FRONT COVER

Strongylocentrotus purpuratus, the purple sea urchin, resting on a bed of giant kelp (Macrocystis pyrifera). Photo taken by B. Neufeld off Pt. Loma, San Diego during a collection trip to obtain gravid deep-water experimental animals for use in research in developmental biology.

BACK COVER

Schwann cell (red) and fibroblasts (green) identified by fluorescent antibodies in dissociated cultures of rat sciatic nerve. A REPORT FOR THE YEAR 1977-78

ON THE RESEARCH AND OTHER ACTIVITIES

OF THE

DIVISION OF BIOLOGY

AT THE

CALIFORNIA INSTITUTE OF TECHNOLOGY

PASADENA, CALIFORNIA ii

STAFF OF BIOLOGY 1978 Bernita K. Larsh: Compilation and typing.

Elizabeth T. Hanson: Copy editing.

The helpful assistance of Barbara Cathey, Jane Chacon, JoAnn Chikahiro, Geraldine Cranmer, and Lody Kempees is greatly appreciated.

Pasteup: Caltech Graphic Arts.

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 1977-June 1978, 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. Characterization of HeLa cell mitochondrial 7S DNA • • • • • • • • •• 17 2. Sequence analysis of the origin of replication of human mitochondrial DNA • 17 3. Transcription map of the mitochondrial DNA from HeLa cells ••••••• 19 4. Localization of promoter sites on HeLa cell mitochondrial DNA • • • • . • 19 5. Characterization of polypeptides synthesized on mitochondrial ribosomes. • 19 6. Isolation of myeloma lines secreting antibodies to human cytochrome oxidase • 20 7. Mutants of human cells deficient in mitochondrial protein synthesis • • 20 8. Mitochondrial DNA in somatic cell hybrids • • • • • • • • • • . . • 20 9. Regions of homology of mitochondrial DNA from human and mouse cells 21

James Bonner - Summary. • • • • • • • • • • • • • 22 10. The role of histone acetylation in gene expression • • • 24 11. Molecular cloning of rat chromosomal DNA fragments . 25 12. Isolation of cloned ribosomal RNA coding sequences • • • • • 25 13. Analysis of rat long repetitive DNA sequences by recombinant DNA methods • 25 14. Immunochemical isolation and purification of rat albumin and et-fetoprotein mRNAs 26 15. Synthesis of full length cDNAs from purified rat albumin and a-fetoprotein mRNAs 26 16. Determination of the number of albumin and a-fetoprotein genes in rat DNA and hepatoma DNA • • • • • • • • • . • • • • . . • • • • . 27 17. The nuclear RNA precursors of albumin mRNA and a-fetoprotein mRNA 27 18. Purification and mapping of slime mold ribosomal DNA •••••• 27 19. An R-loop map of rat rRNA genes isolated from total genomal DNA • 28 20. Foldback DNA in the rat genome . • • . • • • • • . . • • ••• 28 21. Chromatin reconstitution • • • . • • • • . . • • • • • . . • • 29 22. Nucleosome structure of newly replicated chromatin •••..••• 29 23. The effect of single base mismatch on synthetic DNA-0Xl 7 4 DNA duplexes. 29 24. A comparison of mammalian and slime mold histones • • • • . • • • 30 25. Computerized analysis of thermal denaturation of DNA and chromatin • 30

Eric H. Da~n - Summary • • • • • • • • • • • • . • • • • 32 26. Studies of nucleic acid reassociation kinetics . • • • • . . . • • 32 27. Sequence content of the bi thorax region of the Drosophila genome. 32 28. Complexity of Drosophila egg RNA • • • • • • • . . . • • • • 33 29. RNA synthesis in sea urchin oogenesis • • . • • • • • . • • • • • •. 33 30. Evidence for maternally inherited poly(A) containing RNA in mouse eggs • 33 31. Cloned sea urchin DNA complementary to oocyte RNA. I. CS0088 • . 34 32. Cloned sea urchin DNA complementary to oocyte RNA. 1L PSC34 • . • 34 33. Repetitive sequence transcripts in the sea urchin oocyte . • • . •••• 34 34. Maternal mRNA localization and utilization in sea urchin development. • 35 35. Sea urchin maternal mRNA sequences also synthesized during embryogenesis 35 36. Nonmaternal messenger RNA sequences in sea urchin embryos • • • . • • 36 37. Sea urchin embryo mRNA sequences in the nuclear RNA of adult tissues .• 36 38. Repetitive sequence transcripts in sea urchin nuclear RN As. • . . . • • • 36 39. Repeated sequences in the vicinity of expressed single copy sea urchin DNA 37 40. Primary structure of repetitive DNA sequences in the sea urchin genome . 37 41. Selection of cloned sea urchin DNA coding for mRNA .••• 37 42. Single copy DNA sequence polymorphism. . • • • • • . . . • 38 43. Evolutionary change in sea urchin repetitive DNA • • . • . . . 38 44. Sequence organization of individual repeated sequence families • 39 45. Single copy DNA sequence differences among related sea urchins 39

William J. Dreyer - Summary • • • • • • • • • • • • • • • • • • 40 46. Fractionation and analysis of proteins displayed on the surface of tumor cells . 41 4 7. Identification of cell-surface molecules that react with anti-tumor serum 41 48. Studies of the structure and function of a family of cell-surface recognition molecules . 41 49. Establishment of an advanced laboratory for studies of proteins • • • • • •• 42 50. A new, extraordinarily sensitive analytical system for research and medicine • 42 51. Is developmentally programmed gene splicing used only in the immune system? 43 iv

Leroy E. Hood - Summary • • • • . . . . • . . . • • • . 44 52. Sequence analysis of a closely-related set of mouse VK regions 45 53. Sequence studies of a-1,3 dextran-binding immunoglobulins .. 46 54. Sequence studies of levan-binding immunoglobulins • • . . • 46 55. Construction of immunoglobulin cDNA plasmids .•..••• 47 56. The construction of mouse DNA libraries and the characterization of immunoglobulin genes. 47 57. Determination of the structure of mouse lgM • • • • . • • • • . . • 47 58. Comparison of the structure of soluble and membrane-bound mouse lgM 48 59. Characterization of a putative T cell receptor ...... 48 6 0. Analysis of the diversity of mouse transplantation antigens • 49 61. Site of polymorphism in mouse H-2 transplantation antigens 49 62. Direct sequence studies of mouse transplantation antigens • 49 63. Biochemical analysis of mutant H-2 antigens • . . . . • • 49 64. Ia on cells in the epidermis • • . . . • • ...... 50 65. Chemical characterization of Ia antigens of the mouse major histocompatibility complex (MHC). • . . • • . . . . • • . • • • • . • 50 66. Chemical characterization of la antigens from murine tumor cells • 51 67. Development of automated protein microsequencing techniques . 51

Norman H. Horowitz - Summary...... 52 68. Laboratory simulations of Viking results . • • • . • • . . . . 52 69. Siderophore binding to Neurospora cytoplasmic membranes . . . 53 70. Experiments with a siderophore-deficient mutant of Neurospora. 53 71. Siderophores of Aspergillus and Penicillium. 54

Tom Maniatis - Summary. . • • • . . • • 54 72. Construction of libraries of eukaryotic DNA 55 7 3. Characterization of linked 13-globin genes in rabbit 56 7 4. Restriction mapping and nucleotide sequence analysis of cloned 13-globin genes 56 75. Other rabbit globin genes ..•••...•••• 56 76. Isolation and characterization of human globin genes •..• 57 77. Y chromosome genes of Drosophila ..••...•••. 57 7 8. The alcohol dehydrogenase gene of Drosophila melanogaster 57

Herschel K. Mitchell - Summary. • • • . . • 58 79. Heat shock phenocopies in Drosophila • • . • 58 8 0. Heat shock and protein synthesis in Drosophila 59 81. Purification of a heat shock protein . . . • • 59 82. Mapping of the coding region for two heat shock proteins. 60 83. Development and protein synthesis of the flight muscle of Drosophila melanogaster 60 84. The aromatic cross-link in the cuticle of Drosophila 60 85. Teratogens and phenocopies in Drosophila • . • • • • . • • • . • • • • 61

James H. Strauss Jr. - Summary...... 61 86. Amino terminal sequence analysis of the structural proteins of Sindbis virus. 62 87. Amino acid compositions of the structural proteins of Sindbis virus 62 88. Production and analysis of cyanogen bromide fragments of Sindbis proteins 63 89. Host polypeptides present in purified HR and ts103 8indbis virions • • • 63 90. Growth and release of several alpha-viruses in chick and BHK cells • • 63 91. Characterization of E3 in the 8indbis virus infection • • . • • • • • • 64 92. Investigation of the cleavages of the structural proteins of 8indbis virus 64 93. Structural studies on 8indbis virus . • . • • • • . • • • • . • • 64 94. Antibodies against Sindbis structural proteins • • • • • . • • • • • , 65 95. Biophysical studies on the cyclization of 498 RNA of 8indbis virus ••• 65 96. Location of 5-methylcytidine residues in 8indbis virus RNAs • • • • . 66 97. The determination of the 5'-terminal and 3'-terminal sequences of 8indbis 498 and 268 RNAs 66

CELLULAR BIOLOGY AND BIOPHYSICS

Charles J. Brokaw - Summary. • • . . • • • . • • . . • . • • • • . • • • • • • • • • • 69 98. Flagellar hydrodynamics: A comparison between resistive force theory and slender-body theory • 69 99. Inhibition of flagellar movement by components of the ATP system 69 100. Reactivation of demembranated Ciona spermatozoa • • . • • 69 101. Flagellar asymmetry and its control by calcium ••••.•• 70 102. Flagellar oscillation and bilateral arrest in Ciona spermatozoa 70 103. Anti-tubulin antibodies • 71

Max Delbriick - Summary. 71 104. Fluorescence studies of Phycomyces membrane fractions. 72 105. Riboflavin metabolism in Phycomyces • • . • • • • • • 72 v

106. Photoresponse of roseoflavin-doped sporangiophores of Phycomyces 73 107. Induction by light of 6--carotene synthesis in Phycomyces. 73 108. Microinjection of Phycomyces sporangiophores ••• 73 109. Avoidance and wind responses in Phycomyces • • • . 74 110. Phycomyces: Exploring the avoidance response signal 74

Elias Lazarides - Summary • • • • • • . • • • • . 75 111. Specificity of desmin to avian and mammalian muscle cells. • • • . . • • • . • . • 75 112. Characterization and function of desmin, the subunit of smooth muscle 100 ! filaments 76 113. Fluorescent labeling of myofibril proteins catalyzed by transglutaminase • . • • 77 114. Covalent cross-links between myofibril proteins •••••••••••.••• 77 115. The existence of a Z disc scaffold in chicken skeletal muscle • • • • • . . • • 77 116. Immunofluorescent mapping of major structural proteins in isolated Z disc sheets 78 117. The localization of membrane sites in skeletal muscle fibers ••...•• 78 118. Localization within myofibrils of proteins from Kl-extracted chicken muscle 79

Jean-Paul Revel - Summary • • . • • • • . . • • • • • . . . • 79 119. Isolation and characterization of gap junctions from rat livers. • . • 80 120. Identification of low molecular weight bands from avian gap junctions 80 121. Phylogeny of gap junctions • • • • . . • • • • . . • • • • • • . 80 122. Effect of phenoxybenzamine on gap junctions in regenerating rat liver 80 123. Gap junction formation in rat liver • ~ •••••••..•..• 81 124. Establishment of synchrony between heart cells is blocked by protease inhibitors 81 125. The formation of tight junctions and development of transepithelial resistance in a culture system • • • • • • • • • 81 126. Electrical coupling and gap junction formation • • • . • • • • 82 127. Gap junctions in myelin • • • • • . • • • • . . . • • • • . 82 128. The fine structure of dicyemids • . • • • • • . • • • • • • . 82 129. Ultrastructure of cellular junctions in Drosophila salivary glands 82 130. Examination of a Triton-insoluble cytoskeleton by SEM. 83

Anthonie Van Harreveld • • • • • • • • • • • • • 83 131. Rate of freezing of thin sections of aqueous material 83 CELLULAR NEUROBIOLOGY

Jeremy P. Brockes - Summary 87

A. James Hudspeth - Summary • 88 132. Responses of hair cells to mechanical stimulation in three dimensions 88 133. Stereocilia mediate transduction in vertebrate hair cells 89 134. Response latency of vertebrate hair cells. • • • • 89 135. Ionic activities in the inner ear of the bullfrog • • • • 90

Henry A. Lester - Summary. • . • • • • • . • • • • • • 91 136. Numerical reconstruction of the quantal event at nicotinic synapses 91 137. Role of voltage-sensitive receptors in nicotinic transmission ••• 91 138. Dose-response relations for photoisomerizable nicotinic agonist •• 91 139. ACh receptor channels begin to open within 30 µsec after agonist is applied. 92 140. Response of acetylcholine receptors to photoisomerizations of bound agoilist molecules. 92 141. Pharmacology of Bis-Q • • • . . • • • • • • • • • • • 92 142. Light-activated blocker of acetylcholine-receptor channels • 93 143. A tethered, photoisomerizable cholinergic agonist • 93 144. Ion redistribution in Electrophorus electroplaques 94 145. Kinetics of curare action in the synaptic cleft 94 146. Affinity for curare of acetylcholine receptors. • 94

Felix Strumwasser - Summary. • • • • • • • • • • • • • • 95 147. Purification and partial amino acid sequence of egg-laying hormone (ELH) of Aplysia californica • • • • • • • • • • • • • • • • • • • • • • • 96 148. Neurosecretion of egg-laying hormone and other peptides from electrically active bag cell neurons of Aplysia • . • • • • • • • . • • • • • • • • 96 149. Neuronal sites of action of a neurosecretory peptide, egg-laying hormone, in Aplysia californica • • • • • . • • • • • • • • • • • • • • • 96 150. Isolation of egg-laying factors from the atrial gland of Aplysia californica •• 97 151. Neurotransmitter and cAMP control of afterdischarge in neuroendocrine bag cells • 97 152. The peptidergic bag cell neurons: Morphological and electrophysiological studies of dissociated cells in culture • • • • • • • • • • • • • • • • • • • • • 98 153. Effects of ionizing radiation on the circadian oscillator in Aplysia eyes. • • • • • 99 vi

154. Phase shifting the circadian rhythm in the eye of Aplysia with pulses of 2-deoxy-D-g!ucose (2DG). • • • • • • • • • • • • • • • • . • • • • 99 155. Circadian release of presumed peptides from the isolated eye of Aplysia californica 99 156. The effects of ca++;Mg++ on the properties of the burst generator in R15 •••. 100

NEUROBIOLOGY AND BEHAVIORAL BIOLOGY

John M. Allman - Summary • • • . • 103 157. The physiology of primate visual perception 103 158. Quantitative analysis of single unit data • • • • 104 159. Functional localization of neuronal response properties in extrastriate visual cortex of the owl monkey • • • • • • . . . • • • • • • • • • • . • • • • 105 160. Organization of the visual system in a strepsirhine primate • • • . . • • • • • • 105 161. The pattern of interhemispheric connections in visual cortex of Gal.ago senegalensis 105 162. Cortical control of facial muscles in macaque monkeys. 106 163. Behavioral systems analysis 108

Derek H. Fender • . • . • 109 164. Assessing macular function in vitrectomy candidates. 109 165. Spatio-temporal course of excitation in the human brain 109 166. Identifying brain function through evoked scalp potentials 109 167. Clinical studies of the human ERG using nonlinear analysis 110 168. System modeling of human oculomotor system 110 169. Hysteresis in human binocular fusion. • • . • • • • . • 110 170. Nonlinear analysis of cat ERG . . • • • • . • • • • • 111 171. Motion mechanisms in human vision . • • • • . . . • • 111 172. Binocular hysteresis and fusion with voluntary eye motions 111 173. Eye movement in reading • • • . • • • . . . • . •. 112 17 4. Auditory evoked responses to speech. • • • • . . • • • 112 17 5. A component analysis of the ERG using nonlinear analysis 112

Masakazu Konishi - Summary • • • • • • • • • • • • • 113 176. Species specificity of song learning in the white-crowned sparrow 113 177. The oilbird: Hearing and echolocation .••••..•••.. 113 178. Auditory receptive fields: Center-surround organization • • . . 114 179. Development and sexual differentiation of the song system in the zebra finch • 114

Marianne E. Olds - Sommary . . . . . • • • . . . • • • . . . • . • . • • 115 180. Associative neural changes in medial geniculate and inferior colliculus of the rat during conditioning • • • • . . • • • • . . • • • . • • • • . . 115 181. Single unit analysis of learning-specific changes in rat medial geniculate ••• 116 182. Positive reinforcement of rat behavior produced by intrahypothalamic infusion of morphine . • • • • . . • • • • . . • • • . . . • • . • 116 183. Action of hedonic substances on neural activity in structures supporting brain-stimulation reinforced behavior • • • . . • • • . • • • 117 184. Action of hedonic substances on brain-stimulation evoked neural responses in hypothalamus • • • • . . . • • • . . • • • . 117 185. Neural substrate for conditioned emotional behavior in the rat 117 186. Locus coeruleus neuronal activity in freely-behaving rats. • • 118

John D. Pettigrew - Summary. • • • • . . • • • • . • • • • 119 187. Norepinephrine maintains visual cortical plasticity in kittens and cats 119 188. The recovery fro1n abnormal visual experience: Effects of NE and 6-0HDA •••• 120 189. Catecholaminergic terminals in kitten visual cortex: Fluorescence histochemistry. 120 190. Diffusion of norepinephrine and 6-0HDA perfused locally in cat visual cortex • 120 191. A determination of the extent of the critical period in the owl • • . . . • 121 192. The effects of alternate monocular deprivation in the owl and cat ••••• 121 193. The cortico-geniculate pathway in cats • • • • . . . . • . . • • • •• 121 194. Binocular influences on single cell responses in the lateral geniculate nucleus 122 195. A study of development in area 18 of the visual cortex of the cat • 122 196. A natural nature-nurture experiment on the oilbird ••..•.•••.• 123

R. W. Sperry - Summary • • . . . • . . . • . • • . • • • • . • • • • • 124 197. Verbal identification of left visual field numbers in commissurotomized humans • 124 198. Visual cross-integration of numbers in commissur.otomized humans. 125 199. Visual cross-integration of patterns in_commissurotomized humans. 125 200. An analysis of memory in left and right cerebral hemispheres • 126 201. Cross-cultural differences in brain organization? • • 127 202. Syntax in the right hemisphere. • • • . . • • • . . • • • . 127 vii

203. Long-term psycholinguistic changes in the isolated hemispheres • 128 204. Meaningfulness in dichotic listening • • • • • • • • • 128 205. Preferences for complex stimuli in split-brain monkeys. 128 206. Behavioral functions of extrastriate visual areas 129 207. Interocular equivalence in split-brain monkeys • • • • 129 208. Extent of midbrain vision in cats • • • • • • • . • • 130 209. Interocular transfer of a conditioned food aversion in chicks • • • • 130 210. Nonvisual cues may mediate interocular transfer of a visual food aversion in chicks 131 211. Tests for interocular transfer of a heat-rewarded pattern discrimination in chicks 131 212. Horseradish peroxidase study of optic fibers in goldfish. • • • • • • 132 213. Surgically uncrossing the optic nerve in goldfish. • • • • • • . • • • • 132

David C. Van Essen - Summary • • • • • • • • • • • • • • • • • • • 133 214. Architectonic identification of the middle temporal area in the macaque • 134 215. The projections from striate cortex to areas V2 and V3 in the macaque • • 134 216. The distribution of cells projecting interhemispherically in macaque cortex • 135 217. Suppression of original nerve inputs to skeletal muscle by a foreign nerve. 135 218. The timing of synapse elimination in skeletal muscles of neonatal rabbits • 136

NEUROGENETICS

Seymour Benzer - Summary. • • • 139 219. Studies on the physiology of nap ts. 139 220. Cytogenetic studies of nap ts • • • • • 139 221. Genetic interactions among mutants with neurophysiological defects. 140 222. Search for mutant gene products • • • • • • • 140 223. Na+-channel-specific toxin binding studies ••• 141 224. Learning in individual Drosophila • • • • • . • 141 225. A second allele at the dunce locus in Drosophila. 141

Ronald J. Konq>ka - Summary . • • . • • . . 142 226. Transplantation of a circadian oscillator in Drosophila 142 227. Search for neural correlates of the locomotor activity rhythm in adult D. melanoagaster 142 228. Per locus genetics I: Clock phenotypes of chromosome breaks in the per region 143 229. Per locus genetics II: Gene dosage analysis. • 143 230. Activity rhythms of adult Drosophila mosaics. • . • • • . • • • • • • • . 144 231. In vivo and in vitro Drosophila brain culture • • • • • • . • • • • . • • . 144 232. Serotonin N-acetyltransferase in Drosophila . • • • • • • • • • • • . • • 144 233. Characterization of a temperature-sensitive cell line derived from shibire embryos 144 234. Effects on CNS of homeotic mutations affecting the antennae in Drosophila 145 235. Analysis of the syndrome of the mutant Drosophila "drop-dead" 145 236. Actin mutants in Drosophila • • • • • . • • . . • • • • • • • . • • • 146

DEVELOPMENTAL GENETICS AND IMMUNOGENETlCS

FAward B. Lewis - Summary • . • • • • • • • • • • • • • • • • • 149 237. A new cis-regulatory mutant of the bithorax complex • • • • • . • • 149 238. An ultra-abdominal mutant in Drosophila producing gonadal dysgenesis • 150 239. Polycomb: A major regulator gene of the bithorax complex ••..•• 150

Ray D. Owen - Summary • • • • • • • • • • • • • • • • • • • • • 151 240. Characterization of transplantation antigens associated with the MHC of the rat 151 241. Antigens controlled by the Ir region of the rat • • • • • . • • • 151 242. Analysis of the diversity of mouse transplantation antigens • • • . . • • . • • 152 243. Studies of alpha-fetoprotein in the mouse • • • • • • . • • • . . • • . . • 152 244. The immune response in experimental myasthenia gravis . • • • . • • • . • • 153 245. Immunochemical studies on the induction of experimental autoimmune myasthenia gravis. 153 246. Developmental studies of the humoral aspects of murine autoimmune disease • • 154 247. Nature and functions of immunoglobulins in the soft-pellet coprophagous system of the rabbit • • • • • • • • • • • • • • • • • • • • • • • • . • 154

BIOLOGY/CHEMISTRY GRADUATE STUDENTS

248. In vitro studies of plasmid DNA replication. • • • • . • • • . • • • 155 249. Lipid ordering and membrane protein aggregation • • • • • • • • • • 155 250. Structural studies of an acetylcholine receptor from Torpedo californica 155 251. Synaptic vs. extrasynaptic acetylcholine receptors. 156 252. Reconstitution of an acetylcholine receptor • • . • • • • • • • • . 157 viii

Visiting Lecturers ...... 161 Graduates ...... 163 Financial Support ...... 164 Author Index (by page number) •• 168 Financial Support Index (by page number) 170 INTRODUCTION 2

Brockes 3

THE FACULTY

Appointments

We welcome to our faculty Dr. Jeremy P. Brockes, who arrived during the past year from University College, London. Dr. Brockes received his Ph.D. at Edinburgh University. He works principally on the Schwann cell, which is responsible for producing the myelin sheath that surrounds some peripheral nerve fibers. His research report in the present volume identifies the specific phenomena he is investigating and the methods he employs. An illustrative example of the latter adorns the back cover of this volume.

We are happy to announce that Dr. Howard Berg, cufrently Professor of Molecular, Cellular, and Developmental Biology at the University of Colorado, will be joining our faculty in the fall of 1979.

Honors

Professor Seymour Benzer has won the Warren Triennial Prize of the Massachusetts General Hospital. Professor Benzer was also awarded honorary D.Sc. degrees by Brandeis University and the City University of New York.

Professor Jean-Paul Revel has been named the Albert Billings Ruddock Professor of Biology.. During the past year, he was Chaffee Memorial Lecturer at the University of Otago (New Zealand) and Distinguished Lecturer at the University of New Mexico.

Professor Roger Sperry has been elected a member of the Pontifical Academy of Sciences.

Professor David Van Essen was awarded a Sloan Fellowship for Basic Research.

Benzer Revel

Sperry Van Essen 4

LECTURERS

The eighth Jean Weigle Memorial Lecture was presented by Dr. Niels K. Jerne on February 10, 1978. Dr. Jerne is founder and director of the Basel Institute for Immunology. He spoke on "A Description of the Immune System."

The eighth Albert Tyler Memorial Lecture was presented by Dr. Lionel Jaffe, Professor of Biology at Purdue University on April 17, 1978. Professor Jaffe, who was a graduate student of Albert Tyler's, lectured on "Bioelectric Controls of Growth and Development."

Jaffe

ANNIVERSARY

This year, 1978, marks the 50th anniversary of the founding of the Division of Biology at Caltech. A symposium celebrating this event is being held in November of this year and will be reported in next year's annual report. In anticipa­ tion of the occasion, the following brief account of the founding of the Division was prepared by Professor Charles Brokaw.

50 Years Ago From the early 1920s, the founders of modern Caltech--G. E. Hale, R. A. Millikan, and A. A. Noyes--were committed to a role for biology which would be on a par with the lnstitute's developing reputation in the physieal sciences. Millikan's file on biology in the Caltech archives reveals several proposals which came to him for establishment of major research programs in vertebrate biology, to extend the work in paleontology and vertebrate biology already under way in the Division of Geology; in nutritional biochemistry; and in medical sciences. Of particular note is a report prepared in 1925 by a group of physicians which cited the need for a major center of medical education and research in the Los Angeles area. However, even then "the very great danger that in the future the main work of the Institute in the basic sciences and in engineering would be so dominated by the immense medical development that its prestige as a scientific school would be lost" (1) was recognized.

In June, 1927, Caltech decided that the man who should establish the Division of Biology was Thomas Hunt Morgan, a geneticist with a distinguished career at Columbia University and then president of the National Academy 'of Sciences. In his letter of acceptance a few months later, Morgan outlined his plan for developing biology in six areas: Genetics and Evolution, Experimental Embryology, General Physiology, Biophysics, Biochemistry, and Experimental Psychology.

As his initial faculty, Morgan recruited Alfred H. Sturtevant from Columbia to head his group in genetics, and Sterling Emerson and Ernest G. Anderson from the University of Michigan. All four became Caltech faculty members on July 1, 1928, but the summer of 1928 was spent at Woods Hole while the first increment of the Kerckhoff Laboratories was being completed in Pasadena. 5

Caltech's offer to Morgan carried with it the promise to raise the funds necessary to endow the program in Biology and house it in suitable buildings. This was accomplished within a few months, largely because of the generosity of two Caltech trustees. William G. Kerckhoff (1856-1929) came to Los Angeles to make his fortune in 1879, at the beginning of a period of tremendous growth. Beginning with a lumber business, he became involved in 1890 with the first application of hydro­ electric power in Los Angeles, using the flow of the San Gabriel River in Azusa to generate electricity for an ice-making plant. Later hydroelectric developments spread as far as the Kern River, and in 1913 Kerckhoff's firm was responsible for bringing the first natural gas pipeline to Los Angeles, from the Bakersfield area. By the time Kerckhoff sold his interest in 1927, this had become the Southern California Gas Company. Kerckhoff became a Caltech trustee, and in 1928 gave 1 million dollars to Caltech for building biological laboratories to house the program made possible by two other 1 million dollar gifts for endowment, from another trustee, Allan Balch, and from the General Education Board (forerunner of the Rockefeller Foundation). The Kerckhoff Laboratories in Pasadena were built in two stages, partly because Morgan wanted to i;>lan the second stage after choosing the researchers who would occupy it, and partly because the Institute wanted to have a building available for Morgan and his initial colleagues by October, 1928. A decision to proceed was made before Kerckhoff's gift was assured, and site preparation for the first phase began in November, 1927. Buildings were simpler in those days, and construction was completed in September, 1928. The William G. Kerckhoff Marine Laboratory at Corona del Mar was purchased in 1930, and the second phase of the Kerckhoff Laboratories in Pasadena was completed in 1938, at which time the laboratories were formally dedicated.

Morgan, Sturtevant, Emerson, and Anderson moved into their new laboratories in the autumn of 1928, along with two research fellows (Yoshitaka Imai and Theodosius Dobzhansky} and three graduate students from Columbia who were employed as teaching fellows (Albert Tyler, Russell Biddle, and William Hetherington). Morgan also brought Calvin Bridges from Columbia as a research associate of the Carnegie Institution of Washington. Jack Schultz came in a similar capacity the following year. During its first year, the Biology Division offered a one-term introductory survey course in Biology for undergraduates as well as a program of seminar courses for its graduate students. A more extensive curriculum of courses for Biology majors was begun in 1929-30.

The faculty grew by the addition of Karl Belar, Henry Borsook, Herman Dolk, and Albert Tyler (who had just become Caltech's first Ph.D. in Biology) in 1929, and T. Dobzhansky, Robert Emerson, and Kenneth Thimann in 1930. By 1938 the teaching faculty had grown to a total of 16, with the additions of Hugh Huffman, George MacGinitie, Frits Went, Johannes Van Overbeek, Cornelis Wiersma, Anthonie Van Harreveld, James Bonner, and Arie Haagen-Smit. George Beadle had also served on the faculty during part of this period. In 1937, Max Delbri.ick was a Research Fellow of the Rockefeller Foundation, and Norman Horowitz was a graduate student. (1) "Plan for the Development of Biology at California Institute and its Relation to a Medical School in Los Angeles," 1925. Robert A. Millikan Papers, Box 18, Folder 7, California Institute of Technology Archives.

BIOLOGY DIVISION STAFF

Instruction Research

Administrative

9

STAFF OF INSTRUCTION AND RESEARCH DIVISION OF BIOLOGY

Norman H. Horowitz, Chairman Charles J. Brokaw, Executive Officer Masakazu Konishi, Executive Officer

Board of Trustees Professor of Biology Emeritus

Max DelbrUck, 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 Cornelis A. G. Wiersma, Ph.D. • • • • Biology

Professors

Giuseppe Attardi, M.D. . • . . • • • . • • . • . . . • • • • Biology Seymour Benzer, Ph.D., D.. Sc. James G. Boswell Professor of Neuroscience 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 Norman H. Horowitz, Ph.D. • • . • • • . • • . • • • . • Biology Masakazu Konishi, Ph.D. . . • • • • • • . • • • . • • • Biology Edward B. Lewis, Ph.D. . • Thomas Hunt Morgan Professor of Biology Herschel K. Mitchell, Ph.D. • • • • • • • • • . • • . • • Biology Ray D. Owen, Ph.D., Sc.D. • • • • • • • • . • • • • • • Biology 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

Gosney Visiting Professors

Bertil Rasmuson, Ph.D. • • • Biology Marianne T. Rasmuson, Ph.D. Biology

Sherman Fairchild Distinguished Scholar

Thomas R. Tosteson, Ph.D. • • . • • Biology

Senior Research Associate

Roy J. Britten, Ph.D. Biology

Research Associates

Charles R. Hamilton, Ph.D. . Biology Peter H. Lowy, Doctorandum Biology Marianne E. Olds, Ph.D. Biology Helen R. Revel, Ph.D. . • • Biology

Gosney Visiting Associate

Howard Gest, Ph.D. . . • • • • • . • • . • • • • . • • • • • • . Indiana University 10

Visiting Associates

Paul B. Bell Jr., Ph.D. University of California, Los Angeles Hans-J. Bischoff, Dr. rer. nat•. UniversitB.t Bielefeld Raymond P. Briggs, Ph.D•. • • • • . • Chaffey College C. B. G. Campbell, Ph.D. University of California, Irvine John E. Dever Jr., Ph.D. • . • • . Fort Lewis College Arturo Eslava, Ph.D. . . FacuJtad de Biologia, Leon, Spain Patrice L. French, Ph.D. University of California, Los Angeles Jeffrey J. Hubert, Ph.D. Scripps Clinic and Research Foundation Felicia A. Huppert, Ph.D. Cambridge University Toru Itakura, M.D .. Wakayama Medical School Rolf H. Joho, Ph.D. Stanford University Stanley Klein, Ph.D. Claremont College Evelyn Lee-Teng, Ph.D •. University of Southern California Hans-J. Leppelsack, Dr. rer. nat. Ruhr-Universitiit Bochum Fee-Chon Low, M.Sc. Rubber Research Institute, Malaysia Antonio Montalvo-Correa, M.D. University of Santander Tamotsu Ootaki, Ph.D .• . • • • • • Yamagata University Lajos Pike, D.V.M •.•• Veterans Administration Hospital Margaret Y. Scott, Ph.D. University of California, Los Angeles John Trimble, Ph.D. . University of Chicago Norma Williams, Ph.D. Howard University Kaori Yoshida, M.S. . Yamagata University

Associate Professors

John M. Allman, Ph.D. • • • • Biology A. James Hudspeth, Ph.D., M.D. Biology Henry A. Lester, Ph.D. . • . Biology Tom Maniatis, Ph.D. . • . . Biology John D. Pettigrew, M.B., B.S. Biology James H. Strauss Jr., Ph.D. Biology

Assistant Professors

Jeremy P. Brockes, Ph.D. Biology Ronald J. Konopka, Ph.D. Biology Elias Lazarides, Ph.D. • Biology David C. Van Essen, Ph.D. Biology

Gosney Senior Researeh Fellow

Nancy S. Petersen, Ph.D. • . . . . • • . • . • • • • . • . • • • . . • • • . • • • . . . . • • • • • Biology

Spencer Senior Researeh Fellows

Eri Heller, Ph.D. • • • . . Biology Leonard K. Kaczmarek, Ph.D. Biology John C. Woolum, Ph.D. . • . Biology

Senior Research Fellows

Dorwin L. Birt, Ph.D. Biology Karel Grohmann, Ph.D. Biology Barbara R. Hough-Evans, Ph.D. Biology Michael W. HunkapiUer, Ph.D •• Biology Takuji Kasamatsu, M.D. Biology William H. Klein, Ph.D. Biology Amy Shiu Lee, Ph.D. Biology Minnie McMillan, Ph.D •. Biology Ronald L. Meyer, Ph.D .. Biology Ellen G. Strauss, Ph.D. Biology Jung-Rung Wu, Ph.D .• Biology Eran Zaidel, Ph.D. . . Biology 11

Instructor

Rona Pettigrew, Ph.D.

Gosney Research Fellows

Rasika M. Harshey, Ph.D. Gordon C. Machray, Ph.D. Richard M. Maizels, Ph.D. Richard M. Lawn, Ph.D.

Spencer Research Fellows Timothy D. Field, Ph.D. Joel Myerson, Ph.D. Chun-Fang Wu, Ph.D.

Weizmann Fellows

James F. Baker, Ph.D. Gary G. Blasdel, Ph.D. Ze'ev Lev, Ph.D.

Research Fellows

David M. Anderson, Ph.D. Karen F. Greif, Ph.D. Manfred K. Otto, Ph.D. Robert C. Angerer, Ph.D. Eva B. Griepp, M.D. William R. Pearson, Ph.D. Marylouise Ary, Ph.D. Terrence J. Hall, Ph.D. Gene R. Petersen, Ph.D. James P. Ary, Ph.D. Alfred M. Handler, Ph.D. David N. Radin, Ph.D. Eugene T. Butler III, Ph.D. Ross C. Hardison, Ph.D. Carol Readhead, Ph.D. Palmiro Cantatore, Ph.D. Henry V. Huang, Ph.D. Jose M. Sala-Trepat, Ph.D. J. Michael Cecka, Ph.D. Michael T. Hyson, Ph.D. James Shen, Ph.D. Mark C. Citron, Ph.D. Makkuni Jayaram, Ph.D. Nancy L. Shinowara, Ph.D. Loring G. Craymer III, Ph.D. Robert Johnson, Ph.D. Thomas H. Stanton, Ph.D. Ginny W. Darr, Ph.D. Lawrence M. Kauvar, Ph.D. Murray F. Tei tell, Ph.D. Laura De Francesco, Ph.D. Eric I. Knudsen, Ph.D. Terry L. Thomas, Ph.D. Susan G. Ernst, Ph.D. Florence Lafay, Ph.D. R. Bruce Wallace, Ph.D. Sandra J. Ewald, Ph.D. Christian G. Merkel, Ph.D. John E. Wiktorowicz, Ph.D. Malcolm E. Finbow, Ph.D. Gordon P. Moore, Ph.D. Andrew Wiseman, Ph.D. Edward F. Fritsch, Ph.D. Menasche M. Nass, Ph.D. Barbara Yancey, Ph.D. Barry S. GanetzkY, Ph.D. Makoto Okuno, Ph.D.

Graduate Fellows and Assistants

David L. Armstrong, B.A. David A. Goldberg, B.S. William T. Newsome III, B.S. David J. Asai, M.S. Richard H. Gomer, B.A. Bruce J. Nicholson, B.Sc. Eileen A. Bagdonas, B.S. Bruce L. Granger, B.A. Charles E. Novitski, B.A. Antony C. Bakke, B.S. Steven H. Green, B.S. Dominic Orr, B.Sc. John R. Bell, B.S. Karen F. Greif, A.B. Jing-hsiung James Ou, B.S. John L. Bixby, B.A. Mark Gurney, B.A. Richard C. Parker, B.S. Elizabeth P. Blankenhorn, A.B. Alvin J. Hill Jr., B.S. Vann P. Parker, B.S. Duncan Byers, B.A. Henry V. Huang, A.B. Steven E. Petersen, M.A. Edwin P. Ching, A.B. Bruce D. Hubbard, B.S. James W. Posakony, B.S. Arlene Y. Chiu, M.Sc. Kent R. Jennings, B.Sc. David E. Presti, M.S. Anne Chomyn, B.S. Larry E. Johnson, B.S., M.D. Antonio A. Reyes, B.S. Toni R. Claudio, A.B. Nelson D. Johnson, B.S. Charles M. Rice III, B.S. Susan E. Conrad, B.S. Gary S. Jones, B.A. Thomas D. Sargent, A.B. Michael L. Cooper, B.S., M.S. Marilyn R. Kehry, B.S. James W. Schilling, A.B. David P. Corey, B.A. Michael W. Klymkowsky, B.S. Brian S. Seed, B.S. Frank Costantini, B.S. Mitchell Kronenberg, B.A. Robert E. Sheridan Jr., B.S. Stephen T. Crews, B.A. Mauri E. Krouse, B.S. Sandra L. Shotwell, A.B. Mark M. Davis, B.A. Elizabeth H. Lacy, B.A. Mavis Shure, B.S. Philip W. Early, B.S. Jean-Francois Lafay, B.S. Gek Kee Sim, B.A. Jay Edelman, M.S. Joyce E. Lauer, A.B. Randall F. Smith, B.S. Jay W. Ellison, B.S. Katherine Dai-Li Lee, B.S. Barbara L. Stitt, B.A. John Frelinger, B.S. Loveriza S. Lipps, B.S. Duncan K. Stuart, B.S. Teryl K. Frey, M.S. Kenneth L. Marton, B.S. William E. Stumph, B.S. Karl J. Fryxell, B.A., B.S. John H. R. Maunsell, B.S. David Tang, A.B. Jonathan S. Fuhrman, B.A. Jeffrey T. Mayne, S.B. Betty A. Vermeire, B.S. David L. Gard, B.S. James S. Mccasland, B.S., B.A. Chung Wang, M.S. Karen E. Gaston, M.A. Galina Moller, M.A. Barbara J. Wold, B.S. Robert A. Gelfand, M.A. Robert F. Murphy, B.A. Wilson c.-s. Wu, B.A. Sheila Gillard-Crewther, M.Sc. 12

Student Assistants

Jonathan D. Adler Steven T. Eckmann John T. Rising Vivian R. Albert Douglas N. Ericson Eric Saund Alan H. Barr Bradley s. Francis Leon E. Schwartz James H. Bayless Susan S. Gilley George D. Shaffer Clifford Beall Leila M. Gonzalez Carol Shotwell Margaret M. Beloian Kenneth M. Gray Paul A. Shupak Scott T. Bishop Paul A. Gutierrez Sandra K. Sigmund Geoffrey A. Blake Michael Kozlowski David W. Sivertsen Rebecca F. Bodor Cary Lai John C. Terrell William E. Bratton II Kerry Laing-Renay Kerin Thompson Walter G. Bright Martin E. Mann Steven G. Trabert Jeffrey D. Carpenter Kevin B. Martin Christopher D. Vestuto Jefferson W. Chen Paul W. Meyer John C. Wathey Timothy E. Cushing James G. Moore Josef B. Zwass Robert G. Dergarabedian Walter D. Niles JII Peter A. Dewees Tony B. Ramey

Research Staff

Michel E. Adams Robin M. Hamilton Jeanette Navest Cynthia Akutagawa Ronald T. Hardgrove Dana B. Nelson Eugene Akutagawa Nancy Harris Robert F. Nickerson Richard Axel David B. Helphrey Robert Nienhuis David R. Balzer Jr. George L. Hobby Don J. Nishiguchi Ingelore S. Bonner George Horn Peter E. Nolan Caroline M. Carter Suzanna J. Horvath Catherine D. O'Connell Paul K. Cartier III Nancy Hwang Edward Ogawa Francisca A. Castorena Richard A. Jacobs Deanna K. Ojala. Anita S. Cetta Geri D. Joelson Stella Olive Margaret E. Chamberlin Mary Jo Johnson Wanda L. Owens Gisela Charlang Jeannette Johnstone Robin W, Paltis John Wen-Kiang Chu Bertha E. Jones Jeanne P. Patalano Monica B. W. Cooper Gertrude Jordan Carol K. Peck Adriana Cortenbach Susan E. Kass Robert 0. Piehl Alice I. Cox Benneta Keeley Ellen Potter Madeline A. Crosby Peggy L. Kelly Diana H. Quon Samuel D. Culpovich Sarah E. Kennedy Jane Rigg Larry C. Cummings Chin Sook Kim Joan Roach Gloria C. Davis Rosina K. T. Kinzel Richard M. Rohan Maria A. De Bruyn Phyllis F. Knudsen Eileen P. L. Roy Ursino E. Del Valle Patrick F. KOen Helga Schiff-Sertorio Barbara L. DeOgny Baruch Kuppermann Floyd Schlechte Arger L. Drew Edward D. Kusby Sydne A. Schurmeier Wanda J. Dunn Patrick s. Leahy Dennis R. Shelby David W. Easter Edith M. Lenches Thomas F. Simoni ck Jean Edens Ilga Lielausis Amelia M. Smit Eveline Eichenberger Deborah P. V. Loeppky Robert W. Tajima Leah Ellenberg Peter Sin-Yi Lu Stephen I. Vass Gloria Engel Eva H. Lujan Gudrun Vener K. Jeffrey Eriksen Harriett L. Lyle Caroline Vermaes Vincent R. Farnsworth Lois E. MacBird Daniel P. Viele Doris Finch Josephine Macenka Linda S. Vock Sue M. Fitch Mary S. Martin Steven M. Wells Susan E. Gerber EveLynn McGuinness Eva Westmorland Roberta Gerson Francis M. Miezin Gayle-Linda Westrate Ana L. Gharzeddine Charlotte A. Miller Donna R. Williams Robert L. Gimlich K. Elaine Miller Kendrick N. Williams Eef Goedemans Catherine R. Morris Nancy K. Wischhusen Maria R. Gomez Donald D. Morris Leslie L. Wolcott Judith S. Greengard Steven D. Mueller Annette S. Yuen Debra Dison Hall Orland D. Mylander Elizabeth R. Zimmerman 13

ADMINISTRATIVE STAFF

Michael Miranda, Administrator Geraldine S. Cranmer, Division Secretary

Accounting Kerckhoff Marine Laboratory

Lody Kempees, Supervisor Robert K. Blue, Superintendent Sandra L. Carta Joe R. Deem, Superintendent Ruth M. Erickson Peggy R. Bierer Howard J. Sheldon Animal Rooms William Smith

Milton Grooms, Supervisor Machine Shop E. Larry Baldwin Roberto De La Vega Frank L. Ostrander, Supervisor Murat Erduran Thomas Barrigan Julie Mangan Richard J. Broderick Gildardo Vega Donnie Weatherton Secretarial

Beckman Laboratories Jane Chacon, Supervisor Constance R. Katz JoAnn Chikahiro - Accounting Bonnie D. Kimble Elizabeth T. Hanson - Secretarial Elizabeth F. Koster William F. Lease - Stockroom Bernita K. Larsh Dale D. Linder - Animal Rooms Donna J. Lathrop Michael Walsh - Electronics Shop Renee Thorf David C. Hodge Stockroom mectrical Shop Jane C. Keasberry, Supervisor Harold A. Brennan Larry Baker Christopher M. Ingram Grants Louis A. Knust-Graichen Linda Mccasland Isabella Lubomirski, Supervisor Elizabeth A. Sandoz Barbara Cathey Elizabeth Vagner

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

Herschel K. Mitchell

James H. Strauss Jr.

