Manu­ Investigators: Peter H. Lowy, Hersehel K. Mitchell script in preparation. The cuticle of insects consists of protein and the 67

Associate Professor: James H. Strauss Jr. reported on preliminary experiments to determine the Visiting Associate: Keh-Chi Ling basis for the large plaque (LP) or small plaque (SP) Senior Research Fellow: Ellen G. Strauss Research Fellow: Toni R. Claudio phenotype of Sindbis virus. We had found that the Graduate Students: John R. Bell, Thomas E. Crowley, precursors to the envelope glycoprotein E2 (PE2) from LP Jeffrey T. Mayne, Jing-hsiung James Ou, Charles M. Rice Ill and SP-virus-infected cells had different electrophoretic Research Staff: Edith M. Lenches, Cecilia Mong migrations on Laemmli gels. PE2 from the SP strain Laboratory Staff: Jeannette Johnstone, Caroline Vermaes migrated more rapidly and this difference was also seen in Support: The work described in the following research cells infected with ts mutants which had been derived reports has been supported by: Earle C. Anthony Fellowship from LP and SP, respectively. During the final step of Biomedical Research Support Grant (NIH) Sindbis maturation, PE2 is processed, leaving the mature Califomia Foundation for Biochemical Research Charles B. Corser Fund for Biological Research molecule E2 in the virion and releasing a small protein, Intemational Rice Research Institute, Philippines National Institutes of Health, USPHS E3, into the culture fluid. We have examined the E3s National Science Foundation from LP and SP by electrophoresis and have been unable to detect any differences. After treating E3 with summary: We wish to understand the molecular biology neuraminidase to reduce carbohydrate heterogeneity, we of replication of Sindbis virus. Although not pathogenic to examined the E3s by isoelectric focusing and found them man, this alphavirus is a close relative of many other to be similar. Also, HPLC of tryptic peptides of E3 gives alphaviruses which are important human or veterinary a very simple pattem which is identical for LP and SP. pathogens. In addition, the virus provides a useful model We have examined the tryptic peptides of PE2 and E2 membrane system. Two virus-encoded glycoproteins are on HPLC and have been unable to detect any differences made after infection which migrate from the site of between LP and SP, but the patterns are very complex and synthesis in the endoplasmic reticulum through the Golgi one altered peptide might be missed. We suspect that the to the cell surface. The virus matures when the situation is complicated by heterogeneity in glycosylation nucleocapsid, formed in the cytoplasm, buds through the since we find 6 or 7 glycopeptides from E2 on the modified cell plasma membrane and acquires an outer columns, whereas the carbohydrate content indicates only membrane layer. 2 chains per molecule. However, differences in glycosyl­ The following reports describe various approaches we ation cannot completely explain the difference between are using in the study of this system. These include LP and SP, for the PE2s from cells infected with SP or LP genetic approaches, electron microscopic studies of in the presence of tunicamycin (an antibiotic which infected cells, and immunologic approaches, as well as prevents glycosylation) are still separable on SDS-con­ protein chemistry and nucleic acid chemistry. Efforts to taining gels. This indicates that the major difference is in sequence the proteins and nucleic acids of the virus are the protein moiety. proving fruitful. Sequencing of the 3'-end of the RNA has It is possible that these molecules differ from one revealed a conserved set of nucleotides among alpha­ another in the location of the cleavage separating PE2 viruses which must have an important recognition from El in the precursor polyprotein, or from a putative function. Sequencing of the mRNA for the virus glyco­ signal sequence on the other end. We are continuing to proteins and the virus capsid protein, together with the study this problem. corresponding protein sequence, has revealed details involved in the biosynthesis, processing, and transport of 94. GENETIC EXCHANGE BETWEEN SINDB!S VIRUS the proteins and their function in the virus life cycle. AND WESTERN EQUINE ENCEPHALITIS VIRUS Comparative studies of alphavirus strains are proving Investigator: Ellen G. Strauss useful in these studies. Western equine encephalitis virus (WEE) is an alpha­ virus serologically related to Sindbis virus. It is an 93. BIOCHEMICAL STUDIES OF LARGE PLAQUE endemic human pathogen throughout the Western United AND SMALL PLAQUE SINDB!S VIRUS States. Recently, Dr. Bunsiti Simizu1s laboratory in Japan Investigator: Ellen G. Strauss has isolated a series of ts mutants of WEE and grouped Last year in this Report (Biology 1979, No. 103), we them by complementation. This was the first report of 68

successful complementation between alphavirus mutants 96. CLONING OF DNA SEQUENCl!S other than our studies with mutants of Sindbis virus (HR COMPLEMENTARY TO THE SINDBJS VIRUS 26SRNA strain). To examine the relatedness between these Investigators: Thomes E. Crowley, Charles M. Rice ill viruses, I went to Tokyo this year to do mixed comple­ mentation studies. Unfortunately we failed to demon­ The Sindbis virus 268 RNA codes for all three of the strate efficient complementation between Sindbis and viral structural proteins. In order to examine the WEE. This may be due in part to technical difficulties. translation of these proteins, we want to determine the However, the viruses do appear to be sufficiently closely­ primary sequence of the 268 RNA. These data could be related to share genetic material and under appropriate used in conjunction with protein sequence data to deter­ conditions will produce phenotypically mixed particles. mine the exact location of the coding regions for these These particles can contain either genome. Some of the proteins. virions produced contain a mixture of the glycoproteins In order to accomplish this, cDNA was synthesized encoded by Sindbis and by WEE on their surface, i.e., they with the viral 268 RNA as template, using avian myelo­ are more effectively neutralized with a mixture of anti­ blastosis virus reverse transcriptase. The full-length Sindbis and anti-WEE antiserums than by either antiserum cDNA/RNA hybrids were isolated and inserted into a alone. Additional controls are still being performed by bacterial plasmid via the homopolymer tailing method. the Japanese workers. The plasmids were used to transform E. coli, and bacteria containing plasmids with inserts were selected by drug resistance tests and Grunstein-Hogness screening. Although we selected full-length hybrid molecules for 95. COMPARATIVE STUDIES OF THE 3'-TERMINAL SEQUENCl!S OF SEVERAL ALPHAVIRUS RNAs insertion into the plasmids, the plasmids which were Investigator: Jing-hsiung James Ou amplified in E. coli were found to contain a wide range of insert sizes, from 0.5 kb to 4.2 kb (the 268 RNA is The 3'-termini of 268 RNA of Sindbis virus and of the ..r4.5 kb). Restriction enzyme analysis of the various virion 498 RNAs of 8indbis virus, Semliki Forest virus and cloned inserts did not reveal any consistent pattern. Middelburg virus have been sequenced by using a chain­ These results indicate t11at some type of deletion or terminating sequencing method with reverse tran­ rearrangement of the inserts probably occurred in the scriptase. Both 498 and 268 RNAs of Sindbis virus have process of insertion into the plasmid and amplification in identical 3'-terminal sequence for at least 145 nucleotides E. coli. from the poly(A) tail. 268 RNA is known to be a Southern blot and Northern blot analyses were per­ subgenomic RN A located at or near the 3•-end of 498 formed employing a cDN A synthesized from the Sindbis RNA; our present results indicate that 268 and 498 RNA virion 498 RNA, which contains the entire 268 RNA are coterminal. The RN As of all three viruses contain a sequence. The results of these experiments indicated that region of 20 nucleotides adjacent to their poly(A) tracts the various inserts contain mostly viral-specific which is highly conserved. This conserved sequence could sequences, but that some may also contain nonviral be the replicase recognition site and/or a recognition site sequences. for encapsidation. After this conserved region, the Comparison of the restriction enzyme analysis data sequences of Sindbis and of Semliki Forest virus diverge described above with that obtained from uncloned single­ significantly but there does appear to be some residual stranded cDNAs synthesized from the 268 RNA may sequence homology in these regions. The 3'-termini of provide some insight into the arrangement of the cloned Sindbis virus and 8emliki Forest virus RN As contain a high sequences. content of A and U; of th~ first 50 nucleotides adjacent to the poly(A) tract, over half are U and over one-quarter 97. ANALYSIS OF eDNA TO SINDBIS 26S RNA are A. In addition, repeating sequences are present in the Investigator: Charles M. Rice ill 3'-termini. As previously mentioned, hybrid cloning of Sindbis 268 RNA has produced a series of clones which have been difficult to characterize using conventional restriction 69 endonuclease analysis. DNA complementary to 268 RNA 99. STRUCTURAL STUDIES OF S!NDBIS VIRUS GLYCOPROTEINS has been made using either fragments of calf thymus DNA Investigator: Charles M. Rice ill or using oligo(dT) as primers, and this cDNA cut with restriction enzymes which act on single-stranded DNA. We have been studying the membrane-associated We are constructing a map of the 268 cDNA with such hydrophobic tails of the Sindbis virus glycoproteins after enzymes to serve as a basis for analyzing our cDN A digestion of virions with cx-chymotrypsin (see Biology clones. In addition, single-stranded restriction fragments 1979, No. 98). The hydrophobic "root" of each glyco­ can be end-labeled, and sequenced directly using the base­ protein contains associated fatty acids (probably specific chemical cleavage method of Maxam and Gilbert. covalently bound), and is partially soluble in organic Using DNA and structural protein sequence data, we hope solvents. Amino acid compositions have shown that REl to precisely localize the structural protein coding (the 5 K dalton root from El) is rich in hydrophobic amino sequences in the 268 RNA, and the sites of proteolytic acids, while RE2 (the 10 K dalton root from E2) contains a processing between them. larger proportion of charged residues. Preliminary amino acid sequence data obtained in collaboration with Michael Hunkapiller and Lee Hood indicate that both REl and RE2 98. LOCATING THE PANHANDLE STRUCTURE ON SEMIJKI FOREST VIRUS VIRION RNA contain unique amino termini. The sequences thus far Investigator: James Jing-hsiung Ou obtained (.r20 residues) contain some charged amino acids in the first 10 residues followed by an uninterrupted Electron microscopy (Hsu, Kung and Davidson, 1973; stretch of neutral amino acids. We plan to continue Kennedy, 1976) as well as biophysical studies (Frey, Gard sequence analysis of these hydrophobic tails after further and Strauss, 1979) have shown that the virion RNA of chemical or enzymatic cleavage. Sindbis virus or of Semliki Forest virus is able to form a hydrogen-bonded circle. In the electron microscope, the 100. CARBOXY-TERMlNAL SEQUENCING OF THE complementary sequences which are responsible for SlNDBIS STRUCTURAL PROTEINS cyclization are located near the ends of the RNA and the Investigators: Thomas E. Crowley, Charles M. Rice ill panhandle formed by cyclization of the molecule has a The sequence of four to five residues at the C-termini length of about 250 ± 50 nucleotides (Hsu, Kung and of the capsid protein, El and E2 of Sindbis virus has been Davidson, 1973). In our previous sequencing experiments, determined using carboxypeptidase Y and carboxypepti­ it was found that nucleotide 105 from the 3' end after the dase A. Reactions were done with .r10 nmoles of protein, poly(A) tract of Semliki Forest virus RNA is a strong and released amino acids identified and quantitated on an terminating site for reverse transcription. It is very likely automated amino acid analyzer. These results were that there is a tight secondary structure adjacent to confirmed by digestion of radiolabeled proteins with nucleotide 105 and this prevents the reverse transcriptase carboxypeptidase Y and quantitation of released, radio­ from reading through this region. Comparing the length labeled amino acids. of the panhandle structure and the length of the poly(A) It has been proposed that the "roots" of El and E2, tail (60 nucleotides in length on average), it is possible which are embedded in the viral membrane, are derived that this secondary structure is the region of base pairing from the c-termini of El and E2, respectively. In order which holds the molecule in a circle. Subgenomic to verify this, we are now in the process of comparing the fragments of the virion RNA, prepared in several ways, c-terminal sequences of the roots with their parent are being sequenced to test this possibility. If sequences proteins. This is being done with carboxypeptidase Y, after nucleotide 105 constitute the circle structure, then using radiolabeled amino acids and high pressure liquid the disappearance of the strong terminating site at chromatography. nucleotide 105 will be expected. The Sindbis structural proteins are coded for by the References: 268 RNA. Determination of the primary sequence of the Frey, T. K., Gard, D. L. and Strauss, J. H. (1979) J. Mo!. Biol. 132: 1-18. 268 RNA will allow location of the coding regions for the Hsu, M. T., Kung, H. J. and Davidson, N. (1973) Cold three proteins using the C-terminal sequences determined Spring Harbor Symp. Quant. Biol. 39: 943-950. Kennedy, S. I. T. (1976) J. Mo!. Biol. 108: 491-511. as described above, and the N-terminal sequences of El 70

and E2 determined by Bell et al. (1978). The three 102. PURlFlCATION AND CHARACTERIZATION OF E3 IN THE SINDBJS VIRUS INFECTION proteins are derived from a single precursor; therefore, Investigators: Jeffrey T. Mayne, Charles M. Rice III, this information will allow determination of the sequence Ellen G. Strauss structure of the proteolytic processing sites.. The two best studied alphaviruses are Sindbis virus and Reference: Semliki Forest virus (SFV). One of the major differences Bell, J. R., Hunkapiller, M. W., Hood, L. E. and Strauss, J. H. (1978) Proc. Nat. Acad. Sci. USA 75: 2722-2726. between the two viruses is the presence of E3 (a small, heavily glycosylated protein) in the membrane of the SF virion and its lack in the Sindbis virion. In SFV, E3 is 101. N-TERMINAL SEQUENCES OF produced at a late stage of virus maturation when PE2 is BLOCKED PROTEINS OF SINDBIS VIRUS cleaved to produce E2 and E3. But in the Sindbis Investigator: John R. Bell infection, PE2 is cleaved to produce E2 only, and the One mRNA in Sindbis infected cells is translated into a fragment equivalent to E3 is lost. single, large polyprotein which is processed. by proteolytic E3 has been isolated from the culture fluid of Sindbis- cleavage to produce all of the structural proteins of the infected cells by our lab and other investigators (Welch virus. The capsid protein is derived from the N-terminal and Sefton, 1979). Pulse-chase and tryptic digestion end of this polyprotein, and I have previously shown that experiments indicate that it is cleaved from PE2 and this protein contains a modified, or blocked, terminal released into the culture fluid as PE2 is processed to E2. amino group, which renders it resistant to the usual E3 runs as multiple (3-5) closely-spaced species on SDS technique of protein sequencing by sequential Edman acrylamide gels. Initial evidence suggests that this is due degradation. I am now attempting to determine the N­ to differential degrees of glycosylation. terminal sequence of the Sindbis capsid protein. I have A purification scheme has been devised which purifies isolated blocked peptides after exhaustive enzymatic E3 20-40,000-fold. This scheme uses differential digestion of the capsid protein, and from the amino acid precipitation by ethanol, gel filtration, ion exchange compositions of these peptides and their chromatographic chromatography, and a lectin affinity column. behavior I have tentatively identified the N-terminal The purified E3 is being used to do biochemical sequence as Ac-met-asx-. I have also shown that I can studies, including amino acid compositions and sequencing. specifically radiolabel the acetyl group by growing the By comparison of sequences of the carboxy terminus of virus in the presence of tritiated acetate under appro­ E3, the amino terminus of E2, and the appropriate CNBr priate conditions. This acetate label can now be used to peptide of PE2, we hope to determine some of the fine identify larger peptides after less extensive enzymatic structure of the cleavage of PE2 + E2 + E3. digestion, and these peptides can be further digested to We are also investigating the kinetics of synthesis and obtain additional N-terminal sequence information. the location of E3 during the infection cycle in mosquito In the course of these studies on the capsid protein, it cells. In chick cells, the virus buds through the plasma was found that PE2, the p;~cursor to one of the envelope membrane and is released into the medium. In mosquito glycoproteins of the virus, also incorporates radioactive cells, in contrast, the virus seems to be assembled in acetate, and that a peptide can be derived from it which cytoplasmic "factories," although there is some contro­ also has the properties of a blocked peptide. Since an N­ versy over this point. The location of E3, whether terminal sequence has been obtained from _this protein in extracellular or intracellular, will help resolve this this laboratory and elsewhere, this discovery of a blocked question. fraction of PE2 may indicate the existence of a previously Reference: unknown processing step in the maturation of this protein, Welch, w. J. and Sefton, B. M. (1979) J. Virol. 29: although alternative explanations cannot be excluded at 1186-1195. present. I am continuing to study the sequence of this 103. ELECTRON MICROSCOPIC STUDll!S OF blocked fraction of PE2. SINDBIS VIRUS MATURATION Investigator: Toni R. Claudio

An electron microscopic comparison of the maturation 71

of Sindbis virus in chronically infected mosquito cells and the nonpermissive temperature, and this defect is not cytopathically infected chick embryo fibroblast cells is reversible upon shift to 30° in the absence of protein currently in progress. The objective is to determine how synthesis. The second group (ts126 and ts227) showed persistent infections are maintained in host cells. A positive reactions at both 40°C and after the shift to 30°C. second closely-related project uses electron microscopy to Thus, these mutants are able to insert glycoprotein into trace the synthesis and migration of viral proteins through the plasma membrane at 40°. The third group (ts 23 and the host cell to the plasma membrane by synchronizing ts252) had less radioactivity bound at 40"C than after a infection and protein synthesis. Besides leading to a shift to 30"C. Hence, the mutants in this group may be better understanding of virus replication in cytopathic reversible, and proteins synthesized at the nonpermissive infections, information gained about the process of temperature migrated to the plasma membrane after the budding for this family of viruses should be applicable to shift to the permissive temperature. all families (including the RNA tumor viruses) which Reference: mature by budding. In both studies, ferritin-labeled Strauss, E. G., Lenches, E. M. and Strauss, J. H. (1976) antibodies specific to each of the three virus structural Virology 74: 154-168. proteins are being used to locate these viral components throughout the different stages of host cell infection. 105. MONOCLONAL ANTIBODIES TO SlNDBIS PROTEINS Besides the direct applicability of these findings to the Investigators: Jeffrey T. Mayne, Charles M. Rice ID mechanisms of virus maturation, much can also be learned about general mechanisms of glycoprotein biosynthesis We have isolated 10 clones of hybrid mouse cells secreting antibodies specific for Sindbis proteins (see and membrane topology of cell surfaces. Biology 1979, No. 100). Nine of these lines react with El, 104. REVERSIBILITY OF ENVELOPE and one with the capsid protein. Seven lines produce IgG GLYCOPROTE!N MUTANTS OF (representing subclasses IgGl, IgG2a, IgG2b), and three SINDBIS VIRUS lines produce IgM antibodies. Five of the anti-El lines Investigators: Keh-Chi Ling, Ch81'les M. Rice ID react with intact virions, and also precipitate radiolabeled Temperature-sensitive (ts) mutants of Sindbis virus El solubilized from virions with Tri-ton X-100 or SDS. All have been grouped into 7 different complementation of the antibodies thus far examined react more strongly groups (Strauss, Lenches and Strauss, 1976). We are with SDS-treated El than Triton X-100 solubilized El. interested in studying mutants defective in glycoprotein Attempts to produce monoclonal antibodies specific synthesis and/or transport of the nonpermissive tempera­ for E3 by immunizing with infected cell membranes have ture. A radioimmune assay, involving rabbit antiserum been unsuccessful. Using purified E3 and different against Sindbis glycoproteins and 1251-labeled goat anti­ immunization strategies, we are continuing our attempt to rabbit serum, has been developed. This assay was used to isolate hybrid cell lines producing antibodies specific for examine the presence of virus envelope glycoproteins in E3. the plasma membrane of chick embryo fibroblasts in monolayer culture after infection with various ts mutants PUBLICATIONS of the virus and incubation at the permissive and/or Bell, J. R., Strauss, E. G. and Strauss, J. H. (1979) Amino acid composition and molecular weights of the struc­ nonpermissive temperatures. To test for the reversibility tural proteins of Sindbis virus. Virology 91: 287-294. of RNA+ ts mutants, infected cultures were incubated at Frey, T. K., Gard, D. L. and Strauss, J. H. (1979) 40°C for 5.5 hr, treated with cycloheximide to prevent Biophysical studies on circle formation by Sindbis virus 49S RNA. J. Mo!. Biol.132: 1-18. further protein synthesis, and then further incubated at Ou, J. H., Strauss, E. G. and Strauss, J. H. (1980) 40"C or were shifted to 30"C for I hr. Based on the Comparative studies of the 3'-terminal sequences of several alphavirus RN As. Submitted for publication. radioactivity bound to the cell surface, the tested mutants Strauss, E. G., Lenches, E. M. and Martin, M. A. (1980) appear to fall into three groups. The mutants tsl32 and Growth and release of several alphaviruses in chick and BHK cells. J. Gen. Virol. In press. ts246 showed no detectable reaction either at 40°C or Strauss, E. G. and Strauss, J. H. (1980) Mutants of alphaviruses: Genetics and physiology. In: The after the shift to 30°C. Thus, these mutants do not Togaviruses, Chapter 14, R. W. Schlesinger (Ed.). integrate virus glycoprotein into the plasma membrane at Academic Press, New York. In press.

CELLULAR BIOLOGY AND BIOPHYSICS

Howard C. Berg

Charles J. Brokaw

Max Delbrilck

John J. Hopfield

Elias Lazarides

Jean-Paul Revel

Anthonie Van Harreveld

75

Professor: Howard C. Berg Chlamydomonas. Its eyespot appears to function as an Visiting Associate: I. Richard Lapidus optical antenna, specifically, as a quarter-wave reflector. Senior Research Fellow: Michael D. Manson Graduate Student: Jeffrey E. Segall Special Graduate Student: Steven M. Block 106. ADAPTATION IN E. COLI CHEMOTAXIS Research Staff: M. Patricia Conley, Robert D. Smyth Investigator: steven M. IDoek SUpport: The work described in the following research reports has been supported by: Bacteria perform chemotaxis in a gradient of Charles B. Corser Fund attractant or repellent by sensing the gradient per se; National Institutes of Health, USPHS National Science Foundation metabolism and/or transport of the substance detected is Alfred P. Sloan Foundation not required. Cells sense temporal variations in concen­ Stevens Institute of Technology tration which result when they move through the gradient. By making timewise comparisons, they are able to bias summary: We arrived in December 1979 and occupied a their movement in such a way as to migrate to a more niche prepared for us at the south end of the top floor of favorable environment. Bacteria are thus sensitive to the Beckman Laboratories, where we are continuing work changes in ambient level of chemoattractant. Corre­ begun at the University of Colorado on the motile spondingly, they are relatively insensitive to the absolute behavior of microorganisms, particularly bacteria. level: they adapt. Adaptation affords the cells a Two of the bacteria under study, Escherichia coli and heightened sensitivity to changing situations and a large Streptococcus strain V4051, swim as in a random walk. dynamic range over which successful chemotaxis can take The cells move in nearly straight lines for about a second place. (run), jump about erratically for a small fraction of a In an effort to better understand the physiology of the second (tumble), and then move off in another direction. adaptation process, preparations of tethered E. coli are The runs correspond to counterclockwise rotation of the being subjected to programmed changes in concentration. flagella (as seen by an observer standing on the surface of Bacteria are tethered in a tiny cell through which a the cell looking at the shaft of the f!agellar motor as it continuous flow of solution takes place. The flow is emerges from the cell wall), the tumbles to clockwise constant, laminar, and slow enough to allow free rotation rotation. The rotation can be observed directly by fixing of the cells. The solution contains an attractant or one of the flagellar filaments protruding from a cell to a repellent which is mixed into the buffer in a small, high­ glass slide: the cell body spins alternately counter­ speed mixing chamber located upstream of the flow cell. clockwise and clockwise. Cells fixed in this way are The degree of chemoattractant present in the mix is called tethered cells. determined by a variable peristaltic pump, controlled by If chemicals are added to the medium surrounding an electronic programmer. The programmer is capable of tethered cells, either by bulk flow or by an iontophoretic producing steps, linear and exponential ramps, and pulse, chemotactic responses can be elicited. We are sinusoidal variations in the pump speed. Data are taken in using these techniques to determine how cells adapt to the form of a videotape record; the directions of rotation programmed stimuli and to learn how long it takes cells to of the cells, which provide a measure of their state of process chemotactic information. Tethered-cell prepara­ adaptation, are determined later off-line. This set-up is tions also are being used to study the energetics and the currently being used to investigate the adaptation process dynamics of flagellar rotation. in the small-signal (physiological) range. We are interested in more exotic forms of motility, as well. Some bacteria, e.g., cellulose-metabolizing orga­ 107. SIGNAL-PROCF.SSING TIMES IN E. COLI CHEMOTAXIS nisms found in the soil, fail to swim when in free Investigators: Jeffrey E. Segall, Michael D. Manson suspension, but move steadily in the direction of their long axes when in contact with a solid surface. The means by We are using iontophoretic techniques to deliver brief which such gliding bacteria move is a complete mystery_. pulses of attractants and repellents to tethered cells in They do not have flagella or other known organelles of order to learn how long it takes a cell to detect, process locomotion. We are studying the phototactic behavior of and respond to chemotactic stimuli. Wild-type E. coli one eukaryotic organism: the unicellular green alga, responds in about 0.2 to 0.3 sec when suddenly exposed to 76

-3 -3 10 M a-methylaspartate (an attractant) or 10 M flagellar motors are rigidly engaged. This dilemma can be benzoate (a repellent). resolved if several protons must act in concert to advance The observable variables in these experiments include the motor. the delay time before the response, the nature of the A pmf of reversed sign, which drives protons out of the response, and the duration of the response. We plan to cell, also supports flagellar rotation (Manson, Tedesco and determine the effects on these variables of: varying pulse Berg, 1980). As before, the cells spin· .clockwise, but length and size, prior adaptation to attractants or attractants are not able to effect counterclockwise repellents, and successive pulses of attractants and rotation. It is not clear whether inward or outward proton repellents. Mutants defective in different components of currents are preferentially associated with either the chemosensory pathway (Parkinson, 1977) also will be direction of rotation. The problem is complicated, studied. Our goal is to learn more about how signals are because the procedures used to generate pmf also transmitted from the chemoreceptors to the flagella. generate chemotactic signals. As described in the next report, we have isolated generally non-chemotactic Reference: Parkinson, J. S. (1977) Ann. Rev. Genetics 11: 397-414. mutants from strain V4051 to help circumvent this difficulty. 108. ENERGETICS OF FLAGELLAR ROTATION References: Investigator: Michael D. Manson Manson, M. D., Tedesco, P., Berg, H. C., Harold, F. M•. and van der Drift, C. (1977) Proc. Nat. Acad. Sci. USA 74: The rotation of bacterial flagella is driven by a 3060-3064. protonmotive force (pmf); the energy is derived from the Manson, M. D., Tedesco, P. M. and Berg, H. C. (1980) J. Mo!. Biol. 138: 541-561. difference in electrochemical potentials of a proton on either side of the cytoplasmic membrane. We demon­ strated this directly using the motile Streptococcus strain 109. SELECTION OF NON-CHEMOTACTIC V4051, which lacks an endogenous energy reserve (Manson MUTANTS OF STREPTOCOCCUS V4051 Investigators: M. Patricia Conley, Michael D. Manson et al., 1977). These cells can be starved and rendered immotile simply by removing exogenous energy supplies, We have successfully isolated generally non­ without recourse to metabolic poisons. Motility can be chemotactic mutants of Streptococcus strain V4051, some restored by inducing a potassium; diffusion potential (cell of which should help to establish, in a chemotaxis-free exterior positive) or by acidifying the external medium. background, the linkage between the direction of proton In either case, the entry of protons is coupled to flagellar current and the direction of flagellar rotation. The rotation. The response is transient, because the incoming mutants selected after treatment of the cells with EMS protons dissipate the pmf. ATP is not required, nor is the were of three basic types: (1) those that tumble infre­ movement of ions other than H+ necessary. quently or not at all (smooth-swimming mutants), (2) those By examining tethered cells, we learned that flagella that tumble excessively or exclusively (tumbly mutants), rotate clockwise (in the tumble direction) in response to and (3) those that appear to have wild-type motility in an artificial pmf, but can go counterclockwise if a liquid culture but fail to form normal chemotactic swarms chemoattractant is present (Manson, Tedesco and Berg, in semi-soft agar.

1980). We also found that the angular velocity of tethered The smooth-swi~ming mutants were tethered and cells is proportional to the pmf over a wide range of exposed to attractants and repellents. They could be values and that the flagellar motor operates at a constant divided into three classes with respect to their responses torque when the pmf is at a given value. These results to repellent stimuli: (1) those that never spin clockwise suggest that a fixed number of protons carries the motor (that is, in the tumble direction), (2) those that make through one revolution. abortive attempts to spin clockwise, and (3) those that The threshold pmf for flagellar rotation is low, spin very briefly clockwise. The chemotactic responses of certainly less than kT, the energy of thermal fluctuation. the other types of mutants also will be tested in this At first glance, one would predict an activation energy of fashion. at least kT, because de-energized cells do not undergo The chemotactic behavior of wild-type Streptococcus free rotational diffusion but behave as though their strain V4051 is similar to that of Escherichia coli and 77 other peritrichously flagellated bacteria. Our confidence completing more than one revolution). These cells, like that the chemotactic mechanisms actually are similar is the species studied by Pate and Chang (1979), actively reinforced by our finding both tumbly and smooth­ propel small polystyrene latex spheres along their swimming mutants; these two mutant types also have been surfaces. We are using video techniques to analyze these found in other peritrichous species. Some of our mutants, maneuvers quantitatively, in order to obtain insights into particularly those making abortive tumbles and those that possible motility mechanisms. appear to have normal behavior in liquid culture, may Reference: prove to be novel. Pate, J. L. and Chang, L.-Y. E. (1979) Current Microbiol. 2: 59-64.

110. SEARCH FOR STEPS IN FLAGELLAR 112. LIGHT ANTENNAS IN PHOTOTACTIC ALGAE ROTATION Investigator: Robert D. Smyth Investigator: Howard C. Berg Most microscopic flagellated algae are photosynthetic The flagellar rotary motor is on the order of 200 ll. in and are also phototactic, meaning that they regulate their diameter and is composed of a small number of structural elements. We have reason to believe that it is a stepping exposure to sunlight by swimming toward the light or motor (Berg, 1976). This premise is being examined with away from it. Dr. Kenneth Foster and I have reexamined the the aid of a microscope containing special optical filters literature on phototaxis from a physical point of view, and that can measure the angular position of the image of a have formulated a new interpretation (Foster and Smyth, tethered cell with a very short time resolution. The data 1980). Ultrastructural studies show that the different are being analyzed by computer-averaging techniques kinds of phototactic algae possess a variety of eyespots involving the fast Fourier transform. If the motor steps, and associated structures that act as wide aperture it appears to do so more than 100 times per revolution. antennas for detecting visible light. Rotation of an This lower limit is set not by the resolution of the organism at about 2 Hz around its direction of swimming apparatus but by smoothing of the signal due to elastic causes the antenna to scan the environment at an angle of deformation of the element linking the driveshaft of the 9()° to the rotation axis. This generates a periodic signal motor to the glass slide {in the present case, the proximal at the antenna's sensor that serves as an error signal for hook, a structure some 500 A long, normally located be­ tracking the light. Matched-filter processing differen­ tween the driveshaft and the flagellar filament). This tiates the error signal and produces a response that is smoothing can be reduced if the torque generated by the phased with the organism's orientation to the light. This motor is reduced. The torque can be reduced by the gives reliable tracking over a wide range of environmental addition of uncouplers {substances that lower the proton­ light intensities, even when the error signal is noisy. motive force). To insure that the cells spin as rapidly as Antenna directionality is shaped to produce proportional possible, the experiment will be done with cells tethered control of tracking, so that a large deviation from the to 0.2 µm glass beads. light direction produces a large response. Reference: This analysis should provide a better theoretical base Berg, H. C. (1976) In: Cell Motility, R. Goldman, P. Pollard and J. Rosenbaum (Eds.), Vol. 3, pp. A47-A56. for ultrastructural, physiological, and ecological studies of Cold Spring Harbor Conferences on Cell Proliferation, algae. Cold Spring Harbor, New York. Reference: 111. MECHANISM OF GLIDING MOTILITY Foster, K. W. and Smyth, R. D. (1980) Invited paper for Microbiological Reviews. In preparation. Investigator: L Richard Lapidus PUBLICATIONS Cytophaga strain U67 is a rod-shaped organism about Berg, H. C. and Turner, L. (1979) Movements of micro- 0.5 µm in diameter and 4 µm in length. The cells glide on organisms in viscous environments. Nature 278: glass at speeds in excess of 1 µm/sec. They move in the 349-351. Manson, M. D., Tedesco, P. M. and Berg, H. C. (1980) direction of their long axes, stop, hesitate or back up, and Energetics of flagellar rotation in bacteria. J. Mol. occasionally pivot rapidly about one end {sometimes Biol. 138: 541-561. 78

Professor: Charles J. Brokaw suppress flagellar bending, have little or no effect on Visiting Associate: Edward F. Pate disintegration by ATP-induced active sliding. Research Fellows: Makoto Okuno, Winfield S. Sale Graduate Student: David J. Asai These results suggest that anti-dyneins act by Research Staff: Thomas F. Simonick preventing the ATP-induced detachment of dynein cross­ Support: The work described in the following research bridges, and that anti-tubulins have a completely different reports has been supported by: effect. We have now made direct measurements of the National Institutes of Health, USPHS Rockefeller Foundation stiffness, or bending resistance, of flagella in the presence Helen Hay Whitney Foundation of these antibodies. As expected, anti-dyneins increase the stiffness of the flagella towards the value character­ summary: Evidence has accumulated which indicates 2 istic of rigor flagella in the absence of MgATP -, which is that the bending movements of cilia and flagella are consistent with the interpretation that anti-dyneins inter­ generated by a sliding microtubule process, similar to the fere with the detachment of cross-bridges by MgATP2-. sliding filament process responsible for muscle contrac­ In the presence of anti-tubulins, the flagella show visco­ tion. Control mechanisms are needed to cause oscillatory elastic bending resistance. The immediate stiffness is bending, to maintain the phase differences between bending in different regions which are required for high, but the flagella relax within a few seconds to the position expected for relaxed flagella. Further study is propagated bending waves, and to determine the required to determine which of several possible interpre­ parameters of particular bending patterns. tations of this viscoelastic behavior is correct. Our work makes particular use of ATP-reactivated movements of demembranated sperm flagella as a source 114. THE DYNEIN CROSS-BRIDGE CYCLE of experimental data, and computer programs which Investigators: Makoto Olamo, Charles J. Brokaw simulate the movement of model flagella to relate Experiments with the flagellar inhibitor, vanadate, theoretical mechanisms to experimental data. We hope to have been extended to include some observations with identify the types of control mechanisms which actually another inhibitor, AMPPNP. We have been unable to exist in flagella and cilia, to understand how parameters confirm the report of Penningroth and Witman (1978) that of movement such as frequency, wavelength, and bend this ATP analog causes flagellar relaxation. This pub­ angle are controlled, and to 'Use this understanding to lished result now appears to be incorrect (S. Penningroth, enable detailed study of the active process which personal communication) and our measurements of generates sliding in flagella and muscle. flagellar stiffness in the presence of AMPPNP now 113. FURTHER WORK WITH ANTl-TUBULINS provide the strongest available evidence that AMPPNP Investigators: Makoto Oktmo, David J. Asai, binding does not lead to the detachment of dynein arms. Charles J. Brokaw We also find that, in contrast to our earlier finding We previously found that anti-tubulins obtained by that vanadate enhances the competitive inhibition of injecting rabbits with flagellar outer doublet tubulin and flagellar beat frequency by ADP, vanadate does not then purifying the resulting antisera with a tubulin­ enhance the competitive inhibition of frequency of affinity column had a characteristic effect on the motility AMPPNP. This provides strong support for our suggestion of demembranated sperm flagella. They reduce the that vanadate inhibition occurs by formation of a dynein­ amplitude of beating, without any immediate effect on ADP-vanadate complex after dephosphorylation of ATP. the beat frequency. This result has been obtained with However, this complex retains the detached cross-bridge anti-tubulins obtained from four independently-produced state, whereas the complex formed when vanadate and antisera. It contrasts markedly with earlier results ADP are added to flagella must be an attached state. obtained with antibodies against dynein, which had a clear This suggests the presence of at least two states in the inhibitory effect on flagellar beat frequency. dynein cross-bridge cycle which contain bound ADP. We examined the effects of these antibodies on the All of the detailed information obtained with these sliding disintegration of elastase-digested axonemes. inhibitor studies is consistent with the assumption that the Anti-dyneins inhibit sliding, as reported previously, but dynein ATPase cross-bridge cycle is identical with the anti-tubulins, under conditions where they completely actin-myosin cycle, except for the observation that dynein 79

2 forms complexes with ADP and vanadate at a much higher ca + concentrations, and become more asymmetric when 2 rate than myosin. ca + is increased. Potentially symmetric spermatozoa, which swim with symmetric bending waves at low Ca2+ Reference: 2 Penningroth, S. and Witman, G. B. (1978) J. Cell Biol. 79: and become asymmetric as the ca + is increased, can be 2 2 827-832. obtained by exposing the flagella to a high ca +/Mg + ratio, either dufing or subsequent to demembranation. 115. ASYMMETRICAL BEATING OF DEMEMBRANATED SEA URCIIlN The rate of this conversion is an increasing function of SPERM FLAGELLA temperature and Triton concentration. Potentially sym­ Investigators: Makoto Okuno, Charles J. Brokaw metric spermatozoa can be reconverted to potential 2 2 One of the interesting ideas about the bending wave asymmetry, if the exposure to high Ca + /Mg + is brief, asymmetry which can be obtained when demembranated and is terminated by addition of EGTA and Mg2+ before sea urchin spermatozoa are reactivated in the presence of diluting the spermatozoa. The conversion to potential 5 2 high (10- M +) ca + concentrations is that the flagellum symmetry may involve removal of a labile component may contain a Ca-driven contractile system analogous to from the axoneme. ciliate spasmonemes and myonemes. A conclusive demon­ stration of this would require a demonstration that 116. MOVEMENT OF SPERMATOZOA IN VISCOUS ENVIRONMENTS changes in the shape of the flagellum can be produced by Edward F. Pate, Charles J. Brokaw Ca 2+ in the absence of ATP. This is difficult or Investigators: impossible, since in the absence of ATP flagella are in a Some of the fundamental data on flagellar. movement rigor state that will resist deformation. has been obtained by examining movement at increased We have now found that when flagella are in a relaxed viscosities, using methyl cellulose to increase the viscos­ state produced by 5 µM vanadate in the presence of ity. A recent paper by Berg and Turner (1979) demon­ MgATP2-, they can be reversibly bent and straightened by strates that the movement of bacteria at increased changing the ca2+ concentration. This demonstrates that viscosities depends strongly on the nature of the macro­ the normal MgATP 2--driven active sliding mechanism is molecules used to obtain increased viscosities. In probably not involved in the effects of ca2+. However, a particular, results obtained with methyl cellulose were CaATP2--driven mechanism, possibly involving the dynein contrasted with results obtained with Ficoll. We have arms, is not excluded, and in fact is suggested by the compared the effects of Ficoll and methyl cellulose on the observations that higher concentrations of vanadate-50 movement of demembranated sea urchin spermatozoa. At to 100 µM-are effective in inhibiting the response to equivalent macroscopic viscosities, the decrease in beat Ca2+ concentration, and are required to completely frequency caused by Ficoll is about 40% greater than that 2 inhibit the CaATP --mediated oscillation of flagella that caused by methyl cellulose. The effect on wavelength can be observed in the absence of Mg. shows less difference, suggesting that some of the 2 These observations do not suggest that Ca +-induced additional effect of Ficoll may be an osmotic or chemical asymmetry is mediated by calmodulin, although effect associated with the much higher concentration of calmodulin has now been found in cilia and flagella from macromolecules which is required to obtain a given Tetrahymena and Chlamydomonas. The activation of macroscopic viscosity. Nevertheless, there is a difference ATPase activity which is obtained when Ca2+ and which suggests caution in interpreting the data obtained calmodulin are added to extracted dynein from Tetra­ at high viscosities with methyl cellulose. hymena cilia is inhibited by chlorpromazine (J. J. Blum, Reference: personal communication). However, we find no effect of Berg, H. C. and Turner, L. (1979) Nature 278: 349-351. chlorpromazine on any of the effects of ca2+ on demem­ branated sea urchin sperm flagella. 117. COMPUTER SIMULATION OF CROSS-BRIDGE Potentially asymmetric spermatozoa are obtained BEHAVIOR IN RIGOR MUSCLE AND FLAGELLA when spermatozoa are demembranated in the presence of Investigators: Edward F. Pate, Charles J. Brokaw a low ca2+ /Mg2+ ion concentration ratio. They swim with Properties of the rigor state in muscle can be asymmetric bending waves even when reactivated at low explained· by a simple cross-bridge model, of the type 80 which has been suggested for active muscle, in which PUBLICATIONS detachment of cross-bridges by ATP is excluded. Two Asai, D. J. and Brokaw, C. J. (1979) Anti-tubulin anti­ attached cross-bridge states, with distinct force vs. bodies which inhibit flagellar movement do not inhibit microtubule sliding. J. Cell Biol. 83: 340a. distortion relationships, are required, in addition to a Asai, D. J. and Brokaw, C. J. (1980) Effects of antibodies detached state, but the attached cross-bridge states in against tubulin on the movement of reactivated sea urchin sperm flagella. J. Cell Biol. In press. rigor muscle appear to differ significantly from the Brokaw, C. J. (1979) Calcium-induced asymmetrical attached cross-bridge states in active muscle. The beating of Triton-demembranated sea urchin sperm flagella. J. Cell Biol. 82: 401-411. stability of the rigor force maintained in muscle under Brokaw, C. J. (1979) Digestion of sperm flagella with isometric conditions does not require exceptional stability elastase. J. Cell Biol. 83: 175a. Brokaw, C. J. (1980) Elastase digestion of demembranated of the attached cross-bridges, if the positions in which sperm flagella. Science 207: 1365-1367. attachment of cross-bridges is allowed are limited so that Brokaw, C. J. and Goldstein, S. F. (1979) Asymmetrical oscillation of sea urchin sperm flagella induced by the attachment of cross-bridges in positions which have calcium. In: The Spermatozoon: Maturation, Motility minimum free energy is excluded. This new explanation and Surface Properties, D. W. Fawcett and J. M. Bedford (Eds.), pp. 153-156. Urban and Schwarzen­ of the stability of the rigor state may also be applicable berg, Baltimore, Maryland. to the maintenance of stable rigor waves on flagella. Okuno, M. (1980) Inhibition and relaxation of sea urchin sperm flagella by vanadate. J. Cell Biol. 85: 712-725. Okuno, M. (1979) Relaxation and inhibition of flagella by vanadate. J. Cell Biol. 83: 175a. Okuno, M. and Brokaw, C. J. (1979) Inhibition of move­ ment of Tri~on-dem~mbranated sea urchin sperm flagella by Mg +,ATP -, ADP and P .. J. Cell Sci. 38: 105-123. l Pate, E. F. and Brokaw, c. J. (1980) Movement of spermatozoa in viscous environments. J. Exptl. Biol In press.