17

Professor: Giuseppe Attardi mit-DNA [Ojala and Attardi, 1978]), has been further Visiting Associate: Antonio Montalvo-Correa characterized by analyzing 78 molecules labeled at the Research Fellows: Palmiro Cantatore, Laura De 32 Francesco, Christian G. Merkel, Andrew Wiseman 5'-end with [y- PJATP and T4 polynucleotide kinase. 78 Graduate Students: Edwin P. Ching, Stephen T. Crews, DNA, labeled either after separation by sucrose gradient Robert A. Gelfand Research Staff: Doris Finch, Benneta Keeley, Don J. centrifugation or without prior purification from closed Nishiguchi, Deanna K. Ojala circular mit-DNA, was purified by nitrocellulose chroma­ Laboratory Staff: Arger L. Drew, Gloria Engel, Maria R. Gomez, Rosina K. T. Kinzel, Stella Olive, Wanda L. tography and electrophoresis through urea slab gels. The Owens slab gels demonstrated the existence of a major com­ Support: The work described in the following research ponent (70 to 95% in different preparations) corresponding reports has been supported by: to a size of about 680 bases, and two minor components American Cancer Society Consiglio Nazionale delle Ricerche migrating slightly faster. mectrophoretic analysis after National Institutes of Health, USPHS National Science Foundation treatment with RNase or pronase, and rerun of individual components through agarose CH HgOH slab gels indicated University of Bari 3 Helen Hay Whitney Foundation that the observed heterogeneity is not due to a covalently Summary: Our efforts to probe the organization and linked RNA primer, DNA-associated proteins, or differ­ expression of human mitochondrial DNA have been pur­ ences in secondary structure, but reflects, presumably, sued at the molecular and cellular level. An approxi­ differences in length. mately 220-base long segment of HeLa cell mitochondrial The major component was isolated from a urea gel DNA containing the origin of replication has been se­ and subjected to sequencing reactions (see Biology 1978, quenced and aligned with the 5'-end-proximal portion of No. 2) and 5'-end analysis, using for the latter Pl nuclease 78 DNA, which is the heavy chain initiation sequence; this digestion and two-dimensional chromatography on poly­ has allowed the precise localization of the origin in the ethylenimine thin-layer plates. The results indicated that DNA sequence. Progress has been made in the construc­ the major component is substantially homogeneous at the tion of a detailed transcription map of HeLa cell 5'-end, with 90 to 95% of the 5'-terminus nucleotides mitochondrial DNA. At the level of expression of the being represented by dTMP. mitochondrial genome, a much larger number of discrete Reference: mitochondrial protein products than previously known has Ojala, D. and Attardi, G. (1978) J. Mo!. Biol. 122: 301. been identified. By using human cells partially depleted of mitochondrial DNA and mutagenized by treatments 2. SEQUENCE ANALYSIS OF THE ORIGIN OF thought to be specific for the organelle DNA, a new class REPLICATION OF HUMAN MITOCHONDRIAL DNA of mitochondrial DNA mutants has been isolated and Investigators: Stephen T. Crews, Deanna K. Ojala, partially characterized: besides a gross deficiency in Don J. Nishiguchi, James W. Posakony, mitochondrial prOtein synthesis, which is their most Giuseppe Attardi distinctive phenotype trait, these mutants exhibit resis­ Mitochondrial DNA (mit-DNA) synthesis in HeLa tance to chloramphenicol and glucose dependence. An cells begins at a site on the genome corresponding to the analysis of the sequence homology between human and 5'-end of the 7S DNA. We have sequenced the 5'-terminal mouse mitochondrial DNA has revealed that the ribosomal region of the 78 DNA and a restriction fragment of HeLa genes and probably the tRNA genes are the most cell mit-DNA that contains the origin of replication, in conserved portions of the genome. order to obtain some structural information regarding this important region. 5'-end-labeled 7S DNA, prepared as 1. CHARACTERIZATION OF HeLa CELL described by Ojala et al. (Biology 1978, No. 1), was MITOCHONDRIAL 7S DNA sequenced using the chemical procedure of Maxam and Investigator: Deanna K. Ojala Gilbert (1977), and the reaction products were fraction­ HeLa cell mitochondrial 7S DNA, whose 5'-terminus ated on a 25% polyacrylamide/7 M urea gel. Approxi­ marks the position of the origin of mit-DNA H strand mately 25 nucleotides could be unambiguously read. replication (which has been previously localized precisely Previously, it had been demonstrated (Ojala and with respect to the Hpa II restriction map of HeLa cell Attardi, 1978) that the 5'-end of the 78 DNA is located in 18 fragment 8 of the HeLa cell mit-DNA Hpa II restriction approximately 150 nucleotides from the 5' end, and that map, at approximately 80 nucleotides from the cutting corresponding to the heavy strand coul be read for site between fragments 8 and 17. Hpa II fragment 8 can approximately 120 nucleotides, thus providi a 50 nucleo­ be cleaved with another restriction enzyme, Hae III, to tide overlap. The molecular weight of 6.8b ae was found generate two fragments approximately 560 and 200 to be 221 base pairs and the 7S DNA was ound to lie at nucleotide pairs long. The 200 base pair long fragment, exactly 87 nucleotides from the Hpa II cutting site. designated ti.8bHae, contains the sequences of the 5'-ter­ Figure 1 shows the position of the origin or replication in minal portion of 78 DNA. This entire fragment was the genetic map of HeLa cell mit-DNA, he nucleotide sequenced by labeling the 5'-ends with polynucleotide sequence around it, and a predicted sta le secondary 32 kinase and [y- PJATP, separating the two single­ structure that might form when this regi n of DNA is stranded segments of DNA on a 6% polyacrylamide gel unwound, and conceivably could be invol ed in protein and submitting the two strands to the chemical sequencing recognition or some other function. procedure mentioned above. The reaction products were References: fractionated on either 25% polyacrylamide/7 M urea gels Maxam, A. and Gilbert W. (1977) Proc. N t. Acad. Sci. USA 74: 560-564. or 10% polyacrylamide/7 M urea gels. The strand cor­ Ojala, D. and Attardi, G. (1978) J. Mo!. Biol 122: 301. responding to the mit-DNA light strand could be read for

0 ! - 12 I I \7 I \ I \ / \ / \ I \ / \ / \ / \ / \ 3 I \ I \ 10 5' ...... T·G-T-T·A-T-T-A-T-T-A-T-G-T-C T-A·T·T·G·A-A·C·G·T·A·G·G·T·G 3' 3' A-G-A-A -T -A-A-T-A-A- T-A-C-A-G, .A-T-A-A-C-T-T -G-C-A-T-C-C-A-C 5 1 60 '° ~: + 7o L >~ond 9 !: ~ ''° T· ~ t:r"' 4 p.r•-•-..¥: g

110 \,•• _ ••,·~: r· ~:~ ~o 100 T • A G• c;o t: i ''r-c"

4

6

Figure 1. The diagram shows the current genetic map of human mitochondrial DNA with the positio of the genes for the 12S and 16S ribosomal RNA species on the heavy (H) strand, and for the 4S RNA genes (most or all of hich encode tRNA) on the H strand (numbered 1 to 12) and the light (L) strand (numbered 1 to 7). The location of the rigin of DNA replication is indicated by the vertical arrow (0) and the direction of H strand synthesis by the rightward row. In the center the nucleotide sequence of the DNA segment surrounding the origin is shown; this is folded to give a theoretically stable secondary structW'e that might form in this region when the double-stranded DNA is melted, and con eivably could be involved in protein recognition or some other function. This is the first origin of replication of eukaryot c DNA to be precisely localized and sequenced. 19

3. TRANSCRIPTION MAP OF THE MITOCHONDRIAL 4. LOCALIZATION OF PROMOTER SITES ON DNA FROM HeLa CELLS HeLa CELL MITOCHONDRIAL DNA Investigators: Christian G. Merkel, Deanna K. Ojala Investigator: Palmiro C&ntatore

Evidence from this laboratory has indicated the The study of the location of promoter sites on HeLa existence of at least 18 discrete poly(A)-containing RNA cell mitochondrial DNA (mit-DNA), has been undertaken components coded for by mitochondrial DNA (mit-DNA) in order to obtain information useful for understanding the (Amalric et al., 1978). These RNA components have been mechanism of mit-DNA transcription. To accomplish this resolved by agarose slab gel electrophoresis in the goal, nascent transcripts of the light (L) mit-DNA strand, presence of methylmercuric hydroxide as a denaturing isolated from cells labeled with a very short pulse of 3H­ agent and have molecular weights in the range of 9.3 x uridine, were fractionated in a sucrose gradient under de­ 4 6 10 to 3.4 x 10 daltons. In the present study, we have naturing conditions, and the different size classes were isolated these discrete poly(A)-containing RN As from purified by affinity chromatography on heavy (H) strand agarose-CH HgOH slab gels in order to map the location mit-DNA cellulose, reduced to a uniformly small size by 3 of their sequences in mit-DNA by RNA-DNA hybridiza­ controlled alkali treatment, and finally hybridized with tion. A detailed physical map of the HeLa cell mit-DNA isolated Hpa II restriction fragments of mit-DNA. Under has been constructed by ordering the 21 DNA fragments these conditions, the fragments closer to a transcription generated by the endonuclease Hpa II (Ojala and Attardi, initiation point should be recognized for the higher 1977). These DNA fragments have been isolated and hybridization with the labeled segment of the shortest hybridized to the in vivo 32P- or 3H-labeled discrete RN A chain. The results of these experiments suggest that poly(A}-containing RN As under conditions which allow at least two regions are involved in the initiation of the DNA-RNA hybridizatidn in the absence of any appreciable transcription of the L strand mit-DNA. DNA-DNA reassociation. In addition, individual RNA In order to obtain further information, experiments 32 species labeled in vitro with [y- PJATP and T4 poly­ are being performed using a different approach. After nucleotide kinase, with or without prior fragmentation by hybridization of the nascent RN A with Hpa Il restriction controlled alkali trea:,tment, have been hybridized to fragments, labeled in vitro by nick translation with 32 Hpa II restriction endonuclease digests of HeLa cell [Cl- P] deoxynucleoside triphosphates, and treatment with mit-DNA which had been transferred to nitrocellulose Sl, the sample is analyzed on a polyacrylamide gel. Under strips after agarose gel electrophoresis. Using these these conditions, if the fragment containing a transcrip­ techniques, it has been possible to map the location of tion initiation site is small relative to the size of the transcription of a number of these RNA species with transcripts, it should produce a band, whose dimension respect to the position of the rRN A and tRNA genes and indicates the distance between the initiation site and the of the origin of replication. Studies are currently being end of the fragment. carried out using the technique of Berk and Sharp (1977) to locate precisely the position of transcription for the 5. CHARACTERIZATION OF POLYPEPTIDES SYNTHESIZED ON MITOCHONDRIAL RIBOSOMES individual RNA species. It should also be possible using Investigators: Edwin P. Ching, Giuseppe Attardi these techniques to determine the possible presence of ''leader sequences" in the RN A and the occurrence of RN A By appropriate use of antibiotics specifically affect­ splicing. The information gained by these studies will help ing translation by cytoplasmic ribosomes, it has long peen us in understanding the mechanism of transcription and possible to selectively incorporate radioactively-labeled processing of mit-RNA. amino acids into mitochondrially translated polypeptide References: species. Upon analysis of these translation products by Amalric, F., Merkel, C., Gelfand, R. and Attardi, G. high fesolution SDS polyacrylamide gel electrophoresis, (1978) J. Mo!. Biol. 118: 1-25. we have identified a significantly larger number of species Berk, A. J. and Sharp, P. A. (1977) Cell 12: 721-732. Ojala, D. and Attard!, G. (1978) Plasmid 1: 78-105. than the ten previously characterized (Biology 1974, No. 84). We are currently investigating the possibility of prectFsor to product relationships between the different species. 20

6. ISOLATION OF MYELOMA IJNl!S SECRETING and rutamycin-sensitive ATPase activitie , and grows ANTIBODIES TO HUMAN CYTOCHROME indefinitely in the iresence of a concent ation of the OXIDASE mitochondrial protein synthesis inhibitor c ramphenicol Investigators: Andrew Wiseman, l!dwin F. Ching, Giuseppe Attardi (CAP, 100 µg/ml) which is lethal to the pa ental VA -B 2 cells. Unlike wild-type cells, the viability o all mit-PS- Milstein and his co-workers (KOhler and Milstein, mutants tested is low when grown in medi containing 1975, 1976) have recently described techniques for genet­ 9 µg/ml of glucose, while it is normal or nea to normal in ically constructing clones of established mouse myelorna medium containing 4.5 mg/ml glucose. Fusio s of enucle­ cell lines which secrete monospecific antibodies directed ated cells (cytoplasts) of seven independe tly isolated against particular antigens. These clones are extremely mit-PS- mutants to the nucleated thiogua ·ne-resistant valuable for the large-scale production of monoclonal VA -B derivative TG-6 have yielded num rous cybrid antibodies, because they can be grown indefinitely in 2 culture and secrete the antibody into the medium. clones which gTOW in CAP plus thioguanine; in contrast, almost no clones resulted from fusions f nucleated Following their techniques, we are presently preparing to mit-PS- cells to TG-6, as expected from he recessive isolate myeloma clones which secrete antibodies to human cytochrome c oxidase which we have purified from human character of the nuclear mutation conferri thioguanine placentas (Hare, Ching and Attardi, submitted to J. Biol. resistance. These results suggest that the genetic deficiency or deficiencies which enable mit- S- mutants Chem.). Spleen cells prepared from mice that have been immunized against human cytochrome c oxidase will be to grow in CAP are coded by cytoplas ic genetic fused to the established secreting myeloma line MPC-11 determinants, possibly mit-DNA. Additional studies have demonstrated that these CAP-resistant cybri clones are that is resistant to thioguanine, and spleen-MPC-11 also deficient in mitochondrial protein synth is, suggest­ hybrids will be selected in HAT medium. These hybrid clones will then be screened for antibody against cyto­ ing that this deficiency is probably the result of the same chrome c oxidase and characterized as to subunit speci­ cytoplasmic mutation. Experiments are in progress to characterize these mutants biochemically b an analysis ficity of the antibody. of their mit-DNA, ribosomes, and RNA transc ipts. References: Kohler, G. and Milstein, C. (1975) Nature 256: 495. Kohler, G. and Milstein, C. (1976) Eur. J. Immunol. 6: 511. 8. MITOCHONDRIAL DNA IN SOMATIC C L HYBRIDS

7. MUTANTS OF HUMAN CELLS DEFICIENT IN Investigator: Laura De Francesco MITOCHONDRIAL PROTEIN SYN IHl!SIS A series of mouse-human hybrid cell ines which Investigators: Andrew Wiseman, Giuseppe Attardi were made by Carlo Croce of the Wistar lnstit te, is being We have previously shown that the human cell line examined for the retention of mitochondrial NA of each VA -B can grow in the presence of 20 ng/ml of ethidium parental species. The hybrid lines are of two ypes, some 2 bromide in the absence of nearly any detectable mito­ which segregate human chromosomes from t e nucleus, chondrial DNA (mit-DNA) replication, so that, after three and some which segregate mouse chromoso es. These cell doublings, cells thus treated have at most 10% of hybrids have been found by Croce and his to their normal DNA content (Wiseman and Attardi, sub­ express certain properties of only one parent in spite of mitted to Mol. Gen. Genet.). It was hoped that rare the presence of, in some cases, a large number of mit-DNA mutations could be more readily selected from chromosomes of the second (segregating) pare (Croce et cells whose mit-DNA content was greatly reduced. al., 1977; Huebner et al., 1977). In all hybri analyzed We have recently isolated a large number of thus far, the mitochondrial DNA of the s cies being mit-DN A mutants deficient in mitochondrial protein segregated from the nucleus is undetectable, o synthesis (mit-PS-) from VA -B by subjecting cells 2 very small amount. This was determined partially depleted of their mit-DNA to mutagenic treat­ Southern blots of restricted mitochondrial DN from the ments thought to be specific for mit-DNA. Each of these hybrids, and hybridizing it to nick-translate parental mit-PS- mutants has less than 10% of the wild-type rate mitochondrial DNA probes. The level of det ction with of protein synthesis, exhibits reduced cytochrome oxidase this method is of the order of a few percent; f thermore, 21 these experiments would have allowed the recognition of PUBLICATIONS recombinant DNA molecules. There is no evidence thus Amalric, F., Merkel, C., Gelfand, R. and Attardi, G. far for extensive retention of mitochondrial DNA of the (1978) Fractionation of mitochondrial RNA from HeLa cells by high resolution electrophoresis under dormant genome, nor for recombination between the two strongly denaturing conditions. J. Mol. Biol. 118: species of mitochondrial DNA. More hybrid cell lines are 1-25. Attardi, G. and Ching, E. (1978) Biogenesis of mitochon- being examined for these properties. drial proteins in HeLa cells. Methods in Enzymology-Biomembranes D-Vlll-8. In press. References: Attardi, G., Costantino, P., England, J. M., Lynch, D., Croce, C., Talavera, A., Basilico, C. and Miller, O. J. Ojala, D., Posakony, J. W. and Storrie, B. (1977) (1977) Proc. Nat. Acad. Sci. USA 74: 694-697. Huebner, K., Shander, M. and Coce, C. (1977) Cell 11: Mitochondrial and nuclear gene interaction in HeLa cells. In: Genetic Interaction and Gene Transfer, 25-33. Brookhaven Symposia in Biology, No. 29, pp. 16-35. Attardi, G., Crews, S., Nishiguchi, J., Ojala, D. and Posakony, J. (1978) Nucleotide sequence of a frag­ 9. REGIONS OF HOMOLOGY OF MITOCHONDRIAL ment of HeLa cell mitochondrial DNA containing DNA FROM BUMAN AND MOUSE CELLS the precisely localized origin of replication. Cold lnvestigator: Laura De Francesco Spring Harbor Symp. Quant. Biol. 43. In press. Carre, D. and Attardi, G. (1978) Biochemical and electron In hybridizations between highly labeled mitochon­ microscopic characterization of DNA-RNA com­ drial DNAs of two distantly related species (human and plexes from HeLa cell mitochondria. Biochemistry 17: 3263. mouse), extensive cross-hybridization was observed in Ching, E., Costantino, P. and Attardi, G. (1977) In vivo experiments involving Southern blots and nick-translated incorporation of different amino acids into electro­ phoretically characteristic polypeptides synthesized parental mitochondrial DNA probes. The cross-reacting by HeLa cell mitochondria. Biochem. Biophys. Res. regions appear to be of two types according to their Commun. 79: 451-460. England, J. M., Costantino, P. and Attardi, G. (1978) stability. One region is stable under very stringent Mitochondrial RNA and protein synthesis in enucle­ conditions of washing (0.3X SSC) and corresponds to the ated African green monkey cells. J. Mo!. Biol. 119: 455-462. ribosomal RNA genes. That the ribosomal genes are in Grohmann, K., Amalric, F., Crews, S. and Attardi, G. fact responsible for this stable heteroduplex was shown by (1978) Failure to detect neap" structures in mito­ chondrial DNA-coded poly(A)-containing RNA from competition hybridization experiments with purified, cold HeLa cells. Nucleic Acids Res. 5: 637-651. mitochondrial ribosomal RNAs. The remainder of the Mitchell, C. and Attardi, G. (1978) Cytoplasmic location of the genetic information for chloramphenicol cross-reacting sequences is less stable and is scattered resistance in a human cell line. Somatic Cell Genet. around the genome. It is likely that transfer RNAs are In press. Ojala, D. and Attardi, G. (1977) A detailed physical map involved in these regions (Dawid, 1972; Jakovcic et al., of HeLa cell mitochondrial DNA and its alignment with the positions of known genetic markers. Plas­ 1975). This possibility is being examined by similar mid 1: 78-105. competition hybridization experiments. Ojala, D. and Attardi, G. (1978) Precise localization of the origin of replication in a physical map of HeLa cell References: mitochondrial DNA and isolation of a small frag­ Dawid, I. B. (1972) Devel. Biol. 29: 139-151. ment that contains it. J. Mol. Biol. 122: 301. Jakovcic, S., Casey, J. and Rabinowitz, M. (1975) Bio­ Wiseman, A. and Attardi, G. (1978) Reversible tenfold chemistry 14: 2037. reduction in mitochondrial DNA content of human cells treated with ethidium bromide. Mol. Gen. Genet. Submitted for publication. Wiseman, A. and Attardi, G. (1978) Mitochondrial DNA mutants of a human cell line deficient in mitochon­ drial protein synthesis. Somatic Cell Genet. Sub­ mitted for publication. 22

Professor: James Bonner Gottesfeld and Partington (1977) have sh wn that the Visiting Associates: John E. Dever Jr., Fee-Chon Low globin gene is in the DNase II excisable rtion of the Senior Research Fellow: Jung-Rung Wu Gosney Research Fellow: Gordon C. Machray genome. Preliminary studies of the ri soma! genes Research Fellows: Jose M. Sala-Trepat, Murray F. Teitell, R. Bruce Wallace, John E. Wiktorowicz indicate that they are caused to become urned off by Graduate Students: Antony C. Bakke, Robert F. Murphy, induction of Friend cells by DMSO and a e transferred Antonio A. Reyes, Thomas D. Sargent, William E. from the expressed portion of the genome to the nonex­ Stumph Research Staff: Ingelore S. Bonner, Caroline M. Carter, pressed portion of the genome. Merrie Jo Johnson, Ellen Potter Laboratory Staff: Sue M. Fitch, Stella Olive The fact that it is possible thus to exc e selectively the expressed portions of the genome has m de it possible Support: The work described in the following research to determine the chemical and physic differences reports has been supported by: American Cancer Society between expressed genes or expressed DNA equences and Centre National de la Recherche Scientifique nonexpressed genes or DNA sequences. he principal Fort Lewis College Gosney Fund differences are: the DNA in the expressed rtion of the Medical Research Council of Canaada genome is low melting; that is, the DNA is not stabilized National Institutes of Health, USPHS National Science Foundation against melting by histones to any consid able degree, Damon Runyon-Walter Winchell Cancer Fund the DNA of the expressed portion is exten ed (as judged Summary: We have known for some time that the DNA by circular dichroism [CDJ, hyperchromici y, etc.), and of all eukaryotes is complexed with histone molecules. finally the DNA of the expressed portion of he genome is The histones have not only been separated from one selectively attacked not only by DNase II bu also by other another (there are five species) (Fambrough and Bonner, nucleases, in particular DNase I. In additi n, the N-ter­ 1966; Fambrough et al., 1968) but all have been sequenced minal peptides of the four core histones ar more rapidly in one or more creatures (DeLange et al., 1969; Elgin et attacked by trypsin in the DNase II-excised rtion of the al., 1971). Their primary structures are highly conserved genome than is true of the genome as a whol and four of the species present in equimolecular amounts Billing and Bonner (1972) showed that he expressed (Fambrough et al., 1968) interact to form the octameric portion of the genome is selectively attack d by DNase I core of the nucleosome (D'Anna and Isenberg, 1974), the which rapidly degrades the expressed seque ces to acid­ basic structure of the chromosome, containing as it does soluble material. Thus, DNaSe II excises long sequences of about 200 base pairs of DNA (Hewish and Burgoyne, 1973). the expressed portion of the genome, se uences long The complexing of histones to DNA is true of the enough to be hybridizable and thus studyabl as shown by nonexpressed, that is, nontranscribed, portion of the Gottesfeld et al. (1974, 1976), while DNase reduces the genome, as well as of the transcribed or expressed portion same set of sequences to acid nonprecipitabl nucleotides. of the genome. That this is so became particularly clear An interesting experiment in this conn ction is that as a result of the work by Marushige and Bonner in 1971, of attacking chromatin with DNase I until bout 15% of which showed that the expressed portion of the genome the DNA is rendered acid-soluble. The D ase I is then can be preferentially excised from the remainder by the inactivated and the remaining DNA recov red as pure nuclease, DNase II, which makes double-stranded clips in DNA. In a separate experiment, chromatin i attacked by DNA. DNase II attacks the expressed sequences of the DNase II and the DNase II-excisable sequenc s recovered, genome several times more rapidly than it attacks the deproteinized, and used as driver to drive the DNA of nonexpressed portions of the genome. By this method, it DNase I-treated chromatin. (Both are fr m rat liver is possible to selectively excise expressed sequences chromatin.) The DNase I-treated chromatin has lost the (Marushige and Bonner, 1971; Gottesfeld et al., 1974). sequences which hybridize to the DNase II- cisable se­ The fractionation method applies not only for the ex­ quences; that is, the two enzymes attack exa tly the same pressed sequences as a whole but also and equally for sequences of liver chromatin, with the di ference, as individual coding sequences studied with specific cDNA noted above, that DNase II excises sequenc of hybrid­ probes. Thus in the study of Friend cells, in which the izable length while DNase I reduces them to acid-soluble globin genes are inducible with low concentrations of portions. dimethylsulfoxide (DMSO}, Wallace et al. (1977a) and The fact that nucleases attack chromati selectively 23 has not escaped notice. Thus, Gare! and Axel (1976) and becomes low melting, that is, histones no longer stabilize Weintraub and Groudine (1976) have both shown that chromosomal DNA against melting, the CD spectrum of attack of chromatin by DNase I removes the sequences the DNA returns to that of extended B-form DNA, the which hybridize to specific cDNA probes. In chromatin chromatin becomes transcribable by RNA polymerase, from tissues in which the same gene is not expressed, the etc. relevant sequences are not destroyed. Therefore with the Our findings concerning acetylation of histones and use of cDNA probes, also, it is apparent that DNase II and its direct relation to gene activation and alteration of the DNase I selectively remove expressed sequences of the physical state of nucleosomal RNA have been rapidly and genome. The same is not true of micrococcal nuclease, widely confirmed. Thus, Levy-Wilson et al. (1977) have which appears to more or less nonselectively cleave all fractionated the chromatin of cultured Drosophila cells chromatin into nucleosomal particles. (cultured in labeled acetate) by the DNase II method and It has been shown in a series of articles which we found that the histones of the template-active fraction in­ have summarized in earlier reports (Gottesfeld et al., corporate acetate 40% more rapidly than do the histones 1974, 1976) that the expressed portion of the genome, of the· inactive fraction. We have found the same with namely that excised by DNa~e II, is organized into induced Friend cells (Wallace et al., personal communica­ approximately 200 base pair long units, just as is the tion). More interesting are the steady-state compositions nucleosomal nonexpressed portion of the genome. It has of the histones of the active and inactive fractions. Davie been shown repeatedly that the histones of chromatin and Candido (1978) have found with trout testis chromatin remain associated with the DNA when DNA sequences are that essentially all of the di, tri, and tetra acetylated H4 caused to become expressed (instead of remaining re­ is in the template-active fraction, as judged by polyacryl­ pressed); that is, histones do not leave DNA when DNA amide gel electrophoresis. (Separation of acetylated from becomes expressible. A major problem of our time is, nonacetylated forms of other histones was not achieved.) therefore, what chemical mechanism causes histone­ Recently Riggs et al. (1977) found that incubating complexed DNA to become template-active and expressed cells in low concentration of Na butyrate appears to as RNA transcripts? inhibit deacetylation, i.e., reduces acetyl turnover. This There have been many hints concerning the answer in turn results in relatively massive accumulation of to this question. The most direct hints have come from multiply-acetylated forms of H3, H2a, and H2b, as well as (1) the phenomenological observations of Allfrey et al. of H4 (Sealy and Chalkley, 1978). These workers carried (1964) that when cells are caused to become more active, their investigation no further. Vidali et al. (1978a,b) have the histone associated with the DNA of such cells shown further that butyrate not only causes accumulation becomes more acetylated and (2) the direct demonstration in chromatin of multi-acetylated histones, but also that by Marushige in 1976 that acetylation of chromatin with the chromatin whose histones a.re thus acetylated become acetic anhydride, which acetylates the e:-amino groups of more susceptible to DNase I digestion (i.e., more like the lysines of the N-terminal peptides (only) of the template-active chromatin). Their conclusion parallels histones of chromatin, causes such chromatin to become ours; to wit, acetylation of histone N-terminal peptides readily transcribable by RNA polymerase, even though the causes relaxation of his tone binding to DNA. his tones remain associated with the DNA. This matter It is thus increasingly clear that the final act of the has formed the subject of intensive scrutiny by our group turning-on of gene expression is caused by histone during the past three years. We have found that acetylation. Thus, acetylation of the N-terminal peptides acetylation of chromatin with acetic anhydride, which of histones causes the histones to lose their grip on the mimics (so far as sites of acetylation, amounts of DNA and therefore allows the DNA to relax and extend acetylation, histones acetylated, etc.) the acetylations itself into an extended conformation. This conformation which occur in vivo, causes all of the physical changes is recognized as the B-form. It is transcribable by RNA associated with the conversion of template-inactive to polymerase. Transcribability is caused by acetylation of template-active chromatin that have been described histones. The only remaining questions are what directs above (Bonner, 1977; Bonner et al., 1977; Wallace et al., the histone acetylase, a ciu'omosomal protein, to acety­ 1977b). As a result of acetylation the DNA of chromatin late the histones complexed with particular sequences of 24

DNA? What is the way in which the acetylase is kept in Sealy, L. and Chalkley, R. (1978) Cell 14: 115 121. Vidali, G., Boffa, L., Bradbury, E. M. and Allfrey, V. leash when its action is not required? This is the problem (1978a) Proc. Nat. Acad. Sci. USA 75: 2 39-2243. which we are attacking and the ways in which we are Vidali, G., Boffa, L., Mann, R. and Allfrey V. (1978b) Biochem. Biophys. Res. Commun. 82: 22 -227. attacking it are described below. Wallace, R. B., Dube, S. K. and Bonner, J. (19 7a) Science The problems of the control of gene expression in 198: 1166-1168. Wallace, R. B., Sargent, T. D., Murphy, R. F. and Bonner, higher eukaryotes appear to be too complex to approach J, (1977b) Proc. Nat. Acad. Sci. USA 74: 3244-3248. on the level of the entire genome. We therefore are Weintraub, H. and Groudine, M. (1976) S ience 193: 848-956. dissecting the genome in fragments, each fragment long enough (15-20 kb) to contain a coding sequence and 10. THE ROLE OF HllTONE ACEl'YLATIO IN adjacent elements. The fragments, isolated and multi­ GENE EXPRESSION plied by recombinant DNA technology, are then being lnvestigator: John E. Wiktorowicz reconstituted into mini-chromosomes consisting of 75 to In the nucleosomal model of chromati structure, 100 nucleosomes. We then determine which nuclear histones of four classes, 2a, 2b, 3, and 4 are c mbined into elements are required to turn on an expressed (in liver) globular structures (nucleosomal core par icles} sur­ gene and which will not at the same time turn on a rounded by about 140 base pairs of DNA ( ewish and nonexpressed (in liver) gene. Burgoyne, 197 3; Kornberg and Thomas, 19 4). These General References: globular regions contain DNA in a represse (nontran­ Allfrey, V. G., Faulkner, R. M. and Mirsky, A. E. (1964) Proc. Nat. Acad. Sci. USA 51: 786-794. scribable) state and render the DNA insensit ve to mild Billing, R. J. and Bonner, J. (1972) Biochim. Biophys. nucleolytic attack (Sahasrabudde and Van H Ide, 1974; Acta 281: 453-462. Bonner, J. (1977) ln: Molecular Human Cytogenetics: Billing and Bonner, 1972). Chemical modifica ion of this ICN-UCLA Symposia on Molecular and Cellular template-inactive chromatin with acetic anhy ide results Biology, R. s. Sparkes, D. E. Comings and C. F. Fox (Eds.), Vol. VII, pp. 53-64, Academic Press. in an increase in the template activity of th chromatin Bonner, J., Wallace, R. B., Sargent, T. D., Murphy, R. F. without significant removal of the histones Marushige, and Dube, s. K. (1977) Cold Spring Harbor Symp. Quant. Biol. In press. 1976; Wallace et al., 1977). Furthermor , in situ D'Anna, J. and lsenberg, I. (1974) Biochemistry 13: acetylation using radiolabeled acetyl coe 4992-4997. Davie, J, and Candido, E. P. (1978) Proc. Nat. Acad. Sci. sodium acetate has been demonstrated to be cific for USA. 1n press. the e::-amino group of the lysines in the -terminal DeLange, R. J., Fambrough, D. M., Smith, E. L. and Bonner, J. (1969) J. Biol. Chem. 244: 5669-5679. regions of the histones present in the n cleosomal Elgin, S. C. R., Froehner, S. C., Smart, J. E. and Bonner, structure. Since it is thought that the nucleo J, (1971) In: Advances in Cell and Molecular Biology, E. J. DuPraw (Ed.), Vol. 1, pp. 2-57, DNA by virtue of the ionic interaction of the -terminal Academic Press, New York. lysines of the histones and the phosphate back Fambrough, D. M. and Bonner, J. (1966) Biochemistry 5: 2563-2570. DNA, modification of these lysines by acetyla ion should Fambrough, D. M., Fujimura, F. and Bonner, J. (1968) lead to a general loosening and unfolding of the DNA Biochemistry 7: 575-585. Gare!, A. and Axel, R. (19 76) Proc. Nat. Acad. Sci. USA nucleosome interaction and to the observed i crease in . . 73: 3966-3970. template activity. In order to understand n cleosomal Gottesfeld, J. M., Bagi, G., Berg, B. and Bonner, J. (1976) Biochemistry 15: 2742-2482. modification and its relationship to gene derepr sion, we Gottesfeld, J. M., Garrard, W. T., Bagi, G., Wilson, R. F. are examining the role of the chromosomal enzy es which and Bonner, J. (1974) Proc. Nat. Acad. Sci. USA 71: 2193-2197. acetylate the histones in the nucleosome. Histone Gottesfeld, J. M. and Partington, G. (1977) Cell 12: acetyltransferase (HAT) is such a chromosom 1 enzyme 1953-1962. Hewish, D. and Burgoyne, L. (1973) Biochem. Biophys. (Candido, 1975). Res. Commun. 52: 504-510. We have accomplished a 200-fold purifica ion of the Levy-Wilson, B., Gjerset, R. A. and McCarthy, B. J. (1977) Biochim. Biophys. Acta 475: 168-175. enzyme by a combination of ammonium sulfate ractiona­ Marushige, K. (1976) Proc. Nat. Acad. Sci. USA 73: tion, gel exclusion chromatography, ion exchang chroma­ 3937-3941. Marushige, K. and Bonner, J, (1971) Proc. Nat. Acad. Sci. tography, and affinity chromatography. Thi enzyme USA 68: 2941-2944. utilizes acetyl coenzyme A as the acetate nor and Riggs, M., Whittaker, R., Neumann, J. and Ingram, V. (1977) Nature 268: 462-464. seems to exhibit a specificity for the arg ·ne-rich 25

histones, H3 and H4. It is an acidic protein (pl = 5.9) and 12. ISOLATION OF CLONED RIBOSOMAL RNA CODING SEQUENCES in crude preparations may be found bound via ionic Investigators: Antonio A. Reyes, R. Bruce Wallace, interaction to the histones. Its comparatively high James Bonner molecular weight (.r90,000) facilitates its separation from Two recombinant phage containing 188 rRNA coding the histones (molecular weight ll,000 to 23,000) via gel sequences have been isolated from a Charon 4A library of exclusion chromatography in high salt. Preliminary cloned rat DNA fragments by the plaque screening experiments indicate that crude nuclear homogenates technique. The EcoRI restriction patterns of the two contain material which either inhibits the activity of HAT clones were compared. Both cloned rat rDNA fragments or reverses the acetylation of the histones. A nuclear contain two EcoRI sites yielding three restriction frag­ deacetylase has been shown to catalyze the deacetylation ments upon digestion with EcoRI. In one clone (RROl) of the histones and may be present in this crude fraction. these fragments are 8.5 kb, 3.3 kb, and 2.3 kb; in the other Experiments to examine this property of crude nuclear (RR02) these fragments are 6.7 kb, 4.9 kb, and 3.1 kb. preparations are in progress. The EcoRI restriction fragments of the two clones and References: EcoRI digested rat DNA were separated by electropho­ Billing, R. J. and Bonner, J. (1972) Biochim. Biophys. Acta 281: 453-462. resis, transferred to nitrocellulose filters by the blot Candido, E. C. M. (1975) Canad. J. Biochem. 53: 796-803. technique of Southern (1975), and hybridized with Hewish, D. and Burgoyne, L. (1973) Biochim. Biophys. 32 Acta 474: 141-153. P-cDNA prepared either from 18S rRNA or 28S rRNA Kornberg, R. and Thomas, J. (1974) Science 184: 865-868. by the technique of Berns and Jaenisch (1976). Marushige, K. (1976) Proc. Nat. Acad. Sci. USA 73: 3937-3941. Two major restriction fragments of total DNA Sahasrabudde, C. G. and Van Holde, K. E. (1974) J. Biol. hybridize with the 18S cDNA, 9.0 kb and 6.0 kb. In Chem. 249: 152-156. Wallace, R. B., Sargent, T., Murphy, R. and Bonner, J. addition, several smaller restriction fragments hybridize (1977) Proc. Nat. Acad. Sci. USA 74: 3244-3248. to a lesser extent with the 188 cDNA. Two of the three EcoRI restriction fragments of both clones hybridize with 11. MOLECULAR CLONING OF RAT CHROMOSOMAL DNA FRAGMENTS 18S cDNA, 8.5 kb and 3.3 kb for RROl and 4.9 kb and Investigators: R. Bruce Wallace, Thomas D. Sargent, 3.1 kb for RR02. None of these restriction fragments James Bomer corresponds to major fragments from the rat DNA. They A library of cloned rat chromosomal DNA has been either represent minor members of the rDNA repetitive prepared in order to make any gene sequence of the rat family which have a different distribution of EcoRI sites available for detailed study. High molecular weight rat and/or inserted sequences, or represent rearrangements in DNA was prepared from the liver of one animal, partially the DNA sequences during propagation of the recombinant digested with EcoRI, and the resulting material separated phage. Neither clone hybridizes with 288 cDNA. One by sucrose density gradient centrifugation. The 10,000 to major restriction fragment of the rat DNA, 6.0 kb, 20,000 nucleotide pair fraction was recovered and ligated hybridizes with 28S cDNA. This fragment is probably with cloning fragments of Charon 4A DNA (the left and identical to the smaller of the two fragments hybridizing right arms of the DNA prepared by digestion with EcoRI with 188 cDNA as has been observed in the case of human and removal of the two central restriction fragments). and mouse rDNA sequences. The ligated DNA was packaged and the library amplified References: in DP50 SupF. A total of 2.8 x 106 independent Berns, A. and Jaenisch, R. (1976) Proc. Nat. Acad Sci. USA 73: 2448-2452. recombinant phage were created from 6 µg of rat DNA. Southern, E. (1975) J. Mo!. Biol. 98: 503-517. This library should contain greater than 99.9% of the rat single copy complexity on purely statistical grounds. DNA from the library was isolated and used to drive single copy 13. ANALYSIS OF RAT LONG REPETITIVE DNA SEQUENCES BY RECOMBINANT DNA METHODS tracer prepared from rat liver DNA. The library DNA is Investigators: Thomas D.. Sargent, James Bonner indistinguishable from total rat DNA in its ability to drive this tracer. Experiments are in progress to attempt to It has been shown that repetitive sequences make up about 35% of the rat genome (Pearson et al., 1978). When isolate the gene sequences for rat serum albumin and rat alpha-fetoprotein from this library. isolated by Sl nuclease digestion of partially renatured rat 26