Prof""80r: Max Delbriick mad mutants. Sherman Fairchild Distinguished Scholar: William Hayes Visiting Associate: Tamotsu Ootaki The mutants were tested for their response to high 2 Senior Research Fellow: M. Viswanath Reddy fluence irradiation (200 J/m to 3,000 J/m2). The madB Research Fellows: Paul A. Galland, Eliot M. Herman, mutant was most affected, showing less than 50% the Makkuni Jayaram, Leslie S. Leutwiler, Manfred K. Otto maximal response of the wild type. The madA and madD Research Staff: Laura E. Kochevar, Nancy K. Wischhusen mutants were affected to a much smaller extent, showing Laboratory Staff: Teresita U. Legaspi, Jeanette Navest 75% the maximal response of wild type. It may be that the alterations of the LICS effects observed with the SUpport: The work described in the following research reports has been supported by: madA and madD mutations are indirect effects and have Deutsche Forschungsgemeinschaft Fairchild Foundation nothing to do with signal handling for LICS or they may be National Science Foundation the consequence of additional mutations present in these Research in Biology strains. The double mutant, madA madB (L51), and the triple 118. LIGHT-INDUCED CAROTENE SYNTHESIS IN MAD MUTANTS OF PHYCOMYCES mutant, madA madB madC (L 72), showed results indica­ Investigators: Makktmi Jayaram, Leslie S. Leutwiler, tive of a multiplicative effect. The maximal response of Max Delbriick the madA madB mutant was about 35% of the wild type. Several mad mutants were tested for their responses in The madA madB made mutant was only slightly more light-induced carotene synthesis (LICS). Using our in vivo defective in keeping with the fact that the madC assay (Jayaram, Presti and Delbriick, 1979), we sought to mutation by itself has no effect on LICS. ascertain (1) how the different mutants respond to well­ The sensitivity range for each of the mutants and the defined fluences, and (2) how LICS is affected in multiple wild type were plotted in order to compare the sensitivity 81 range of LICS with that for phototropism and photo­ mutant and wild type were compared. When grown in the initiation of sporangiophores. All single, double and triple presence of vitamin A, a protein, which is present in the mutants along with wild type show closely similar absence of vitamin A, is induced in both carA and wild sensitivity ranges lying between that of the madB strains type. This protein has a pl and molecular weight very and the double mad strain (madA madB) for phototropism. similar to that which is missing in the carAR mutant. If We conclude that the sensory channel to LICS must further investigation confirms that this protein is the lack the amplification step(s) characteristic of the path­ carA gene product, then this system would be useful for ways leading to phototropism and sporangiophore studies on the mechanism of regulation by vitamin A. initiation. References: Eslava, A. P., Alvarez, M. I. and Cerdti-Olmedo, E. (1974) Reference: Eur. J. Biochem. 48: 617-623. Jayaram, M., Presti, D. and Delbrlick, M. (1979) Exptl. Mycol. 3: 42-52. Jayaram, M., Presti, D. and Delbriick, M. (1979) Exptl. Mycol. 3: 42-52. Lopez-Dfaz, I. and Cerda-Olmedo, E. (1980) Planta. In 119. IDENTIFICATION OF PROTEINS INVOLVED IN press. CAROTENOID BIOSYNTHESIS BY TWO-DIMENSIONAL GEL ELECTROPHORESIS 120. PROTEIN PHOSPHORYLATION IN Investigator: Leslie S. Leutwiler PHYCOMYCES AND ITS BEHAVIORAL Blue light and vitamin A each act independently to MUTANTS stimulate the synthesis of B-carotene in Phycomyces; Investigator: Paul A. Galland their mode of action is dependent on de novo protein In many biological systems, cAMP seems to act as an synthesis (Jayaram, Presti and Delbri.ick, 1979; Eslava, effector which regulates protein kinase. Since light Alvarez and Cerda'.-Olmedo, 1974). Phycomyces offers a induces a transient change in the cAMP pool in Phyco­ relatively simple system to detect the enzymes involved myces sporangiophores, we have initiated a study of in carotene biosynthesis, since they are well characterized protein phosphorylation and its regulation. Differences by four different classes of carotenoid mutants: those between the wild type and mutants might be expected, which produce only trace amounts of the end product, B­ since in several mutants the phosphodiesterase activity carotene, but which can be induced by vitamin A to drastically deviates from that in the wild type (see produce normal amounts (carA}; those which produce only Biology 1980, No. 121). Conditions have been worked out the earliest carotenoid of the biosynthetic pathway, to show phosphorylated proteins on one-dimensional slab phytoene (carB); those which produce the intermediate, gels with autoradiography. No differences were detected lycopene (carR); and those which produce undetectable in the myceliar phosphoproteins between wild type and a amounts of carotenoids and cannot be induced by either behavioral triple mutant (madA madB madC). Sporangio­ light or vitamin A (car AR). In order to identify the phores of the wild type were analyzed under a number of proteins responsible for carotenoid biosynthesis, protein illumination programs suitable to test for the involvement extracts of wild type and of several of the mutants have of phosphorylation in the process of adaptation. It seems been subjected to two-dimensional gel electrophoresis. that none of the approximately 30 phosphoproteins is It has been proposed (LOpez-Dfaz and Cerdtl-Olmedo, playing a role in adaptation. Analysis of other behavioral 1980) that the carA and carR genes are transcribed and mutants is under way. translated together as a single bifunctional gene product and then cleaved to the individual proteins. A nonsense or 121. ROLE OF cAMP IN THE SENSORY TRANSDUCTION PROCESS IN PHYCOMYCES frameshift mutation in the proximal carR segment would Imrestigator: M. Viswanath Reddy create a double mutant lacking both gene products (genotype carAR). Therefore, extracts from light- and In sporangiophores of Phycomyces blakesleeanus, dark-grown mycelium of such a carAR mutant were cAMP levels decrease to about 60% of the initial dark compared with those from wild type. A protein with a pl value within 1 minute of blue light stimulation and return • 4 and molecular weight approximately 130,000 appears to prestimulation level at about the time growth response to be missing in the extracts of a carAR mutant, both ensues, suggesting a role for cyclic AMP in the sensory light- and dark-grown. Additionally, extracts from a car A transduction process (Cohen, 1974). This transient 82 decrease in cAMP levels in response to light stimulation is defect specific to the photoreceptor. Such a mutant probably brought about either by the effect on cyclic AMP would help when chasing the photoreceptor spectrophoto­ biosynthesis via cAMP cyclase or through the breakdown metrically during purification. of cAMP by cAMP phosphodiesterase. Indeed, it has been The rationale for the isolation of photochromic found that the phosphodiesterase is activated by blue light mutants is simple: the phototropic Phycomyces in Phycomyces (Cohen and Atkinson, 1978), although the sporangiophores are kept in balance by illumination with precise mechanism is not yet known. Several mutants, blue light (450 nm) from one side and green light (530 nm) designated as mad, defective in the sensory transduction from the other side. Since the quantum efficiency for pathway, have been well characterized genetically, but green light is about a thousand times lower than that for the biochemical defects in these mutants have not yet blue light, a thousand-fold higher green light intensity has been identified. An attempt is being made to see if any of to be given to achieve balance for the wild type. Mutants these mad mutants have defects in cAMP metabolism. We displaying a chromic shift towards either blue or green found several mad mutants that have either drastically light can be recognized by their bending to the blue or reduced or increased levels of cAMP phosphodiesterase. green light source, respectively. A box with a tuneable In one madA (madA7) mutant (C21), the level of the blue light and a fixed green light source was constructed. enzyme is 20% of the wild type. However, in another The combination of various plexiglasses and a layer of madA allele (madA35) (C47), the enzyme level is normal. copper sulfate solution gives rather pure light with half­ This raises the question whether the strain C21 is a double band widths of 40 nm and 30 nm, respectively. The box mutant with two different mutant loci, namely, madA and allows the incubation of 300 plates at a time, i.e., testing phosphodiesterase. In all double and triple mad mutants of about 20,000 mutagenized colonies bearing sporangio­ with madA7 allele (C21), the phosphodiesterase levels are phores. So far, 150,000 single colonies were checked reduced to less than 50% of the wild type, suggesting a without finding any stable mutant. defective regulatory gene for phosphodiesterase in C21. We are trying to answer this question by isolating 123. STUDIES WITH RIBOFLAVIN ANALOGUES Investigators: Manfred K. Otto, Laura E. Kochevar revertants of C21 to wild-type photoresponse. In other systems, cAMP phosphodiesterase is regulated To characterize the blue light photoreceptor in Phyco­ by either an inhibitor or an activator, although the myces, we seek to replace the supposed photopigment, genetics of the system is practically unknown. We hope riboflavin, by a flavin analogue with different absorbance that our combined study of purification and characteri­ properties thereby altering the action spectrum of light­ zation of cAMP phosphodiesterase coupled with the induced responses. For our studies, we use C322 (rib Bl genetic analysis of C21 will provide some answers to the C-1), a riboflavin auxotroph. In such an auxotroph, the role of cAMP in the sensory transduction process in added analogue competes only with added riboflavin and Phycomyces. not with endogenous riboflavin. If the photochromophore is indeed riboflavin, it is very likely that the photo­ References: Cohen, R. J. (1974) Nature 251: 144-146. receptor has evolved from one of a great number of Cohen, R. J. and Atkinson, M. M. (1978) Biochem. Biophys. flavoenzymes, all of which contain either FMN and/or Res. Comm. 83: 616-621. FAD as cosubstrate. 51-Phosphorylation of both riboflavin and analogue would then be an essential step in the 122. SEARCH FOR PHOTORECEPTOR MUTANTS biosynthesis of the flavin pigment, a reaction which is Investigator: Paul A. Galland catalyzed by riboflavin kinase. Despite numerous efforts, the molecule mediating the Phosphorylation of the analogue, on the other hand, various blue light responses in plants, fungi and bacteria may also lead to growth inhibition due to interference has remained elusive. We know that in Phycomyces it is a with general flavin metabolism. Purified riboflavin kinase flavoprotein. The main problem in biochemical isolation (see Biology, 1979, No. 114) phosphorylates only those is to distinguish it from the bulk of other flavoproteins in flavin analogues that inhibit growth of Phycomyces. The the cell. Behavioral mutants with an altered action toxicity of the respective analogue parallels the photo­ spectrum (photochromic mutants) should result from a phosphorylation rate in vitro. 83

We have been quantitating growth inhibition by extensively by DelbrUck and Ootaki (1979). The dar gene analogues to find conditions where growth may be is a nuclear gene; its wild-type allele codes for riboflavin partially inhibited but expression of the photoresponse is permease, the dar mutants fail to take up riboflavin and still possible. Flavin requirements and analogue inhibition its analogues, including 5-carbon-5-dea~ riboflavin (to be of C322 were assessed with a racing plate technique that referred to as deaza), an analogue which inhibits germi­ measures the growth rate of the mycelial front as a nation and growth at 1 µM concentration. function of riboflavin and analogue concentration. The The dar mutations occur spontaneously at a high rate, rate of mycelial growth of C322 increases linearly with are all allelic (do not complement), and in heterokaryotic 8 riboflavin concentration, half saturates at 3 x 10- Mand situations are highly recessive to dar+. Delbriick and saturates at a rate of 0.65 mm/hour. Sporangiophores Ootaki (1979) reported that the dar mutants spontaneously 7 form at higher riboflavin concentrations (>2 x 10- M). revert to dar+ even faster than the forward mutation rate. The concentration required for half maximum growth rate We have made further observations on the reversion of the mycelium is similar to the apparent Km of rates of dar mutants, and on a new phenomenon found permease, suggesting that flavin utilization in the auxo­ with dar mutants, called "Abortive Growth" (Abo): for troph is limited by permease. Of 20 riboflavin analogues many dar spore stocks, a variable and often high percen­ tested, 5-deaza-5-carboriboflavin (3), roseoflavin (13), and tage of young colonies cease to grow when transferred to 8-chloro-8-demethylriboflavin (22) are inhibitory to both a new medium under any conditions we tried, including + wild-type and riboflavin auxotroph. The numbers in very rich media. Unlike dar mycelia on .deaza, they parentheses give the ratio analogue: riboflavin for 5096 cannot be subcultured. growth inhibition of the auxotroph. 6-Hydroxyriboflavin We first noted that seven dar mutants freshly isolated (3) and 6-aminoriboflavin (J'J.5) inhibit the auxotroph only. from old spore stocks showed very low or zero reversion These analogues compete with riboflavin at permease, but rates, in strong -contrast to the findings reported by are apparently not further processed within the cell. 6- DelbrUck and Ootaki (1979). This stability was traced to Hydroxyriboflavin is poorly phosphorylated by riboflavin the fact that the dar mutants had been isolated from kinase in vitro. spore stocks that had been stored in the refrigerator for Replacement of photopigment due to analogue doping several months. When isolations were made from fresh can be detected by changes in threshold intensities spore stocks, high reversion rates were found, as reported required for tropism towards unilateral light. Undoped by DelbrUck and Ootaki (1979). In addition, these dar C322 sporangiophores have a threshold identical with wild spore stocks showed medium to high Abo rates, not type. The threshold does not vary with variations of correlated with the reversion to dar+ rates. riboflavin in the growth medium. For doping experiments, In all cases, the reversion rates dar to dar+ and the we chose the highest analogue/riboflavin ratio that allows Abo rates were highly erratic. They were neither sporangiophore formation. Doping with .6-aminoriboflavin, characteristic of the particular dar+ stock from which the 6-hydroxyriboflavin, 8-methylamino-8-demethylriboflavin, dar mutants were isolated, nor of the particular dar or 8-chloro-8-demethylriboflavin, did not substantially mutant spore stocks. Different subclones from a given change the threshold of phototropism. A significant dar spore stock differed widely. Even when different threshold shift was observed with roseoflavin. Doped pieces of mycelium from individual dar colonies were used sporangiophores need about five times higher light to produce separate dar spore stocks, these differed intensity to show phototropism. This suggests that a widely in reversion and Abo rates. substantial amount of photopigment is replaced by roseo­ It is clear that the dar mutation and the Abo syndrome flavin. Further experiments are needed to clarify to what are linked in some way but the nature of this linkage and extent roseoflavin can exert the photopigment function. the nature of the very wide fluctuations in the rates is still obscure. 124. STABLE AND UNSTABLE DAR ALLELES OF Reference: PHYCOMYCl!S Delbrilck, M. and Ootaki, T. (1979) Genetics 92: 27-48. Investigators: William Hayes, Max Delbriick: (Note: Figures 1 and 4 are interchanged in this paper.)

The dar gene of Phycomyces has been described 84

125. NUCLEAR BEHAVIORS IN 126. SOME NEW ASPECTS OF THE AVOIDANCE PHYCOMYCilS ZYGOSPORilS RllSPONSE OF PHYCOMYCES Investigator: Tamotsu Ooteki Investigators: Fonda Wu*, Max Delbriick

In the sexual life cycle of Phycomyces, thousands of We have found that the avoidance response of the nuclei get into the zygospore from gametangia of opposite sporangiophores of Phycomyces does not occur in dry air, mating types (gametangiogamy). After a long dormancy although the growth rate is normal. We have checked the period, thousands of spores are produced through inter­ phototropic response in dry air and have found it to be the mediate stages which include effectively only one diploid same as at 65% humidity, with the same lower intensity nucleus. Although many cytological and genetic studies threshold. Russo, Halloran and Gallori (1977) discovered have been done in the past to clarify the nuclear behavior that a step-up in ethylene concentration in the air yields a in the zygospore, many problems have been left unsolved positive transient growth response of the sporangiophore. concerning the time of karyogamy and meiosis. We have confirmed this effect and have found that it We tried to study the timing problems genetically by occurs equally in dry air and at 65% humidity. using a microinjection technique. The protoplasm from The avoidance response appears to be similar for the zygospore formed in a cross between the heterokaryon barriers of any material tested. We have tested this [carA*carR(-)] and one of the yellow wild types, similarity more stringently by using opposing vertical UBC21(+), or a wild type isogenic with the (-) parents, barriers with grossly different gas adsorption properties: ASS(+), was sucked into a capillary pipette and injected stainless steel versus glass; bibulous paper versus stainless into a decapitated stage I sporangiophore of the auxo­ steel; glass versus wax. In no case did the sporangiophores trophic strain C271 [carA nicA(-)], here used as a host show a preference for either barrier but grew straight in cell to induce the production of asexual spores. the middle between the barriers, as they do in the case of The spores were collected from the sporangia which barrier pairs of the same material regenerated on the host cell, and were plated on minimal When this experiment is performed with a sporangio­ medium (to exclude the auxotrophic host nuclei), acidified phore between two horizontal barriers (of the same to pH 3.2 to produce colonial growth. The spores that material), it bends upward toward a barrier 1 mm away, contained nuclei sucked from the zygospore were able to never making contact with the barrier, however. When gE!rminate and grow on the medium, producing various the top or bottom barriers are alternately removed to a colors depending on the nuclear ratio of the car mutants distance >5 mm, the sporangiophore avoids the remaining represented in them. barrier after a latency of 2 min at a rate of 1-3°/min. Nuclei from zygospores less than 10 days old were thus Reference: recovered but no nuclei from older ones (up to 37 days) Russo, V. E. A., Halloran, B. and Gallori, E. (1977) Planta 134: 62-67. were recovered. Most of the nuclei in the zygospores may lose their physiological functions around 10 days after *Undergraduate, University of California, Berkeley, California. crossing and/or the nuclei might be less easily removed from the more viscous peripheral cytoplasm. These PUBLICATIONS questions may be clarified by the observation of nuclei Delbrtick, M. (1980) Was Bose-Einstein statistics arrived stained with the fluorescent nuclear strain mithramycin. at by serendipity? J. Chem. Educ. 57: 467-474. No colonies which appeared on the minimal media- showed Jayaram, M., Leutwiler, L. and Delbrtick, M. (1980) Light­ induced carotene synthesis in mutants of Phycomyces any evidence of genetic recombination, indicating that with abnormal phototropism. Photochem. Photobiol. meiosis occurs later. 32: 241-245. Professor: John J. Hopfield identifying characteristics involving non-stoichiometric use of energetic substrates or cosubstrates. Because Support: The work described in the following research report has been supported by the National Science many processes (e.g., mammalian DNA polymerases) of Foundation. high accuracy do not show these characteristics, we have made a theoretical search for other methods of proof­ Summary: The general area of interest is the chemical reading. One such has been found, involving an enzyme physics of how biological processes take place. We attempt to abstract from biology a defined property which with configurational memory of its recent past states. can be expressed in terms of quantitative chemistry; to This use of "dynamic cooperativity" (or a ''hyperetic 11 understand, through theory and experiments how these enzyme ) can be used to yield proofreading without the symptoms of kinetic proofreading and without the excess properties may come a~out; and what they have to do with static and dynamic molecular structures; and to then take energy consumption kinetic proofreading involves. The these results back into biological systems to test our symptoms of this new and more efficient means of understandings. Other areas in which such studies have proofreading are subtle. There are several places where been going on include the molecular mechanism of circumstantial evidence suggests this mechanism might be cooperative oxygenation in hemoglobin; the mechanism of operative, but new experiments designed to search for the electron transfer processes central to most of bioener­ new symptoms are essential. getics; and the problem of accuracy in the synthesis of PUBLICATIONS biological molecules-its limits, mechanisms to enhance Cho, K. C. and Hopfield, J. J. (1979) Spin equilibrium and accuracy, and its origin. quaternary structure change in hemoglobin A. Experi­ ments on a probe of cooperativity. Biochemistry 18: 5826-5833. 127. PROOFREADING IN BIOCHEMICAL PROCESSES Hopfield, J. J. (1979) Nonadiabatic electron tunneling: Investigator: John J. Hopfield Implications for photosynthesis. In: Tunneling in Biological Systems, B. Chance, D, C. DeVault, H. DNA replication and protein synthesis both require a Frauenfelder, R. A. Marcus, J. R. Schrieffer and N. well-developed ability to discriminate between similar Sutin (Eds.), pp. 417-430. Academic Press, New York. Hopfield, J. J. and Yamane, T. (1980) The fidelity of substrates. "Kinetic proofreading" is used in simple protein synthesis. In: Ribosomes: Structure, Function, and Genetics, Chambliss et al. (Eds.), pp. polymerases and in some aspects of protein synthesis to 585-596. University Park Press, University Park, obtain high accuracy in recognition. It is recognized by Pennsylvania.

Assistant Professor: Elias Lazarides Summary: Intermediate filaments comprise a diverse Senior ReseBl'Ch Fellow: Makoto Okuno class of cytoplasmic filaments whose diameter (..!'100 A) is Research Fellows: David J. Asai, Leah T. Raimo, Clare M. O'Connor, Elizabeth A. Repasky intermediate to those of actin filaments and microtubules. Graduate Students: David L. Gard, Jeff D. Gelles*, Although similar in morphology, they are quite hetero­ Richard H. Gomer, Bruce L. Granger, Chung Wang Research Staff: Ilga Lielausis, Janet Sauer geneous by chemical and immunological criteria. During Laboratory Staff: Margaret M. Griffith the past year, we have continued our studies on the *Division of Chemistry and Chemical Engineering, biochemical characterization of two of the major subunits California Institute of Technology. of these filaments, desrnin and vimentin, in muscle and Support: The work described in the following research nonmuscle cells. Since both the biochemical and bio­ reports has been supported by: American Cancer Society logical characterizations of these molecules were American Heart Association, Los Angeles Affiliate virtually nonexistent, a considerable amount of our efforts Biomedical Research Support Grant (NIH) Muscular Dystrophy Association of America was channelled towards their thorough biochemical char­ National Institutes of Health, USPHS acterization. Through the ~evelopment of new techniques National Science Foundation Research Career Development' Award (NIH) for the fractionation and isolation of skeletal muscle Z discs, we could show that these two subunits are components of the peripheries of the Z discs. Their prove useful for gaining insight into the expression of immunological localization at the periphery of these desmin and vimentin in different tissues. structures indicated that desmin- and vimentin-containing intermediate filaments may play an important role in 128. DESMIN AND VJMENTIN COEXIST AT THE PERIPHERY OF THE MYOFIBRIL Z DISC maintaining the characteristic striated phenotype of Investigator: Bruce L. Granger myofibrils by laterally integrating these structures at their Z discs. Following this process in vitro during Two-dimensional gel electrophoresis has revealed that muscle morphogenesis in tissue culture, we showed that vimentin, the predominant subunit of intermediate fila­ the two intermediate filament subunits are synthesized ments in cells of mesenchymal origin, is a component of noncoordinately after fusion. Vimentin is expressed in isolated skeletal myofibrils. It thus coexists in mature both myoblasts and myotubes, while the synthesis of muscle fibers with desmin, the major subunit of muscle desmin increases severalfold after fusion. In the early intermediate filaments. Antisera to desmin and vimentin, stages of myogenesis, desmin and vimentin are deposited shown to be specific for their respective antigens by two­ in cytoplasmic filaments independent of the assembling dimensional immunoautoradiography, have been used in myofibrils. Approximately seven days after fusion, these immunofluorescence to demonstrate that vimentin has the two proteins begin to transit to the Z disc. same distribution as desmin in skeletal muscle. Both One of the proteins that may regulate this transition is desmin and vimentin surround each rnyofibril Z disc and filamin. This high molecular weight protein apparently form honeycomb-like networks within each Z plane of the functions in vivo to laterally crosslink actin filaments. muscle fiber. This distribution is complementary to that We have found that filamin disappears from the cells upon of a-actinin within a given Z plane. Desmin and vimentin myoblast fusion and reappears along the Z lines shortly may thus be involved in maintaining the lateral regis­ before the transition of desmin and vimentin to this tration of sarcomeres by transversely linking adjacent structure. We are currently investigating the mechanism myofibrils at their Z discs. This linkage would support and whereby filamin synthesis is turned off and on again integrate the fiber as a whole, and provide a molecular during myogenesis. basis for the cross-striated appearance of skeletal muscle. The regulation of polymerization of desmin and vimentin into intermediate filaments is pursued along 129. THE SYNTHESIS AND DISTRIBUTION OF three lines. (1) We are trying to isolate monomeric DESMIN AND VlMENTIN DURING MYOGENESIS IN VITRO desmin and vimentin, a task by no means simple since the proteins are insoluble and tend to remain as polymers. Investigator: David L. Gard (2) We have purified a high molecular weight protein Electrophoretic and autoradiographic analyses of the which copurifies and interacts very tightly with desmin, incorporation of ~ 5 S]methionine into newly synthesized and are investigating the role that the protein might play proteins during myogenesis reveal that presumptive in the polymerization of desmin. (3) We have observed chicken myoblasts synthesize primarily one intermediate that both desmin and vimentin are phosphorylated in vivo. filament protein, vimentin. Desmin synthesis is initiated Purification of the kinases that phosphorylate these two at the onset of fusion. Synthesis rates of both filament molecules has shown that they are the cAMP-dependent subunits increase during the first three days in culture, kinases. We are currently in the process of identifying the relative to the total protein synthesis rate. The observed peptides that are phosphorylated on the two molecules and increase in the rate of desmin synthesis (at least 10-fold) establishing any homology between them by direct is significantly greater than that observed for vimentin, sequencing analysis. and is responsible for a net increase in the cellular desmin The degree of homology between desmin and vimentin, content relative to vimentin. Both filament subunits their number of genes as well as their evolutionary continue to be synthesized through at least 20 days in conservation are not known. To answer some of these culture. Immunofluorescent staining using desmin- and qtiestions, we are currently in the process of constructing vimentin-specific antisera supports the conclusion that cDNA probes for these two molecules from mRNA desmin is synthesized only in fusing or multinucleate cells. isolated from muscle cells. These probes also should These results indicate that the synthesis of the two 87 filament subunits is not coordinately regulated during dimensional gels and trypsin for two-dimensional gels. myogenesis. The distributions of desmin and vimentin in Immunofluorescence using anti-230K on primary cultures multinucleate chicken myotubes are indistinguishable, as of rnyogenic chick cells reveals a pattern characteristic of determined by double immunofluorescence techniques. In intermediate filaments in early myotubes, and a Z line early myotubes, both proteins are found in an intricate distribution in late myotubes, similar to that of desmin network of free cytoplasmic filaments. Later in myo­ and vimentin. Fibroblasts remain nonfluorescent. genesis, several days after the appearance of 0;-actinin­ Immunofluorescence of mature skeletal myofibrils and containing Z line striations, both filament proteins isolated Z disc sheets show that 230K again has the same become associated with the Z lines of newly assembled distribution as desmin and vimentin, at the periphery of myofibrils, with a corresponding decrease in the number the myofibril Z disc. Double immunofluorescence using of cytoplasmic filaments. This transition corresponds to anti-230K and anti-desmin gives indistinguishable patterns the time when the a-actinin-containing Z lines become in early and late myotubes, even after aggregation of aligned laterally. These data suggest that the two intermediate filaments with Colcemid. Fluorescence can intermediate filament systems, desmin and vimentin, have be blocked by preadsorption of the antisera with their an important role in the lateral organization and regis­ respective antigens, but not with the heterologous anti­ tration of myofibrils and that the synthesis of desmin and gens. These results suggest that 230K specifically assembly of desmin-containing intermediate filaments associates with desmin and plays an important role in the during myogenesis is directly related to these functions. functioning of muscle intermediate filaments. Current These results also indicate that the Z disc is assembled in studies are aimed at purifying 230K in order to determine at least two distinct steps during myogenesis. its solubility properties and to determine if it has an effect on the polymerization of desmin. Since it is 130. ASSOCIATION OF A IDGH MOLECULAR WEIGHT apparently absent from nonmuscle cells, it is also of PROTEIN WITH AVIAN MUSCLE DESMIN interest to see if it interacts with vimentin as well as Investigator: Bruce L. Granger desmin in muscle cells. A protein with an approximate molecular weight of 230,000 (230K) copurifies with chicken gizzard desmin 131. THE SYNTHESIS AND DEPLOYMENT OF FILAMIN IN CIDCKEN SKELETAL MUSCLE through cycles of depolymerization in acetic acid and repolymerization or precipitation upon neutralization. Investigator: Richard H. Gomer This protein has a mobility on SDS-polyacrylamide gels In addition to a-actinin, actin, and the two that is different from avian myosin, filamin, fibronectin, intermediate filament subunits, desmin and vimentin, collagen, or spectrin. Desmin and 230K comigrate in filamin (molecular weight 250,000) has been detected as a molecular sieving columns in the presence of 1 M acetic component of vertebrate muscle Z discs (Bechtel, 1979). acid or 6 M urea, but not in the presence of 6 M Immunofluorescence performed on isolated myofibrils and guanidinium chloride. On overloaded isoelectric focusing Z disc sheets indicates that filamin has the same gels in 9. 7 M urea, desmin and 230K focus in complexes distribution as desmin and vimentin; it surrounds each that are more basic than desmin itself; desmin spots myofibril Z disc and forms honeycomb-like networks excised from these complexes on two-dimensional gels can within each Z plane of the muscle fiber. However, unlike be refocused to the normal positions of a and a desmin. desmin and vimentin, the filamin fluorescence is removed Antisera have been raised against desmin and 230K by extensive extraction in buffers containing 0.6 M KI. As purified by SDS-polyacrylamide gel electrophoresis; in judged by d0uble immunofluorescence, metabolic pulse double immunodiffusion, anti-230K precipitates 230K but labeling with r3 5s]methionine and immunoautoradiography not desmin, while anti-desmin precipitates desmin but not of SDS-polyacrylamide gels, filarnin exists in myoblasts 230K. Immunoautoradiography of proteins fixed in SDS­ and early fused cells, associated with a-actinin-containing polyacrylamide gels or transferred to diazotized paper filament bundles. Approximately one day after cell confirms this result. That 230K is not a multimeric fusion, and before the development of a-actinin-con­ aggregate of desmin is also supported by peptide mapping taining Z line striations, filamin disappears from the cells. using Staphylococcus aureus protease VS for one- Later in myogenesis, several days after the appearance of 88

a-actinin-containing Z line striations, filamin reappears subunit protein of intermediate filaments from cells of and accumulates in the cells. The newly appearing filamin mesenchymal origin (Lazarides, 1980). Skeletal muscle localizes now to the myofibril Z line and is visible there RN A directs the synthesis of approximately equal amounts shortly before vimentin (or desmin) transits to the Z line; of desmin and vimentin, each of which constitutes less filamin may thus be involved in the transition of desmin than 1% of the total synthesis. On the other hand, desmin and virnentin to the Z disc. The removal of filamin from synthesis represents between 1 and 5%. of the total the cells results from cessation of its synthesis coupled synthesis from gizzard mRNA. _ Vimentin synthesis is with metabolic turnover. This phenomenon suggests that undetectable in translates of total gizzard mRNA. How­ either filamin may not be required or may actually ever, a small amount of vimentin synthesis is detectable interfere with a necessary process during the early stages when a size cut of gizzard mRNA is used to prime the of sarcomere morphogenesis. We are currently trying to translation system. understand the molecular events which regulate the Neither desmin nor vimentin is derived from a larger disappearance and reappearance of filamin during myo­ precursor in the reticulocyte lysates, since desmin and genesis. vimentin-specific antibodies precipitate only the mature proteins. Reference: Bechtel, P. (1979) J. Biol. Chem. 254: 1755-1758. References: Lazarides, E. (1980) Nature 283: 249-256. Levin, D., Ernst, V. and London, I. M. (1979) J. Biol. 132. IN VITRO TRANSLATION OF INTERMEDIATE Chem. 254: 7935-7941. FILAMENT SUBUNIT PROTEINS O'Connor, C. M. and Lazarides, E. (1979) J. Cell Biol. 83: Investigators: Clare M. O'Connor, David J. Asai 314a. ·

Previous work from this laboratory has shown that there are two major isoeleetric variants of desmin in 133. CHARACTERIZATION OF PHOSPHORYLATION muscle cells. The more acidic one, a-desmin, is a SITES OF Dl!SMIN AND VIMENTIN Investigators: David L. Gard, Clare M. O'Connor phosphorylated protein, which is phosphorylated in vitro by the cAMP-dependent protein kinases (O'Connor and We have previously reported that the intermediate Lazarides, 1979). Approximately equal amounts of a- and filament proteins of avian muscle, desmin and vimentin, B-desmin are detected in muscle tissue. are phosphorylated in vivo (see Biology 1979, No. 117), and To determine if a-desmin is derived from the that the endogenous phosphorylating agent has many of nonphosphorylated variant, B-desmin, by posttranslational the characteristics of the muscle cyclic AMP-dependent phosphorylation, we have translated desmin mRNA in protein kinase. We have been continuing our investi­ vitro using a rabbit reticulocyte cell-free system. RNA gations into the nature and function of this posttrans­ has been extracted from neonatal chicken breast (skeletal) lational modification of the intermediate filament and gizzard (smooth) muscle and separated into poly(Af proteins. Two-dimensional (high voltage electro- and poly( A)+ fractions by oligo(dT) cellulose chromatog­ phoresis/thin layer chromatography) tryptic peptide 32 raphy. The desmin mRNA, which is enriched in the analysis of desmin labeled in vivo with PO reveals two 4 poly( A)+ fraction, directs the synthesis of predominantly phosphopeptides, suggesting the presence of two phos­ B-desmin in vitro. Less than 15% of the total desmin phorylation sites within the desmin polypeptide. Acid 32 translated in vitro is a-desmin. The production of a­ hydrolysis of either PO -labeled desmin, or the isolated 4 desmin may be due to the presence of endogenous cAMP­ phosphopeptides reveals o-phosphoserine, consistent with dependent kinase activity in the lysates (Levin, Ernst and the known specificity of the cAMP-dependent kinases. London, 1979), since supplementing the lysates with Tryptic peptide analysis indicates that vimentin contains additional kinase augments the amount of a-desmin phosphopeptides which comigrate with those found in produced. Our attempts to separate specific a- and B­ desmin, indicative of at least limited sequence homology desmin mRNA:; have been unsuccessful. between these two filament proteins. These data support Desmin mRNA is much more prevalent in the poly(At our previous conclusions based upon one-dimensional RNA from gizzard than in skeletal mRNA. RNA from peptide analysis. The degree of homology and the size of both tissues also directs the translation of vimentin, the these peptides remain undetermined. We are currently 89 isolating the phosphopeptides from desmin and vimentin 135. THE 68,000 DALTON NEUROFILAMENT­ for amino acid composition and sequence analysis to ASSOClATED POLYPEPTIDE IS A COMPONENT OF NONNEURONAL CELLS answer these questions. Further protein sequencing Investigators: Chung Wang, David J. Asai studies may also be carried out to determine the extent of homology between desmin and vimentin. Purified preparations of 10 nm neurofilaments from rat spinal cord and bovine or porcine brain contain a predominant 68,000 dalton polypeptide. This polypeptide 134. IDENTIFICATION OF ENDOGENOUS is also a major component of the neurofilaments that SUBSTRATES FOR CffiCKEN MUSCLE TRANSGLUTAMlNASE copurify with brain tubulin isolated by cycles of poly­ Investigator: David L. Gard merization and depolymerization. A protein that has the The transglutaminases are a class of enzymes which same isoelectric point and molecular weight as the neurofilament-associated polypeptide has also been identi­ catalyze the formation of covalent <( y-glutamyl) lysine fied as a cytoskeletal protein in a variety of avian and linkages between protein molecules. These enzymes have mammalian cell types, including baby hamster kidney been implicated in many diverse functions such as the clotting of blood, crosslinking of the stratus corneum, and (BHK-21), mouse 3T3, Novikoff rat hepatoma, chicken recently, in the process of receptor-mediated endocytosis. fibroblast, and chicken muscle cells. This protein is also a We have previously demonstrated that skeletal muscle component of isolated chicken skeletal myofibrils. One­ myofibril proteins, particularly those of the Z line, may dimensional peptide maps of the 68,000 dalton proteins serve as substrates for exogenous guinea pig liver trans­ purified by two-dimensional isoelectric focusing/SD8-gel glutaminase. We have now demonstrated the presence of electrophoresis from myofibrils, cycled tubulin, purified an endogenous transglutaminase in sonicated extracts of neurofilaments, and various cultured cell types were cultured embryonic chicken myotubes. The enzyme was identical. In immunofiuorescence, this protein was identified by its ability to covalently incorporate the associated with cytoplasmic intermediate filaments and lysine analogue r3H]putrescine into cellular proteins, and myofibril Z discs. These results indicate that the by its requirement for ca2+ ions and a reducing agent. neurofilament-associated polypeptide is a conserved pro­ The activity is inhibited by ca2+ chelators such as EGTA, tein that is present in many different cell types in by the oxidizing agent diamide, and by primary amines. addition to neuronal cells. Because some of these cells The proteins serving as amine acceptors have been contain the major components of two other intermediate identified by the incorporation of dansyl cadaverine, a filament classes, desmin and vimentin, a given cell type fluorescent lysine analogue. Incubation of sonicated may contain the subunits of at least three distinct myotubes in the presence of ca2+ ions, glutathione, and intermediate filament types. dansyl cadaverine results in the incorporation of label into PUBLICATIONS desmin, vimentin, and y-actin, and an unidentified a- Falke, J. and Lazarides, E. (1980) Staining of viable and protein of approximately 230,000 daltons. The specificity nonviable myotubes and of myofibrils by the fluores­ cent dye merocyanine 540. Differentiation. In press. of the reaction is indicated by the relative lack of Gard, D. L. and Lazarides, E. (1980) The synthesis and incorporation of dansyl cadaverine into other prevalent distribution of desmin and vimentin during myogenesis myotube proteins such as a-actin, myosin, and the in vitro. Cell 19: 263-275. Granger, B. L. and Lazarides, E. (1979) Desmin and tropomyosins. These data suggest that transglutaminase­ vimentin coexist at the periphery of the myofibril Z disc. Cell 18: 1053-1063. catalyzed covalent crosslinks may be involved in the Lazarides, E. (1980) Desmin and intermediate filaments in assembly and stabilization of the myotube cytoskeleton. muscle cells. In: Results and Problems in Cell We are currently in the process of isolating and character­ Differentiation, Proceedings of the Third International Conference on Differentiation. Springer-Verlag, izing the myotube transglutaminase in an attempt to Berlin. In press. define its role in myogenesis more carefully. Lazarides, E. (1980) Fluorescent analysis of cell motility. Internat. Rev. Cytol. In press. Lazarides, E. (1980) Intermediate filaments and cell structure. In: Gene Families of Collagen and Other Structural Proteins, D. J. Prockop and P. Champe (Eds.). Elsevier/North Holland Biomedical Press. In press. 90

Lazarides, E. (1980) Intermediate filaments as mechanical Lazarides, E., Hubbard, B. D. and Granger, B. L. (1979) integrators of cellular space. Nature 283: 249-256. Studies on the structure, interaction with actin, and Lazarides, E. (1980) Intermediate filaments in muscle function of desmin and intermediate filaments in morphogenesis. In: Proceedings of the International chicken muscle cells. In: Cell Motility: Molecules Congress on Cell Biology. Berlin. In press. and Organization, S. Hatano, H. Ishikawa and H. Sato Lazarides, E. (1980) The cytoskeleton. In: Encyclopedia (Eds.), pp. 521-543. Tokyo University Press. of Science and Technology. McGraw-Hill, New York. O'Connor, C. M. and Lazarides, E. (1980) Intermediate In press. filament protein phosphorylation by cAMP-dependent Lazarides, E. (1981) Antibody production and immuno­ protein kinases. Submitted for publication. fluorescent characterization of actin and contractile Strader, C. D., Lazarides, E. and Raftery, M. A. (1980) proteins. In: Methods in Cell Biology, L. Wilson (Ed.), The characterization of actin associated with . post­ Volume 23, Part A. Academic Press, New York. In synaptic membranes from Torpedo californica. press. Biochem. Biophys. Res. Comm. 92: 365-373. Lazarides, E. (1981) Biochemical and immunocytological Wang, c., Asai, D. J. and Lazarides, E. (1980) The 68,000 characterization of intermediate filaments in muscle dalton neurofilament-associated polypeptide is a com­ cells. In: Methods in Cell Biology, L. Wilson (Ed.), ponent of nonneuronal cells and of skeletal myofibrils. Volume 23, Part B. Academic Press, New York. In Proc. Nat. Acad. Sci. USA 77: 1541-1545. press.

Professor: Jean-Paul Revel derived from rat liver preparations. A 20-amino acid N­ Senior Researeh Fellow: S. Barbara Yancey terminal sequence for mouse liver junctions is virtually Research Fellow: David J. Meyer Graduate Student: Bruce J. Nicholson identical. There is, however, no homology between N­ Research Staff: Peter Devrees, Jean Edens, Les B. terminal sequences of liver junctional polypeptides and Grim, Patrick F. Koen the N-terminal sequence of lens gap junctions (25 amino Support: The work described in the following research acids). After trypsinization of junctional membrane reports has been supported by: Earle C. Anthony Fellowship fractions, one can recover a polypeptide of apparent Biomedical Research Support Grant (NIH) molecular weight 10,000 which has an N-terminal region National Institutes of Health, USPHS Northwest Area Foundation identical to that found in "intact" junctions and, like the Albert Billings Ruddock Fund latter, contains a region of largely hydrophobic residues Summary: The year has been marked by some very starting at amino acid 23. It is probable that this region is encouraging advances in our understanding of the molec­ present as a-pleated structure and that it represents a ular biology of gap junctions and their role in cellular transmembrane portion of the peptide chain. communication. While J.-P. Revel was on sabbatical at New York A carefully correlated quantitative study comparing University with Drs. T. Morimoto and D. Sabatini, Barbara junction number and size to electrical or dye coupling Yancey did wonders in keeping the laboratory running. suggests close correlation between the two. The sur­ The work described below was hers, David Meyer's and prising result is that very few junctions are needed for Bruce Nicholson's_ with the collaboration of M. Hunkapiller electrotonic coupling. One needs to carry out careful of the Hood group and L. Takemoto of Kansas State studies to detect the subtle changes in ionic coupling University. The contributions made by Pat Koen, Jean which result from even drastic changes in number of Edens and Les Grim to the research described was recognizable junctions. Studies of dye transfer gave a extremely valuable and is much appreciated. much more readily perceived reflection of the states of the junctional complement. 136. AN ANALYSIS OF THE PROTEIN COMPONENTS From a biochemical standpoint, evidence has OF RAT LIVER GAP JUNCTIONS accumulated that radioactively-labeled methionine is Investigators: Bruce J. Nicholson, Les B.. Grim, Michael W. Hunkapiller, Jean-Paul Revel rapidly incorporated into junction protein, thus opening the way for metabolic studies. The molecular weight of Several proteins of different molecular weights have the junction protein appears to be close to 28,000. We been claimed in the past to be the major components of have succeeded in obtaining a sequence of 58 amino acids liver gap junctions. Even now, when a 28-26 kilodalton at the apparent N-terminal of the junctional polypeptide peptide is generally recognized as the major protein, all 91

preparations of gap junctions published show additional has been established as the major component of gap protein components. If proteases are used in the junctions isolated from liver (bovine, rat and mouse). preparation protocol, a 10 kilodalton peptide is the major However, antibodies raised against the lens gap junctional component of the gap junctional fraction. protein do not cross-react with the protein of liver gap Using two-

138. A QUANTITATIVE ANALYSIS OF consistent with the hypothesis that gap junctions are INTERCELLULAR COMMUNICATION IN aggregates of intercellular channels. NORMAL AND REGENERATING RAT LIVER Investigators: David J. Meyer, S. Barbara Yancey, Jean-Paul Revel 139. AN IlSTIMATE OF THE CONDUCTANCE OF A SINGLE CONNEXON IN RAT LIVER Gap junctions are thought to be composed of Investigators: David J. Meyer, S. Barbara Yancey, aggregates of intercellular channels (connexons). A Jean-Paul Revel reduction in the number or size of gap junctions should In view of the evidence that gap junctions are therefore result in a decrease in the permeability of the aggregates of intercellular channels, we ha.Ye attempted intercellular pathway to ions and small molecules. As to estimate the conductance of these putative channels reported previously (Biology 1979, No. 127), we have been using a combination of physiological and morphological using the regenerating rat liver to test this prediction. techniques. A cable analysis of the spatial dependence of Between 29 and 35 hr after partial hepatectomy, gap electrotonic potentials in normal rat liver yielded an junctions virtually disappear from the hepatocyte surface. estimate of Ri (cytoplasmic + junctional resistances) of Measurements performed on freeze-fracture replicas 2500 ncm. Scanning electron microscopy of rat liver demonstrate a 100-fold reduction in the overall area of reveals that hepatocytes can be described as flattened, gap junctions in regenerating as compared with normal hexagonal prisms, approximately 20 µ across. Each of the liver. six facets that form the perimeter of a hepatocyte is 2 . We have used scanning electron microscopy to examine about 100 µ in area. Freeze-fracture of rat liver 2 intercellular relationships in rat liver and to estimate the demonstrates that about 3 µ of each facet is occupied by area of contact between adjacent hepatocytes. By gap junctions. Gap junctions only occur on these facets. combining these results with our freeze-fracture data, we The electrical resistance of a hepatocyte to radial current have shown that in normal rat liver a hepatocyte forms at (current injected into the network of hepatocytes from a least one gap junction with every hepatocyte it contacts point source such as a microelectrode) is Ri (~) where 1 is (about 6). In regenerating liver, a hepatocyte may form the distance and a is the area through which current gap junctions with only one or two adjacent hepatocytes, flows. If current traverses a hepatocyte by way of gap if one presumes that the junctions are randomly junctions, then 1 is 10 µ on average (1/2 the ''length" of a 2 distributed throughout the cell population. cell) and a is 200 µ (the area of two facets). The 6 Our physiological investigations reveal that the inter­ resistance is then calculated to be 1.25 x 10 n. In cellular spread of fluorescent tracer molecules and elec­ glutaraldehyde fixed material, there are 11,000 con­ tric current is restricted in regenerating as compared with nexons/µ 2 of gap junction. Hence, the resistance of 1.25 normal liver. An analysis of the spatial dependence of the x 106n may be attributed to 6.6 x 104 connexons. The 10 electrotonic potentials reveals that there is a 20- to resistance of a single connexon is then about 8 x 10 n. 40-fold increase in intercellular resistance in regenerating This estimate is similar to estimates derived from purely liver. If each connexon seen in freeze-fracture is an physiological measurements and therefore provides intercellular channel, then the resistance increase should additional support for the hypothesis that the connexon is be 100-fold. the intercellular channel. In normal liver, all hepatocyte pairs tested were electrically coupled, while in regenerating liver only about 140. JK VIVO INCORPORATION OF 90% were coupled. The distribution of gap junctions is r•s]METIDONINE INTO GAP JUNCTIONAL PROTIDN IN RAT LIVER consistent with the incidence of coupling in normal liver Investigators: S. Barbara Yancey, Peter Dewees, but is not sufficient (by a factor of 2 to 3) to account for Jean-Paul Revel the pattern of coupling in regenerating liver. A simple, rapid method employing proteolytic enzymes We feel that in view of the simplifying assumptions has been devised for the isolation of gap junctional made. in the analysis of the data, the discrepancies fractions from as little as two to three grams of rat liver. between our physiological and morphological results are Analysis of these fractions by SDS-polyacrylamide gel relatively small and we conclude that our results are electrophoresis shows a major component at 10,000 93

daltons along with a number of bands at higher molecular cause and effect relationship between these two events weights. Mapping of peptides generated from this 10,000 during regeneration or whether the correlation is merely dalton component by digestion with trypsin or chymo­ coincidental. trypsin indicates complete homology with the 10,000 We are currently exploring this problem further by dalton peptide characteristic of very clean fractions of making use of the observation that mitotic activity may gap junctions isolated by our previously published protocol also be induced in rat liver by a single injection of (Finbow et aL, 1980). We have now been able to follow thioacetamide. Between 24 and 54 hr after injection, 5 the incorporation of r3 s]methionine into gap junctional approximately 50% of the cells enter mitosis with the protein after a single intraperitoneal injection of radio­ mitotic frequency reaching a peak between 36 and 48 hr active isotope. Comparing incorporated counts estimated (Reddy, Chiga and Svoboda, 1969). Our preliminary by autoradiography and scintillation counting of bands observations after quantitative analysis of the area obtained on polyacrylamide gel microslabs with the occupied by gap junctions on freeze-fracture faces of intensity of Coomassie staining of the bands, we firid that hepatocyte cell membranes in thioacetamide-treated rats maximum specific activity of the junctional peptide from compared to controls indicates that there is a decrease in normal liver is reached within 3 hr. The specific activity the mean junctional area detectable by 34 hr, a further of the protein in the 10,000 dalton band is higher than that decrease to a minimum reached between 43 and 46 hr, and in any other component on the gel. Less than 0.5 µg of a return to near normal levels by 49 hr. The overall junctional protein is required for gel electrophoresis in decrease in gap junctional area appears to be significant Order to obtain levels of radioactivity three to four times (about 7-fold by 43 hr), but the loss of gap junctions background 3 hr after injection of isotope. The incorpor­ induced by thioacetamide does not appear to be as marked ation of amino acid is surprisingly rapid, suggesting that as that initiated in response to partial hepatectomy where there is a relatively high rate of synthesis of gap there is a 50-fold reduction in gap junctional area. junctional protein in normal rat liver. Preliminary results References: from studies of regenerating liver show a similar rapid Klinge, 0. (1968) Virchows Arch. Abstr. B ZellpathoL 1: 342-345. incorporation of amino acid into junctional protein during Merk, F. B. and McNutt, N. S. (1970) J. Cell Biol. 47: the first 8 hr after partial hepatectomy, before the 666-688. Reddy, J., Chiga, M. and Svoboda, D. (1969) Lab. Invest. disappearance of the junctions 28 hr postoperatively 20: 405-411. (Biology 1977, No. 131) as well as after the reappearance of gap junctions during the third day of regeneration. 142. FORMATION OF GAP JUNCTIONS DURING These experiments provide the basis for continuing studies EMBRYONIC DEVELOPMENT of the in vivo synthesis and turnover of gap junctional OF THE CIDCK LENS protein. Investigators: Jean F.dens, S. Barbara Yancey, Jean-Paul Revel Reference: Finbow, M., Yancey, S. B., Johnson, R. and Revel, J.-P. The vertebrate lens is an avascular tissue made from (1980) Proc. Nat. Acad. Sci. USA 77: 970-974. only one cell type, epithelial cells which in the mature lens form a monolayer on the anterior surface of the lens 141. TIDOACETAMIDE-INDUCED CHANGES IN GAP JUNCTIONAL MORPHOLOGY and which during the life of the animal differentiate, Investigators: Peter Dewees, S. Barbara Yancey, elongating to form the lens fibers. In the mature lens, gap Jean-Paul Revel junctions, similar in morphology to those found in many At present there is contradictory evidence as to other vertebrate tissues, occur between the epithelial whether mitotic activity need be associated with changes cells (Benedetti et al., 1976). The gap junctions which are in gap junctional morphology. Dividing ovarian granulosa found in profuse numbers between the lens fibers, how­ cells appear to retain their gap junctions (Merk and ever, differ from typical gap junctions not only in McNutt, 1970). On the other hand, in regenerating liver, structural organization (Benedetti et al., 1976) but in their the first major peak in mitotic activity (Klinge, 1968) is protein components (see Biology 1980, No. 137), antigenic temporally correlated with a drastic loss of gap junctions properties of the junctional protein (N. B. Gilula, personal (Biology 1977, No. 131). It is not known whether there is a communication) and behavior in response to conditions 94 which favor uncoupling (Goodenough, 1979). In order to PUBLICATIONS gain some further understanding of the rclationship Bell, P. B. and Revel, J.-P. (1980) Scanning electron between these two types of gap junctions and how they microscope application to cells and tissues in culture. Jn: Biomedical Research Applications of Scanning evolve, we are currently using freeze-fracture techniques Electron Microscopy, G. M. Hodges and R. C. Hallowes to study the formation of gap junctions between the cells (Eds.), pp. 1-63. Academic Press, London. Finbow, M., Yancey, S. B., Johnson, R. and Revel, J.-P. of the developing lens during early stages of ernbryo­ · (1980) Identification of a major junctional protein in genesis of the chick. gap junctions. Proc. Nat. Acad. Sci. USA 77: 970-97 4. Kaczmarek, L. K., Finbow, M., Revel, J.-P. and By day three of development, small aggregates of Strumwasser, F. (1979) The morphology and coupling of polygonally-packed gap junctional particles can be found Aplysia bag cells within the abdominal ganglion and in cell culture. J. Neurobiol. 10: 535-550. between the epithelial cells. By day four, the stage Meyer, J. D., Yancey, s. B. and Revel, J.-P. (1980) encompassing the first definitive steps in formation of Intercellular communication in the regenerating rat liver. Submitted for publication. primary lens fibers, the elongating, differentiating cells Miller, M. M., Strader, B. D. and Revel, J.-P. (1980) are joined by large numbers of gap junctions which appear Hemocyanin-protein A: An immunochemical reagent for scanning and transmission electron microscopy. In: to be in different stages of formation, varying from SEM/1980, O. Johari (Ed.). SEM, Inc., Chicago, isolated linear arrays of junctional particles to more lliinois. Revel, J.-P., Yancey, S. B., Meyer, D. J. and Finbow, M. elaborate arrangements wherein many linear arrays inter­ s. (1980) Behavior of gap junctions during liver sect or closely approach to enclose small areas of regeneration. In: Communications of Liver Cells, H. Popper, L. Bianchi, F. Gudat and W. Reutter. MTP membrane or to encircle clusters of gap junctional Press Limited, Lancaster, England. In press. particles. Larger, more compact but still disordered Revel, J.-P., YanCey, S. B., Meyer, D. J. and Nicholson, B. (1980) Cell junctions and intercellular communication. aggregates of gap junctional particles are also abundant. In Vitro. In press. Complementary views of all these formations can be seen Shinowara, N. L., Beutel, W. B. and Revel, J.-P. (1980) Comparative analysis of junctions in the myelin sheath as arrays of pits on the corresponding outer membrane of central and peripheral axons of fish, amphibians, faces. and mammals. A freeze-fracture study using comple­ mentary replicas. J. NeurocytoL 9: 15-38. References: Strader, C. D., Revel, J.-P. and Raftery, M. A. (1979) Benedetti, E. L., Dunia, 1., Bentzel, C. J., Vermorken, A. Demonstration of the transmembrane nature of the J. M., Kibbelaar, M. and Bloemendal, H. (1976) acetylcholine receptor by labeling with anti-receptor Biochim. Biophys. Acta 457: 353-384. antibodies. J. Cell Biol. 83: 499-510. Goodenough, D. A. (1979) Invest. Ophthalmol. Visual Sci. Yancey, S. B., Easter, D. and Revel, J.-P. (1979) Cyto­ 18: ll04-ll22. logical changes in gap junctions during liver regeneration. J. Ultrastruct. Res. 67: 229-242.