DNA, these repetitive sequences are found to be of two bumin and AFP, raised in goats, were reacted with rat types: short (300 to 400 bp) and divergent (about 10% liver polysomes (for isolation of albumin mRNA) and mismatch in a typical family) on the one hand, and long Morris hepatoma 7777 polysomes (for AFP mRNA). The (greater than 1000 bp) conserved (less than 1 to 2% mis­ antibody polysome complexes were immunoprecipitated by match) on the other. While these two forms exhibit using a purified burro anti-goat IgG fraction. Polysomal clearly different physical properties they share virtually RN A was extracted from the immunoprecpitates and the complete sequence homology (Wu et al., 1977). mRNAs isolated by poly(U)-Sepharose 4B chromatography. It is our supposition that clarification of the In vitro translation analysis in wheat germ extracts relationship between long and short repetitive DNA indicated that the immunoprecipitation and affinity chro­ sequences will provide important insights into the overall matography steps afforded a 400- to 500-fold purification structure of the rat genome. We have isolated cloned for both mRNAs over the respective total polysomal fragments of long repetitive rat DNA. These will be used RNAs. The two specific mRNAs were then further as probes to screen our rat lambda recombinant DNA purified by sedimentation through preparative dimethyl.., library. The genome fragments isolated by this method sulfoxide-sucrose density gradients. The albumin transla­ will enable us to examine a few families of repeated tional activity was found associated with a main 178 RNA sequences in detail, including both ''long" and "short" species in the gradient. A similar result was obtained for versions of a particular repeated sequence. Application of the AFP translational activity in the AFP mRNA gradient. standard techniques of sequence analysis (restriction In vitro translation of the density gradient purified mapping, blots, DNA reassociation kinetics, etc.) to these mRNAs, followed by analysis of the products by sodium clones should lead to useful conclusions regarding the dodecyl sulfate/polyacrylamide gel electrophoresis, has organization of the rat genome. shown that they code for single polypeptides that can be identified immunologically as albumin and AFP. References: Pearson, W. R., Wu, J.-R. and Bonner, J. (1978) Biochem­ istry 17: 51-59. Wu, J.-R., Pearson, W.R., Posakony, J. W. and Bonner, J. 15. SYNTHl!SIS OF FULL LENGTH eDNAs FROM (1977) Proc. Nat. Acad. Sci. USA 74: 4382-4386. PURIFIED RAT ALBUMIN AND o-FETOPROTEIN mRNAs Investigators: Jose M. Sala-~t, Thomas D. Sargent 14. IMMUNOCHEMICAL ISOLATION AND PURIFICATION OF RAT ALBUMIN DNA complementary (cDNA) to albumin mRNA and AND o-FETOPROTEIN mRNAs to AFP mRNA was synth~sized with avian myeloblastosis Investigators: Jose M. Sala-~t, John E. Dever Jr. virus reverse transcriptase. Reaction conditions were Albumin and a-fetoprotein (APP) are two major adapted from the optimal conditions found in our previous plasma proteins synthesized by the liver which are W1der studies concerning the synthesis of cDN A complementary developmental control. In rats, the concentration of to the total poly(A)-containing RNA population from rat albumin in fetal sera increases during ontogenesis and liver polysomes (see Biology 1977, No. 21). Yield of cDNA early weeks of postnatal life, to reach a high constant was usually between 20 and 30% of the input albumin level in the serum of adult animals. Contrarily, AFP is mRNA and AFP mRNA templates. The cDNAs were only found in trace amounts in the sera of normal adult characterized by alkaline sucrose density gradient sedi­ rats but in greatly elevated concentrations in the sera of mentation and by electrophoresis under denaturing condi­ fetal, newborn, pregnant, and hepatoma-bearing animals. tions in alkaline agarose gels. The albumin mRNA To study the control processes which underlie the expres­ transcript showed three main discrete size products sion of these two related proteins we have first isolated corresponding to 820, 1351, and 1907 nucleotides. T.he the corresponding messenger RN As in a pure homogeneous largest transcript (1907 nucleotides) corresponded to the state. This has been achieved by a combination of full length of the mRNA template and comprised about immunoprecipitation of polysomes synthesizing these pro­ 20% of the total products. Transcripts of the AFP mRN A teins, poly(U)-Sepharose 4B chromatography, and sucrose preparation encompassed molecules of different sizes up gradient centrifugation. to the full length of the template. Highly purified monospecific antibodies against al- The kinetics of hybridization of these cDNAs with 27 an excess of the albumin mRNA and APP mRNA tem­ 17. THE NUCLEAR RNA PRECURSORS OF ALBUMIN mRNA AND 1.5 kb) and short formed by reacting purified 18S and 28S rRNAs with (0.25-0.40 kb) which is similar to the size distribution of unfractionated rat DNA. The molecules containing middle repetitive DNA. Also there is a considerable R-loops were isolated by two cycles over an affinity degree of sequence homology between the foldback and column that was constructed with antibodies specific to middle repetitive fractions. The only unique quality of DNA/RNA hybrids covalently attached to Sepharose. The foldback DNA appears to be the intrastrand inverted degree of enrichment was determined by hybridizations of repetition pattern. the test DNA immobilized on filters driven by labeled To further characterize this fraction we have chosen rRNA. An enrichment of approximately 135-fold was to look at two aspects of the flanking sequences of the achieved for rRNA coding sequences relative to whole foldbacks, the first being the kinetic components and the genomal DNA. In addition, approximately 30% of the second the sequence complexity. The criteria for fold­ original rRNA genes were recovered in the enriched back formation were long DNA (>20 kb) at a concentra­ fraction. tion no greater than 0.25 µg/ml (in 0.12 M PB), incubated -4 This DNA was then prepared for electron micros­ at 60"C for 1.5 to 6 min (Cot ~10 ), and bound to copy and used to obtain an R-loop map of rat rDNA. hydroxyapatite. The adjacent sequences were then iso­ Fifteen molecules have thus far been measured ranging in lated from the foldback fraction and driven by an excess overall length from 11 to 46 kb (kilobase pairs), with an of whole rat DNA. The results indicated that the adjacent average length of 21 kb. The sizes of the 18S and 28S sequences contained three kinetic components in the same rRNA R-loops have been determined to be on the average proportions as the total rat genome. This seems to imply 1.62 and 4.37 kb respectively. The interloop spacer that the foldback adjacent sequences may be randomly averaged 2.25 kb in length. Since the measured lengths of distributed throughout the genome. the R-loops were only about 80% as long as the lengths of We are currently in the process of using the adjacent the respective rRNAs, the interloop transcribed spacer sequences to renature themselves and also to drive may actually be only about 1.5 kb when corrected for the different kinetic components, which will enable us to shortened R-loop distances. determine the sequence complexity and whether they are We have also tentatively determined that the re- randomly distributed or a subset of the genome. 29

constant size (approximately 140 base pairs), and a Reference: Britten, R. J. and Smith, J. (1970) Carnegie Inst. Wash. "spacer" of varying size (40 to 80 base pairs). Efforts to Yearbook 68: 378-386. distinguish (in a single cell type) between the spacers of specific genes and bulk DNA, and between "active" and 21. CHROMATIN RECONSTITUTION ninactive" genes, as well as satellite and main band DNA, investigator: Gordon C. Machray have been unsuccessful. Recombinant DNA technology has given us the We have investigated the structure of chromatin ability to isolate relatively large amounts of the DNA from mouse Friend cells which have been labeled for one sequence of a single gene. A method for reconstituting to two generations with 14c-thymidine, followed by 10 or such DNA with histones to yield a nucleosomal structure 30 min with 3H-thymidine. Newly replicated chromatin similar to that of chromatin Constitutes an essential step has a smaller "spacer" region (approximately 20 nucleo­ in the elucidation of the elements controlling gene tides shorter), and is less rapidly digested by staphylo­ transcription. coccal nuclease (about half as rapidly). This is the first Multiples of 200 base pair DNA have been generated demonstration of a difference in nucleosome arrangement by staphylococcal nuclease digestion of rat liver nuclei, between different chromatin fractions in a single cell separation of oligomers of nucleosomes in isokinetic type, and may indicate that nucleosome replication sucrose gradients, and isolation of the DNA of each consists of "core" creation (using an octamer of histones), oligomer. Such DNA is then mixed in 1.2 M NaCl buffer followed by 11spacer" elongation as histone Hl and/or with histones H2a, H2h, H3, and H4 (isolated by nonhistone proteins are deposited. It is unlikely that our Bio-Rex 70 column chromatography of an acid extract of data can be explained with out recourse to the assumption rat liver chromatin). The salt concentration is lowered that nucleosome "sliding" is possible. stepwise over a 2 hr period to 0.2 M NaCl by addition of buffer solution containing no salt. This 11gradient dilution" 23. THE EFFECT OF SINGLE BASE MISMATCH ON method has reconstituted mononucleosomes and dinucleo­ SYNTHETIC DNA-0X174 DNA DUPLEXES somes frorn 200 and 400 base pair DNA respectively. The Investigators: R. Bruce Wallace, George D. Shaffer*, complexes produced by longer stretches of DNA are also T. Hirose**, K. Itakura**, James Bonner under analysis. Oligodeoxyribonucleotides have been chemically Recent results from workers using Xenopus (Laskey synthesized which are complementary to the region of the et al., 1977) and Drosophila (Nelson et al., 1978) have 0Xl 7 4 genome encompassing the am3 point mutation. indicated the presence at certain developmental stages of These oligonucleotides, 11, 14, and 17 bases long, are these organisms of a factor which can promote reconstitu­ complementary to the wild-type phage DNA. The 32 tion of chromatin under physiological salt conditions. oligonucleotides were labeled with P in the 5'-end and Since no similar activity has been found in rat liver, use of hybridized to either wild type or am3 0X174 DNA that the above organisms to achieve a physiological reconstitu­ had been immobilized on nitrocellulose filters. All tion of chromatin is being investigated. hybridizations and washing of the filter are done in 6X References: SSC ([Na •1 = 1.2 M). The 11-, 14-, and 17-mer hybridize Laskey, R. A., Mills, A. D. and Morris, N. R. (1977) Cell to a 5-fold higher level with wild type than with am3 10: 237-243. Nelson, T. M., Hsieh, T-S. and Brutlag, D. L. (1978) Fed. DNA. The thermal denaturation behaviors of the duplexes Proc. 37: 1464. were compared. The Tm of the 11-, 14-, and 17-mer •

22. NUCLEOSOME STRUCTURE OF NEWLY wild-type DNA duplexes are 34°C, 41°C, and 54°C respec­ REPLICATED CHROMATIN tively, while the Tm of the 11-, 14-, and 17-mer • am3 lnvestigators: Robert F. Murphy, R. Bruce Wallace DNA duplexes are 22°C, 3o"C, and 43°C respectively. Eukaryotic chromatin is packaged into a repeating These results, when the Tm data are corrected for percent structure, the unit of which is the nucleosome. The G+C of the duplexes, suggest that one mismatch destabi­ pattern of nucleosomal DNA sizes (observed on polyacryl­ lizes a duplex by about 3 to 4 base pairs. 32 amide gels) differs between organisms, but the data The hybridization of the P-labeled 14-mer to wild indicate that the nucleosome consists of a "core" of type and to am3 0X174 DNA separated by electrophoresis 30 and transferred to nitrocellulose filters by the technique (Elder et al., 1977). More than half of the spots in the of Southern (197 5) was compared. If hybridization was slime mold H3- and H4-like proteins comigrate with spots performed at 12"C and the filter washed at 4°C followed from the respective mammalian histones. Only 25% of by autoradiography, hybridization to both DNAs was the spots in the Hl proteins are homologous and almost observed although the hybridization to am3 DNA was none of the spots from slime mold H6 comigrate with much less than to wild type. If the filter was subsequently either mammalian H3, H2a, or H2b peptides. washed at 37°C, 4°C below the Tm of the wild-type The -slime mold histones apparently have functions duplexes but 7°C above the am3 duplexes, hybridization to similar to mammalian ones since the structures of their the wild-type DNA was unaffected while hybridization to chromatins are similar (Bakke et al., 1978). These results am3 DNA was virtually eliminated. indicate that three of the slime mold histones are homologous to Hl, H3, and H4 in their primary structure. Reference: Southern, E. (1975) J. Mo!. Biol. 98: 503-517. However, the fourth (H6) is different, although it may *Undergraduate, California Institute of Technology. function like the slightly lysine-rich histones. **Staff member, City of Hope, Duarte, California. References: Bakke, A. C., Wu, J.-R. and Bonner, J. (1978) Proc. Nat. 24. A COMPARISON OF MAMMALIAN AND SLlME Acad. Sci. USA 75: 705-709. MOLD HISTONES Elder, J. H., Jensen, F. C., Bryant, M. L. and Lerner, R. A. (1977) Nature 267: 23-28. Investigator: Antony C. Bakke

The chromatin of the cellular slime mold, Dictyo­ 25. COMPUTERIZED ANALYSIS OF THERMAL stelium discoideum, contains equal masses of DNA and DENATURATION OF DNA AND CHROMATIN histones. There are only four histones instead of the five Investigators: Robert F. Murphy, James Bonner found in higher eukaryotes. One of the four comigrates We have developed programs for analysis of thermal with mammalian H3, but the other three do not comigrate denaturation profiles of DNA and chromatin. At present, with any histone on either acidic, neutral, Or basic two methods are used. The first involves derivatizing electrophoresis gels. To determine their homology with evenly X-incremented data using a polynomial smoothing mammalian histones, they have been cut out of gels, their program, followed by fitting the data to a set of amino acid compositions determined, and their peptide Gaussians. This is done with a nonlinear least squares fragments mapped. The amino acid compositions are from fitting program using band matrices to minimize the a 24 hr acid hydrolysis. Preliminary results indicate that amount of memory required. The program was developed the slime mold histone migrating near Hl is a lysine-rich for resolving bands in polyacrylamide gel profiles and has histone with large amounts of alanine and proline. The also been used to resolve peaks on sucrose and cesium values are not as high as those for Hl, but they are chloride gradients. Using a PDP-11/34 with 16 K words of similar. The histone co migrating with H3 has large and memory, we can currently fit up to 1000 data points using nearly equal amounts of lysine and arginine and high up to 20 curves. This method has the advantage of amounts of glutamic acid, alanine, and leucine. It closely accentuating differences between components in the resembles H3 but is also similar to H2a. The smallest resulting plot, but requires evenly spaced points and slime mold histone, which migrates near H4, also re­ considerably more data than the second approach. It is sembles H4 in its amino acid composition. It contains thus best suited to automated optical melts. large amounts of lysine, arginine, and glycine. The most The second method avoids the derivatizing step, and unusual slime mold histone (called H6) migrates between hence the restriction on evenly spaced data. Instead the Hl and H3 and appears to be present in twice the quantity data are fit to a set of error functions. (The error of the slime mold H3- and H4-like proteins. Its function is the integral of the Gaussian or normal composition is most similar to H2b. probability function.) This allows rapid determination of The peptide maps reveal further homologies between melting temperatl.ll'es and transition breadths, especially slime mold and mammalian histones. The proteins are for melts done on filters or hydroxyapatite, where it is radioiodinated, digested with either trypsin or chymo­ difficult to collect more than 10 to 30 points. The trypsin, and displayed on thin-layer chromatography plates program generates and decomposes a partial derivative 31 matrix dimensioned 3n*3n, where n is the number of Pearson, W. R., Smith, S. L., Wu, J.-R. and Bonner, J. (1978) Kinetic determination of the genome size of components (which must be less than or equal to five). the pea. Plant Physiol. In press. Both fitting programs give parameter error esti­ Pearson, W. R., Wu, J.-R. and Bonner, J. (1978) Analysis of rat repetitive DNA sequences. Biochemistry 17: mates and calculate the contribution of each component 51-59. to the total hyperchromicity. An interactive command Sala-Trepat, J. M., Dever, J. E. Jr. and Bonner, J. (1978) Determination of the number of albumin and a-feto­ language gives the user control over all aspects of the protein genes in rat DNA and hepatoma 7777 DNA. fitting process, including editing of parameters between Cell Biol. (Abstract). In press. Savage, M. J. and Bonner, J. (1977) Structural analysis of fits and setting constraints on acceptable fits. These native and reconstituted chromatin by nuclease programs are available on request. digestion. In: The Molecular Biology of the Mammalian Genetic Apparatus, Paul Ts'o (Ed.), Vol. II, pp. 149-162, Elsevier/North-Holland Biomedical PUBLICATIONS Press. Savage, M. J. and Bonner, J. (1978) Fractionation of Bakke A. C., Wu, J.-R. and Bonner, J. (1978) Chromatin chromatin into template-active and template-in­ ~tructure in the cellular slime mold Dictyostelium active portions. In: Methods in Cell Biology, discoideum. Proc. Nat. Acad. Sci. USA 75: 705-709. Chromatin and Chromosomal Protein Research III, Bonner, J. (1977) Cell differentiation in microorganisms, G. Stein, J. Stein and L. J. Kleinsmith (Eds.), Vol. plants and animals. Leopoldina Symposium, L. XVIII, pp. 1-21, Academic Press, New York. Nover and K. Mothes (Eds.), pp. 76-93, VEB Gustav Savage, M. J., Sala-Trepat, J. M. and Bonner, J. (1978) Fischer Verlag, Jena. Measurement of the complexity and diversity of Bonner J. (1977) My life as a chromosomologist. In: The poly(adenylic acid) containing messenger RNA from Molecular Biology of the Mammalian Genetic Ap­ rat liver. Biochemistry 17: 462-476. paratus, Paul Ts'o (Ed.), Vol. II, pp. 317-326, Stumph, W. E., Wu, J.-R. and Bonner, J. (1978) An R-loop Elsevier/North-Holland Biomedical Press. map of rat ribosomal DNA isolated from whole Bonner, J. (1977) Some progress in our understanding of genomal DNA by affinity chromatography. Cell chromatin organization. ICN-UCLA Symposia on Biol. (Abstract). In press. Molecular and Cellular Biology, R. S. Sparkes, D. E. Stumph, W. E., Wu, J.-R. and Bonner, J. (1978) Gene Comings and C. F. Fox (Eds.), Vol. VIII, pp. 53-64. enrichment using an affinity resin with specificity Bonner J. (1977) The evolution of underabundance. The for RNA/DNA hybrids. J. Cell Biol. In press. Next 80 Years Conference, California Institute of Wallace, R. B., Dube, s. K. and Bonner, J. (1977) Technology (April), pp. 13-24. Localization of the globin gene in the template­ Bonner, J. (1978) How can we most rapidly increase the active fraction of chromatin of Friend leukemia production of natural rubber? Centenary Inter­ cells. Science 198: 1166-1168. national Rubber Conference, Sri Lanka (December Wallace, R. B., Sargent, T. D., Murphy, R. F. and Bonner, 1976). In press. J. (1977) Physical properties of chemically acety­ Bonner, J., Sala-Trepat, J. M., Pearson, W. R. and Wu, lated rat liver chromatin. Proc. Nat. Acad. Sci. J.-R. (1978) Mammalian chromatin: Structure, USA 74: 3244-3248. expression, and sequence organization. In: The Cell Wilkes, M. M., Pearson, W. R .. and Bonner, J. (1978) Nucleus, H. Busch (Ed.), Vol. VI. In press. Sequence organization of the rat genome by electron Bonner, J., Wallace, R. B., Sargent, T. D., Murphy, R. F. microscopy. Biochemistry 17: 60-69. and Dube, S. K. (1978) The expressed portion of Wu, J.-R., Pearson, W.R., Posakony, J. W. and Bonner, J. eukaryotic chromatin. Cold Spring Harbor Symp. (1977) Sequence relationship between long and short Quant. Biol In press. repetitive DNA of the rat: A preliminary report. Douvas, A., Harrington, C. and Bonner, J. (1978) Selective Proc. Nat. Acad. Sci. USA 74: 4382-4386. dissociation of nonhistone proteins by NaCl and Wu, J.-R., Pearson, W. R., Wilkes, M. M. and Bonner, J. urea. Biochemistry. In press. (1977) Analysis of sequence structure of the rat Dube, s. K., Wallace, R. B. and Bonner, J. (1978) State of genome. In: The Molecular Biology of the Mamma­ globin gene in Friend cell. Cold Spring Harbor lian Genetic Apparatus, Paul Ts'o (Ed.), Vol. II, pp. Symp. Quant. Biol. In press. 51-62, Elsevier/North-Holland Biomedical Press. Hayashi, H., Iwai, K., Johndon, J. D. and Bonner, J. (1977) Pea histones H2a and H2b. J. Biochem. 82: 503-510. 32

Professor: Eric H. Davidson tion of RN As of oocytes, embryos, and adult tissues. A Sherman Fairchild Distinguished Scholar: Thomas R. major fraction of the research in this laboratory is carried Tosteson Senior Research Associate: Roy J. Britten* out with recombinant DNAs. A new and potent tool in Visiting Associate: Lajos PikO** this research is a recently constructed rec.ombinant DNA Senior Research Fellows: Barbara R. Hough-Evans, William H. Klein, Amy Shiu Lee library which includes almost the whole of the sea urchin Weizmann Research Fellow: Ze'ev Lev genome. This library greatly facilitates detailed examina­ Research Fellows: David M. Anderson, Robert C. Angerer, Susan G. Ernst, Terrence J. Hall, Gordon P. tion of sequence organization around expressed structural Moore, William R. Pearson, Terry L. Thomas genes and other regions which may be important for Graduate Students: Frank Costantini, Jay W. Ellison, James W. Posakony, Richard H. Scheller***, Barbara understanding genome function. J. Wold Research Staff: Clifford Beall, Peggy R. Bierer, Anita S. Cetta, Margaret E. Chamberlin, Susan E. Gerber, 26. STUDIES OF NUCLEIC ACID REASSOCIATION Roberta Gerson, Robert L. Gimlich, Michael KINETICS Kozlowski, Cary Lai, Patrick S. Leahy, Peter Sin-Yi Investigators: Margaret E. Chamberlin, Glenn A. Galau•, Lu, James G. Moore, Walter D. Niles II, Peter E. Roy J. Britten, Eric H. Davidson Nolan, Jane Rigg, Richard M. Rohan, Linda S. Vock Laboratory Staff: Edward D. Kusby, Robert 0. Piehl, Measurements were made of the kinetics of nucleic Sydne A. Schurmeier acid strand pair reassociation where the complementary *Concurrently a member of the staff of the Carnegie strands are of different lengths and are present in Institution of Washington. different concentrations. Rate constants for the reaction **Veterans Administration Hospital, Sepulveda, Cali­ fornia. of labeled fragments ("tracer"} with excess complemen­ ***Division of Chemistry and Chemical Engineering, California Institute of Technology. tary strands ("driver"} were determined, both for driver fragment length greater than tracer fragment length and Support: The work described in the following research for the reverse case. Second-order reactions and pseudo­ reports has been supported by: American Cancer Society first-order reactions utilizing strand-separated drivers and Earle C. Anthony Fellowship Carnegie Institution of Washington tracers were studied. The nucleic acids which served for Danforth Fellowship this investigation were 0X174 DNA and RNA, plasmid March of Dimes RSF2124 DNA, and E. coli DNA. Approximate empirical McCallum Fund National Institutes of Health, USPHS expressions relating driver and tracer fragment lengths National Science Foundation with the observed rate constants were obtained for Veterans Administration Weizmann Fellowship practical use. In long tracer-short driver reactions the Summary: The work of this laboratory deals with the observed rate constant for the tracer reaction increases molecular biology of animal development, and with gener­ proportionately with tracer length. In long driver-short al questions of genomic sequence organization and gene tracer reactions the rate of tracer reaction is retarded. control. A theoretical framework for some of the The latter result is unexpected and appears to represent a research is provided by the Britten-Davidson model for departure from standard interpretations of the renatur­ gene regulation in eukaryotes. Previous studies have ation reaction. shown that the DNA is organized in such a way that single *Present address: Department of Biochemistry, Univer­ copy sequences (including most of the structural genes} sity of Georgia, Athens, Georgia. are interspersed with repetitive DNA sequences. The repeated sequences are postulated to be concerned in the 27. SEQUENCE CONTENT OF ml! BITHORAX control of transcription of the adjacent genes. REGION OF ml! DROSOPHILA GENOME Problems which are central to our research include Investigators: Margaret E. Chamberlin, Eric H. Davidson, the structure, function, and evolution of repetitive DNA Edward B. Lewis families, and the utilization of genes in early develop- In order to study the DNA of the bithorax region of ment. Questions of evolutionary changes are being Drosophila melanogaster, we are preparing to hybridize examined by extensive comparison of DNA sequences of wild-type tracer with DNA isolated from embryos which several species of sea urchins. Other studies involve are homozygously deficient for the bithorax region, and analysis of the sequence content and sequence organiza- harvest the unhybridized sequences. We have isolated 33

8 wild-type DNA from Drosophila embryos which have been copy complexity of about 1.7 x 10 nucleotides of unique 13 15 3 fed on a medium containing c, N, and H-thymidine sequence was extracted from nuclei of previtellogenic (a generous gift of Dr. Robert M. Grainger, University of oocytes. These nuclear RN A sequences from young Virginia). This DNA is significantly denser than the oocytes were found to consist of transcripts of a set of bithorax mutant DNA previously isolated, and thus affords single copy DNA sequences which significantly overlapped a further purification step by cesium chloride centrifuga­ the set which is expressed later in the hnRNA of gastrula tion. We also plan to end label the tracer DNA with stage embryos. The nuclear RNA of vitellogenic oocytes y-AT32P. Once we have obtained DNA enriched for appears to contain a class of more prevalent single copy bithorax sequence we can screen an existing bacterio­ transcripts that are not, however, the same as the phage lambda ''library" of Drosophila DNA sequences, and maternal mRNA sequences that are found stored in investigate the positive plaques by in situ hybridization to mature oocytes. This was determined by reacting the Drosophila salivary gland chromosomes. Having thus vitellogenic oocyte nuclear RN A with a single copy identified the clones containing bona fide bithorax se­ 3H-DNA fraction enriched for sequences complementary quence, we will proceed to examine its sequence content. to the maternal single copy sequence set (oDNA). oDNA was also reacted with cytoplasmic RNA of previtellogenic 28. COMPLEXITY OF DROSOPHILA BGG RNA oocytes, and it was found that less than half of the final Investigators: Barbara R. Hough-Evans, Bric H. egg RNA complexity of 37 million nucleotides has been Davidson, Marcelo Jacobs-Lorena• accumulated in the cytoplasm in early stages of sea urchin The eggs of a variety of animals previously exam­ oogenesis. ined each contain about 20,000 different kinds of stored "maternal messenger" RNA. We carried out a similar 30. EVIDENCE FOR MATERNALLY INHERITED POLY(A) CONTAINING RNA IN MOUSE EGGS measurement for an animal with a much smaller genome, Investigators: Lajos Piko*, Kerry B. Clegg" using isolated single copy Drosophila DNA tracer, and RNA extracted from ovulated but unfertilized Drosophila Maternal mRNA has been shown to be synthesized eggs. From the extent of hybridization of the DNA, and and·' stored during oogenesis in sea urchins for use in knowing its complexity, it was calculated that the protein synthesis after fertilization. In order to deter­ Drosophila egg contains only 4000 to 5000 RNA message mine if maternally derived poly(A)+ RNA is stored in the sequences. Similar experiments to determine the RNA mouse oocyte and persists until after fertilization, we complexity of several stages of developing Drosophila have studied the content and distribution of poly(A) eggs are planned, and will allow us to follow the course of stretches in oocytes and fertilized and unfertilized eggs 3 maternal mRNA accumulation through oogenesis. by two procedures: hybridization of H-poly(U) to phenol­ extractable nucleic acids and in situ hybridization of *Case Western Reserve School of Medicine, Cleveland, 3 Ohio. H-poly(U) followed by autoradiography. Preovulatory germinal vesicle stage oocytes contained about 1 pg of 29. RNA SYNTHESIS IN SEA URCHIN OOGENESIS poly(A). The poly(A) content of ovulated unfertilized eggs Investigators: Barbara R. Hough-Evans, Susan G. Ermt, decreased slightly to about 0. 7 pg/egg, but remained EricH.Da~ constant after fertilization throughout the 1-cell stage. During the reproductive season, ovaries of the This finding suggests that there is no large increase in the purple sea urchin contain small previtellogenic oocytes overall length of existing poly(A) tracts upon fertilization and growing vitellogenic oocytes as well as mature eggs. as has been observed in sea urchin eggs. The ratio of Only previtellogenic oocytes are found in "out of season" poly(A) to total cellular RNA (0.3 ng/egg determined ovaries. We extracted nuclear RNA and cytoplasmic RNA spectrophotometrically) in the mouse egg is about 0.25% from ovaries containing only previtellogenic oocytes, and which is similar to the values reported for other eggs. In nuclear RNA from preparations enriched for vitellogenic situ hybridization of 3H-poly(U) to ovary sections indi­ oocytes. These oocyte RNAs were characterized by cated that the relative concentration of poly(A) remained hybridization reactioflS with radioactively-labeled single approximately constant during oocyte growth, i.e., that copy sea urchin DNA. A typical nuclear RNA of single poly(A) was accumulating progressively during oogenesis. 34

The germinal vesicles were heavily labeled up to the time polysomes by the 16-cell stage of embryogenesis. These of meiotic maturation indicating synthesis and/or storage data implicate cloned DNA such as CS0088 as bona fide of poly(A)+ RNA. These data as well as protein synthetic structural genes. As a consequence, these cloned DNA patterns reported for unfertilized and fertilized mouse sequences are being employed to investigate a number of eggs support the hypothesis that maternal mRN As are interesting aspects of gene expression during sea urchin preserved and function in protein synthesis in the mouse development. egg after fertilization. *Present address: Department of Biology, University of Rochester, Rochester, New York. *Veterans Administration Hospital, Sepulveda, California.

32. CLONED SEA URCHIN DNA COMPLEMENTARY 31. CLONED SEA URCHlN DNA COMPLEMENTARY TO OOCYTE RNA. II. PSC34 TO OOCYTE RNA. L CS0088 Investigators: Amy Shiu Le~ Bric H. Davidson Investigators: Terry L. Thomas, Robert C. Angerer*, Ze'ev Lev, Robert L. Gimlich, We have previously described a sea urchin recombi­ Erie H. Davidson nant clone PSC34 which contains three short repeat We have constructed recombinant bacterial plasmids sequences interspersed with single copy sequences varying containing DNA sequences from the sea urchin. Of more from a few hundred to two thousand nucleotides long (Lee than 100 tested, 8 to 12 were shown to contain DNA et al., 1978). Restriction enzyme fragments from this sequences complementary to oocyte RNA. Several of 7000 nucleotide sea urchin DNA fragment were tested for these cloned DNA sequences have been analyzed in transcriptional activity. The HhaIA fragment, which is a greater detail. In addition, restriction enzyme maps have nonrepetitive sequence and is 2000 nucleotides long, was been generated showing the position of the codogenic found to hybridize with mature oocyte RNA. Transcrip­ regions with respect to restriction fragments containing tion is asymmetric. From the observed hybridization repetitive and nonrepetitive DNA sequences within the rates, it can be estimated that there are about 1000 cloned DNA fragment. copies of this transcript in the mature oocyte, which is The major class of cloned DNA fragments isolated fairly typical of maternal RNA belonging to the single by this approach contained both repetitive and nonrepeti­ copy sequence set of the oocyte. This sequence also tive DNA. This class is typified by the clone designated hybridizes to polysomal RNA isolated from gastrulae, and CS0088. CS0088 is 5800 nucleotides in length. Mapping initial results indicate that it also exists in the cytoplas­ data places the codogenic region in the terminal third of mic RNA population isolated from sea urchin intestine. the cloned fragment. The RN A encoded by this fragment The HhaIA sequence can be detected in the hnRNA is present at about 6000 copies per oocyte and represents population from gastrula cells, and is transcribed asym­ only one strand of the DNA, i.e., the transcript is metrically. There are one to two copies of RNA asymmetric. The codogenic region is nonrepetitive and is transcripts of this sequence present in each cell nucleus at about 1200 nucleotides in length. Several distinct the gastrula stage, and one to two copies per cell in the repetitive sequence elements interspersed with nonrepeti­ polysomal RNA as well. The orientation of transcription tive DNA sequences are located in the remaining two­ for this sequence has also been determined. thirds of the cloned DNA fragment. The transcript is Reference: initiated either outside of the cloned DNA fragment or Lee, A. s., Britten, R. J. and Davidson, E. H. (1978) Cold within a couple of hundred nucleotides of the fragment Spring Harbor Symp. Quant. Biol. 62. In press. terminus. Of some significance is the isolation of cloned DNA 33. REPETITIVE SEQUENCE TRANSCRlPTS IN fragments complementary to nonprevalent maternal RNA. THE SEA URCHIN OOCYTE This class of mRNA accounts for about 90% of the Investigators: Frank Costantini, Richard He Scheller, different mRNA species present in the oocyte. Like many Erie H. Davidson other known structural genes, these transcripts are en­ The expression of interspersed repetitive sequences coded by nonrepetitive DNA and are asymmetric. In in the RN A of mature sea urchin oocytes was investi­ 3 addition, the RNA encoded by CS0088 is loaded on gated. H-DNA tracers representing short interspersed 35 repetitive sequences a few nundred nucleotides long, and sequence complexity of 37 million nucleotides, which is long repetitive sequences about 2000 nucleotides long, equivalent to about 20,000 structural genes. Hybridiza­ were prepared from genomic DNA of the sea urchin. tion of cytoplasmic 16-cell embryo RNA with single copy These tracers were reacted with excess RN A from the DNA sequences complementary to the complex maternal mature oocyte. About 80% of the reactable short repeat RNA stored in the egg (oDNA) shows that 100% of the tracer and 35% of the long repeat tracer hybridized. sequences stored in the egg remain in the cytoplasm of Therefore most of the repetitive sequence families in the the 16-cell embryo. About 73% of the maternal sequences short repeat tracer are represented in oocyte RN A, and stored in the egg is expressed on polysomes at this early transcripts complementary to both strands of many repeat cleavage stage. The RN A from the cytoplasm of isolated sequences are present. The kinetics of the reaction show micromeres was found to represent the full complexity that some transcripts are highly prevalent, while others stored in the egg. Preliminary measurements with oDNA are rare. Nine cloned repetitive sequences were labeled, have indicated that these small cells are also translating strand-separated, and reacted with the oocyte RN A. all of these maternal sequences. This is in contrast to the Transcripts of both strands of all nine repeats were found cells of the rest of the embryo which are making proteins in the RN A. The prevalence of transcripts of the cloned from a subset of the stored maternal messenger RNA repeat families varied from about 3000 to 100,000 copies population. per oocyte. Studies with both cloned and genomic tracers show that transcript prevalence is independent of the 35. SEA URCHIN MATERNAL mRNA SF.QUENCES genomic reiteration frequency of the transcribed repeti­ ALSO SYNTHESIZED DURING EMBRYOGENESIS Investigators: Ze'ev Lev, Eric H. Davidson tive sequences. Most of the families represented by prevalent transcripts have less than 200 copies per haploid Previous work indicates that embryo structural gene genome. The RNA molecules with which the cloned transcriptiou accounts for most or all of the polysomal repeats react are at least 1000 to 2000 nucleotides in mRNA by gastrula stage. However, the same sequences length. Other experiments show t~at a majority of the transcribed in the gastrula are in general also represented members of repeat families represented by prevalent in the maternal RNA of the unfertilized egg (Galau et al., transcripts in the oocyte RNA are interspersed among 1976, 1977). It appears therefore that at a certain stage single copy sequence elements in the genome. We are between fertilization and gastrulation, transcription of currently attempting to isolate the oocyte repetitive mRNA from genes whose product was already present in sequence transcripts and examine their sequence organi­ the egg is turned on, and the newly synthesized mRNA zation directly. molecules are added to identical maternal molecules. In more advanced stages the maternal mRNA is degraded 34. MATERNAL mRNA WCALIZATION AND and disappears from the cells, so that at the gastrula UTILIZATION IN SEA URCHIN DEVELOPMENT stage all the mRNA in the cells is in fact newly Investigators: Susan G. Ermt, Eric H. Davidson synthesized. To follow this replacement at the level of a Sea urchin eggs divide into cells equal in size until single gene, we are utilizing the recombinant DNA the fourth division. This unequal division produces the 16- plasmid, designated CS0088, which contains sea urchin cell stage embryo with three distinguishable cell types. sequence complementary to egg RNA (Biology 1978, No. Micromeres, one of the three cell types of defined cell 31). Hybridization of excess RNA with the labeled lineage present in 16-cell stage embryos, are responsible plasmid DNA will indicate the quantity of the specific for the formation of the primary mesenchyme that gene product at various stages, both newly synthesized produces the embryonic skeleton. and maternal. By labeling the mRNA of the embryos, the Protein synthesis in early cleavage stages of the sea plasmid probes can be used to detect the appearance of urchin is coded for by maternal messenger RNA. In this new transcripts of the structural gene. So far, asym­ study we are investigating the messenger RN As found in metric transcripts of the putative structural gene included the 16-cell embryo to determine if there is specific in CS0088 have been found in polysomal RNA from 16-cell localization of expression of these maternal messenger embryos, in a quantity of 600 molecules per embryo. This RN A sequences. The egg contains RN A equal to a amount is about 20% of their prevalence in unfertilized 36 eggs. Clll'rent studies are being done to determine the RNAs, while 40% of the blastula sequences are repre­ amount of these transcripts in other stages of sea" urchin sented in gastrula mRNA. 3H-mDNA was hybridized with embryogenesis and in adult tissues. excess nuclear RNA from coelomocytes, intestine, and gastrula embryos. Within our limits of detection, all References: Galau, G. A., Klein, W. H., Davis, M. M., Wold, B. J., mD NA sequences were hybridized by each of the nuclear Britten, R. J. and Davidson, E. H. (1976) Cell 7: RNAs. We conclude from this result that virtually all of 487-505. Galau, G. A., Lipson, E. D., Britten, R. J. and Davidson, E. the blastula mRNA sequences are transcribed in three H. (1977) Cell 10: 415-432. heterologous tissues in which the transcripts are not utilized as messenger RN As. The kinetics of hybridization 36. NONMATERNAL MESSENGER RNA SEQUENCES IN SEA URCHIN EMBRYOS show that the blastula structural gene sequences are Investigators: Barbara J. Wold, Erie H. Davidson present in each nuclear RNA at about the same concen­ tration as are the majority of the complex single copy The vast majority of messenger RNA sequences nuclear transcripts. expressed in sea urchin embryos are also present in the maternal RNA of mature oocytes. To detect and 38. REPETITIVE SEQUENCE TRANSCRIPTS IN quantitate embryo messenger RNA sequences that are not SEA URCHIN NUCLEAR RNAs homologous with maternal RNAs, a labeled single copy Investigators: Richard H. Scheller, Jay W. Ellison, DNA fraction entirely devoid of oocyte RNA complemen­ Eric H. Davidson tary sequences was prepared. This selected DNA, termed Nine cloned repetitive sequences were labeled, null oDNA, was reacted with excess polysomal mRNA strand-separated, and individually hybridized with RN A from 16-cell, blastula and gastrula embryos. Nonmaternal extracted from nuclei of gastrula stage sea urchin sequences could not be detected in the mRNAs from 16- embryos and of adult sea urchin intestine cells. The cell and gastrula stage embryos. Mesenchyme blastula concentration of transcripts complementary to each mRNA, however, hybridized about 3.6 million nucleotides cloned s~uence was measured by RN A excess hybridiza­ of null oDNA sequence which is sufficient to code for tion kinetics and by a DNA excess titration method. approximately 2000 mRNAs of average size. These Transcripts of certain of the repeat families are present nonmaternal mRNAs are complex class sequences present at over 100 times the concentration of transcripts of on blastula polysomes at about the same concentration other families in each RNA. The set of repetitive (500 copies per embryo) as are most maternal sequence sequence families highly represented in intestine nuclear mRNAs. Thus, a significant number of new, nonmaternal RNA is different from that highly represented in gastrula structural genes are expressed in blastula stage embryos. nuclear RNA. Together with results obtained with mature oocyte RNA (see abstract No. 33), these findings show 37. SEA URCHIN EMBRYO mRNA SEQUENCES IN that quantitative patterns of repetitive sequence repre­ THE NUCLEAR RNA OF ADULT TISSUES sentation in RNA are specific to each cell type. Both Investigators: Barbara J. Wold, William H. Klein, strand; of all of the ~e cloned repeats are represented Barbara R. Hough-Evans, Erie H. Davidson at some level in all the RN As studied. Usually though not always the concentrations of transcripts complementary Because the sea urchin displays large differences I to the two strands of each repeat do not differ by more between embryo and adult tissue messenger RNA se­ than a factor of two. The cloned tracers do not react quence sets, it is possible to test the proposition that with polysomal messenger RNA, and the nuclear RNA structural gene sequences are transcribed only in cells molecules with which they hybridize are many times where the transcripts are translated. A 3H-labeled single larger than the repetitive sequences themselves. copy DNA highly enriched for sequences complementary to blastula polysomal messenger RNA was prepared. This selected DNA fraction, referred to as mDNA, represents about 26 million nucleotides of embryo messenger RNA sequence. A maximum of 1696 of the blastula sequences are present in coelomocyte and intestine messenger 37

39. REPEATED SEQUENCES IN THE VICINITY OF fragments containing members of the chosen repetitive EXPRESSED SINGLE COPY SEA URCHIN DNA sequence families (see Biology 1978, No. 39). These in­ Investigators: David M. Anderson, Richard H. Scheller, dividual repetitive sequences were isolated from the large James W. Posakony, Eric H. Davidson Charon 4 inserts by restriction enzyme digestion and gel The studies under way involve an investigation of electrophoresis. the relationships between repetitive DNA sequences that Comparing the nucleotide sequences of several occur adjacent to expressed single copy regions in the members of several repetitive families will shed consider­ genome of the sea urchin. Our approach has been to clone able light on a number of interesting but heretofore long (15 kb) EcoRI DNA fragments utilizing a lambda inaccessible problems, e.g. (1) the internal organization of vector system. By cloning long fragments, we are assured repetitive sequences, (2) the type and location of the of being able to study the DNA sequences flanking a given nucleotide substitutions which we observe as sequence transcribed single copy region. The use of the lambda divergence within repetitive families, (3) the nature of the vector system has provided an efficient cloning vehicle. "boundary" between repetitive sequence elements and To date, we have cloned 90% of the sea urchin genome as other sequences. measured in hybridization reactions utilizing single copy The nucleotide sequences of several repetitive plas­ 3 H-DNA driven by DNA extracted from the total clone mid clones (Scheller et al., 1977) have already been set. determined. Common features include tandem repetitions In order to select individual cloned sequences for of simple sequences and regions unusually rich in A-T or detailed study, we have screened the total lambda clone G-C base pairs. set with several cloned repetitive DNA fragments pre­ References: viously described in Scheller et al. (1977). The repetitive Maxam, A. M. and Gilbert, W. (1977) Proc. Nat. Acad. families chosen differ in their representation in the RNA Sci. USA 74: 560-564. Scheller, R. H., Thomas, T. L., Lee, A. S., Klein, W. H., populations of egg, embryonic, and adult tissues. To date, Niles, W. D., Britten, R. J. and Davidson, E. H. we have selected 10 to 20 different members of three (1977) Science 196: 197-200. repetitive sequence families. One family is highly represented in intestine nuclear RNA, one in gastrula 41. SELECTION OF CLONED SEA URCHIN DNA hnRNA, and one in the total oocyte RNA population. CODING FOR mRNA From five members of each of these families, we have Investigators: Jay W. Ellison, Eric H. Davidson obtained 3 to 5 kb fragments which carry the repetitive Our previous studies of structural gene expression DNA element used in the initial screening. We are now in during sea urchin development have revealed that the the process of identifying those which have adjacent presence on polysomes of certain mRNAs is specific with single copy regions that are expressed. respect to developmental stage. These studies relied on Reference: complexity measurements of mRNA populations, and the Scheller, R. H., Thomas, T. L., Lee, A. s., Klein, W. H., conclusions pertain to the rare or "complex11 class of Niles, W. D., Britten, R. J. and Davidson, E. H. (1977) Science 196: 197-200. mRNAs. We wish to make similar studies of the abundant or 11prevalent11 class of mRNAs, to see if the appearance 40. PRIMARY STRUCTURE OF REPETITIVE DNA of these messages is developmentally regulated. This type SEQUENCES IN THE SEA URCHIN GENOME of investigation is feasible if recombinant DNA clones are Investigators: James W. Posakony, Erie H.. Davidson available that contain sequences coding for prevalent The sequencing technique of Maxam and Gilbert mRNAs, beca\.Se individual species could then be studied. (1977) is being used to determine the nucleotide sequence This project concerns the isolation of such clones of several members of selected repetitive sequence from the sea urchin DNA library of recombinant lambda families in sea urchin DNA. phage. The prevalent mRNA probe used to screen the Plasmid clones of individual repetitive sequence library is in vivo labeled polysomal mRNA from sea urchin elements (Scheller et al., 1977) were used to screen a blastula. Embryos were grown in phosphate-depleted 32 "shotgun library" of the sea urchin genome, carried in 15 seawater and labeled with P-orthophosphate for 9 hr, 6 to 20 kb pieces by the phage vector Charon 4, for which results in mRNA specific activities of about 10 to 38

7 32 10 cpm/µg. P-RNA extracted from polysome gradi- 43. EVOLUTIONARY CHANGE IN SEA URCHIN REPETITIVE DNA ents was hybridized to filter-bound DNA from the cloned Investigators: Gordon P. Moore, Roy J. Britten library, and individual clones were plll'ified by several rounds of screening. The frequency of occurrence of particular repetitive With the use of restriction ~nzymes the long (15 to sequence families has been estimated in the DNA of the 20 kb} sea urchin DNA inserts are being analyzed to three sea urchin species, Strongylocentrotus purpuratus, confirm the presence of sequences coding for mRNAs. Strongylocentrotus franciscanus, and Lytechinus pictus, Such sequences could be used as hybridizing probes to using individual cloned S. purpuratus repetitive sequence study the expression of the respective genes in different elements. Estimates have also been made of frequency sea urchin cell types. changes of many repetitive families by measurement of the reassociation of labeled repetitive DNA fractions with 42. SINGLE COPY DNA SF.QUENCH POLYMORPHISM total DNA from other species. In each reciprocal Investigators: Roy J. Britten, Richard M. Rohan comparison the labeled repetitive sequences reassociate Measurements of the· differences in average single more slowly with DNA of other species than with DNA of copy DNA sequence between individual sea urchins have the species from which they were prepared. Thus it been continued. The thermal stability of duplexes formed appears that the dominant repetitive sequence families in between DNA derived from two or more individuals is each species' DNA are present at lower frequencies in compared with that for DNA from one individual. closely related species DNA. No greater differences have been observed in the Thermal stability measurements have been made of single copy DNA sequences between individuals collected repetitive DNA fractions from S. purpuratus and S. 2000 kilometers apart than between individuals collected franciscanus when reassociated with homologous or heter­ from under one boulder off Point Loma, California. The ologous DNA, and indicate that repetitive DNA sequences measurements indicate that the two genomes of a diploid change more slowly during evolution than single copy DNA individual normally differ about as much as any two sequence. This supports the concept of a functional role genomes present in the population of this sea urchin for repetitive DNA. (Strongylocentrotus purpuratus) on the Pacific coast of Measurements of thermal stability have been made North America. of individual S. purpuratus cloned repetitive sequences The melting temperature for duplexes formed be­ reassociated with S. franciscanus DNA or s. purpuratus tween the single copy DNA sequences of two genomes is DNA. Most families have changed both in frequency and about 4° below that for perfect DNA duplexes, indicating sequence. Some, however, have changed little in sequence a 4% average sequence difference. About one quarter of but show great changes in frequency. These frequency the single copy DNA sequences appear to differ in about differences are therefore not due to divergence and 10% of their nucleotides, while the least divergent quarter failure of duplex formation under our conditions. We of the DNA differs in 1 % or less, and the majority of the conclude that during evolution member sequences are sequences have intermediate degrees of divergence. If we added or deleted from families of repeated sequences. make the logical assumption that the evolutionary rate of Repeated sequence families grow or shrink in the DNA of incorporation of base substitutions leading to poly­ one species and, in general, different families change morphism is the same as that leading to interspecies most rapidly in size in other closely related species DNA. sequence divergence, we may conclude that much of the Since families of short interspersed repetitive sequences polymorphism is due to sequence differences between very share in these frequency changes it appears ttiat some ancient versions of single copy sequences in the sea urchin unexpected events of insertion or sequence rearrangement genome. This conslusion is based on measurements must occur at significant rates during the evolution of the summarized below which indicate that the evolutionary genome. rate of change in sea urchin single copy DNA is about 0.25% per million years. A preliminary measurement suggests that mouse single copy DNA polymorphism is much less than that of the sea urchin. 39

44. SEQUENCE ORGANIZATION OF INDMDUAL sequences. Single copy DNA sequence relationships form REPEATED SEQUENCE FAMILIES a useful baseline for comparison with other evolutionary Investigators: William R. Pearson, Roy J. Britten changes. The measurements reported here represent a The length distribution and sequence organization of significant extension of older work in precision and ability four repeated DNA sequence families have been examined to measure more distant relationships. In a new technique in two sea urchin species, using cloned repeated sequence a series of samples are heated in a solvent (2.4 M elements and the "Southern blot" hybridization technique. tetraethyl ammonium chloride) which suppresses the With gel blots of EcoRI-digested Strongylocentrotus pur­ effect of base composition on thermal stability, and the puratus DNA, it was shown that a part of each family is strand-separated DNA is digested with Sl nuclease. With organized in long repetitive regions. In two cases (clones this approach Strongylocentrotus purpuratus single copy 2034 and 2133) an individual band dominated the gel labeled DNA reassociated with S. franciscanus DNA melts electrophoresis pattern, indicating that a large fraction of 14°C below perfect duplexes and the reassociation is the members of these families is organized in one type of nearly complete (81%). In previous measurements reas­ long repeated sequence, possibly a tandem array. For sociation proceeded to less than 50% and only about half clones 2007 and 2101 the majority of the hybridization is of this Tm reduction was observed. Similar measurements with a wide distribution of fragment lengths, and thus between S. purpuratus single copy labeled DNA and S. interspersed sequences make up a majority of these drobachiensis DNA show a melting temperature about 7°C families. With S. franciscanus DNA EcoRI gel blots a below perfect duplexes. Considering the effects of the reduced level of hybridization was seen as expected from sequence polymorphism of S. purpuratus DNA this result is the frequency differences of the repetitive families very close to the previous measurements. Thus, as represented by the clones. expected, the inclusion of more divergent sequences has a The same cloned repeats were hybridized with gel greater effect for the more distantly related pair of blots made from reassociated repetitive DNA digested species. When these data are compared with the fossil with Sl single strand-specific nuclease, to assay the record we estimate that single copy DNA sequences have length distribution of the reassociated repetitive regions been changing at a rate of about 0.25% per million years containing family members. In the case of one predomi­ in each line, since the last common ancestors of $. nantly interspersed family, 2101, a large fraction of the purpuratus and S. franciscanus existed about 15 to 20 hybridization occurs in two bands at about 300 and 400 million years ago and of S. purpuratus and S. drobachiensis nucleotides, supporting the conclusion that interspersed about 7 million years ago. sequences tend to be of a given length. Bands also are observed in Sl nuclease digest gel blots for the two families which are dominated by long or tandem repeti­ tion. Clone 2133 shows a band at almost the same length PUBLICATIONS as with the EcoRI digest blot, as well as a "ladder11 of Britten, R. J., Cetta0 A. and Davidson, E. H. (1978) The bands at 2, 3, 4, and 5 times this length. Thus 2133 may single copy DNA sequence polymorphism of the sea urchin S. purpuratus. Cell. Submitted for publica­ be organized in a long or tandem array which reassociates tion. with regularly spaced partially paired regions which are Chamberlin, M. E., Galau, G. A., Britten, R. J. and Davidson, E. H. (1978) Studies on nucleic acid often but not always digested by Sl nuclease. reassociation kinetics: V. Effects of disparity in tracer and driver fragment lengths. Nucleic Acids Res. In press. 45. SINGLE COPY DNA SEQUENCE DIFFERENCES Clegg, K. and Pik6, L. (1977) Size and specific activity of AMONG RELATED SEA URCHINS the UTP pool and overall rates of RNA synthesis in Investigators: Terrence J. Hall, Roy J. Britten early mouse embryos. Devel. Biol. 58: 76-95. Costantini, F. D., Scheller, R. H., Britten, R. J. and The majority of the single copy DNA sequences of Davidson, Ew H. (1978) Repetitive sequence tran­ scripts in the mature sea urchin oocyte. Cell. In higher organisms does not appear to consist of structural press. genes, and the sequences change rapidly in evolution. The Hough-Evans, B. R., Wold, B. J., Ernst, S. G., Britten, R. J. and Davidson, E. H. (1977) Appearance and effect of selection on these changes is not known, but is persistence of maternal RNA sequences in sea considerably less than on changes in average protein urchin development. Devel. Biol. 60: 258-277. 40

Klein, W. H., Thomas, T. L., Lai, C., Scheller, R. H., Moore, G. P., Scheller, R. H., Davidson, E. H. and Britten, Britten, R. J. and Davidson, E. H. (1978) Character­ R. J. (1978) Evolutionary change in the repetition istics of individual repetitive sequence families in frequency of sea urchin DNA sequences. Cell. In the sea urchin genome studied with cloned repeats. press. Cell. In press. Pik6, L. (1977) Immunocytochemical detection of a murine Leahy, P. S., Tutschulte, T. c., Britten, R. J. and leukemia virus-related nuclear antigen in mouse Davidson, E. H. (1978) A large-scale laboratory oocytes and early embryos. Cell 12: 697-707. maintenance system for gravid purple sea urchins Scheller, R. H., Costantini, F. D., Kozlowski, M. R., (Strongylocentrotus purpuratus). J. Exptl. Zoo!. 204: Britten, R. J. and Davidson, E. H. (1978) Specific 369-380. representation of cloned repetitive DNA sequences Lee, A. S., Britten, R. J. and Davidson, E. H. (1978) Short in sea urchin RN As. Cell. In press. period repetitive sequence interspersion in cloned Wold, B. J., Klein, W. H., Hough-Evans, B. R., Britten, R. fragments of sea urchin DNA. Cold Spring Harbor J. and Davidson, E. H. (1978) Sea urchin embryo Symp. Quant. Biol. 42. In press. mRNA sequences expressed in the nuclear RNA of adult tissues. Cell. In press.

Professor: William J. Dreyer Research Felli>w: Carol Readhead cutting and splicing during the development of B-lympho­ Graduate Student: Henry V. Huang cytes are not unique to these cells. For example, very Research Staff: John Wen-Kiang Chu, 8amuel D. similar gene splicing events might help place recognition Culpovich, Ursino E. Del Valle, Barbara L. DeOgny, David B. Helphrey, Suzanna J. Horvath, Eva H. molecules of high specificity on cells of the developing Lujan, Catherine R. Morris, Jeanne P. Patalano, Stephen I. Vass, Gayle-Linda Westrate, Elizabeth R. nervous system so as to aid in the assembly of the brain. Zimmerman While a random gene scrambling and splicing mechanism might feasibly result in a functioning immune system with Support: The work described in the following research reports has been supported by: the necessary array of antibody diversity, it is unlikely The Ahmanson Foundation that such a random process could result in the precise Leland Fikes Foundation, Inc. The Max C. Fleischmann Foundation neural arr~ements of a developing brain. If on the other Litton Bionetics, Inc. hand antibody genes are spliced in a highly ordered John A. Mccarthey Foundation National Institutes of Health, USPHS manner under genetic command, then it would not be so National Science Foundation surprising to find similar processes used elsewhere in the Summary: Long before Caltech began, scientists had embryo. Last year we reported studies which suggest that marveled at the extraordinary precision with which the the immune system of developing chicl< embryos is indeed cells of embryos move and position themselves during programmed in a highly ordered manner. The embryos development. They attempted to understand the process seem to use an orderly process of antibody gene expres­ but found it was not possible. Before our current students sion to generate cells which synthesize very similar were even born, Caltech Professors Albert Tyler (1946) antibody molecules from individual to individual. This and Roger Sperry (1951, 1963) had both studied and work has been extended this year. speculated about the amazingly high specificity of ce!l­ We are now focusing most of our attention on the surface interactions. Today, despite the ever increasing use of tumors other than those of the immune system. appreciation of the remarkable genetic and molecular Tumors provide us with large quantities of homogeneous events which are responsible for generating large numbers cell populations that bear on their surface molecules of diverse cell types, each committed to a particular related to those of individual fetal or stem cells. position and role in the developing organism, we still know Comparison of a series of different tumors should then almost nothing about these events. Studies of tumors of permit us to begin to understand the molecular basis for the immune system have provided a glimpse of strange surface variation among cells and also for cell-cell gene splicing events that help explain the embryos' ability recognition processes that occur during normal embryo­ to produce hundreds of thousands of different clones of logical development. We may also learn something about cells, each making one specific antibody molecule dif­ abnormal molecules of importance in cancer research and ferent in its sequence of amino acids from the others. We tumor immunology-. have speculated (Dreyer et al., 1967; Hood et al., 1977) The surface of a cell represents a very small part of that the chromosomal programs which lead to gene the total cell mass. Accordingly, even though we can 41 obtain hundreds of grams of cells, the quantity of any RNA tumor viruses expressed on tumors because of single cell-surface protein which can be isolated is activation of germline genes of value to the mouse or are exceedingly small. It is for this reason that we have the molecules expressed as a result of infection with devoted considerable effort to developing new highly viruses? effective and sensitive methods for analysis of cell­ We have isolated cell-surface membranes from a surface proteins. Whenever possible, methods and instru­ series of different UV-induced fibrosarcomas and from ments have been adapted from thase proven effective by normal cells. Membrane proteins were fractionated using others. However, in some cases new systems were needed lectin affinity chromatography and the various fractions in order to achieve the required sensitivity. We have were analyzed by means of two-dimensional gel electro­ joined with Leroy Hood's group to set up an advanced phoresis. We have found a number of interesting families facility for analysis of cell-surface proteins. A number of of molecules and we are in the process of further the new procedures and instrument systems are now in use characterizing them using a variety of immunological and and others are in an advanced testing phase. The facility protein chemical techniques. has already proven extremely effective in the analysis of one class of surface recognition molecules (gp70) isolated 47. IDENTIFICATION OF CELirSURFACE MOLECULES THAT REACT WITH from the surface membranes of RNA tumor viruses. ANTI-TUMOR SERUM Related molecules are also found on certain normal fetal Investigators: Carol Readhead, Jeanne P. Patalano, and adult cells. There are a number of intriguing Barbara L. DeOgny, William J. Dreyer questions which we ask about this family of molecules and Last year we reported that we had prepared and the genes which code for them. We expect that the new tested antisera directed against a series of different facility will help answer questions about this and other tumor lines (Biology 1977, No. 52). We have now used families of cell recognition molecules. The answers to these antisera to identify protein molecules in the tumor these questions are expected to help gain insights into membrane fractions. The sera react with only some of both normal and abnormal cell growth. the molecules within one of the families of related Generi.11. References: proteins. We are anxious to learn the exact nature of the Dreyer, W. J., Gray, W.R. and Hood, L. (1967) Cold Spring relationships among the proteins in this family. Peptide Harbor Symp. Quant. Biol. XXXII: 353-367. Hood, L., Huang, H. V. and Dreyer, W. J. (1977) J. mapping protein sequencing and other methods are now Supramolec. Struct. 7: 531-559. being used to obtain definitive answers to the many Sperry, R. W. (1951) Chicago Med. School Quart. 12: 66-73. questions raised. Sperry, R. W. (1963) Proc. Nat. Acad. Sci. USA 50: 703-710. Tyler, A. (1946) Growth 10: 7-19. 48. STUDIES OF THE STRUCTURE AND FUNCTION OF A FAMILY OP CELlrSURFACE RECOGNIDON MOLECULES 46. FRACTIONATION AND ANALYSIS OF PROTEINS Investigators: carol Readhead, Michael W. Hunkapiller*, DISPLAYED ON THE SURFACE OF TUMOR Leroy E. Hood, William J. Dreyer CELLS The host range of a particular RN A tumor virus is Investigators: Carol Readhead. Jeanne P. Patalano, Barbara L. DeOgny, Elizabeth R. determined by struc~ure of a recognition molecule dis­ Zimmerman, William J. Dreyer played on the membranes of that virus. These glyco­ We have undertaken a study of the proteins that are proteins vary somewhat in size, but a common class of displayed on the surface of tumor cells. There are several such molecules (gp70) has an apparent molecular weight of questions which we would like to answer concerning these about 70 K. molecules: (1) Are some of them important for cell-cell We are intrigued by observations which indicate that recognition in normal growth? (2) Are some of them a number (perhaps a large number) of genes coding for abnormal proteins, which are important in studies of related molecules is carried in the germline of many cancer? (3) How do these molecules relate to the diverse vertebrates. Such genes seem to have been processes of immunological self-recognition and tumor conserved during evolution for long periods of time. It is rejection? (4) Are gp7 0 and other molecules related to hard to accept the view that genes of this type have 42 survived only to generate RNA tumor viruses. Indeed, a both peptides and proteins, (5) a flow-through scintillation number of reports have appeared demonstrating the counter for monitoring radioactively-tagged derivatives expression of related antigens on normal fetal and adult from the sequenators as well as proteins and peptides tissues (Huebner et al., 1970; Del Villano et al., 1975). after separation by HPLC. Since such molecules clearly play a role in highly specific A prototype of the advanced design, ultra-high virus-cell interactions some feel that they may well sensitivity mass spectrometer system is now operational function in cell-cell interaction during normal ernbryo­ at the Jet Propulsion Laboratory (see Biology 1978, No. genesis. 50). We have determined the amino-terminal sequence of This new facility with its combination of instru­ gp70 from Rauscher leukemia virus (in collaboration with ments and other new procedures now permits users of the R. Gilden and S. Oroszlan of the Frederick Cancer facility to carry out studies which have never before been Research Center). The new sequencing facility allowed us possible. to sequence through the first 46 amino acid residues using *Division of Chemistry and Chemical Engineering, Cali­ only a few nanomoles of protein. This sequence informa­ fornia Institute of Technology. tion penetrates more than twice the distance into the protein than ever before possible with any sequenator. We 50. A NEW, EXTRAORDINARILY SENSITIVE ANALYTICAL SYSTEM FOR RESEARCH are now using the systems to study related molecules in an AND MEDICINE effort to learn more about this fascinating class of Investigators: Charles E. Giffm.•, Heinz G. Boettger*, recognition molecules. David D. Norris•, David B. Helphrey, Aron Kuppermann**, Michael W. References: Hunkapiller••, Leroy E. Hood, Del Villano, B. c., Nave, B., Croker, B. P., Lerner, R. A. William J. Dreyer and Dixon, F. J. (1975) J. Exptl. Med. 141: 172-187. Huebner, R. J., Kelloff, G. J., Sarma, P. s., Lane, W. T., Advances in basic research as well as in clinical Turner, H. C., Gilden, R. V., Oroszlan, S., Meier, H., medicine are often greatly facilitated by the introduction Myers, D. D. and Peters, R. L. (1970) Proc. Nat. Acad. Sci. USA 67: 366-376. of new analytical systems. We believe that the new type of mass spectrometer which we are developing is an *Division of Chemistry and Chemical Engineering, Cali­ fornia Institute of Technology. example and that it will find many important applications. The new instrument is similar to early mass spec­ 49. l!STABLISHMENTOF AN ADVANCED trometers which ionized molecules, separated them by LABORATORY FOR STUDIES OF PROTmNS size, and detected the various ions by exposing a photo­ Investigators: Michael W. Hunkaplller*, David B. graphic plate. The position and darkness of the spots Helphrey, Suzanna J. Horvath, Nelson D. Johnson, Stephen L Vess, produced by the ions indicated their size and quantity. Leroy E. Hood, William J. Dreyer The new instrument replaces the photographic plate with Remarkably little is known about the structure and an electrcroptical image detector so sensitive that it can function of protein molecules displayed on cell surfaces sense individual ions which enter any one of tens of despite very great interest in such molecules. The reason thousands of channels within the detector array. It is for is that it is generally exceedingly difficult if not impos­ this reason that the new system is more than one thousand sible to obtain such molecules in quantities suitable for times more sensitive than previous instruments. The data analysis by means of conventional methods. are rapidly collected, processed by a minicomputer and The new laboratory, designed to allow analysis of plotted in a useful form. scarce proteins and peptides, includes a number of Progress has been substantial during the past year. instruments: (1) a newly constructed, advanced design We continue to carry out preliminary studies of amino spinning cup sequenator, (2) a new type of miniature solid acid derivatives using the prototype instruments located phase sequenator suitable for use with very small amounts at JPL; however, we anticipate that in about two years it of sample, (3) automated, high pressure/performance will be possible to acquire a suitable version of the new liquid chromatographs (HPLC) for analysis of amino acid instrument for installation in the Caltech protein chem­ derivatives generated by the sequenators, (4) an HPLC and istry laboratory. new procedures for rapid, high resolution separation of We also plan a series of feasibility studies aimed at 43 performing clinical assays similar to those now carried out developmental programs, and the generation of diverse by means of radioimmunoassays. The extreme speed and antibodies must occur in a systematic and reproducible sensitivity of the new mass spectrometer opens up many fashion even in different embryos. We found that very other possibilities in the field of clinical chemistry. similar antibody light chains were made by different References: inbred chickens bursectomized at slightly different but Dreyer, W. J., Kuppermann, A., Boettger, H. G., Giffin, C. early developmental times. The evidence indicates that E., Norris, D. D., Grotch, S. L. and Theard, L. P. the genetic rearrangements which occur in the developing (1974) Clin. Chem. 20: 998-1002. Giffin, C. E., Boettger, H. G. and Norris, D. D. (1974) Int. immune system are nearly identical in different embryos, J. Mass Spectrom. Ion Phys. 15: 437-449. and that there is an orderly readout of germline genes *Jet Propulsion Laboratory (numerous other members of during ontogeny. Thus the immune system develops the JPL staff have contributed substantially to this according to a set program. It will be interesting to learn program). **Division of Chemistry and Chemical Engineering, Cali­ whether gene splicing also occurs in other systems as fornia Institute of Technology. proposed.

51. IS DEVEWPMENTALLY PROGRAMMED GENE Reference: SPLICING USED ONLY IN THE IMMUNE Hood, L., Huang, H. V. and Dreyer, W. J. (1977) J. SYSTEM? Supramolec. Struct. 7: 531-559. Investigators: Henry V. HWlJlt, William J. Dreyer PUBLICATIONS We have posulated that antibodies are evolutionarily Dreyer, W. J. (1978) A progress report on two highly related to cell-surface molecules which mediate cell-cell sensitive, nonisotopic assay systems. Antibiotics recognition during embryogenesis, and that both kinds of Chemother. 26. In press. molecules are coded for by separate variable- and Dreyer, W. J. (1978) A theoretical and experimental approach to the study of cell-cell recognition. In: constant-region genes which are spliced together to Manipulation of the Immune Response in Cancer, A. Mitcheson (Ed.), Academic Press. In press. generate functional genes (Hood et al., 1977). These Hood, L., Huang, H. V. and Dreyer, W. J. (1977) The area­ events are believed to help explain the stability of code hypothesis: The immune system provides clues developmental programs in different cell lineages. A key to understanding the genetic and molecular basis of cell recognition during development. J. Supra­ prediction is that if the genetic rearrangements seen in molec. Struct. 7: 531-559. the developing immune system are representative of other Huang, H. V. and Dreyer, W. J. (1978) Bursectomy in ovo blocks the generation of immunoglobulin diversity. systems they must be under the stringent control of J. Immunol. In press. 44

Professor: Leroy E. Hood are analyzing the gene products from normal antibody­ Visiting Associates: Jeffrey J. Hubert, Rolf H. Joho Senior Research Fellows: Michael W. Hunkapiller*, Minnie producing cells that have been fused to drug-resistant McMillan myeloma cell lines. These hybrid cells are permanent cell Research Fellows: J. Michael Cecka, Sandra J. Ewald, Henry V. Huang, Florence Lafay, Thomas H. Stanton lines that secrete normal antibody polypeptides. Although Graduate Students: Mark M. Davis, Philip W. Early, John these studies are in a very preliminary stage, they should Frelinger, Jonathan S. Fuhrman, Nelson D. Johnson, Marilyn R. Kehry, Mitchell Kronenberg, Carol A. allow us to analyze the patterns of diversity in normal Oken*, James W. Schilling antibody responses and will make available a new spec­ Research Staff: Paul K. Cartier lll, Vincent R. Farnsworth, Judith S. Greengard, David B. Helphrey, trum of probes for nucleic acid analysis. Chin Sook Kim During the past year we have begun to isolate the Laboratory Staff: Ronald T. Hardgrove, Bertha E. Jones, Eva Westmorland antibody genes encoding the response to phosphorylcholine in mice. The phosphorylcholine system is attractive *Division of Chemistry and Chemical Engineering, Cali­ fornia Institute of Technology. because we have determined the complete sequence of the variable region of eight heavy chains (V H) from myeloma SUpport: The work described in the following research reports has been supported by: proteins binding phosphorylcholine. These VH regions American Cancer Society constitute a set of closely-related sequences. Moreover, Earle C. Anthony Fellowship B. W. Foundation certain of these myeloma proteins constitute the pre­ Ethel Wilson Bowles and Robert Bowles dominant response when antibodies to phosphorylcholine Professorship Cancer Research Institute, Inc. are raised by normal immunization techniques. In The Camille and Henry Dreyfus Foundation, Inc. addition, we can look at additional components in the The Max C. Fleischmann Foundation National Institutes of Health, USPHS normal library of the phosphorylcholine response by the National Science Foundation cell fusion technique mentioned earlier. To date we have Gordon Ross Medical Foundation Smith, Kline and French constructed several cDNA probes to phosphorylcholine Stanford University light and heavy chains from myeloma proteins binding Universite de Paris-Sud phosphorylcholine, have generated germline, embryo, and ·summary: Our laboratory is interested in studying the myeloma genomic libraries in A bacteriophage using organization, expression, and evolution of several differ­ partial Rl digests, and have proceeded to isolate about 20 ent multigene families that have immune-related func­ genomic clones of light and heavy chains. We are tions. These include the antibody gene families as well as interested in determining how the organization and se­ several gene families in the major histocompatibility quences of the antibody gene segments (i.e., V, J, and C) complex. We employ the techniques of protein and change during differentiation (i.e., in the various germ­ nucleic acid chemistry as well as those of molecular line, embryo, and somatic gene libraries we have con­ immunology. structed). The amino acid sequence analysis of myeloma We also are interested in the lgM receptor on B cell immunoglobulins and homogeneous antibodies from this membrane that triggers proliferation and differentiation and other laboratories has provided important insights into upon interaction with a complementary antigen. Our the structure of antibody molecules and the organization initial efforts are directed at comparing the secreted IgM and evolution of their genes. During the pa.st year, the heavy chain (µ)and its membrane-bound counterpart. To nearly complete variable (V) region analysis of 22 closely­ this end we have determined the complete amino acid related myeloma light chains has suggested that the sequence of a secreted µ chain (MOPC-104E) and are in mouse K chain is encoded by three separate DNA the process of isolating sufficient membrane-bound µ so segments: V or variable (residues 1-99); J or joining that we can determine whether membrane and secreted µ (residues 100-112); and C or constant (residues 113-219). chains are identical or whether they differ in amino acid Furthermore, the combinatorial joining of different V and sequence, in attached carbohydrate, or in both. We also J segments may be a fundamental mechanism for produc­ plan to study the orientation of the lgM molecule in the ing antibody diversity (Weigert et al., 1978). membrane and its relationships to other membrane pro­ To analyze the normal antibody repertoire for two teins. This IgM receptor is an ideal model for studying the natural antigens, phosphorylcholine and a-1,3 dextran, we anatomy and physiology of membrane receptors in gen- 45 eral. We also have begun a series of studies on the T cell subgroups, as yet lacking multiple members. Five of our receptor, an entity that has remained surprisingly elusive six subsets contain two or more identical sequences. This over the past six years. observation has important implications for the mechanism We are studying several gene families (transplanta­ of the generation of diversity. Ea.ch distinct subgroup is tion antigens and Ia antigens) encoded by the major generally believed to be encoded by one or more separate histocompatibility complex (MHC). The MHC is perhaps germline V genes. Thus it appears this set of closely­

the central focus of modern cellular immunology and related K chains is encoded by at least six and potentially immunogenetics. These gene products carry out immune­ more germline V genes. DNA saturation studies suggest related functions, although their precise roles are poorly that there are five to six germline genes for this set of understood. Our interest in these gene families is to use proteins (Valbuena et al, 1978). Thus the DNA estimate microsequence analyses to characterize their gene prod­ agrees with the number of subgroups that have currently ucts so as to learn more about the organization, expres­ been defined. However, additional subgroups will probably sion, and evolution of their genes. In the process we also be defined If so, the DNA saturation experiments may come to understand better the function of these underestimate the number of germline genes or a single V molecules. The preliminary analyses of these molecules gene can generate two or more distinct subgroups of V clearly indicate that the genetic organization of these sequences. This latter alternative would necessitate families is far more complex than expected (Silver and repeated identical (or parallel) somatic mutations, a Hood, 1976; Maizels et al., 1978). requirement that is considered unattractive by most We have spent a great deal of effort on microse­ geneticists. This conflict could be resolved by direct quencing techniques. These efforts have included rede­ nucleic acid sequencing of germline DNA coding for these signing and modifying our commercial sequenators, per­ proteins. fecting the Edman chemistry, and improving the analytic As classically defined, the variable region of these instrumentation for detection of the phenylthiohydantoin proteins comprises residues 1-112. In examining our amino acid derivatives (Hunkapiller and Hood, 1978). In sequences it is apparent that this classical V region addition, we are just finishing the construction of a newly consists of two independently assorting portions. We designed sequenator that should significantly improve our propose to call the major portion from residues 1-99 the V microsequencing capacities. Thus we are interested in de­ segment and the minor portion from residues 100-112 the veloping ever more sophisticated instrumentation for the J or joining segment. Preliminary nucleic acid sequencing analysis of various multigene families. studies indicate that V and J regions are not contiguous in germline DNA. The functional significance of the J General References: Hunkapiller, M. and Hood, L. (1978) Biochemistry 11: region is not known. It could conceivably be involved in 2124-2133. V-C joining or the generation of antibody diversity Maizels, R. M., Frelinger, J. A. and Hood, L. (1978) Immunogenetics. Submitted for publication. (Weigert et al., 1978). Silver, J. and Hood, L. (1976) Cont. Topics Molec. This work was carried out in collaboration with Dr. Immunol. 5: 35-68. Weigert, M., Gatmaitan, G., Loh, E., Schilling, J. and Martin Weigert at the Institute for Cancer Research, The Hood, L. (1978) Nature. Submitted for publication. Fox Chase Cancer Center, Philadelphia, Pennsylvania. References: 52. SEQUENCE ANALYSIS OF A CLOSELY-RELATED Valbuena, o., Marcu, K. B., Weigert, M. and Perry, R. SET OF MOUSE Voc REGIONS (1978) Nature. Submitted for publication. Weigert, M., Gatmaitan, G., Loh, E., Schilling, J. and Investigator: James W. Schilling Hood, L. (1978) Nature. Submitted for publication. We have determined the nearly complete variable (V) region sequences of 22 closely-related myeloma kappa (K) chains from the NZB mouse (Biology 1977, No. 58). Based on shared amino acid substitutions at specific positions we are able to divide 18 NZB sequences into 6 subgroups with 2 to 5 VK sequences in each. The four additional NZB sequences probably define four additional 53. SEQUENCE STUDIES OF a-1,3 DmITRAN-filNDING set of heavy chains we hope to be able to make important JMMUNOGLOBULINS inferences regarding the mechanism of the generation of Investigator: James W. Schilling diversity and also perhaps find evidence for a J region We have extended our investigation of immuno­ similar to what we have found in K chains (see abstract globulins binding a-1,3 dextran through the complete No. 52). sequence analysis of the variable region of a heavy chain This work is carried out in collaboration with Drs. (V H) from a myeloma protein binding this hapten Brian Clevinger and Joe Davie at the Department of (MOPC-104E). This heavy chain has a carbohydrate Microbiology, Washington University Medical School, St. moiety attached to the second hypervariable region. This Louis. is to our knowledge the first report of carbohydrate attached to a hypervariable region in a heavy chain. Since 54. SEQUENCE STUDIES OF LEVAN-mNDING the hypervariable regions comprise the walls of the JMMUNOGLOBULINS antigen-binding crevice of the immunoglobulin, it is Investigator: Nelson D. Johnson possible that the hypervariable region carbohydrate is Primary sequence data from BALB/c myeloma pro­ involved in antigen binding. Alternatively, the carbo­ teins have yielded a large amount of information on both hydrate may lie outside the crevice and exposed to the the nature of antibody-antigen complementarity and the external medium. patterns of antibody variability. Myeloma proteins with The heavy chain from a second myeloma protein­ known specificity are particularly interesting in this binding a-1,3 dextran, J558, has also been partially regard and several such immunoglobulins have been sequenced. It is identical to MPOC-104E H for the Investigated (Hood et al., 1976; Vrana et al., 1978). N-terminal 54 residues. Purification of cyanogen bromide We have completed a study of the heavy chains of fragments from J558 is now under way and we hope to two levan-binding proteins and have analyzed most of the complete the V region sequence shortly. As previously sequence of the corresponding light chains. In comparing reported (Biology 1977, No. 60) J558 and MPOC-104E these sequences with those of other levan-binding proteins each possess a unique V region antigenic determinant (the (Vrana et al., 1978), we find a pattern of variability idiotypic determinant) which allows them to be distin­ different from that seen in other sets of antigen-binding guished by anti-idiotypic antisera. They also share an proteins; namely, in the heavy chain, amino acid differ­ additional common idiotypic determinant. Immunization ences occur outside the antigen-binding site while there is with a-1,3 dextran causes some mice to produce anti­ virtual identity within the binding site (or hypervariable dextran antibodies which completely lack the J558 and regions). This observation is interesting in light of the MPOC-104E-specific idiotypes and have a low level of the various antigen-binding properties exhibited by the levan­ common idiotype. We have sequenced both IgM and IgG binding proteins studied and in light of the more extensive heavy chains from such a response and found both heavy variability seen in the antigen-binding crevice of the chains identical to MPOC-104E for the 35 and 52 residues myeloma proteins binding phosphorylcholine (Hood et al., sequenced, respectively. There was no evidence of 1976). sequence heterogeneity in these normally induced anti­ References: lx>dies. We have previously reported that natural anti­ Hood, L., Loh, E., Hubert, J., Barstad, P., Eaton, B., dextran antibody with a high level of MPOC-104E-specific :Early, P., Johnson, N., Kronenberg, M. and Schilling, J. (1976) Cold Spring Harbor Symp. Quant. Biol. 41: idiotype is also identical to MPOC-104E at all residues 817-836. sequenced. Thus we are presently unable to identify any Vrana, M., Rudikoff, S. and Potter, M. (1978) Proc. Nat. Acad. Sci. USA 75: 1957-1961. structural correlates for any serologically defined idio­ typic determinants. We have begun to characterize anti-dextran anti­ bodies obtained from myeloma-splenocyte hybrid cell lines. This will enable us to examine a potentially unlimited number of homogeneous antibody molecules directed against a-1,3 dextran. From this closely-related 47

55. CONSTRUCTION OF IMMUNOGLOBULIN eDNA level for both variable-constant region joining and heavy PLASMIDS chain constant region switching during lymphocyte devel­ Investigator: Philip W. Early opment. In order to examine these and other phenomena The study of immunoglobulin proteins has raised (such as the antibody diversity, as mentioned in abstract several important questions, including the orig.in of anti­ No. 55) we have constructed mouse DNA libraries in the body diversity, best answered by examining the antibody modified lambda phage, Charon 4a. These libraries genes themselves. Does antibody diversity arise from consist of EcoRI partially-digested sperm, embryonic, or many germline variable region genes, or from the somatic myeloma (from the phosphoryleholine binding myeloma generation of new variable regions? A second type of tumor M603} DN As. The average insert size appears to be question is related to the differentiation of antibody­ 15,000 to 16,000 (range from 12 to 19 kb). We are now producing cells. In heavy chains, a single variable region characterizing several Charon 4a-M603 clones comple­ appears to be sequentially expressed in association with mentary to both heavy and light chain double-stranded different constant regions. Does this process involve cDNA plasmids to test models of variable and constant successive DNA rearrangements of the heavy chain region joining. We also are interested in the germline constant region genes? diversity of the heavy chain genes encoding phosphoryl­ Recombinant DNA techniques make it possible to choline binding, the sequence organization of the heavy isolate immunoglobulin genes from mouse genomes (see chain constant region, and postulated differences between Biology 1978, No. 56). In order to do this, a hybridization variable region genes in sperm DNA and embryonic or probe must be available for the genes of interest. In oU.r nonlymphoid adult DNAs (Kabat et al., 1978).

case, the genes of interest are the heavy chain constant References: regions, the variable regions of the phosphorylcholine­ Honjo, T. and Kataoka, T. (1978) Proc. Nat. Acad. Sci. 75: 2140. binding heavy chains (see Biology 1977, No. 63), and the Kabat, E. A., Wu, T. T. and Bilofsky, H. (1978) Proc. Nat. region of the variable-constant junction in kappa light Acad. Sci. USA 75: 2429. Tonegawa, S., Brack, C., Hozumi, N. and Pirrotta, V. chains (see Biology 1978, No. 52). To this end, I have (1977) ICN-UCLA Symposium, pp. 43-55. prepared many immunoglobulin messenger RNAs. In order to have completely pure probes available in unlimited 57. DETERMINATION OF THE STRUCTURE OF MOUSElgM quantity, I have constructed recombinant plasmid clones Investigators: Marilyn R. Kehry, Jonathan S. Fuhrman from some of these messengers. We are now using plasmids from alpha heavy chain, a gamma-3 heavy chain, The lgM class of immunoglobulins, phylogenetically and one containing the complete amino acid coding the first to appear in vertebrates, is the first to appear sequence of the M167 kappa light chain. With these and during an organism's development, and shows a highly other plasmid clones still under construction, we are conserved primary structure. The IgM molecule fulfills a isolating cloned portions of the mouse genome which dual role. In the serum, it exists as a 198 pentamer should enable us to answer some of the questions raised by where, upon binding antigen, it fixes complement. It also the sequence analysis of antibody proteins. is found as a 78 monomer on the surface of immature lymphocytes, where its interaction with specific antigen leads to the proliferation and differentiation of that 56. THE CONSTRUCTION OF MOUSE DNA LIBRARIES AND THE CHARACTERIZATION OF IMMUNO­ lymphocyte. Such distinct functions might be expected to GLOBULIN GENES impose rather different structural requirements on the Investigators: Marl< M. Davis, Philip W. Early, molecule, and this led us to determine the amino acid Rolf H. Joho sequence of a mouse IgM heavy chain. With the recent advances in DNA technology many The lgM heavy ( µ) chain secreted by the MOPC- very basic and interesting questions can now be answered 104E plasmacytoma was purified and its complete amino regarding the organization of immunoglobulin genes and acid sequence was determined. This was facilitated by changes that may occur during differentiation. In first isolating each of nine cyanogen bromide fragments particular, the work of Tonegawa et al. (1977) and Honjo and then using additional chemical or enzymatic cleavage and Kataoka (1978) implicates rearrangements at the DNA techniques to generate smaller peptides. 48

Comparing the mouse µ chain sequence to those of fragments from the secreted and membrane µ chains human and dog µ chains reveals a striking gradient of which differ in their primary amino acid structure and/or homology from the N-terminal region (37% identical) to attached carbohydrate moieties. Additional differences the c-terminal region (89% identical). This high degree will be delineated by peptide mapping studies of the of sequence conservation at the C-terminus suggests fragments. This structural characterization of the anti­ there are strong selective constraints, perhaps relating to gen receptor may help us understand how a membrane­ conformational requirements for pentamer formation or bound receptor triggers the immature lymphocyte to complement fixation. The mouse µ chain also has five proliferate and differentiate. constant region sites of carbohydrate attachment which These studies are being carried out in collaboration are placed identically to those on the human µ chain. with Dr. Carol Sibley at the Department of Genetics of Again, although their function is unknown, the conserva­ the University of Washington, Seattle. tion of attachment sites clearly indicates a strong selection for their placement. 59. CHARACTERIZATION OP A PUTATIVE T CELL The secreted µ chain sequences lack any stretch of RECEPTOR uncharged amino acids capable of spanning the lympho- Investigators: Mitchell Kronemerg, Nelson D. Johnson cyte membrane. Thus, if the membrane-bound and The molecular nature of the antigen-specific re­ secreted µ chains are identical, the 78 receptor cannot be ceptor on T lymphocytes is one of the major controversies a transmembrane protein. Alternatively, these two in immunology. Although it has long been known that B molecules may differ in primary amino acid sequence. lymphocytes use immunoglobulin (lg) as their antigen­ With this in mind, we are currently examining the specific receptor, the classical techniques for detecting polypeptide structure of the membrane-bound µ chain immunoglobulin have generally given negative results from a nonsecreting B cell tumor line (see Biology 1978, when applied to T cells. No. 58). Recently (Szenberg et al., 1977), it was reported that an antiserum made in chickens to a fragment of 58. COMPARISON OF THE STRUCTURE OF SOLUBLE mouse Jg [(Fab) J will react with normal mouse T cells and AND MEMBRANE-BOUND MOUSE JgM 2 with a number of T lymphomas. This antiserum will Investigators: Marilyn R. Kehry, Sandra J. Ewald precipitate from T cells a molecule with subunits similar The IgM molecule is secreted into the serum as a in molecular weight to Jg light and heavy chains. 198 pentamer and also exists as a membrane-bound 78 In order to reconcile the previous failure to detect monomer on the surface of immature lymphocytes. The lg on T cells with these new results, N-terminal sequenc­ membrane lg~ is an antigen receptor that triggers small ing of the heavy and light chains of the Wehi 22 molecule lymphocytes to divide and differentiate into antibody­ will be carried out. In addition, comparative peptide secreting plasma cells. This class of molecules can analyses of the cyanogen bromide fragments of this therefore exist in soluble form and apparently as an molecule will be compared to those of IgD and lgM, the integral membrane protein. We are examining how these major immunoglobulin classes of murine cell-surface dual functions are carried out by comparing the detailed antibodies. structure of a secreted IgM myeloma heavy(µ} chain with Reference: the 78 membrane-bound l.1 chain from a nonsecreting B 8zenberg, A., Marchalonis, J. J. and Warner, N. L. (1977) lymphocyte cell line. Proc. Nat. Acad. Sci. USA 74: 2113-2117. The complete structure and peptide fragmentation patterns of the mouse myeloma µ chain MOPC-104E have been elucidated (see Biology 1978, No. 57). We are purifying the radiolabeled membrane-bound l.1 chains from the nonsecretor mouse cell line Wehi 279. These membrane-associated µ chains will be cleaved by cyano­ gen bromide and the fragments sequenced at their ___ N-termini. By this direct comparison, we hope to identify 49

60. ANALYSIS OF THE DIVERSITY OF MOUSE 62. DIRECT SEQUENCE S'roDIES OF MOUSE TRANSPLANTATION ANTIGENS TRANSPLANTATION ANTIGENS Investigator: Florence Lafay Investigators: Henry V. Huaqr, Miehael. W. Hunkllpiller Within the major histocompatibility complex of the With the dramatic improvement in sequenator capa­ mouse (H-2 region), two loci (Kand D) code for molecules bilities (see Biology 1978, No. 67) it is now possible to primarily responsible for transplantation reactions. To perform sequence studies on much smaller amounts of understand the evolution and the polymorphism of these transplantation antigens. This obviates the cost and the loci, the structural characterization of several allelic limitations of microsequencing using radioactive pre­ forms of K and D molecules has been undertaken. The cursors, and involves relatively modest nurnbers of cells. partial N-terminal sequence analysis of several K and D We are at present screening different cell lines for ease of molecules has led to several observations (e.g., species­ growth, for quantities of transplantation antigens pro­ associated residues, complex allotypes, and no K- or duced, and for ease of separation of the products of the K D-specific residues) that raise provocative genetic and and D loci. Preparative techniques are being developed in evolutionary questions about the genes encoding these parallel to permit rapid isolation of pure transplantation molecules. This project, a comparative tryptic peptide antigens from the K and D loci. The purified antigens will map analysis of several K and D molecules, is aimed at then be used for direct sequence studies. determining whether the above observations are true of the entire transplantation antigen. For example, are 63. BIOCHEMICAL ANALYSIS OF MUTANT H-2 there some peptides characteristic of the K or D ANTIGENS Investigator: Sandra J. Ewald molecules? Are there some peptides shared by both K and D molecules? For this purpose tryptic maps in two Histocompatibility mutations have been detected in dimensions are being used. The first dimension is a number of mouse strains. Some of these mutations have performed by ion exchange chromatography. Certain been mapped to the major histocompatibility complex (the peptide peaks are subsequently analyzed by high pressure H-2 region). The H-2 region contains two genes, K and D, liquid chromatography. which code for the two major transplantation antigens of This project is being carried out with Dr. J. A. the mouse. These antigens have been shown to be Frelinger at the Department of Microbiology, The Uni­ glycoproteins of 45,000 daltons, which by N-terminal se­ versity of Southern California, who is providing the quencing and peptide analysis are homologous but differ alloantisera and strains of mice necessary for the study. by multiple amino acid residues. I have been comparing biochemically the K region 61. SITE OF POLYMORPHISM IN MOUSE H-2 products of two H-2 mutant strains to their wild-type TRANSPLANTATION ANTIGENS counterparts. The molecules are first labeled by incubat­ Investigator: Henry V. H~ 3 ing spleen cells of mutant or wild-type origin with H­ The major transplantation antigens in mammals are arginine or lysine. The cells are then solubilized, and the extremely polymorphic. For example, in inbred strains of H-2K molecules isolated by precipitating them with mice at least 21 different alleles at two loci have been antisera specific for H-2K, followed by a Staphylococcus found by serological techniques. We are interested in aureus preparation. After elution of antigen from the determining the location of this variability in the trans­ Staphylococcus aureus, the H-2K molecules are separated plantation antigens. Are there variable and constant from other components by electrophoresis on 10% poly­ regions analogous to those in immunoglobulins? Alterna­ acrylamide gels. Tryptic peptides of the molecules are tively, is the variability distributed throughout the mole­ prepared and separated on an ion exchange column and cule? Our approach is to use high resolution peptide compared with similar preparations from wild-type mole­ mapping techniques to compare different alleles cleaved cules. Thus far, I have shown that one H-2K mutant at various residues. Besides providing information on the differs from its parent by one tryptic peptide, and another site(s) of variability in the transplantation molecules these mutant differs from its parent by two. These studies may studies will be informative for the generation and purifi­ illuminate the relationship between structural features of cation of peptides for more detailed sequence studies. the H-2K molecules and their serological and functional properties. 50