Professor Emeritus: Anthonie Van Harreveld bathing the retina (Van Harreveld and Fifkova, 1973). The Research Staff: Marjorie Sturgis effect of these compounds on the histological structure of Support: The work described in the following research the retina was investigated both with light and electron reports has been supported by the National Science microscopy after fixing -the tissue either by freeze­ Foundation. substitution of the tissue in acetone at -79°C or by conventional glutaraldehyde-osmiumtetroxide fixation. 143. MECHANISM OF TRANSPARENCY CHANGES IN THE RETINA The freeze-substituteq preparations showed a marked Investigator: A. Van Harreveld swelling of a glial element, the Millier fibers, which run The isolated chicken retina exhibits marked changes in radially through the retina. In control preparations transparency. Such changes occur during spreading (treated with MgC1 to prevent spreading depression), the 2 depression, which is characterized by an increase in MUiler fibers have a mean diameter of 0.2 µM. In the retinal transparency progressing slowly over the retina. glutaraldehyde treated retainas, the diameter grew to Comparable transparency changes can be elicited by 0.4 µM and in KC! treated ones to 0.6 µM. It is suggested exposing the retina for 2 min to potassium chloride that the transparency changes are due to this effect on (30 mM) or glutamate (1 mM) applied in the medium the Miiller fibers which may act as light guides. 95

In contrast to the large swelling of the Milller fibers freeze-dried central nervous tissue were continued. An elicited by KCl and glutamate application observed in improved lyophilizing apparatus was built which allowed freeze-substituted materials, only a small increase in the drying to proceed at predetermined temperatures. diameter of the structures was found in chemically fixed The effect of drying on the tissue structure has been preparations of retinas exposed to 30 mM KCl or 1 mM varying from rather well-maintained to poor. Attempts glutamate. The diameter of the fibers in these prepara­ are being made to determine the parameters which result tions was only 30% larger than in the controls. It can be in satisfactory tissue structure preservation after drying. surmised that the Miiller fibers in the KCl and glutamate The use of a silver compound of a crown ether treated preparations were swollen at the moment of (Benzo-15-Crown-5) dissolved in acetone to precipitate fixation. The reduction of this swelling during chemical the chloride as silver chloride has been shown to be fixation is another example of the distorting effect of reliable, depositing the silver in the expected locations. chemical fixatives on the fluid distribution in a tissue, The AgCl crystals precipitated are quite small, visible making this mode of fixation unsuitable for the study of only at high magnification with the electron microscope. the water distribution in nervous tissue (Van Harreveld However, by precipitating silver on the crystals by a and Khattab, 1968, 1969). method used in photographic intensification the particles

References: can be made large enough to become visible at moderate Van Harreveld, A. and Fifkova, E. (1973) J. Neurobiol. 4: magnifications. 375-387. Van Harreveld, A. and Khattab, F. I. (1968) J. Cell Sci. 3: 579-594. PUBLICATIONS Van Harreveld, A. and Khattab, F. I. (1969) J. Cell Sci. 4: 437-453. Van Harreveld, A. (1979) Effect of L-proline, some proline analogs and other amino acids on spreading depression in the retina. Soc. Neurosci. Abstracts 5: 325. Van Harreveld, A. (1980) L-Proline as a glutamate 144. A HISTOLOGICAL METHOD FOR THE ELECTRON MICROSCOPIC LOCALIZATION antagonist at a crustacean neuro-muscular junction. J. OF CHLORIDE Neurobiol. In press. Van Harreveld, A., Gherkin, A. and Davis, J. L. (1980) Investigator: A. Van Ilarreveld Amnestic potency of proline analogs correlates with anti-spreading depression potency. Pharmacol. The attempts to determine the position of chloride in Biochem. Behav. 12: 533-541.

CELLULAR NEUROBIOLOGY

Jeremy P. Brockes

A. James Hudspeth

Henry A. Lester

Felix Strumwasser

99

Assistant Professor: Jeremy P. Brock es the zebra finch, and the effect of diphtheria toxin on Del Webb Research Fellow: Mark E. Gurney cultured rat Schwann cells. The latter project (a Research Fellow: Katherine A. Stygall Graduate Students: Karl J. Fryxell, Greg Erwin Lemke collaboration with Professor Alvin Pappenheimer) has Research Staff: David R. Balzer Jr., Teresa M. Stevens Laboratory Staff: Caroline Vermaes disclosed two singular features in the way that these cells respond to the binding of the toxin and mutant proteins. Support: The work described in the following research reports has been supported by: Biomedical Research Support Grant (NIH} The Kroc Foundation 145. PURIFICATION OF GIJAL GROWTH FACTOR National Institutes of Health, USPHS (GGF), A NEW COMPONENT OF THE National Multiple Sclerosis Society BRAlN AND PITUITARY National Science Foundation Pew Memorial Trust Investigators: Jeremy P. Brockes, David R. Balzer Jr., Greg Erwin Lemke Del E. Webb Foundation In a normal tissue culture medium containing 10% Summary: We work with identified and purified neural fetal calf serum, purified rat Schwann cells divide very cells in tissue culture, and we are particularly interested slowly. We have previously reported that the cells are in the molecular basis of interactions between these cells. stimulated to divide by an activity present in extracts of One major focus of activity is a novel and potent growth the brain and pituitary (Brockes, Fields and Raff, 1979), factor /hormone, almost certainly a product of nerve cells and this activity appears to be both novel and restricted in in the brain and pituitary, which acts on Schwann cells, its distribution (Raff et al., 1978). The activity, which astrocytes and muscle fibroblasts, but not on oligodendro­ can be assayed by the incorporation of [1251]iododeoxy­ cytes, microglia or 3T3 cells. We have made significant uridine into DNA of Schwann cells growing in micro wells, progress during the last year in purifying and character­ has been purified over 4000-fold from a pool of 10 kg of izing this molecule. It may be important in that subset of frozen glands and 4000 lyophilized anterior lobes. It had 4 "neurotrophic" phenomena which involves nerve­ an apparent molecular weight of 6 x 10 on gel filtration stimulated mitosis (for example in muscle development or and adhered strongly to cation exchange resins. The most limb regeneration), or in division of glial cells on injury. purified fraction was analyzed on SDS gel electrophoresis We hope to investigate these possibilities after raising and had a major band of 3 x 104 molecular weight which monoclonal antibodies to the molecule. was approximately 25% of the stained material. When A rather different influence is exerted by neurons on analyzed by native gel electrophoresis at pH 4.5 followed Schwann cells in the peripheral nervous system. The by a second dimension of SDS gel electrophoresis, the major peripheral myelin proteins, in particular. the Po activity was consistently associated with this component, glycoprotein, are not made by the Schwann cell unless it is suggesting that the native species is a dimer. triggered by contact with an appropriate axon. We have The effect of the phosphocellulose fraction on prolif­ set up this interaction in culture between pure sensory eration of central glial cells in dissociated cultures of the neurons of the dorsal root ganglion and pure Schwann rat corpus callosum was also investigated by using cells, and we are attempting to identify the molecules fluorescent antiera to identify the cells and involved (on the surface of the nerve and Schwarm cell) by [3H Jthymidine autoradiography to assay proliferation. using biochemical, serological and genetic approaches. The oligodendrocytes and microglia were not stimulated, The characterization of monoclonal antibodies to the but the astrocytes were stimulated over the same range of dorsal root cells is important for this project and also for concentration as Schwann cells. The activity against technical studies on the use of this methodology to astrocytes and that against Schwann cells comigrated on disclose different classes of nerve cells. The initiation of native gel electrophoresis at pH 4.5, providing strong myelination by particular (myelinated) axons is an evidence that the same molecule acts on both cell types. expression of neuronal "recognition" which may be more The factor had no significant effect on the proliferation amenable to biochemical analysis than the familiar of Swiss mouse 3T3 cells but stimulated rat muscle example of synaptogenesis. fibroblasts; the activity against fibroblasts also migrated In two collaborative projects, we are studying the with that against Schwann cells on electrophoresis at pH influence of steroid hormones on cultured song neurons of 4.5. 100

We are currently attempting to derive a monoclonal since it makes up more than half of the protein in antibody by immunizing with the purified fractions. This peripheral myelin, and is not synthesized in detectable reagent will be important in projected studies on the amounts by SC cultured alone (Brockes et al., 1980). purification, assay, localization and functional role of Since last year's abstract (Biology 1979, No. 147), we GGF. have improved our techniques for culturing purified DRG neurons. Some of the changes include: an initial References: Brockes, J. P., Fields, K. L. and Raff, M. C. (1979) Brain enrichment of neurons by centrifugation through a density Res. 165: 105-118. gradient; culture on a dried collagen layer of standardized Raff, M. c., Abney, E., Brockes, J. P. and Hornby-Smith, A. (1978) Cell 15: 813-822. thickness that is coated with purified LETS protein; and the addition of carrier protein to the serum-free media in 146. GLlAL GROWTH FACTOR IN BRAIN which the neurons are maintained (Bottenstein and Sato, Investigator: Greg Erwin Lemke 1979). These cultures can now be maintained in good condition for at least seven weeks. Glial growth factor (GGF)-like activity is present in Preliminary results, using either enzyme-linked brain. Crude extracts of whole bovine brain contain immunoassays or indirect immunofluorescence, confirm mitogenic activity toward Schwann cells at a level that Po is produced by cocultures, and not by neurons or approximately 30% of that of pituitary extracts. This SC cultured separately. We are presently working on a activity exhibits a distinct regional variation-extracts radioimmunoassay (O'Keefe and Bennett, 1980) in Order to prepared from ·ctifferent brain areas vary in specific improve the quantitation of Po levels. We hope to use this activity over an approximately 6-fold range; one region, assay to resolve issues such as: (1) the time course of Po the caudate nucleus, has an even higher specific activity production after SC are added; (2) the time course with than the pituitary. which DRG neurons become competent to induce myelin­ The caudate activity is similar in its biochemical ation after they are introduced into culture; and (3) properties to the pituitary mitogen. It shows similar whether or not any of the antisera that we have produced binding to and elution from phosphocellulose ion-exchange will block myelination. resin, and it comigrates with the pituitary activity on pH In addition, we have begun to study the mouse mutant, 4.5 native gel electrophoresis. As is the case with Trembler. Trembler has a specific deficit of peripheral pituitary GGF, it shows activity against astrocytes as well myelin; in vivo experiments indicate that the deficit is as Schwann cells, but is not active against oligodendro­ due to the SC rather than the axon (Aguayo, Bray and cytes or macrophage-like microglia. The pituitary and Perkins, 1979). We can culture purified SC from wild-type brain factors are thus closely related, if not identical and Trembler mice, and intend to characterize some of molecules. More detailed studies on the properties, the differences in their biochemistry and interactions with localization and role of this activity should be possible rat DRG neurons in vitro. after derivation of monoclonal antibodies to the pituitary factor. References: Aguayo, A. J., Bray, G. M. and Perkins, S. C. (1979) Ann. N.Y. Acad. Sci. 317: 512-531. 147. PREIJMINARY STUDIES OF MYELINATION Bottenstein, J. E. and Sato, G. H. (1979) Proc. Nat. Acad. IN VITRO Sci. USA 76: 514-517. Brockes, J. P., Raff, M. C., Nishiguchi, D. J. and Winter, Investigators: Karl J. Fryxell, David R. Balzer Jr., J. (1980) J. Neurocytol. 9: 67-77. Jeremy P. Brockes, Teresa M. Stevens O'Keefe, E. and Bennett, V. (1980) J. Biol. Chem. 255: 561-568. A major, long-term goal of this laboratory is to develop a quantitative assay for myelination in vitro and to exploit it for molecular studies of the early events in 148. GENERATION OF MONOCLONAL ANTIBODIES myelination. We can do this by culturing purified dorsal TO SPECIFIC NEURAL CELLS Investigator: Katherine A. Stygall root ganglion (DRG) neurons and Schwann cells (SC) separately, and then combining them and measuring the Conditions for the long-term culture of purified rat resulting induction of myelin proteins. We have concen­ dorsal root ganglion_ neurons have been developed in this trated on measuring the peripheral myelin protein Po, laboratory (Biology 1980, No. 147). Our principal interest 101 in such cultures lies in the identification of membrane daltons which has three functionally distinct regions. The structures important during myelination. As one approach C-terminal region binds tightly to an uncharacterized to this, we are making monoclonal antibodies against receptor that is present on the surface of most animal cultured dorsal root ganglion cells. Our aim is to produce cells. There is a hydrophobic sequence that is possibly antibodies that will interfere wi_th in vitro myelination, an involved in translocating the molecule through the mem­ assay system for which is being developed (Biology 1980, brane and finally an N-terminal region which has No. 147). Such antibodies would be invaluable for the enzymatic activity. This region splits NAD and transfers purification of membrane components important during the ADP-ribose moiety onto elongation factor .2, thus myelination and for a study of their appearance in dorsal blocking protein synthesis and killing the cell (for review, root ganglion cultures. In addition, we hope that this see Pappenheimer, 1977). technique will produce antibodies that bind specifically to Rat and mouse cells are unusually resistant to the neuronal subpopulations, for example those using distinct toxin (see Table 1) both in vitro and in vivo. Nevertheless, neuropeptides as transmitters. minute doses (.r1 nanogram) will cause demyelination if Many functionally important molecules have been injected into the sciatic nerve or intracerebrally into found to be structurally conserved. For this .reason, rodents. We have therefore investigated the effect of the interspecies immunization may fail to produce antibodies toxin and various mutant proteins on protein synthesis in against such important molecules, including those involved cultured rat Schwann cells. As shown below, Schwann in myelination. In an attempt to overcome tolerance, we cells are abnormally sensitive to the action of DTx, as are using a method developed by Leech and Mitchison compared to other rat cells. They are also unusually (1976) in which T lymphocytes are primed to recognize a sensitive to CRM 45, an amber mutant missing the C­ hapten, arsanilic acid. Such T cells then recognize haI_>ten terminal receptor reacting determinants which th~refore conjugated to the tolerogen and help B lymphocytes has to insert via the hydrophobic sequence in some ill­ respond to the tolerogen. We are immunizing primed mice understood fashion. The native toxin is competed by prior with dorsal root ganglion and Schwann cells derivatized incubation with CRM 197, a missense mutant in the with arsanilic acid. The diversity and titer of the enzymatic region which binds to the receptors but is antibodies so produced is being investigated. nontoxic. After competition with CRM 197, the toxin

Reference: curve becomes superimposable with that of CRM 45 with Leech, S. H. and Mitchison, N. A. (1976) Eur. J. Immunol. or without CRM 197. These studi_es define two singular 6: 810-816. features of the rat Schwann cell: firstly, it is unusual relative to other rodent cells (with the possible exception 149. EFFECT OF DIPHTHERIA TOXIN AND MUTANT of the oligodendrocyte) in its ability to internalize DTx; PROTEINS ON CULTURED RAT SCHWANN CELLS secondly, it is unusual relative to all other cells in the Investigators: Alvin M. Pappenheimer Jr.*, high toxicity of CRM 45. The structural bases of these Jeremy P. Brockes singularities are unknown. Diphtheria toxin (DTx) is the product of corynephage Reference: Pappenheimer, A. M. Jr. (1977) Ann. Rev. Biochem. 46: 69-94. $, a temperate phage which resides in Corynebacterium diphtheriae. It is secreted as a single polypeptide of 60 K *The Biological Laboratories, Harvard University.

Table 1. Toxicity of DTx and related proteins for various cell types

Toxin + Species Cell Toxin a CRM 45 CRM 197 Fragment Ab l\.1onkey Kidney 0.005 5,000 ND ND Rat Fibroblasts 5,000 10,000 ND ND Rat Schwann 50 500 500. >10,000 cells aConcentration (nanogram/ml) for 50% inhibition of r3Hl or r14c]Ieucine incorporation. bFragment A is an enzymatically inactive fragment derived by tryptic digestion. 102

150. STUDIES ON THE EFFECTS OF GONADAL differentiation of these neurons in vivo, and we seek to HORMONES UPON THE DIFFERENTIATION OF achieve the same effects in vitr"o. DISSOCIATED NEURONS IN VITRO Investigator: Marie E. Gurney PUBLICATIONS Enduring sex differences in behavior and reproductive physiology are established during development through the Brockes, J. P. (1979) Consequences of signal reception. action of gonadal steroids. Such differences in the In: The Role of Intercellular Signals: Navigation, Encounter, Outcome, J. G. Nicholls (Ed.), pp. 285-300. outward signs of the brain's function have been taken to Dahlem Konferenzen, Berlin. Brockes, J. P. and Raff, M. C. (1979) Studies on cultured imply sex differences within the brains of various bird and rat Schwann cells. II. Comparison with a rat Schwann mammalian species. There is encouragement for believing cell line. In Vitro 15: 772-778. that the brain is the target of gonadal steroid action Brockes, J. P. (1980) Identification and purification of cultured Schwann cells, and a purified factor con­ during development since these hormones can exert their trolling their proliferation. In: Proceedings of the First International Conference on Spinal Cord Recon­ developmental effect when locally implanted into the struction, c. C. Kao (Ed.). Raven Press, New York. In hypothalamus of neonatal rats, and since small bits of press. Brockes, J. P., Lemke, G. E. and Balzer, D. R. Jr. (1980) neonatal mouse hypothalamus exhibit steroid-dependent Purification and preliminary characterization of a glial growth in vitro. However, it is presently unclear whether growth factor from the bovine pituitary. J. Biol. Chem. In press. neurons, non-neuronal cells, or both, are the cellular Brockes, J. P., Raff, M. c., Nishiguchi, D. J. and Winter, targets of steroid action. We seek to address this question J. (1980) Studies on cultured rat Schwann cells. III. Assays for peripheral myelin proteins. J. Neurocytol. by putting identified populations of neurons from the 9: 67-77. zebra finch song system into culture in the absence of Fryxell, K. J. (1980) Synthesis of sulfatide by cultured rat non-neuronal cells, and we will then assess the ability of Schwann cells. J. Neurochem. In press. Pappenheimer, A. M. Jr., Harper, A. A. and Brockes, J.P. gonadal steroids to affect their subsequent growth and (1980) Effect of route of injection on the toxicity of diphtheria toxin and related proteins for certain differentiation. Gurney (Biology 1980, No. 182) has animal species: their cytotoxicity for cultured cells characterized the effects of gonadal hormones upon the derived from them. J. Cell Biol. In press.

Associate Professor: A. James Hudspeth understanding of the interaction of light, sound, acceler­ Graduate Students: David P. Corey, Ruth A. Eatock, Richard S. Lewis ation, pressure, molecular concentration, etc., with their Research Staff: Richard A. Jacobs respective receptor cells; in addition, it aids in appreci­ Support: The work described in the following research ating how our senses shape our perception of the world. reports has been supported by: Our research group is investigating the transduction The Deafness Research Foundation The Hearst Foundation process of hair cells, the receptors which enable the inner National Institutes of Health, USPHS ear to respond to sound and to acceleration. These are National Research Council Canada Fellowship Ann Peppers Foundation columnar, epithelial cells each endowed with a specialized Pew Memorial Trust receptor organelle, the hair bundle, which. characteris­ Gordon Ross Medical Foundation tically comprises a single true cilium or kinocilium, and a Summary: For each of the various senses, the body fascicle of 50-200 stout microvilli termed stereocilia. possesses specialized receptor cells responsible for con­ Acoustical and accelerational stimuli are mechanically verting the relevant stimulus energy into an electrical converted into forces which displace the hair bundle and, signal that the nervous system can interpret. Such in so doing, elicit responses. We wish to understand how receptor cells are remarkably sensitive: some photo­ this comes about. receptors can detect a single photon, and certain Our earlier investigations have established that: (a) it olfactory sensory cells respond to individual odorant is bending of the hair bundle which produces responses; (b) molecules. Study of the transduction processes whereby the range of hair bundle deflection over which a cell stimuli engender responses can provide a biophysical responds is remarkably small-an angular extent of about 103

±2°; (c) hair cells are correspondingly very sensitive, tion. In order to use the preparation for quantitative giving measurable responses in vitro to stimuli smaller studies of transduction by hair cells, we have analyzed the than 0.5 nm; (d) the cells possess an adaptation mechanism generation of the microphonic response in terms of the which keeps them highly sensitive even in the presence of various currents through hair cell membranes. large static stimuli; (e) it is the stereocilia, rather than Three phenomena affect the relationship between hair the kinocilium that mediate mechanosensitivity; (f) the bundle displacement and microphonic response. Depolar­ intracellular electrical response is associated with a ization following the increase in transducer conductance change in the membrane conductance of the cell, implying acts to decrease the receptor current by decreasing the that ion channels are opened and closed by mechanical driving force on the ions that carry this current. Depolar­ stimuli; (g) the channels so opened are relatively non­ ization also activates a voltage-sensitive potassium selective in their ionic permeability, and will pass cations conductance (delayed rectification). Finally, an adaptive of molecular dimensions less than about 0.6 nm; and (h) shift of the hair cell's operating curve causes a relaxation the channels open following a stimulus with a very short of the transducer conductance toward its resting value. latency-less than 13 µsec at 37°C. When mathematical expressions for each phenomenon are We are presently seeking to determine more precisely combined and solved with stepwise integration, the solu­ where on the hair cell's surface the transduction event tion adequately fits the measured microphonic response. takes place. One approach to this issue involves the use The model has also been successfully tested against of procedures that interfere with the transduction process experimental results from preparations in which pharma­ as probes for understanding the normal operation of hair cological and ionic methods have eliminated some of these cells. Aminoglycoside antibiotics, which are potently phenomena and thus simplified the response. ototoxic drugs in common clinical use, block transduction and eventually kill hair cells. Overstimulation also inactivates and destroys hair cells. We are studying the 152. ADAPTATION IN THE RESPONSE OF HAlR CELLS TO SUSTAINED STIMULI physiological bases of these two types of trauma and Investigators: Ruth A. Ee.tock, David P. Corey, seeking their ultrastructural bases. Richard A. Jacobs, Donald c.-T. Lo*, A. James Hudspeth

151. ANALYSIS OF THE MICROPHONlC RESPONSE Adaptation is the decline with time in the sensation RECORDED FROM THE SACCULUS IN VITRO evoked by a maintained stimulus of constant intensity. Investigators: David P. Corey, A. James Hudspeth Impulse frequency in many first-order sensory neurons Recording of microphonic potentials, small extra­ also declines during a sustained stimulus; adaptation is cellular signals generated by current flow through hair thus at least partly attributable to processing in the sense cells, has been a mainstay of auditory physiology for fifty organs and at the synapses between receptor cells and years. The recording method is less invasive, and so primary nerve fibers. We have found that in the bullfrog provides greater stability, than intracellular recording, sacculus, as in visual organs, the receptor cells themselves but is consequently less direct as well: receptor currents adapt, and that this adaptation is a property of the cells through the mechanically sensitive, transducer conduc­ rather than of the accessory structures within the organ. tance are summed with currents through time-, voltage-, The microphonic receptor current in response to a and ion-dependent conductances. The assumption of most maintained step displacement of the otolithic membrane researchers that the microphonic potential is instanta­ relaxes to a steady-state value over a few tens of neously proportional to hair bundle displacement is not milliseconds. Determination of the relationship between only simplistic but diverts attention from the relationship response and displacement of the preparation before and most expressive of the transduction mechanism. at various times during the step indicates that the We have developed an in vitro preparation of the adaptation is not simply fatigue. The hair cells continue bullfrog sacculus which involves recording an extracellular to be fully responsive but the range of displacements to microphonic response. This preparation complements our which they respond shifts in such a way as to enhance studies on hair cells with intracellular recordings, and has progressively their sensitivity at the new hair bundle the advantages of greater stability and temporal resolu- position. This shift is reversible and occurs for steps in 104

both directions, although it is initially faster for steps 153. ELECTRICAL RESPONSES FROM SOLITARY HAIR CELLS toward the kinocilium than for those in the opposite Investigators: Richard S. Lewis, A. James Hudspeth direction. Optical monitoring of the positions of the stimulus probe and otolithic membrane indicates that the We have developed techniques for the isolation of observation is not an artifact resulting from slippage intact, solitary hair cells from the saccular macula of the between the probe and membrane. Independent evidence bullfrog. Partial digestion of the saccular epithelium in against this possibility comes from intracellular vitro with papain, followed by a period of several hours at recordings from cells whose hair bundles are individually 4°C in low-calcium saline solution, yields a preparation in deflected with a glass probe (see Figure 1, left panel); a which the connections among cells have been weakened to similar shift in their displacement-response curves follows the point at which gentle teasing with a dissecting needle a maintained step stimulus. will free individual cells (see Figure 1, center panel). Adaptation confers. upon hair cells a high-pass filter Each cell may be partially sucked into a micropipette so characteristic; the cells retain their responsiveness to that the cell is lodged with the hair bundle accessible transient stimuli in the presence of static or low­ from the bath and with its membrane forming a tight frequency stimuli. This may be particularly useful in a electrical seal of 15 MS"i! with the glass electrode walls vestibular organ for which head and body movements (see Figure 1, right panel). Mechanical deflection of the would provide large static stimulation and would, in the hair bundle with a piezoelectric micromanipulator and absence of an adaptive mechanism, render the organ measurement of currents entering the pipette from the insensitive to superimposed transient stimuli. cell show that the cells can transduce stimuli in this condition, producing currents of up to 50 pA. *Undergraduate, California Institute of Technology.

Figure 1. Ueft) Scanning electron micrograph of the apical surface of the bullfrog saccular epithelium. From the top of each hair cell protrudes a hair bimdle consisting of several dozen cylindrical stereocilia and a single kinocilium, whose distal end forms a bulbous enlargement. In this and the subsequent panels, the length of the hair bundles is about 8 µm. The hair bundle of one cell (arrow) has been deflected with a probe in order to elicit an electrical response and has been fixed in this stimulated position. (center) Scanning electron micrograph of a hair cell isolated from the sacculus of the bullfrog by enzymatic treatment. While supporting cells and nerve endings have been removed from the cell, its surface membrane and hair bundle appear to be intact. (right) Light micrograph of an isolated hair cell about to be pulled into a suction electrode for recording of extracellular receptor potentials. 105

We hope to puncture the basal membrane of such an roughly exponential approach to steady state and the bell­ immobilized cell and to measure receptor currents while shaped variation of time constant with displacement imply varying the ionic constitution of the solutions within and an energy barrier to transition between the states of surrounding the cell. This preparation offers several 12 kcal/mo! whose height is influenced by hair bundle advantages for the study of the molecular events under­ displacement. Thus, energy states of the transduction lying transduction. (a) The availability of low-resistance channel appear to be directly and continuously modified access to the cell's interior may allow one to measure by hair bundle position. fluctuations associated with the opening and closing of The two-state model is only qualitatively correct, single transduction channels, providing important evidence however: a sigmoidal rise at the onset of current, a two­ for the existence of discrete channels. (b) The ability to phase fall at the offset, and an asymmetry in the define rigorously the internal ionic envirOnment of the displacement-response curve are all inconsistent with two cell through internal perfusion should help in investi­ states and a single transition. Good quantitative agree­ gations of the ionic dependences of various transduction­ ment requires a model with three states. The transition related phenomena. (c) The ability to perfuse the cell from the first, nonconducting state to the second, may also be useful in selectively modifying various nonconducting state is relatively slow and very displace­ cellular structural elements in an effort to deduce the ment-sensitive; the subsequent transition to the third, mechanical linkage between hair bunclle and receptor conducting state is faster and less displacement-sensitive. channel. 155. AN IN VITRO STUDY OF OTOTOXICITY DUE TO AMINOGLYCOSIDE ANTIBIOTICS 154. GATING KINETICS OF THE TRANSDUCTION ELEMENT IN VERTEBRATE HAIR CELLS Investigators: Bonnie L. Blamick*, Richard A. Jacd>s, David R. Mathog*, A. James Hudspeth Investigators: David P. Corey, A. James Hudspeth

A large variety of ionic channels in cell membranes As a preliminary step toward locating the site of action of the aminoglycoside drugs against hair cells, we can be thought of as existing in either open or closed have tested the efficacy of various antibiotics in blocking states. The probability of a channel being in a given state is determined by the relative energy of that state, and the the in vitro response of bullfrog hair cells to mechanical stimulation. When superfused over the apical surfaces of relative energies are changed-the channel is gated-by the cells, the drugs streptomycin, dihydrostreptomycin, factors such as voltage and binding of ligands. We have gentamicin, netilmicin, neomycin, and several of their evidence that the opening of transduction channels in derivatives all rapidly block the receptor current. The vertebrate hair cells involves a redistribution between states; here the energy change is effected by displace­ relationships between logarithm of drug concentration and remnant response are sigmoidal and consistent with ment of the hair bundle. An in vitro microphonic preparation was used wherein blockage by combination of the drugs with a single class of receptor sites. Half-blocking concentrations of the a small portion of a bullfrog sacculus was stimulated and various antibiotics are in the range of 2-50 µM, levels the transepithelial receptor current was measured. similar to those encountered in the inner ear fluids of Following a 0.1-µm step displacement of the otolithic patients treated with the drugs to combat bacterial sepsis. membrane in the positive direction, toward the kino­ If the drugs are removed from the experimental prepara­ cilium, the current increases with a roughly exponential time course with a time constant of 400 µsec. Larger tion within a few minutes of their application, the displacements elicit larger currents and shorter time receptor current recovers to essentially its control level; more prolonged drug exposure, however, produces a constants; with displacements larger than about 0.5 µm, permanent blockage of responsiveness in hair cells. The the current saturates but the time constant continues to extent of this permanent effect by various drugs is decline. Negative displacements decrease the current but correlated with their relative ototoxicities in clinical also decrease the time constant. The relationship between steady-state current and displacement (the dis­ applications; the in vitro assay may accordingly prove useful in screening antibiotics for low ototoxicity. placement-response curve) is roughly sigmoidal, consistent with a probability distribution between two states. The *Undergraduate, California Institute of Technology. 106

PUBLICATIONS

Corey, D. P. and Hudspeth, A. J. (1979) Ionic basis of the Corey, D. P. and Hudspeth, A. J. (1980) Mechanical receptor current in a vertebrate hair cell. Soc. stimulation and micromanipulation with piezoelectric Neurosci. Abstracts 5: 18. bimorph elements. Submitted for publication. Corey, D. P. and Hudspeth, A. J. (1980) Gating kinetics of Eatock, R. A., Corey, D. P. and Hudspeth, A. J. (1979) the transduction element in a vertebrate hair cell: Adaptation in a vertebrate hair cell: stimulus-induced evidence for a three-state model. Soc. Neurosci. shift of the operating range. Soc. Neurosci. Abstracts Abstracts 6. In press. 5: 19.

Associate Professor: Henry A. Lester 156. PERFUSION OF CELLS CONTAINING Professor: Jerome Pine* ACETYLCHOLINE RECEPTORS Visiting Associate: Joel Nargeot Investigator: Maori E. Krouse Senior Research Fellow: Menasche M. Nass Research Fellows: Jeanne M. Nerbonne, Robert E. Sheridan Jr., Martin M. Weinstock Nicotinic acetylcholine receptors are routinely studied Graduate Student: Mauri E. Krouse with voltage-clamp techniques in extracellular solutions Research Staff: Donna R. Williams of varying composition. We are developing a technique *Division of Physics, Mathematics and Astronomy, for varying the composition of the intracellular solution as California Institute of Technology. well as for controlling the voltage across the cell Support: The work described in the following research membrane. reports has been supported by: Muscular Dystrophy Association of America One would like to perfuse and voltage-clamp a cell National Institutes of Health, USPHS containing acetylcholine receptors in much the same way NATO Fellowship Pew Memorial Trust a squid axon is perfused and voltage-clamped. University of Tours, France The most promising cell appears to be the electro-­ plaque from the electric eeL Each electroplaque is highly Summary: Most of our recent work concerns the control convoluted and surrounded by collagenous connective of membrane properties by neurotransmitters and other tissue. After removing the connective tissue, we hoped to small molecules. We have continued to exploit the swell the cell osmotically into a less convoluted shape. nicotinic synapse, where impulses are transmitted from a Collagenase treatment exposed the cell surface but nerve to a muscle or electric organ. These experiments subsequent osmotic shock did not change its contours. involve the use of photoisomerizable molecules to produce It therefore appears that the structure of the cell is 11concentration-jumps11 of drugs near membranes under determined from the inside. An injection of 0.5 M KC! electrophysiological investigation. We also applied this and 10 mM EGTA liquifies the cytoplasm of Myxicola procedure to study the muscarinic acetylcholine receptor, axons. We find that this solution also liquifies the which functions on a time scale thousands of times slower cytoplasm of eel electroplaques. Thus, it appears possible than the nictonic receptor. to replace the cytoplasm with artificial solution. We are We are developing molecules that release calcium, presently using electrophysiological measurements to protons, or cyclic nucleotides upon absorption of a photon. check the health and viability of the perfused cell. These molecules, in conjunction with electrophysiological If the cell membrane remains intact for an hour or recording and the light-flash technique, may prove useful more, we will set up a system to control the voltage in studying the control of membrane properties by across the cell membrane while perfusing the cell. This intracellular messengers. system will allow us to study (a) how internal solutions Finally, we are exploring the physiology of cells affect the opening and closing of the receptor channels, developing in culture. The emphasis is on advanced (b) how ions permeate the open channel, and (c) how local techniques for electrical stimulation and recording, with anesthetics block the open channel. the aim of monitoring and influencing the development of synaptic connections. 107

157. NUMERICAL RECONSTRUCTION OF chemistry has been studied extensively, comparatively RELAXATION AND FLUCTUATION little is known about the lifetime of the excited state of EXPERIMENTS AT NlCOTINlC SYNAPSES the molecules and hence the rate at which the stereo­ Investigator: Mauri E. Krouse isomerization proceeds to completion. This information is Several variables, such as voltage, agonist concen­ of critical importance as we are using these compounds to tration, and temperature, influence the number of open probe fast processes at the nicotonic receptor. We have acetylcholine receptor channels in a postsynaptic mem­ therefore begun to study the time course· of azobenzene brane. A rapid change in any of these variables causes a photoisomerizations in aqueous solutions. relaxation from one equilibrium state to another. This Photoisomerizations were initiated with a 1 µsec pulse relaxation is a single exponential function of time. This from a dye laser at 440 nm while the absorbance at fact has led several investigators to conclude that a 340 nm was continuously monitored with a weak light unique step in the channel activation mechanism limits source arranged at right angles to the actinic flash. For the rate of channel opening. The two most likely Bis-Q, absorbance decreased during trans to cis photo­ candidates for the rate-limiting step are the binding of isomerizations and increased during net cis to trans the second agonist molecule to the receptor and the photoisomerizations. The time course of both types of subsequent conformational change from the closed to the isomerizations closely paralleled the integrated photon open state. flux during the laser pulse, with a delay of less than We are presently using computer programs to simulate 200 nsec. Similar results were obtained for photoisomeri­ the consequences of these two different hypotheses. zations of QBr, 2BQ, and EW-1. Thus, it is clear that These programs are modifications of that employed by azobenzene photoisoinerizations are complete within 200 Wathey, Nass and Lester (1979) to simulate the release of nsec and that the photochemical manipulations occur a quantum of acetylcholine into the synaptic cleft and the instantaneously on the time scale of channel gating. subsequent diffusion, hydrolysis, and binding of the acetyl­ choline molecules. Certain parameters are fixed to the 159. PHOTOCHEMICAL PROBES OF THE values determined experimentally; other parameters are AGONlST-RECEPTOR COMPLEX allowed to vary until the best fit to all experimental data Investigators: Robert E. Sheridan Jr., Henry A. Lester is reached. The best-fit simulations will be compared to In these studies, voltage-clamped Electrophorus see which molecular hypothesis most closely describes the electroplaques are exposed to the photoisomerizable experimental data. These simulations should enable us to nicotinic agonist, trans-Bis-Q. Some acetylcholine recep­ decide which step is rate-limiting and also which tors bind trans-Bis-Q molecules; as a result, some parameters are critical for the functioning of the synapse. receptor channels are open. When the preparation is The most important feature of the simulation is the exposed to a flash of light, Bis-Q molecules undergo (cis extent to which the changes in acetylcholine concen­ to trans) and (trans to cis) photoisomerizations. In tration within the cleft are distorted by the buffering particular, some trans-Bis-Q molecules bound to receptors action of receptors themselves. are converted to the cis isomer, which is not an agonist. Reference: As a result, a fraction of the receptor channels close. The Wathey, J. C., Nass, M. M. and Lester, H. A. (1979) membrane conductance decreases. This decrease, called Biophys. J. 27: 145-164. phase 1, is so rapid (time constant <100 µsec) that it can be studied independently of later signals caused by the 158. PHOTOJSOMERIZATION OF AZOBENZENES flash. We are employing phase 1 to determine how many Investigators: Robert E. Sheridan Jr., Henry A. Lester Bis-Q molecules are necessary to keep the receptor in an A variety of photoisomerizable azobenzene derivatives open configuration. This is accomplished by comparing are useful in the study of the nicotinic acetylcholine the fraction of Bis-Q molecules which undergo isomeri­ receptor. These compounds undergo stereoisomerization zation to the fraction of receptors which close. The after absorbing a photon and thus change their potency as present method has several advantages over methods agonists, antagonists, or open-channel blockers, depending based on examining the shape of dose-response curves. upon the derivative used. Although azobenzene photo- The measurements are made on preexisting agonist- 108

receptor complexes and thus are independent of agonist dependence on membrane voltage, and similarities diffusion, binding rates, and receptor desensitization. between light-flash and voltage-jump relaxations. According to a number of proposed models for the *University of Tours, Tours, France. receptor function, a short, weak pulse of light should close a fraction of receptors equal to nK where K is the t t probability of a trans to cis isomerization and n is the 161. HOW PHOTOISOMERIZABLE AZOBENZENE number of trans-Bis-Q molecules bound to the receptor. COMPOUNDS AFFECT ACETYLCHOLINE In our experiments with a 1 µsec laser flash at 440 nm, RECEPTORS OF FISH MUSCLE the value for n is close to 2. The same value is obtained Investigators: Mauri E. Krouse, Henry A. Lester, Martin M. Weinstock for the Hill coefficient of dose-response curves with Bis-Q. Two molecules of agonist are required to open a Previously we have studied the effects of azobenzene receptor channel. compounds on Electrophorus electroplaques. This work extends those investigations to the skate, Raja erinacea. Photoisomerizable drugs were applied to the depressor 160. THE MUSCARINIC ACETYLCHOLINE RECEPTOR OF FROG ATRIAL FIBERS rostri muscle. Effects of these drugs on endplate Investigators: Joel Nargeot,. Marie-Christine Nargeot*, potentials and endplate currents (recorded under voltage Henry A. Lester clamp) were examined. Trans-Bis-Q is a potent agonist at Acetylcholine slows the heart by modifying membrane the eel electroplaque (KD = 150 nM at -150 mV). A 30 µM properties. One such effect, mediated by muscarinic solution of Bis-Q containing predominantly the inactive receptors, is a hyperpolarization caused by an increase of cis isomer was added to the voltage-clamped muscle. A potassium conductance. Previous studies with iontopho­ light flash increased the concentration of the trans isomer retie application of acetylcholine show that the from about 5 µM to 11 µM and thus induced a net inward muscarinic actions of acetylcholine occur on a time scale current of 15 nA. This increased inward current was, several orders of magnitude slower than the nicotinic however, transient even though both isomers are stable in actions. There is an initial delay of several tens of the dark. Ten µM 2BQ, a competitive antagonist on the eel electroplaque (K = 150 nM for the cis isomer), milliseconds before the first detectable hyperpolarization; 1 the peak response occurs several hundred msec later. We reduced the amplitude of the endplate current to about are employing light-activated drugs to study temporal 25% of its control value. This effect was seen with both aspects of the response to muscarinic drugs in voltage­ the cis and trans configurations of 2BQ. Ten µM benzyl clamped atrial trabeculae from frog hearts. Bis-Q inhibits the response to carbachol in the electric eel Several photoisomerizable azobenzene compounds but in the skate muscle the effect is to increase the block the increase of potassium conductance produced by amplitude of the endplate potential and to prolong its muscarinic agonists. We are studying the interaction decay, suggesting that this drug is an acetylcholinesterase between carbachol and Bis-Q. Trans-Bis-Q shifts the inhibitor. A small antagonist effect would not have been equilibrium dose-response curve for carbachol to the seen. EW-1, a local anesthetic (in its cis isomer) on the eel electroplaque (K = 25 µM), has no effect on endplate right, without changing the maximal response. At a 1 concentration of 20 µM trans-Bis-Q, the carbachol con­ currents with concentrations as high as 100 µM. Thus, centration must be increased by 3-fold to produce the there are differences between eel electroplaque and skate half-maximal response. The cis configuration is several­ muscle in regard to drug action and potency. fold less potent as a muscarinic blocker. Trans-Bis-Q is also a potent agonist on muscles of the In fibers exposed to carbachol and cis-Bis-Q, light South American gymnotid fishes, Hypepomus and flashes convert Bis-Q molecules to the trans isomer and Eigenmannia. A concentration of 100 nM produces a the acetylcholine-induced current decreases as a result. depolarization of tens of mV. We are currently developing The final 80% of relaxation has an exponential time the Hypopomus neuromuscular junction as a preparation course. The time constant is about 600 msec at 22°c and for the study of light-activated drugs. increases with te~perature; the Q is about 3. We are 10 now investigating the initial part of the relaxation, the 109

162. PHOTOCHEMICALLY LABILE PRECURSORS toxin binding studies indicate that the effect is being OF CALCIUM, PROTONS, AND CYCLIC duplicated in our cultures. We are examining the tissue NUCLEOTIDES and species specificity of these effects. Freeze-fracture Investigator: Jeanne M. Nerbonne studies are planned to look for anatomical correlates of We are continuing work on the design, synthesis, and this enhanced receptor production. biological evaluation of photolabile chelates of small A collaboration has been established with a group from molecules and ions. In the area of photoactivatable the State University of New York at Stony Brook who are calcium chelates, 4,4'-bis-{iminodiacetic acid) azotoluene developing a soft X-ray scanning microscop.e. This (1) was prepared and tested. We find that, although this device, which capitalizes on recent developments in X-ray 2 ~olecule binds ca +, the affinity constant (Ka " 1 mM) source technology and X-ray optics, has great promise for for binding is too low to be useful. The low affinity the study of cultured cells. It seems likely that wet, 2 constant of ! for Ca + implies that only two (rather than unstained, whole cells can be observed with good contrast the desired four) acetic acid moieties participate in at a resolution of roughly 100 Angstroms. There is also a binding. This result provided the structural rationale for possibility that the X-ray exposure will not kill the cells. the design of other drugs in which four acid groups should Nerve and muscle cells are now being cultured on thin . ff" . t c 2+ participate in binding, thus rendering e 1c1en a (1000 !!.) substrates, and an attempt will be made to make chelation properties (K ; 0.01-1.0 µM) on the molecule. a crude micrographs of these cells during microscope tests The synthesis of 4,4'-bis-[2-(iminodiacetic acid)ethoxy] in June 1980. azobenzene has recently been completed and its calcium A second-generation version of the "electronic petri binding properties are under investigation. dish" (Pine, 1980) is being produced. This dish will have 61 . . . t 11 la c 2+ In many systems, alterations 1n m race u r a extracellular microelectrodes in a close-packed array concentration alter pH (and vice versa) and, in addition, covering an area about 500 microns in diameter. With changing intracellular pH causes diverse physiological these electrodes, it is anticipated that it will be possible effects. Therefore, it has been of interest to us to to stimulate any individual cell of a microculture growing develop molecules which would liberate H+ rapidly and on the area covered by the array and to record impulses efficiently upon irradiation. The synthesis of one series of from any cell. Developmental studies are planned on compounds with the desired chemical properties has been microcultures of a small number of neurons. The effects accomplished. For example, irradiation of nitrobenzyl of chronic stimulation, synaptic competition, and time­ acetate in unbuffered solution results in the rapid correlated stimulus patterns will be investigated. liberation of H + (quantum efficiency equal to 0.25) and a References: subsequent drop in pH. The biological applicability of Jessel, T. M., Siegel, R. E., Fishbach, G. D. (1979) Proc. these molecules is presently under investigation. Nat. Acad. Sci. USA 76: 5397-5401. Pine, J. (1980) J. Neurosci. Meth. 2: 19-31.

PUBLICATIONS 163. STUDIES OF NERVE AND MUSCLE IN CELL CULTURES Armstrong, D. and Lester, H_. A. (1~79) !he ki_ne~ics of Investigators: Jerome Pine, Jeanne M. Nerbonne curare action and restricted d1ffus1on w1th1n the synaptic cleft. J. Physiol. 294: 365-386. Much of the effort during the past year has gone into Krouse, M. E., Lester, H. A., Nass, M. M., Nerbonne, Je M., Wassermann, N. H. and Erlanger, B. F. (1979) _AC_h equipping a lab for growing cells and studying their receptors begin to open within 10 µsec after agon1st is electrophysiology. Rat sympathetic neurons and chick applied. Soc. Neurosci. Abstracts 5: 483. Krouse, M. E~, Lester, H. and Weinstock, M. (1979) How skeletal muscle are now being cultured, and three projects photoisomerizable azobenzene compounds. affect have begun. acetylcholine receptors of skate muscle. Biol. Bull. 157: 376. The effect of brain extracts on the synthesis and Krouse, M. E., Nass, M. M., Nerbonne, J. M., Lester, H. incorporation of acetylcholine receptors on developing A., Wassermann, N. H. and Erlanger, B. F. (1980) Agonist-receptor interaction is only a small component chick skeletal muscle is being investigated. Jessel, Siegel in the synaptic delay of nicotinic transmission. In: and Fishbach (1979) reported a dramatic enhancement of Receptors for Neurotransmitters, Hormones and Pheromones in Insects, D. B. Sattelle, L. M. Hall and J. receptor production during early development if the G. Hildebrand (Eds.), pp. 17-26. Elsevier/North muscles are treated with chick brain extract. Bungaro- Holland. 110

Lester, H. A., Krouse, M. E., Nass, M. M., Wassermann, N. Lester, H. A., Nass, M. M., Krouse, M. G., Nerbonne, J. H. and Erlanger, B. F. (1979) Light-activated drug M., Wassermann, N. H. and Erlanger, B. F. (1980) confirms a mechanism of ion channel blockade. Electrophysiological studies with photoisomerizable Nature 280: 509-510. cholinergic compounds. Review and progress report. Lester, H. A., Krouse, M. E., Nass, M. M., Wassermann, N. Ann. N.Y. Acad. Sci. 346: 475-490. H. and Erlanger, B. F. (1980) A covalently bound Pine, J. (1980) Recording action potentials from cultured photoisomerizable agonist. Comparison with revers­ neurons with extracellular microcircuit electrodes. J. ibly bound agonists at Electrophorus electroplaques. J. Neurosci. Meth. 2: 19-31. Gen. Physiol. 75: 207-232. Lester, H. A., Krouse, M. E., Nass, M. M., Wassermann, N. H. and Erlanger, B. F. (1979) A covalently bound photoisomerizable agonist at Electrophorus electro­ plaques: equilibria, kinetics, and stoichiometry. Soc. Neurosci. Abstracts 5: 484.

(such as TRF) which were thought to be uniquely localized Professor: Felix Strumwasser Del E. Webb Visiting Associate: Sudarshan Malhotra to the hypothalamus also occur in other regions of brain Senior Research Fellows: Eri Heller, Leonard K. Kaczmarek where they may operate as synaptic transmitters or Graduate SID.dents: Arlene Y. Chiu, Kent R. Jennings, Joanne M. Yeakley modulators. If ELH turns out to have such a dual function Research Staff: Mary M. Nousek, Floyd Schlechte, John (hormone and transmitter), then this feature would repre­ M. Scotese, Delilah A. Stephens, Daniel P. Viele, Annette S. Yuen sent an old pattern in evolution likely to be found in

Support: The work described in the following research invertebrates other than molluscs. reports has been supported by: Arlene Chiu and Jim Host (undergraduate student) Lawrence A. Hanson Foundation have developed an ELISA (enzyme-linked immunosorbent National Institutes of Health, USPHS Pew Memorial Trust assay) method for quantitating ELH release during bag Gordon Ross Medical Foundation Evelyn Sharp Fellowship cell afterdischarge. Their preliminary results indicate Del E. Webb Foundation that about 2.5 µg of ELH is released during one afterdis­ charge from an intact abdominal ganglion. Since there is Summary: Research continued on the mechanisms of two good evidence that all bag cells fire synchronously during slow central nervous system electrical processes which afterdischarge, we estimate the release of between 2.5 to have behavioral consequences: afterdischarge in the 3.0 ng (or about 0. 7 pico moles) of ELH per single bag cell. peptidergic bag cell neurons, and the circadian rhythm of Afterdischarge is a long-lasting discharge of nerve impulses in the eye of Aplysia. Afterdischarge is a cells after a brief excitatory synaptic stimulus. _The bag response mode of the bag cells that ensures maximal cells, which are normally silent, afterdischarge for about release of the egg-laying hormone (ELH) which in turn 30 minutes after a few seconds of synaptic input. Pre­ initiates egg-release from the ovotestis and the behavioral vious studies from our laboratory have shown that this programs associated with egg-laying. The eyes of Aplysia afterdischarge is correlated with a rise in cAMP in the contain a neuronal circadian oscillator system that is bag cells and that certain cAMP analogs, such as 8- dominant in controlling circadian locomotion. benzylthio cAMP can initiate an "afterdischarge" in the Arlene Chiu has raised antibodies to pure ELH (coupled absence of synaptic stimulation. Kent Jennings and to thyroglobulin) in rabbits and used these for specific Leonard Kaczmarek have determined that at least one immunocytochemical staining (Sternberger's peroxidase­ protein (with apparent molecular weight of 33,000 daltons) antiperoxidase method) of the bag cells and their undergoes enhanced phosphorylation (approximately 80% processes. This is the first use of immunocytochemistry increase) at two minutes into afterdischarge. for a natural neuronal peptide in an invertebrate. It was Kaczmarek has tested the protein phosphorylation surprising to find ELH immunoreactivity in the pleural and model of intracellular events stimulated by cAMP by cerebral ganglia rather than exclusive restriction to the injecting highly purified beef heart protein kinase bag cell system. However, in mammals it is now well catalytic (PKC) subunit (obtained from Professor Paul accepted that certain hypothalamic releasing factors Greengard's laboratory, Yale University) into single bag 111

cells in primary cell culture. Such intracellular enzyme 164. LOCALIZATION OF ELH-LIKE injections increase membrane excitability as evidenced by JMMUNOREACTIVJTY IN THE APLYSIA NERVOUS SYSTEM enhanced rate of rise and amplitude of action potentials Investigators: Arlene Y. Chiu, Felix Strumwasser which in bag cells appear to be primarily ca2+-mediated since they are insensitive to tetrodotoxin but are sensitive The neuropeptide, egg-laying hormone (ELH) of 2+ . t o C o • Since PKC catalyzes protein phosphorylation in Aplysia californica is known to be synthesized by the crude bag cell membrane preparations, it is likely that the neurosecretory bag cell organ of the abdominal ganglion positive effects on membrane excitability by intra­ and released via an array of processes in the vascularized cellularly-injected PKC actually are due to increased connective tissue sheath. We have mapp~d the extent of protein phosphorylation. The specific phosphorylated the neurohemal surface of this system and the distribution proteins that account for the increase in membrane of the neuropeptide by conventio~al immunohistochemical excitability, after PKC injection, remain to be deter­ methods. mined. Rabbit antibodies which specifically recognized ELH Our circadian studies, in the last year, have concen­ were generated using pure ELH, bonded to a carrier trated on investigating the effects of lithium (Li) on the molecule (Tg), as an immunogen. These antibodies were period of the neuronal circadian oscillator in the eye of employed to localize sites containing ELH or ELH-like Aplysia. Lithium is the most effective drug in the immunoreactivity in the cerebral, buccal, pleural, pedal treatment of manic-depressive illness in humans. It has and abdominal ganglia of A. californica. Cryostat been suggested by Franz Halberg (University of sections of fixed and frozen ganglia were stained by the Minnesota) and Goodwin and Weir (NIMH) that manic­ peroxidase-antiperoxidase method of Sternberger (1979) depressive illness may be a malfunction of the human for light microscopic viewing. circadian system. Some evidence exists that lithium slows Three main areas of staining were noted: (1) The the free-running locomotor rhythm in rats (D. Kripke, extensive bag cell system of the abdominal ganglion UCSD) but there has never been a direct test of Li on a consisting of two homogeneous clusters of ELH-bearing neuronal circadian oscillator. Dan Viele and I find that Li neurons sending out a network of neurosecretory processes lengthens the period of the circadian oscillator in the into the connective tissue sheath over most of the Aplysia eye in a dose-dependent fashion. The effect is ganglionic surface. Bag cells also send "cuffs11 of profound with a lengthening of period from a control value processes, which wrap around n~rve bundles, and a of 23.8 to 32.6 hr at 37 .5 mM Li. This is clear evidence sea ttering of stained fibers within the neuropil of the that Li has marked effects on a neuronal circadian ganglion. (2) 1-4 stained neurons, in each of the pleural oscillator system and it may well turn out that its ganglia, whose· somal size and processes resemble those of therapeutic effects in human manic-depressive disorders the bag cells. (3) Strongly immunoreactive fiber tracts could be primarily related to this action. within the neuropil of the cerebral ganglion apparently We have also started work on mammalian brain slices originating from two bilaterally symmetric clusters of which we hope will eventually lead to in vitro studies of small neurons which are very unlike bag cells in the hypothalamic suprachiasmatic nuclei, the location of a morphology. No processes associated with the connective mammalian neuronal circadian oscillator. Professor Van tissue sheath are seen in the cerebral ganglion. Harreveld and I have used the excitatory monosynaptic While the role of the bag cells in inducing egg laying pathway in the hippocampal slice between Schaffer has been well established both in vivo and in vitro, the collaterals and pyramidal cell dendrites to explore the participation of the two smaller systems with ELH-like action of L-proline on glutamate receptors. We demon-· irnmunoreactivity in the egg-laying program remains to be strate that L-proline is an effective reversible blocker of investigated. the excitatory population postsynaptic potential and that Reference: during such block, 4-aminopyridine can relieve this block Stern?erger, L. A. (1979) Immunocytochemistry. John Wiley and Sons, New York. by lengthening the duration of the presynaptic spike which presumably enhances the natural transmitter (glutamate) release. 112

165. MICROINJECTION OF PROTEIN KlNASE lNTO injection. Control experiments were carried out with CULTURED BAG CELL NEURONS either the carrier media alone (0.3 M K phosphate, pH 6.8 Investigators: Leonard K .. Kaczmarek, or KC! with 10 mM 8-mercaptoethanol) or heat­ Felix Strumwasser inactivated enzyme preparations. Our past work on the neuropeptidergic bag cell neurons Our experiments have shown therefore that the in the abdominal ganglion of Aplysia has provided a injection of the catalytic subunit of cAMP-dependent number of lines of evidence that the excitability of these protein kinase into bag cell neurons increases their cells is controlled by the intracellular concentration of excitability. In addition we have shown, by immunocyto­ adenosine 3',51 cyclic monophosphate (cAMP). One piece chemical techniques, that cultured bag cells contain of evidence is that the extracellular application of a material which cross-reacts with the regulatory (cAMP­ membrane-permeant cAMP analogue, 8-benzylthio-cAMP, binding) subunit of bovine heart protein kinase. An to isolated bag cell neurons in primary culture induced identity of action of injected catalytic subunit with that changes in their electrical properties, including the onset of exogenously applied cAMP analogues, however, remains of spontaneous discharge and subthreshold oscillations in to be determined. membrane potential, enhanced spike broadening and increased input resistance. The intracellular messenger 166. PROTIDN PHOSPHORYLATION 1N cAMP is widely thought to exert its ceUular effects by BAG CELL NEURONS causing the phosphorylation of cellular proteins through Investigators: Kent R. Jennings, Leonard K. Kaczmarek, the action of the enzyme cAMP-dependent protein kinase. Felix Strumwasser We have tested this hypothesis directly by microinjection Our previous work on the mechanism of bag cell of the catalytic subunit (C) of this enzyme into cultured excitability has demonstrated that the cyclic nucleotide, bag cell neurons. This work was carried out in collabora­ cyclic AMP, may be involved_ in the genesis of after­ tion with Drs. A. C. Nairn, U. Walter, F. D. Wilson and P. discharge. Afterdischarge is the term applied to the long­ Greengard (Yale University) who purified the catalytic lasting (30 min) postsynaptic neural activity that occurs in subunit from bovine heart. these cells subsequent to a short triggering electrical Intracellular application was through pressure (5-40 stimulus applied to a synaptic pathway in the pleuro­ psi) applied to intracellular microelectrodes (tips 0.5-1.0 abdominal connective nerve. µm) filled at the tip with C (0.2-1.0 mg/ml). Electrical The currently accepted model for the mode of action recordings were carried out either with the same elec­ of cyclic AMP postulates that the nucleotide stimulates a trode or through a second electrode in the same cell. The protein kinase enzyme to phosphorylate cellular proteins. most prominent effect of successful injection was en­ We therefore have been examining cAMP-stimulated hanced spike genesis. Both the slope of the rising phase protein phosphorylation events occurring in bag cell tissue and the height of the action potential in response to a homogenates and crude membrane preparations and after­ constant depolarizing current were markedly enhanced discharge-dependent phosphorylation in the intact bag relative to the preinjection control (11 of 16 experiment.s). cells. 6 4 In two experiments, tetrodotoxin (5 x 10-5 M) was added Cyclic AMP (10- M-10- M) has been found to to the medium and found to have no effect on these stimulate the phosphorylation of a number of bag cell­ enhanced action potentials. They could however be associated proteins in a dose-dependent manner in crude abolished by 12 mM CoCl2' indicating that calcium ions membrane preparations. are probably the major component of the enhanced spikes. At least one of these phosphoproteins appears to This effect of C was usually accompanied by an increase undergo phosphorylation changes during afterdischarge of 32 in input resistance and, in several cases, enhanced spike the bag cells. In abdominal ganglia prelabeled with P as broadening with repetitive depolarizing current pulses. In sodium orthophosphate, there is a significant (80%) three of the eleven positive experiments, subthreshold enhancement of phosphorylation of a bag cell-associated oscillations in membrane potential resulted from the protein at a time point of 2 minutes into the after­ injections, and in one case the cell generated a repetitive discharge. Estimates from SDS-acrylamide gels indicate discharge that continued for several minutes following this protein has a molecular weight (m.w.) of 33,000 113 daltons rather than 22,000 daltons as reported last year the atrial peptides do not, or there might be an inhibiting (Biology 1979, No. 160), factor blocking the action of these peptides in crude Crude membrane preparations labeled with AT32P extracts. have been shown to contain a bag-cell specific phospho­ References: protein of m.w. 22,000 daltons. Although this protein Arch, S., Smock, T., Gurvis, R. and McCarthy, C. (1978) J. undergoes cAMP-dependent phosphorylation in vitro, we Comp. Physiol. 128: 67-70. Heller, E., Kaczmarek, L., Hunkapiller, M. W., Hood, L. E. have been unable to show a significant change in the and Strumwasser, F. (1980) Proc. Nat. Acad. Sci. USA phosphorylation state of this protein during after­ 77: 2328-2332. Toevs, L. (1979) Ph.D. Thesis, California Institute of discharge. Technology, Pasadena, California. Further studies are under way to elucidate the significance of the afterdischarge-dependent phosphoryl­ 168. L-PROLINE BLOCKS HIPPOCAMPAL ation events. Two atrial peptides (purified from the atrial EXCITATORY SYNAPTIC ACTIVITY gland, part of the reproductive tract of Ap)ysia) have been Investigators: A. Van Harreveld, Felix Strumwasser shown to initiate an afterdischarge in the intact bag cell Field potentials, elicited by stimulation of the preparation (Heller et al., 1980). Experiments are in Schaffer collaterals, were led off from the stratum progress (in collaboration with J. Yeakley, L. Brown and J. radiatum of CAl in rat hippocampal slices suspended on a Ibers) to investigate whether there exists an atrial nylon net in a chamber perfused with a physiological salt peptide-sensitive adenylate cyclase specific to bag cell solution. The slices were completely submerged. L­ tissue. A bag cell-specific cyclase would provide evidence proline in suitable concentrations was applied in the salt for a direct action of these atrial peptides on the bag solution. The responses to the stimulus consisted of a cells. negative synaptic potential and one or more population spikes preceded by a small spike ascribed to conduction in Reference: Heller, E., Kaczmarek, L. K., Hunkapiller, M. W., Hood, the Schaffer collaterals. In a concentration of 2 mM, L­ L. E. and Strumwasser, F. (1980) Proc. Nat. Acad. Sci. proline had no marked effect, but 3 mM enhanced the USA 77: 2328-2332. response, the population spikes becoming larger and more 167. ATRIAL GLAND EXTRACTS FROM numerous. At 5 mM or more the synaptic responses were APLYSlA BRASILlANA reversibly abolished but the presynaptic spike was not Investigators: Joanne M. Yeakley, Felix Strumwasser affected. This effect was counteracted by simultaneously Extracts of the atrial gland of Aplysia californica have applied 4-amino pyridine (0,5 mM), which prolonged the been shown to cause egg laying (Arch et al., 1978) and bag presynaptic spike and was postulated to increase the cell afterdischarge (Heller et al., 1980). Two active release of the transmitter, believed to be L-glutamate and peptides from atrial extracts have been purified and L-aspartate (see e.g., Nadler et al., 1976). This can be sequenced (Heller et al., 1980). As a part of preliminary expected to favor the binding of the transmitter to the experiments investigating the action of these peptides and postsyneptic membrane in its competition with L-proline as a source of material, atrial glands were collected from for glutamate receptors. These observations support the Aplysia brasiliana in the Gulf of Mexico (courtesy of Dr. postulate that L-proline is a glutamate antagonist, as has David McAdoo). Unfortunately, extracts from A. brasiliana been suggested, for instance, by its blocking effect on the atrial glands have not shown activity in A. californica. An responses of hippocampal neurons to applied glutamate extract of up to five A. brasiliana atrial glands does not (Segal, 1976), and on the crustacean neuromuscular junc­ induce egg laying (n ; 3) or bag cell afterdischarge (n ; 6) tion, which is believed to be glutamate-mediated (Van in A. califomica, although the extract contains a small Harreveld, 1980), molecular weight fraction on a gel exclusion column It has been suggested that L-proline binding with similar to that containing the active peptides in extrasynaptic glutamate receptors causes a moderate A. californica. Continuing experiments will be performed increase in Na+ permeability of the dendritic membrane on imported Aplysia brasiliana as a comparative study. and a release of K+ (Van Harreveld, 1979). The While it is known that ELH cross-reacts between enhancement of the synaptic response to Schaffer A. brasiliana and A. californica (Toevs, 1969), apparently collateral stimulation in 3 mM proline may be due to this 114

K+ release, which was supported by the observation of a Kaczmarek, L. K., Jennings, K. R. and Strumwasser, F. similar enhancement by an increase in the K+ concen­ (1979) Cyclic AMP analog generates afterdischarge in Aplysia bag cell neurons. Soc. Neurosci. Abstracts 5: tration in the perfusion fluid. However, for the time 249. being, the possibility that the enhancement is due to an Kaczmarek, L. K. and Strumwasser, F. (1980) Subthreshold oscillations underlie the cAMP-induced discharge of initial agonistic effect of L-proline (Shank and Freeman, Aplysia peptidergic neurons. Soc. Neurosci. Abstracts 1976) cannot be excluded. D-proline even in 10 mM 6. In press. Kandel, E. R., Krasne, F. B., Strumwasser, F. and Truman, concentration had no effect on the responses to collateral J. W. (1979) Cellular mechanisms in the selection and stimulation. modulation of behavior. Neurosci. Res. Program Bull. 17: 523-710. References: Strumwasser, F. (1979) Neuropeptides controlling behavior Nadler, J. V., Vaca, K. W., White, W. F., Lynch, G. and in Aplysia. J. Gen. Physio!. 74: 3a-4a. Cotman, C. W. (1976) Nature 260: 538-540. Strumwasser, F., Alvarez, R. B., Viele, D. P. and Woolum, Segal, M. (1976) Br. J. Pharmacol. 58: 341-345. J. C. (1980) Structure and function of a neuronal Shank, R. P. and Freeman, A. R. (1976) J. Neurobiol. 7: circadian oscillator system. In: Biological Rhythms 23-26. and their Central Mechanism, M. Suda, O. Hayashi and. Van Harreveld, A. (1979) J. Neurobio!. 10: 355-365. H. Nakagawa (Eds.), Naito International Symposium, Van Harreveld, A. (1980) J. Neurobiol. In press. Tokyo, Japan, August 30-September 2, 1978, pp. 41-56. Elsev1er/North-Holland Biomedical Press. Strumwasser, F., Kaczmarek, L., Chiu, A., Heller, E., PUBLICATIONS Jennings, K. and Viele, D. (1980) Peptides controlling behavior in Aplysia. In: Peptides: Integrators of Cell Chiu, A. Y., Hunkapiller, M. w., Heller, E., Stuart, D. K., and Tissue Functions, F. Bloom (Ed.), pp. 197-218. Hood, L. E. and Strumwasser, F. (1979) Purification Raven Press. and primary structure of the neuropeptide egg-laying Strumwasser, F. and Viele, D. P. (1980) Lithium increases hormone of Aplysia califomica. Proc. Nat. Acad. Sci. the period of a neuronal circadian oscillator. Soc. USA 76: 6656-6660. Neurosci. Abstracts 6. In press. Chiu, A. Y., Hunkapiller, M. and Strumwasser, F. (1979) Strumwasser, F., Viele, D. P. and Scotese, J. M. (1979) The__ neu~opepti~e, e~g-laying hormone of Aplysia: Dissection of a neuronal circadian oscillator system in Pur1f1cat1on, amino acid sequence and antibodies. Soc. t~e eye of Aplysia: Intracellular recording from single Neurosci. Abstracts 5: 243. disconnected photoreceptors in cell culture. Soc. Heller, E., Kaczmarek, L. K., Hunkapiller, M. W., Hood, L. Neurosci. Abstracts 5: 809. E. and Strumwasser, F. (1980) Purification and Stuart, D. K., Chiu, A. and Strumwasser, F. (1979) primary structure of two neuroactive peptides that ~eurosecretion of egg-laying hormone and other pep­ cause bag cell afterdischarge and egg-laying in tides from electrically active bag cell neurons of Aplysia. Proc. Nat. Acad. Sci. USA 77: 2328-2332. Aplysia. J. Neurophysiol. 43: 488-498. Heller, E., Kaczmarek, L. K., Hunkapiller, M. and Stuart, D. K. and Strumwasser, F. (1979) Neuronal sites of Strumwasser, F. (1979) Afterdischarge in bag cell action of a neurosecretory peptide, egg-laying hor­ neurons is initiated by peptides from the atrial gland mone, in Aplysia californica. J. Neurophysiol. 43: of Aplysia. Soc. Neurosci. Abstracts 5: 248. 499-519. Jennings, K. R., Kaczmarek, L. K. and Strumwasser, p. Van Harrev~ld, A. and Strumwasser, F. (1980) L-proline (1979) Protein phosphorylation during afterdischarge of blocks h1ppocampal excitatory synaptic activity. Soc. the neuroendocrine bag cells in Aplysia. Soc. Neurosci. Abstracts 6. In press. Neurosci. Abstracts 5: 249. Woolum, J. C. and Strumwasser, F. (1980) The differential Kaczmarek, L. K. (1979) Flux of labeled compounds in effects of ionizing radiation on the circadian oscillator biochemical oscillations. Am. J. Physiol. 237: R350- and other functions in the eye of Aplysia. Proc. Nat. R354. Acad. Sci. USA 77. In press. Kaczmarek, L. K., Finbow, M., Revel, J.-P. and Woolum, J. C. and Strumwasser, F. (1979) Dissection of a Strum_wasser, F. (1979)_ The morphology and coupling of neuro?al _circadian oscillator system in the eye of Aplys1a bag cells withm the abdominal ganglion and in Aplysia with X-rays. Soc. Neurosci. Abstracts 5: 814. cell culture. J. Neurobiol. 10: 535-550. NEUROBIOLOGY AND BEHAVIORAL BIOLOGY

John M. Allman

Derek H. Fender

Masakazu Konishi

Marianne E. Olds

John D. Pettigrew R. w. Sperry David C. Van Essen

117

Associate Professor: John M. Allman In addition to this work, we are studying the responses Visiting F. Baker, EveLynn McGuinness, Associates: James of neurons in the visual cortical areas while the monkey is Joel Myerson Graduate Students: William T. Newsome III, Steven E. experiencing ?erceptual rivalry. We are also studying the Petersen, David W. Sivertsen control of the muscles of facial expression in primates. Research Staff: Francis M. Miezin Laboratory Staff: Carl Nicholson, Margaret S. Norton

Support: The work described in the following research reports has been supported by: The Helen G. and Arthur McCallum Fund National Institutes of Health, USPHS National Science Foundation Pew Memorial Trust Pitzer College University of California, Los Angeles University of California, Riverside .....···r·· ..... l +: + ...... summary: Most of the mammalian cerebral neocortex is : i ! ••H••-•i""""""""! comprised of a series of multiple, discrete, topographic \.. -1-.1 ...... ,;...... representations of the sensory domains of vision, somates­ thesia and audition. Since each representation contains a discrete map, each area is likely to be a functional entity and a fundamental unit in the organization of neocortex. One of the clearest delineations of the cortical represen­ tations has been achieved in the owl monkey (Figure 1). The owl monkey was selected as a research subject because it is a higher primate that possesses relatively few fissures in its neocortex, a feature that greatly facilitates functional studies. Recent studies indicate Figure 1. The representation of the sensory domains in that many of the areas fowtd in the owl monkey are also the cerebral cortex of the owl monkey. Above is a view is present in other primate species. Our goal has been to ventromedial of the left hemisphere; below a dorsolateral view. The perimeter chart on the left shows determine, through studies of the response properties of the visual field. The symbols in this chart are super­ imposed neurons, what functional differences exist among the on the surface of the visual cortex. Pluses indicate upper quadrant representations; minuses, lower cortical visual areas and ultimately to understand the quadrants; dashed lines, borders of areas that correspond contribl).tion of each area to perception and/or visuomotor to the representation of the relatively peripheral parts of the visual field, but not to the extreme periphery. The coordination. Figure 2 illustrates our findings for four row of Vs indicates the approximate anterior border of visual areas: DL (Dorsolateral), MT (Midclle Temporal), visually responsive cortex. The dotted line broken by a question mark indicates an uncertain border. Each DM (Dorsomedial), and M (Medial). The relative size of auditory area contains a representation of the frequency domain. area represen­ the squares beneath each area indicates the relative Each somatosensory contains a tation of the body. AI, First Auditory Area; AL, strength of the selectivity of neurons in that area for each Anterolateral Auditory Area; CC, Corpus Callosum; DI, Crescent stimulus parameter tested. Neurons in l\i1T, DM and M are Dorsointermediate Visual Area; DL, Dorsolateral Visual Area; DM, Dorsomedial Visual Area; IT, Infero­ strongly orientation selective in that they respond temporal Corter; M, Medial Visual Area; MT, Middle Temporal Visual Area; ON, Optic Nerve; OT, Optic optimally to some bar orientations and poorly to others. Tectum; PL, Posterolateral Auditory Area; PP, Posterior Neurons in DL are less selective for bar orientation but Parietal Corter; R, Rostral Auditory Area; VA, Ventral are strongly selective for particular spatial dimensions Anterior Visual Area; VP, Ventral Posterior Visual Area. The cortical visual areas were mapped by Allman and (length and width) of stimuli; neurons in the other areas Kaas (Science 191: 572-575, 1976); the somatosensory areas by et (J. Comp. are poor at discriminating spatial dimensions. Neurons in were mapped Merzenich al. Neural. 181: 41-74, 1978); the auditory areas were mapped by Imig MT are very selective for direction of movement and et al. (J. Comp. Neurol. 171: 111-128, 1977). respond well to visuhl texture (random dot arrays); neurons in the other areas are poorer at discriminating direction of movement and respond much less well to moving random dot arrays. 118

STIM. DL MT OM M ORIENTATION TUNING [i] • DIMENSIONAL SELECTIVITY 00 • • • DIRECTIONALITY INDEX ~ • • • BEST RANDOM DOT RESPONSE .::-;:•...... BEST MOVING BAR RESPONSE ·.:i:.~· • • • % OF AREA DEVOTED TO CENTRAL 10° OF VISUAL FIELD D t> D ~ D 73% 10% 22% 4% Figure 2. Functional specificity· of four visual areas in the owl monkey.

169. NEURONS SELECTIVE FOR STIMULUS neurons had a wide range of preferred sizes, from 1° to DIMENSION IN THE DORSOLATERAL AREA ::S0° in length, and from ! 0 to 7° in width, and these OF THE OWL MONKEY preferences appeared independent of each other when Investigators: Steven E. Petersen, James F. Baker, John M. Allman both dimensions were tested on the same cell. The dimensional selectivity of DL neurons suggests that DL We have found that the Dorsolateral Crescent (DL) has contributes to form perception. This hypothesis is a high proportion of neurons that are selective for the consistent with the observation that DL has the most spatial dimensions of visual stimuli within excitatory expanded representation of the central visual field receptive fields that are generally much larger than the (Allman and Kaas, 1974) where the most acute recognition preferred stimulus dimensions (see Figure 3). An index of of form takes place, and the recent discovery that DL is stimulus dimension selectivity was calculated using the the main source of input to the inferotemporal cortex formula: (Weller and Kaas, 1980). Inferotemporal cortex has been _ response to the largest stimulus tested 1 strongly implicated in the analysis of complex visual response to the optimal stimulus tested stimuli and the leaming of visual form discriminations For length and spot diameter, DL cells were significantly (Gross, 1973). more selective than cells in DM, M, and MT, and were References: significantly more selective to width than cells in MT Allman, J. M. and Kaas, J. H. (1974) Brain Res. 81: (Figure 4). The dimensional selectivity of DL cells was 199-213. Gross, C. G. (1973) Handbook of Sensory Physiology, Vol. independent of the sign of contrast in the receptive field, VIl/3B: 451-482. being equally selective to both light-on-dark and dark-on­ Weller, R. E. and Kaas, J. H. (1980) Soc. Neurosci. Abstracts. In press. light stimuli (Figure 3), the amount of contrast (similar response over a 1.5 log unit change in intensity), and the position of the stimulus within the receptive field. The optimal length and width were typically considerably smaller than the excitatory receptive field (Figure 3). DL 119

5 40 l ,...... 4 DL 0 \ 30 z f OM N2DM280 § 3 i• ~ i u~ ~ 2 20 II:! i 0 w~ ,.,. .-.A DL HEDL53A " I 2 ~"' .>-..·'· 10 0 ,~

100 20° 30° 40° 50° .1 .3 .5 .7 .9 ~1.0 N.S. BEST LENGTH BAR LENGTH EXCITATORY RECEPTIVE FIELD LENGTH

5 40

4 HEDL67A 0 30 z L-ON-0 "'°"''" 3 8w ~ D-ON-L- § u ~ 2 20 ~ 0 ~w 0 1 z "Bi 10 0 ~ ..... lil'""'""'""'""'""'""i::'""'""'""'""'""'::i

50 100 150 20° .3 .5 .7 .9 ~1.0 N.S. BEST LENGTH SPOT DIAMETER EXCITATORY RECEPTIVE FIELD LENGTH

Figure 3. (AJ Responses of two units to different bar lengths. Each data point is the average of 5 stimuli presented in pseudorandom order. HEDL53A (solid line) was recorded from DL and shows marked selectivity. N2DM28D (dashed line) illustrates the typical response profile for cells outside of DL, in which the cell summates up to a certain value, whereupon the response levels off. The length of the excitatory receptive field for both cells was 20°. (BJ Responses of a DL neuron to light (open circles) and dark (closed circles) spots of different diameters. Responses to stimuli of the same size were virtually the same regardless of contrast. (C and D) Optimal bar length is expressed as a percentage of the comparable dimension of the excitatory receptive fields. Cells with a length selectivity index of less than 0.5 were considered to be nonselective and are represented by the bins at the right. The average length of the excitatory receptive fields in DL was 20.3° with a standard deviation of 9.5°, while the combined average for the other areas was 15° with a standard deviation of 7.4°.

tations each presented 10 times in pseudorandom order. 170. ORIENTATION SELECTIVITY AND The dotted line indicates the level of spontaneous activity RESPONSIVENESS TO MOVING RANDOM DOT ARRAYS IN MT, DL, DM AND M for each neuron. Neuron A was broadly tuned; B through Investigators: Steven E. Petersen, James F. Baker, K all strongly preferred horizontally oriented bars; L was John M. Allman strongly inhibited by horizontal bars and thus was the Neurons in MT, DL, DM and M were tested for negative complement of the other cells in the penetration. orientation selectivity with flashed stationary bars pre­ These data, together with other data we have obtained, sented at different orientations in pseudorandom order. suggest the presence of vertical columns of orientation The great majority of neurons in MT, DM and M were selective neurons in MT such as have been described for sharply orientation selective; DL neurons were less selec­ the primary visual area (V-1) by Hubel and Wiesel (1977). tive for bar orientation. The responses of a series of The graphs on the left show the average response of each single neurons in _a penetration in MT, which histological neuron to moving bar stimuli crossing the receptive field reconstruction showed to be perpendicular to the cortical in 12 different directions. In each case the bar was surface and nearly parallel to the radial fibers, is oriented perpendicular to the direction of movement. illustrated in Figure 5. The graphs on the right show the Neurons A through F responded optimally to a horizontally average response of each neuron to 6 different orien- oriented bar approaching from 270° (straight down); 120

MT - neurons G through K responded optimally to a horizontally DL M oriented bar approaching from 90° (straight up). One or DM ~ more of these abrupt 180° shifts in preferred direction of MT- X=.31 A X=.s9 SD=.26 movement have been observed in virtually every long SD=.39 M X=.27 20 20 penetration we have made in MT. These data suggest that § SD=.31 with u OM X "'.28 groups of neurons diametrically opposed preferred so=.33 directions of movement lie juxtaposed within a larger ~ 10 10 ~ system sharing the same orientation preference. , z0 In spite of the clear-cut orientation selectivity of most 0 0 0 .25 .s .75>.75 0 .25 .s .75>.75 MT neurons, the majority actually respond better to a RESPONSE TO MAXIMUM LENGTH moving array of random dots, which lacks orientation 1 RESPONSE TO OPTIMUM LENGTH altogether, than to an optimally oriented bar, moving at MT X=.33 8 X=.70 SD=.31 the preferred direction at the best velocity. In this SD=.45 20 20 M x =.43 respect, MT neurons differ markedly from neurons in DL so=.33 and DM which respond poorly to moving random dot OM X =.40 so:.20 arrays. Hammond and McKay (1977) have found that 10 10 complex cells located in layer V in primary visual cortex

(V-1) in the cat respond well to moving random dot arrays. 0 .25 .5 .75 >.75 0 .25 .5 .75 ::>:-75 The giant Meynert cells of layer V project to MT in owl - RESPONSE TO MAXIMUM WIDTH 1 RESPONSE TO OPTIMUM WIDTH monkey (C. S. Lin, J. Wall and J. Kaas, in preparation). There is also evidence that MT projects to the pontine MT X=.21 X=.63 so=.25 visual nuclei (Glickstein et al., 1980), which are also very c so=.27 M X=.31 20 20 responsive to moving random dot arrays (Baker et al., so=.27 OM X=.21 1976), and in turn project to the cerebellum and probably so=.31 are involved in the visual guidance of body and eye 10 10 movement. The presence of orientation selectivity and strong 0 .25 .5 .75 >.75 0 .25 .5 .75 ::>:"75 responsiveness to moving random dot arrays indicates that - RESPONSE TO MA.XJMUM DIAMETER 1 RESPONSE TO OP'TIMUM DIAMETER MT neurons are capable of encoding two seemingly Figure 4. Distributions of stimulus dimension selectivity incompatible types of information and that they may indices for length (A), width (B), and spot diameter (C). participate in more than one mode of visual information The distributions of DL cells are on the left, and the distributions for MT.Mand DM cells are on the right. processing. Statistics comparing these distributions; df for length = 3,94; width = 3,66; spot diameter = 3,81. S-values for References: length, width and spot diameter selectivity, respectively: Baker, J. F., Gibson, A., Glickstein, M. and Stein, J. (1976) DL vs. MT = 3.42*, 3.91*, 8.66*; DL vs. M = 3.14*, 1.53, J. Physiol. 255: 415-433. 3.44*; DL vs. DM = 2.76*, 1.55, 4.94*; MT vs. M = 0.05, Glickstein, M., Cohen, J., Dickson, B., Gibson, A., Hollins, 0.21, 0.29; MT vs. DM = 0.03, 0.07, 0.01; M vs. DM =0.01, M., LaBossiere, E. and Robinson, F. (1980) J. Comp. 0.02, 0.21. (*, p < 0.05.) Neurol. In press. Hammond, P. and McKay, D. M. (1977) Exptl. Brain Res. 30: 275-296. Hubel, D. H. and Wiesel, T. N. (1977) Proc. Roy. Soc. Lond. B 198: 1-59. 121

,,,,. 171. INNERVATION AND CONTROL ~,-.r.. OF THE FACIAL MUSCLES T- Investigator: EveLynn McGuinness ·­'" Our earlier research on the neural control of facial :b~-L expression in primates concentrated on the represen­ . ~ ~1--:e--­ ,,, ·­ tations of the face in precentral motor cortex. We are -~ ~-- : ~ -~::::::·__ ------:1~-- currently studying the representation of muscles in the •00" 'IJ? ...... - ... ' " .. - .. , -,,, brainstem motor nucleus of the facial nerve. We also :J--"'-L'>__ _ hope to determine the pattern of projections from motor Jb-~--- _,. ' ' , " od' ,.,. - ...... - .. cortex and subcortical structures to the facial nucleus in primates. Preliminary research on the facial nucleus thus '~I-/~ __ far has been carried out in the rat although we hope to begin work on primates again in the near future. In higher ':j___/\__ primates such as the macaque, the facial muscles are ...., ' ' ... - .. } specialized for the expression and communication of :1--~--- emotion. The rat facial muscles are also specialized, but 1 ' "' - , I for ear and vibrissae movements that are used to help :j_../\... ___ orient the animal in space. The comparison with the rat ... ' ' .. - .. ; gives us an example of an animal which makes extensive use of its facial muscles but for quite a different purpose :1 __ _/\____ than do primates. The rat work also served the function ...., ' " ... - .. , of allowing us to perfect techniques and determine optimal procedures on a less valuable animal than a J~ monkey. Our current work falls into two sets of experiments. ~l-..J\--~--- One approach we have used is to determine the " 00' .... """ .... l~ topography of representation of the facial muscles in the '~t<\=-~-:::-- facial nucleus by injecting the muscles themselves with " 00° ,,,,. - - Jl~--­ horseradish peroxidase (HRP). Such injections, typically 2-5 µI of 20% HRP in water, produce dense labeling of ~I--\=;:;-- small groups of cells in the facial nucleus. The t ' ... - ... ' BAR (DIRECTION) FLASH (ORIENTATION) topography in the rat is more complex than was previously Ft.gw-e S. Direction and orientation selectivity for a thought. The buccolabial branch of the facial nerve series of neurons recorded in a single penetration nearly innervating the vibrissae has its origin in the lateral perpendicular to the surface of MT. A pair of graphs are illustrated for each unit (HEMT69 A through L). The subnucleus but the innervation of the lips comes from the depth beneath the surface at which each cell was recorded intermediate and ventromedial portions. is given beneath each identifying number. The graphs on the left illustrate the average response of each cell to 5 The second project is to map electrophysiologically the presentations of a 20° :x: 1° light bar at 12 different angles. facial nucleus by microstimulation through a pipette filled The graphs on the right illustrate the average response of the cells to 10 presentations of a flashed bar at the with HRP using negative currents. Observed responses on orientations shown. All of the cells except the last prefer the face are recorded during microstimulation. At a the horizontal orientation; the last was inhibited by horizontal bars. The direction of movement was 90° to selected location, positive current is passed through the the bar orientation, thus the preferred directions of 270" same pipette causing HRP to be iontophoretically (down) and 90° (up) are consistent with the preferred horizontal orientation. The receptive field centers were deposited at that site. By manipulation of current located an average of 16.6° below the horizontal meridian parameters, we can routinely produce an injection site with a standard deviation of 0.9°, and an average of 6.7° temporal to the vertical meridian with a standard 200-500 µ in diameter. In this way we are able to study deviation of 3.3°. the inputs to different subnuclei of the facial nucleus. 122

PUBLICATIONS Miezin, F. M., Myerson, J., Julesz, B. and Allman, J. M. (1979) Evoked potentials to dynamic random-dot Allman, J. (1980) Evolution of the cortical visual areas in correlograms in monkey and man: A test for primates. Am. J. Physical Anthropol. In press. cyclopean perception. Soc. Neurosci. Abstracts 5: 798. Allman, J. M., Baker, J. F., Newsome, W. T. and Petersen, Miezin, F. M., Myerson, J., Julesz, B. and Allman, J. M. S. E. (1980) The cortical visual areas of the owl (1980) Evoked potentials to dynamic random-dot monkey: Topographic organization and functional correlograms in monkey and man: A test for correlates. In: Multiple Cortical Somatic Sensory­ cyclopean perception. Vision Res. Accepted for Motor, Visual and Auditory Areas and Their publication. Connectivities, C. N. Woolsey (Ed.). Humana Press, Myerson, J. and Miezin, F. M. (1980) The kinetics of Clifton, New Jersey. In press. choice: An operant systems analysis. Psycho!. Rev. Allman, J., Campbell, C. B. G. and McGuinness, E. (1979) 87: 160-174. The dorsal third tier in Galago senegalensis. Brain Myerson, J., Miezin, F. M. and Allman, J. M. (1980) Res. 179: 355-361. Binocular rivalry in macaque monkeys and humans: A Baker, J. F., Petersen, _S. E., Newsome, W. T. and Allman, psychophysical comparison. In preparation. J. M. (1980) Visual response properties of neurons in Newsome, W. T. and Allman, J. M. (1980) The inter­ four extrastriate visual areas of the owl monkey hemispheric connections of visual cortex in the owl (Aotus trivirgatus): A quantitative comparison of the monkey, Aotus trivirgatus, and the bushbaby, Galago medial (M), dorsomedial (DM), dorsolateral (DL), and senegalensis. J. Comp. Neurol. Accepted for publi­ middle temporal (MT) areas. J. Neurophysiol. Sub­ cation. mitted for publication. Petersen, S., Baker, J. F. and Allman, J. M. (1980) McGuinness, E., _Sivertsen, D. and Allman, J. M. (1980) Dimensional selectivity of neurons in the dorsolateral Organization of the face representation in macaque visual area (DL) of the owl monkey. Brain Res. In motor cortex. J. Comp. Neural. Accepted for press. publication. Petersen, S. E., Baker, J. F., Rockland, K. S. and Allman, McGuinness, E., Sivertsen, D. W. and Allman, J. (1979) J. M. (1979) Visual response properties of single Two macro-representations of the facial muscles in neurons in the dorsolateral crescent (DL) in the owl the precentral gyrus of macaque monkeys. Soc. monkey: Selectivity for stimulus size, direction, and Neurosci. Abstracts 5: 377. orientation. Soc. Neurosci. Abstracts 5: 803.