These studies are carried out in collaboration with 65, CHEMICAL CHARACTERIZATION OF la Dr. Jan Klein, Department of Immunogenetics, Max ANTIGENS OF mE MOUSE MAJOR HISTOCOMPATIIIILlTY COMPLEX (MHC) Planck Institute for Biology, Tubingen, Germany. Investigators: J. Michael Cecka, Minnie McMillan

64. la ON CELLS IN THE EPIDERMIS We have used highly specific alloantisera to isolate Investigator: John Frelinger Ia Omrnune response region associated) antigens from The I region of the major histocompatibility complex lymphoid cells of mice. The Ia antigens consist of two of the mouse is involved in regulating the immune noncovalently associated polypeptides with apparent mo­ response to antigens, graft versus host reactivity, mixed lecular weights ranging from 31,000-35,000 (ex) and lymphocyte reactivity, T-B collaboration, and graft rejec­ 27,000-29,000 (6) de(lYale University, New Haven, Connecticut. 51

66. CHEMICAL CHARACTERIZATION OF Ia as addition of a series of filters to remove trace ANTIGENS FROM MURINE TUMOR CELUI impurities in the argon used to provide an inert reaction Investigator: Thomas H. Stanton atmosphere. It will also be compatible with chemistry The murine immune response associated antigens (Ia) used for automated conversion of the thiazolinones re­ are encoded in the central portion of the major histo­ leased during the Edman degradation to amino acid compatibility complex. These antigens are found on methylamides and with gas phase Edman chemistry (Biol­ lymphocytes of both B and T type, macrophages, and on ogy 1977, No. 54). epidermal cells. In some cases, Ia antigens have been We are also making further refinements in the implicated as products of immune response genes, and analytical phase of the sequencing process. These include they are either associated with or constitute antigen­ development of gradient systems for use with HPLC specific and antigen-nonspecific helper and suppressor columns having nitrile functionalities and the evaluation factors. It is still an open question as to whether or not of a flow-through liquid scintillation counter for continu­ the Ia molecules expressed on different cell types and on ous monitoring of radioactivity in the column effluent. different clones of the same cell type are identical. The Reference: Ia molecules on spleen cells and epidermal cells appear to Hunkapiller, M. W. and Hood, L. E. (1978) Biochemistry 17: 2124-2133. be identical (see Biology 1978, No. 64). In addition, sequence analysis of Ia from spleen cells suggests that for each Ia subregion (A or E) only one molecule is present. In order to further investigate whether different PUBLICATIONS clones of B cells express different Ia molecules, we are Barstad, P., Hubert, J., Hunkapiller, M., Goetze, A., investigating la molecules found on tumor cells. The cell Schilling, J., Black, B., Eaton, B., Richards, J., Weigert, M. and Hood, L. (1978) Immunoglobulins line 2PK3 derived from BALB/c (H-2d) expresses Ia with hapten-binding activity: Structure-function molecules from two subregions A and E. The expression correlations and genetic implications. Eur. J. Immunol. In press. of the A subregion molecules by the criteria of incorpora­ Bell, J. R., Hunkapiller, M. w., Hood, L. E. and Strauss, J. 3 tion of H-amino acids is much less than the expression of H. (1978) Amino-terminal sequence analysis of the structural proteins of Sindbis virus. Proc. Nat. E subregion molecules. We are further analyzing the 1-E Acad. Sci. USA 75: 2722-2726. molecules by determining the amino acid sequence of Blankenhorn, E. P ., Cecka, J. M., Goetze, D. and Hood, L. (1978) The partial N-terminal amino acid sequence their N-terminal region, and by the comparative analysis of rat transplantation antigens. Nature 27 4: 90-92. of tryptic peptides. Cecka, M., McMillan, M., Murphy, D., McDevitt, H. O. and Hood, L. (1978) Partial N-terminal sequence anal­ yses of Ia molecules e'ifoded by tfe I-A subregion 67. DEVELOPMENT OF AUTOMATED PROTEIN from mice of the H-2 and H-2 haplotypes. J. MICROSEQUENCING TECHNIQU~ Immunol. Submitted for publication. Cecka, M., McMillan, M., Murphy, D., Silver, J., Investigator: Michael W. Hunkapiller McDevitt, H. and Hood, L. (1978) Partial amino acid sequence analyses of Ia molecules. In: Ir Genes and Improvements in automated sequencing techniques la Antigens, H. 0. McDevitt (Ed.), p. 275, Academic have allowed primary structure determination of proteins Press, Inc., New York. Frelinger, J. G., Wettstein, P. J., Frelinger, J. A. and and polypeptides at the subnanomole and nanomole level, Hood, L. (1978) Epidermal la molecules from the I-A respectively (see Biology 1977, No. 75, and Hunkapi!ler and I-EC subregions of the mouse H-2 complex. Immunogenetics 6: 125-135. and Hood, 1978). These improvements have included Hood, L. (1978) Antibody genes and antibody molecules. modifications of commercial sequenators, the use of Clin. Chem. In press. Hood, L., Huang, H. and Dreyer, W. J. (1977) The area­ Polybrene as a carrier to prevent protein washout from code hypothesis: The immune system provides clues the reaction· cup, and the development of rapid and to understanding the genetic and molecular basis of cell recognition during development. J. Supramolec. sensitive analytical techniques based upon high pressure Struct. 7: 407-435 (Review). liquid chromatography (HPLC). Hood, L., Kronenberg, M., Early, P. and Johnson, N. (1977) Nucleic acid chemistry and the antibody problem. We have completed construction of a new spinning In: Immune System: Genetics and Regulation, E. E. cup sequenator specifically designed for microsequencing Sercarz, L. A. Herzenberg and C. F. Fox (Eds.), pp. 1-27, Academic Press. applications. It includes refinements in the vacuum Hood, L., Weissman, I. and Wood, W. B. (1978) Immunol­ system, reagent delivery system, and programmer as well ogy, W. A. Benjamin, Inc., Menlo Park, California. 52

Hunkapiller, M. and Hood, L. (1978) Direct microsequence McMillan, M., Cecka, J. M., Murphy, D. B., McDevitt, H. analysis of polypeptides using an improved sequen­ o. and Hood, L. (1978) Partial amino aejd sequences ator, a nonprotein carrier (Polybrene), and high of murine Ia antigens of the I-EC subregion. pressure liquid chromatography. Biochemistry 11: Immunogenetics 6: 137-147. 2124-2133. Regier, J., Kafatos, F. C., Goodfliesh, R. and Hood, L. Loh, E., Black, B., Riblet, R., Weigert, M., Hood, J. M. (1978) Silkmoth chorion proteins: Sequence analysis and Hood, L. (1978) Comparisons of myeloma pro­ of the products of a multigene family. Proc. Nat. teins from NZB and BALB/c mice: Structural and Acad. Sci. USA 75: 390-394. functional differences of light chains. Proc. Nat. Weigert, M., Gatmaitan, G., Loh, E., Schilling, J. and Acad. Sci. USA. In press. Hood, L. (1978) Rearrangement of genetic informa­ Loh, E., Hood, J. M., Riblet, R., Weigert, M. and Hood, L. tion may produce immunoglobulin diversity. Nature. (1978) Comparisons of myeloma proteins from NZB Submitted for publication. and BALB/c mice: Structural and functional dif­ Weiner, S., Lowenstam, H. A. and Hood, L. (1977) Discrete ferences of heavy chains. J. Immunol. In press. molecular weight components of mollusk shell or­ Maizels, R. M., Frelinger, J. A. and Hood, L. (1978) ganic matrices. J. Exptl. Marine Biol. Ecol. 30: Partial amino acid sequences of mouse transplanta­ 45-51. tion antigens. Immunogenetics. Submitted for Weissman, I., Hood, L. and Wood, W. B. (1978) Essential publication. Concepts in Immunology, W. A. Benjamin, Inc., McMillan, M., Cecka, J. M., Murphy, D. B., McDevitt, H. Menlo Park, California. 0. and Hood, L. (1977) Structure of murine Ia antigens: Partial N-terminal amino acid sequences of products of the 1-E/l-C subregion. Proc. Nat. Acad. Sci. USA 74: 5135-5139.

Professor: Norman H. Horowitz water requirements. Curiously enough, iron chemistry has Gosney Visiting Associate: Howard Gest Visiting Associate: Norma Williams turned out to be a central aspect of this investigation as Research Staff: Paul K. Cartier Ill, Gisela Charlang, well. In this case, it is the transport of iron across the George L. Hobby, George Horn plasma membrane that is most strikingly affected by Support: The work described in the following research reduction of the ambient water activity. Recent results reports has been supported by the National Aeronautics and Space Administration. of our studies of this relationship are summarized below.

Summary: We report this year some results of attempts 68. LABORATORY SIMULATIONS OF VIKING to reproduce in the laboratory the weak positive results RESULTS obtained in the Carbon Assimilation experiments per­ Investigators: George L. Hobby, Paul K. Certier Ill, formed on Mars during the Viking mission. The Martian Norman H. Horowitz environment is much better understood now than it was The Carbon Assimilation Experiment detected a before the Viking mission. With respect to these very small but significant fixation of atmospheric carbon experiments, the most important information relates to (co2 or CO)--presumably into organic matter--in the the elemental composition of the Martian surface mate­ surface material of Mars during the Viking mission rial. We know now that it contains about 13% of iron, and (Horowitz et al., 1977). Since the fixation was quite this knowledge has guided our choice of materials for the thermostable, a biological source was excluded. In order simulation experiments. Much of importance is still to verify this conclusion and, if possible, to understand the unknown, of course, and this includes the mineralogy of nature of the reaction that took place, we are performing the surface material and the nature of the oxidant whose simulations of the Martian experiment using a laboratory presence in the surface was detected by the Viking breadboard of the Viking instrument. instruments. Without this additional information, it is The main finding to date is that the basic Martian doubtful if a complete simulation of the Martian surface results have been reproduced by using iron-rich minerals chemistry will ever be attainable. The results reported to simulate the Martian surface material. The following here nevertheless suggest that the major features of the have shown activity of the right magnitude under differ­ Carbon Assimilation data can be reproduced by the use of ent conditions: hematite, oc-Fe ; maghemite, y-Fe ; 2o 3 2o3 iron-containing minerals to represent the Martian surface. and magnetite, Fe3o4• Hematite is inactive by itself, but The Neurospora experiments reported here had their becomes active in the ~esence of water vapor. origin in the Mars project as an investigation of microbial Maghemite is also inactive by itself, but becomes active if 53 pretreated with ammonia gas. Magnetite is active by of slime and causes a tighter binding of ferricrocin. This itself; addition of water vapor increases the activity. possibility is being investigated. All of these minerals are plausible for Mars. Meghemite is the best candidate for the magnetic material detected on the surface of Mars by the Viking 70. EXPERIMENTS WITH A SIDEROPHORE-DEFICIENT MUTANT OF NEUROSPORA instruments (Hargraves et al., 1977). Ammonia was a Investigators: Gisela Charlang, Norman H. Horowitz major_ exhaust product of the hydrazine-fueled retro­ engines of the Viking landers. The tests are continuing. The siderophores of Neurospora crassa and other fungi are trihydroxamates derived from ornithine. If the References: synthesis of ornithine were blocked, no siderophores would Hargraves, R. B., Collinson, D. W., Arvidson, R. E. and be produced, but since there are several sources of Spitzer, C. R. (1977) J. Geophys. Res. 82: 4547-4558. ornithine in Neurospora, no single-gene mutant is orni­ Horowitz, N. H., Hobby, G. L. and Hubbard, J. S. (1977) J. thine-deficient. A triple· mutant blocked in the known Geophys. Res. 82: 4659-4662. ornithine-producing reactions has been constructed by Prof. Rowland Davis (University of California, Irvine), 69. SIDEROPHORE BINDING TO NEUROSPORA who kindly provided it to us. This strain is deficient in CYTOPLASMIC MEMBRANES arginase, ornithine transaminase, and N-acetyl-ornithine Investigators: Gisela Charlang, Norman H. Horowitz transaminase. Neurospora crassa produces two principal iron­ The triple mutant grows slowly without ornithin~ if transport peptides (siderophores): coprogen, which is arginine and putrescine are supplied. This implies either secreted into the medium of growing cultures, and that the iron-transport function of siderophores is taken ferricrocin, which is retained in the cells. Considerable over by another constituent of the medium, or that some evidence indicates that a fraction of the total ferricrocin growth can occur without iron. We have found that the of conidia is located in the plasma membrane, where it mutant does not grow without ornithine in media in which functions in iron transport. This ferricrocin is released glycerol, rather than sucrose or glucose, is the carbon from the membrane in media of low water activity (aw). source. Since sugars are known to form soluble complexes We are examining plasma membrane preparations in with Fe(Ill) (Spiro and Saltman, 1969), this result could order to understand the molecular mechanisms that mean that some Fe-transport is occurring by means of underlie (1) the release of ferricrocin at low aw and (2) these complexes, or alternatively, that sugars, but not the physical separation of the two major siderophores. glycerol, can yield some carbon and energy by Fe-free Plasma membranes prepared from a cell wall-less mutant metabolism. This question remains to be answered. (slime) have been incubated with isotopically labeled Besides ornithine, siderophores or ascorbic acid ferricrocin or coprogen. All of the preparations bind support growth of the triple mutant in glycerol media. ferricrocin, and some have also bound coprogen. Bound Ascorbate presumably functions by reducing ferric iron to ferricrocin is released from the membranes when aw is ferrous. The latter is quite soluble and, evidently, is lowered to 0.9 by addition of glycerol to the buffer, but readily transported into the cells. The triple mutant can not if NaCl is used to lower aw. Under some conditions, be grown through an indefinite number of vegetative use of NaCl results in an increase of ferricrocin binding. generations on ascorbate. Assays on conidial extracts of The responses of these membranes thus differ from those such cultures have shown that about 596 of the wild-type observed with conidia in vivo, where loss of ferricrocin level of siderophores is present in conidia after four occtn's at low aw regardless of whether NaCl or glycerol is transfers on glycerol-ascorbate medium. A trace quantity used to effect the lowering. of coprogen has been identified in the medium of the The above results suggest that the active uptake triple mutant growing in the presence of glucose without mechanism for siderophores that is detectable in growing ornithine. These results indicate either that the mutant is cultures, but not in freshly shed conidia, influences the slightly leaky or that a minor, undetected, metabolic responses of membranes prepared from growing cultures source of ornithine exists. Despite the fact that the 54

mutant is not quite free of siderophores, the conclusion nidulans and P. chrysogenum, although the identification seems justified that siderophores have no essential func­ of ferrirhodin is not complete at this writing. Cell tion that cannot be replaced by other means of iron extracts, however, contain ferricrocin only (no ferri­ transport. rhodin) in the case of Aspergillus and ferrichrome (tenta­ tive identification) in the case of Penicillium. Reference: Spiro, T. G. and Saltman, P. (1969) Struct. Bonding 6: Like Neurospora, Aspergillus and Penicillium lose 116-156. conidial siderophores in media of low water activity, and the loss delays conidial germination. Aspergillus loses 71. SIDEROPHORJ!S OF ASPERGILLUS AND PENICILLIUM ferricrocin, and Penicillium loses ferrichrome. Both these lnvestigators: Norman H. Horowitz, Gisela Charlang, fungi are more xerotolerant than Neurospora, and their George Hom loss of siderophores occurs at correspondingly lower water Studies in our laboratory, still incomplete, of Asper­ activities. gillus nidulans and Penicillium chrysogenum indicate that These findings show that iron transport in fungi is more complex than had been previously supposed. these species resemble Neurospora crassa in that they produce intracellular siderophores that are different from References: those secreted into the medium. Fifteen years ago, Horowitz, N. H., Charlang, G., Horn, G. and Williams, N. P. (1976) J. Bacteriol. 127: 135-140. Zahner et al. (1963) reported finding ferricrocin and Zahner, H., Keller-Schierlein, W., Htitter, R., Hess­ Leisinger, K. and Deer, A. (1963) Arch. Mikrobiol. ferrirhodin in the medium of A. nidulans grown under iron­ 45: 119-135. deficient conditions; they found coprogen in Fe-deficient media of P. chrysogenum. Cell extracts were not tested. PUBLICATIONS In view of our recent finding of different siderophores in Charlang, G. and Williams, N. P. (1977) Germination­ cell extracts and medium, respectively, of N. crassa defective mutant of Neurospora crassa that responds (Horowitz et al., 1976), we are examining the siderophore to siderophores. J. Bacteriol. 132: 1042-1044. Horowitz, N. H. (1977) The search for life on Mars. production of Aspergillus and Penicillium. We have Scientific American 237: 52-61. confirmed the findings of Zahner et al. (1963) with respect Horowitz, N. H., Hobby, G. L. and Hubbard, J. S. (1977) Viking- on Mars: The carbon assimilation experi­ to the siderophores secreted into the medium by A. ments. J. Geophys. Res. 82: 4659-5662.

Summary: Our primary interest is in the molecular basis Associate Professors Tom Maniatis Visiting Scientist: Richard Axel of differential gene activity in eukaryotes. Our immedi­ Gosney Research Fellow: Richard M. Lawn Research Fellows: Eugene T. Butler III, Edward F. ate goal is to isolate and characterize segments of Fritsch, Ross C. Hardison, James Shen eukaryotic chromosomal DNA which carry genes that are Graduate Students: David A. Goldberg, Elizabeth H. Lacy, Joyce E. Lauer, Richard C. Parker, Mavis expressed during particular stages of development. To Shure, Gek Kee Sim accomplish this goal we are using a combination of Research Staff: Catherine D. O'Connell, Diana H. Quon Laboratory Staff: Adriana Cortenbach, Maria A. De Bruyn biochemical, genetic, and recombinant DNA techniques. The availability of well-defined DNA fragments provides Support: The work described in the following research reports has been supported by: the opportunity for studying the structure and function of Rita Allen Foundation individual transcriptional units which comprise different American Cancer Society Jane Coffin Childs Memorial Fund for developmental pathways. Questions such as the precise Biological Research location of transcription initiation and termination sites Gosney Fund National lnstitutes of Health, USPHS with respect to structural gene sequences, the timing of National Science Foundation synthesis and turnover of specific gene transcripts, and Northwest Area Foundation President's Venture Fund the chromosomal organization of coordinately or sequen­ Damon Runyon-Walter Winchell Cancer Fund tially expressed genes are being addressed. 55

72. CONSTRUCTION OF LIBRARIES OF EUKARYOTIC either by shearing followed by 81 nuclease treatment or DNA by a nonlimit endonuclease digestion with restriction Investigators: Joyce E. Lauer (Drosophila); Gek Kee Sim* (Silkmoth); Ross c. Hardison, Elizabeth H. enzymes generating molecules with blunt ends. Molecules Lacy, Catherine D. O'Connell, Diana H. of approximately 20 kb are selected by preparative su­ Quon, Argiris Efstratiadis** (Rabbit); Edward F. Fritsch, Richard M. Lawn, crose gradient centrifugation and rendered EcoRl-resis­ Catherine D. O'Connell (Human) tant by treatment with EcoRI methylase. Synthetic We and other laboratories at Caltech have estab­ decamer DNA linkers bearing EcoRI recognition sites are lished procedures for eukaryotic gene isolation which covalently attached to the eukaryotic DNA by blunt-end involve the construction and screening of cloned libraries ligation using T4 ligase; cohesive ends are then generated of genomic DNA. The procedure we used is illustrated in by digestion with EcoRI. These molecules are covalently Figure 1. High molecular weight DNA is fragmented joined to phage A DNA and the hybrid DNA molecules

RI RI RI > 100 kb Charon 4A(4} High Molecular Weight I I I DNA Eukaryotic DNA l Fragment l Anneal Cohesive Ends i Size Fractionate XXXXXXXXXXX 20kb DNA Q- I Eco RI Methylose + to Block Eco RI Sites l Eco RI c~ CH3 CH3 Ends I Blunt End Ligation to + Synthetic Eco RI Linkers +

Internal ~CH3 CH3 ~ ~ Fragments

l Eco RI Digestion l Size Fractionate CH3 CH3 ~ Eco RI Cohesive Ends 31 kb CH3 CH3

1Cohesive End Ligation ~~~~~~~~>=~~~~~==<(~~~~~~~~~ cH3 cH3 CH3 CH3 i In-vitro Packaging

I AmplificOtion '+ and Screening

Figure 1. Schematic diagram illustrating the strategy used to construct libraries of random eukaryotic DNA fragments. 56

packaged into viable phage particles in vitro. Amplifica­ 14. RESTRICTION MAPPING AND NUCLl!OTIDE tion of these phage produces a permanent library of SEQUENCE ANALYSIS OF CLONED 11-GLOBIN GENES eukaryotic DNA sequences. These libraries have been Investigators: Elizabeth H. Lacy, Argirls Efstratiadis*, successfully screened for a number of different single James Shen, Diana H. Quon copy structural genes. Nucleotide sequence analysis of the adult a-globin References: gene in the rabbit a-globin clone designated RaG-1 has Blattner, F. R., Williams, B. G., Blechl, A. E., Thompson, K. D., Faber, H. E., Furlong, L. A., Grunwald, D. J., shown that the large (approximately 585 bp) intervening Kiefer, D. 0., Moore, D. D., Schumm, J. W., sequence reported by Jeffreys and Flavell (1977) is Sheldon, E. L. and Smithies, 0. (1977) Science 196: 161-169. located between codons for amino acids 104 and 105, Maniatis, T ., Hardison, R., Lacy, E., Lauer, J., O'Connell, exactly as is found in the mouse a-globin gene (Tilghman C., Quon, P., Sim, G. K. and Efstratiadis, A. (1978) et 1978). A second intervening sequence (approxi­ Cell. In press. al., Sternberg, N., Tiemeier, D. and Enquist, L. (1977) Gene 1: mately 125 bp) has been located between the codons for 255-280. amino acids 30 and 31. Using cDNA clones for embryonic *Present address: Department of Cellular and Develop­ and a-minor globin messages as hybridization probes, we mental Biology, Harvard University Biological Labora­ plan to determine whether the a-related genes in RaG-2 tories, Cambridge, Massachusetts. ~·*Department of Biological Chemistry, Harvard Univer­ and RaG-5 (see Biology 1978, No. 73) also have inter­ sity Medical School, Boston, Massachusetts. vening sequences. Further fine structure restriction mapping and blot­ 73. CHARACTERIZATION OF LINKED 11-GLOBIN ting experiments done on the a-related genes should GENES IN RABBIT permit the isolation of gene-specific restriction fragments Investigators: Elizabeth H. Lacy, Eugene T. Butler Ill, to use as hybridization probes to follow the expression of James Shen, Diana H. Quon a-globin genes during erythropoiesis. Four independent a-glob in genomic DNA clones References: were isolated from a screen of 750,000 recombinant Jeffreys, A. J. and Flavell, R. M. (1977) Cell 12: phage. Data from restriction enzyme analyses and 1097-1108. Tilghman, S. M., Tiemeier, D. C., Seidman, J. G., Peterlin, blotting experiments revealed that two clones (designated B. M., Sullivan, M., Maize!, J. and Leder, P. (1978) RaG-2 and RaG-5) contain a-globin-related sequences Proc. Nat. Acad. Sci. USA 75: 725-729. linked to the adult a-globin gene. Restriction enzyme *Department of Biological Chemistry, Harvard University mapping of RaG-2 places the a-related gene in this clone Medical School, Boston, Massachusetts. 9.1 kb to the 5' side of the adult gene. The direction of transcription of this a-related gene relative to that of the adult a-globin gene has not yet been determined. To 75. OTHER RABBIT GI.OBIN GENES decide whether the linked genes are embryonic S-globin Investigators: Ross C. Hardison, Diana H. Quon and/Or adult B-minor globin genes, we are preparing In addition to the four clones containing a-globin cDNA clones from mRNA isolated from the blood islands gene(s), five other clones containing globin genes were of 12-day rabbit fetuses and from the bone marrow of isolated from a screen of 750,000 recombinant phage that anemic adult rabbits. Once the a-related genes are comprise the rabbit library. Data from restriction identified, it will then be possible to establish the order of enzyme analyses indicate that each of these clones linkage of the genes in the rabbit a-globin family. contains two regions that hybridize to globin probes; these Furthermore, it should also be possible to determine regions are separated by about 8 kb of DNA. We are whether there are more linked a-like genes by using currently trying to identify these genes and study their terminal fragments from the four a-clones to rescreen sequence arrangement using cloned cDNA hybridization the library. probes corresponding to embryonic and adult rabbit globin mRNAs. Reference: Lacy, E., Lawn, R. M., Fritsch, E., Hardison, R., Parker, R. C. and Maniatis, T. (1978) In: Cellular and Molecular Regulation of Hemoglobin Switching, G. Stamatoyannopoulos and A. Nienhuis (Eds.), Grune and Stratton, New York. 57

76. ISOLATION AND CHARACTERIZATION OF 77. Y CHROMOSOME GENES OF DROSOPHILA HUMAN GLOBIN GENES Investigator: Joyce E. Lauer Investigators: Edward F. Fritsch, Richard M. Lawn, Catherine D. O'Connell, Richard C. Genetic and cytological characterization of the Y Parker, Geoffrey Blake* chromosome of Drosophila has revealed a set of seven The human globin gene system presents the possi­ 11 fertility factor" genes which are necessary for normal bility of using mutants to study the relationships between sperm maturation and thus for male fertility. These genes the structure and organization of globin genes and the are expressed only in primary spermatocytes (where the Y mechanisms of their differential expression during devel­ chromosome develops structures analogous to the lamp­ opment. To study human globin genes a library of cloned brush loops of amphibian oocytes) on which are visible human DNA fragments has been constructed in a bacterio­ transcripts of about 30,000 nucleotide length. We are in phage >. vector (Maniatis et al., 1978). Two recombinant the process of isolating chromosomal DNA clones contain­ clones containing linked o- and S-globin genes have been ing these genes by screening a Drosophila library with a isolated from the library using the rapid screening probe derived from testes RNA. Once Y-specific clones technique of Benton and Davis (1977). These clones have are identified, we will map the positions of messenger been characterized by mapping restriction endonuclease RNA and of total nuclear transcripts. By correlating sites, and by hybridization of radioactive copies of structural aberrations in the DNA Of fertility factor 8-globin mRN A to these restriction enzyme fragments mutants with defects in the molecular biology of spermio­ following agarose gel electrophoresis and transfer to genesis, we hope to identify: (1) particular structural nitrocellulose filters (Southern, 1975). The results indi­ features shared by the seven genes which might be related cate that each of the cloned human DNA fragments to their coordinate expression; (2) the relationship of very contains two linked globin genes (o and S) separated by large nuclear transcripts to mRNA; and (3) regions 5500 base pairs of intervening DNA. In addition, both S important for control of transcription. and 0 genes were shown to possess at least one internal Reference: noncoding sequence interruption of approximately 900 Williamson, J. H. (1976) Jn: The Genetics and Biology of Drosophila, M. Ashburner and N. Novitsky (Eds.), base pairs. Vol. 16, p. 667, Academic Press, New York. The structure of the globin genes located in the recombinant bacteriophage is consistent with results that 78. THE ALCOHOL DEHYDROGENASE GENE OF we and others have obtained by restriction endonuclease DROSOPHILA MELANOGASTER digestion and filter hybridization analysis of wild-type and Investigator: David A. Goldberg mutant human DNA. We will continue the study of the We are studying the alcohol dehydrogenase (Adh) globin gene family by further screening of the human DNA gene-enzyme system of Drosophila. Adh is an abundant library for a- and y-globin sequences. We hope to protein comprising 1 to 3% of the total soluble protein of construct a complete linkage map for the y-, 0-, and the adult fly. The enzyme is small and well character­ a-globin genes, which are known to be closely linked by ized. The complete amino acid sequence of 214 residues genetic evidence. We will also use fragments of cloned (24,000 molecular weight) has been determined, as well as genomic DNA to probe the DNA sequence organization of the amino acid replacements in a number of variants. Adh the human genome within and surrounding the globin is among the best genetically characterized enzyme genes. DNA from patients with hereditary diseases of systems in Drosophila. The Adh locus has been mapped to hemoglobin synthesis (thalassemias) will be a source of within two bands (35B2-3) on the second chromosome. mutant genetic material to compare with results obtained Flanking visible and lethal loci have been identified and from studies of normal DNA. their positions determined using deletion mapping of References: induced mutations. Adh from a variety of strains may be Benton, W. D. and Davis, R. W. (1977) Science 196: 180-182. distinguished by electrop~oretic mobilities of the enzyme Maniatis, T., Hardison, R., Lacy, E., Lauer, J., O'Connell, on starch gels. A putative control variant that produces C., Quon, D., Sim, G. K. and Efstratiadis, A. (1978) Cell In press. low levels of Adh protein has been identified. The Southern, E. M. (1975) J. Mo!. Biol. 98: 503-517. isolation of individual flies that express or lack Adh *Undergraduate, California Institute of Technology. activity is possible because both positive and negative 58 selections have been devised. Maniatis, T. and Efstratiadis, A. (1978) Fractionation of low molecular weight DNA or RNA in polyacryl­ Adh activity varies during development and displays amide gels containing 98% formamide or 7 M urea. tissue specificity. Adh activity is first detected during In: Methods in Enzymology, Nucleic Acids and the first larval lnstar, increases throughout larval devel­ Protein Synthesis, Part H, K. Moldave and L. Grossman (Eds.), Academic Press. In press. opment, and gradually declines during the pupal stage. Maniatis, T., Hardison, R. C., Lacy, E., Lauer, J., O'Connell, C., Quon, D., Sim, G. K. and Efstratiadis, Adh activity rises just prior to adult emergence and peaks A. (1978) The isolation of structural genes from 5 to 10 days later. libraries of eukaryotic DNA. Cell. In press. Maniatis, T., Sim, G. K., Kafatos, F. C., Villa Komaroff, We hope to identify the molecular elements that L. and Efstratiadis, A. (1977) An approach to the determine the developmental regulation of Adh by com­ study of developmentally regulated genes. In: Molecular Cloning by Recombinant DNA, W. A. bining both recombinant DNA and classical genetic tech­ Scott and R. Warner (Eds.), pp. 173-203, Academic niques. Press. Parker, R. C. and Seed, B. (1978) Two-dimensional agarose References: gel electrophoresis, "sea plaque" agarose dimension. O'Connell, J., Mandel, H., Krauss, M. and Sofer, w. (1977) In: Methods in Enzymology, Nucleic Acids and Genetics 86: 533. Protein Synthesis, Part H, K. Moldave and L. Thompson, J., Ashburner, M. and Woodruff, R. (1977) Grossman (Eds.), Academic Press. In press. Nature 270: 363. Sim, G. K., Efstratiadis, A., Jones, C. W., Kafatos, F. C., Koehler, M., Kronenberg, H. M., Maniatis, T., Regier, J. C., Roberts, B. F. and Rosenthal, N. PUBLICATIONS (1978) Studies on the structure of genes expressed during development. Cold Spring Harbor Symp. Lacy, E., Lawn, R. M., Fritsch, E., Hardison, R. c., Quant. Biol. 42: 933-945. Parker, R. C. and Maniatis, T. (1978) Isolation and characterization of mammalian globin genes. In: Cellular and Molecular Regulation of Hemoglobin Switching, G. Stamatoyannopoulos and A. Nienhuis (Eds.), Grune and Stratton, New York.

Professor: Herschel K. Mitchell ited rather suddenly in the later stages of metamorphosis. Research Associate: Peter H. Lowy Gosney Senior Research Fell.ow: Nancy S. Petersen All of these systems show time-specific on and off Graduate Students: Anne Chomyn, Loveriza S. Lipps, patterns of transcriptional and/or translational ac~ivities. Galina Moller We continue also with investigations on chromo­ support: The work described in the following research somal proteins in relation to gene function in salivary reports has been supported by: Josephine V. Dumke Fund glands and on the chemical structure of cuticle. Recently Gosney Fund increasing attention has been given to studies on the National Institutes of Health, USPHS National Science Foundation effects on Drosophila of chemicals that are teratogenic in Rockefeller Foundation mammals. Thus far such substances tested, which include Gordon Ross Medical Foundation Spencer Foundation Fund colchicine, eserine, caffeine and ethanol, also cause abnormalities in Drosophila although heat shock remains Summary: Studies concerned with the biochemistry of the easiest to control and its effects are more easily the developmental process, particularly with Drosophila, defined than those of various injected teratogens. The continues as the center of attention in our research group. system does appear suitable for further studies along We have made extensive use of the heat shock phenom­ these lines. enon for investigations on the rapid changes that occur in transcriptional and translational activities during the differentiation of several tissues and structures. These 79. HEAT SHOCK PHENOCOPIES IN DROSOPHILA Investigators: Herschel K. Mitchell, Loveriza s. Lipp; include the salivary glands in which transcriptional activ­ ity can be visualized directly in the polytene chromo­ The bristles of the adult fly of Drosophila are somes, epithelial cells which give rise to bristles and hairs sensory structures derived through a complex program of during metamorphosis, the brain as a tissue which survives differentiation of single cells. Many mutations are known metamorphosis, and the flight muscles which are depos- which modify bristle structure and many of these modifi- 59 cations can be mimicked (phenocopied) by application of a In all cases, protein synthesis that is in progress is heat shock to the animals at specific stages of develop­ drastically reduced and development is arrested for the ment before the bristles are formed. We have shown the first 4 hr after heat shock; the maximum labeling of the production of a phenocopy of the mutant hook at 36 hr heat shock proteins (Tissieres et al., 197 4) occurs between (after puparium formation), a second distinct type 4 and 7 hr. During this latter period (4 to 7 hr), the (smooth) at 40 hr, and a third (spear) at 45 hr. Each normal pattern of protein synthesis resumes. Neither phenocopy appeared to result from a single independent recovery of the normal protein synthesis nor regression of event and this has been confirmed by production of double the synthesis of the heat proteins occurs coordinately. In phenocopies such as hook-smooth and hook-spear. The addition, initial suppression of normal protein synthesis doubles result from double heat shocks but only with varies in degree between tissues and within a tissue at appropriate timing to allow for a 4 to 6 hr period of different stages. These variables in developmental se­ suspended development. That is, a heat shock at 36 hr quences may be sufficient to account for phenocopy yields the hook phenocopy.. If this is followed by a second production but no specific components have yet been shock at 41 hr and even a third at 46 hr only the hook identified as being responsible. phenocopy is obtained. However if the second shock is We have also analyzed the effect on protein synthe­ given at 46 hr then the double hook-smooth is obtained. sis of a second heat shock, given either 5 or 10 hr after The suspension of development following a heat the first. In both cases, the effect of the second heat shock is correlated with shutdown of at least 99% of RNA shock is similar to that of the single treatment, but the synthesis and a similar but less drastic shutdown of reduction in total protein synthesis immediately after the protein synthesis. The heat shock-induced proteins and second heat shock is not as severe as the first, and the their mRNAs are produced during the suspended state. It maximum heat shock protein synthesis occurs about 2 hr appears that the specific biochemical events which give after the heat shock. Also, the normal pattern of protein rise to specific phenocopies are involved in the processes synthesis is resumed sooner. of recovery from the heat shock and depend on the Reference: pattern of transcriptional activity in effect at the time of Tissi~res, A., Mitchell, H. K. and Tracy, U. M. (1974) J; stress. Mo!. Biol. 84: 389-398.

References: 81. PURIPICATION OF A HEAT SHOCK PROTEIN Mitchell, H.K. (1966) J. Insect. Physiol. 12: 755-765. Mitchel~ H. K., Chen, P. s., Lipps, L. S. and Moller, G. Investigators: Anne Chomyn, Herschel K. Mitchell (1978) Insect Biochem. 8: 29-35. Mitchell, H. K. and Lipps, L. S. (1978) Cell. In press. The 84,000 dalton molecular weight protein, one of Mitchel~ H. K., Lipps, L. S. and Tracy, U. M. (1977) the eight major heat shock proteins described by TissiE!res Biochem. Genet. 15: 575-587. TissiE!res, A., Mitchell, H. K. and Tracy, U. M. (1974) J. et al. (197 4), has been purified from Drosophila melano­ Mo!. Biol. 84: 389-398. gaster prepupae by ammonium sulfate precipitation, hy­ droxyapatite chromatography, and two-dimensional iso­ 80. HEAT SHOCK AND PROTEIN SYNTHESIS IN DROSOPHILA focusing-SDS electrophoresis. Antibodies against this Investigators: Anne Chomyn, Galina Moller, protein have been produced in rabbits. Herschel K. Mitchell It has been shown that most, if not all, of the heat We have made use of high-resolution gradient gels shock proteins that are produced from a short pulse are for the analysis of changes in patterns of protein synthesis present in the nuclei, probably on the chromosomes, of following a heat shock of Drosophila pupae. Three stages salivary glands after a heat shock (Mitchell and Lipps, of development (34, 53, and 76 hr after puparium forma­ 1975). Experiments are now in progress to determine by tion) and two tissues (brain and thoracic epithelium) have indirect immunofluorescence (Jamrich et al., 1977; Silver been examined in detail. The stress conditions (40.2°C for and Elgin, 1976), whether any specific binding sites exist 40 min) are known to yield stage-specific phenocopies on the polytene chromosomes of salivary glands for the (abstract No. 79). The tissues were dissected from the 84,000 dalton molecular weight protein. The antibodies pupae at intervals following the heat shocks, and im­ are also being used to determine whether this protein 35 mediately pulse labeled (30 min) with s-methionine. occurs normally in larval and prepupal tissues. 60

35 Referenees: and pulse labeling with s-met or 3H-leu. Samples were Jamrich, M., Greenleaf, A. L. and Bautz, E. K. F. (1977) Proc. Nat. Acad. Sci. USA 74: 2079-2083. analyzed by gel electrophoresis of labeled proteins, Mitchell, H. K. and Lipps, L. S. (1975) Biochem. Genet. 13: Coomassie staining, and autoradiography. The first visible 585-602. appearance of flight muscle in quantity takes place at Silver, L. M. and Elgin, S. C. R. (1976) Proc. Nat. Acad. Sci. USA 73: 423-427. about 65 msec after puparium formation. A very rapid Tissieres, A., Mitchell, H. K. and Tracy, U. M. (1974) J. Mo!. Biol. 84: 389-398. linear increase in total amount of protein commences about 7 hr later and reaches the maximum (more than 82. MAPPING OF THE CODING REGION FOR TWO 3-fold increase) at 92 hr. This increase of total amount of HEAT SHOCK PR

LD of about 50 mg/kg for Drosophila larvae and at near References: 50 Anderson, s. 0. and Barrett, F. M. (1971) J. Insect Physiol. lethal levels it induces puffing in salivary gland chromo­ 17: 69-83. Driskell, W. J. (1974) Ph.D. Thesis, California Institute of somes at some of the same positions that puff by heat Technology, Pasadena, California. shock (especially regions 87A, 87BC, and 93D). Further­ Kimura, s., Strout, H. v. and Lipke, H. (1975) Insect Biochem. 6: 65-77. more, the treatment induces synthesis of the proteins that have been shown to be coded for at the heat shock puff 85. TERATOGENS AND PHENOCOPIES IN sites (TissiE:res et al., 1974; Ashburner and Bonner, 1978). DROSOPHILA Investigator: Herschel K. Mitchell References: Ashburner, M. and Bonner, J. J. (1978) Cell. In press. Heat shock is one of the many stress conditions that Cockroft, D. C. and New, A. T. (1978) Teratology 17: 277-284. induces developmental abnormalities when applied during Shepard, T. H. (1973) Catalog of Teratogenic Agents, 209 embryogenesis in mammals and birds (for example see pp., Johns Hopkins University Press, Baltimore and London. Cockroft and New, 1978). The same is true for Drosophila Tissieres, A., Mitchell, H. K. and Tracy, U. M. (1974) J. (see abstract No. 79) but in addition one can observe in Mo!. Biol. 84: 389-398. the fly that heat shock induces a specific pattern of puffs on salivary gland chromosomes and the corresponding PUBLICATIONS specific patterns of transcriptional and translational activities. Thus, the current investigation is addressed to Chen, P. S. and Mitchell, H. K. (1978) Tyrosine-0-glu­ coside from Drosophila. In press. the question of whether studies of phenocopy production Mitchell, H. K. (1978) Ernst Hadom. Am. Rev. Genet. 12: in Drosophila may be used as a model for investigating 1-3. Mitchell, H. K., Chen, P. S., Lipps, L. S. and Moller, G. mechanisms of teratogenesis in general. (1978) Separation of Drosophila RNAs on acrylamide Besides heat shock a number of unrelated chemicals gels in formamide. Insect Biochem. 8: 29-35. Mitchell, H. K. and Lipps. L. S. (1978) Heat shock and are known to induce puffs in salivary gland chromosomes phenocopy induction in Drosophila. Cell. In press. of Drosophila. These include salicylate, azide, amytal, Mitchell, H. K., Lipps, L. S. and Tracy, U. M. (1977) Transcriptional changes in prepupal hypoderm in rotenone, and arsenite (Ashburner and Bonner, 1978} and Drosophila melanogaster. Biochem. Genet. 15: all of these substances are teratogens (Shepard, 1973). 575-587. Mitchell, H. K., Tracy, U. M. and Lipps, L. S. (1977) The Our recent explorations have added another known prepupal salivary glands of Drosophila melanogaster. teratogen, colchicine, to this list. This substance has an Biochem. Genet. 15: 563-573.

and glycosylated by the host cell's normal membrane Associate Professor: James H. Strauss Jr. Senior Research Fellow: Ellen G. Strauss protein synthesizing system, eventually appearing in the Graduate Students: John R. Bell, Teryl K. Frey, Jeffrey host cell plasmalemma. The third protein in the virus, the T. Mayne, Jing-hsiung James Ou, Vann P. Parker, Charles M. Rice III capsid protein, is associated with the viral RNA in a Research Staff: Edith M. Lenches, Mary S. Martin nucleocapsid. The binding between a preformed nucleo­ Laboratory Staff: Jeannette Johnstone, Caroline Vermaes capsid and those regions of the glycoproteins exposed to Support: The work described in the following research the cytoplasmic face of the plasmalemma is responsible reports has been supported by: Earle C. Anthony Fellowship for the final maturation _of the virus particles. This The Merck Company Foundation binding results in the nucleocapsid budding through the National Institutes of Health, USPHS National Science Foundation modified host cell plasmalemma, thereby acquiring its Research Corporation envelope of El and E2 and lipid during its release from the Summary: Sindbis virus is an enveloped virus with a cell. Thus in addition to our interest in the structure and particularly simple protein composition. The two en­ maturation of the virus itself, Sindbis is a promising velope glycoproteins, El and E2, have been shown to be system for the study of the more general questions of the integral membrane proteins, and one or both extends synthesis and structure of integral membrane proteins and completely through the bilayer formed by the viral lipids. the interaction of these proteins with other structures in During virus infection El and E2 appear to be synthesized the cytoplasm of the cell. 62

Sindbis virus is also similar to a wide variety of viruses results in the creation of a common necessary other animal viruses in that the viral structural proteins structure at the amino terminal end of the product. undergo post-translational modification by specific pro­ The glycoproteins of Sindbis also appear to b~ teolytic cleavage. In the case of Sindbis, all three unusual, when compared with other membrane glycopro­ structural proteins are derived in a series of steps from teins for which sequence data is available, in that there is one precursor polypeptide, and the molecular details of no carbohydrate attached to these molecules in the first this processing, such as the number of proteolytic en­ 40 amino terminal residues. The viral capsid protein was zymes involved and the factors responsible for the found to have a blocked amino terminus. This is delineation of the cleavage sites, are unknown at present. consistent with the observation that viral proteins derived The availability of mutants in which the processing of the from the amino termini of precursor molecules are often virus proteins or the maturation of the virus particle is blocked. aberrant is an additional advantage in our studies of the *Division of Chemistry and Chemical Engineering, Cali­ Sindbis structural proteins. fornia Institute of Technology. We are also engaged in studies of the structure and replication of the viral RNA. The facts that the virion 87. AMINO ACID COMPOSITIONS OF TIIE 49S RNA is capable of forming hydrogen-bonded circles, STRUCTURAL PROTEINS OF SINDBIS VIRUS and that both the viral 49S RNA and 26S RNA contain Investigators: John R. Bell, Ellen G. Strauss, methylated cytidine residues, raise interesting questions James H. Strauss Jr. as to the function of these structures. The development of a procedure for the purification of large-scale (tens of milligrams) amounts of Sindbis 86. AMINO TERMINAL SEQUENCE ANALYSIS OF mE structural proteins and the determination of the amino STRUCTURAL PROTEINS OF SINDB!S VIRUS acid compositions of these proteins has been completed. Investigators: John R. Bell, Michael W. Hunkapiller*, Leroy E. Hood, James H. Strauss Jr. The composition data show that the hydrophobic regions of the envelope glycoproteins, responsible for anchoring More than 50 amino acid residues (J'l3% of the these proteins in the lipid bilayer, probably do not account total) have been sequenced from the amino terminal end for much of the mass of these proteins (in agreement with of each of the Sindbis glycoproteins in single sequencer the results of other work in our lab). As expected from its , runs using only 25 to 50 nm of material. More limited function, the capsid protein contains a high proportion of data are available for some proteins of other viruses that basic amino acids. It is in_teresting to note in this respect are similar to Sindbis in two respects: the amino termini that there is a remarkable similarity with the capsid of the proteins are produced by proteolytic cleavage of protein of Semliki Forest virus, a closely-related virus. larger precursor molecules, and one such cleavage is a The molecular weights of the viral proteins have necessary final step in the maturation of infectious virus been estimated from the composition data and from the particles. The protein conformation at the cleavage sites relative extent of incorporation of radioactive amino would be expected to play a role in defining these sites acids. These polypeptide molecular weights are 41,000 for and, in turn, the amino acid sequences on both sides of El, 44,000 for E2, and 30,000 for capsid protein. Previous these sites must contribute to the protein conformation. molecular weight estimates have depended upon the rate However, no homology is found between the amino termini of migration of the proteins in SDS-containing acrylamide (created by proteolytic cleavage) of these various proteins gels. Although this method works well for most proteins beyond the observation that they tend to be quite glycoproteins are known to give anomalous results, and hydrophobic. Sindbis virus is an exception even to this these molecular weight estimates for the Sindbis glyco­ generalization, _as our data show that the amino termini of proteins are about 20% lower than the estimates from gel El and E2, which are created by cleavage events, are electrophoresis. quite hydrophilic. It is thus of interest that many of these viruses, including Sindbis, can replicate in the same host cells. Our observations also exclude the possibility that the final proteolytic cleavage in the maturation of these 63

88. PRODUCTION AND ANALYSIS OF CYANOGEN particles. We have performed an accmate quantitation of BROMIDE FRAGMENTS OF SINDBIS PROTmNS the amount and type of host polypeptides found in purified 35 Investigator: John R. Bell mature virions. Cells were labeled with s-methionine I am involved in studies directed toward the produc­ for several cell generations before infection, and labeled 3 tion of defined fragments of Sindbis proteins. In addition post-infection with H-methionine. Virions were purified to being necessary for the eventual determination of the with two cycles of alternating velocity and isopycnic complete amino acid sequence of these proteins, the centrifugation and the proteins analyzed on cylindrical analysis of such fragments is expected to provide informa­ Laemmli gels. HR virions contain as little as 0.2% host tion on the structure and function of certain regions of polypeptide (or approximately 1 molecule/virion) which is these molecules. For example, one part of the capsid found primarily in a small number of discrete polypep­ protein is probably involved in the interaction of this tides. Virus-sized particles of mutant ts103 contain protein with the viral RNA, another in the binding to the significantly more host material and the multiploid par­ envelope glycoproteins, and yet a third part may be ticles from ts103 infection contain up to 12% host involved in protein-protein interaction in the nucleo­ prelabeled protein. It appears, therefore, that host capsid. Similarly, the envelope glycoproteins have do­ polypeptides are almost completely excluded from areas mains which interact with the solvent and are expected to of plasmalemma where HR Sindbis is maturing, whereas be hydrophilic, domains which interact with the lipids and the budding of ts103 permits inclusion of host material. are expected to be hydrophobic, and domains which These results support our hypothesis that the interaction interact with the capsid. between envelope glycoproteins and capsid is very strong One approach is cyanogen bromide cleavage of the in HR Sindbis, whereas tsl03 has an altered capsid protein proteins. This chemical cleavage is very specific for that leads to weaker interactions with the glycoproteins methionine residues, and the amino acid compositions of during budding. the proteins and their molecular weights indicate that a 90. GROWTH AND RELEASE OF SEVERAL ALPHA­ reasonable number of fragments should be produced. VIRUSES IN CHICK AND BHK CELLS Preliminary experiments have been encouraging, yielding Investigator: Ellen G. Strauss results useful in other experiments and supporting the The final maturation and release of Sindbis virus notion that this method of analysis will be productive. from infected cells is known to be inhibited by incubation For example, the leucine (a hydrophobic amino acid) of in media of reduced ionic strength; the entire virus yield the capsid protein is found almost entirely in only two is subsequently rapidly released following a shift to cyanogen bromide fragments. The improvement of an SDS isotonic or hypertonic medium. These results have been gel electrophoresis system for the separation of the extended by comparing the release of several budding fragments is a major priority, since although the separa­ viruses in the same cell lines as a function of ionic tion system used so far, isoelectric focusing, has some strength. In primary chick cell monolayers the effect was advantages, it may not retain all the fragments and found to depend upon the particular virus and the strains cannot be used to estimate molecular weights. In used could be ordered from those where inhibition was addition, the problem of incomplete reaction at specific minimal (i.e., no additional virions are released after a methionine residues, presumably related to the avail­ shift to high ionic strength) to the opposite extreme ability of only small amounts of proteins for use in the (virions are released only after a shift to high ionic reaction, must be overcome. strength) as follows: minimum inhibition of release ~

vesicular stomatitis virus ~ Middelburg virus < Semliki 89. HOST POLYPEPTIDES PRESENT IN PURIFIED HR AND tsl03 SINDBJS VIRIONS Forest virus < wt Sindbis virus < HR Sindbis virus < Investigators: Ellen G. Strams, Edith M.. Lenches, Sindbis mutant ts103 = maximum inhibition. Mary S. Martin It was also found that the properties of the infected We have continued to compare the maturation and cells were important in determining the magnitude of the budding of the HR strain of Sindbis virus with the aberrant inhibitory effect. The effect of ionic strength was much behavior of the mutant ts103, which produces multiploid reduced in hamster cells as compared to chick cells. 64

Furthermore, the serum in which the cells are grown some controversy over this point. The location of E3, affects the magnitude of the ionic strength effect. whether extracellular or intracellular, will help resolve Finally, serial passage of chick cells leads to changes in this question. the ionic strength optimum for both virus production and subsequent release. 92. INVESTIGATION OP mE CLEAVAGES OP mE STRUCTURAL PROTEINS OP SINDBIS VIRUS These results indicate that the final maturation Investigators: Jeffrey T. Mayne, Jolm R. Bell, steps in virus budding, which involve a cleavage event in Charles M. Rice m one of the viral polypeptides, and probably configurational The three structural proteins of Sindbis virus (El, rearrangement of the viral structural proteins, followed E2, and C) as well as polypeptide E3 are translated from by pinching off of the virion and release into the culture the viral 268 RNA into a large precursor protein (molec­ fluid, are affected not only by changes in the virion ular weight 130,000) which is cleaved to yield the final polypeptides but also by changes in the host plasmalemma. proteins. The capsid protein (30,000 molecular weight) is There must be interactions between the virion proteins cleaved from the amino-terminal end of the precursor and the host plasmalemma that are important in virus while it is nascent. The second cleavage event (again maturation which are at least partially ionic. while the polypeptide is nascent) produces PE2 (54,000 molecular weight) from its amino-terminal end and El 91. CHARACTERIZATION OP E3 JN THE SINDBIS VIRUS INFECTION (48,000 molecular weight) from its carboxyl-terminal end. Investigator: Jeffrey T. Mayne Finally, PE2 is cleaved just before virus maturation to give E2 (48,000 molecular weight) and E3 (about 5000 The two best studied alphaviruses are Sindbis virus molecular weight, presumably from the amino-terminal and Semliki Forest virus (SFV). One of the major end of PE2). Uncleaved precursors can be obtained by differences between the two viruses is the presence of E3 using mutants or by growing the virus in cells where some (a small, heavily glycosylated protein) in the membrane of of the cleavages are inefficient. the SF virion and its lack in the Sindbis virion. In SFV, E3 The nature of the cleavage events is not known. It is produced at a late stage of virus maturation when PE2 is unclear whether these events are single cuts which is cleaved to produce E2 and E3. But in the Sindbis conserve the amino acids on either side, or if a stretch of infection PE2 is cleaved to produce E2 only, and the amino acids is excised. In addition, it is unclear where the fragment equivalent to E3 is lost. "signal" sequences required for insertion of the glycopro­ A protein can be isolated from the culture fluid teins into the membrane are found. Our approach to this from Sindbis-infected cells which is believed to be E3. It problem is to obtain N-terminal sequence data for a appears to be released from the cells after it is cleaved number of the uncleaved precursor polypeptides, as well from PE2. We are in the process of characterizing and as for the virion structural proteins. It is hoped that purifying this putative E3 and plan to compare its amino enough sequence overlap can be obtained to answer these acid sequence and/or cleavage map with that of PE2. questions. Since the cleavage of PE2 to E2 and E3 is a late event in the maturation of the virus, we are interested in investigating the kinetics of production of E2 (and of PE2) 93. STRUCTURAL STUDIES ON SINDBIS VIRUS during the infection process, including any possible tran­ Investigator: Charles M. Rice fil sient appearance of E3 in the cell plasma membrane. We I am investigating the structure and orientation of hope to be able to accomplish this in part with the use of the viral polypeptides El and E2 in the lipid bilayer of the antibodies specific to E3. virion and of the infected cell. This should yield We are also investigating the kinetics of synthesis information on how the glycoproteins might have arrived and the location of E3 during the infection cycle in at their conformation in the bilayer, and also may tell us mosquito cells. In chick cells the virus buds through the something about their function in the budding process and plasma membrane and is released into the medium. In the mature virion. mosquito cells, in contrsst, the virus seems to be The initial efforts in this area have involved assembled in cytoplasmic 0 factories," although there is a-chymotryptic digestion of isolated Sindbis virions and 65 characterization of the resultant particle-associated poly­ antisera to Sepharose 4B and used this affinity resin for peptides. By adjusting the digestion conditions I can partial purification of radiolabeled PE2 for microsequenc­ isolate particles of a homogeneous buoyant density that ing. A similar approach will be useful for isolating contain virtually no intact El and E2 while the capsid nanomolar quantities of the BHK protein, a polypeptide protein remains undigested. Two small hydrophobic from Sindbis-infected BHK cells containing the tryptic polypeptides Rl (apparent molecular weight of .rl0,000 peptides of PE2 and El. daltons) and R2 (apparent molecular weight .r5000 daltons) These specific antibodies should also be useful for are found in addition to the capsid protein when these looking at the subunits of 8indbis virus. Previous studies particles are solubilized and run on SDS gels. R2 is very with Semliki Forest virus, a closely related alphavirus, soluble in several lipophilic solvent systems while Rl have shown that the virion can be delipidated by treat­ shows more limited solubility under the same conditions. ment with the nonionic detergent Triton X-100, even­ In addition to differential solubility in organic solvent tually forming detergent-glycoprotein complexes. These systems and electrophoretic resolution, Rl and R2 have complexes can be cross-linked and appear to be dimeric at also been separated by gel filtration and SOS hydroxy­ high detergent concentrations. By using reversible cross­ apatite chromatography. Comparative tryptic and chym

96. LOCATION OF 5-MEfHYLCYTIDINE RESIDUES IN sequences of 49S RNA in an effort to find the sequences SINDBIS VIRUS RNAs responsible for cyclization. In addition, these regions of Investigators: Teryl K. Frey, David L. Gard the molecule contain ribosome binding sites and binding Sindbis virus intracellular 26S and 49S RNA contain sites for the viral replicase, and knowledge of these 5 3 internal 5-methylcytidine (m c) residues. In H-methyl­ sequences is important in our understanding of the control methionine-labeled Sindbis virus RNA, an average of 19% meqhanism operative during the replication cycle of the 3 5 of the H-methyl label in 26S RNA is in m C while in virus. 3 intracellular 49S RNA an average of 8% of the H-methy We also intend to sequence the 5'-terminal regions 5 label is in m c, the remainder of the label being in the of Sindbis virus 26S RNA, using the same techniques, for 5 cap. Sindbis virion 49S RNA contains much less m c than the same reasons. intracellular 49S RNA extracted from the same cells. In 5 268 RNA, m c residues occtll' in five oligonucleotides, PUBLICATIONS four of which have been identified as having base compositions of C A UG, C A UG, C A G, and C AG Bell, J. R., Hunkapiller, M. W., Hood, L. E. and Strauss, J. 4 4 6 2 3 2 2 H. (1978) Amino-terminal sequence analysis of the that found distributed between two locations, one approxi­ structural proteins of Sindbis virus. Proc. Nat. mately 4000 nucleotides from the 3'-end and the other Acad. Sci. USA 75: 2722-2726. Bell, J. R., Strauss, E. G., Hood, L. E. and Strauss, J. H. about 1200 nucleotides from the 3'-end (out of a total (1978) Amino acid composition and molecular 5 length of 5000 nucleotides). The m C containing nucleo­ weights of the structural proteins of Sindbis virus. In preparation. tide C AG is found only at the former location and the 2 Frey, T. K., Gard, D. L. and Strauss, J. H. (1978) oligonucleotide of composition A UG (which contains Biophysical studies on circle formation by Sindbis c4 4 5 virus 49S RNA. J. Mo!. Biol. In press. 40-50% of the total m C in 26S RNA) is restricted to the Frey, T. K., Gard, D. L. and Strauss, J. H. (1978) latter location; the distribution of methyl label between Replication of Sindbis virus VII. Location of 5-methylcytidine residues in virus-specific RNA. the two locations suggests that each region contains at Virology. In press. least two of the methylated sequences. Although there Frey, T. K. and Strauss, J. H. (1978) Replication of Sindbis virus VI. Poly(A) and poly(U) in virus-specific RNA appear to be five specific sites for methylation on the 268 species. Virology 86: 494-506. RNA, only a minority of these sites is modified, as each Strauss, E. G. (1978) Mutants of Sindbis virus III. Host polypeptides present in purified HR and ts103 virus 268 RNA molecule contains an average of less than one particles. J. Virology. In press. m5c. Sindbis virus 26S RNAs isolated from both Strauss, E. G., Birdwell, C. R., Lenches, E. M., Staples, S. E. and Strauss, J. H. (1977) Mutants of Slndbis virus. polysomes and nonpolysomal ribonucleproteins contain II. Characterization of ts103, a maturation-defec­ equal amounts of m5c. 49S RNA isolated from polysomes tive mutant. Virology 82: 122-149. 5 Strauss, E. G., Leaches, E. M. and Stamreich-Martin, M. contains 60-80% more m c than does 49S RNA which is A. (1978) Growth and release of several alphaviruses isolated primarily from nucleocapsids. The m 5c residues in chick and BHK cells. In preparation. Strauss, E. G. and Strauss, J. H. (1978) Genetics of appear somehow to be involved .in translating the viral alpha viruses. In: The Toga viruses, R. W. RNA. Schlesinger (Ed.), Chap. 12, Academic Press, New York. In preparation.

97. THE DETERMINATION OF TilE 5'-TERMINAL AND 3'-TERMINAL SEQUENCES OF SINDBIS 498 AND 268 RNAs Investigator: Jing-hsiWlg James Ou It is known that Sindbis virus 49S RNA is capable of cyclization via complementary sequences near the end of 32 the molecule. By using y- P-ATP end-labeling tech­ niques and partial ribonucleotide substitution during syn­ thesis of cDNA with reverse transcriptase, we are now trying to determine both the s•-terminal and 3'-terminal CELLULAR BIOLOGY AND BIOPHYSICS

Charles J. Brokaw

Max Delbrlick

Elias Lazarides

Jean-Paul Revel

Anthonie Van Harreveld

69

Professor: Charles J. Brokaw calculated by the two methods is almost identical. The Research Fellows: Robert Johnson, Makoto Okuno resistive force theory is completely satisfactory for use in Graduate Student: David J. Asai Research Staff: Thomas F. Simonick analysis of mechanisms for the control of flagellar Support: The work described in the following research bending, at the current level of sophistication of this reports has been supported by the National Institutes of analysis. The addition of a cell body to one end of the Health, USPHS. flagellum, which modifies the flow field experienced by Summary: Evidence has accumulated which indicates the flagellum, was also examined. This interaction, which that the bending movements of cilia and flagella are is not considered by resistive force theory, is probably generated by a sliding microtubule process, similar to the insignificant for small cell bodies, such as the heads of sliding filament process responsible for muscle contrac­ simple spermatozoa, but for larger cell bodies, or cell tion. Control mechanisms are needed to cause oscillatory bodies which have large-amplitude motions transverse to bending and to maintain the phase differences between the swimming direction, use of slender-body theory is bending in different regions which are required for required for accurate analysis. propagated bending wavesa Two quite independent mech­ anisms for flagellar oscillation have been proposed; both 99. INHIBmON OF FLAGELLAR MOVEMENT BY mechanisms also lead to bending wave propagation under COMPONENTS OF THE ATP SYSTEM Investigator: Makoto Okwto at least some conditions. Our work makes particular use of ATP-reactivated Beat frequency, bend angle, and wavelength of ATP- movements of demembranated sperm flagella as a source · reactivated, demembranated flagella from the sea urchin, of experimental data, and computer programs which Lytechinus pictus, were measured. Previous work demon­ simulate the movement of model flagella to relate strated a linear double-reciprocal relationship between 2 theoretical mechanisms to experimental data. We hope to beat frequency and MgATP - concentration. This is now identify the types of control mechanisms which actually found to be true for both swimming and attached exist in flagella and cilia, to understand how parameters spermatozoa. It was not possible to determine whether of movement such as frequency, wavelength, and bend MgATP 2- is directly controlling the beat frequency or the angle are controlled, and to use this understanding to velocity of sliding between flagellar microtubules. enable detailed study of the active process which gener­ MgADP-, ADP3-, and HPo 2- act as competitive 4 ates sliding in flagella and muscle. inhibitors. Addition of these inhibitors changes the movement in the same way as a reduction in MgATP2- 98. FLAGELLAR HYDRODYNAMICS: A COMPARISON concentration. BETWEEN RESISTIVE FORCE THEORY AND ATP4- is a competitive inhibitor of beat frequency, SLENDER-BODY THEORY and also causes a reduction in bend angle which is not seen Brokaw 2 Investigators: Robert Johnson, Charles J. when MgATP - concentration is reduced. We investigated the accuracy of the simple resistive Mg2+ is an uncompetitive inhibitor of beat fre­ force theory (Gray and Hancock method) which is com­ quency, and also enhances the bend angle, so that there is monly used for hydrodynamic analysis of swimming fla­ no 41lhibition of the velocity of sliding between flagellar gella. A comparison was made between the forces, micro tubules. bending moments, and shear moments calculated by These three distinct patterns of inhibition require resistive force theory and by a newly-developed slender­ more complex models for the action of flagellar cross­ body theory for large-amplitude, planar, waveforms com­ bridges. puted for a flagellar model. By making an upward adjustment (by about 35%) of the classical drag coeffi­ 100. REACTIVATION OF DEMEMBRANATED CIONA SPERMATOZOA cient values used for resistive force theory calculations, Investigator: Stan Shipley* good agreement can be obtained between the distributions of the forces and moments along the length of the Spermatozoa from the tunicate, Ciona, are easily flagellum calculated by the two methods. Once this obtainable and have proven to be useful material for adjustment is made, the rate of energy expenditure experiments with live spermatozoa. However, the pro- 70

cedures for demembranation and reactivation which work The initiation of synchronous sliding during a portion well with sea urchin spermatozoa do not appear to be of the beat cycle in asymmetrically beating flagella may suitable for reactivation of Ciona spermatozoa. Two be related to the initiation of synchronous sliding at the conditions were found which improve the reactivation of beginning of the effective stroke in cilia. Ciona spermatozoa: (1) motility of the spermatozoa needs References: to be activated by histidine-seawater before demembrana­ Brokaw, C. J., Josslin, R. and Bobrow, L. (1974) Biochem. tion, and (2) excess Mg2+ and ATP4- in the reactivation Biophys. Res. Commun. 58: 795. Goldstein, S. F. (1976) J. Exptl. Biol. 64: 173. solution should be avoided. Results so far suggest that the Goldstein, S. F. (1977) J. Exptl. Biol. 71: 157. 2 effect of ca + on the asymmetry of flagellar beating, *Department of Genetics and Cell Biology, University of seen with sea urchin spermatozoa, is not found with Ciona Minnesota, Minneapolis, Minnesota. spermatozoa.

*Undergraduate, California Institute of Technology. 102. FLAGELLAR OSCILLATION AND BILATERAL ARREST lN CIONA SPERMATOZOA Investigator: Charles J. Brokaw 101. FLAGELLAR ASYMMETRY AND ITS CONTROL BY CALCIUM I have previously discussed two distinct mechanisms Investigators: Charles J. Brokaw, Stuart F. Goldstein* for flagellar oscillation: (1) dynein-tubulin cross-bridges Moving-film flash photography has been used to in flagella are regulated by the curvature of the flagel­ record the flagellar waveforms of spermatozoa swimming lum, establishing a feedback loop leading to oscillation next to glass surfaces. Detailed measurements were made (Brokaw, 1971); (2) dynein-tubulin cross-bridges are rapid­ on spermatozoa of the sea urchin, Strongylocentrotus ly deactivated when moved in a direction opposite to the purpuratus. direction in which they normally generate force (Brokaw, Previous work (Goldstein, 1976) demonstrated that 1975). These can be called the (1) curvature-feedback in symmetrically beating flagella the two bends closest to mechanism and the (2) self-oscillatory cross-bridge mech­ the base of the flagellum grow in angle at equal and anism. Mechanism (2) predicts that in a symmetrical opposite rates, so that the initiation of bends near the system no net force will be generated at 0 velocity. base of the flagellum is independent of any sliding Sperm flagella are frequently found in arrested between microtubules in more distal regions of the states, which maintain the flagellum in a bent configura­ flagellum. tion. An actively generated sliding force would appear to Observations on live spermatozoa with asymmetrical be required to maintain a bent state during arrest. This bending patterns (Goldstein, 1977) demonstrate that dur­ would be consistent with the self-oscillatory cross-bridge ing a portion of the beat cycle the principal bend at the mechanism only if the cross-bridge system is not sym­ base of the flagellum is growing at a much more rapid metrical. However, photographs have been obtained rate than the preceding reverse bend. This net increase in which demonstrate that Ciona spermatozoa can become angle of the two growing bends requires that synchronous arrested in either direction of bending. This observation sliding between microtubules must be superimp9sed on the is not rea_dily explainable by the self-oscillatory cross­ bend propagation occurring in distal regions of the bridge mechanism. It suggests instead that the dynein­ flagellum. This occurs with no detectable alteration in tubulin cross-bridges are regulated by a switching mech­ the propagation of bends in the distal region. anism which selects an active set of cross-bridges. Arrest These observations have now been extended to can occur when this switching mechanism gets decoupled demembranated spermatozoa, where the asymmetry can from curvature and gets stuck in either of its two possible be controlled by regulating the exposure of the axoneme states. to ca2+ ion (Brokaw et al., 1974). The same type of 2 References: asymmetry is observed. As the Ca + concentration is Brokaw, C. J. (1971) J. Exptl. Biol. 55: 289. Brokaw, C. J. (1975) Proc. Nat. Acad. Sci. USA 72: 3102. increased, growth of the reverse bend terminates earlier in the beat cycle, and there is also a progressive increase in the ratio of the growth rates of the principal and reverse bends. So far, we have not discovered a causal relationship between these two changes. 11

103. ANTI-TUBUIJN ANTIBODIES imally until only about one-third of the flagellum is able Investigator: David J. Asai to move. We have not been able to completely stop the Anti-tubulin antibodies are obtained from rabbits movement using this antibody. Spontaneous anti-tubulins injected with SOS-denatured purified sea urchin (S. pur­ from nonimmune serum have no effect on the frequency, puratus) flagellar outer doublet microtubules as well as bend angle, or symmetry of reactivated spermatozoa. We from nonimmune rabbit serum. Purification of both types are currently investigating the biochemical basis for the of antibodies is accomplished by sea urchin tubulin differences between these two apparent anti-tubulins. affinity chromatography. Approximately one-third as much "spontaneous11 anti-tubulin (per unit volume whole PUBLICATIONS serum) is obtained as compared to "induced" anti-tubulin. Brokaw, C. J. (1977) co2-inhibition of the amplitude of Both preparations appear to be specific for tubulin and do bending of Triton;Iemembranated sea urchin sperm flagella. J. Exptl. Biol. 71: 229-240. not cross-react with any other sea urchin flagellar Brokaw, c. J. and Rintala, D. (1977) Computer simulation proteins. The induced anti-tubulin has no effect on the of flagellar movement. V. Oscillation of cross­ bridge models with an ATP-concentration-dependent frequency but has a pronounced effect on the bend angles rate function. J. Mechanochem. Cell Mot. 4: and symmetry of reactivated sea urchin (S. purpuratus and 205-232. Brokaw, C. J. and Simonick, T. F. (1977) Motility of L. pictus) spermatozoa. The amplitude of the bend form Triton;iemembranated sea urchin sperm flagella is reduced followed closely by a steady reduction in during digestion by trypsin. J. Cell Biol. 75: 650-665. symmetry. After a few minutes the distal tip of the Goldstein, S. F. (1977) Asymmetric waveforms in echino­ flagellum ceases to bend; this paralysis proceeds prox- derm sperm flagella. J. Exptl. Biol. 71: 157-170.

Professor: Max Delbruck Jayaram and Hershey (abstract 107) took on the Visiting Associates: Arturo Eslava, Tamotsu Ootaki, Kaori Yoshida study of another response to light Of Phycomyces (and Gosney Research Fellow: Rasika M. Harshey other fungi): the induction of carotene synthesis. They Research Fellows: Makkuni Jayaram, Manfred K. Otto, David N. Radin made the remarkable discovery that this response to light Graduate Students: Jean-Francois Lafay, David E. Presti is a mixture of two distinct ones: a highly efficient, Research Staff: Doris Finch, Robin M. Hamilton, Harriett L. Lyle, Nancy K. Wischhusen quickly-saturating response not requiring mRNA or pro­ Laboratory Staff: Jeanette Navest tein synthesis, and a slower response requiring both Support: The work described in the following research classes of macromolecule synthesis. Methods are de­ reports has been supported by: veloped to pursue especially the latter on the level of Lucy Mason Clark Fund Deutsche Forschllllgsgemeinschaft molecular genetics. Gosney Fund Phycomyces has a unique feature which might make National Institutes of Health, USPHS National Science Foundation it eminently suitable as a host for recombinant DNA work Research in Biology using plasmids: the sporangiophore is a single cell of Summary: Several approaches were used to zero in on sufficient size to be handled surgically. Thus material of the photoreceptor molecule of Phycomyces, believed to any kind, including plasmids, can be injected, followed by carry riboflavin as its chromophore. Presti (abstract 104) regeneration. Lafay (abstract 108) has worked out discovered a flavin fluorescence in a Phycomyces plasma procedtll'es for such injections which look encouraging. membrane fraction whose short lifetime makes it a Phycomyces sporangiophores sense, in complete promising candidate for the receptor pigment. Otto darkness, the presence in their vicinity of a barrier (abstract 105) initiated a study of the enzymes in flavin (avoidance response). Our previous notions regarding the metabolism: synthase, permease, kinase, and pyrophos­ mechanism of this sensing were based on the idea that the phorylase. Jayaram et al. (abstract 106) obtained evi­ avoidance response is a response to wind speed gradients. dence that an analogue of riboflavin (roseoflavin), which is This idea had to be dropped (abstract 109) when it was also a natural antibiotic, can get into the photoreceptor found that reversal of wind gradients did not reverse the and function there as such, although with very low response and that a "wind-blind" mutant still avoided. It efficiency. 72 has now been shown (abstract 110) that very slow winds Pill-Soon Song of the Department of Chemistry, Texas perpendicular to the barrier stop avoidance, both in wild Tech University, Lubbock, Texas. type and in the "wind-blind" mutant. These and other findings suggest strongly that the barrier creates a 105. RIBOFLAVIN METABOLISM IN PHYCOMYCES concentration gradient by acting as a diffusion boundary. Investigators: Manfred K. Otto, Harriett L. Lyle, Paul Shupak*

104. FLUORESCENCE STUDIES OF PHYCOMYCES The photoreceptor pigment of Phycomyces contains MEMBRANE FRACTIONS a riboflavin derivative as the chromophore. We have Investigator: David E. Presti begun a study of the ability of substances structurally Riboflavin is believed to be the photoreceptor for related to riboflavin to cross the cytoplasmic membrane the physiological responses of Phycomyces to light. and their ability to compete successfully with the respec­ Because of the widespread occtn"rence of flavins in every tive riboflavin derivatives at the various enzymatic steps living organism, no specific assay for the photoreceptor leading to the synthesis of the photoreceptor. We are flavin has yet been developed. Most flavoproteins are studying (1) the transport of riboflavin and analogues nonfluorescent or only very weakly fluorescent, probably across the cytoplasmic membrane, (2) the regulation of as a result of quenching of the flavin excited states by riboflavin biosynthesis, and (3) the enzymes of riboflavin binding site interactions with various amino acids. The metabolism. photoreceptor flavin should not have its excited state The transport of riboflavin across the cytoplasmic quenched by chromophore-protein interactions since it membrane is studied by measuring the incorporation of 14 will need to use excitation by light to initiate signal 2- c-riboflavin into the ·mycelium of Phycomyces. 5 transduction. Thus it is possible that the photoreceptor Above 10- M riboflavin the uptake rate is saturated. The -6 flavin might be relatively fluorescent but with a lifetime apparent Km -value is ca. 2 x 10 M. which is short (relative to free flavin), indicative of an In some organisms external riboflavin represses the efficient depopulation of the excited singlet state via a synthesis of riboflavin synthase (the last enzyme in photochemical mechanism. riboflavin biosynthesis). Our results show that riboflavin Fluorescence emission spectra indicative of flavin offered in the growth medium does not influence the were measured from relatively crude cell wall plus specific activity of this enzyme in Phycomyces. membrane preparations from wild-type Phycomyces. Riboflavin derivatives (FMN, FAD) rather than free Identical flavin fluorescence was measured for wall­ riboflavin serve as coenzymes in most of the flavo­ membrane preparations from the phototropically deficient enzymes. 5'-Phosphorylation by the riboflavin kinase is strains C47 madA and Cl14 madB. Measurement of probably also the first step in the biosynthesis of the fluorescence lifetimes indicated a heterogeneous mixture photoreceptor pigment. Riboflavin kinase of Phycomyces of fluorescent species with lifetimes less than 2 ns was isolated to study the interaction of the enzyme with (nanosecond) and longer than 5 ns. Further purification of riboflavin and analogues. Riboflavin kinase phosphor­ the wild-type wall-membrane fraction by sonication and ylates riboflavin with a Km of about 5 x 10-6 M. Optimal repeated centrifugation enriches for plasma membrane conditions for the reaction are pH 7.5 and 45°C. We found and eliminates possible contamination by mitochondria that each of those riboflavin analogues which inhibit and soluble flavin. The resulting plasma membrane­ Phycomyces are also converted to the respective FMN enriched fraction exhibited flavin fluorescence with a analogue. Riboflavin concentrations in the growth medi­ lifetime indicating that an increase in the relative um above 10-5 M increase the specific activity' of contribution of the short-lifetime component may have riboflavin kinase up to 20-fold compared to growth been achieved. The short component lifetime appears to conditions without external riboflavin. be about 1 ns, significantly shorter than free riboflavin, *Undergraduate, California Institute of Technology. flavin mononucleotide (FMN), and flavin adenine dinucleo­ tide (FAD). Fluorescence lifetime spectroscopy was done in collaboration with Dr. Robert Fugate and Professor 73

106. PHOTO RESPONSE OP ROSEOPLA VIN-DOPED broad-band blue light. There is a response which shows SPORANGIOPHORHS OP PHYCOMYCHS half-saturation at the very low dose of approximately Investigators: Makkuni Jayaram, Robin M. Hamilton, 0.7 mJ/cm2, which is less than a tenth of the expected Max Delbriick saturating energy flux, if riboflavin were the photorecep­ The action spectra for the growth and tropic tor. We have not yet looked into the possibility of an response of Phycomyces sporangiophores to light suggest alternate photoreceptor for this quickly saturating re­ that the primary photoreceptor is riboflavin. If a flavin sponse to blue light. This response does not seem to be moiety does form the chrom-ophore of the photoreceptor it affected by actinomycin D or cycloheximide. The other 2 might be possible to replace it by a flavin analogue. If response is half-saturated at §1.34 mJ/cm and is sensitive this analogue has absorption peaks different from those of to both actinomycin D and cycloheximide. riboflavin and if the new receptor is functional, one could As LICS to long exposures requires fresh synthesis of expect changes in the action spectrum as compared to RNA and proteins, attempts are being made to identify undoped controls. We have found evidence for such specific RNA and protein molecules which might be changes when the analogue roseoflavin is added to the induced by light and which, in turn, are responsible for growth medium. In this analogue the methyl group in enhanced carotenogenesis. Conditions have been stan­ position 8 of riboflavin is replaced by a dimethyl-amino dardized for isolation of polysomes, so that polysomal group. It has a very strong absorption peak at 505 nm as mRNAs could be isolated and subjected to electrophoretic against the much weaker ones of riboflavin at 480 nm and separation on agarose gels. Separation and detection of 445 nm. In addition roseoflavin has an absorption mini­ proteins could be achieved by the high-resolution two­ mum near 380 nm where riboflavin has a maximum. dimensional electrophoretic method of O'Farrell (1975). It Our data show a small decrease in sensitivity at is planned to radioactively pulse-label RNA and proteins 380 nm and small changes in the neutrality points when in dark~own and irradiated mycelia of mutants which 380 nm light is matched against 509 nm and 530 nm light are blocked at specific steps in the carotenogenic pathway in phototropic or photogrowth balance experiments. as well as those that overproduce ~-carotene. The The data can be interpreted by assuming that in 50% labeled molecules thus obtained could be resolved by the of the photoreceptor molecules riboflavin is replaced by above techniques to locate (hopefully) specific macro­ roseoflavin and that the modified photoreceptor is weakly molecules responsible for LICS. active (somewhat below the 1% level). Further experi­ ments will be needed to check into the possibility that Reference: O'Farrel~ P.H. (1975) J. Biol. Chem. 250: 4007-4021. some of the differences found are due to a photoproduct of riboflavin produced during the experiments. 108. MICROINJECTION OP PHYCOMYCHS SPORANGIOPHORHS 107. INDUCTION BY LIGHT OP ll-CAROTENE SYNTHESIS IN PHYCOMYCHS Investigator: Jean-Francois Lafay Investigators: Makkuni Jayaram, Rasika M. Harshey The purpose of this project was to find a method for Phycomyces, grown in the dark, synthesizes B­ injecting various materials into Phycomyces sporangio­ carotene, but light causes significant enhancement of the phores and to derive cultures from these injected sporan­ amount of B-carotene synthesized. The mycelium of the giophores. Techniques have been developed which allow wild-type strain, grown in light, is significantly more injection of small volumes (in the nanoliter range) into yellow than that grown in the dark. In order to see decapitated stage I sporangiophores. Normally these whether a single shot of light can cause a stimulation of mutilated sporangiophores regenerate a smaller sporangio­ B-carotene synthesis in the dark~own mycelium, meth­ phore and sporangium containing spores, but we found ods have been developed which permit in vivo measure­ conditions where we can induce them to grow into a ments of the B-carotene contents of mycelia grown on mycelium. The material injected into albino (carA) solid minimal medium in petri plates. In the wild type, sporangiophores was either protoplasm from a red (carR) light-induced carotene synthesis (LICS) shows a biphasic strain stage I sporangiophore or "nuclear fractions11 iso­ response to irradiation for varying lengths of time by lated from the same kind of carR sporangiophores 74 intracellularly fractionated by centrifugation (Zalokar, the sporangiophore environment is the wind velocity 1969). Virtually any kind of solution could be injected. distribution. The sporangiophore failed to respond to this Usually the injected material, clearly visible inside the steep gradient, thus ruling out the wind gradient hypothe­ sporangiophore, does not mix with the receiver protc:r sis. plasm. In 20% of the cases we detected and recovered (by Another approach was to study "wind blind" mutants. plating the spores from the regenerated sporangium) some After X-ray mutagenesis of the wild type, such a mutant viable carR nuclei from such injected sporangiophores. was found (Fl). After 7 hr in a wind tunnel, the wild type Regenerating mycelia from injected sporangiophores did and Fl sporangiophores are bent about 55 and 8 degrees not give better results; neither did the injection of nic+ into the wind respectively. In contrast, the avoidance and protoplasm into a nic- receiver and selecting on minimum light responses of both types of sporangiophores are medium. Thus these techniques are not yet reliable similar. Therefore, the wind response and the avoidance enough to replace such procedures as the grafting method response appear to be mediated by two different signals. (Ootaki, 1973) for heterokaryon formation. Reference: References: Cohen, R. J., Jan, Y.-N., Matricon, J. and Delbrlick, M. Ootaki, T. (1973) Mo!. Gen. Genet. 121: 49-56. (1975) J. Gen. Physiol. 66: 67-95. Zalokar, M. (1969) J. Cell Biol. 41: 494-509.

109. AVOIDANCE AND W1ND RllSPONSllS IN no. PHYCOMYCES: EXPLORING THE AVOIDANCE PHYCOMYCllS RllSPONSE SIGNAL Investigator: Jean-Francois Lafay Investigators: Jean-Francois Lafay, Paul Meyer* The sporangiophore of Phycomyces grows away from The Phycomyces sporangiophore bends away from any object placed in its vicinity, and its growth rate is any object or "barrier" placed within 1 mm of its growing affected by wind. A positive growth response is associ­ zone. Previous studies have related this response to the ated with a drop in wind velocity. It has been proposed wind response, a bending into a lateral wind, postulating (Biology 1974, No. 12; Cohen et al., 1975) that the specifically that the barrier acts by modifying in its avoiding sporangiophore in fact responds to a wind vicinity the distribution of random winds. However, the velocity gradient. It is observed that barriers calm relation between the two responses must be more subtle, natural winds flowing parallel to the barrier. It was since the avoidance response is not reversed when the assumed that this gradient disturbs the spatial distribution wind gradient is reversed and is still observed in a mutant of a growth-IX"omoting gas and induces the sporangiophore which does not bend into the wind (nwind blind") (see to react by growing away from the calmer side and Biology 1978, No. 109). We have now found that a very towards the windier side. slow air flow perpendicular to the surface of a barrier (a To test this hypothesis, different moving barriers 1 cm/sec wind), produced by forcing air through a narrow­ were designed so as to reverse the wind velocity gradient. mesh wire screen, and which by itself induces almost no The expected reversal of the tropic avoidance response bending, is sufficient to prevent avoidance, both in the was not found. The failure to induce a "negative wild type and in the "wind blind" mutant. In contrast, a avoidance response" could be interpreted either as indi­ longitudinal air flow parallel to the barrier must be faster cating independence of the wind and barrier signals or as than 12 cm/sec in order to prevent avoidance. Within due to insufficient reversal of the wind gradient. 2 min after the air flow is stopped, the sporangiophore To decide between these hypotheses, we increased avoids the barrier at a rate of 2 to 5°/min in both cases the wind velocity gradient by reversing periodically the (wind parallel or perpendicular to the surface). direction of the movement of plane barriers to prevent In another set of experiments, sporangiophores any steady regime in the induced air pattern. For growing vertically between two identical parallel flat instance, a sporangiophore was placed between two barriers exhibit no tropic response. If one of the barrier parallel barriers, 8 mm apart, one fixed and the other surfaces is coated with cytoplasm from about 10 sporan­ moving at up to 10 to 20 cm/sec and alternating its giophores, a bending of approximately 0.5°/min away from movement every 2 sec; the only permanent disymmetry in the coated surface occurs. 75

These results suggest that the sporangiophore "sees" PUBLICATIONS barriers by sensing, at the surface of its growing zone, the Delbrlick, M. (1978) Virology revisited. In: Molecular concentration of some self-produced growth-promoting Basis of Host Virus Interaction, M. Chakavorty {Ed.), volatile substance. A nearby barrier restricts the dif­ Science Press. Delbrilck, M. and Ootaki, T. (1978) An unstable nuclear fusion and reflects this gas, thus creating a concentration gene in Phycomyces. Genetics. In press. gradient across the sporangiophore and thereby eliciting Presti, D. and Delbrlick, M. (1978) Photoreceptors of biosynthesis, energy storage, and vision. Plant, Cell an avoidance response. A slow air flow (1 cm/sec) and Environment 1: 81-100. Presti, D., Hsu, W.-J. and Delbrlick, M. (1977) Photo­ perpendicular to the barrier surface would greatly inter­ tropism in Phycomyces mutants lacking a-carotene. fere with such a sensory mechanism, in agreement with Photochem. Photobiol. 26: 403-405. our observations. *Undergraduate, California Institute of Technology.