Professor: Derek H. Fender 172. BINOCULAR HYSTERESIS AND FUSION Visiting Associates: Raymond P. Briggs, Patrice L. WITH VOLUNTARY EYE MOTIONS French, Dennis J. Hocevar, Stanley Klein Investigators: Michael T. Hyson, Bela Julesz*, Senior Research Fellow: James P. Ary* Derek H. Fender Research Fellows: G. Wesley Davis*, Michael T. Hyson, Ross L. Larkin* If the images to the two eyes are stabilized on the Graduate Students: Michael J.-W. Chen*, K. Jeffrey Eriksen*, Harrison Leong* retinae, the left eye and right eye images can be misaligned horizontally up to 2° before the sensation of *Division of Engineering and Applied Science, California Institute of Technology. depth in random dot stereograms is lost. To regain the depth, the images must be almost realigned, thus showing Support: The work described in the following research reports has been supported by: that the detection of depth and the tolerance of the National Institutes of Health, USPHS system for alignment errors depends on its prior history of Pew Memorial Trust stimulation and thus shows hysteresis. Summary: This group is concerned with information We have now measured this effect during normal vision processing in the human visual system. The techniques of with voluntary eye motion. We used scleral contact lenses Wiener analysis are being developed and applied to the to monitor the positions of both of the subject's eyes while identification of the nonlinear processing of the normal they viewed a random dot stereogram which was slowly visual system and to the identification of visual abnor­ misaligned horizontally in the temporal direction. mality. Three areas are of specific interest: (1) the Fusion and perception of depth were maintained for processes which produce potentials on the surface of the image misalignments of up to 8.5°. Divergence motions of human head and eyes; (2) the processes which control the both eyes accounted for 6° of this while 2° was the result movement of human eyes; and (3) the processes whereby of cortical neural mechanisms which dynamically correct the images presented to the two retinae generate the for vergence errors during which the images will fall on percept of depth. noncorresJ?Qnding points of the two retinae. This toler­ ance is similar to that found under stabilized image conditions. 123

The vergence of most subjects shows a constant, slow 174. RAPID ADAPTATION 1N THE RETINA (0.5 Hz) oscillation (± 1°l about the point of image align­ Investigators: Ross M. Larkin, Stanley Klein, Derek IL Fender ment. This movement is not seen by the subject whose perceptions are corrected by the cortical shift When light enters the eye it initiates a complicated mechanisms described above. chain of events which eventually leads to the sensation Once fusion is lost, the images appear double until of vision. The electroretinogram (ERG) is a widely used they have been brought into closer alignment. We find measure of the retinal portion of the visual system. To that fusion is regained when the vergence movements of function over a very wide range of light intensities, the the eyes reduce alignment errors to zero, i.e., the images retina continuously modifies its characteristics to accom­ are refused when they fall at corresponding points on the modate the current conditions. We have chosen to study retinae. the dynamic behavior of this ongoing adjustment, called In summary, we now are able to measure vergence and rapid adaptation. vergence errors during binocular vision so as to. understand We use a variation of white-noise analysis technique to the dynamic coordination of eye motions and cortical study rapid adaptation, which has proven very useful in fusion mechanisms. the study of the dynamics of rapid adaptation. The output This is a preliminary report of work in progress and as of this technique is called a kernel. First-order kernels yet no written reports are available for distribution. are similar to impulse or flash responses and are used to provide the basic system response and to probe the wide *Head, Sensory and Perceptual Processes, Bell Laboratories, Murray Hill, New Jersey. range of static ad8.ptation. With the second-order kernel we have characterized the dynamics of the rapid adapta­ tion of the two major components of the ERG in normal 173. EYE MOVEMENTS IN READING and also in some abnormal subjects and obtained basic Investigators: Michael T. Hyson, Raymond P. Briggs, information about the internal organization of the system. Patrice L. French, Derek IL Fender The third-order kernel characterizes suppression-recovery The goal of this project is to understand the role of in the photopic ERG. eye movements in reading. We have now begun to use these techniques in the We have studied the fixation times for subjects looking clinic for further studies on their usefulness. Preliminary at displays that are varied in complexity and meaning. In results indicate that they will provide additional useful general, subjects can fixate each work or symbol in a information about the condition of patients which would meaningless display quite accurately but cannot do so if not otherwise be available. The major obstacle which has the words form a meaningful sentence. Fixation times are prevented the introduction of white-noise into the clinic much longer for a subject viewing identical or meaningless has been the much higher computational burden which the symbols. These results suggest that accurate fixation technique presents. We have designed a special-purpose requires longer processing time. This mode appears to be computer which performs the necessary calculations and overridden by reading-like behavior in the case of data presentation in real-time. meaningful materials. Here, less positional accuracy is found in the eye movements and fixation times are 175. LOCAIJZATION OF DEPTH PERCEPTION PROCESSES IN THE MONKEY reduced. These results imply that redundant linguistic Investigators: Geoffrey D. Rubin*, James P .. Ary, structures in text allow the brain to decode the printed K. Jeffrey Eriksen, Francis M. Miezen, message with less visual information than if no structure John M. Allman, Derek H. Fender or meaning is present. The presence of meaning evokes Lehmann and Julesz (1978) have shown that dynamic eye movements that differ from those evoked by word binocular random dot patterns produce a visually-evoked patterns without meaning. scalp potential (VESP) when uncorrelated dot patterns This is a preliminary report of work in progress and as change to correlated patterns. The goal of this project is yet no written reports are available for distribution. to identify the location of the source of the "pure depth" VESP by measuring its potential distribution over the head and then applying a localization algorithm to determine 124

the parameters of the "equivalent" current dipole. These noise followed by another period of coherence. The parameters will be used to guide the search for the single response is measured from the start of the incoherence neurons which presumably constitute the cyclopean retina, "pulse.11 Variants of this stimulus include (a) noise pulses the place where information from two eyes becomes one to individual ears, (b) coherent noise pulses to both ears, percept. Because of the invasive nature of single-cell (c) incoherent noise pulses to both ears, and (d) continuous work, a trained macaque monkey, whose visual cortex has noise to one ear- with a pulse of incoherence to the other. been mapped, will be the subject. Preliminary comparisons of the responses to these stimuli _ This is a preliminary report of work in progress and as show both differences and similarities. yet no written reports are available for distribution. This is a preliminary report of work in progress and as Reference: yet no written reports are available for distribution. Lehmann, D. and Julesz, B. (1978) Vision Res. 18: 1265-1271. *Sherman Fairchild Distinguished Scholar.

*Undergraduate, California Institute of Technology. 178. SUPERPOSmON OF RESPONSES TO 176. LGN AND DEEP-SOURCE LOCALIZATION PARTIAL-FIELD VISUAL STIMULATION THROUGH EVOKED POTENTIALS Investigators: Terrance M. Darcey*, K. Jeffrey Eriksen, Ary, Investigators: Michael J.-w. Chen, James P. Ary James P. Derek H. Fender

A bright xenon flash transilluminating a red and black Various investigators of sensory-evoked potentials checkerboard pattern is sufficiently powerful to evoke have suggested that a major part of these responses is reliable early peaks in the visually-evoked scalp potential nonspecific in origin, i.e., not directly related to the (VESP). When such flashes are presented rapidly in a sensory modality stimulated. Any stimulus would then be random sequence, several peaks are developed in the first­ equally potent in activating the response. Utilizing multi­ order response before 60 msec. The sources for the most electrode arrays, we have found a simple, stimulus­ prominent of these peaks can be located by applying dependent relation between early components of the electric field theory and least-squares fitting algorithms. visually-evoked scalp potential elicited by a pulsed­ The resultant location for the largest of these early peaks patterned stimulus. is deep within the head, presumably originating from the Potentials were measured simultaneously at 40 lateral geniculate nucleus or other thalamic structures. locations on the human scalp and plotted as equipotential maps. Responses to the various quarters, halves and the

177. AUDITORY SCALP POTENTIALS whole of a centrally presented 10° circular field were EVOKED BY MONAURAL/BINAURAL compared. Visual inspection of the maps shows that COHERENT/INCOHERENT NOISE STIMULATION responses to half-fields are very similar to the sum of the Investigators: Donald M. Mackay*, K. Jeffrey Eriksen, James P. Ary, Michael J.-w. Chen, responses to the corresponding quarter-fields, and that the Derek IL Fernier response to whole-field stimulation is very similar to the

The auditory system seems to act in part as a sum of the responses to left and right or top and bottom correlator of stimuli presented to the two ears. Psycho­ half-fields. Thus, the principle of superposition of partial­ physically it is difficult to separate aspects of a response field responses is suggested by the data but this would not that may be produced by a correlator from aspects be expected if there were a large nonspecific response to produced by intensity- or frequency-specific components. arbitrary stimuli. However, preliminary evoked potential studies have shown Statistics are being developed to estimate the degree a clear response to the onset of correlated noise and we of similarity in the maps. An estimate can then be made are attempting to locate the source and determine its on the size of the residual response, which could be relationship to other aspects of auditory processing. nonspecific or alternately the result of a nonlinear One subject has been tested for auditory evoked scalp interaction between partial-stimulus fields. potentials (AESPs) to various forms of correlated and *Department of Neurology, Kantonsspital Zurich, noncorrelated noise. The stimulus consists of a period of Switzerland. coherent noise to both ears, then a period of incoherent 179. LOCALIZATION OF VESP FOR A Ary, J. P., Klein, S. A. and Fender, D. H. (1980) Location NONSPHERJCAL HEAD of sources of evoked scalp potentials: correction for skull and scalp. EEG Journal. Submitted for publi­ Investigators: Stanley Klein, Harrison Leong, cation. Derek H. Fender Ary, J. P. and Stow, R. W. (1979) Measuring on op amps common-mode impedance. Electronics, March 1, 1979. The potential on the surface of a homogeneous Chen, M. and Ary, J. P. (1979) LGN and deep-source conducting sphere caused by an embedded dipole source localization through evoked potentials. Am. Acad. Optometry Abstracts. has an exact analytic expression. If the outer boundary is Darcey, T. M., Ary, J. P. and Fender, D. H. (1979) nonspherical, there is no known analytic formula for the Methods for the localization of electrical sources in the human brain. In: Proceedings of the 5th surface potential. We have developed an approximation International Symposium on Electrical Potentials scheme which is adequate to handle the case of cortical Related to Motivation, Motor Old Sensory Processes of the Brain, H. H. Kornhuber and L. Deecke (Eds.). sources for nonspherical heads. This new technique may Darcey, T. M., Ary, J. P. and Fender, D. H. (1980) Dipole be especially valuable for recent experiments on visually­ source representation of evoked cortical activity from partial-field retinal stimulation. In preparation. evoked scalp potential (VESP) of monkeys. Darcey, T. M., Ary, J. P. and Fender, D. H. (1980) Spatio­ This is a preliminary report of work in progress and as temporal visually-evoked scalp potentials in response to partial-field retinal stimulation. EEG Journal. yet no written reports are available for distribution. Submitted for publication. Eriksen, K. J., Darcey, T. M., Ary, J. P. and Fender, D. H. (1980) Superposition of scalp potentials evoked by 180. COMBINED SPATIAL AND TEMPORAL partial-field retinal stimulation. In preparation. LOCALIZATION OF VESP SOURCES Hadani, I., Gur, M., Meiri, Z. and Fender, D. H. (1979) Investigators: Stanley Klein, Harrison Leong, Hyperacuity in the detection of absolute and differ­ Derek H. Fender ential displacements of random dot patterns. Vision Res. Submitted for publication. The sources of visually-evoked scalp potentials (VESP) Hou, R. L. and Fender, D. H. (1979) Processing of direction and magnitude by the saccadic eye-move­ can be localized by measuring the potentials at many ment system. Vision Res. 19: 1421-1426. scalp locations at a given instant after a visual stimulus Hyson, M. T. (1980) Sunlight reflections from solar power satellites or solares mirrors should nQt harm the eyes. and then searching for that cortical dipole which best fits In: Proceedings of DOE's SPS Assessment Conference, the spatial distribution of the evoked potential. If, Lincoln, Nebraska, April 24-28. Larkin, R. M., Klein, S., Ogden, T. E. and Fender, D. H. however, several sources are active at any instant, then (1979) Nonlinear kernels of the human ERG. Bio. the solutions which provide a good fit to the data are not Cybernetics 35: 145-160. Ogden, T. E., Larkin, R. M., Klein, S. and Fender, D. H. unique. In order to better constrain the dipole param­ (1979) Nonlinear analysis of the ERG: I. A study of the eters, we make the assumption that the location and normal primate. In: Proceedings of the U.S.-Japan Joint Seminar on Advanced Analytical Techniques direction of the dipoles are fixed and only the magnitude Applied to the Vertebrate Visual System. Tokyo, changes in time. A nonlinear least squares program then Japan. Ogden, T. E., Larkin, R. M., Fender, D. H., Cleary, P. E. fits the spatial and temporal behavior of the dipoles to and Ryan, S. J. (1979) Nonlinear analysis of the ERG: match the data. In this manner we are able to determine II. An experimental private study. In: Proceedings of the U.S.-Japan Joint Seminar on Advanced Analytical a seven dipole fit in which up to four dipoles are Techniques Applied to the Vertebrate Visual System. temporally overlapped. This technique offers the possi­ Tokyo, Japan. Ogden, T. E., Larkin, R. M·., Fender, D. H., Cleary, P. E. bility of measuring small time delays between almost and Ryan, S. J. (1980) The use of nonlinear analysis of simultaneous sources. the primate ERG to detect retinal dysfunction. Exptl. Eye Res. In press. Williams, R. A. and Fender, D. H. (1979) Velocity PUBLICATIONS precision in smooth pursuit eye movements. Vision Res. 19: 343-348. Ary, J. P., Darcey, T. M. and Fender, D. H. (1979) The Williams, R. A. and Fender, D. H. (1980) Nonlinear effect of stimulus locus on the topography and sources analysis of the human smooth pursuit acceleration of the VESP. Am. Acad. Optometry Abstracts. response. IEEE Trans. Biomed. Engng. Submitted for Ary, J. P., Darcey, T. M. and Fender, D. H. (1980) publication. Methods for locating scalp electrodes in spherical coordinates. In preparation. Ary, J. P., Fry, T. L. and Biersdorf, W. R. (1979) A multiplexing system for multichannel signal averaging of the visual evoked response. IEEE Trans. Biomed. Engng. BME-26. _.1asakazu Konishi exogenous androgen. Research Fellow: Andrew Moiseff Graduate Students: Mark E. Gurney, Lawrence C. Katz, Reference: Daniel Margoliash*, James S. McCasland Nottebohm, F. (1980) In: Sexual Differentiation of the Special Graduate Student: Sachie Yoshida Brain, R. w. Goy and B. S. McEwen (Eds.), pp. 115-119. Research Staff: Eugene Akutagawa, Jamie P. Leverette MIT Press. Laboratory Staff: Cynthia Akutagawa

*Division of Engineering and Applied Science, California 182. THE CELLULAR NATURE OF THE CRITICAL Institute of Technology. PERIOD FOR SEXUAL DIFFERENTIATION JN THE ZEBRA FINCH SUpport: The work described in the following research Investigator: Mark E. Gurney reports has been supported by: Bing Chair of Behavioral Biology Within telencephalic brain nuclei which participate in The Helen G. and Arthur McCallum Fund National Institutes of Health, USPHS the efferent motor pathway for song in the zebra finch National Science Foundation (Poephila guttata), it has proved possible to follow over Pew Memorial Trust time the ability of individual neurons to undergo morpho­ logical sexual differentiation as a consequence of 181. ANDROGEN INCREASES PROTEIN SYNTHESIS wrrmN THE AVIAN BRAIN SONG CONTROL exposure to 17 S-estradiol (E ). The number of neurons in 2 SYSTEM the nucleus robustus archistriatalis (RA) which are able to Investigators: Masakazu Konishi, Eugene Akutagawa differentiate under the influence of exogenous E declines 2 In most song birds it is sexually mature males that sing exponentially with increasing age, while the magnitude of during the breeding season. They cease to sing as their their growth response does not change. Such data may testes regress during other times of the year. Adminis­ suggest that the critical period transition of RA neurons tration of androgen can stimulate song in nonbreeding and from E -responsive to E -unresponsive is autonomous to 2 2 castrated males as well as in normally mute females. In these cells. oscine passerines song is controlled by a discrete neural Exposure of female zebra finch chicks to E at 2 system consisting of nuclei and fiber tracts. There is hatChing masculinized the cytoarchitecture of RA. When m8.rked sexual dimorphism in these nuclei; they are larger administered to one-year-old, gonadally intact adult in males than in females. Steroid autoradiography shows females on the other hand, E had no effect on RA's 2 that testosterone or its metabolites concentrate in most cytoarchitecture. To investigate the temporal constraints of these nuclei. In the adult female canary, testosterone on responsiveness to E , female finches were implanted 2 triggers dramatic cell growth in some of the nuclei, as the subcutaneously with a silastic pellet which contained . behavioral effect, singing, gradually manifests itself 50 µg of E either at hatching, at 3, 6, 9, 15, 30, or at 40 2 (Nottebohm, 1980). These cellular and correlated days of age, and then were sacrificed for histological behavioral responses make the bird song control system a examination when sexually mature at 90 days of age. The convenient preparation for studying how hormones act on morphological assay of the ability of RA neurons to nerve cells to produce behavior. respond to E consisted of measurement of their maxi­ 2 Most steroid hormones act on their target cells by mum somal diameter and average packing in Nissl-stained inducing the synthesis of new species of proteins as well material. The distribution of somal diameter for RA as by increasing the overall level of protein synthesis in neurons in either males or females was unimodal and them. An increase in the rate of protein synthesis in nonoverlapping. In males the somal diameter of RA target cells exposed to androgen may be revealed by neurons averaged 17 .9 µm and in normal females averaged pulse-labeling with radio-amino acids combined with 8.2 µm. In females exposed to E at hatching, the 2 autoradiography. Our results show that increased rates of distribution of somal diameter remained unimodal, but protein synthesis in the brain song control areas precede was shifted to an average diameter of 15.9 µm. In the induction of song by androgen in female song birds. females exposed to E at 40 days of age, the pattern of 2 These biochemical and behavioral responses are androgen­ response to E was quite different: neurons which 2 specific. The method also reveals that the vocal control responded to E2 with an increase in somal size comprised as well as some other brain areas maintain higher rates of only a fraction of the total neuronal population in RA and protein synthesis than does surrounding tissue without were confined within the core of the nucleus. The 127

distribution of somal diameter was thus bimodal with a In the coming year we plan to implement a telemetry peak of responsive neurons at 14.6 µm while unresponsive system and the Herb Adams Microdrive, which should neurons remained 8. 7 µm in diameter. The maximum allow us to obtain single unit data during song from our hand-raised mockingbirds. somal diameter attained by E2-responsive neurons in RA was equivalent in all of the different treatment groups, References: while the number of E -responding neurons within RA fell Nottebohm, F. and Nottebohm, M. E. (1976) J. Comp. 2 Physiol. 108: 171-192. exponentially with increasing age. The half-life with Nottebohm, F., Stokes, J. M. and Leonard, C. H. (1976) J. which RA neurons lost E -responsiveness was 17 .3 days. Comp. Neurol. 165: 457-486. 2

183. NEUROPHYSIOLOGICAL STUDIES OF 184. USE OF PHASE BY THE SINGING filRDS OWL AUDITORY SYSTEM Investigator: James S. McCssland Investigators: Andrew Moiseff, Masakazu Konishi The goal of this project is to uncover basic information A class of cells in owl's midbrain auditory nucleus about the neuronal mechanisms by which a bird produces (MLD) is known to respond to sounds from restricted the learned motor patterns of song. We are presently regions of space. These cells have small spatial receptive concentrating our efforts on three important aspects of fields, and are arranged anatomically to form a "neural song production: neural lateralization of motor activity; map" of auditory space. We have exploited the orgai;ii­ specializations in production of timing cues by different zation of these cells to examine the role of interaural­ nuclei in the brain; and topogra:phically organized time, -phase, and intensity differences in the extraction specializations of motor activity. of directional information from sound. In this study we Several preliminary conclusions can be drawn from combine free-field and dichotic sound stimuli. available data concerning these issues. Thus far our Single units were recorded with glass insulated Pt-Ir multiple unit data fail to show hemispheric specializations microelectrodes. The receptive fields were mapped using in the telencephalon; our recordings tend to show simulta­ a free-field loudspeaker. Threaded plugs containing neous bilateral activity of roughly equal magnitudes above miniature speakers were then inserted into threaded tubes baseline. We hope to demonstrate lateralization in the previously implanted into the ear canals allowing the motor neurons which control song production; such a presentation of dichotic stimuli. The phase of tone (in the result would be expected since unilateral section of the range of 6-8 kHz) or noise stimuli delivered to each ear motor nerve has a markedly greater effect on the left side was varied, keeping intensity and arrival time constant, than on the right (Nottet>0hm and Nottebohm, 1976). and the number of spikes resulting from each stimulus We have seen temporally modulated, song-related counted. activity in both HVc and RA of zebra finches and The results indicated that the responses of all MLD canaries; these two nuclei are known through lesion cells with restricted receptive fields varied as a function studies to be necessary for normal song production of interaural-phase differences. For example, cells with (Nottebohm, Stokes and Leonard, 1976). We have not yet frontal receptive fields preferred zero phase difference seen any song-related activity in area X of adults; between the two ears, whereas cells having lateral destruction of X does not affect song. The first receptive fields preferred the phase of the signal recordings from MAN show weak but clearly modulated delivered to the ear contralateral to the receptive field to activity, which appears to lag behind that in HVc arid RA. lag behind the signal delivered to the ipsilateral ear. The Our recordings from canary HVc show activity for only magnitudes of the preferred phase differences, which some of the elements of a typical song bout. By contrast, were within the physiologically relevant range, were electrodes in canary RA show activity for virtually all correlated with the azimuth of the receptive fields. song elements. We do not yet know whether all elements These data indicate that the owl auditory system is are represented within HVc at different locations; if so, capable of using interaural-phase differences to determine we may conclude that HVc represents a topography or the direction of high-frequency sounds. "motor map11 of song. Otherwise, it is very likely that RA specifies the temporal cues for the remaining elements. 128

PUBIJCATIONS Knudsen, E. I., Blasdel, G. G. and Konishi, M. (1979) Sound localization by the barn owl (Tyto alba) measured with Gurney, M. and Konishi, M. (1980) Hormone-induced the search coil technique. J. Comp. Physiol. 133: sexual differentiation of brain and behavior in zebra 1-11. finches. Science 208: 1380-1383. Knudsen, E. I. and Konishi, M. (1979) Mechanisms of sound localization in the barn owl (Tyto alba). J. Comp. Physiol. 133: 13-21.

Research Associate: Marianne E. Olds modify the reinforcing effects of brain stimulation. In Visiting Associates: Theo B. Sonderegger, Mamoru Umemoto recent years, three systems have been singled out in Senior Research Fellow: Dorwin L. Birt relation to brain reinforcement-the central noradren­ Research Fellows: James L. Fobes, Kendrick N. Williams Graduate Student: Gary Aston-Jones* ergic, the dopaminergic, and the enkephalinergic systems. Laboratory Staff: Maria A. Clancy, Jeffrey D. Carpenter, The distribution of these substances in the brain matches Ronald M. Satchell to some extent the distribution of sites rewarding to brain *The Salk Institute, La Jolla, California. stimulation, and interference with transmission in these Support: The work described in the following research systems abolishes the behavior maintained by brain stimu­ reports has been supported by: lation. Japanese Education Department National Institutes of Health, USPHS We have approached this problem using three method­ National Science Foundation ologies. With respect to the role of the catecholamines in University of Nebraska the brain, we are working with a preparation in which the Summary: Experiments are being carried out which are central levels are permanently altered in a known way, part of a long-range program to investigate the neuro­ owing to certain treatments carried out at birth that anatomical bases of reinforcement and learning. The produce in the adult rat a virtual depletion of the experiments on reinforcement were begun with the dis­ substances in the forebrain, where their terminal fields covery that animals can be trained to stimulate their are, and a dramatic elevation in their concentration in the brains electrically and that, therefore, t~e brain stimulus hindbrain, where these substances are contained in the functioned in these situations in the manner of cell bodies that give rise to the catecholaminergic conventional rewards to reinforce certain behaviors at the pathways. With respect to the enkephalinergic systems, expense of others. This discovery suggested the presence we use the method of self-administration of opioids to in the brain of neural systems perhaps specialized to determine whether these substances have reinforcing mediate reinforcing effects. Since the initial observations properties when they are delivered intracerebrally, and, of this phenomenon were made, much of the research when we identify these sites, to establish their relation­ effort in this field has gone into the search for these ship to brain stimulation reinforcing sites. With respect systems. In the process, considerable information has to the two classes of transmitters, catecholaminergic and been obtained about the sites where electrical stimulation peptidergic, we are interested in establishing their effect produces these effects, about the neurotransmitters that at the single unit level in regions where reinforcement has influence the properties of the brain stimulus, and about been demonstrated, either with opioids or with brain the neural activity that correlates with the behavioral stimulation, our ultimate goal being to obtain information effects. However, the demonstration that these sites, as concerning the neural activity that underlies reinforce­ a. whole, constitute a functional system has not as yet ment. been accomplished. The experiments concerned with the neural basis of Some systems, identified through the transmitter associative learning arise from the observation that substance they contain, appear to be candidates on the animals do not respond alike to all environmental stimuli. basis of evidence that alteration in the metabolism of They learn to respond to those that are behaviorally these substances, or the induction of various other significant while ignoring those that are not. In some changes through experimental procedures, profoundly region of the brain, during the learning process, the 129

response of neurons must reflect the fact that the stimuli the noradrenergic innervation of the locus coeruleus was have acquired this behavioral significance. permanently enriched as a result of treatment at birth We have recently shown that neurons in a restricted with a toxic substance which destroyed the remote part of the caudal medial geniculate body (MGB) of rats terminal fields of these cells and produced sprouting and respond to a broad range of auditory stimuli, but that the regeneration in regions closer to the cell bodies lying in magnitude of the neural response to a particular tone is the locus coeruleus. It has been demonstrated greatly modified by varying the behavioral significance of biochemically that this type of treatment leads to the tone. rrhis variation is produced either by following abnormally high levels of norepinephrine in the locus or by not following the tone presentation with food coeruleus and reduced levels in the hypothalamus. We delivery.] Neurons in the rest of the MGB seem to be have found that adult rats treated neonatally in this uninfluenced by variations in the significance of auditory manner show abnormally high rewarding properties to stimuli. It therefore seemed likely that neurons in other brain stimulation applied in the region of the locus regions of the brain that are heavily interconnected with coeruleus and abnormally low properties in the the caudal region of the MGB might be part of the hypothalamus. Tests with noradrenergic receptor blockers circuitry involved in evaluating the behavioral signifi­ such as phenoxybenzamine, phentolamine, and yohimbine cance of external stimuli. These regions are being show that the rewarding properties of brain stimulation in identified by use of retrograde neural tracing techniques. the region of the locus coeruleus are probably mediated Also, multiple and single unit neuron recording experi­ presynaptically by an adrenoceptor of the alpha type. ments are in progress to study the process of neural These studies are continuing with the aim of finding out response changes during learning in regions identified as whether other adrenergic cell groups of the pontine region being interconnected with the caudal MGB. can similarly be enriched with norepinephrine and also lead to increased reward. 185. THE ROLE OF THE WCUS COERULEUS IN STIMULATION REWARD 186. ANATOMICAL BASIS FOR THE RmNFORCING Investigators: Mamoru Umemoto, Marianne E. Olds EFFECTS OF OPIOIDS Investigators: Marianne E. Olds, Kendrick N. Williams Anatomical evidence based on mapping studies of positive reinforcement produced by electrical stimulation We have shown previously that rats can be made to of the brain has implicated the locus coeruleus region in self-administer morphine or a synthetic analogue of the reinforcing effects obtained there and in remote methionine-enkephalin when these substances are given regions receiving projections from it. Pharmacological directly in the brain, at a site in the posterior lateral and biochemical evidence also indicates that this nucleus, hypothalamus. This same region is known to support some which is almost exclusively constituted by cell bodies that of the highest rates of self-stimulation in the rat brain contain norepinephrine, is an important factor in deter­ thus far demonstrated. These data suggest that the mining the reinforcing properties of brain stimulation. On euphoric effects of opiates might be mediated by activa­ the other hand, lesions of this region followed by tests for tion of the same structures that are directly activated reinforcement in the hypothalamus, a principal focus of during electrical self-stimulation behavior. To obtain the reinforcing effects, seemed ineffective, and treat­ evidence for this hypothesis, during this past year we have ment of the subjects with drugs which inhibit the synthesis investigated sites other than the hypothalamus for of norepinephrine, and thus presumably depletes the locus evidence of opiate-reinforcing properties. In our experi­ coeruleus of norepinephrine, had no effects on the ments, the subject is a rat implanted with a combination stimulation reward. Much controversy therefore sur­ cannula-stimulation probe. The animal is prescreened for rounds the question of whether norepinephrine, and in reinforcing effects of electrical stimulation at the particular the norepinephrine system which originates implanted site, and then is trained to self-administer the with the norepinephrine-containing cell bodies of the locus test substance using a paradigm that involves the repeated coeruleus, is essential for stimulation-induced reinforce­ reversal of the lever the animal has to operate to obtain ment. delivery at the implanted site of the brain of a minute We have investigated this question in animals in which amount of the substance tested. If the subject 130

successfully "learns" to reverse and presses more often on platinum recording electrode and fine gauge injection the "hot" lever than on the "cold11 one and at a rate higher needle connected via polyethylene tubing to a Hamilton than that observed when a control solution is given, the microsyringe containing the test substance. Electrode and site in the brain yielding this effect is classified as an needle can be moved simultaneously to sample unit opiate-reinforcing site. activity and to inject amounts of solution as small as 8 Three pathways are of particular interest. The first is nanoliters at a time near the tip of the electrode, along a the nigrostriatal pathway, and for several reasons: it track starting at the cortex and terminating in the basal contains opiate receptors in high density; it yields high region of the brain. The experiments and the injections rates of self-stimulation in the substantia nigra, the are under computer control. The paradigm consists in region containing the cell bodies that send projections to presenting a series of trials to a single neuron, each trial the striatum; and finally, considerable evidence links the comprising the injection of 8 nanoliters or more of a transmitter dopamine,. found in the cell bodies _and the solution containing a test substance. Analysis of changes striatal terminal fields, to both self-stimulation and to in the rate of discharge produced by the series of trials opiate-receptor activation. The second pathway is the (mimicking the pattern of local injection that occurs coerulo-cortical system because, again, this system is during self-administration) is effected by comparing histo­ viewed as a neural substrate of stimulation-induced grams before and after injection. Thus far, we have reinforcement and it contains opiate receptors. The third obtained evidence of decreased firing when morphine is pathway of interest is the mesolimbic system which injected in the hypothalamus. These studies are still in originates with the dopamine-containing cells in the progress. ventral tegmentum and terminates in regions classically associated with the regulation of emotions and moods. 188. ONTOGENY OF THE STIMULANT EFFECTS Brain-stimulation reward has been demonstrated for the OF MORPffiNE AND METffiONINE-ENKEPHALIN region of origin and for the regions containing the IN RATS terminal fields of these neurons, the nucleus accumbens, Investigators: James L. Fobes, Marianne E. Olds the amygdala, and the septum. In adult animals, opiates produce stimulant effects at The findings of this past year support the notion of a low doses and depressant effects at high doses. The common substrate for the electrical and chemical re­ stimulant effects have been related to activation of warding effects in that (1) neither effect was obtained in structures in the forebrain, the depressant effects to the caudate, the region of nigral terminal fields and high activation of structures in the hindbrain. Also, the density opiate receptors, and (2) moderate effects were stimulant effects, and perhaps also the depressant effects, obtained in the nucleus accumbens, the region that of opiates have been related to dopaminergic trans­ contains the terminal fields of the mesolimbic system and mission. In the adult animal, the two effects are not that also contains opiate receptors. Tests for opiate always distinct, and it is not uncommon to find that rewarding affects in the regions giving rise to these stimulant effects follow depressant effects. For these projects are being carried out. reasons, the neural bases of both effects have been investigated in the neonate. In this preparation, it has been established that regions containing cell bodies of the 187. OPIATE SENSITIVITY PROFILES OF NEURONS IN REGIONS SUPPORTING dopaminergic systems are fully mature at birth, whereas REWARDING BRAIN STIMULATION the regions that receive dopaminergic input mature during Investigators: Marianne E. Olds, Kendrick N. Williams the first few weeks after birth. It is possible that a Single unit studies in the freely-behaving rat are being similar process takes place for the neurons containing carried out to identify pS:tterns of changes in spontaneous peptides with opiate-like properties. unit activity procedures by the local injection in the brain Two experiments were carried out. In the first, rat of minute amounts of opiates. Our aim is to duplicate the pups were tested repeatedly, starting with the fourth day local environment of the cell during self-administration of after birth and then every fourth day until day 40 after the drug. birth, for the effects on locomotor activity of morphine or The subjects are rats prepared with a combination of an analogue of methionine-enkephalin as measured with a stabilimeter. The observation period was 30 min. The applied to the contralateral face and anterior portion of results showed that until day 16 only depressant effects the body. The majority of units showed a selective were produced, and that after day 16 depressant and increase in response to the tone frequency paired with stimulant effects were seen. These results are compatible food pellet presentation during conditioning. These with the view that two systems, maturing at different increases started as early as the period 14-28 msec after times, mediate the behavioral effects of these substances. stimulus onset and persisted throughout the interval However, in these experiments the dose tested may have between tone onset and pellet presentation. The early been too high for neonates, which are known to be more latency unit response changes were only very _loosely sensitive than adults to toxic substances, or the repeated correlated in magnitude and latency with learned move­ treatments may have produced tolerance, or the obser­ ments of the head and body toward the pellet dispenser, vation period may have been too short to observe the while longer latency unit changes were more highly stimulant effects if they followed the depressant effects. correlated with movements of the head and body. Similar The second experiment was designed to take _these factors unit changes were not observed when head or body into account. The observation period was extended to movements of similar magnitude were made in the 6 hr: each animal was tested only once, and two doses absence of the auditory stimulus. These results support a were tested, each being in the low range for adult rats. role for the intermediate and deep layers of SC in the The results show that stimulant effects appeared on day 7, neural circuitry that evaluates and responds to the but only after depressant effects were seen in the same behavioral significance of sensory stimuli. test session. However, the latency of the stimulant Units recorded in the external nucleus of inferior effects decreased with the age of testing, a finding that colliculus also responded to a broad range of tone supports the notion that the two effects are mediated by frequencies and to somatosensory stimulation. These systems which mature at different times. These experi­ units, however, were not affected by differential ments are being pursued to find out whether with conditioning. maturation the system responsible for depression becomes less sensitive, or whether the stimulant system, also 190. AFFERENTS TO MGB AND DEEP LAYERS OF THE having matured, makes demands on the opiate ligands that SUPERIOR COLLICULUS STUDIBD WITH THE reduce the amount available for the depression-sensitive HORSERADISH PEROXIDASE METHOD Investigator: Derwin L. Birt system. We have previously reported that the responses of 189. PLASTICITY DURING AUDITORY CONDffiONING neurons in a region near the caudal tip of rat medial IN THE DEEP LAYERS OF THE SUPERIOR geniculate (MGB) are affected by differential conditioning COLLICULUS BUT NOT IN THE INFERIOR COLLICULUS procedures in a manner quite different from that of Investigators: Dorwin L. Birt, Kendrick N. Williams, neurons in the remainder of MGB. It was therefore of Marianne E. Olds interest to see the extent to which the connections of this Multiple unit auditory evoked responses have been region differed from the more well known connections of recorded from deep and intermediate layers of rat the ventral division of medial geniculate. The enzyme superior colliculus (SC) and external nucleus of inferior horseradish peroxidase (HRP) was deposited in caudal colliculus during a differential appetitive conditioning and MGB by microelectrophoresis. HRP histochemistry reversal experiment in which different frequency tones revealed that the heaviest transport of HRP is to the deep were used as the conditioned stimuli. These two regions and intermediate layers of ipsilateral superior colliculus are among those which horseradish peroxidase tracing (SC), to the external and pericentral regions of inferior experiments showed project heavily to the portion of colliculus (IC), to a narrow band along the medial-most medial geniculate where associative unit changes were border of IC, and to the portion of the cuneiform nucleus found. underlying the IC. Less dense labeling was seen in the Most units in superior colliculus responded phasically contralateral caudal MG and contralateral deep and to a wide range of auditory frequencies and were also intermediate SC. Label was virtually absent in the responsive to somatosensory stimuli, particularly those central nucleus of IC. HRP has also been injected into the intermediate and Olds, M. E. and Fobes, J. L. (1980) Activity responses to deep layers of SC. Cells labeled by retrograde transport morphine and amphetamine in rats treated neonatally with 6-hydroxydopamine. Psychopharmacology. Jn of HRP are seen ipsilaterally in the external nucleus of press. IC, the dorsal and ventral nucleus of the lateral lemniscus, Olds, M. E. and Fobes, J. L. (1980) The central basis of motivation: self-stimulation studies. Ann. Rev. the parabigeminal nucleus, and scattered sites throughout Psycho!. Jn press. the mesencephalic tegmentum. Contralaterally, label is Olds, M. E., Fobes, J. L. and Albert, V. (1980) Catechol­ amine-serotonin interaction effects on activity in rats seen in the cuneiform nucleus, the parabigeminal nucleus, treated neonatally with 6-hydroxydopamine. Psycho­ and the ventral nucleus of the lateral lemniscus. pharmacology. In press. Olds, M. E. and Williams, K. N. (1980) Hyperactivity and deficits in discrimination of auditory signals induced PUBLICATIONS by chronic ventricular and hypothalamic injections of morphine in the rat. Psychopharmacology. Submitted Birt, D. and Olds, M. E. (1980) Response properties predict for publication. Olds, M. E. ~nd Williams, K. N. (1980) Self-administration associative change in rat medial geniculate during of D-Ala -met-enkephalinamide at hypothalamic self­ differential appetitive conditioning. J. Neurophysiol. In press. stimulation sites. Brain Res. 194: 155-170. Fobes, J. L. and Olds, M. E. (1980) Locomotor responses in Olds, M. E., Williams, K. and Birt, D. (1980) Unit neonatal rats to acute and chronic administration of responses in subdivisions of the inferior colliculus of opioids. Brain Res. Submitted for publication. the rat during differential conditioning and reversal. Fobes, J. L. and Olds, M. E. (1980) The effects of neonatal J. Neurophysiol. In press. treatment with 6-hydroxydopamine on behavioral activity during development and adulthood. Psycho­ pharmacology. Submitted for publication.

Associate Professor: John D. Pettigrew the visual system, to the auditory system. It has been Visiting Associates: Hermes Bravo, Kunio Nakai Senior Research Fellow: Takuji Kasamatsu known for some time that there is an orderly represen­ Weizmann Research Fellow: David E. Presti tation of sound frequency in the auditory cortex with what Research Fellows: Marylouise Ary, Vilayanur S. Ramachandran have been called isofrequency lines, along which all cells Graduate Students: Baruch Kuppermann, Kenneth L. have the same characteristic frequency running dorso­ Marton ventrally at right angles to the anterior-posterior axis of Research Staff: Maria A. Clancy, Robert W. Tajima* Laboratory Staff: Jay Venti the brain along which the characteristic frequency varies *Deceased April 30, 1980 systematically. The big puzzle concerns what is repre­ sented along the dimension at right angles to the Support: The work described in the following research reports has been supported by: representation of frequency. Using the apparatus and Fogarty International Center (NIH) concepts which have proved so successful in elucidating National Institutes of Health, USPHS National Science Foundation auditory localization in the owl auditory system, in the Pew Memorial Trust past year Middlebrooks and Pettigrew have looked at the President's Venture Fund possibility that sound location might be an important Evelyn Sharp Fellowship Weizmann Fellowship variable along the cortical dimension at right angles to Whitehall Foundation Robert E. and May R. Wright Foundation frequency. Sound localization does appear to be a very important variable and a large number of cells were found Summary: Our laboratory uses the central visual pathway whose receptive fields were sharply localized in auditory of vertebrates as a model system for tackling some space along ~he acoustical axis of the pinna. central problems in the integrative organization of the Developmental plasticity of the visual system nervous system. Progress in the last year has continued continues to be an important focus of the group and in the despite the frequent absences of Professor Pettigrew who past year we have extended observations in this area in a is on leave in Australia. number of new species including the owl, which appears to An important new departure in the last year has been have ocular dominance columns in its cortex which are to try to apply principles which have been so successful in obvious only after deprivation, and the sheep, whose visual 133

system during development may prove to be an even more References: Held, R., Ingle, D., Scheider, G. E. and Trevarthen, c. B. valuable model system than that of the cat. Our (1967) Psychologische Forsch. 31: 42-43. understanding of the interaction between the catechol­ Hodos, W. and Karlen, J. H. (1966) Exptl. Brain Res. 2: 151-167. aminergic system and visual cortical plasticity continues Karlen, J. H. and Hodos, W. (1970) J. Comp. Neurol. 150: to increase with a clear picture of the ultrastructural 253-278. basis of the interaction between catecholaminergic inputs *Work carried out while on leave at the National Vision and visual cortical neurons and also with microiontopho­ Research Institute of Australia, 386 Cardigan street, Carlton, Vic. Australia 3053. retic studies where catecholamines are applied directly to visual cortical neurons.