Assistant Professor: Elias Lazarides of techniques including electron microscopy, immuno­ Research Fellow: Karen F. Greif fluorescence, and biochemical analysis. Graduate Students: David L. Gard, Bruce L. Granger, Bruce D. Hubbard (5) Muscle cells provide a good system to look for Research Staff: David R. Balzer Jr., Ilga Lielausis mutants affecting specific stages of assembly. SUpport: The work described in the following research (6) The Z disc of sarcomeres provides a well-defined reports has been supported by: model system where the interaction of actin filaments American Cancer society Muscular Dystrophy Association of America with the plasma membrane {transverse tubule system in National Institutes of Health, USPHS chicken) can be investigated. National Science Foundation Alfred P. Sloan Foundation summary: Our laboratory is concerned with the under­ 111. SPECIFICITY OF DESMIN TO AVIAN AND standing of the biochemistry of the cytoplasmic fila­ MAMMALIAN MUSCLE CELl.8 mentous systems that are responsible for maintaining a Investigators: Elias Lazarides, David R. Balzer Jr. cell's motile activities. In particular we are interested in The extent of invariance and heterogeneity in 0 two filamentous systems: actin filaments and desmin desmin, the major component of the muscle form of 100 A (100 A) filaments. Our long-term aim is to understand (1) filaments, has been investigated in avian and mammalian the assembly of actin and desmin filaments, {2} the muscle and nonmuscle cells with two-dimensional gel cytoplasmic factors which regulate their assembly, and (3) electrophoresis and indirect immunofluorescence. Desmin the mode of interaction of actin and desmin filaments from chick, duck, and quail cells (smooth, skeletal, and with the plasma membrane as well as with each other. As cardiac muscle) is resolved into two isoelectric variants, a an experimental system we use primarily chicken muscle and a, with each possessing the same charge and cells. The reasons for choosing muscle as a model system electrophoretic mobility in all three avian species irre­ are as follows: spective of muscle type. Guinea pig and rat muscle (1) The contractile components of muscle have been desmin resolves into only one variant; it also possesses the thoroughly investigated. same charge and electrophoretic mobility in the two (2) The ultrastructure of muscle is well docu­ mammalian species, but it is more acidic and slower in mented. electrophoretic mobility than the two avian variants. (3) Chick muscle cells can be grown easily in tissue In immunofluorescence, desmin is localized together culture and induced to differentiate. Thus questions of with a-actinin along myofibril Z lines. Antibodies to assembly processes can be approached rather easily in chick smooth muscle desmin, prepared against the protein vitro. purified by preparative SDS gel electrophoresis prior to (4) Differentiated muscle sarcomeres are very well immunization, cross-react with myofibril Z lines in all defined structurally, and structural analysis of their three avian species. These antibodies do not cross-react individual components can be approached using a variety with either rat or guinea pig myofibril Z lines. Similarly, 76 they do not cross-react with avian or mammalian non­ Antibodies were made against desmin and used to muscle cells grown in tissue culture and known to contain characterize the localization of desmin in skeletal muscle 0 cytoplasmic 100 A filaments. by immunofluorescence. (The structure of skeletal muscle These results demonstrate that desmin is highly is much more suitable for microscopy than is that of conserved within avian muscle cells and within mamma­ smooth muscle.) Desmin was found both in the Z lines and lian muscle cells. However, it is both biochemically and where the Z lines come into apposition with the plasma immunologically distinguishable between avian and mam­ membrane. Desmin was also found in the Z lines and malian muscle cells and between muscle and nonmuscle intercalated discs of cardiac muscle. These are all sites cells. We conclude that there are biochemically and where actin structures are linked together or to mem­ immunologically specific forms of desmin for avian and branes. From these distributions, we concluded that mammalian muscle cells. Furthermore, within a particu­ desmin forms a network in muscle cells which interlinks lar vertebrate species, there are at least two separate individual myofibrils, at their Z discs, into a single 0 classes of 100 A filaments: the muscle class whose major integrated mechanical unit and also functions in the component is desmin and the nonmuscle class whose major linkage of this unit to the plasma membrane. component is distinct from desmin. Taking into consider­ We have subsequently extracted desmin from smooth ation the immunological specificity reported by other muscle and studied its properties. The solubility prop­ 0 laboratories for the 100 A filaments in glial cells, for erties of both desmin and the small amount of actin that 0 neurofilaments, and for the epidermal 80 A keratin fila- remains KI insoluble with it are the same: they are both ments, we propose that a given vertebrate species insoluble in high concentrations of salt, but are solubilized contains at least four major distinguishable classes of at low pH or by agents that dissociate hydrophobic bonds. 100 A filaments: muscle 100 A filaments (desmin fila­ Desmin may be extracted and purified by repeated cycles ments), glial filaments, neurofilaments, and epidermal of solubilization by 1 M acetic acid and subsequent keratin filaments.. precipitation by neutralization to pH 4. During this process, a constant but nonstoichiometric ratio of actin to 112. CHARACTERIZATION AND FUNCTION OF desmin is attained. DESMIN, THE.$l!BU.NIT OF SMOOI'H Gel filtration on Ultrogel AcA34 in the presence of MUSCLE 100 A FILAMENTS 0.5% Sarkosyl NL-97 reveals a nonmonomeric fraction of investigator: Bruce D. Hubbard actin and desmin that co-migrate through the column. The millions of sarcomeres in a muscle cell are Gel filtration on BioGel P-300 in the presence of 1 M linked together, end to end and side to side in structures acetic acid reveals that both actin and desmin are nearly called Z lines. They are also linked to the plasma monomeric under these conditions. membrane. This serves to mechanically integrate their If the acetic acid is removed from actin and desmin motion and to transmit it through the plasma membrane. by dialysis against water, a gel forms that is composed of The links themselves appear to be mediated by the tangled, intertwining filaments with diameters of 120 to 0 interaction of actin and a variety of poorly characterized 130 A. These filaments react uniformly with both anti- actin cross-linking proteins. Our research is on the actin and anti-desmin antiserum. These results suggest formation and structure of these links. that desmin is the major subunit of 100 Asmooth muscle We began this research by investigating the 100 A filaments and that it may form a complex with actin. The filaments of smooth muscle. These filaments, of unknown existence of such an actin-desmin complex would provide function, are often associated with regions where actin both a physical basis for the hypothesized linking function 0 filaments are linked together or to membranes. 100 A of desmin and an approach for understanding how such filaments are insoluble in 1 M KI, whereas most other links are constructed. proteins, including actomyosin, are soluble in it. In 0 References: smooth muscle that has been enriched in 100 A filaments Cooke, P. (1976) J. Cell Biol. 68: 539-556. by KI extraction, only actin and desmin, a 50,000 Lazarides, E. and Hubbard, B. D. (1976) Proc. Nat. Acad. Sci. USA 73: 4344-4348. molecular weight protein, remain in significant amounts. 77

113. FLUORESCENT LABELING OF MYOFIBRIL called transglutaminases, which have been isolated from PROTEINS CATALYZED BY TRANSGLUTAMINASE erythrocytes, epidermal tissue and liver, and which are Investigator: David L. Gard present in many cell and tissue types (see review by Folk We have developed a technique for fluorescently and Chung, 1973). labeling proteins of the myofibril Z line using the covalent Several observations have led us to postulate that incorporation of the fluorescent lysine analog dansyl­ certain myofibril structures, particularly the Z line, may cadaverine, as catalyzed by guinea pig liver transgluta­ be stabilized by covalent cross-linking of their constituent minase. This enzyme catalyzes a calcium dependent acyl proteins. Chung (1972) has reported the presence of a transfer reaction, linking the primary amine of the transglutaminase activity in smooth, cardiac, and striated fluorescent probe to the y position of glutamine residues muscle, and Loewy (as cited by Folk and Chung, 1973) has of substrate proteins. Epifluorescence microscopy reveals detected the E-(y-glutamyl) lysine dipeptide in isolated that dansylcadaverine is specifically and exclusively in­ myofibrils. This, along with the observation of significant corporated at or near the myofibril Z line. SDS amounts of a high molecular weight material in myo­ polyacrylamide gel electrophoresis, and two-dimensional fibrils, and the specific labeling of myofibril Z lines by an isoelectric focusing/polyacrylamide gel electrophoresis exogenous transglutaminase, suggested that an endogenous indicate that two known Z line proteins, cx-actinin and transglutaminase activity may act to covalently link Z desmin, are labeled by the transglutaminase reaction, as line proteins during myofibril assembly. well as actin, tropomyosin, and a series of proteins with We are pursuing this hypothesis using two ap­ molecular weights near 145,000. In addition some proaches: first, we are attempting to identify the intensely fluorescent material is seen which does not glutamyl-lysine dipeptide cross-link in the high molecular enter a 4% acrylamide gel, suggesting that it possesses a weight protein, on the assumption that this material may very high molecular weight. Since microscopy indicates represent cross-linked Z line proteins; in addition, we are that dansylcadaverine is incorporated exclusively at the Z using a technique for in vitro culture of developing muscle line, the proteins labeled, including the high molecular cells to study the levels and localization of transgluta­ weight material, must be of z line origin. minase during the differentiation process from prefusion This technique is useful, in that it provides evidence mononucleate myoblasts to striated multinucleate myo­ for the Z line localization of several myofibril proteins, tubes. independent of the problems associated with antibody References: specificity. The Z line nature of a-actinin and desmin has Chung, S. I. (1972) Ann. N.Y. Acad. Sci. 202: 240-255. Folk, J. E. and Chung, S. I. (1973) Adv. Enzymol. 38: been confirmed by transglutaminase labeling, and several 109-191. new putative Z line proteins have been identified. We are currently investigating the nature of the high molecular 115. THE EXISTENCE OF A Z DISC SCAFFOLD IN CHICKEN SKELETAL MUSCLE weight material and the 145,000 dalton protein, to Investigator: Bruce L. G.._ confirm their localization using immunofluorescence. The extraction of glycerinated chicken skeletal 114. COVALENT CROSS-IJNKS BETWEEN MYOFIBRIL muscle with 0.6 M KI leaves a framework of insoluble PROTEINS structural components within each fiber. This residual Investigator: David L. Gard material maintains its integrity even though most of the The formation of covalent cross-links between actomyosin has been solubilized. Light and electron structural proteins appears to be a widespread method for microscopy reveal that this framework is composed of the stabilization of cellular and extracellular structures. transverse planes of material associated with the Z discs, Examples of one type of cross-link, between the £ amino with each Z disc increased in thickness due to the collapse group of lysine residues, and the y carbon of protein­ of material onto it during the extraction with KI. When bound glutamine have been found in keratin, fibrin, and sheared in a blender, there is a preferential cleavage of the plasma membrane of erythrocytes and cultured L this framework along planes perpendicular to the longitu­ cells. The formation of such E-(y-glutamyl) lysine cross­ dinal axis of the muscle fiber; this results in the links is catalyzed by a class of Ca regulated enzymes production of large sheets of interconnected, closely 78 packed Z discs in a honeycomb-like array. Cleavage of a-Actinin is localized within the Z disc; en face extracted fibers occurs in regions formerly occupied by views of Z disc sheets show that a-actinin and desmin the A bands, which have been weakened by the removal of have exactly complementary distributions. myosin. The existence and stability of these planar Z disc Actin is present predominantly within the Z disc, but arrays demonstrate the presence and strength of lateral also between neighboring discs, as is desmin. Actin may connections between adjacent myofibrils. This view is thus be the basis for the underlying framework of z disc supported by transmission electron micrographs of whole scaffolds, forming the backbone elements upon which all mounts of myofibril bundles that have been extracted with other proteins are arranged. KI, which show filamentous bridges spanning between Residual myosin comprises the outer edges of the neighboring Z discs of adjacent fibrils. SDS polyacryl­ material that collapses onto the Z disc during extraction amide gel electrophoresis shows that this insoluble scaf­ with KI. Tropomyosin is also part of this collapsed fold consists primarily of actin and desmin, with lesser material, but is more closely apposed to the Z disc itself. amounts of a few proteins including a-actinin, myosin, and These distributions suggest that actin and desmin tropomyosin. Since the interfibrlllar Z bridges appear to are largely responsible for the intracellular order, align­ be connected through the T system to the outer plasma ment, end structural and functional integrity of skeletal membrane, this scaffold may be an ideal system for the muscle. study of filament-membrane interactions. The Z bridges may thus serve to integrate myofibrils, mechanically or 117. THE LOCALIZATION OP MEMBRANE SITES IN SKELETAL MUSCLE PIBERS structurally, to align or secure the T system and the Investigators: Elias Lazarides, Bruce L. G~r junctional complexes of the T system and SR (triads) to the Z line, or to help assemble and hold the myofibril Di-dansyl derivatives of amino acids and N-phenyl- together. 1-napthylamine are used to localize membrane hydro­ phobic sites in glycerol-extracted chicken skeletal muscle fibers. Epifluorescence microscopy reveals that such sites 116. IMMUNOPLUORl!SCENT MAPPING OP MAJOR STRUCTURAL PROTEINS IN ISOLATED coincide with the distribution of the transverse tubular (T) Z DISC SHEl!TS system and the sarcoplasmic reticulum (SR) in unex­ Investigator: Bruce L. Gr~ tracted muscle. They are specifically associated with Sheets of interconnected Z discs representing cross­ myofibril Z lines and extend from one Z plane to the next sections of muscle fibers are readily produced from longitudinally along the muscle fiber in between myo­ glycerinated chicken skeletal muscle that has been ex­ fibrils. The hydrophobic probes interact noncovalently tracted extensively with 0.6 M potassium iodide, as with the Z lines and their induced fluorescence can be described previously. Indirect immunofluorescence car­ eliminated by exposure of the myofibrils to ionic deter­ ried out on these Z disc sheets in conjunction with that gents, nonionic detergents, or phospholipase C, before or carried out on extracted myofibrils (thus presenting both after addition of the hydrophobic label. face-on and side views of the Z disc) reveals the Extraction of glycerinated skeletal muscle fibers distribution of proteins that comprise this insoluble with 0.6 M KI removes the majority of sarcomeric actin framework. and myosin and leaves a scaffold of longitudinally inter­ 0 Desmin, the major subunit of 100 A filaments, connected Z planes. Membrane fluorescence remains constitutes the periphery of each Z disc. It does not tightly associated with these Z planes as well as with the appear to be an integral part of the Z disc matrices, but material that interconnects the planes. Shearing of such exists as a network of proteinaceous collars that surround scaffolds results in the cleavage of the longitudinal each and every disc. Desmin may thus form the Z bridges connections and the production of large sheets of inter­ that interconnect neighboring Z discs of adjacent myo­ connected, closely packed Z discs in a honeycomlrlike fibrils; it might also aid in maintaining the structural array. Comparison of the localization of two Z disc integrity of the Z disc, and take part in filament­ proteins, desmin and a-actinin, with that of the membrane membrane interactions involving the T system, sarcoplas­ material reveals that a-actinin is localized in the interior mic reticulum, and plasma membrane. of each myofibril Z disc while both desmin and the 79 membrane material surround each disc. Thus, glycerina­ amounts of protein (typically 10 to 20 µg). It is tion and KI extraction of muscle fibers leave remnants of hypothesized that the two unknown proteins will also be T system and SR membranes tightly associated with the Z localized to the Z line structure, suggesting that they disc honeycomb lattice. Since the Z discs are connected might be part of the actin-desmin complex. at their peripheries through the T system to the plasma membrane, desmin and this membrane structure appear to be connected throughout the whole Z plane up to and PUBLICATIONS including the plasma membrane. The congruent localiza­ Eckert, B. S. and Lazarides, E. (1978) Localization of tion of desmin and the T system strongly suggests that actin in Dictyostelium amebas by immunofluores­ cence. J. Cell Biol. In press. this molecule mediates the adhesion of this membrane Gard, D. L. and Lazarides, E. (1978) Specific fluorescent system around each and every Z disc. labeling of chicken myofibril Z line proteins cata­ lyzed by guinea pig liver transglutaminase. J. Cell Biol. Submitted for publication. 118. LOCALIZATION WITHIN MYOFIBRILS OF Granger, B. L. and Lazarides, E. (1978) The existence of a PROTEINS FROM Kl-EXTRACTED Z disc scaffold in chicken skeletal muscle. Cell. CHICKEN MUSCLE Submitted for publication. Investigators: Karen F. Greif, David R. Balzer Jr. Granger, B. L. and Lazarides, E. (1978) Immunofluorescent mapping of major structural proteins in isolated Z Two-dimensional (2-D) polyacrylamide gel electro­ disc sheets. Cell. Submitted for publication. Hubbard, B. D. and Lazarides, E. (1978) The copurification phoresis of Kl-extracted chicken muscle yields a charac­ of actin and desmin from chicken smooth muscle and teristic protein pattern enriched in actin and desmin. their copolymerization to intermediate filaments. J. Cell Biol. Submitted for publication. Both are major protein constituents of the Z lines of Lazarides, E. (1978) Comparison of the structure, cystribu­ myofibrils. The retention of these proteins after high salt tion, and possible function of desmin (100 A) fila­ ments in various types of muscle and nonmuscle extraction suggests that actin and desmin may form a cells. In: The Molecular Basis of Cell-Cell relatively insoluble complex within the Z line (Granger Interaction, R. A. Lerner and D. Bergsma (Eds.), The National Foundation - March of Dimes Birth De­ and Lazarides, in press). Also present on the 2-D gels are fects: Original Article Series, Vol. XIV, No. 2, pp. 41-63, Alan R. Liss, Inc., New York. two unidentified proteins of molecular weight inter­ 0 Lazarides, I!. (1978) The distribution of desmin (100 A) mediate to that of actin and desmin, one relatively acidic filaments in primary cultures of chick embryonic and the other basic. We are attempting to apply indirect cells. Exptl. Cell Res. 112: 265-273. Lazarides, E. and Balzer, D. R. Jr. (1978) Specificity of immunofluorescence techniques to localize these proteins desmin to avian and mammalian muscle cells. Cell within chicken myofibrils. By using 2-D gel electropho­ 14: 429-438. Lazarides, I!. and Granger, B. L. (1978) The fluorescent resis, it is possible to obtain highly purified proteins with localization of membrane sites in glycerinated skel­ etal muscle fibers and their relationship to the a minimum of purification steps. Protein spots are cut protein composition of the Z disc. Proc. Nat. Acad. from the gels and used to immunize rabbits via injection Sci. USA. In press. into the popliteal lymph nodes. This method of injection Lazarides, E. and Revel, J.-P. (1978) Cell motility and the maintenance of cell shape. Scientific American. In putatively maximizes immunological response to small press.

Professor: Jean-Paul Revel Visiting Associate: Paul B. Bell Jr. Summary: There is a large body of evidence to suggest Research Fellows: Ginny W. Darr, Malcolm E. Finbow, that gap junctions play a very important role in the cell Eva B. Griepp, Nancy L. Shinowara, Barbara Yancey Graduate Students: Richard H. Gomer, Bruce J. economy and may well be involved in such processes as Nicholson, Chung Wang the control of growth and development, malignancy, and Research Staff: David W. Easter, Jean !!dens, Patrick F. Koen, Charlotte A. Miller neural integration, although just how is not yet clear. We are now beginning to acquire a reasonable Support: The work described in the following research reports has been supported by: understanding of junction organization and formation with Harle c. Anthony Fellowship the development of techniques for the isolation of McCallum Fund National Institutes of Health, USPHS relatively intact gap junction proteins in good yield, the Northwest Area Foundation development of criteria for the recognition of gap Ruddock Fund University of California, Los Angeles junction proteins, the availability of several approaches 80 for rapid screening for gap junctions, and the emerging used to obtain large amounts of gap junctions, pure by understanding of factors affecting their formation. With morphological criteria, using chicken livers as starting those advances well under way we can hope to character­ material. Analysis by SDS-PAGE shows a characteristic ize junction proteins biochemically and to develop im­ doublet around 10,000 daltons as well as some material of munological reagents which may give us specific answers slightly higher molecular weight. The isolated material is about this highly conserved intercellular structure. therefore similar to the gap junction proteins found by SDS-PAGE in fractions of purified junctions from several 119. ISOLATION AND CHARACTERIZATION OF GAP mammalian livers (Biology 1977, No. 129). JUNCTIONS FROM RAT LIVERS Investigators: Malcolm B. Finbow, David W. Easter, 12L PHYLOGENY OF GAP JUNCTIONS Jean-Paul Revel Investigators: Richard H. Gomer, Jean-Paul Revel We have devised a rapid method for the isolation of We are confirming the long-felt suspicion that gap large amounts of gap junctions from rat liver without need junctions are ubiquitous throughout the animal kingdom. of the proteolytic step which was previously required to Using freeze-fracture electron microscopy, we are making obtain highly purified junctions (Biology 1977, No. 129). In a systematic search of the various animal phyla. So far, the present approach mitochondria, lysosomes, and other we have observed gap junctions in Rotifers, Nematodes, contaminating materials are removed from the crude and Echinoderms, and confirmed previous observations in homogenate by low speed centrifugation. The pelleted Molluscs, Annelids, and Chordates. We are trying to use material containing plasma membranes is treated with the computers to correlate plasma membrane phospholipid detergent Sarkosyl NL97 and gap junctions as well as composition and aspects of the morphologies of the gap other Sarkosyl insoluble material are pelleted by high junctions seen in the various phyla. This has led to other speed centrifugations. This pellet is then treated with experiments on the effect on gap junction morphology of 6 M urea and high pH to remove collagen and uricase, certain drugs that alter phospholipid lateral motility after which the junctions are purified on sucrose gradi­ within the plasma membrane. ents. Analysis of the gap junction fractions by SDS polyacrylamide gel electrophoresis shows two intensely staining bands at 27 ,OOO and 25,000 daltons with somewhat 122. EFFECT OF PHENOXYBENZAMINE ON GAP less intense bands at 37,000 and 43,000 daltons and very JUNCTIONS IN REGENERATING RAT LIVER diffuse bands between 54,000 and 60,000 daltons. A mild Investigators: Barbera Yancey, Jean Edens, Jean-Paul Revel trypsin treatment causes disappearance of the doublet at 27 ,000 and 25,000 as well as of the other, more diffuse Surgical removal of two-thirds of the liver mass of bands with the concomitant appearance of a band at weanling rats results in the loss and subsequent reappear­ approximately 10,000 daltons, similar to that shown ance of hepatocyte gap junctions (Yee and Revel, 1978). previously to be degraded junctional proteins. Preliminary By freeze fracturing and morphometry we have shown evidence from cyanogen bromide treatment shows that that gap junctions are present at near normal levels and polypeptides of identical molecular weight can be found size until 24 hr and are rare or totally absent during the after treatment of either 10,000 or 27 ,ooo dalton bands. span of 28 to 35 hr. They begin to reappear 36 hr after hepatectomy. It does seem likely therefore that the high molecular weight bands seen are one of the major components of rat MacManus et al. (1973) have shown that liver cell liver gap junctions. cyclic AMP levels are elevated throughout much of the first 24 hr after hepatectomy and that a S-adrenergic 120. IDENTIFICATION OF LOW MOLECULAR WIDGHT blocker, phenoxybenzamine, can reduce this increase. BANDS FROM AVIAN GAP JUNCTIONS Livers of animals treated with phenoxybenzamine 8, 11, Investigators: Richard H. Gomer, Malcolm E.. Finbow, and 18 hr after hepatectomy have been examined by Jean-Paul Revel freeze cleaving. We find that the junctions are still We have now developed isolation procedures appli­ present in very large number at 28 hr, often beyond what cable to the purification of gap junctions from tissues of is seen in the normal liver. Besides a population of large, animals in a wide range of phyla. These procedures were relatively normal gap junctions, there are numerous Very 81 small junctions grouped together in particle-poor areas of 124. ESTABLISHMENT OF SYNCHRONY BETWEEN membrane or closely associated with tight junctions. HEART CELIS IS BLOCKED BY PROTEASE INHIBITORS While the number of junctions is elevated, the overall Investigators: Eva P. Griepp, Jean-Paul Revel proportion of pericanalic-ular membrane occupied by gap junctions is actually somewhat decreased when phenoxy­ We are using the acquisition of synchronous beating benzamine-treated hepatectomized rats are compared between 7-day chick embryo heart cell aggregates and with normal animals, but not nearly as markedly as in the layers to investigate gap junction formation (Biology 1977, case for hepatectomized rats not treated with the drug. No. 130). As reported (Griepp and Bernfield, 1978) there is normally a gradual increase in the percentage of It would thus appear that there may be a relationship between levels of cyclic AMP and the presence of gap synchronous aggregate-layer pairs, but pretreatment with trypsin gives a high initial rate of synchronization. Other junctions. enzymes such as chymotrypsin and pronase have less Referenees: MacManus, J. P., Braceland, B. M., Youdale, T. and marked effects, while thrombin, collagenase, and neur­ Whitfield, J. F. (1973) J. Cell Physiol. 82: 157-164. aminidase have essentially none. We have now found that Yee, A.G. and Revel, J.-P. (1978) J. Cell Biol. 78: 544. phenylmethylsulfonyl fluoride (PMSF) or aprotinin (Trasylol), both protease inhibitors which do not prevent 123. GAP JUNCTION FORMATION IN RAT LIVER the beating of heart cells, cause a complete inhibition of Investigators: Barbara Yancey, Jean-Paul Revel synchronization. Even the enhancing effect of trypsin During response of weanling rats to partial hepa­ pretreatment is blocked, suggesting that the initial trypsin tectomy, hepatocyte gap junctions disappear and sub­ treatment does not obviate the need for further or more sequently re-form. The first evidence of the re-forma­ specific proteolysis. In a single experiment we found that tion is the appearance of small aggregates of particles tunicamycin, which interferes with the synthesis of which are preferentially located within the pericanalicular glycoproteins, decreases the rate of synchronization. network of tight junctional strands and at the free end of Since concanavalin A also inhibits, a glycoprotein may be the strands extending from this network. A number of involved at some stage. We are investigating the possible aggregates also form in areas of pericanalicular mem­ complex relationship between these biological effects and branes remote from tight junctions. These small junctions the formation of intercellular gap junctions. may be separated from the other surrounding intramem­ Reference: brane particles by rather narrow particle-free halos or Griepp, E. B. and Bernfield, M. R. (1978) Exptl. Cell Res. appear grouped together within relatively large particle­ 113: 263-272. poor areas. Occasionally, rounded, uniformly-sized parti­ cles appearing slightly larger than those within the 125. THE FORMATION OF TIGHT JUNCTIONS AND DEVELOPMENT OF TRANSEPITHELIAL junctions are found within these particle-poor areas, an RESISTANCE IN A CULTURE SYSTEM occurrence very reminiscent of the "formation plaques" Investigators: Eva B. Griepp, David Sabatini*, Jean-Paul Revel seen in other systems (Johnson et aL, 197 4). Many of the small aggregates found in regenerating liver exhibit the One can measure the electrical resistance and even close particle packing typical of gap junctions, but there follow the development of transepithelial permeability are also somewhat more loosely organized structures with barriers (tight junctions) across confluent monolayer rather widely spaced particles. Further growth of gap cultures of dog kidney epithelial cells (MU6K) grown on junctions may be by accretion since at later times well­ collagen discs. As in the case of synchronization of formed junctions often exhibit areas of loosely arranged beating heart cell layers and aggregates, we find that particles at their periphery. Trasylol, a reversible inhibitor of proteolysis, partially Reference: prevents the establishment of such barriers. Phenyl­ Johnson, R., Hammer, M., Sheridan, J. and Revel, J.-P. methylsulfonyl fluoride (PMSF) has only a transient (197 4) Proc. Nat. Acad. Sci. USA 71: 4536-4540. inhibitory effect, and then actually increases the trans­ epithelial resistance. We are further studying the effect of other, more specific inhibitors (antipain, lenpeptin, 82

etc.) on the formation of tight junctions. turns of myelin. The precise nature of these structures is *Department of Cell Biology, New York University School not known at the present time. of Medicine.

128. THE PINE STRUCTURE OF DICYEMIDS 126. ELECTRICAL COUPLING AND GAP JUNCTION FORMATION Investigators: Chung Wung, Jean-Paul Revel Investigators: Nancy L. Shinowara, Eva B. Griepp, A. James Hudspeth, Jean-Paul Revel ln last year's report (Biology 1977, No. 133), we have shown that gap junctions are found between the axial cell The appearance of electrical coupling in cultures of and the peripheral cell of dicyemids, a very primitive 7-day-old heart cells was studied in aggregates placed side organism, parasitic in molluscs. We have continued these by side and allowed to adhere to each other for 7-1/2 hr. investigations trying to establish the presence of other The extent of coupling was examined by simultaneously cell junctions and to define the architecture of the recording from single cells in both aggregates and within organism more clearly. For this purpose we have analyzed single, isopotential aggregates while passing 5 to 10 nA the fine structure in thin sections under a variety of hyperpolarizing current pulses through either intracellular conditions of fixation. The images obtained suggest that electrode. The ratio of aggregate-to-aggregate coupling developing dicyemicls actually form within the vacuoles of to intra-aggregate coupling was 0.4 under control condi­ the axial cell. There is some evidence for cell-cell tions. We examined the effect of various drugs which attachments other than gap junctions but these cannot be have previously been shown to affect the rate of acquisi­ clearly classified as either septate junctions, desmosomes, tion of synchrony between heart cell layers and aggre­ or tight junctions. In spite of numerous different gates. In the presence of cycloheximide, aggregate-to­ techniques of fixation and staining, all that can be aggregate coupling was down to 50% of control values. A observed is a slight increase in density of the extracellular proteolysis inhibitor (Trasylol) also reduced the extent of space in regions where membranes seem to be held some coupling although PMSF, which had proved very effective 0 500 A apart from each other. Attempts at maintaining in abolishing the establishment of synchrony, did not seem the dicyemids in culture, and experiments aimed at to have an effect on coupling between aggregate cells. studying electrotonic coupling between the cells are The results obtained using Trasylol and PMSF in the presently in progress. several approaches described (see Biology 1978, Nos. 124 and 125) suggest that proteolytic steps are involved in intercellular junction formation. 129. ULTRASTRUCTURE OF CELLULAR JUNCTIONS IN DROSOPHILA SALIVARY GLANDS 127. GAP JUNCTIONS IN MYEIJN Investigators: Bruce J. Nicholson, Jean-Paul Revel

Investigators: Nancy L. Shinowara, Jean-Paul Revel The Drosophila salivary gland has been used exten­ In freeze-fractured samples of spinal cord of the sively in studies of cell-to-cell coupling and seems to be electric eel and of the rainbow trout, gap junctions can be an excellent system for the study of the modulation of found in the outermost tongue of myelin wrapping. The junctional activity by modifying intracellular calcium ion identity of the cells forming the junctions with the myelin concentrations (Rose and Loewenstein, 1975). Although is not yet clear, but they are probably of glial nature, and there have been several electron microscopic studies of resemble fibrous astrocytes in the trout samples ex­ thin sections of both Drosophila and Chironomus salivary amined At the present time we have one example (in the glands there is still much controversy as to precisely what eel) of a cell of very different appearance which makes a junctions exist in these organs. We have decided to study contact with both myelin and the process of a putative the salivary gland by freeze-fracturing and have been able fibrous astrocyte. While they are found in central myelin, to show the existence of structures characterized by a gap junctions have not been observed in peripheral myelin collection of intramembrane particles which have pre­ of the fish. There are, however, occasional clusters of viously been identified as gap junctions in arthropods. regularly shaped particles of uniform size associated with Because these structures do not exactly match mam­ extensive areas of tight junctional strands in the outer malian gap junctions in their appearance we are continu- 83 ing these studies to ascertain that we are indeed dealing PUBLICATIONS with gap junctions. Bell, P. B., Miller, M. M., Carraway, K. L. and Revel, J.-P. (1978) SEM-revealed changes in the distribu­ Reference: Rose, B. and Loewenstein, W.R. (1975) Nature 254: 250. tion of the Triton-insoluble cytoskeleton of Chinese hamster ovary cells induced by dibutyryl cyclic AMP. Scanning Electron Microscopy/1978/Vol. II, 130. EXAMINATION OP A TRlTON-INSOLUBLE pp. 899-906. CYTOSKELETON BY SEM Bell, P. B. and Revel, J.-P. (1978) Scanning electron microscopy of cells in culture. In press. Investigators: Paul B. Bell Jr., Marcia M. Miller*, Brown, S. S. and Revel, J.-P. (1978) Cell surface labeling Kermit L. Carrawa~•, Jean-Paul Revel for the SEM. In: Advanced Techniques in Biological Electron Microscopy, J. K. Koehler (Ed.), pp. 65-88, We have developed a method for gently removing Springer-Verlag, Berlin-Heidelberg. the membranes of cells in culture with Triton X-100 Griepp, E. B., Peacock, J., Bernfield, M. and Revel, J.-P. (1978) Morphological and functional correlates of detergent that exposes an insoluble cytoskeletal network synchronous beating between embryonic heart cell for examination in the scanning electron microscope aggregates and layers. Exptl. Cell Res. 113: 273-282. (SEM). We have used this method to observe changes in Johnson, G., Johnson, R. J., Miller, M. M., Borysenko, J. the distribution of the Triton-insoluble cytoskeleton of and Revel, J.-P. (1977) Do cellular slime molds form intercellular junctions? Science 197: 1300. Chinese hamster ovary (CHO) cells following treatment Revel, J.-P. (1978) Biological scanning electron micros­ with dibutyryl cyclic AMP (Bt cAMP). In untreated CHO copy for physicists and engineers. Scanning Electron 2 Microscopy/1978/Vol. I, pp. 829-834. cells, the cytoskeletal material is largely condensed Revel, J.-P., Darr, G., Griepp, E. B., Johnson, R. and around the cell nucleus in a fibrous mat. The fibrous Miller, M. M. (1978) Cell movement and intercellular contact formation. In: Molecular Basis of Cell-Cell material spread on the substratum away from the nucleus Interaction, R. Lerner (Ed.), pp. 67-81, Alan R. Liss, is without obvious order. In CHO cells treated with Inc., New York. Revel, J.-P., Griepp, E. B., Finbow, M. and Johnson, R. Bt2cAMP the nucleus is only sparsely covered with (1978) Possible steps in gap junction formation. cytoskeletal fibers and the cytoplasmic fibers are spread Differentiation. In press. Revel, J.-P., Hemming, U. and Fox, F. F. (Eds.) (1977) over the substratum in a linear pattern. The cytoskeletal Cell shape and surface architecture. In: Progress in fibers in these preparations are composed largely of actin Clinical and Biological Research, Vol. 17, Alan R. Liss, Inc., New York. and a 55,000 dalton molecule tentatively identified as the Revel, J.-P. and Solursh, M. (1978) Ultrastructure of subunit protein of 10 nm filaments. Thus, it appears that primary mesenchyme in chick and rat embryos. Scanning Electron Microscopy/1978/Vol. II, pp. the previously reported cAMP-stimulated changes in 1041-1046. microtubule distribution are accompanied by parallel Yee, A.G. and Revel, J.-P. (1978) Loss and reappearance of gap junctions in regenerating livers. J. Cell Biol. changes in the distribution of microfilaments and 10 nm 78: 544. filaments. *City of Hope Medical Center, Duarte, California. **Department of Biochemistry, Oklahoma State Univer­ sity, Stillwater, Oklahoma.

Professor Emeritus: Anthonie Van Harreveld al., 1974) and for the fixation of stages in transmitter release of synaptic vesicles (Heuser, 1976). During fusion SUpport: The work described in the following abstract has been supported by Divisional Funds. the boundary between a frozen surface layer and the unfrozen tissue moves into the material. It has been 131. RATE OP FREEZING OP TIIIN SECTIONS OP proposed to use this progression to arrest successive AQUEOUS MATERIAL stages in the transmitter release from presynaptic endings Investigator: A. Van Ilarreveld stimulated at the moment when or slightly before the Rapid freezing followed by substitution fixation or tissue is frozen (Trubatch and Van Harreveld, 1977). The fracturing for electron microscopy holds great promise for rate of fusion is not precisely known. Estimates have the investigation of fast physiological processes. It has been made ranging from 1 µm/msec (Van Harreveld et al., been used to arrest muscle contraction (Van Harreveld et 1974) to 15 µm in 0.3 msec (Heuser, from equations by A. F. Huxley). 84

The method used to determine the rate of fusion was freezing is high during the first msec (4 µm/msec) but based on the large difference of the dielectric constants slows down markedly during the ensuing 10 msec. At the of water (!:80 cgse units) and ice (_:!:2.5 cgse units at end of freezing the rate of fusion increases again, often -21°C). Thin slices (50 to 60 µm) of a gelatin gel were very markedly. This unexpected finding may be due to used as the dielectric in a plate condenser. The slice was undercooling of the unfrozen part of the section which placed on one of the metal plates of the condenser and may then freeze not by a progressing plane of fusion, but then dropped on a silver freezing surface kept at about "en masse.11 -200"C which formed the other plate of the condenser. A References: 20,000 cycle alternating current was passed through the Heuser, J. E. (1976) Pathogenesis of human muscular condenser. At the moment the gelatin makes contact dystrophy. In: Proc. 5th Internat. Scientific Con­ ference of the Muscular Dystrophy Association, L. with the silver surface a large ionically conducted current P. Rowland (Ed.), pp. 61-72. passes through the condenser. In about 0.5 msec the Trubatch, J, and Van Harreveld, A. (1977) Internat. Congr. Physiol. Sci., Paris 1977. surface freezes over and the much reduced current is now Van Harreveld, A., Trubatch, J, and Steiner, J. (1974) J. conducted capacitatively. In the ensuing 15 to 30 msec Microscopy 100: 189-198. the current decreases, reaching a stable minimum when PUBLICATIONS the entire section is frozen. Considering the freezing assembly as two condensers in series, one with the frozen, Trubatch, J., Loud, A. V. and Van Harreveld, A. (1977) Quantitative stereological evluation of KCl-induced the other with the unfrozen material as dielectric, it can ultrastructural changes in frog brain. Neuroscience be shown that the thickness of the frozen layer is a 2: 963-974. function of the reciprocal of the current passing through Van Harreveld, A. (1978) Two mechanisms for spreading depression in the chicken retina. J. Neurobiol. In the condenser. The rate of fusion can therefore be press. plotted against time. As could be expected the rate of CELLULAR NEUROBIOLOGY

Jeremy P. Brockes A. James Hudspeth Henry A. Lester Felix Strumwasser

87

Assistant Professon Jeremy P. Brockes vations, are being used to investigate the interaction of Research Staff: Don J. Nishiguchi Schwann cells with inert fibers of an appropriate diam­ Support: The work described in the following summary eter, with neuronal cell lines that form thick neurites in was supported by the Medical Research Council (U .K.). vitro, and with cultured sensory neurons of the rat dorsal Summary: This laboratory is concerned with under­ root ganglion, which are known to be myelinated in vivo standing the cellular and molecular basis of various and in vitro. interactions that occur in the peripheral nervous system Schwann cells also undergo an interaction with (PNS). We are particularly interested in the Schwann cell, muscle which induces another differentiated function. the principal glial cell in the PNS, which is responsible for After denervation of a skeletal muscle, the Schwann cells elaborating the myelin sheath in concert with certain migrate into the old end plate and begin to release nerve axons. During the last two years we have applied acetylcholine. This is believed to reflect induction of the immunological methods to both identify and purify the synthetic enzyme choline acetyltransferase in the Schwann cell in tissue culture. In cultures of dissociated Schwann cell. This interaction presents a relatively neonatal rat sciatic nerve, the Schwann cells display a accessible model for investigating the developmental specific surface antigen called Ran-1, while the fibro­ commitment to a particular transmitter. We are cur­ blasts of the nerve connective tissue carry another rently investigating the synthesis of acetylcholine by surface antigen called Thy-1. All of the cells are either purified Schwann cells in isolation, and in co-culture with + - - + Ran-1 Thy-1 , or Ran-1 Thy-1 ; thus by treating the adult denervated or primary embryonic muscle. cultures with antibody to Thy-1 and complement, we are Knowledge of how the Schwann cell is induced to able to remove the fibroblasts, leaving pure Schwann differentiate in these two situations is relevant for our

cells. In the presence of an activity in extracts of the understanding of normal neuronal~lial and synaptic rela­ bovine pituitary, the Schwann cells grow and divide in tionships, and of demyelinating disease. culture, allowing us to study their properties in isolation and in combination with other cell types. PUBIJCATIONS In the PNS, the Schwann cell may either simply enfold an axon to give an unmyelinated fiber, or wrap Brockes, J. P., Fields, K. L. and Raff, M. C. (1977) A surface antigenic marker for rat Schwann cells. around it to produce the concentric organization of the Nature 266: 364-366. myelin sheath. There is now decisive evidence from in Brockes, J. P., Fields, K. L. and Raff, M. C. (1978) Studies on cultured rat Schwann cells. I. Fstablishment of vivo experiments that this distinction is a property of the purified populations from cultures of peripheral nerve. Myelinated axons therefore possess some quality nerve. Brain Res. In press. Brockes, J. P. and Hall, Z. W. (1977) Studies on the origin which is absent from unmyelinated axons and which and identity of acetylcholine receptors in normal triggers the Schwann cell into myelination. We wish to and denervated muscle. In~ Pathogenesis of the Human Muscular Dystrophies, L. Rowlands (Ed.), pp. use the purified Schwann cells to determine what this 171-178, Excerpta Medica. quality is. The myelin sheath has a simplified protein Brockes, J. P. and Raff, M. c. (1978) Studies on cultured rat Schwann cells. II. Comparison with a rat composition, and we find that Schwann cells growing in Schwann cell line. Submitted for publication. isolation in culture, or in association with unmyelinated or Brockes, J. P., Raff, M. C. and Winter, J. (1978) Studies on cultured rat Schwann cells. III. Assays for premyelinated axons in vivo, do not express detectable Schwann cell function. Submitted for publication. levels of the major proteins as assayed by immunofluo­ Fields, K. L., Brockes, J.P., Mirsky, R. and Wendon, L. M. B. (1978) Cell surface markers for distinguishing rescence and immunochemical techniques. It appears, different types of rat dorsal root ganglion cells in therefore, that the nerve is able to induce the synthesis of culture. Cell 14: 43-51. Raff, M. C., Hornby-Smith, A. and Brockes, J. P. (1978) these proteins at the onset of myelination. These assays, Cyclic AMP as a mitogenic signal for cultured rat together with other light and electron microscopic obser- Schwann cells. Nature 273: 672-673. 88