192. CO-EVOLUTION OF NOCTURNAL VISION AND FRONTALLY-PLACED EYES? 191. THE TWO FOVEAE OF THE HAWK'S RETINA Investigator: John D. Pettigrew SUBSERVE TWO VISUAL PATHWAYS Investigator: John D. Pettigrew* It is commonly assumed that frontal placement of the eyes, an arrangement characteristic of primates, serves in The notion of two visual systems was first put forward the interests of binocular depth discrimination. Study of a by Karten and Hodos (Hodos and Karten, 1966; Karten and number of avian visual systems reveals two counter­ Hodos, 1970) on the basis of their findings that massive examples to this view. Forward rotation of the eyes in lesions in the geniculostriate pathways of pigeons led to the head is linked to the nocturnal niche rather than to barely discernible visual deficits, whereas tiny lesions in binocular depth discrimination. the tectofugal pathway led to obvious deficits in color and (1) The nocturnal oilbird, with frontally-placed eyes pattern discrimination. The notion was subsequently which give it the appearance of a predator, is in fact a widely proselytized by Held et al. (1967) and we now know fruit-eater with no neural substrate for stereopsis. No that there are in fact more than two parallel visual binocular interaction can be found in those parts of the pathways. oilbird's forebrain where stereoscopic mechanisms have The peak of differentiation of this division of labor been observed in the owl. among the visual pathways appears to be reached in the (2) Diurnal birds of prey with laterally-placed eyes diurnal birds of prey, which have two foveae in each eye. and a laterally-directed, hyperacute, "monocular" fovea in The present investigations, using neurophysiological and each eye, also have forward-directed "binocular foveae, 11 neuroanatomical tracing techniques on Falco, Circus and whose axes are more than 40° from the optical axis of the Milvus species, show that the geniculostriate pathway eye. By neurophysiological criteria, these "binocular utilizes information largely derived from the binocular foveae" mediate stereoscopic vision equal to that of owls. foveae situated in temporal retina, whereas ganglion cells One way to account for these apparent exceptions is to in the monocular fovea project almost exclusively into the suppose that optical aberrations in the large-aperture, contralateral optic tectum. nocturnally-adapted eye impose constraints on the amount This arrangement can be demonstrated in a by which optical and visual axes can diverge. A diurnal particularly striking way using retrograde transport of hawk may be able to retain good image quality along the horseradish peroxidase (HRP). Tectal injections lead to a binocular visual axis, despite optical axes directed 40° dense burst of labeled ganglion cells around the contra­ laterally, only because of the high optical quality of its lateral "monocular fovea" in central retina, with only small-aperture eye. scattered cells in the vicinity of the binocular fovea in Support for this proposal comes from a number of new temporal retina. In contrast, thalamic injections lead to a observations on nocturnal and diurnal birds from diverse dense burst of labeled ganglion cells in the 11binocular geographical sites. These include the tawny frogmouth, fovea," with only scattered labeled cells in central retina. the laughing jackass, the letter-winged kite (which is a totally nocturnal bird in an otherwise diurnal genus) and the black-shouldered kite, all from the Australian region. 134

(Presti and Pettigrew, 1980). One important implicaton of 193. AVIAN GEOMAGNETIC FIELD PERCEPTION a muscle spindle-based magnetic field sensor is that Investigators: David E. Presti, John D. Pettigrew natural motion of the muscle may be requisite for the Behavioral data gathered over the past several years perception of magnetic stimuli. A detection mechanism by a number of investigators have established that pigeons of this kind could account for the difficulties encountered and other birds can employ the earth's magnetic field to in conditioning immobile homing pigeons to magnetic field obtain directional information during navigation. The changes (Kreithen and Keeton, 197 4; Beaugrand, 1976) and mechanism of geomagnetic field detection, however, is for the puzzling requirement of movement in other totally unknown. We are engaged in both neurophysio­ behavioral experiments involving pigeons and magnetic logical and anatomical studies directed toward elucidating fields (Bookman, 1977). the nature of the magnetic receptor and transduction pathway in the pigeon (Columba livia). References: Beaugrand, J. P. (1976) J. Comp. Physiol. 110: 343-355. Using the superconducting magnetometer in the Blakemore, R. (1975) Science 190: 377-379. Bookman, M.A. (1977) Nature 267: 340-342. Caltech Geology Division, we have determined that Frankel, R. B., Blakemore, R. P. and Wolfe, R. S. (1979) several parts of the pigeon head and neck musculature are Science 203: 1355-1356. reproducibly magnetic (Walcott, Gould and Kirschvink, Kreithen, M. L. and Keeton, W. T. (1974) J. Comp. Physiol. 91: 355-362. 1979; Presti and Pettigrew, 1980). Magnetic remanence Presti, D. and Pettigrew, J. D. (1980) Nature 285: 99-101. similar to that measured in pigeons was also detected in Walcott, C., Gould, J. L. and Kirschvink, J. L. (1979) Science 205: 1027-1028. migratory white-crowned sparrows (Zonotrichia leucophrys) and a number of other species of birds, 194. PROLONGING THE "CRITICAL PERIOD" FOR suggesting that a variety of birds either synthesize or PLASTICITY IN THE VISUAL CORTEX accumulate magnetic material. Anatomical studies of Investigators: Vilayanur S. Ramachandran, these areas have produced magnetic material charac­ Baruch Kuppermann terized as iron-rich by electron microprobe analysis. If one eye of a kitten is closed early in life, that eye We are also using single neuron recording techniques to becomes functionally disconnected from visual cortical record from the pigeon brain while the ambient magnetic neurons. Normally this effect can be produced only during field is varied to mimic changes which might occur while a "critical period11 restricted to the first 3 months of life the pigeon is orienting in flight. Deoxyglucose labeling (Hubel and Wiesel, 1970)0 but if kittens are dark-reared experiments directed toward determining which areas of for 6-12 months and subsequently exposed to a normal the pigeon brain are active when the magnetic field is visual environment with one eye closed, most of their varied are also under way. Thus far, none of these cortical neurons are found to be dominated by the techniques has yielded neurons which respond to changes experienced eye (Cynader and Mitchell, 1980). in the ambient magnetic field. There are two possible interpretations of this effect. Geomagnetic field sensitivity in animals may arise First, individual cortical neurons may be actually shifting from the coupling of small permanent magnets to sensory their allegiance to the experienced eye and there may be structures which originally evolved to serve other func­ an 11expansion11 of columns devoted to that eye. Alterna­ tions. For example, strings of magnetic crystals, such as tively, the findings may simply be the result of selective those found in magnetotactic bacteria (Blakemore, 1975; activation of one eye's connections by the visual Frankel, Blakemore and Wolfe, 1979), coupled to sensory experience restricted to that eye. hairs like those present in the receptor structures of Using anatomical and physiological techniques, we vertebrate acoustico-lateralis organs, would provide a have been doing experiments to distinguish between these feasible mechanism for detection of the geomagnetic two possibilities and also to try to elucidate further the field. Similarly, magnetic crystals properly coupled to a nature of the "critical period." sensitive muscle receptor, such as a spindle, so that the (1) Two cats were dark-reared from birth until 6 torque exerted on the grain ensemble in the geomagnetic months, then monocularly deprived (M.D.) and exposed to field is sufficient to stimulate the muscle receptor, might light for 10 days and 3.5 weeks, respectively. The first form an effective basis for magnetic field sensitivity showed a breakdown of binocularity while the second 135 showed a marked shift in ocular dominance toward the Recordings soon after stimulation· showed a small but open eye. This conforms to the normal sequence of significant increase in the extent to which neurons could changes that one observes in M.D. kittens. (2) The second be driven by the deprived eye. Similar results were cat was subsequently reverse-sutured, that is, the obtained in a fifth animal which was not anesthetized or originally experienced eye was closed, while the initially paralyzed during stimulation but was fixed in a head deprived eye was opened for 3 months. We found that this holder. (Two control kittens had their deprived eyes caused a strong shift back to the newly-opened eye. exposed to a white screen for 4 hr instead of rotating (3) Another cat was dark-reared from birth until 6 lnonths gratings and no change in ocular dominance was observed.) and then allowed 2 weeks of binocular visual experience However, if four days of darkness were allowed to elapse after which one eye was sutured for 6 weeks. This animal after grating stimulation, the degree of recovery was presumably had all his visual cortical cells "reactivated11 greater than if the recording was done immediately after to a normal state by the initial binocular visual experience stimulation. and yet the ocular dominance had now shifted toward the Another two kittens were revived from anesthesia and nonsutured eye. We conclude that dark-rearing can the deprived eye was stimulated on 4 successive days for actually preserve the plasticity of cortical cells into 1 hr each day. They were kept in total darkness whenever adulthood and that this plasticity can survive at least 2 they were not being stimulated so that the deprived eye weeks of binocular visual experience. (4) 6-0H-Dopamine received a total of only 4 hr of experience as in the was used to deplete catecholamines from one hemisphere previous animals. In these animals, recovery of functional of a dark-reared adult cat which was then monocularly connections through the deprived eye was even more deprived for 6 weeks. Shifts in ocular dominance were striking and in fact the two eyes became almost equally seen only in the untreated control hemisphere. This dominant. A third control kitten was kept in total suggests that preserving plasticity into adulthood may at darkness for four days (i.e., without grating stimulation) least partially involve the continued activity of ascending and no significant recovery was seen. We conclude that catecholamine pathways. Perhaps there are some cortical (1) very brief periods of visual experience (3-4 hr) can areas even in normal adult cats which remain indefinitely modify the eye preference or ocular dominance of cortical plastic in this manner. neurons; (2) even after active synaptic stimulation has We are currently exploring the time cOurse and ceased, some time-dependent process akin to "consoli­ mechanisms by which visual experience can nswitch off11 dation" must continue to occur in the dark. Since it is the plasticity of neurons in the visual cortex. now known that ocular dominance shifts can be induced even in adult cats (Biology 1980, No. 194), it may not be References: Cynader, M. and Mitchell, D. E. (1980) J. Neurophysiol. In too fanciful to suggest that these changes may be press. analogous to changes thought to be involved in memory Hubel, D. and Wiesel, T. N. (1970) J. Physiol. 206: 419-436. and learning in the adult mammalian brain. References: 195. BRIEF VISUAL EXPERIENCE CAN MODIFY Hubel, D. and Wiesel, T. N. (1970) J. Physiol. 206: OCULAR DOMINANCE OF 419-436. CORTICAL NEURONS IN Kl'ITENS Olson, C. R. and Freeman, R. D. (1975) J. Neurophysiol. Investigators: Vilayanur S. Ramaehandran, 38: 26-32. Marylouise Ary Ramachandran, V. S., Rao, V. M., Martin, K. A. L. and Whitteridge, D. (1979) Nature 277: 391-393. Kittens 5-6 weeks of age were used to study the effects of brief visual experience on the ocular dominance 196. BINOCULAR NEURAL MECHANlSMS IN of cortical neurons. Kittens were deprived monocularly GOATS AND SHEEP for 2-3 days and, as in earlier studies (Hubel and Wiesel, Investigators: Vilayanur S. Ramachandran, Hermes Bravo, John D. Pettigrew 1970; Olson and Freeman, 1975), the ocular dominance shifted almost completely toward the experienced eye. We have been studying binocular neural mechanisms in Four of these kittens had their deprived eye stimulated by goats and sheep using anatomical and physiological tech­ rotating high contrast gratings (Ramachandran et al., niques. We used sheep because their large interocular 1979) for 3-4 hr while still anesthetized and paralyzed. distance magnifies binocular parallax and correspondingly 136

magnifies the range of disparity-selective cells pronounced in the binocular segment of the LGN but no encountered (up to ± 13° can occur in V2 of the lamb obvious anatomical changes were seen in the cortex. cortex; Ramachandran, Clarke and Whitteridge, 1977). However, even two days of monocular deprivation caused Although ocular dominance columns can be identified striking shifts in ocular dominance measured physio­ _physiologically, we find that after monocular injections of logically. tritiated proline, there is more or less uniform labeling in Recording from cells in Vl and V2, we find that the layers IV and VI (Figure 1) and no well-defined dominance vertical horopter in goats is tilted toward the animal's columns of the kind seen in cats and rhesus monkeys. We feet as in cats and owls. Multiple unilateral HRP also find strong labeling in V2 suggesting direct lateral injections into the LGN revealed the nasotemporal geniculate nucleus (LGN) input to that area, and were able division of the retinothalamic pathway. The ipsilateral to confirm this physiologically in two animals by removing decussation was sharp while the contralateral decussation Vl and demonstrating receptive fields in V2. In coronal was diffuse and the decussation lines of the two eyes sections of the ipsilateral hemisphere, there seemed to be seemed tilted in a direction appropriate to the tilted a discontinuity in labeling between Vl and V2. The horopter. Horizontal disparities were encountered in V2 contralateral superior colliculus (stratum opticum and in two baby goats but we found no evidence of well­ griseum superficiale) was also strongly labeled but the defined "depth columnsn of the kind seen in adults. The ipsilateral projection was confined to the lateral 2.5 mm. conversion of immature OR-gate binocular neurons Also, after c3HJproline injections in Vl, -we found antero­ (drivable through either eye) to AND-gates requires six grade labeling in the claustrum, the dorsal LGN (including weeks of maturation and it is possible that the emergence monocular segment), V2, and the superior colliculus. of depth columns is similarly delayed. Our eventual aim is to find out whether there is any systematic relationship between physiologically identified ocular dominance columns and 11depth columns" labeled by using 2-deoxyglucose.

Reference: Ramachandran, V. S., Clarke, P. G. H. and Whitteridge, D. (1977) Nature 268: 333-335.

197. OCULAR DOMINANCE COLUMNS IN THE Figure 1. Dark field autoradiograph of the primary visual VISUAL CORTEX OF MONOCULARLY cortex of the sheep. Radioactively-labeled praline was DEPRIVED OWLS injected into one eye of an anesthetized sheep. The Investigator: Hermes Bravo, John D. Pettigrew praline was taken up by retinal cells and transported to the cortex via the lateral geniculate nucleus--a relay In owls, the whole retina is represented in the station en route to the cortex. A parasagittal section of the contralateral hemisphere is shown here and the label contralateral lateral geniculate nucleus (LG N). This can be seen clearly in both layers N and VI of the striate nucleus sends its projections towards the ipsilateral and cortex. There is no clear-cut segregation of the two eyes' inputs into "ocular dominance columns" of the kind seen in contralateral visual cortex where three targets have been cats and rhesus monkeys. The striate cortex of the sheep described (Bravo and Pettigrew, 1980). The projection to is about 15 cm long anteroposteriorly (when unfolded) and about 3-4 cm wide transversely. The lateral half of this the granular and infragranular layer is continuous as seen area receives inputs from bath eyes while the remaining with autoradiographic techniques. Since there is electro­ half ("the monocular crescent") receives an exclusively contralateral input. physiological evidence that each eye has a preferential columnar projection towards the cortex (Pettigrew, 1979), Horseradish peroxidase {HRP) injections in V2 also monocular lid suture was performed in two baby owls to revealed a direct input to that area from the medial enhance the columnar projection to the cortex (Le Vay, 14 interlaminar nucleus (MIN) and LGN and also from layers Stryker and Shatz, 1978). Three months later, r CJ­ 2, 3 and 5 of an area medial to the striate cortex. proline was injected in the deprived LGN in order to trace Monocular deprivation for three months (starting from the projections toward the visual cortex. Autoradio­ three weeks) caused cell shrinkage which was very graphic techniques were carried out in serial sections of 137

the brain. The examination of the sections revealed the clear cases of axons ending very superficially in the presence of four to five bands of labeling in the granular supragranular layer of the cortex. This new finding layer, alternate with areas without labeling. The width of further supports the strong parallel between mammalian the bands measure 500 to 600 µm wide, while the areas and avian cortical visual processing, since avian cortico­ without labeling measure 800 to 850 µm wide. This geniculate neurons lie superficially and could thus receive finding supports the neurophysiological studies in the owl's direct thalamic input like their mammalian (infragranular) visual cortex (Pettigrew, 1979) and shows that, as seen in counterparts. some mammals, segregation of the inputs to cortex from each eye is altered by visual deprivation.

References: Bravo, H. and Pettigrew, J. D. (1980) Proceedings of the Topical Meeting on Recent Advances in Vision. Optical Society of America, Sarasota, May 1980, p. Th A7. Le Vay, S., Stryker, M. P. and Shatz, C. J. (1978) J. Comp. Neurol. 179: 223-244. Pettigrew, J. D. (1979) Proc. Roy. Soc. Lond. B 204: 435-454.

198. FURTHER ANATOMICAL FEATURES IN THE Figure 2. Corona! section of a burrowing ow! brain VlSUAL SYSTEM OF OWLS (Speotyto cunicularia). J;E• right lateral geniculate nucleus was injected with [ C]pro!ine. The ipsi- and con­ Investigators: Hermes Bravo, John D .. Pettigrew tralateral projections to the visual cortex end in three Patterns of retinal, lateral geniculate nucleus (LGN) targets: (1) the granular layer or intercalatus hyper­ striatum accessorium (IHA) which has two sublayers, one and tectum opticum (TO) connections in the visual system internal and the other external; (2) the infragranular layer of owls, Speotyto cunicularia and Tyto alba, have been or hyperstriatum dorsale (HD) and (3) the supragranular layer or hyperstriatum accessorium (HA). The gap compared, using retrograde and anterograde anatomical without labeling shown in the left side of the figure is the tracing techniques. area of monocular representation of the ipsilateral visual field. In both species of owl, the bulk of the ganglion cells projecting to the LGN is distributed in the temporal 199. BINOCULAR FACILITATION AND INIDBITION retina, mainly around the fovea (Speotyto) or in the area IN THE LATERAL GENICULATE NUCLEUS centralis (Tyto). Isodensity lines of ganglion neurons form OF THE CAT irregular concentric circles, with the highest densities at Investigators: Kenneth L. Marton, John D. Pettigrew the center (16,000 cells/mm2 for Speotyto and 4000 The lateral geniculate nucleus (LGN) relays visual cells/mm2 for Tyto). The subpopulation of ganglion cells information from the retinas to the cortex, segregating which produces the horizontal visual streak did not appear input from each eye into separate laminae. The LGN to send projections to the LGN in either species. receives an equally large input back from the visual Retinal ganglion cells projecting to the tectum have a cortex, whose cells are driven from both eyes. Therefore, preferential distribution in the horizontal streak. Cells binocular interactions in the LGN were studied by with projections to tectum are also found in the temporal systematically varying visual stimuli known to fire retina, but with different density and morphology com­ cortical neurons. Binocular and monocular responses were pared with those projecting to LGN. compared by interleaving them using computer-driven Highest densities after tectal injections were 5000 shutters in order to eliminate errors due to LGN cell 2 2 cells/mm for Speotyto and 11,000 cells/mm for Tyto. response variability. Full statistical analysis was used to Anterograde transport of r14cJproline from the LGN identify significant binocular facilitation and inhibition. shows that the granular layers of the visual cortex are the Significantly more and stronger binocular feedback principal target. We also found light labeling of both the (BF) was seen with this approach than in previous studies. infragranular layers and supragranular layers of the cortex The vast majority of LGN cells showed both binocular (see Figure 2). This pattern was confirmed with antero­ facilitation and inhibition; as many as half showed BF grade horseradish peroxidase (HRP) tracing; here, we saw amplitudes exceeding 50% of their typical monocular 138

firing rate. Importantly, BF was found to be well-tuned to with the pattern of binaural interaction bands demon­ velocity, relative retinal disparity, and sweep direction, strated with diotic stimulation (Middlebrooks, Dykes and parameters known to profoundly affect cortical firing. Merzenich, 1980). Multiple regions of BF were found for most cells, with the In correlative acoustical experiments, a probe majority located near the monocular receptive fields in microphone was introduced from behind the pinna into the visual space. Regions of facilitation required zero retinal acoustic meatus. Sound levels were recorded while disparity twice as often as inhibition. Further, most BF varying the location of the loudspeaker. Isointensity reached maximum amplitudes at 6°/sec to 12°/sec. These contour plots were derived and compared with the rf's results are a strong indication that the BF is cortical in determined physiologically. Many of the characteristics origin. of the measured rf's could be inferred from such contour It is likely that this BF has a role in highlighting visual plots, but neither the H nor the A rf!s can be explained features on the plane of fixation. Because BF is very entirely by the passive acoustics of the head and pinna. sensitive to parameters of motion, it is also conceivable There was no indication of an orderly map of sound that it is involved in interpreting the visual signals space in AI of the cat. All well-circumscribed rf's lay on generated by eyes constantly in motion. the acoustical axis of the pinna. The results suggest the presence of at least three independent processing systems 200. FUNCTIONAL CLASSES OF AUDITORY in AI. One system, the O units, does not appear to play a CORTICAL NEURONS DISTINGUISHED BY SENSITIVITY TO SOUND LOCATION role in sound localization. The H units may be involved in Investigators: John C. Middlebrooks*, the approximate localization of sounds, while the A units John D. Pettigrew are suited for active scrutiny of relevant sound stimuli. We have explored the possible role of the primary References: auditory cortex (AI) of the cat in the representation of Knudsen, E. K., Konishi, M. and Pettigrew, J. D. (1977) sound location. We determined the direction (location) Science 198: 1278-1280. Middlebrooks, J. C., Dykes, R. W. and Merzenich, M. sensitivity of cortical neurons and compared these (1980) Brain Res. 181: 31-48. properties with the passive acoustics of the cat's head and *Department of Physiology, University of California, San pinna. Francisco, California. Tonal stimuli were generated by a loudspeaker that could be positioned in any direction a constant distance 201. CELL SHRINKAGE IN GENICULATE NEURONS from the animal; the animal and loudspeaker were FOLLOWING BRIEF UNILATERAL BLOCKADE enclosed in an anechoic chamber (Knudsen, Konishi and OF RETINAL GANGLION CELL ACTIVITY Pettigrew, 1977). The activity of single units was Investigators: Baruch Kuppermann, Takuji Kasamatsu recorded from AI of cats lightly anesthetized with Tonic background activity in visual afferents is Ketamine. The spatial receptive fields (rf's) of neurons indispensable for maintenance of synapses in the adult were plotted by noting the area of the sound field in which brain (Lund and Lund, 1971). By brief reversible blockade a tone of characteristic frequency elicited a reliable of tonic retinal discharges, the functional connectivity of response. Units were grouped into three classes on the geniculo-cortical synapses can be modified in binocular basis of spatial location sensitivity. Omnidirectional units cortical cells of kittens (Kasamatsu, 1976). (O units) responded to sounds presented anywhere in the Currently, we have studied morphological changes in sound field. Hemifield units (H) responded only to sounds the dorsal lateral geniculate nucleus (LGN) and physio­ presented in the contralateral sound hemifield. Axial logical changes in the primary visual cortex of kittens units (A) had circular or elliptical rf's (as small as 30") induced by monocular blockade of tonic retinal activity. located on the acoustical axis of the contralateral pinna We injected tetrodotoxin (TTX) intraocularly, 10 µg per (approximately 30° lateral to the· midline and 10-20° dose given twice, 3 days apart, to 6-week-old kittens. above horizontal). In radial electrode penetrations, all TTX reversibly blocks ganglion cell firing in the retina. units tended to show the same class of rf. Along Five kittens received unilateral intraocular injections of tangential penetrations, sequences of O, H, and A units TTX and were kept either in the light or in a dark room were segregated from each other in a manner consistent for a week-long period of TTX-induced monocular 139

deprivation. After one week, histological examination of ment (T. Kasamatsu and P. Heggelund, unpublished). the celloidin-embedded LGN showed cell shrinkage in the These two findings led me to study effects of direct deprived laminae in the animals kept both in the light and manipulation of the intracellular cyclic nucleotides. An in the dark. The magnitude of cell shrinkage in deprived increase in intracellular cyclic AMP, for example, is laminae of the dark-animals was remarkable despite the expected to be correlated with enhancement of synaptic brief, one-week blockade of tonic retinal discharges. Cell plasticity. Preliminary studies with dibutyryl cyclic AMP shrinkage in the deprived monocular segment was also have produced results which seem to be consistent with observed, as expected with a pure deprivation effect my hypothesis (Kasamatsu, 1980). (Hickey, Spear and Kratz, 1977). References: Single unit recordings were made on five kittens Kasamatsu, T. (1979) ARVO 1979 Abstracts, p. 135. Kasamatsu, T. (1980) Soc. Neurosci. Abstracts, 6. In subjected to unilateral intraocular TTX (10 µg per dose in press. 2 injections 3 days apart) and kept in the light or in a dark room until the recording session. The duratio_n of effects 203. SINGLE CELL RESPONSES IN CAT VISUAL of TTX on ganglion cell firing was determined by CORTEX: THElR MODULATION BY IONTOPHORESIS OF NE physiological recording in the cortex. The end of the Investigators: Paul Heggelund*, Takuji Kasamatau effect coincided with reappearance of the pupillary reflex of the injected eye. The period of suppression was found Single cells in the cerebral cortex may increase or to last from 11 to 13 days. In all recordings, a striking decrease their firing frequency in response to norepineph­ loss of binocular cells was observed, with a larger number rine (NE) applied iontophoretically (Krnjevic, 1964). Most of cells driven exclusively through the contralateral eye previous studies were concerned with changes in the level than through the ipsilateral eye. of "spontaneous" activity during application of NE. How­ In summary, we found that total blockade of tonic ever, the majority of cortical cells especially in cat visual retinal discharges could induce striking morphological cortex has a very low spontaneous firing rate (the mean changes in the LGN after only one week of treatment and and SD, 2.8 ± 4.9/sec in resting arousal; the median, also resulted in a decrease of the number of binocular 0.75/sec) (Kasamatsu and Adey, 1974). Therefore, it cells in the cortex. These changes were found in animals seems to be more sensible to measure visually-evoked which were kept in the dark as well as in the light discharges against which effects of iontophoretically­ throughout the TTX treatment. applied NE can be estimated properly. A minicomputer controlled the presentation of optimal visual stimulus References: Hickey, T. L., Spear, P. D. and Kratz, K. E. (1977) J. sweeping across the receptive fields of individual cells and Comp. Neurol. 172: 265-282. also did the collection of subsequent spike trains. Kasamatsu, T. (1976) Exptl. Brain Res. 26: 487-494. Lund, R. D. and Lund, J. S. (1971) Science 171: 804-807. We found three classes of responses by cortical cells in response to exogenous NE: enhancement, suppression and 202. CYCLIC NUCLEOTIDES IN THE SYNAPTIC indifference. PLASTICITY OF VISUAL CORTEX References: Investigator: Takuji Kasamatsu Kasamatsu, T. and Adey, W. R. (1974) Physiol. Behav. 13: 101-112. I have suggested a possible role for B-adrenergic KrnjeviC, K. (1964) Internat. Rev. Neurobiol. 1: 41-98. receptors in the synaptic plasticity in kitten visual cortex *Neurobiological Laboratory, University of Trondheim, (see Biology 1979, No. 199). Continuous perfusion of the Trondheim, Norway. visual cortex with a B-adrenergic receptor blocker, propranolol, blocked the ocular dominance shift which 204. BRIEF PERFUSION OF NOREPINEPHRINE INCREASED THE NEURAL PLASTICITY usually follows monocular lid suture (Kasamatsu, 1979). IN KITTEN VISUAL CORTEX A recent study has shown that localized perfusion of a Investigators: Takuji Kasamatsu, Paul Heggelund* B-adrenergic receptor stimulant, isoproterenol, restored synaptic plasticity to visual cortex which had lost A high level of norepinephrine (NE) (0.5 mM in the plasticity due to the prior depletion of intracortical cannula-minipump) facilitated the synaptic plasticity in catecholaminergic terminals by 6-hydroxydopamine treat- kitten visual cortex as determined in experimental 140

paradigms which led either to an increase (recovery from Reference: Itakura, T., Kasamatsu, T. and Pettigrew, J. D. (1980)­ monocular deprivation) or a decrease (monocular depriva­ Neuroscience. Submitted for publication. tion) in the number of normal binocularly driven cells. It was shown that if a sufficient amount of NE was available 206. COEXISTENCE OF CATECHOLAMINERGIC to postsynaptic cells, changes in the ocular dominance TERMINALS AND ACETYLCHOLINESTERASE­ SENSITIVE NEURONS IN CAT VISUAL CORTEX distribution could take place very quickly (<24 hr) in Investigators: Kunio Nakai, Takuji Kasamatsu response to brief alterations of binocular afferents. Normal kittens and cats were prepared for single cell A previous study demonstrated the rich innervation of recordings. Twenty-four hours after repeated iontopho­ cat visual cortex with catecholamine-containing fibers retic applications of NE in the visual cortex, the and terminals (ltakura, Kasamatsu and Pettigrew, 1980). proportion of normal binocular cells decreased signifi­ Single cells in the neocortex change their activity in cantly. The result was interpreted as due to the exposure response to iontophoretically injected acetylcholine. A of highly plasticized synapses to discordant binocular recent study, using an indirect immunohistochemical input resulting, in the paralyzed preparation, from the approach, showed an equally rich innervation of cat visual misalignment of the normally converged visual axes (cf. cortex with choline acetyltransferase-sensitive terminals squint effect of Hubel and Wiesel, 1965; Heggelund and throughout the cat neocortex (H. Kimura et al., personal Kasamatsu, 1980). communication). We are interested in studying by light and electron microscopy a possible interaction of these References: Heggelund, P. and Kasamatsu, T. (1980) Proceedings of two neurotransmitter systems in cat cerebral cortex. As the Satellite Symposium of the 29th International Congress of Physiological Sciences on "Cellular a first step, we are trying to establish a method to analogues of conditioning and neural plasticity." In visualize simultaneously both catecholamine terminals and press. acetylcholinesterase-containing neurons in the Hubel, D. H. and Wiesel, T. N. (1965) J. Neurophysiol. 28: same 1041-1059. sections. This is a modification of the method developed for the blood vessels in the brain (Imai et al., 1980). *Neurobiological Laboratory, University of Trondheim, Trondheim, Norway. References: Imai, H., Nakai, K., Kamei, I., Itakura, T., Komai, N., Kimura, H., Nagai, T., Imamoto, K. and Maeda, T. 205. MORPHOLOGY OF CATECHOLAMINERGIC (1980) Cell. Molec. Biol. In press. TERMINALS IN KITTEN VISUAL CORTEX Itakura, T., Kasamatsu, T. and Pettigrew, J. D. (1980). Investigators: Ktmio Nakai, Talruji Kasamatsu Neuroscience. Submitted for publication.

The distribution pattern of norepinephrine-containing terminals in kitten visual cortex has been studied pre­ 207. REGROWTH OF CATECHOLAMINE TERMINALS IN CAT VISUAL CORTEX FOLLOWING viously (Itakura, Kasamatsu and Pettigrew, 1980). As an LOCALIZED LESIONS extension of the above study, we are further studying the Investigators: GOsta Jonsson*, Toru Itakura**, spatial relationship between identified aminergic terminal Takuji Kasamatsu boutons and identified neuronal elements of the visual Monoamine systems are known for their vigorous cortical neurons in area 17, under electron microscopy. capability of terminal regrowth following lesions, if the The former are tagged by intraventricular or local somatas and the proximal part of axons are kept intact injection of catecholamine-specific vesicle markers such {Moore, BjOrklund and Stenevi, 197 4; Sachs and Jonsson, as 5-hydroxydopamine or 6-hydroxydopamine. The latter 1975). are labeled by the electron dense reaction product of First, kittens were implanted with a cannula-minipump peroxidase following extracellular microinjection of which contained 4 mM 6-hydroxydopamine (6-0HDA) at horseradish peroxidase (HRP) into known structures in the various postnatal days (newborn./'13-week-old). O, 2, 4, 6 central visual pathways. The lateral geniculate nucleus, and 16 weeks after the end of 6-0HDA perfusion which the superior colliculus and area 18 of the visual cortex are lasted for a week, the kittens were subjected to either injected with HRP to label, by retrograde transport, catecholamine (CA) fluorescence histochemistry {Itakura, neurons in layer 6, layer 5, and layers 2 end 3, Kasamatsu and Pettigrew, 1980) or biochemical assay of respectively. endogenous monoamines using high pressure liquid 141 chromatography combined with an electrochemical regis­ both the initial lid suture and the later reverse suture; tration (Keller et al., 1976). The preliminary results have here we obtained some cells (20%) which responded in shown shrinkage of the size of the CA-depleted area in favor of stimulation of the initially deprived eye. proportion to the survival time of animals after 6-0HDA References: perfusion. Blakemore, C. and Van Sluyters, R. (1974) J. Physiol. 237: We are also trying to establish a profile of normal 195-216. Wiesel, T. N. and Hubel, D. T. (1963) J. Neurophysiol. 26: changes in endogenous monamines in kitten visual cortex 1003-1017. as kittens mature postnatally. *Smith-Kettlewell Institute of Visual Sciences, San References: Francisco, California. Itakura, T., Kasamatsu, T. and Pettigrew, J. D. (1980) Neuroscience. Submitted for publication. Keller, R., Oke, A., Mefford, I. and Adams, R. (1976) Life Sci. 19: 995-1004. Moore, R., BjOrklund, A. and Stenevi, U. (1974) The Neurosciences. Third Study Program, pp. 961-977. PUBLICATIONS The MIT Press, Cambridge, Massachusetts. 8achs, C. and Jonsson, G. (1975) In: Chemical Tools in Blasdel, G. G. and Pettigrew, J. D. (1979) Degree of Catecholamine Research, G. Jonsson, T. Malmfors and interocular synchrony required for maintenance of Ch. Sachs (Eds.), VoL 1, pp. 163-171. North Holland binocularity in kitten's visual cortex. J. Neurophysiol. Publishing Co., Amsterdam. 42: 1692-1710. Bravo, H. and Pettigrew, J. D. (1980) Further anatomical *Department of Histology, Karolinska Institute, Stock­ features in the visual system of owls. In: Recent holm, Sweden. Advances in Vision. Technical Digest, p. Th AS, **Department of Neurosurgery, Wakayama Medical Sarasota. College, Wakayama, Japan. Cooper, M. L. and Pettigrew, J. D. (1979) The decussation of the retinothalamic pathway in the cat with a note on the major meridians of the cat's eye. J. Comp. 208. TOTAL DEPRIVATION OF VISUAL AFFERENTS Neurol. 187: 285-311. FROM ONE EYE: A COMPARISON WITH Cooper, M. L. and Pettigrew, J. D. (1979) The retino­ CONVENTIONAL MONOCULAR LID SUTURE thalamic pathways in Siamese cats. J. Comp. Neurol. Investigators: Takuji Kasamatsu, filbert Magoon*, 187: 313-348. Gillard-Crewther, S., Crewther, D., Peck, C. and Arthur Jampolsky* Pettigrew, J. D. (1980) The visual cortical effects of Monocular lid suture (Wiesel and Hubel, 1963) has been rearing cats with monocular and binocular cyclo­ torsion. J. Neurophysiol. In press. used as a simple way to induce imbalance between the two Heggelund, P. and Kasamatsu, T. (1980) Exogenous sets of visual afferents impinging on binocular cortical noradrenaline increases the neuronal plasticity in cat visual cortex: Localized, continuous microperfusion cells. A clinical observation of patients with amblyopia and iontophoresis. In: Proceedings of the Satellite suggests that an incomplete or partial patching of one eye Symposium of the 29th International Congress of Physiological Sciences on "Cellular Analogues of Con­ of children may produce much more profound effects in ditioning and Neural Plasticity", o. Feher and F. JOO later life on the reversibility of the effects of monocular (Eds.). Szeged, Hungary. In press. Imai, H., Nakai, K., Kamei, I., ltakura, T., Komai, N., deprivation than total depletion of visual afferents by the Kimura, H., Nagai, T., Imamoto, K. and Maeda, T. complete occlusion of one eye. The occlusion-amblyopia (1980) Simultaneous detection method of amine and acetylcholine esterase containing nerve fiber in pial may in fact be much more remedial than the diffusion­ vessels. 1. Light microscopic study, and 2. Electron amblyopia. Physiological recordings have been carried out microscopic study. Cell. Molec. Biol. In press. Itakura, T., Kasamatsu, T. and Pettigrew, J. D. (1980) to test the above hypothesis in kitten visual cortex. We Norepinephrine terminals in kitten visual cortex: employed the reverse-suture paradigm (Blakemore and Laminar distribution and ultrastructure. Submitted for publication. Van Sluyters, 1974) as a test for the reversibility of the Kasamatsu, T. (1980) A possible role for cyclic nucleotides effects of monocular deprivation. In our preliminary in plasticity of visual cortex. Soc. Neurosci. Abstracts, 6. In press. studies, we found no cells responding to stimulation of the Kasamatsu, T. (1980) Central noradrenaline fibers in previously deprived eye 5 weeks after the reverse depriva­ neocortical plasticity. In: Proceedings of the Satellite Symposium of the 29th International Congress of tion which took place at 9 weeks of age. In this kitten, Physiological Sciences on "Visual Mechanisms in Pri­ one eye was initially lid sutured with a clear soft mates and Lower Mammals" (Abstract). Exptl. Brain con~t Res. Suppl. In press. lens and was later reverse sutured with an opaque Kasamatsu, T., ltakura, T. and Jonsson, G. (1980) Intra­ (pigmented) lens. This result contrasts with that found in cortical spread of exogenous catecholamines: Effective concentration for modifying cortical the other kitten in which an opaque soft lens was used for plasticity. In preparation. 142

Kasamatsu, T., Pettigrew, J. D. and Ary, M. (1980) Pettigrew, J. D. (1980) Co-evolution of nocturnal v1s1on Cortical recovery from effects of monocular depriva­ and frontally-placed eyes? In: Recent Advances in tion: Acceleration with norepinephrine and Vision. Technical Digest, p. Th AS, Sarasota. suppression with 6-hydroxydopamine. Submitted for Pettigrew, J. D. (1980) The two foveae of the hawk's publication. retina subserve two visual pathways. In: Recent Kuppermann, B. and Kasamatsu, T. (1980) Cell shrinkage Advances in Vision. Technical Digest, p. SB 13, in geniculate neurons following brief unilateral block­ Sarosota. ade of retinal ganglion cell activity. Soc. Neurosci. Presti, D. and Pettigrew, J. D. (1980) Ferromagnetic Abstracts, 6. In press. coupling to muscle receptors as a basis for geo­ Lipson, E. and Presti, D. (1980) Graphical estimation of magnetic field sensitivity in animals. Nature 285: cross sections from fluence-response data. Photo­ 99-101. chem. Photobiol. In press. Ramachandran, V. S. and Ary, M. (1980) Brief periods of Marton, K. L. (1980) Binocular facilitation and inhibition visual experience can modify ocular dominance of in the lateral geniculate nucleus of the cat. Ph.D. cortical neurons in kittens. Soc. Neurosci. Abstracts, Thesis, California Institute of Technology. 6. In press. Middlebrooks, J. and Pettigrew, J. D. (1980) Functional classes of auditory cortical neurons distinguished by sensitivity to sound location. Soc. Neurosci. Abstracts, 6. In press.

Professor: Roger W. Sperry Sherman Fairchild Distinguished Scholar: Donald M. 209. SOMATOGNOSTIC ABJLfflF.S OF THE RIGHT Mac Kay AND LEFT CEREBRAL HEMISPHERF.S Research Associate: Charles R. Hamilton Del Webb Visiting Associate: Evelyn L. Teng Investigators: Gilles Plourde, R. W. Sperry Visiting Associates: Ronald L. Meyer, Eran Zaidel It is accepted that in right-handed persons certain Researeh Fellow: R. Gilles Plourde Graduate Students: Alice M. Cronin, Karen E. Gaston, types of body schema disorders characteristically result Larry E. Johnson, Jay J. Myers, Betty A. Vermeire from lesions of the right cerebral hemisphere while other Research Staff: Eef Goedemans, Lois E. MacBird, Edward Ogawa, Julia J. Wolfe types appear mainly following left-sided lesions. For example, hemiasomatognosia (unawareness of the left Support: The work described in the following research reports has been supported by: hemi-body) results from right hemisphere damage while Earle C. Anthony Fellowship autotopagnosia (loss of the ability to localize or name Biomedical Research Support Grant (NIH) Fairchild Foundation body parts) results from left-sided lesions. These Frank P. Hixon Fund generalizations have been based principally on symptoms Medical Research Council of Canada National Institute of Mental Health, USPHS produced by unilateral cerebral lesions. lt has been National !ntitutes of Health, USPHS suggested that the picture derived from unilateral lesions National Science Foundation Pew Memorial Trust may be misleading in regard to the functional potential of Del E. Webb Foundation the intact hemisphere because the damaged hemisphere Summary: Work in psychobiology deals with the higher may be transmitting suppressive influences that prevent behavioral functions of the nervous system and has expression of latent functions otherwise present in the continued to focus on problems related to the following: intact hemisphere. An investigation of body schema (1) hemispheric specialization and functional organizatjon disorders was therefore undertaken in patients with in human commissurotomy, hemispherectomy and other complete section of the forebrain commissures where the neurosurgical patients with occasional study of normal same complications are not present and where each subjects and children; (2) mechanisms and pathways for disconnected hemisphere can be assessed separately. visual processing and interhemispheric transfer and recall Results showed that the right hemisphere of these of visual information in cats and monkeys and the problem patients could readily identify and recognize bodily parts of hemispheric functional asymmetry in monkeys; (3) and that their left hemisphere could easily obey factors underlying learning and interocular transfer of instructions ("raise your left hand," "touch your left ear") conditioned food aversion in newly hatched chicks; and (4) that patients with hemiasomatognosia often fail to conceptual developments in mind-brain relations and execute. The findings reinforce the contention that, in implications for value theory. the presence of commissures, a cerebral lesion restricted 143

to one hemisphere may impair the functioning of the readily accounted for by the neglect tendency itself. It intact hemisphere. It is inferred that the classical body was also found that the seven patients who did not show schema disorders described above arise from the defective neglect on clinical examination likewise chose most of function of the brain as a whole rather than, as long . their answers on the right side of the page while the assumed, from an intrinsic inability of the undamaged answers given by the controls were equally distributed on hemisphere. both sides. These findings suggest that this test may also be used to detect subtle neglect tendencies.

210. UNILATERAL SPATIAL NEGLECT *Medical Director, Casa Colina Hospital for Rehabilita­ AND SPATIAL DISORIENTATION tive Medicine, Pomona, California; Adjunct Associate Investigators: Gilles Plourde, Robert M. Tager* Professor, Gerontology, Unive1•sity of Southern California, Los Angeles, California. The syndrome of unilateral spatial neglect consists of a lack of awareness for one half of surrounding space and generally results from lesions of the right cerebral 211. GOAlrCONFIJCT IN SPIJT BRAINS Investigators: Donald M. MaeKay, Valerie MacKay• hemisphere. Patients showing neglect may, for instance, draw only the right side of a clock or daisy, fail to pick up The aim of this project was to investigate the types of objects on the left side, or even fail to eat the food on the goal-conflict that can arise between the left and right left half of their plate. When tested by conventional halves of a callosum-sectioned human brain. Work by procedures, such patients tend to have more severe spatial R. W. Sperry and collaborators (Sperry, 1974) has estab­ cognitive deficits than other patients with right hemi­ lished that in such cases each hemisphere can pursue sphere lesions but without neglect symptoms. However, different goals and show diametrically opposed since patients with neglect ignore the left half of the preferences for target objects. Sperry (1966) has referred testing material, it is difficult to determine whether their to this as "having two free wills ••• inside the same cranial inferior performance results from neglect itself or from a vault." more pronounced deficit of cognitive spatial abilities. The present experiments were designed to make it This study attempts to clarify this question by diminishing possible for one hemisphere-system to engage in various the effect of neglect on the evaluation of spatial abilities. forms of active struggle with the other, in order to see at A spatial relation test that can be correctly performed how deep a level signs of "twoness of will0 could be even if the subject ignores the left half of the testing demonstrated (MacKay, 1980). The plan was first to train material was therefore devised. The test requires the each hemisphere-system in turn (or simultaneously) to patient to perform various mental rotations of a geo­ pursue goals in active conflict with an experimenter; and metric pattern composed of dissimilar right and left then to cross-couple the signals to and from the two half­ halves. However, the right half of each pattern contains systems so that each could now engage in conflict with enough information to allow the patient to perform the the other. task. The patient must select the answer from two It was found that the two hands could in this way be multiple-choice arrays in which the correct answer is induced systematically to struggle with one another in presented on both the right and left halves of the_ page. pursuit of disparate physical goals flashed to left and right The test therefore permits independent evaluation of visual half-fields. Left and right digits could also be used spatial abilities and of neglect tendency. simultaneously to signal the results of different Ten patients with right cerebral hemisphere damage, arithmetical calculations flash-presented simultaneously of whom three showed unilateral spatial neglect, have to the two half-fields. thus far been tested along with five controls. Patients On the other hand, although the subject's left hand with neglect chose practically all their answers on the could be trained to rate an experimenter's verbal descrip­ right side of the page and although the design of the test tions of visual stimuli presented to the left half-field as improved their overall score, they still performed worse "correctn or "incorrect,11 it showed no similar disposition than most patients with no neglect. These preliminary to rate the subject's own verbal guesses (controlled findings suggest that unilateral neglect is associated with presumably by the left hemisphere). In competitive games more pronounced spatial cognitive deficits that are not pitting left hand against right, moves on both sides 144

seemed controlled by the speaking hemisphere. than RVF stimuli, irrespective of accuracy. 1n summary, although the two half-systems in split­ None of the four patients tested was able to success­ brain subjects could grapple with one another in pursuit of fully cross-compare bilateral facial photographs as conflicting goals, and evaluate simultaneously-perceived expressing the same emotion or not. In addition, although situations accordi!}g to conflicting criteria, they gave no the subjects also had difficulty comparing facial emotions evidence of ability to engage in mutual conflict at the during trials with intrahemispheric controls (two photos level of criteria of evaluation. Such "twoness of will" as within one visual field), all nevertheless responded more was suggested by the evidence seemed confined to the accurately during LVF control presentations, and LVF executive rather than the normative level. responses were again as fast as or faster than RVF responses. References: MacKay, D. M. (1980) In: Consciousness and the Physical These results support other studies suggesting that the D. World, B. Josephson and V. S. Ramachandran (Eds.), right hemisphere is specialized for facial and emotional pp. 95-113. Pergamon Press. Sperry, R. W. (1966) In: Brain and Conscious Experience, recognition. Several commissurotomy subjects are more J. C. Eccles (Ed.), 293-313. Springer-Verlag, New pp. accurate in identifying LVF facial emotions than other York. Sperry, R. W. (1974) ln: Third Neurosciences Study kinds of stimuli (e.g., numbers or letters) but these results Program, F. 0. Schmitt and F. G. Worden (Eds.), pp. do not explain the ability of some subjects to name either 5-20. MIT Press, Cambridge. stimuli from large sample populations or stimuli that are *Department of Communication and Neuroscience, not easily or naturally labeled emotionally. University of Keele, Staffordshire, ST5 5BG England.

213. DICHOTIC EAR DlFFERENCE 1S A POOR 212. IDENTIFlCATION AND CROSS-COMPARISON MEASURE OF HEMISPHERIC DOMINANCE OP F ACl'll BY HUMAN COMMISSUROTOMY PATIENTS lnvestigator: Evelyn Lee Teng lnvestigator: Larry E. Johnson The dichotic input procedure involves simultaneous Some forebrain commissurotomy patients can cross­ presentations of two different stimuli, one to each ear, compare bilateral visual stimuli presented close to the via stereophonic headphones. It has been widely used to fixation point and name stimuli presented exclusively measure hemispheric dominance, where the obtained ear within the left visual field (LVF) (Biology, 1978, Nos. 197, advantage on a given task is taken to indicate the 198, 199; Biology, 1979, No. 215). Earlier studies on these dominance of its processing by the contralateral hemi­ patients have suggested that information about emotional sphere. stimuli may transfer between the "disconnected" hemi­ The validity and the reliability of the dichotic ear spheres. Perhaps the ability of these patients to correctly difference score are critically examined. It is shown that name LVF stimuli is due to the transfer of affective a given hemispheric dominance may be associated with information from the right hemisphere to left hemisphere various degrees and even different directions of ear vocal centers? To test this hypothesis, schematically advantage, depending on the extent and direction of input drawn faces and photographs of human faces representing asymmetry. Common interest has concentrated on indi­ three emotions (happy, angry, surprised) were selected to vidual variations in hemispheric asymmetry, to the examine whether split-brain patients could name visual general neglect of natural variations in input asymmetry. stimuli thought to have a high emotional content faster or Neurological evidence is cited to show that some more accurately than other kinds of stimuli. individuals do have a reverse dominance of the ipsilateral Three of four split-brain subjects (NG, LB, AA) were over the contralateral ear-to-hemisphere input. Numer­ able to name unilaterally presented (150 msec) LVF ical illustrations are presented to demonstrate that schematic faces, and one (LB) was able to name the individual variations in ear advantage are in fact attribut­ emotion on LVF human facial photographs. Unlike able more to input asymmetry than to hemispheric previous findings with non-facial stimuli where split-brain asymmetry. patients were found to name LVF stimuli significantly Empirically, a study on the reliability of dichotic ear more slowly than right visual field (RVF) stimuli, LVF advantage on four commonly used verbal tests has been facial stimuli are generally named as fast as or faster conducted. The results show that ear advantage on one 145

test has little predictive value for either results on information transsynaptically from more peripherally another test given in the same test session, or results on located cells in the visual hemifield and relay this the same test given at another time. "digested" information to the opposite hemisphere by mechanisms more complex than those so far suggested by physiological recordings from these cells or their commis­ 214. FURTHER ANATOMICAL FEATURES OF INTERHEMISPHERIC CONNECTIONS sural axons. The anterior commissure is also capable of Investigators: Charles R. Hamilton, Gary Moel

two hemispheres of split-brain monkeys in their ability to PUBLICATIONS learn a task requiring comparison of two sequentially­ Dimond, S. J. (1979) Tactual and auditory vigilance in presented stimuli. Further analysis of these data with an split-brain man. J. Neurol. Neurosurg. Psychiat. 42: 70-74. increased number of subjects now shows that even though Ellenberg, L. and Sperry, R. w. (1979) Capacity for the overall mean hemispheric difference for this task is holding sustained attention following commissurotomy. Cortex 15: 421-438. not significantly different from zero, a significant dif­ Gaston, K. E. (1978) Interocular transfer of pattern ference is found between the hemispheres contralateral discrimination learning as a function of age in the developing chick. Soc. Neurosci. Abstracts 4, No. 803, and ipsilateral to the preferred hand as determined p. 259. preoperatively. Specifically, left-handed monkeys learned Gaston, K. E. (1979) Differential food aversion learning by faster with the right hemisphere and right-handed the two half-brains of monocularly trained chicks. Soc. Neurosci. Abstracts 5, No. 1040, p. 316. monkeys learned faster with the left hemisphere. Thus, Gaston, K. E. (1979) Lack of interocular transfer of pattern discrimination learning in chicks. Brain Res. the hemisphere opposite the preferred hand appears 171: 339-343. dominant for processing sequentially-presented stimuli, Gaston, K. E. (1980) Evidence for separate and concurrent which is the reverse of the results reported in the avoidance learning in the two hemispheres of the normal chick brain. Behav. Neural Biol. 28: 129-137. preceding abstract for processing pictorial information. Greif, K. and Scott, M. Y. (1980) Interocular transfer of color discrimination after tectal lesions in goldfish. Thus monkeys, like man, appear from these two studies to Exptl. Neurol. 67: 504-512. have some degree of hemispheric specialization, the Greif, K. and Scott, M. Y. (1980) Intraretinal transfer of a color discrimination task after tectal and telenceph­ direction of which is similar to that of man if handedness alic lesions in goldfish. Exptl. Neurol. 67: 492-503. of the subjects is taken into account. Johnson, L. E. (1979) Left visual field identification and bilateral cross-integration in split-brain humans. Soc. 218. RECOGNITION OF INDIVIDUAUI AND FACIAL Neurosci. Abstracts 5, No. 1048, p. 318. EXPRESSION BY THE TWO HEMISPHERES OF Meyer, R. L. (1978) Evidence from thymidine labeling for SPLIT-BRAIN MONKEYS continuing growth of retina and tectum in juvenile goldfish. Exptl. Neurol. 59: 99-111. Investigators: Charles R. Hamilton, Anne L. Erdmann•, Meyer, R. L. (1979) Retinotectal projection in goldfish to Betty A. Vermelre an inappropriate region with a reversal in polarity. Science 205: 819-821. Split-brain rhesus monkeys learn to discriminate Meyer, R. L. (1980) Mapping the normal and regenerating photographs of the faces of conspecifics about equally retinotectal projection of goldfish with autoradio­ well when using either their left or their right hemisphere graphic methods. J. Comp. Neurol. 189: 273-289. Meyer, R. L. (1980) Ordering of retinotectal connections: (Biology 1976, No. 154), unlike human subjects in whom A multivariate operational analysis. Current Topics in Develop. Biol. In press. facial recognition is performed better by the right Peck, C. K., Crewther, S. G. and Hamilton, C. R. (1979) hemisphere. However, we now have preliminary evidence Partial interocular transfer of brightness and move­ that dominance for discriminating faces is positively ment discriminations by split-brain cats. Brain Res. 163: 61-75. correlated with the monkeys' weight, and therefore age, Peters, A. M. and Zaidel, E. (1980) The acquisition of homonymy. Cognition. In press. at the time of split-brain surgery. This suggests that, as Sperry, R. W. (1979) Bridging science and values: A in humans, laterality may develop with age. Future unifying view of mind and brain. Reprinted in Zygon experiments on lateralization will include a broader 14(1): 7-21. Sperry, R. W. (1979) Consciousness, freewill and personal spectrum of ages at surgery. identity. In: Brain, Behavior and Evolution, D. A. Evidence from human subjects indicates that the Oakley and H. C. Plotkin (Eds.), pp. 219-228. Methuen and Co., Ltd., London. recognition and assessment of facial expression and Sperry, R. W. (1980) Mind-brain interaction: Menta!ism, Yes; Dualism, No. Neuroscience 5(2): 195-206. emotion may be more strongly lateralized than is facial Sperry, R. W., Zaidel, E. and Zaidel, D. (1979) Self recognition of individuals. A series of experiments is now recognition and social awareness in the deconnected under way to study the relative abilities of the two minor hemisphere. Neuropsychologia 17: 153-166. Vermeire, B. A. and Hamilton, c. R. (1979) Selective hemispheres in recognizing specific facial expressions as attention in mirror image discriminatiOn by monkeys. well as individuals. Complementary experiments are Soc. Neurosci. Abstracts 5, No. 2748, p. 813. Vermeire, B. A. and Hamilton, C. R. (1980) Hemispheric directed toward studying the effects of unilateral lesions specialization in split-brain monkeys. Soc. Neurosci. Abstracts. In press. in various cortical visual areas of split-brain monkeys on these same discriminations.

*Undergraduate, California Institute of Technology. 147

Zaidel, E. (1979) Long-term stability of hemispheric Token Zaidel, E. (1980) Hemispheric intelligence: The case of Test scores following brain bisection and hemi­ the Raven Progressive Matrices. In: Intelligence and decortication. In: Auditory Comprehension: Clinical Learning, M. P. Frieman (Ed.), Proceedings of the and Experimental Studies with the Token Test, F. NATO Conference, York, England, July 16-20. Plenum Boller and M. Dennis (Eds.), pp. 135-159. Academic Press, New York. In press. Press, New York. Zaidel, E. (1980) The structuring of language: Clues from Zaidel, E. (1979) Of apes and hemispheres: Commentary hemispheric specialization. In: Signed and Spoken in the special issue on Cognition and Consciousness and Language: Biological Constraints on Linguistic Form, Nonhuman Species. Behav. Brain Sci. 1: 607-609. U. Bellugl and M. Studdert-Kennedy (Eds.). Dahlem Zaidel, E. (1979) On measuring hemispheric specialization Konferenzen, Weinheim/Deerfield Beach, FL/Basel, in man. In: Advanced Technobiology, B. Rybak (Ed.), Verlag Chemie. In press. pp. 365-403. Proceedings of the NATO-ASI, Paris, Zaidel, E. (1980) Performance on the ITPA following July 30, Noordhoff, Lejden. cerebral commissurotomy and hemispherectomy. Neuropsychologia. In press.

Associate Professor: David C. Van Essen ture, connections with other areas, or functional Research Fellow: William T. Newsome 111 properties of its constituent neurons. We suspect that the Graduate Students: John L. Bixby, Herman J. Gordon, John H. R. Maunsell complexities in topographic organization reflect impor­ Research Starr: Carol Shotwell tant aspects of. the information processing occurring in Support: The work described in the following research different areas. Hence, we intend to continue the analysis reports has been supported by: of this problem in conjunction with other experiments National Institutes of Health, USPHS National Science Foundation aimed at understanding the functional organization of Pew Memorial Trust Alfred P. Sloan Foundation different visual areas. A separate facet of the research in this laboratory summary: Our research on the primate visual system concerns the process of synapse elimination that occurs involves the use of anatomical and physiological tech­ during normal maturation of the neuromuscular junction. niques to characterize the higher visual areas in the Our major goal this past year has been to determine cerebral cortex of the macaque monkey. This past-lyear whether neuromuscular activity has any influence on the most of our effort has been directed at elucidating the likelihood that particular synaptic connections will survive location, organization and connections of four areas in the during the period when other connections are being lost; occipital lobe: V2, V3, MT (the Middle Temporal area) and our preliminary results suggest that activity may play a VP (the Ventral Posterior area). An important outcome of significant role in this process. We are hopeful that this these studies is the realization that the representation of experimental system will provide a valuable model for the visual field in higher cortical areas is fundamentally understanding the role of experience in the refinement of more complex than heretofore suspected. It has generally pathways in other parts of the nervous system. been presumed that each visual area in the cerebral hemisphere contains a complete, orderly representation of 219. AREAL BOUNDARIES AND the contralateral half of the visual field. However, TOPOGRAPffiC ORGANIZATION OF THE VENTRAL POSTERIOR AREA (VP) exceptions have been reported in other species, and we Investigators: William T. Newsome Ill, have found that none of the areas in the macaque John H. R. Maunsell, David C. Van Essen examined to date conform to this simple pattern. Some Last year (Biology 1979, No. 225), we reported a areas, in particular V3 and VP, contain only partial marked asymmetry in the projections of striate cortex in representations of the visual hemifield. Other areas, in the macaque monkey. Whereas dorsal striate cortex particular V2 and MT, contain representations which are projects to three cortical targets-V2, V3 and the Middle complete, but which are markedly disordered-much less Temporal area (MT)-ventral striate cortex projects only regular than is found, for example, in Vl, the primary to V2 and MT. We have studied the cortex adjacent to the visual cortex. Despite these irregularities, though, each ventral half of V2 and have found that it contains a well­ area is a well-defined entity whose borders can be defined visual area which we have called the Ventral accurately delineated on the basis of cortical architec- Posterior area (VP) because of its apparent homology with 148

VP in New World primates (Newsome and Allman, 1980). teristic pattern of myelination.. Our analysis has been VP has a long, narrow shape (25-30 mm by 2-3 mm) and greatly facilitated by the use of a new silver impregnation contains a topographic representation of only the upper stain which reveals a sharper, more consistent pattern of half of the visual field. Electrophysiological mapping myelinated fibers than is seen with conventional stains. experiments have shown that VP shares a common We have found that V3 is a densely myelinated area, representation of the horizontal meridian with V2 along similar in this respect to MT. We have utilized two its posterior boundary, whereas its anterior boundary is independent anatomical approaches to show that this formed by a representation of the superior vertical densely myelinated region indeed corresponds to V3. meridian. Thus, the representation of the upper visual First, the sites of transported label in V3 following quadrant in VP forms a mirror image of that of ventral injections of rHJproline in Vl fall within the myelo­ V2, much as the representation of the lower visual architectonic borders of V3. Also, a long strip of callosal quadrant in V3 forms a mirror image of that of dorsal V2. projections to the site of representation of the inferior However, because of the difference in connections with vertical meridian forms a good marker for the anterior Vl as well as a difference in myeloarchitecture, it seems boundary of V3. In hemispheres in which the pattern of reasonable to regard VP as a visual area distinct from V3. callosal inputs has been visualized with appropriate stains, The representation of the upper visual quadrant in VP the architectonically defined anterior border of V3 emphasizes the center of gaze to about the same degree parallels, and is often precisely coincident with the long as is found for Vl and V2. A narrow band of callosal strip of callosal inputs. inputs coincides precisely with the vertical meridian We have mapped V3 completely in one hemisphere. It representation along the anterior boundary of VP and thus is an elongated area lying immediately anterior to the serves as an effective guide in locating VP. fH]proline dorsal half of V2, 40 mm '1ong and 1-4 mm wide. The injections into ventral V2 have shown that VP can be observation that the anterior border of V3 occasionally independently defined by a topographically organized fails to fall within callosal-recipient cortex suggests that projection which it receives from V2. Occasionally, V2 the visual topography of V3 is complex. Myeloarchitec­ injections give rise to multiple sites of transported label tonically defined V3 does not extend onto the ventral in VP, suggesting that the topographic organization of VP aspict of the occipital lobe where the upper visual field is is not always simple. Focal HRP injections in V2 represented. This supports the idea derived previously demonstrate a reciprocal projection from VP to V2. from independent data that V3 contains a representation of the lower visual field only and is distinct from the Reference: Newsome, W. T. and Allman, J. M. (1980) J. Comp. Ventral Posterior area (VP) (see Biology 1980, No. 219). Neurol. In press. 221. PATCHY AND NON-PATCHY CONNECTIONS 220. ARCHITECTONIC IDENTIFICATION OF THE BETWEEN V1 AND V2 IN THE MACAQUE BOUNDARIES OF V3 IN THE MACAQUE MONKEY Investigators: David c. Van Essen, Investigators: William T. Newsome III, William T. Newsome Ill, David C. Van Essen John H. R. Maunsell

Although it has been known for a decade that there are Several recent reports have demonstrated an numerous extrastriate visual areas in the macaque's unexpected complexity in the organization of projections occipital lobe, analysis of their various functions and from Vl to V2 in primates, in that discrete injections of connections has been hindered by the lack of reliable labeled amino acids in Vl often reveal a patchy pattern of criteria for distinguishing boundaries between areas. terminations in V2. We have analyzed the inter- However, recent studies (Biology 1978, No. 214) have connections of Vl and V2 in the macaque in greater detail shown that the Middle Temporal area (MT) can be readily using r3H]proline as an anterograde tracer and horseradish recognized as a zone of dense myelination on the posterior peroxidase (HRP) as both an anterograde and retrograde bank of the superior temporal sulcus. We have extended tracer. this analysis of cortical myeloarchitecture to other Focal tracer injections in Vl (1-2 mm in extent for regions of extrastriate cortex and found that another proline, 0.1-0.3 mm in extent for HRP) resulted in extrastriate area, V3, is distinguishable by its charac- anterograde labeling over a variable extent jn V2, ranging 149

from 2-4 mm in width, and 3-8 mm in length. In most projects. Comparing the relative position of projection cases (10 of 12), the projection was distinctly patchy, with sites in MT to the position of sites in VI, we have found a center-center spacing between patches of 1-3 mm. that a comparably large fraction of each area is devoted Cells in V2 labeled by retrograde transport were largely to parts of the visual field near the center of gaze. coextensive with the region of anterograde projections, However, MT underrepresents superior fields relative to but the two distributions were not identical. Vl and overemphasizes that part of the visual field Markedly different results were obtained following inferior to the center of gaze. Physiological recordings HRP injections in V2. In all 7 cases, the projections from confirm the existence of this inferior versus superior field Vl originated in a roughly circular zone 3-4 mm in bias and indicate that about two-thirds of MT contains diameter, with most cells situated in a central core neurons with inferior receptive fields. We have also found 1.5-2 mm across. Within this central region, cells were anatomical evidence of irregularities in the representation usually distributed relatively uniformly, but in one case of the visual field in MT. Sites on the perimeter of Vl the pattern was clearly patchy, with a center-center have occasionally been found to project to foci in MT well spacing of 0.75-1 mm between patches. The distributions away from its perimeter. In addition, sites in Vl do not of anterograde and retrograde labeling were very similar. always project to single loci in MT. In several cases, a These observations are consistent with the notion that V2 restricted site in Vl was found to project to two distinct contains a "repetitiven representation in which most foci in MT centered 2-3 mm apart. points in the visual field are represented at multiple, As MT is further characterized, it may be possible to distinct loci within V2. determine to what extent these features of topographic The internal connections of Vl and V2 revealed by organization in MT are relevant to the role it plays in HRP injections differ markedly in their spatial and processing visual information. laminar distribution. In Vl, layers II and III are densely labeled, but only within 1-3 mm of the injection site; 223. TWO-DIMENSIONAL MAPS OF THE LATERAL deeper layers are labeled more sparsely but over a greater GENICULATE NUCLEUS Investigators: Michael P. Connolly•, David C. Van Essen extent (up to 10 mm in layer VI). In V2, the inter­ connections are more irregular and extensive, with The lateral geniculate nucleus (LGN) is the major relay distinct patches of anterograde and retrograde labeling up center transmitting information from the two eyes to the to 5 mm from the injection site. visual cortex. The inputs from each eye are segregated into sharply-defined cell layers in the LGN, but the 222. THE REPRESENTATION OF THE VISUAL FIELD number of layers varies systematically from two to six in IN_ THE MACAQUE MIDDLE TEMPORAL AREA different parts of the nucleus. In the macaque monkey, as Investigators: John H. R. Meunsell, John L. Bixby, in the human, the layers are folded in such a way that it is David c. Van Essen difficult to determine the precise internal organization of Last year (Biology 1979, No. 223), we described some the LGN from direct examination of histological sections. features of the Middle Temporal visual area (MT) in the We have found that the detailed analysis of LGN organi­ cerebral cortex of the macaque monkey. It was found to zation can be facilitated considerably by making be a very small visual area, having only 396 of the surface 11unfolded" representations of its constituent layers. The area of Vl, the primary visual area. It was also found that procedure for making such two-dimensional maps is MT contains a complete representation of the contra­ identical to that previously developed for making maps of lateral visual field, but that there are substantial convoluted regions of cerebral cortex (see Biology 1979, irregularities in the representation. Using both No. 222). A major advantage of this technique is that it anatomical and physiological techniques, we have investi­ provides an accurate representation of the surface area of gated the topographic organization of MT in greater detail the structure being mapped; such information has hereto­ and compared it to that of Vl. fore been unavailable for the primate LGN. Because Vl has a strong projection to MT, small In the one example studied to date, the surface areas lesions or injections of labeled amino acids in Vl can be of individual LGN layers range from 21 mm2 for the used to determine the regions in MT to which a site in Vl smaller of the two magnocellular layers (layer 1) to 150

49 mm 2 for the largest of the parvocellular layers diffused away, we determine the number of motor axons (layer 6). Two of the parvocellular layers, 4 and 5, are each spinal root supplies to the soleus and the number of much smaller in surface area (13-16 mm 2) because they muscle fibers innervated. (This is accomplished by are present only in the portion of the LGN representing stimulating each root in turn and recording the resultant the central 15-20° of the visual field. extracellular action potential at the soleus nerve and the The surface area of the striate cortex, the sole target corresponding tension elicited from the muscle.) The 2 of the LGN projection, is about 1100 mm , or roughly ratio of muscle fibers to motor axons provides an estimate 20-50 times the area of individual LGN layers. It is of the average motor unit size for each root. interesting that the relative emphasis of central versus The results of the experiments carried out to date peripheral visual fields is markedly greater in the striate have been encouraging. The number of muscle fibers cortex than in the LGN, indicating that the visual innervated by each motor axon in the paralyzed nerves is representation in striate cortex is not simply a uniformly decreased by about a factor of two relative to controls in expanded version of that in the LGN. We are currently which a plug containing no tetrodotoxin had been inserted studying this relationship in greater detail by tracing the in L7. These results are thus consistent with the connections between LGN and striate cortex with focal hypothesis that nerve activity affects the outcome of 3 injections of [ H]proline and horseradish peroxidase. One synapse elimination at the neuromuscular junction. How­ specific goal, which should be answerable with this ever, there is substantial scatter in the values for approach, is to ascertain whether there are significant individual experiments, and further investigation is in differences in the topographic organization of various progress to confirm the presence of an effect and LGN layers. determine its magnitude.

*Undergraduate, California Institute of Technology. PUBLICATIONS

Bixby, J. L. (1980) Ultrastructural observations on synapse elimination in neonatal rabbit skeletal muscle. J. 224. EFFECT OF IMPULSE BLOCKADE ON THE Neurocytol. In press. ELIMINATION OF NEUROMUSCULAR Bixby, J. L., Maunsell, J. H. R. and Van Essen, D. C. SYNAPSES (1980) The effects of motor unit size on innervation Investigators: Herman J. Gordon, John L. Bixby, patterns in neonatal mammals. Exptl Neurol. In press. David C. Van F.ssen Bixby, J. L. and Van Essen, D. C. (1979) Competition between foreign and original nerves in adult mam­ At birth, mammalian skeletal muscle fibers are each malian skeletal muscle. Nature 282: 726-728. Maunsell, J. H. R., Bixby, J. L. and Van Essen, D. C. innervated by more than one motor neuron. Over the (1979) The Middle Temporal area (MT) in the macaque: course of one or two weeks, all but one of the synapses Architecture, functional properties and topographic organization. Soc. Neurosci. Abstracts 5: 796. per fiber are eliminated. While the factors influencing Maunsell, J. H. R., Newsome, W. T. and Van Essen, D. C. the fate of each synapse are not well understood, one (1980) The spatial organization of connections between interesting possibility is that activity plays a role in Vl and V2 in the macaque: Patchy and nonpatchy projections. Soc. Neurosci. Abstracts 6. In press. synapse elimination, with inactive synapses being at a Newsome, W. T., Maunsell, J. H. R. and Van Essen, D. c. competitive disadvantage relative to active synapses. (1980) Areal boundaries and topographic organization of the Ventral Posterior area (VP) of the macaque The rabbit soleus muscle offers a good system for monkey. Soc. Neurosci. Abstracts 6. In press. Van Essen, D. C. and Maunsell, J. H. R. (1980) Two­ testing the activity hypothesis. Motor axons from two dimensional maps of the cerebral cortex. J. Comp. distinct spinal roots, L 7 and Sl, innervate the soleus, and Neurol. In press. Van Essen, D. C., Maunsell, J. H. R. and Bixby, J. L. early in development a substantial percentage of muscle (1979) Areal boundaries and topographic organization fibers receives inputs from both roots. We have developed of visual areas V2 and V3 in the macaque monkey. Soc. Neurosci. Abstracts 5: 812. a procedure for blocking nerve activity in 4-day-old Van Essen, D. c., Maunsell, J. H. R. and Bixby, J. L. rabbits in one of the spinal roots, L 7, while leaving the (1980) The Middle Temporal area (MT) in the macaque: other root unimpaired. The technique involves surgically Architecture, connections, functional properties and topographic organization. Submitted for publication. inserting into L 7 a small Silastic plug containing tetrodo­ Van Essen, D. c., Maunsell, J. H. R. and Bixby, J. L. toxin, a drug which interrupts propagation of action (1980) The organization of extrastriate visual areas in the macaque monkey. In: Multiple Cortical Areas, potentials. Three days later, after the tetrodotoxin has C. N. Woolsey (Ed.). Humana Press, Clifton, New Jersey. In press. NEUROGENETICS

Seymour Benzer

Ronald J. Konopka

153

Professor: Seymour Benzer cell body profile is roughly circular, although a kidney­ Gosney Visiting Associate: Yoshiki Hotta shaped, ventral region stains a bit more intensely. The Visiting Associate: Ruggero Pierantoni Gosney Research Fellow: Alberto Ferrus cell body diameter is about 14 microns. A process extends Research Fellows: Shinobu C. Fujita, Lawrence M. from the cell body ventrally, then turns posterior-medially Kauvar, Mark A. Tanouye Graduate Student: Sandra L. Shotwell to enter the cervical connective as the descending giant Research Staff: Eveline Eichenberger, Devra C. Spurr fiber axon. Just prior to the posterior-medial turn, two SUpport: The work described in the following research branches occur. One of them is ipsilateral and branches reports has been supported by: profusely around the posterior antenna! center. The other The James G. Boswell Foundation for Virus Research branch crosses contralaterally above the esophagus, turns Jane Coffin Childs Memorial Fund for forward and branches near the contralateral antenna! Medical Research E. S. Gosney Fund center. Staining of this contralateral branch was always Muscular Dystrophy Association of America much reduced and varied from animal to animal. A likely National Institutes of Health, USPHS National Science Foundation possibility is that it corresponds to a dye-coupled cell Pew Memorial Trust other than the giant fiber. University of Tokyo The descending giant fiber axon enters the thoracic Summary: Our activities are directed at the mechanisms ganglion, but is devoid of branching until it reaches the underlying behavior, using the fruit fly, Drosophila, as a mesothoracic neuromere, where it sends a short process model organism in which the connections between medially. Continuing beyond this process, the axon turns genetics, biochemistry, physiology and behavior can be dorsolaterally, ending within the neuromere. traced. *Department of Biology, Yale University, New Haven, Drosophila turns out to be excellent for neuro­ Connecticut. physiology, since it contains large muscle fibers that are easily impaled with microelectrodes. The fly also 226. THE PHYSIOLOGY OF SHAKER (Sh) MUTANTS contains 11giant" axons that permit intracellular recordings of action potentials. Various mutants have been demon­ Investigators: Marl< A. Tanouye, Alberto Ferrus strated to be affected in elementary processes such as Physiological studies on Shaker (Sh) mutants indicate axonal conduction, synaptic transmission or muscular that the Sh gene is important for proper neurological contraction. function. Jan, Jan and Dennis (1977) suggested that these At a higher level of behavior, normal flies can learn to mutants may have defective K + channels. avoid selectively a specific odor that has been associated Intracellular recordings from giant axons of normal with electric shock. A mutant, dunce, does not learn this Drosophila melanogaster show a single spike following task, in spite of having essentially normal ability to sense electrical stimulation of the brain. This action potential the odorants and the shock. The defect is due to change is similar in waveform and in time course to that of the of a single gene, and the mutant lacks one of the forms of squid giant axon, the major difference being an apparently cyclic AMP phosphodiesterase, which may implicate that larger ca2+ ·component in Drosophila. Abnormal action enzyme in the process of memory formation. potentials are seen in the giant axons of Sh mutants (Biology 1979, No. 230). Different Sh alleles show 225. PROFILES OF THE GIANT FIBER OF characteristically different abnormalities. Sh KSl33 DROSOPHILA AS REVEALED BY LUCIFER YELLOW INJECTION action potentials have abnormalities including a plateau or Investigators: Alberto Ferrus, Mary L. Koto*, "shoulder" following the initial spike, followed by Marl< A. Tanouye additional spikes. A prolonged depolarization has also The dye Lucifer yellow was introduced iontopho­ been observed on rare occasions. retically into giant fiber axons, using dye-filled micro­ The number and amplitudes of multiple spikes vary pipettes. The giant fiber and its processes were from animal to animal, and are also affected by manipu­ subsequently examined in whole-mount preparations. lation of divalent cation concentration in the saline The cell body of each of the two giant fibers is located solution. However, plateau potentials show little in the lower part of the ipsilateral protocerebrum. The variation among animals and little response to divalent 154

cation changes, suggesting that the plateau potential is acetone mixture, dehydrated in acetone at -25°C, and the characteristic defect in ShKSt33, the multiple spikes dried. This procedure was found to cause no covalent being secondary. modification of sample proteins. Using the recently We are currently investigating the plateau potential in reported silver staining method for electrophoretic gels detail. Since the plateau persists under conditions (Switzer et al., 1979), it was possible to resolve and detect 2 designed to suppress Ca + currents, our results indicate on the 2D gels approximately 900 polypeptide species in that abnormal Ca Z+ channels cannot account for the samples prepared from 5 heads or 10 brains of adult flies. 33 ShKSt defect. Our present experiments are focusing on Analysis of whole heads, dissected brains, dissected measurements of membrane resistance to determine compound eyes, and heads from which brains and com­

whether the plateau is associated with a conductance pound eyes had been completely removed showE~d that increase or decrease relative to resting conditions and nearly 40 polypeptides were largely or exclusively local­ normal giant fiber action potentials. ized in the brain and 14 in the retina of the compound eye. About 20 polypeptides were common to both brain and Reference: Jan, Y.-N., Jan, L. Y. and Dennis, J. (1977) Proc. Roy. compound eye, but not found elsewhere in the head. Soc. Lond. B 198: 87-108. Thoracic ganglia gave a polypeptide composition pattern nearly identical to brain. Preliminary analysis

Both enzymes were purified over 1000-fold relative to the cross-reacts with the two isozymes of PDE present in initial homogenate. No evidence of interconversion of the bovine brain, with inhibition of catalytic activity and two forms has been observed, in contrast to some reports specific precipitation of the purified enzymes. It does not on PDE from mammalian systems. Complete deficiency precipitate calmodulin, a calcium-dependent regulator of for the chromosomal band 304, which was identified by some isozymes of PDE (J. Robinson, personal communi­ Kiger and Golanty (1977) for its effect on the cAMP cation). phosphodiesterase level, leads to complete loss of activity In D. melB.nogaster, one of the two isozymes, form I, is of form II (the smaller isozyme). partially inhibited by the antiserum, but not by preimmune We would like to obtain conclusive evidence that dunce or normal rabbit serum. Form II, the form affected by the is the structural gene for the form II enzyme. Hqwever, dunce mutation, is not specifically inhibited by the all the mutant alleles which retain some activity failed to immune serum. Thus, it appears that PDE form I from D. show any electrophoretic differences. Examination of the melanogaster and the PDE isozymes of bovine brain are various point mutants and the • deficiency by two­ immunologically similar. D. melanogaster form II is dimensional gels is under way. immunologically distinct from both the second isozyme We are attempting to prepare specific antibodies to present in Drosophila, and those found in bovine brain. each of the enzymes, for use in determining their tissue distribution. Histochemical analysis of PDE in mam­ PUBLICATIONS malian brains has shown staining at postsynaptic densities, Byers, D., Davis, R. L. and Kiger, J. A. Jr. (1980) Impaired but in view of the multiplicity of isozymes and their learning in the dunce mutant of Drosophila is associated with a deficit of a cyclic AMP phospho­ known sensitivity to aldehyde fixation, such results must diesterase. Submitted for publication. be interpreted cautiously. In preliminary experiments, we Fujita, S. C. and Hotta, Y. (1979) Two-dimensional electrophoretic analysis of tissue-specific proteins of have obtained specific enzymatic reaction in unfixed Drosophila melanogaster. Proteins, Nucleic Acids, frozen sections of Drosophila. Strong activity appears in Enzymes 24: 1336-1343. · Fujita, S. C. (1980) Biochemical analysis of behavioral the brain and in the thoracic ganglion, but the separate mutations of Drosophila melanogaster. Taisha 17: distributions of forms I and II remain to be determined. 489-494. (In Japanese) Ganetzky, B. and Wu, c.-F. (1980) Nerve membrane References: excitability in Drosophila melanogaster; the role of Byers, D., Davis, R. and Kiger, J. (1980) Manuscript in spatial and genetic interaction in repetitive firing. preparation. Submitted for publication. Dudai, Y., Jan, Y.-N., Byers, D., Quinn, W. and Benzer, S. Kankel, D. R., Ferrus, A., Garen, S. H., Harte, P. J. and (1976) Proc. Nat. Acad. Sci. USA 73: 1684-1688. Lewis, P. E. (1980) The structure and development of Kiger, J. A. and Go!anty, E. (1977) Genetics 85: 609-622. the nervous system. In: The Genetics and Biology of Kiger, J. and Golanty, E. (1979) Genetics 91: 521-535. Drosophila, Vol. 2d, M. Ashburner and T. R. F. Wright (Eds), pp. 295-368. Academic Press, London. Tanouye, M. A. (1979) Mutations affecting the giant fiber system of Drosophila melanogaster. Soc. Neurosci. 232. IMMUNOLOGICAL STUDIF.S OF PDE Abstracts 5: 263. IN DROSOPHILA MELANOGASTER Tanouye, M. A. and Wyman, R. J. (1980) Motor outputs of lnvestigator: Sandra L. Shotwell the giant nerve fiber in Drosophila. J. Neurophysiol. 44: 405-421. Immunological probes would be useful for studying the Tanouye, M. A. and Wyman, R. J. (1980) Inhibition between flight motoneurons in Drosophila. J. Comp. dunce enzyme defect at the biochemical and anatomical Physiol. Submitted for publication. levels. Drs. Jack Robinson and Earl Stellwagon at the Wu, c.-F. and Ganetzky, B. (1980) Genetic alteration of nerve membrane excitability in temperature-sensitive University of Iowa generously provided an antiserum paralytic mutants of Drosophila melanogaster. Nature raised against PDE purified from bovine brain. The serum 286: 814-816. 157

Assistant Professor: Ronald J. Konopka 233. A MEMBRANE MODEL FOR THE Graduate Students: Steven H. Green, Dominic Orr, DROSOPIIlLA CIRCADIAN OSCILLATOR Randall F. Smith Investigators: Ronald J. Konopka, Dominic Orr Research Staff: Thomas E. Lee, Steven M. Wells Laboratory Staff: Rebecca F. Bodor, Caroline Vermaes Mutations at the per locus in the 3Bl-2 region of the X SUpport: The work described in the following research chromosome of D. melanogaster can shorten, lengthen, or reports has been supported by: eliminate the circadian rhythms of eclosion and adult Biomedical Research Support Grant (NIH) Lucy Mason Clark Fund locomotor activity. In wild-type flies, the activity and National Institutes of Health, USPHS rest portions of the activity cycle are approximately equal Pew Memorial Trust President's Venture Fund in duration. Likewise, the light-insensitive portion of the Whitehall Foundation light resetting curve of the circadian pacemaker, corre­ sponding to the subjective day, is equal in duration to the Summary: Our studies of the Drosophila circadian clock light-sensitive portion, corresponding to the subjective have led us to the possible involvement of membranes in night. In contrast, the duration of the active portion of the clock mechanism. We have formulated a membrane the activity cycle in pers (short-period) flies is shortened model for the Drosophila clock as a framewOrk for by an amount roughly equal to the difference in period considering the function of clock gene products as between the rhythms of pers and wild-type flies, while the revealed by mutant clocks. In this model, there are two durations of the rest portions are similar in pers and wild­ major proteins in the clock mechanism, one involved in type flies. The light-insensitive portion of the light the subjective day process and one in the subjective night. resetting curve is similarly reduced in duration by the pers The formation of a gradient across a membrane by an ion mutation, while th.e duration of the light-sensitive portion pump occurs during the subjective day and its dissipation is essentially unaffected. These results suggest that through light-sensitive channels occurs during the subjec­ separate molecular processes are involved in subjective tive night. The prediction that clock gene products are day and subjective night. We propose a model for the membrane proteins is consistent with the observation by Drosophila circadian pacemaker, in which the basic c. P. Kyriacou and J. Hall of Brandeis University that the oscillation consists of the establishment and depletion of per mutants which shorten, lengthen, or abolish circadian an ion gradient across a membrane. The model postulates rhythms also affect a short-term rhythm (with a period of two classes of molecules: an ion pump, most likely with about a minute) in the interpulse interval of the fly ATPase activity, which operates during the subjective courtship song. We are testing the hypothesis that day, and light-sensitive ion channels, which are open membrane function is altered in the per clock mutants by during the subjective night and which can be closed by using ions and pharmacological agents as probes for strong light pulses. It is proposed that the protein coded altered electrical responses in these mutants. for by the per locus is the ion pump. Since processes on two very different time scales are affected by the per mutants, we are investigating possible 234. LONGEVITY OF CLOCK MUTANTS IN effects on other aspects of the temporal organization of ARTIFICIAL ENVIRONMENTAL CYCLES development and behavior. We have found that the Investigators: Steven M. Wells, Thomas E. Lee, arrhythmic mutation increases the longevity of adult flies Ronald J. Konopka under several environmental conditions. We are con­ Previous work by Pittendrigh and Minis (1972) and tinuing to investigate interactions of clock genes, v. Saint Paul and Aschoff (1978), among others, suggests environmental cycles, and longevity. that longevity of flies is reduced if they are maintained in In studies of homeotic genes on the morphology of the non-24 hr light-dark cycles. The interpretation of these central nervous system, Steven Green has found evidence results is that driving the circadian system which has a that the bithorax gene complex specifies positional "natural" period of about 24 hours at ''unnatural" information for the nervous system in addition to its well­ periodicities is detrimental to the organism, causing known effects on external cuticular structures. decreased longevity. Since we have Drosophila clock mutants available with "natural" periods that differ from 24 hours by several hours (19 hr and 29 hr), we are testing 158

the effect of environmental cycles on these mutants to ately adjacent to the miniature locus. determine if their maximum longevity occurs in the In order to characterize the manner in which this environmental cycle closest to their genetically based mutant affects the underlying pacemaker system, we have "natural" periodicity. Thus far we have studied the 19 hr conducted a phase-resetting study of the andante eclosion and 29 hr mutants, an arrhythmic mutant, and wild type, rhythm. We have found that the andante phase-resetting in a 24 hr light-dark cycle at two different temperatures curve to short light pulses is lengthened by approximately and in a 20 hr light-dark cycle. The results indicate that 1.5 hr per cycle but that (1) the 1.5. hr lengthening could the arrhythmic mutant has increased longevity with not be clearly localized to one particular portion of the respect to the other mutants and wild type in every cycle, and (2) the amplitude of the resetting curve is not condition. There does not appear to be a significant significantly different from that of wild type. This is in alteration in the longevity of the period mutants in the contrast to the resetting curve of the pershort clock 20 hr cycle compared with the 24 hr cycle. The abolition mutant which shows both a significant shortening of only of circadian rhythmicity by the arrhythmic mutation is one portion of the cycle, the light-insensitive phase, and a somehow responsible for a slowing down of the processes significant increase in the amplitude of the resetting leading to senescence and death. If the per gene product response (Konopka, 1979). is indeed a membrane protein, it may be that the Reference: membrane has greater stability without the per protein. Konopka, R. J. (1979) Fed. Proc. 38: 2602-2605.

References: Pittendrigh, C. S. and Minis, D. H. (1972) Proc. Nat. Acad. 237. ISOLATION OF NEW CLOCK MUTANTS Sci. USA 69: 1537-1539. v. Saint Paul, U. and Aschoff, J. (1978) J. Comp. Physiol. Investigators: Ronald J. Konopka, Dominic Orr, 127: 191-195. Steven M. Wells Two additional X-linked mutations affecting the adult 235. PHARMACOLOGICAL STUDIBS ON THE locomotor activity rhythm have been isolated after DROSOPIIlLA NERVOUS SYSTEM mutagenesis with ethyl methanesulfonate. One of these lnvestiga tors: Thomas E. Lee, Ronald J. Konopka has a period of 28 hours and is allelic to the long-period Extracellular recordings from the cervical connective, allele of the per locus, per1. We therefore designate it as which links the brain with the thoracic nervous system in per12• In combination with deficiencies for the per locus, the Drosophila adult, can be easily made with a suction per11 exhibits its long-period phenotype. In contrast, with electrode while the nervous system is bathed with Ringer's particular deficiencies, per12 shows an arrhythmic pheno­ solution. These preparations usually last for a few hours. type. Thus, while the hemizygous and homozygous We have been studying the effect of ion replacements and phenotypes of these two alleles are similar, they can be po~sible neurotransmitters and neuromodulators on the distinguished on the basis of their interaction with other electrical activity recorded from the connective. Re­ chromosomes. These two alleles may represent different placement of sodium by lithium decreases electrical mutations within the per gene. The second mutation is at activity, while addition of octopamine stimulates activity. a separate locus and shortens the period of the activity We are currently screening mutant strains for differences rhythm by about 1.5 hr as compared with wild type. in response to these and other agents. Nothing is yet known about the location of this mutation; it is not an allele of per or of the andante locus, which 236. MAPPING AND CHARACTERIZATION OF THE lengthens the period by 1.5 hr. The effects of the period ANDANTE CLOCK MUTANT mutations isolated thus far in Drosophila suggest that Investigator: Randall F. Smith there is some quantization in the determination of We have previously reported the isolation of a clock periodicity by clock genes. mutant which lengthens the free-running period of both the eclosion and adult locomotor activity rhythms of 238. INNERVATION OF ANTENNAE AND LEGS IN Drosophila by about 1.5 hr.. Using deficiency and dupli­ WILD-TYPE AND HOMEOTIC MUTANT FLIES cation mapping, we have localized this mutant (named Investigator: Steven H.. Green andante), to polytene chromosome band 10E3, immedi- Homeotic mutations transform one appendage into 159 another. The innervation of such appendages is compared bithoraxoid and ssa suggest that the sensory neurons are with the wild type in order to study: (1) How does the largely guided to the vicinity of their targets by their path afferent projection select its targets in the CNS? (2) How and will make functionally appropriate connections only if does CNS development depend on an appropriate afferent brought to close proximity with the target. projection? (3) Is position in the CNS determined by the Mutations in the bithorax genes (BX) affect position same sets of genes that determine position in other determination in the thorax and abdomen. The combi­ tissues? nation of BX genes we use transforms metathorax to The mutation spineless-aristapedia (ssa) transforms mesothorax. The motorneurons of the metathoracic distal antenna to tarsus. Filling neurons with cobalt from neuromere of these animals resemble those of the wild­ wild-type and transformed antennae, I find that the type mesothorax in morphology and relative position. projections are identical in spite of the fact that the Therefore, position in the nervous system, the cuticle, and tarsus contains types of sensillae different than antennae. possibly the mesoderm as well is determined in parallel by This is all the more striking considering the extreme this set of genes. dissimilarity between the simple tarsal sensory projection to the thoracic ganglion and the complex antenna! PUBLICATIONS projection to the brain. The mutation antennaless removes one or both antennae. The morphology of brains Konopka, R. J. (1979) Genetic dissection of the Drosophila circadian system. Fed. Proc. 38: 2602-2605. from ssa and antennaless flies appears wild-type in silver­ Konopka, R. J. (1980) Effects of Drosophila photoreceptor stained preparations. mutations on the circadian activity rhythm. Soc. Neurosci. Abstracts 6. In press. HRP fills from each of the three different legs give a Konopka, R. J. (1980) Genetics and development of circadian rhythms in invertebrates. In: Handbook of characteristic pattern of motorneurons and sensory axons Behavioral Neurobiology, Vol. 4, J. Aschoff (Ed.). in the thoracic ganglion. The abdominal legs caused by Plenum Press, New York. In press. the mutation bithoraxoid send their afferents into the Konopka, R. J. and Orr, D. (1980) Clock gene effects on the subjective day and night of the Drosophila activity CNS along an abdominal nerve or, occasionally, along the rhythm. Genetics 94: 855. Konopka, R. and Orr, D. (1980) Effects of a clock metathoracic (third) leg nerve. In either case, sensory mutation on the subjective day: Implications for a neuropil of the metathoracic neuromere is the target but membrane model of the Drosophila circadian clock. In: Development and Neurobiology of Drosophila, J. the precise pattern depends on the route taken. Hall and L. Hall (Eds.). Plenum Press, New York. In Occasionally metathoracic leg motorneurons innervate press. Konopka, R. J. and Wells, S. (1980) Drosophila clock these abdominal legs, initially taking a "normal" route mutations affect the morphology of a brain neuro­ through the metathoracic leg nerve. Results from secretory cell group. J. Neurobiol. 11: 411-415.

DEVELOPMENTAL GENETICS AND IMMUNOGENETICS

Edward B. Lewis

163

Professor: Edward B. Lewis the normal pattern of segments is generated. The rules Visiting Associate: Welcome W. Bender which have been identified thus far may be summarized as Research Fellows: Loring G. Craymer III, Ian W. Duncan Research Staff: Josephine Macenka, Ker-kwa Susan Ou, follows: (1) the more posterior the segment, the more Tony B. Ramey, Jane T. Traub genes of the complex that are turned on; (2) genes once Laboratory Staff: Ana L. Gharzeddine, Nancy Harris, Gertrude Jordan, Eva Westmoreland turned on in a given segment tend to stay on in all segments posterior thereto; and (3) the more proximal a Support: The work described in the following research reports has been supported by: gene is in the complex, the more anterior (with one Biomedical Research Support Grant (NIH) exception) is the segment in which it becomes turned on. National Institutes of Health, USPHS National Science Foundation The way in which the complex controls normal develop­ Edwin H. Schneider Fund ment can perhaps best be understood if there are two Stanford Medical School Helen Hay Whitney Foundation gradients: a gradient along the embyro in a substance produced by Pc+ and a gradient along the chromosome in Summary: The segmentation pattern of Drosophila turns the affinity of the cis-regulatory elements for that out to be an ideal system for studying the genetic control substance. of development. A simple principle underlies the plan of 239. DEVELOPMENTAL EFFECTS OF DELETING THE development of segmental organisms. Early in embryonic POLYCOMB GENE life the organism consists of little more than a linear Investigators: Edward B. Lewis, Loring G. Craymer III, array of redundant segments. Gradually, the segments Josephine Maeenka, Tony B. Ramey, diversify to produce head, thoracic and abdominal seg­ Susan Ker-bwa Ou ments. Much of that diversification appears to be under With the aid of various techniques including a new the primary control of a giant cluster of genes known as method of screening for deficiencies devised by L. the bithorax complex, named after bithorax, the type Craymer, deficiencies have been obtained for what is mutant found by C. B. Bridges in 1915. This complex is believed to be a major regulatory gene for the bithorax composed of at least nine genes of a structural gene type. gene complex, namely, Polycomb (Pc). Our model for the In addition, four cis-regulatory elements have been identi­ action of the wild-type Pc gene is that it produces a fied. We report on a recently discovered locus in the repressor of the bithorax genes. In the absence of the cluster, anterobithorax, a mutant of which when combined repressor, all of the genes of the complex are assumed to with two other mutants of the cluster produces a four­ be derepressed (or activated). Embryos homozygous for winged fly that has the second pair of wings and the Polycomb point mutants which are not accompanied by accompanying segment virtually identical in size and visible deficiencies such as Pc1, the original mutant found external structure with the first pair of wings and its by P. H. Lewis, or Pc 3, a somewhat more extreme allele, accompanying segment. have the thoracic and first seven abdominal segments The main goal of our work is to understand how the transformed so that externally they come to resemble the bithorax genes co.me to be switched on and off during eighth abdominal segment. Does this effect result from development in a programmed way. We have therefore partial or complete inactivation of the Polycomb gene been searching for genes that regulate the functioning of (are the dominant point mutants hypomorphs or amorphs, the bithorax complex and have already identified several: to use the useful terminology of Muller) or do they one, Rg-bx +, acts as a positive regulator of the entire represent some new or antagonistic functions (are the Pc complex, deficiencies for this gene resulting in partial point mutants neomorphs or antimorphs)? The answer can inactivation of the complex; another, Rg-pbx +, acts as a be obtained at least in part by examining the phenotype of regulator that is possibly specific for postbithorax, and animals homozygous for the newly induced Pc deletions. finally Pc+, or the Polycomb gene (named after the type Such animals die in the late embyronic stage and have mutant found by P. H. Lewis), acts as if it produces a essentially the same type of abdominalization of the repressor of the bithorax complex. thoracic and first seven abdominal segments as seen in Pc Each of the genes of the cluster controls a specific embryos homozygous for the point mutants. The results level of segmental development, and, by a sequential clearly support the idea that it is the loss of Pc gene turning on of these genes during very early development, function that leads to a turning on of the genes of the 164 bithorax complex. We are unable to see any qualitative patches observed are transformed to the same structure, difference between the phenotypes of embryos homo­ then that structure must be the anal plate, the most zygous for the Pc point mutants and embryos homozygous posterior structure on the fly. Transformations to for the Pc deficiencies. However, adults heterozygous for genitalia or seventh tergite can be excluded because the point mutants have a Pc phenotype slightly more clones have the same appearance in females (which have extreme than that of adults heterozygous for Pc no large bristles on their genitalia) and males (which have deficiencies, as first found by Denell {1978). One no seventh tergite). Transformations to the sixth abdomi­ possibility is that the Pc point mutants are not amorphs nal tergite are ruled out because clones without ~richomes but antimorphs, as Puro and Nygren (1975) showed for Pc2 occur in the region of this tergite that normally has them. 2 (Pc2 /Pc2 /+ being more mutant than Pc /+). Thus, such These results show that the Pc+ gene is active as lat_e as mutants may produce a new or modified product which the pupal stage and that the ability of a cell to remember competes with the wild-type Pc gene product to produce which segment it belongs to depends on this Pc+ activity. even less of the postulated repressor substance than would One model consistent with these data is that the level of the single dose of the wild-type gene, which would be activity of the Pc+ gene is set early in development such present in heterozygotes involving the Pc deficiency that the amount of activity increases in a graded fashion chromosome and a wild-type chromosome. Another as one proceeds anteriorly in the embryo. After this possibility is that Pc is itself a gene complex ~d that the initial setting, each activity level of the Pc+ gene is point mutants inactivate only a P?rtion of the complex clonally inherited throughout later development. The while the deletions remove all of it. genes of the bithorax complex would then respond continuously to the amount of Pc+ substance present, References: Denell, R. (1978) Genetics 90: 277-289. resulting in a graded activation of bithorax complex genes Puro, J. and Nygren, T. (1975) Hereditas 81: 237-248. as one proceeds posteriorly in the animal.

240. CLONAL ANALYSIS OF POLYCOMB IN THE 241. IDENTIFICATION OF TWO NEW GENJ!S THAT ABDOMEN OF DROSOPHILA APPEAR TO ACT AS BITHORAX COMPLEX lnvestigator: Ian Dunean REPRESSORS

From examination of embryos homozygous for Pc Investigator: Ian Duncan mutants, it is clear that this gene is active during There is by now a considerable amount of evidence embryogenesis. To determine if the Pc+ gene also acts supporting the idea that the Polycomb gene (Pc+) acts to later in development, clones of cells homozygous for an repress the genes of the bithorax complex. To gain a extreme allele, Pc 3, were induced by irradiating larvae of better understanding of bithorax complex repression, a 3 the genotype Pc /trc, where trc is a cell marker mutation search has been made for other genes that are involved in that, like Pc, is located in proximal 3L. Clones of cells this process. The search was done by selecting X-ray and homozygous for Pc3 were identified in the adult abdomi­ EMS induced mutations that act as dominant enhancers of nal tergites as patches of cuticle bare of trichomes that an extreme Pc allele, Pc3• The majority of such frequently occurred in twin to patches of trc cells. It is mutations have turned out to be located in the second clear that these trichomeless patches produce bristles chromosome and define just two genes, mapping in the because when they occur in the first abdominal tergite, right arm at positions 69 and 84.. Recessive lethal alleles which normally has very small bristles, they are associ­ of the distal locus have a dominant phenotype similar to ated with large wavy bristles similar to those that occur that of mutations at the Pc locus. Thus, males carrying on the anal plate. Trichomeless patches did not occur in such alleles in heterozygous condition often have sex­ irradiated Pc 3 /trc siblings that also carried a duplication comb teeth on the mesothoracic basitarsus and patches of for Pc+ carried on the fourth chromosome, indicating that fifth abdominal tergite tissue on the fourth abdominal these patches owe their phenotype to homozygosity for tergite. Also, these alleles act as enhancers of certain Pc3 and not some other mutant on 3L. If it is assumed dominant gain-of-function mutants in the bithorax com­ that these trichomeless clones represent adult tissue plex and Antennapedia region. Two viable alleles have normally found in a more posterior location and that all been recovered at this locus and flies heterozygous for 165 one of these and a lethal allele show transformations of another hypothetical structural gene. A possible all the abdominal segments to a more posterior level as candidate for this latter gene would be the locus of 3 well as an antenna-to-leg transformation and an almost bithorax-like mutants which partially complement bx • complete transformation of the distal mesothoracic and The most promising of such mutants seemed to be an 7 metathoracic legs into distal prothoracic legs. Homo­ X-ray induced mutant, formerly designated bx (Lewis, zygotes for a: lethal allele at this locus die in the late 1980), which not only partially complements bx3 but in 3 embryonic stage but show no segmental transformations. addition exhibits the transvection phenomenon with bx ; The proximal locus is defined by two lethal alleles. When that is, the phenotype of bx7/bx 3 is made more extreme heterozygous with wild type both show, with low by the introduction of structural heterozygosity for the penetrance, sex-comb teeth on the mesothoracic right arm of the third chromosome. Presumably, somatic basitarsae of males. These mutants are weaker enhancers pairing facilitates complementation between the two 3 of Pc than are the lethal alleles of the distal locus and mutants. A recombination analysis was therefore under­ even the stronger of the two has little effect on dominant taken and it has recently been found that bx 7 maps gain-of-function mutants in either the bithorax or extremely close to the left of bx3 (approximately 0.01 Antennapedia regions. Homozygotes for the stronger of centimorgan on the basis of one wild-type and one the two alleles die in the larval stage and show patchy reciprocal double-mutant crossover). The bx7 mutant has transformations of the mesothorax, metathorax and first been renamed anterobithorax (abx). The triple-mutant, abdominal segment to more posterior segments. abx bx3 pbx, when homozygous or hemizygous produces a Thus, it appears that the wild-type alleles of these two four-winged fly in which the third thoracic segment is now genes as well as Pc+ are required for normal repression of virtually as large as the second segment. Indeed, the the bithorax complex genes. The antenna-to-leg or extra­ transformation is so extreme that many but not all of the sex-comb phenotypes of mutations in these genes suggest animals fail to emerge from the puparium. that these loci are also required for proper repression of Reference: genes located outside of the bithorax complex (perhaps in Lewis, E. B. (1980) Drosophila Information Service 55: 207-208. the Antennapedia region) that are directly responsible for these phenotypes. 243. MOLECULAR ANALYSIS OF THE BITHORAX COMPLEX 242. AN IMPROVED FOUR-WINGED FLY Investigators: Welcome W.. Bender, Pierre Spierer*, Investigator: Edward B. Lewis David S. Hogness**, Edward B. Lewis

Two mutants of the bithorax gene complex are We have been able to isolate part of the bithorax required to produce a four-winged fly. One of the complex on recombinant DNA molecules and have begun mutants, bithorax-3 (bx3) of Stern, transforms the to define the lesions responsible for particular mutant anterior, and- the other, postbithorax (pbx), the posterior phenotypes. The bithorax DNA was made accessible by an portions of the haltere-bearing or third thoracic segment inversion which placed part of the bithorax complex next into the corresponding wing-bearing or second thoracic to a DNA region we had already cloned. The inversion, +Rl segment. Although the transformation appears to be called Cbx , breaks the third chromosome at the qualitatively complete, the third segment is distinctly polytene chromosome band 87E1,2 and again within the smaller than the second segment and the prescutum or bithorax complex at 89El,2; the latter breakpoint has an extreme anterior portion of it is not well developed. Ultrabithorax (Ubx) phenotype. We had used a procedure 3 Paradoxically, the bx mutant causes the prescutum of called 0 walking" to collect overlapping· DNA fragments the second thoracic segment to be slightly underdeveloped spanning 300,000 base pairs (300 kb) covering the bands as well. This effect of bx3 acts as if it is a dominant 87011 through 87E5, and we located the Cbx +Rl inversion gain-of-function that cannot be suppressed by duplications breakpoint on the DNA map. We made a new library of which do, however, suppress the effects of bx3 on the recombinant molecules from the inversion stock, and, third thoracic segment. A possible resolution of the using the 87E sequence as probe, recovered a DNA 3 paradox is that bx causes a recessive loss-of-function of fragment spanning the breakpoint, half from 87E, half a structural gene, bx+, but a dominant gain-of-function of from 89E. The 89E DNA has served as the starting point 166

for "walking" within the bithorax complex; to date there segmental control. The bx region and most of the iab are overlapping DNA fragments covering 130 kb. The region are probably outside of the DNA cloned so far, and DNA is apparently all single copy sequence; there are no we therefore expect the whole complex spans over 200 kb large tandem repeat elements and no large copia-like and accounts for most of the DNA in the bands 89El-4. mobile repeat elements. Our immediate goal is to collect the DNA for the rest To align the DNA map with the genetic and cytological of the complex and to correlate the genetic map with the maps, the clearest landmarks are the breakpoints of DNA map. We are starting to identify DNA regions chromosomal rearrangements that have bithorax pheno­ associated with particular genetic functions; we hope to types. Four such bithoraxoid (bxd) breakpoints have been find in vivo RNA transcripts from those regions. We will positioned over a 20 kb region near the starting point of then be able to ask what the gene products are and how our walk. The Ubx breakpoint of Cbx +Rl lies 20 kb to the they control the developmental fates of the various left of the bxd breaks, and other Ubx rearrangements have segments. breakpoints further to the left beyond the region cloned so *Department of Molecular Biology, University of Geneva, far. A translocation with recessive infra-abdominal (iab) Switzerland. **Department of Biochemistry, Stanford University effects (2:3) PlO] breaks about 50 kb to the right of the rr Medical School, Palo Alto, California. bxd region. We have also tentatively located the Cbx1 1 and pbx mutations. These two mutations were produced PUBLICATIONS together on the same chromosome by X-rays; apparently a Craymer, L. (1980) New mutants - D. melanogaster. 17 kb piece of DNA was transposed 40 kb leftward within Drosophila Information Service 55: 200-204. the bithorax complex. This created a deletion (pbx} to the Lewis, E. B. (1980) New mutants - D. melanogaster. Drosophila Information Service 55: 207-208. right of the bxd region and an insertion (Cbx) to the left Lewis, E. B. (1980) Genetic control of body segmentation ,of bxd. We suspect the transposed DNA still makes the in Drosophila and Bombyx by homeotic genes. Inter­ national Congress of Entomology, Kyoto, Japan. In pbx + gene products although no longer under proper press. (Abstract) 167

THE FOLLOWING REPORTS ARE BY GRADUATE STUDENTS IN THE DIVISION OF BIOLOGY WHO ELECTED TO DO THEIR THESIS WORK IN THE DIVISION OF CHEMISTRY AND CHEMICAL ENGINEERING.

244. NATURE OF INTEGRATED FeLV PROVIRUS Drosophila (Fyrberg et al., 1980). Assuming they follow Investigators: Arthur Roach, James L Mullins*, the vertebrate example (Vandekerckhove and Weber, Kathy Bauman Burck*, James W. Casey*, 1978), some of these genes are expressed only in a Norman Davidson* particular type of muscle cell, while others are expressed Support: American Cancer Society National Institutes of Health, USPHS in the cytoplasm of all cell types. The differential Gordon Ross Medical Foundation expression of the six actin genes, as well as their Feline leukemia virus is a retrovirus which infects and evolutionary conservation and ubiquity, make them good causes leukemia in natural populations of cats. During the candidates for a comparative study of gene expression. infection process, the single-stranded RNA genome of the Other workers in this laboratory have initiated studies of virus is copied into a double-stranded DNA molecule which the myosin and other muscle protein genes of Drosophila. integrates into the DNA of the host cells. Viral RNA is It is hoped that this will allow future studies of the then transcribed from some of the integrated viral DNA. coordinate expression of specific muscle protein genes in The general problems of interest are the sequence organi­ specific tissues. zation of the integrated DNA, the functions of the viral All six actin genes were isolated as recombinant DNA genes and the structural factors affecting viral expression. molecules from our lambda library of the Drosophila A cloned cell line in which several copies of viral DNA genome and subcloned into the plasmid vector pBR322. have integrated was chosen for study. Molecular cloning They are being characterized by restriction endonuclease techniques were used to obtain large amounts of 14 of mapping, electron microscopic heteroduplex analysis and these proviral sequences and some of their host-derived DNA sequencing. These studies will allow us to examine flanking DNA in the pure state. Previous studies in this the evolutionary relationships of the genes, and to laboratory have shown that some of these 14 clones are compare Drosophila gene organization with that of other infectious in a tissue culture assay while others are not. eukaryotes. Further, the amino acid sequence of the actin Restriction endonuclease mapping, electron microscopic proteins, as derived from the DNA sequence, should allow heteroduplex analysis and SI-nuclease protection assays of us to classify each gene as coding for a muscle-specific heteroduplexes have all failed to show any differences protein or a cytoplasmic one. between infectious and noninfectious clones. We are The next phase of our work will involve the identifi­ currently constructing molecular recombinants between cation of probes that we can use to identify the specific various clones in order to see whether loss of infectivity messenger RNAs from each gene. The most likely can be localized to a particular region of the viral DNA. candidates for these probes are the DNA segments that code for the 3' untranslated regions of the messages. References: Mullins, J. I., Casey, J. W., Nicolson, M., Burck, K. B. and These segments will be used as probes for in situ Davidson, N. (1980) In: Proceedings of the 3rd hybridization studies on thin sections of the whole International Feline Leukemia Virus Conference, W. J. Hardy Jr., M. Essex and A. J. McClelland (Eds.). organism at various stages of development, as well as for Elsevier-North Holland, New York. "Northern" blots of stage-specific RNAs. These experi­ Mullins, J. I., Casey, J. W., Nicolson, M. and Davidson, N. (1980) Nucleic Acids Res. In press. ments should identify the stage- and tissue-specificity of Mullins, J. I., Nicolson, M., Casey, J. w., Burck, K. B. and • Davidson, N. (1980) Manuscript in preparation. the six actin genes of Drosophila.

*Division of Chemistry and Chemical Engineering, References: Califomia Institute of Technology. Fyrberg, E., Kindle, K., Sodja, A. and Davidson, N. (1980) Cell 19: 365-378. Vandekerekhove, J. and Weber, K. (1978) J. Mo!. Biol. 126: 245. CHARACTERJZATION OF THE ACTIN GENES 783-802. OF DROSOPHILA Investigators: Beverley J. Bond, Katharine S. Mixter, *Division of Chemistry and Chemical Engineering, Erie A. Fyrberg*, N. Davis Hershey*, California Institute of Technology. Norman Davidson* Support: National Institutes of Health, USPHS

There are six known genes for actin in the fruitfly, 168

3 246. CUTICLE GENES polypeptides of molecular weights 40, 50, 60 and 65 x 10 Investigators: Michael P. Snyder, Jay Hirsh*, daltons. The receptor complex in purified membranes Norman Davidson* mediates cation translocation in response to agonist National Institutes of Health, USPHS Support: binding. Affinity alkylating agents of agonists and We are studying the sequence organization and expres­ antagonists both label at least the 40,000 dalton subunit, sion of the larval cuticle genes of Drosophila. Five major indicating that this component carries a physiological cuticle proteins are synthesized and secreted by late recognition site for acetylcholine. The role of the larvae in order to provide a protective pupal coat. This remaining polypeptides is not yet known. gene system is of interest for the study of evolutionarily­ Reconstitution is generally believed to be a .logical related genes, their hormonally-induced developmental approach for dissection studies of the molecular com­ expression and the coordinate control of this expression. ponents and assigning specific functions, including the We have selected a lambda clone from a Drosophila defining of the minimal elements that constitute a recombinant DNA library with an insert which codes for physiological receptor complex. Several laboratories have several of the larval cuticle proteins. This i11:sert contains now reported reconstitution of membranes derived from four genes, three of which are tightly clustered within 4.5 receptor protein of various levels of purity, including kilobases of DNA. All three of these genes are expressed highly homogeneous preparations. In all cases ion trans­ abundantly during the late larval stage of development port was measured on the scale of seconds to minutes, and to. a much lesser extent in the second instar stage, but which is at least three orders of magnitude slower than a to no detectable levels during embryonic, pupal, and adult physiologically significant scale. Thus, to demonstrate stages. In contrast, the fourth gene on this clone appears meaningful reconstitution, a method of measuring ion to be most abundantly-expressed during the adult stage of transport kinetics no slower than milliseconds scale is fly development. essential. Using the techniques of hybridization/selection of Recent efforts in our laboratory (Moore and Raftery, RNA, in vitro translation, immunoprecipitation and two­ 1980) have provided a spectroscopic method that allowed dimensional gel electrophoresis, the following points have studies of ion translocation on a time scale achievable by been established: (a) two of the three clustered genes a stopped-flow instrument, i.e., a few milliseconds. We code for larval cuticle proteins, and (b) one of these two have used this technique to resolve the kinetics of ion genes possesses a sequence closely related to that 9f transport across reconstituted receptor membranes. The another cuticle gene. The third gene of the tight gene preparation used in the experiments has been demon­ cluster, and the remote fourth gene are not yet identified. strated in a recent 22 Na + flux study (Wu and Raftery, Additional work to identify and characterize these genes 1980) to represent reconstituted membranes in which a fully is now being carried out. large fraction of the receptor was functional. It was

*Division of Chemistry and Chemical Engineering, demonstrated from the study of fast ion transport that the California Institute of Technology. reconstituted receptor membranes derived from purified

protein mediated agonist-induced c~tion transport on a 247. MILLISECONDS TIME RESOLUTION OF CATION msec level in a totally reproducible manner. Quantitative TRANSPORT BY RECONSTITUTED PURIFIBD analysis of the fast ion flux kinetics revealed that the ACETYOCHOLINE RECEPTOR Investigators: Wilson C.-S. Wu, Hsiao-ping H. Moore*, reconstituted protein resembles a physiologically active Michael A. Raftery• receptor. Support: American Heart Association References, Muscular Dystrophy Associations of America Moore, H.-P. H. and Raftery, M. A. (1980) Proc. Nat. National Institutes of Health, USPHS Acad. Sci. USA. In press. Wu, W. C.-S. and Raftery, M. A. (1980) Biochemistry. The acetylcholine receptor (AcChR) from Torpedo Submitted for publication electroplax is now agreed by most to be a complex of four *Division of Chemistry and Chemical Engineering, California Institute of Technology. VISITING LECTURERS

GRADUATES

FINANCIAL SUPPORT

171

VISITING LECTURERS

Gert-Jan B. van Ommen, University of Amsterdam, The Andreas Radbruck, Genetics Institute, University of Netherlands: Transcription and RNA splicing in yeast Cologne, West Germany: The class switch of immuno­ mitochondria. globulin variants. Douglas Melton, Medical Research Cancer Laboratory of Fred Wilt, University of California, Berkeley: Expression Molecular Biology, Cambridge, England: Gene injec­ of histone genes in sea urchin embryos. tion into frog ooeytes. Peter MacLeish, Harvard Medical School: Physiology of Chris Kinter, University of Wisconsin: The structure and solitary rod photoreceptors. Epstein bar virus as isolated from vision and trans­ Harry Orr, Harvard University: Primary structure of formed cells. human histocompatibility antigens: Implications for Donald Kennedy, Stanford University: Health and safety their evolution and variability. regulation: Where does science leave off and politics Vann Bennett, Wellcome Research Laboratory, Research begin? Triangle Park, North Carolina: Molecular basis of John Wilson, Baylor College of Medicine, Houston: Path­ cytoskeletal-membrane interaction. ways of genetic recombination in somatic cells. Elio Vannin, University of Wisconsin, Madison: The Susan Litvak, University of Bordeaux, France: Mito- structure of a mouse pseudo-a-globin gene. chondrial DNA polymerase. Studies on the enzymes Masayasu Nomura, Institute for Enzyme Research, isolated from animal, plant and enucleated cells University of Wisconsin, Madison: Autogenous (platelets) and inhibition of enzyme activity by DNA regulation of ribosomal protein gene expression in intercalating drugs. Escherichia coli. Gary Struhl, Medical Research Cancer Laboratory of Shirleen Roeder, Cornell University: DNA rearrange- Molecular Biology, Cambridge, England: Develop- ments associated with a transposable element in yeast. mental compartments in the Drosophila head. Jack McMahan, Stanford Univesity School of Medicine: Volkar Bruns, University of Frankfort, Germany: The The role of basal lamina in regeneration of the inner ear of bats: ultrastructural analysis of adapta­ neuromuscular junction. tions for echolocation. Michael Merzenich, University of California, San Edward M. De Robertis, Medical Research Cancer Francisco: Some thoughts about "topographic maps" Laboratory of Molecular Biology, Cambridge, England: and the genesis of perception in auditory and somato­ Nucleocytoplasmic interactions in frog oocytes. sensory systems. Igor Dawid, National Cancer Institute: Isolation and Donald D. Brown, Carnegie Institution of Washington, structure of vitellogenin gene in Xenopus. Baltimore: In vitro genetics on a purified eukaryotic S. Hagiwara, University of California, Los Angeles: gene. Properties of anomalous rectification in starfish egg Ray Erickson, University of Colorado Medical Center, membrane. Denver: The transforming protein of avian sarcoma w. B. Adams, Friedrich Miescher Institut, Basel, viruses and its homologue in normal cells. Switzerland: Slow currents activated by action Ralph Hinegardner, Thimann Laboratories, Univeristy of potentials in Aplysia cell R15. California, Santa Cruz: Sea. urchin immunology. M. H. P. West, National Institutes of Health: New types Kenneth Robinson, University of Connecticut Health of histone variants: Characterization and comparison Center, Farmington: Electrically directed growth of with known variants. nerve and muscle cells in vitro. Corrado Baglioni, State University of New York at Robert B. Goldberg, University of California, Los Angeles: Albany: The mechanism of action of interferon. Gene expression inplant development. Ann L. Hubbard, Yale University Medical School: Marvin R. Paule, Colorado State University: Purification, Receptor-mediated endocytosis: Fates of ligand and structure, an_d functioning of RNA polymerases during receptor. differentiation of Acanthamoeba. Ken Muller, Carnegie Institution of Washington, Michael Fried, Imperial Cancer Research Fund, London: Baltimore: The specificity of synapse formation after The organization and expression of polyoma virus DNA regeneration in the leech CNS. sequences in transformed cells. Geoffrey Raisman, National Institute for Medical Richard Gregory, MRC, University of Bristol, England: Research, Mill Hill, England: Synapse formation after Visual perception and color. injury in the adult rat brain. Martha Constantine-Patton, Princeton University: Two Bessie P .-H. Huang, The Rockefeller University: Genetic components of synaptogenesis in the visual system. and biochemical studies on eukaryotic flagella. Joseph Bogen, University of Southern California: The Norton Bernard Gilula, The Rockefeller University: Cell­ split brain revisited, with some speculations on art. to-cell communication and gap junctions. Jay Goldberg, University of Chicago: Electrophysiological Anthony Means, Baylor School of Medicine, Houston: studies of the vestibular labyrinth in the monkey. Calmodulin: An intracellular ca++ receptor. John Heuser, University of California, San Francisco: Robert Poyton, University of Connecticut Health Center, Three-dimensional visualization of cell structure using Farmington: Cooperative interaction between mito­ quick freezing techniques. chondrial and nuclear genomes: Cytochrome c oxidase Tasuku Honjo, Osaka University School of Medicine, assembly as a model. Japan: Immunoglobulin heavy chain gene organization John Gerhart, University of California, Berkeley: and developmental rearrangement. Establishment of the dorsal-ventral axis in the Joan A. Steitz, Yale University: snRNPs and scRNPs: Xenopus embryo. New classes of RNA:protein complexes from Colin Pittendrigh, Stanford University: Pacemaker and eukaryotic cells. slave oscillations in a circadian system. Gerald R. Fink, Cornell University: Unusual genetic Richard Lynch, Washington University Medical School, St. events associated with a yeast transposable element. Louis: Regulation of malignant antibody-producing cells. 172

Tom Reese, National Institutes of Health: Application of Torsten N. Wiesel, Harvard Medical School: Morpho­ rapid-freezing to capture fast structural changes logical basis of cortical visual function. underlying secretion. Owen Witte, University of California, Los Angeles: Are Murray Gardner, University of Southern California School protein kinases the transforming gene products of of Medicine: Natural history of retroviruses in wild leukemia viruses? mice and other animals. Wendy Clough, University of Southern California: Arthur T. Winfree, Purdue University: Phase-control of Oncogenesis and herpes viruses. pacemakers: Neural, biochemical, saccadian and ? Angeline Douvas, University of Colorado Medical Center, Andre Sobel, Institut Pasteur, Paris: Reconstitution of a Denver: Nuclear macromolecules and autoimmune functional acetylcholine receptor. diseases. Rami Rahamimoff, Hebrew University Medical School, Gerald Westheimer, University of California, Berkeley: Jerusalem: Calcium and regulation of transmitter Visual hyperacuity. release at the neuromuscular junction. Argiris Efstratiadis, Harvard Medical School: Structure Carl Parker, Stanford University Medical School: An in and molecular evolution of insulin and globin genes. vitro approach to transcription in Drosophila. Harald Biessmann, Max Planck Institut, TUbingen: Char­ Nigel Birdsall, National Institute of Medical Research, acterization of mRNA populations in the development London: Subclasses of muscarinic acetylcholine recep­ of Drosophila melanogaster. tors and their interactions with guanine nucleotides Charlotte Omoto, Princeton University: Rotation of the and adenylate cyclase. central pair of microtubules during ciliary bonding. Marcus Jacobson, University of Utah: Clones and compartments in the vertebrate central nervous system. 173

GRADUAT~

Thirty-five students in Biology were awarded the B.S., M.S. or Ph.D. degree in June 1980. Names, degrees conferred, titles of doctoral theses (with plans of recipients for future work) are as follows:

Bachelor of Science

Bruce Matathias Baskir, B.S. with honor. Kenneth Gray, B.S.

Alan Howard Boyar, B.S. with honor. Lynn Mary Hildemann, B.S. with honor.

Kenneth Hale Britten, B.S. John Bertram Reinitz, B.S.

Richard O'Reilly Brown, B.S. Constance Slavens Royden, S.S. with honor.

Erik Eriksen, B.S. with honor. Kurt William Runge, B.S. with honor.

Henry Hayim Farhi, B.S. Paul Alan ShuJ?ak, B.S.

Susan Shevaun Gilley, B.S. with honor.

Mester of Science

Jay William Ellison, M.S. Katherine Dai-Li Lee, M.S.

Mark E. Gurney, M.S.

Doetor of Philosophy

David John Asai, Ph.D. Thesis: Immunological Mark E. Gurney, Ph.D. Thesis: Sexual differentiation of approaches to flagellar movement. Postdoctoral brain and behavior in the zebra finch (Poephila research, California Institute of Technology. guttatah A cellular analysis. Postdoctoral research, John Lovell Bixby, Ph.D. Thesis: The formation and loss California lnstitute of Technology. of supernumerary synapses in mammalian skeletal Larry E. Johnson, Ph.D. Thesis: lnterhemispheric visual muscle. Postdoctoral research, University of communication in human commissurotomy subjects. California, San Diego. Marilyn Rose Kehry, Ph.D. Thesis: Structure and Andrew Duncan Byers, Ph.D. Thesis: Studies on learning function of murine immunoglobulin M from serum and and cyclic AMP J?hOSJ?hodiesterase of the dunce mutant cell membrane. Postdoctoral research, University of of Drosophila melanogaster. Postdoctoral research, Oregon. European Molecular Biology Laboratory, Heidelberg. Michael William Klymkowsky, Ph.D. Thesis: On the Edwin P. Ching, Ph.D. Thesis: Biochemical and func­ structure of the acetylcholine receptor from Torpedo tional characterization of the products of mito­ californica electroplaques. Postdoctoral research, chondrial protein synthesis in HeLa cells. Postdoctoral University College London. research, The Rockefeller University. Kenneth Lawrence Marton, Ph.D. Thesis: Binocular David Paul Corey, Ph.D. Thesis: A bioJ?hysical a1>1>roach facilitation and inhibition in the lateral geniculate to sensory transduction by vertebrate hair cells. nucleus of the cat. Postdoctoral research, Yale Medical School. Galina Dmitrieyvna Moller, Ph.D. Thesis: Development Franklin David Costantini, Ph.D. Thesis: Studies of and protein synthesis in Drosophila. Postdoctoral repetitive sequence transcripts in the sea urchin. research, California Institute of Technology. Postdoctoral research, Oxford University. Robert Francis Murphy, Ph~D. Thesis: Chromosomal Philip Warren Early, Ph.D. Thesis: Mouse immuno­ protein-DNA interactions. Postdoctoral research, globulin heavy chain gene organization and rearrange­ Columbia University. ment: Genetic bases for antibody diversity and William Thomas Newsome ID, Ph.D. Thesis: Studies on regulated expression. Postdoctoral research, primate extrastriate visual cortex. I. The interhemi­ California lnstitute of Technology. spheric connections of visual cortex in the owl John Gregory Frelinger, Ph.D. Thesis: Studies on the monkey, Aotus trivirgatus, and the bushbaby, Galago major histocompatibility complex of the mouse and senegalensis. II. A functional localization of neuronal rat. Postdoctoral research, California Institute of response properties in extrastriate cortex of the owl Technology. monkey, Aotus trivirgatus. Postdoctoral research, Karen Elizabeth Gaston, Ph.D. Thesis: Behavioral California lnstitute of Technology. studies on learning and interocular transfer in the Betty Anne Vermeire, Ph.D. Thesis: Laterality and domestic chick. Assistant Professor, Dept. of visual perception in monkeys. Postdoctoral research, Behavioral Sciences, Cal Poly, Pomona. University of Washington. Robert Allen Gelfand, Ph.D. Thesis: Studies on RNA metabolism in HeLa mitochondria. Postdoctoral research, Purdue University. 174

FINANCIAL SUPPORT

The financial support available for the work of the Division of Biology comes from many sources: from general Institute endowment and from special endowment funds for broad areas of work; from grants or contracts with individuals, companies, foundations, and U.S. governmental agenices for specific projects; from unrestricted annual gifts; from fellowships for the support of individual scholars; and from contributions to general funds provided by Industrial Associates and Institute Associates, as follows:

Fund--Researeh Support Purpose

The Ahmanson Foundation Research in molecular biology

Rita A.lien Foundation Cancer research

American Cancer Society Cancer research

American Heart Association Cardiac research

Anonymous Gift Fund Research in educational programs

John E. Barber Fund Biological research, particularly .as related to the brain

Louis D. Beaumont Foundation Neuroscience research

Beckman Instruments, Inc. Funds for equipment

Bing Endowment Fund, Inc. Professorship in behavioral biology

Biology Research in Neuroscience Neuroscience research

Biomedical Research Support (NIH) Biomedical research

The James G. Boswell Foundation Professorship in biology

The James G. Boswell Foundation Virus research

Ethel Wilson Bowles and Robert Bowles Professorship in chemical biology

Mrs. Leah Hills Brlice Research in biology

The B.W. Foundation Research in immunology

Norman Chandler Professorship in cell biology and chemistry

Louisa Jane Church Fund Research in biology

Norman W. Church Foundation Research in chemical biology

The Commonwealth Fund General support

Charles B. Corser Fund Research in chemical biology Roberta Crutcher Research in bi_ology

Mr. and Mrs. Allen V. C. Davis Research in neuroscience

The Deafness Research Foundation Deafness research

Mrs. Mary Bruce Delbrlick Research in biology

Josephine V. Dumke Fund Cancer research

E. I. DuPont de Nemours & Co. Interferon research Fairchild Foundation Fairchild scholars program

Leland Fikes Foundation, Inc. Research in molecular biology

The Max C. Fleischmann Foundation Research in molecular biology 175

Gloria Gartz Fund Research and education in biology

E. S. Gosney Fund Research in genetics

The Hearst Foundation Research in auditory anatomy and physiology

Frank P. Hixon Fund Neurobiology, physiological psychology, and related research

Hoag Foundation Medical research program

Irma Hoefly Fund Ganeer research

Hoffman-LaRoche, Inc. Research on barbiturate and minor tranquilizers

The Kroc Foundation Research in biology

Lasker Award Fund Chemical genetics

Louis B. Mayer Foundation Medical research program

The John A. McCarthy Foundation Research in molecular biology

Merck and Company, Inc. Research in biology

The Merck Company Foundation Faculty development

Muscular Dystrophy Associations of America, Inc. Research in biology National Aeronautics and Space Administration Space research • The National Foundation - March of Dimes Research in birth defects

National Institutes of Health, USPHS Studies in basic experimental biology, animal physiology, biochemistry, biological systems analysis, biophysics, developmental biology, genetics, neurobiology, plant and cell biology, psychobiology, and virology; graduate research training, biomedical sciences support grant

National Science Foundation Studies in animal physiology, biochemistry, biophysics, developmental biology, genetics, neurobiology, plant and cell biology; undergraduate research participation program

Northwest Area Foundation Medical science program in biology

Dr. and Mrs. Raymond Pearson Research in chemical biology

The Ann Peppers Foundation Research in auditory physiology and anatomy

Pew Memorial Trust Neuroscience research program

The President's Fund Research and development at JPL

President's Venture Fund Medical science

Research Corporation Genetic research

The Rockefeller Foundation Chemical biology research

Albert Billings Ruddock Fund Professorship in biology

Damon Runyon-Walter Winchell Cancer Fund Cancer research

Edwin H. Schneider Fund Study and research in genetics

Alfred P. Sloan Foundation Neuroscience research

Alfred P. Sloan Fund for Basic Research General support 176

Richard Steele Grace C. Steele professorship of immunology

The Stone Foundation Psychobiology research

A. H. Sturtevant Memorial Fund General support

Sundry donors Chemical biology research

Albert Tyler Memorial Fund Annual lectureship in biological research

Union Oil Company Corona Del Mar Marine Station

Joseph G. Venable Fund Arthritis research

The Del E. Webb Foundation Neuroscience research program

Martin Webster Fund Immunology and virology basic to the problems of multiple sclerosis

Jean Weigle Memorial Fund General support

Whitehall Foundation Neurobiology

Robert E. and May R. Wright Foundation Medical science

The Zanetti Grant Biochemistry, cancer research

• 177

Fund--Fellowship Support

American Cancer Society International Rice Research Institute

Earle C. Anthony Fellowship Japanese Education Department

Arthritis Foundation Mackenzie Foundation

The James G. Boswell Foundation The Helen G. and Arthur McCallum Fund

California Foundation for Biochemical Research The John A. McCarthy Foundation

California State Graduate Fellowship Medical Research Council, Canada

Cancer Research Institute, Inc. Muscular Dystrophy Associations of America, Inc.

Carnegie Institution of Washington National Institutes of Health, USPHS

Centre National de la Recherche Scientifique, France National Multiple Sclerosis Society

Chinese Nationalist Embassy National Science Foundation

Lucy Mason Clark Fund NATO

The Jane Coffin Childs Memorial Fund for Medical Research Prince Charitable Trusts

Consiglio Nazionale delle Ricerca Gordon Ross Medical Foundation

Albert and Kate Page Crutcher Rubber Research Institute of Malaysia

Del Amo Foundation Damon Runyon-Walter Winchell Cancer Fund

Deutsche Forschungsgemeinschaft Evelyn Sharp Fellowship

Deutsche Krebsforschungszentrum Smith, Kline and French Laboratories

The Camille and Henry Dreyfus Foundation, Inc. Spanish Ministerio de Universidad e Investigacion

European Molecular Biology Organization Swiss National Agency

Fairchild Scholars Program Walter and Sylvia Treadway Fund

Federal College Work-Study Program Vern Underwood Undergraduate Scholarship

John E. Fogarty International Research Fellowship Veterans Administration Program for Advanced Study in the Health Sciences (NIH) The Del E. Webb Foundation French National Institute for Medical Research Chaim Weizmann Foundation German National Cancer Center Helen Hay Whitney Foundation E. S. Gosney Fund

Lawrence A. Hanson Foundation 178

AUTHOR INDEX (by page oomber)

Akutagawa, E., 126 Early, P. W., 43 Jacobs, R. A., 103, 105 Allman, J.M., 117-119, 123 Eatock, R. A., 103 Jacobs-Lorena, M., 33 Anderson, D. M., 31, 32 Edens, J., 93 Jagodzinski, L. L., 25 Aranovich, D., 52 Efstratiadis, A., 30, 57 Jampolsky, A., 141 Ary, J.P., 123, 124 Ellert, B. M., 52 Jayaram, M., 80 Ary, M., 135 Ellison, J. w., 36 Jennings, K. R., 112 Asai, D. J., 78, 88, 89 Erdmann, A. L., 146 Johnson, L. E., 144 Attardi, G., 17, 20, 21 Eriksen, K. J., 123, 124 Johnson, N. D., 45 Ernst, S. G., 34 Jonsson, G., 140 Baker, J. F., 118, 119 Ewald, S. J., 48, 49 Julesz, B., 122 Balzer, D.R. Jr., 99, 100 Bell, J. R., 70 Fambrough, D., 49 Kaczmarek, L. K., 112 Belliveau, J. W., 40 Fender, D. H., 122-125 Kasamatsu, T., 138-141 Bender, W.W., 165 Ferrus, A., 153, 154 Kauvar, L. M., 155 Benzer, s., 153, 155 Fobes, J. L., 130 Keeley, B., 20 Berg, H. C., 75, 77 Frelinger, J. G., 51 Kehry, M. R., 48, 49 Birt, D. L., 131 French, P. L., 123 Kim, S. K., 45 Bixby, J. L., 149, 150 Fritsch, E. F ., 56 Klein, s., 125 Blamick, B. L., 105 Fryxell, K. J., 100 Kochevar, L. E., 82 Block, S. M., 7 5 Fujita, S. c., 154, 155 Konishi, M., 126, 127 Boettger, H. G., 40 Fyrberg, E. A., 167 Konopka, R. J., 157, 158 Bond, B. J., 167 Koto, M. L., 153 Bond, M. W., 44 Galland, P. A., 81, 82 Kronenberg, M., 49 Bonnor, J., 23, 25 Gao, B., 34 Krouse, M. E., 106-108 Bravo, H., 135-137 Gard, D. L., 86, 88, 89 Kuppermann, B., 134, 138 Briggs, R. P ., 123 Gay, H., 21 Britten, R. J., 29-31 Gelfand, R. A., 17 Lacy, E. H., 57, 58 Brockes, J.P., 99-101 Giugni, T ., 29 Lapidus, I. R., 77 Brokaw, C. J., 78, 79 Glackin, C. A., 26 Lasky, L.A., 29-31, 35 Burck, K. B., 167 Goldberg, D. A., 62 Lauer, J. E., 59 Butler, E. T. III, 57 Gomer, R. H., 87 Lawn, R. M., 56, 57 Buxbaum, J., 48 GonzEtlez-Cadavid, N ., 20 Lazarides, E., 85 Buzin, C., 66 Goodenow, R. S., 50 Leahy, P. S., 32 Gordon, H. J., 150 Lee, T. E., 157, 158 Cabrera, C. V., 36 Granger, B. L., 86, 87 Lemke, G. E., 99, 100 Calame, K. L., 46 Green, S. H., 158 Leong, H., 125 Campos, K. L., 24 Griffin, c., 40 Lester, H. A., 106-108 Casey, J. W., 167 Grim, L. B., 90 Leutwiler, L. S., 80, 81 Castro-Gonzalez, R., 27 Grula, J. W., 29, 30 Lev, Z., 35 Chamberlin, M. E., 33 Gurney, M. E., 102, 126 Levy, D. E., 38, 39 Charlang, G., 54, 55 Lewis, E. B., 163, 165 Chen, M. J.-w., 124 Hall, T. J., 29, 31 Lewis, R. S., 104 Chiu, A. Y., 111 Hamilton, C. R., 145, 146 Ling, K.-c., 71 Chomyn, A., 19, 22 Hardison, R. c., 57 Livant, D. L., 45 Claudio, T. R., 70 Hayes, W., 83 Lo, D. C.-T., 103 Clegg, K. B., 34 Heggelund, P ., 139 Lowy, P.H., 06 Conley, M. P., 76 Hershey, N. D., 167 Connolly, M. P., 149 Hewick, R. M., 40 Maccecchini, M.-L., 47 Corey, D. P., 103, 105 Hirsh, J., 168 Macenka, J., 163 Costantini, F ., 32 Rogness, D. S., 165 Machray, G. c., 26 Craymer, L. G. Jll, 163 Hood, L. E., 40, 41, 91 MacKay, D. M., 124, 143 Crews, s. T., 43 Hopfield, J. J., 85 MacKay, V., 143 Crowley, T. E., 68, 69 Horowitz, N. H., 54 Magoon, E., 141 Cummings, M., 29 Horvath, S. J., 40 Maniatis, T ., 55, 57 Hotta, Y., 154, 155 Manson, M. D., 75, 76 Darcey, T. M., 124 Hough-Evans, B. R., 33, 34 Marton, K. L., 137 Davidson, E. H., 28-36 Huang, H., 43, 52 Masters, J. N., 20-22 Davidson, N ., 167, 168 Hudspeth, A. J., 102-105 Mathog, D.R., 105 Davis, M. M., 45 Hunkapiller, M. W., 19, 40, 52, 90, 91 Maunsell, J. H. R., 147-149 De Francesco, L., 19 Hunkapiller, T., 47 Mayne, J. T., 70, 71 Delbrlick, M., 80, 83, 84 Hunt, J., 29 McAllister, L. B., 31, 32 DeOgny, B. L., 38 Hyson, M. T., 122, 123 Mccann, G. D., 52 Dewees, P ., 92, 93 Mccasland, J. s., 127 Douglas, R. H., 45, 49 Itakura, T., 140 McGuinness, E., 121 Dreyer, W. J., 37, 39, 40 McMillan, M., 51 Duncan, I., 164 Mellon, P. L., 61 179

Merkel, c. G., 17 Patalano, J.P., 38, 39 Snyder, M. P., 168 Meyer, D. J., 92 Pate, E. F., 79 Sorensen, G ., 45 Meyerowitz, E. M., 63 Petersen, N. S., 64-66 S1>erry, R. W., 142 Middlebrooks, J. C., 138 Petersen, s. E., 118, 119 Spierer, P., 165 Miezen, F. M., 123 Pettigrew, J. D., 132-138 Spurr, D. C., 155 Miller, 0. L., 33 Pierantoni, R., 155 Steinmetz, M., 50 Mitchell, H. K., 64-66 Pik0, L., 34 Stevens, T. M., 100 Mixter, K. S., 167 Pine, J., 109 Strauss, E. G., 67, 70 Mockli, G., 145 Plourde, G., 142, 143 Strauss, J. H. Jr., 67 Moiseff, A., 127 Posakony, J. W., 31, 33, 57 Strumwasser, F., 110-113 Montoya, J., 17, 18 Presti, D. E., 134 Stygall, K. A., 100 Moore, G. P., 35 Proudfoot, N. J., 59-62 Moore, H.-P. H., 168 Tager, R. M., 143 Moore, J., 36 Raftery, M. A., 168 Takemoto, L., 91 Moore, K., 47 Ramachandran, V. s., 134, 135 Tanouye, M. A., 153, 154 Morandi, c., 21, 22 Ramey, T. B., 163 Teng, E. L., 144 Mottes, M., 19 Reddy, M. V., 81 Thomas, T. L., 29, 30 Mullins, J. I., 167 Rembaum, A., 39 Trabert, S. W., 31 Revel, J.-P., 90-93 Nakai, K., 140 Rice, C. M. III, 68-71 Umemoto, M., 129 Nargeot, :'J., 108 Roach, A., 167 Nargeot, M.-c., 108 Roach, J., 65 Van Essen, D. C., 147-150 Nerbonne, J. M., 109 Roberts, J. w., 29, 30 Van Harreveld, A., 94, 95, 113 Newsome, W. T. III, 147, 148 Roman, J. M., 38 Vermeire, B. A., 145, 146 Ng, B., 54 Rubin, G. D., 123 Nicholson, B. J., 90, 91 Wang, C., 89 Sala-Trepat, J. M., 25 Weinstock, M. M., 108 0 1Connell, C. D., 57, 60 Sargent, T. D., 25 Wells, S. M., 157, 158 O'Connor, c. M., 88 Scheller, R. H., 31, 32 Wik:torowicz, J. E., 24 Ojala, D. K., 17, 18 Schilling, J. W., 44 Wilhelm, F.-x., 26, 27 Okuno, M., 78, 79 Segall, J. E., 75 Wilhelm, M. L., 26, 27 Olds, M. E., 128-131 Shander, M. H. M., 61, 62 Williams, K. N., 129-131 Ootaki, T ., 84 Shen, C.-K. J., 59 Williams, N. P., 55 Orr, D., 157, 158 Sher, B. T., 46 Wu, F., 84 Otto, M. K., 82 Sheridan, R. E. Jr., 107 Wu, J.-R., 25 Ou, J. J.-H., 68, 69 Shotwell, s. L., 155, 156 Wu, W. C.-S., 168 Ou, S. K.-H., 163 Silver, M. E., 62 Smith, N. L., 30 Yancey, S. B., 92, 93 Pappenheimer, A. M. Jr., 101 Smith, R. F., 158 Yang, M. Y.-L., 25 Parker, V. P., 62 Smyth, R. D., 77 Yeakley, J.M., 113 180

FINANCIAL SUPPORT INDEX (by page number)

Rita Allen Foundation, 55 National Institute of Mental Health, USPHS, 142 American Cancer Society, 17, 23, 28, 41, 55, 85, 167 National Institutes of Health, USPHS, 17, 23, 28, 37, 41, American Cancer Society, California Division, 41 55, 64, 67' 75, 78, 85, 90, 99, 102, 106, 110, 117' 122, American Heart Association, 17, 85, 168 126, 128, 132, 142, 147, 153, 157, 163, 167, 168 Earle C. Anthony Fellowship, 64, 67, 90, 142 National Multiple Sclerosis Society, 99 Arthritis Foundation, 41 National Research Council of Canada, 102 National Science Foundation, 17, 28, 37, 41, 55, 67, 75, Bing Chair of Behavioral Biology, 126 80, 85, 94, 99, 117, 126, 128, 132, 142, 147, 153, 163 Biomedical Research Support Grant (NIH), 23, 28, 37, NATO, 106 41, 54, 55, 63, 67' 85, 90, 99, 142, 157' 163 The James G. Boswell Foundation for Virus Research, 153 Ann Peppers Foundation, 102 Ethel Wilson Bowles and Robert Bowles Professorship, 41 Pew Memorial Trust, 99, 102, 106, 110, 117, 122, 126, The B. W. Foundation, 41 132, 142, 147, 153, 157 Pitzer College, 117 California Foundation for Biochemical Research, 28, 67 President's Venture Fund, 41, 132, 157 Carnegie Institution of Washington, 28 Prince Charitable Trust, 41 Jane Coffin Childs Memorial Fund for Medical Research, 153 Research in Biology, 80 Norman w. Church Foundation, 23, 28, 37, 55 The Rockefeller Foundation, 64, 78 City of Hope Medical Center, 23 Gordon Ross Medical Foundation, 41, 102, 110, 167 Lucy Mason Clark Fund, 15 7 Albert Billings Ruddock Fund, 90 Charles B. Corser Fund, 28, 67, 75 Damon Runyon-Walter Winchell Cancer Fund, 23, 41, 55

The Deafness Research Foundation, 102 Edwin H. Schneider Fund, 163 Deutsche Forschungsgemeinschaft, 41, 80 Evelyn Sharp Fellowship, 110, 132 Deutsche Krebsforschungszentrum, 28 Alfred P. Sloan Foundation, 75, 147 Josephine V. Dumke Fund, 64 Alfred P. Sloan Fund for Basic Research, 6 3 E. I. DuPont de Nemours & Co., 37, 41 Spanish Ministerio de Universidad e Investigacion, 17 Stanford Medical School, 163 European Molecular Biology Organization, 17, 55 Stevens Institute of Technology, 75 Swiss National Agency, 41 Fairchild Foundation, 23, 80, 142 The Max C. Fleischmann Foundation, 37, 41 Universidad Central de Venezuela, 17 Fogarty International Center (NIH), 132 University of California, Los Angeles, 117 French National Institute for Medical Research, 23 University of California, Riverside, 117 University of California, San Diego, 23 E. S. Gosney Fund, 17, 28, 55, 64, 153 University of Nebraska, 128 University of Tokyo, 153 Lawrence A. Hanson Foundation, 110 University of Tours, France, 106 The Hearst Foundation, 102 University of Virginia, 28 Frank P. Hixon Fund, 142 Hoag Foundation, 41 Veterans Administration, 28, 41

International Rice Research Institute, Philippines, 67 Del E. Webb Foundation, 99, 110, 142 Weizmann Fellowship, 55, 132 Japanese Education Department, 128 Whitehall Foundation, 157 Helen Hay Whitney Foundation, 78, 163 The Kroc Foundation, 99 Robert E. and May R. Wright Foundation, 132

Louis B. Mayer Foundation, 41 The Helen G. and Arthur Mccallum Fund, 117, 126 Medical Research Council of Canada, 142 Muscular Dystrophy Association of America, 85, 106, 153, 168