Associate Professor: A. James Hudspeth hair bundles in three dimensions. Stimuli are delivered by Graduate Students: David P. Corey, John H. R. Maunsell, an electromechanical micromanipulator that can move Sandra L. Shotwell Research Staff: Richard A. Jacobs vertically as well as within the horizontal plane parallel to Support: The work described in the following research the hair cell's apical surface. The stimulus is transmitted reports has been supported by: by a hollow glass capillary tube placed over the hair The Hearst Foundation National Institutes of Health, USPHS bundle's tip. Ann Peppers Foundation Bending the hair bundle towards the kinocilium Alfred P. Sloan Foundation depolarizes the cell, while bending away from the kino­ Summ.ary: Hair cells are specialized epithelial cells cilium causes a hyperpolarization; responses as large as which are the primary receptors of the vertebrate inner 24 mV have been recorded. The operating range of the ear and lateral line organ. Each cell is a mechanoreceptor transducer is less than ~0.3 µm, which corresponds to an which produces electrical signals in response to move­ angular deflection of the hair bundle through !_2°. A line ments of its hair bundle, a cluster of large microvilli and a connecting the kinocilium to the center of the hair bundle single kinocilium projecting from the cellular apex. The coincides with the axis of greatest sensitivity of the cell; nature of the stimulus that evokes hair bundle displace­ this has been designated the Y axis, or D° of stimulus ment determines the modality to which a given hair cell is rotation. Displacement of the hair bundle at 9D° of sensitive: sound, vibration, angular acceleration, linear stimulus rotation, along the X axis, elicits little or no acceleration, or water movement. The electrical response response. The sensitivity of the cell, expressed as the induced in the cell by appropriate stimulation modulates amplitude of response per unit of hair bundle displac~ the release of a chemical transmitter from synaptic sites ment, falls from a maximum of 50 mV /µmat 0° to zero at on the basal and lateral surfaces of the hair cell, and 9D°. The falloff approximates a cosine function, suggest­ thereby controls the firing rate of the postsynaptic ing that the cell transduces only the Y component of any neurons that convey the signal into the central nervous mechanical stimulus in the line parallel with the cellular system. apex. Figure 1 shows the complete two-dimensional We are interested in the transduction process by response curve of a representative cell. which mechanical stimulation evokes an electrical re­ sponse, the receptor potential, from a hair cell. We have developed an in vitro preparation of hair cells from the sacculus of the frog's inner ear with which it is possible to record receptor potentials intracellularly while the hair bundles of single cells are stimulated with a fine probe. By this means, we are able to study how a cell responds to stimuli of known amplitude, direction, and velocity. We are employing this preparation not only to study the basic electrophysiology of the hair cell's membrane, but also to investigate the physiological effects of treatments which produce permanent damage to hair cells and consequent deafness or vertigo: overstimulation (auditory trauma) " and exposure to aminoglycoside antibiotics (streptomycin, gentamycin, etc.). x 0 ... ~y •• 132. RESPONSES OF HAIR CELLS TO MECHANICAL ~ STIMULATION IN THREE DIMENSIONS Investigators: Sandra L. Shotwell, A. James Hudspeth Figure 1. This diagram represents the response of a hair cell to stimuli in the plane parallel to the cell's apical We are investigating the transduction mechanism of surface. The response of the cell, in millivolts, is shown on the vertical axis of the figure; the other axes vertebrate hair cells by recording their intracellular correspond to displacements parallel with the cell's axis of electrical responses to calibrated displacements of their bilateral symmetry (Y axis) or orthogonal to tt (X axis). 89

When a hair cell is stimulated in the third dimension, definite receptor potential. Both static and dynamic along the Z axis perpendicular to the cell's apical surface, stimuli are transduced, yielding responses as large as depression of the hair bundle depolarizes the cell, with 13 mV. As with normal cells, motions directed towards 1 µm of lowering equivalent to approximately 1 µm of the site of the kinocilium produce depolarizations, while displacement towards the kinocilium. deflections in the opposite direction produce hyper­ polarizations; the total operating range is about 0.5 µm. 133. STEREOCILIA MEDIATE TRANSDUCTION 1N A normal receptor potential is also found after complete VERTEBRATE HAIR CELIB removal of the kinocilium. Investigators: A. James Hudspeth, Richard A. Jacobs These observations indicate that the kinocilium is The association of ciliary structures with sensory not obligatorily involved in the transduction process of transduction in various receptor cells, including the hair vertebrate hair cells. Although the kinocilium may be cells of invertebrates, has led to the suggestion that the necessary for ontogenetic or other reasons, its role in kinocilium is an essential component of the transduction transduction may be limited to serving as a linkage apparatus in vertebrate hair cells. We are testing this between the otolithic membrane or related structure and hypothesis by recording from hair cells after microdis- the stereocilia. There is thus a fundamental difference section of their hair bundles. The attachments of between the hair cells of invertebrates, which have only kinocilia to stereocilia ~re broken either by treatment kinocilia, and those of vertebrates, in which stereocilia with collagenase or by inserting a 0.2 µm-tipped glass evidently mediate transduction. probe between the kinocilium and the remainder of the hair bundle and pulling the kinocilium free. Stimuli are 134. RESPONSE LATENCY OF VERTEBRATE then applied to either the kinocilium or the stereociliary HAIRCELIB cluster while the other is fixed by a stationary probe. Investigators: David P. Corey, A. James Hudspeth Scanning electron microscopy is employed after com­ The delay with which a sensory receptor responds to pletion of the recordings to confirm that the kinocilium is a stimulus provides information about the steps involved detached from the stereocilia (Figure 2). in transduction of the stimulus into an electrical signal. The latency of response of hair cells has not been precisely known, in part because stimuli reach the cells through complex mechanical linkages with unknown de­ lays, and in part because analysis of the extracellular receptor potential (microphonic) is confused by complex current paths in the organs. We are investigating the response latency of hair cells with an in vitro system which largely circumvents these problems. The sensory epithelium of the bullfrog sacculus, containing some 3000 hair cells, is removed and mounted Figure 2. A scanning electron micrograph of the as a partition separating two chambers both ionically and apical surface of a hair cell whose kinocilium has been electrically. The potential difference across the epi­ separated from the remainder of the hair bundle. The 8 µm-long kinocilium, which ends in a 1 µm bulb, is mown thelium is measured with an electrode in each chamber; a prostrate in the left half of the figure; the 50 stereocilia second pair is used to pass current to determine the in the remainder of the bundle remain upright. passive electrical properties of the epithelium. The in No significant intracellular response (<1 mV) is vitro microphonic potential recorded in response to observed if the kinocilium's tip is deflected with a stimulation is the product of the net transduction current stimulus probe through distances up to 3 µm. In the across the epithelium and the epithelial impedance. extreme, stimuli may be so large that the kinocilium is Hair cells are stimulated en masse by moving the detached from the hair cell altogether. On the other overlying otolithic membrane, into which the hair bundles hand, application of the stimulus probe to the residual hair insert, with a piezoelectrically-driven stimulus probe. By bundle, which consists wholly of stereocilia, evokes a monitoring with a photodiode the projected image of 90 either the probe or the membrane, we can measure within endolymph of the bullfrog's ear. The tip diameter of these 5 µsand 10 nm the stimulus delivered to the hair cells. electrodes is sufficiently small (about 1 µm) to allow them A fast pulse stimulus (200 µs duration, 400 nm to pass through the epithelial membrane of the labyrinth amplitude) evokes a response with a rapid (J'lOO µs) rising without causing significant leakage. mectrodes sensitive phase and an exponential decay. Because the time to [K +] and [Ca++] have been used in separate experi­ constant of the decay (.r350 µs) exactly matches the RC ments; studies with electrodes sensitive to [Na+] are in time constant of the epithelium, we believe the response progress. waveform is largely determined by the passive electrical The preliminary results of these experiments are properties of the epithelium. After correction for the shown in Table I. The low activity of Ca++ in the epithelial capacitance with an active filter (transfer function: V = Vi + TdVi/dt), the response waveform TABLE I 0 closely mimics that of the stimulus, with slight broadening Ionic Activities in the Inner Ear of the Bullfrog and a distinct delay of 40 to 50 µs. This short latency, Ion Perilymph Endolymph and its modest sensitivity to temperature (Q : .!'2.5), 10 2.69 mM 91.6 mM suggests that there is not a complex series of processes 0.526 mM 0.101 mM intervening between the receipt of a mechanical stimulus and the resulting memt?rane conductance change. The endolymph is particularly interesting, since it implies that short latency excludes, for instance, intermediate mes­ Ca++ is actively extruded from this fluid compartment. sengers diffusing over distances of greater than about The results of these experiments will be used to 500 nm. produce in vitro preparations which closely match the normal environment of the hair cell, and which allow a 135. IONIC ACTIVmES IN THE INNER EAR OF THE BULLFROG more complete interpretation of the role of the ionic Investigators: John H. R. Maunsell, A. James Hudspeth environment in the electrophysiology of the vertebrate hair cell. The hair cells of the bullfrog sacculus, like all vertebrate hair cells, exist in an unusual environment in PUBLICATIONS which their apical and basal surfaces are bathed by fluids of different ionic compositions. The basal surface is Corey, D. P. and Hudspeth, A. J. (1978) Response latency exposed to the high [Na+], low [K+J perilymph which of vertebrate hair cells. Submitted for publication. Hudspeth, A. J. and Corey, D. P. (1978) Controlled surrounds the labyrinth, while the apical surface, with its bending of high-resistance glass microelectrodes. Am. J. Physiol. 234: C56-C57. hair bundle, faces the endolymph, which has low [Na+] and Hudspeth, A. J. and Jacobs, R. (1978) Stereocilia mediate high [K+]. Because all vertebrate hair cells exist in transduction in vertebrate hair cells. Submitted for publication. similar external fluids, it seems likely that this environ­ Hudspeth, A. J., Poo, M. M. and Stuart, A. E. (1977) ment is important to normal hair cell function. Knowl­ Passive signal propagation and membrane properties in median photoreceptors of the giant barnacle. J. edge of the activities of key ions in these fluids will be Physiol. 272: 25-43. important to an understanding of hair cell operation. Hudspeth, A. J. and Stuart, A. E. (1977) Morphology and responses to light of the somata, axons, and terminal Ion-selective microelectrodes are used to measure regions of individual photoreceptors of the giant the activities of ions in both the perilymph and the barnacle. J. Physiol. 272: 1-23. 91

Associate Professor: Henry A. Lester 137. ROLE OF VOLTAGIHIENSITIVE RECEPI'ORS Research Fellow: Menasche M. Nass IN NICOTINIC TRANSMISSION Graduate Students: David L. Armstrong, Mauri E. Krouse, Robert E. Sheridan Jr. Investigators: Henry A. Lester, Donald D. Koblin•, Research Staff: Baruch Kupperman, Donna R. Williams Robert E. Sheridan Jr. Support: The work described in the following research This investigation compares the conductance in­ reports has been supported by: duced by bath-applied acetylcholine (ACh) and by the Muscular Dystrophy Association of America National Institutes of Health, USPHS same transmitter released from nerve terminals at Elec­ Spencer Foundation Fund trophorus electroplaques. For the former case, dose­ Summary: We continue to explore that exquisite bio­ response relations are characterized by the maximal 2 physical machine, the chemical synapse. A numerical agonist-induced conductance, ry (130 mmho/cm ), and by model indicates that we now have a good quantitative the concentration which induces half ~his conductance; this concentration is termed K and equals 50 µM at grasp of the processes that shape the response to a app quantum of acetylcholine. -85 mV. For the latter case, neurally evoked postsynaptic With photochemical tools we are probing details of currents (PSCs) are characterized by the peak conduc­ the interaction between drugs and the acetylcholine tance during strongly facilitated release, gPSC' and by the receptor. And we are attempting to obtain similar rate constant for decay, a. Since gPSC roughly equals ry, information about the control of membrane properties by it is concluded that the PSC activates nearly all available other chemical messengers, particularly calcium ions. receptor channels. These and other data agree with recent estimates that during the growth phase of the 136. NUMERICAL RECONSTRUCTION OF IBE quanta! response, (1) the ACh concentration is at least QUANTAL EVENT AT NICOTINIC SYNAPSES several hundred micromolar; and (2) most nearby channels Investigators: John C. Wathey*, Menasche M. Nass, are activated. However both a and J{ increase during Henry A. Lester app depolarization, at a rate of about e-fold per 86 mV. To test our quantitative knowledge about nicotinic These observations on voltage sensitivity suggest that a transmission, we have synthesized the available data in a suprathreshold synaptic event is rapidly terminated be-' computer model that simulates events occurring after a cause the action potential abruptly releases ACh mole­ quantum of acetylcholine (ACh) appears in the synaptic cules from receptors. cleft. We assume that molecules can diffuse radially *Present address: Department of Anesthesia, University outward in the plane of the cleft; that they can bind of California, San Francisco, California. reversibly to acetylcholinesterase and undergo hydrolysis; that they bind reversibly to receptors; and that when two 138. DOSE-RESPONSE RELATIONS FOR PHOTO­ agonist molecules bind to a receptor, its channel opens. ISOMERIZABLE NICOTINIC AGONBT The following results agree with experimental data: the Investigators: Merua?he M. NllllS, Henry A. Lester, Mauri E. Krouse growth phase of the quanta! event lasts about 200 µsec; between 1000 and 2000 channels open at the peak; the Using the flash-induced "concentration jump" of declining phase is exponential with a time constant that trans-Bis-Q, we have continued to study kinetic and nearly equals the channel dtn"ation. The model correctly equilibrium aspects of the activation of acetylcholine predicts the effects of changing the membrane voltage receptor channels in mectrophorus electroplaques. Mul­ and of pharmacologically inactivating acetylcholin­ tiple flashes generate a dose vs. conductance ctn"ve in a esterase and receptors. The calculations reveal that the few seconds; the curve has a nonlinear start. Channel response is confined to a small area of membrane--two­ opening rates increase linearly with the concentration of thirds of the open channels occtn" within a radius of agonist; the constant of proportionality provides a lower 0.21 µm--and that nearly 80% of the channels within this limit on the bimolecular rate constant of the agonist area are activated. receptor binding. This is interestingly high for Bis-Q: roughly 3 x 109 M-1 sec-l at 25°C (Q .r3). In the *Undergraduate, California Institute of Technology. 10 presence of d-tuboemarine the opening rate decreases, implying similarly rapid binding of curare. 92

139. ACh Rl!CEPl'OR CHANNELS BEGIN TO OPEN phorus electroplaque is exposed to trans-Bis-Q, a potent WITHIN 30 µSEC APTER AGONllT 11 APPLIED agonist. Some channels are open; these receptors have Investigators: Henry A. Lester, M.,.._,he M. Nass, Matri E. Krouse, Norbert H. Wassermann•, bound agonist molecules. A light flash isomerizes 3 to Bernard F. Bri....,r* 3596 of the trans-Bis-Q molecules to their cis form, a far These experiments measure how soon ACh receptor poorer agonist. This causes a rapid decrease of membrane channels begin to open after agonist appears near re­ conductance (phase 1), followed by a slower increase ceptors. Isolated Electrophorus electroplaques are ar­ (phase 2). Phase 1 has the amplitude and wavelength ranged for transcellular recording. The innervated face is dependence expected if the channel closes within 100 µsec bathed in a 1 µM solution of cis-Bis-Q (3,3'-Bis-[a(tri­ after a single bound trans-Bis-Q is isomerized, and if the methylammonium)methyl] azobenzene). This solution con­ photochemistry of bound Bis-Q resembles that in solution. tains less than 196 trans isomer and has no effect on ACh Therefore, the receptor channel responds rapidly, and with receptors. Trans-Bis-Q, on the other hand, is a potent a hundredfold greater closing rate, after this change in agonist; a concentration of 400 nM produces half-maximal the structure of a bound ligand. Phase 2 (the conductance receptor activation at the resting potential. A light flash increase) seems to represent the relaxation back toward increases the concentration of trans-Bis-Q in the solution equilibrium after phase 1, because (a) phase 2 has the to about 200 nM within 40 µsec (individual molecules of same time constant (1 to 5 msec) as a voltage- or cis-Bis-Q are presumably photoisomerized to trans in less concentration-jump relaxation under identical conditions; than 1 µsec after absorbing a photon). At 34°C the cell and (b) phase 2 is smaller if the flash has led to a net detectably begins to depolarize less than 3 0 µsec after the decrease in [trans-Bis-Ql. Still slower signals follow: start of the light flash. More intense light flashes, we phase 3, a decrease of conductance (time constant 5 to presume, will reduce the latency further. The minimum 10 msec); and phase 4, an equal and opposite increase measurable latency increases at lower temperature (several seconds). Phase 3 is abolished by curare and does not depend on the history of the membrane voltage. We (Q1o.".2), probably because the channel opening rate .decreases. On the other hand, the growth phase of the are considering several mechanisms for phases 3 and 4• miniature postsynaptic current (MPSC) has a much lower temperature dependence (Q of 1.2, Gage and McBurney, 141. PHARMACOLOGY OF BIS-Q 10 1975). Therefore the growth phase of the MPSC is Investigators: Henry A. Lester, David L. Armstroiw, Matri E. Krouse, A. Marty*, probably not shaped primarily by the growth of ACh E. Marder*, J. T...... ,- concentration; it may be limited by lateral diffusion of We have tested this photoisomerizable analog of transmitter within the cleft. Our results indicate that acetylcholine on several cholinergic synapses. In addition channels begin to open rapidly when agonist is suddenly to its well-documented agonist effect on Electrophorus applied, and that only a small part of the synaptic delay is electroplaques, trans-Bis-Q is an agonist at the nerve­ due to interaction of agonist and receptor. muscle synapse of several South American fishes and of Reference: the goldfish, at concentrations less than 5 µM. At the Gage, P. W. and McBurney, R. N. (1975) J. Physiol. 244: 385-407. frog nerve-muscle synapse, the drug has no agonist action, but the response to acetylcholine is abolished by Bis-Q at *Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York. 10 to 20 µM. In the pleural ganglion of Aplysia, there are cells with acetylcholine receptors that increase the permeability either to K+, Cl-, or Na+. None of these 140. RHSPONSE OF ACETYLCHOLINE Rl!CEPl'ORS receptors is affected by Bis-Q. At concentrations of 10-5 TO PHOTOllOMERIZATIONS OF BOUND 4 AGONllT MOLl!CULHS to 10- M, Bis-Q inhibits acetylcholine- and carbachol­ Investigators: M.,.._,he M. Nass, Henry A. Lester, induced depolarizations and contractures of the dorsal Matri E. Kl"ouse dilator and gml muscles of the crab, Cancer pagurus. Cis In these experiments, agonist-induced conductance and trans isomers are equally effective. 4 is measured while a sudden perturbation is produced at the At the frog (Rana temporaria) node of Ranvier 10- -3 ' agonist receptor binding site. A voltage-clamped Electro- to 10 M Bis-Q produces a partial blockade of electri- 93 cally excitable K currents when applied externally; Na 143. A TETHERED, PHOTOISOMERIZABLE CHOLINERGIC AGONIST currents are unaffected When applied internally via a cut Investigators: Henry A. Lester, Mauri E. Krouse, internode, 1 to 2 mM Bis-Q shortens Na currents in a Bernard F. Erlanger*, Norbert H. fashion similar to that of pancuronium and several local Wassermann* anesthetics. Both Na and K currents are diminished as Electrophorus electroplaques are exposed to the well. Cis and trans isomers are equally effective and no reducing agent dithiothreitol; trans 3-(a-bromomethyl}- additional relaxations are produced by a light flash. 3'-[a-(trimethylammonium)methylJ azobenzene bromide *D0partement de Neurobiologie, Ecole Normale SupE!riore, (QBr) is then covalently attached to the acetylcholine Paris, France. receptor. Membrane conductance increases and remains elevated when unreacted QBr is washed away (Bartels et 142. LIGHT-ACTIVATED BLOCKER OF ACETYL­ al., 1971). The increase disappears upon reoxidation with CHOLINE-RECEPTOR CHANNELS 5,5'-dithio-bis-(2-nitrobenzoic acid) and reappears upon Investigators: Henry A. Lester, Mauri E. Krouse, Menasche M. Nass, Bernard F. Erlanger*, further exposure to dithiothreitol; the cycle can be Norbert H. Wassermann* repeated several times. Voltage-clamp studies reveal The cis isomer of N-p-phenylazophenyl carbamyl several quantitative similarities between the conductance choline (EW-1), can be formed from the trans isomer by induced by tethered QBr and that induced by reversible agonists. The conductance depends on the membrane H Q + voltage; voltage-jump relaxations have an exponential Q-N=N-Q-N---t'.:-3). 10 10 min in neutral solution at room temperature; the However rate constants are severalfold greater than for spontaneous reisomerization proceeds much more quickly other agonists. at low pH. We have tested EW-1 on acetylcholine The photochemistry of QBr strongly resembles that receptors in voltage-clamped electroplaques from Elec­ of Bis-Q. When QBr is tethered to receptors, flashes of trophorus electricus. The trans isomer (1 to 2 µM) has light lead to changes in the conductance. These changes little or no effect on neurally evoked postsynaptic have the sign, wavelength dependence, and approximate currents (PSCs), or on steady-state conductances and amplitude expected if trans-QBr is a far more potent voltage-jump relaxations during steady agonist applica­ agonist than the cis isomer. Where light-flash relaxations tion. Following a light flash, however, the PSC decay is are dominated by the cis+trans photoisomerization, the faster. If a flash occurs during steady application of relaxation is a conductance increase and the rate constant agonist, the conductance relaxes to a smaller value. The equals that for voltage-jump relaxations; but in contrast relaxation follows an approximately exponential time with the results with reversible agonists, these rate course and the rate constant exceeds that for a voltage­ constants cio not vary with the amount of agonist (trans jump relaxation just before the flash. Currents induced by isomer) present at the membrane. Where light-flash suberyldicholine are diminished more than those induced relaxations are dominated by the trans+cis photoisomer­ by carbachol; relaxation rates are increased about equally ization, the conductance decreases and the time constant for these two agonists. Electrically excitable Na currents equals the duration of the cis-QBr channel, which seems are unaffected. This blockade of PSCs and of steady about one-fourth that of the trans-QBr channel. agonist-induced currents strongly resembles that produced Reference: by similarly low concentrations of local anesthetics such Bartels, E., Wassermann, N. H. and Erlanger, B. F. (1971) as procaine or QX-222. The blockade is graded with the Proc. Nat. Acad. Sci. USA 68: 1820-1823. flash intensity at 300 to 400 nm, suggesting that cis-EW-1 *Department of Microbiology, College of Physicians and is the blocking form and that it need not be present at its Surgeons, Columbia University, New York, New York. site of action before blocking. *Department of Microbiology, College of Physicians and Surgeons, Columbia University, New York, New York. 94

144.. ION REDISTRIBUTION 1N ELECTROPHORUS acetylcholine receptors before escaping. The acetyl­ ELl!CTROPLAQUl!S cholinesterase in the cleft slows diffusion of curare Investigator: Henry A. Lester slightly: when acetylcholinesterase is removed from the Cholinergic agonists came an increase in the mem­ cleft with purified1 collagenase, curare recovery rates I brane permeability to Na and K at the innervated face of increase only twofolkl. Electrophorus electroplaques. Therefore sustained ex­ References: posure to agonist reduces Na and K concentration gradi­ Betz, W. and Sakmann, B. (1973) J. Physiol. 230: 673-688. Dreyer, F. and Peper, K. (1974) Pfliigers Arch. 348: ents. These gradients are monitored with voltage-clamp 263-272. sequences and pharmacological treatments that selec­ Kuffler, S. W. and Yoshikami, D. (1975) J. Physiol. 244: 703-730. tively measure the Nernst potentials for individual ions. McMahan, U. J., Spitzer, N. C. and Peper, K. (1972) Proc. EK is normally near -90 mV but moves toward zero during Roy. Soc. B181: 421-430. bath application of agonist. Depolarizations by bath­ 146. AFFINITY FOR CURARE OF ACETYlr applied agonist measure primarily this shift of EK' not CHOLINE RECEPTORS short-circuiting of EK by the agonist-induced conduc­ Investigator: David L. Armstrong tance. After a rapid jump of agonist concentration, there Bath-applied curare competitively inhibits the re­ is a fast (millisecond) depolarization due to the conduc­ sponse to brief iontophoretic acetylcholine pulses with an tance increase, followed by a much slower additional 6 1 apparent equilibrium affinity constant, KI= 2 x 10 M- • "creep" due to the shift in EK. Sodium replaces the lost On denervated frog muscle cells, extrasynaptic acetyl­ intracellular potassium: ENa' normally very positive, also choline receptors have a lower apparent affinity for moves toward zero. The shifts in EK and ENa are 5 -1 curare: KI = 2 x 10 M ; and after a pulse of curare, normally reversible but become permanent after blockade inhibition recovers tenfold more rapidly at extrasynaptic of the Na-K pump. In the presence of agonist, the shifts sites than at the nerve-muscle synapse. Previously such can be driven further by passing current of the appropri­ data have been interpreted as evidence for a structural ate polarity. Similar ion redistribution occurs with other difference between synaptic and extrasynaptic acetyl­ drugs, such as batrachotoxin and nystatin, that induce choline receptors. However, we suggest that curare prolonged increases in Na permeability. The redistribu­ inhibits synaptic receptors more effectively because the tions cause little net change in the reversal potential of nerve terminal allows the receptors to buffer the con­ the neurally evoked postsynaptic current. centration of curare in the cleft~ Such buffered diffusion 145. KINETICS OF CURARE ACTION 1N THE may resolve the paradox of- competitive binding and slow SYNAPTIC CLEFT kinetics in situ for many antagonists of synaptic re­ Investigator: David L. Armstroqf ceptors. We have extended our studies of curare inhibition at frog nerve-muscle synapses to cutaneous pectoris muscle fibers. When a monolayer of these cells is dissected and PUBLICATIONS

viewed with Nomarski interference contrast optics Armstrong, D. and Lester, H. A. (1977) Kinetics of curare (McMahan et al., 1972; Dreyer and Peper, 1974) individual action at the frog nerve-muscle synapse. Neurosci. Abstracts 3: 369. nonmyelinated nerve terminals can be identified. After Armstrong, D. and Lester, H. A. (1978) The kinetics of exposure to proteolytic enzymes, the nerve terminal can curare action and restricted diffusion within the synaptic cleft. Submitted for publication. be displaced (Betz and Sakmann, 1973) and drugs ionto­ Koblin, D. D. and Lester, H. A. (1978) Local anesthetic phoresed directly onto the exposed postsynaptic mem­ modification of rates and equilibria at the acetyl­ choline receptor of Electrophorus electroplaques. brane (Kuffler and Yoshikami, 1975). When the nerve Submitted for publication. terminal is displaced, recovery from curare inhibition Lester, H. A. (1978) How do neurotransmitters control ion channels in biological membranes? Biophys. J. 21: proceeds twentyfold more quickly than at normal syn­ 154a. apses. Evidently the nerve terminal obstructs the Lester, H. A. (1978) Analysis of sodium and potassium redistribution during sustained permeability in­ diffusion of curare molecules out of the cleft; conse­ creases at the innervated face of Electrophorus quently each curare molecule binds repeatedly to several electrop!aques. J. Gen. Physiol. In press. 95

Lester, H. A., Koblin, D. D. and Sheridan, R. E. (1978) Nass, M. M. and Lester, H. A. (1977) A photochemical Role of voltage-sensitive receptors in nicotinic probe for the nicotinic drug-receptor complex. J. transmission. Biophys. J. 21: 181-194. Gen. Physiol. 70: 13a. Lester, H. A., Nass, M. M., Erlanger, B. F. and Wasser­ Nass, M. M., Lester, H. A. and Krouse, M. E. (1978) mann, N. H. (1978) Light-activated blocker of Response of acetylcholine receptors to photoisomer­ acetylcholine receptor channels. Abst. Sixth Inter­ izations of bound agonist molecules. Biophys. J. In nat. Biophys. Cong. In press. press. LeSter, H. A., Nass, M. M., Krouse, M. E., Wassermann, N. H. and Erlanger, B. F. (1978) ACh receptor channels begin to open within 30 µsec after agonist is applied. Neurosci. Abstracts 4. In press.

Professor: Felix Strumwasser colleagues. The functional relationship of ELH to these Spencer Senior Research Fellows: Eri Heller, Leonard K. Kaczmarek, John c .. Woolum* atrial gland factors remains mysterious. Graduate Students: Arlene Y. Chiu, Kent R. Jennings, Kaczmarek and Jennings have published a detailed Katherine Dai-Li Lee, Duncan K. Stuart Visiting Graduate Student: Orland D. Mylander** study of afterdischarge in the BC neurons. The prolonged Research Staff: Larry C. Cummings, Floyd Schlechte, refractoriness, after an initial afterdischarge, can be Daniel P. Viele, Annette S. Yuen broken by the application of dopamine as well as a number *Department of Physics, California State University, Los of phosphodiesterase inhibitors at the end of the after­ Angeles, California. **California State University, Los Angeles, California. discharge. They have demonstrated that there is an increase of cAMP within the first two minutes of an Support: The work described in the following research reports has been supported by: initial afterdischarge and that certain cAMP analogs will American Heart Association initiate afterdischarge in the absence of synaptic stimula­ California State University, Los Angeles Lawrence A. Hanson Foundation tion. Mccallum Fund National Aeronautics and Space Administration We describe electrical excitability of dissociated National Institutes of Health, USPHS BCs in cell culture, a preparation that offers much Spencer Foundation Fund promise for physiological and biochemical work. Such Summary: We study how neurohormones initiate and dissociated cells appear to have serotonin receptors on modulate behaviors and the structure, function, and their membranes, according to preliminary iontophoretic mechanisms of neuronal circadian oscillators. In this last experiments. year the neuropolypeptide hormone, ELH, of Aplysia We continue work on the neuronal circadian oscil­ californica has been partially sequenced (Chiu et al.). A lator system in the eye of Aplysia. Woolum describes decade ago, in our laboratory, this neuropeptide was blocking the expression of the circadian rhythm of shown to initiate egg-laying and correlated behaviors. compound action potential (CAP) frequency by X-ray ELH has been demonstrated to be released, during irradiation. Doses that block the CR do not appear to afterdischarge in the bag cell (BC) neurons, along with affect the excitability of membranes, phototransduction, several other peptides (Stuart et al.). action potential generation and propagation, synchrony of Highly specific excitatory actions of ELH on neurons CAPs, or the bursting pacemaker property. Miss Lee is of the isolated buccal ganglia have been demonstrated and investigating the influence of such X-rays on macro­ analyzed, by intracellular recordings (Stuart and molecular synthesis. Strumwasser). There is circumstantial evidence that the Viele and I describe the effects of a pulse of timing of these excitatory actions is correlated with the 2-deoxy-D-glucose (2DG) on the CR of the eye. Accord­ onset of inhibition of behavioral feeding brought on by ing to other reports, 2DG can be used to depress ATP injection of ELH in intact hungry Aplysia confronted by levels within mammalian cells. We find systematic phase food. delays in onset and offset of the activity cycle with 5 x 3 Heller has confirmed the presence of another egg­ 10-4 to 5 x 10- 2DG at phase points 180" apart. It may laying factor in the atrial gland, a part of the reproduc­ be that the circadian oscillation requires a constant supply tive tract. He is presently purifying and characterizing of energy to maintain its time-keeping mechanisms. these factors, one of which was first reported by Arch and Finally Mylander and I report on investigations of 96 the bursting pacemaker mechanism in neuron Rl5 of the Arch, S., Earley, P. and Smock, T. (1976) J. Gen. Physiol. abdominal ganglion. The burst rate generator appears to 68: 197-210. Stuart, D. K. (1978) Ph.D. Dissertation, California Insti­ be relatively insensitive to [Ca++] over a 30-fold range, tute of Technology, Pasadena, California. which is somewhat surprising. Toevs, L. (1970) Ph.D. Dissertation, California Institute of Technology, Pasadena, California.

147. PURIFICATION AND PARTIAL AMINO ACID *Division of Chemistry and Chemical Engineering, Cali­ SEQUENCE OF EGG-LAYING HORMONE (ELH) fornia Institute of Technology. OF APLYSIA CALlFORNICA Investigators: Arlene Y. Chiu, Eri Heller, Michael 148. NEUROSECRETION OF EGG-LAYING HORMONE Htmkapiller*, Dlmcan K. Stuart, AND OTHER PEPTIDES FROM ELECTRICALLY Felix Strumwasser ACTIVE BAG CELL NEURONS OF APLYSIA Investigators: Duncan K. Stuart, Arlene Y. Chiu, A four-step procedure has been developed to purify Felix Strumwasser egg-laying hormone, a neurosecretory polypeptide synthe­ Radiolabeled peptides released from electrically sized and released by the bag cell neurons of the active bag cell neurons in isolated bag cell clusters were abdominal ganglion of Aplysia. This 6000 dalton peptide compared with the polypeptide egg-laying hormone (ELH), (Toevs, 1970; Arch, 1972), with an isoelectric point of 9 to 6000 daltons, pl 9.0 to 9.3, as purified from homogenates 9.3 (Arch et al., 1976) induces Aplysia to lay eggs within of bag cen clusters. A labeled peptide which is 2 hr of injection into the animal's hemocoel, and can selectively released from electrically active bag cell activate specific neurons of the buccal and pedal ganglia clusters co-migrates with ELH from cluster homogenates in vitro (Stuart, 1978). on P-6 gel filtration columns and subsequent isoelectric To purify ELH, the supernatant of a homogenate of focusing gels. Other presumed peptides of unknown radioactively labeled and unlabeled bag cell clusters is function are also released. When bag cells are activated, precipitated with ammonium sulfate. The 0 to 0.25 a released factor(s) induces egg-laying and co-migrates saturation pellet is redissolved and excluded on an A-50 with ELH from cluster homogenates on P-6 columns. At column followed by cation exchange chromatography least three other presumed peptides of unknown function (SP C-25) where ELH is eluted with a salt gradient of 0 to are also released, including one of 5000 to 6000 molecular 250 mM NaCl. Finally, the material is fractionated by weight, pl 4.5 to 5.0. These experiments demonstrate that P-6 gel filtration. ELH (6000 molecular weight, pl 9.0 to 9.3) as purified When material recovered by the above procedure from bag cell cluster homogenates is the major, active was analyzed on a guanidine-HCl agarose column, only one form secreted from bag cells that is involved in the peak of radioactivity was seen and this had an estimated induction of egg-laying. This purified ELH can now be molecular weight of 6500. Similarly recovered material, used to study the physiological effects of a secreted separated on isoelectric focusing gels again showed a neurohormone and their relationship to behavior. single radioactive peak and had an isoelectric point of 9 to 9.2. The amino acid composition and a partial amino acid 149. NEURONAL SITES OF ACTION OF A NEURO­ sequence of this material has been obtained: N-trp-ser­ SECRETORY PEPTIDE, EGG-LAYING HORMONE, his-asn-gln-asp-leu-lys-ala-ile-thr-asp-met-leu-leu-thr-glu­ IN APLYSIA CALlFORNICA gln-ile-arg-glu-. Both cysteine and phenylalanine appear Investigators: Dwtcan K. Stuart, Felix Strumwasser to be absent. Egg-laying hormone (ELH) is a polypeptide of about We plan to generate antibodies specific to ELH, 6000 molecular weight synthesized in the bag cell neurons after conjugation to a carrier such as BSA, in order to of the abdominal ganglion of Aplysia. We studied the develop a radioimmunoassay for quantifying the presence effects of ELH on neuronal activity of the attached head of the molecule in tissues and in circulation; and even­ ganglia (buccal, cerebral, pleural, and pedal), on the tually for immunohistochemical studies of its distribution. isolated buccal ganglia, as well as on feeding in intact Pure, radiolabeled ELH will also be used as a probe for Aplysia. Starved animals {n=7) injected with crude locating receptors in neurons and other target tissues. extract containing ELH stopped eating algae at 17 :': 4 min and their eggs first appeared at 29 ::_ 4 min after injection References: Arch, S. (1972) J. Gen. Physiol. 60: 102-119. at 2D°C. This cessation of eating is significant when 97 compared to the seven controls (p<.01). These data equilibrated with 10 mM sodium phosphate, pH 6.5. The clearly indicate that a suppression of feeding activity column was developed with a linear gradient of NaCl. occurs before the appearance of eggs. ELH applied to the Three major peaks emerged, each showing egg-laying paired buccal ganglia in vitro activates a pair of neurons activity. The excluded material from the SP Sephadex into a tonic pacemaker mode (.rl spike/sec). The time for column also showed egg-laying activity. Samples from the full appearance of this activity in vitro correlates well each peak that showed bioactivity and from the void were with the time for suppression of feeding in vivo. Each of run on acid-urea polyacrylamide gels. Several protein these neurons has an ipsilateral axon in buccal nerve 3. bands could be detected in each sample when the gels The neuron has been identified by intracellular recording were stained with amido black. In another experiment, and has been stained with lucifer yellow, a highly the 25% ammonium sulfate pellet was dissolved and fluorescent intracellular dye. ELH increases the rate of applied to a Bio-Gel A 0.5 m agarose column equilibrated firing of a second pair of buccal neurons, each with an with 6 M guanidine HCl. The egg-laying activity was ipsilateral axon in the cerebrobuccal connective. ELH located at a region corresponding to 4000 to 6000 daltons. when applied to the attached head ganglia causes large We intend to isolate and characterize the various bursts of neuronal activity in pedal nerves to the foot, and atrial gland factors that show egg-laying activity, and increased activity in the nerve to the penis; the relevant compare them to the egg-laying neurohormone that has neurons remain to be identified. These in vitro effects been isolated from the bag cell neurons of Aplysia. were produced by ELH partially purified from bag cell Reference: cluster homogenates using ammonium sulfate precipita­ Arch, S., Smock, T., Frost, W. and Naughton, S. (1978) tion, an anion exchange column, and a gel filtration Neurosci. Abstracts 4. In press. column, or by ELH released from activated bag cells in 151. NEUROTRANSMITrKR AND eAMP CONTROL isolated abdominal ganglia and then fractionated by gel OF AFTKRDISCIIARGE IN NEUROENDOCRINE filtration. The isolated buccal ganglia effects have been BAG CELLS confirmed with fully purified ELH. The ELH effects upon Investigators: Leonard K. Kaczmarek, Kent R. Jennings, Felix Strumwasser the in vitro nervous system support the hypothesis that ELH in vivo acts directly on the nervous system to The neuroendocrine bag cells in the abdominal suppress feeding activity, controlled by the buccal and ganglion of Aplysia generate a long-lasting synchronous cerebral ganglia. ELH may also produce characteristic afterdischarge upon brief electrical stimulation of a movements of the head during egg-laying, controlled pleurovisceral connective. Following their afterdischarge probably by the pedal and cerebral ganglia. these cells become refractory to further stimulation. We find that synchrony, afterdischarge, and prolonged re­ 150. ISOLATION OF EGG-LAYING FACTORS FROM fractoriness are properties that can be expressed in the THE ATRIAL GLAND OF APLYSIA CALIFORNICA isolated asomatic neurites of the bag cells. We have Investigator: Eri Heller distinguished two apparently independent types of refrac­ The atrial gland of Aplysia has recently been found toriness. The first (type I) is seen as a failure of action to contain an egg-laying factor. Arch et al. (1978) potentials generated in the tips of bag cell neurites to reported that the substance is sensitive to enzymatic invade cell somata (Dudek and Blankenship, 1977). This proteolysis, is heat stable, and that the egg-laying activity form of refractoriness may be prematurely induced by can be located at two positions (pH 7.4 and 9.3) on increases in temperature or by partial substitution of isoelectric focusing gels. propionate for extracellular chloride ions. The second We are currently trying to isolate the active form of refractoriness (type II) controls the duration of material. Atrial glands were dissected from the large afterdischarge in the neurites such that stimuli after the hermaphroditic duct of Aplysia californica and homoge­ first afterdischarge (mean duration 30.2 min) produce only nized in low ionic strength buffer. The homogenate was very short afterdischarges or fail to elicit an afterdis­ centrifuged and the supernatant was made 25% in ammo­ charge. We have found that the durations of the first and nium sulfate. After centrifugation, the pellet was subsequent afterdischarges in each experiment are lin­ dialyzed and applied to a SP Sephadex C-25 column early related to the mean firing rate within 1 min of the 98 onset of each afterdischarge. Type II refractoriness is between juvenile (5 g, body weight) and adults (200 g). sensitive to serotonin, dopamine, and the methyl xanthine We have used a variety of individual enzymes and phosphodiesterase inhibitors. Extracellularly applied sero­ combinations for dissociation of cells (collagenase, elas­ tonin and certain of its analogs suppress an ongoing tase, neutral protease, pronase, trypsin). Our best results afterdischarge, while dopamine and the phosphodiesterase were obtained incubating abdominal ganglia in neutral inhibitors, when applied at the end of the first afterdis­ protease (1.25% w/v solution in filtered seawater [FSWJ) charge, generate a subsequent afterdischarge of long for 6 hr at 22"C. After removal of the BC clusters from duration _without further electrical stimulation. The the abdominal ganglion, the connective tissue capsule is mean durations of the subsequent afterdischarges induced removed and the bag cells are disaggregated using a by theophylline were found to be 7.5 times longer than Siliclad-eoated pasteur pipet. Typical yields, with neutral those which could be generated by electrical stimulation protease, of single BCs (from 2 BC clusters) vary between at this time. None of these compounds influenced the 250 and 500 cells. We estimate about a 30 to 50% degree of type I refractoriness. We have shown that both recovery during dissociation of the cluster. We also serotonin and dopamine increase cAMP levels in the obtain glial and other unidentified cells. pleurovisceral connectives (Cedar and Schwartz, 1972) and Many of the BCs and other cells attach to the in the bag cell clusters, and that the occurrence of an plastic surface of the dish (Falcon, 35 mm) and develop afterdischarge is also associated with a specific increase neurites, within 24 hr, in Eagle's minimum essential in total cAMP in the bag cell clusters. cAMP levels peak medium made up in FSW. This medium has been shown to 2 min after the onset of afterdischarge and thereafter support normal resting, action, and synaptic potentials in decline to control values. Moreover, we are able to the intact abdominal ganglion (Strumwasser and Ba.hr, initiate an afterdischarge in an unstimulated isolated 1966). It is interesting that some BCs exhibit as many as neurite preparation by extracellular application of the six processes emanating from the cell body indicating that cAMP analog, 8-benzylthio cAMP. Our data suggest that a heteropolar neuronal condition can be expressed, at serotonin and dopamine may control bag cell activity and least in tissue culture, and is not unique to vertebrate that activation of adenylate cyclase may generate the neurons. afterdischarge. Intracellular recordings from BC neurons without neurites in primary culture have allowed us to conclude References: Cedar, H. and Schwartz, J. H. (1972) J. Gen. Physiol. 60: that the soma itself is capable of supporting a re­ 570. generative action potential (AP). The soma also supports Dudek, F. E. and Blankenship, J.E. (1977) J. Neurophysiol. 40: 1312. repetitive firing, with applied transmembrane current. In our best experiments overshoot-to-undershoot values of 152. THE PEPTIDERGIC BAG CELL NEURONS: APs are 80 mV, and firing frequencies to applied currents MORPHOLOGICAL AND ELl!CTROPHYSIO­ are as high as 8 spikes/sec (14°C). The specific membrane LOGICAL STUDIES OF DISSOCIATED CELLS IN CULTURE resistance and specific capacitance of J'60 µm diameter 2 Investigators: Felix Strumwasser, Leonard K. BCs (without neurites) are approximately 30,000 Q• cm Kaczmarek, Daniel P. Viele and 0.5 µF/cm 2• The surface receptors of BC neurons are The bag cell (BC) neurons exhibit interesting dis­ being explored with iontophoretic applications of various charge properties in the intact BC system such as transmitter candidates. We find that many BCs respond prolonged afterdischarge and postactivity refractoriness. to serotonin (creatinine sulfate) with depolarization, and In order to further dissect these interesting electrophysio­ in those BCs with APs serotonin may cause firing. logical properties we have explored the possibility of References: producing primary cell cultures of BC neurons. The BC Coggeshall, R. E. (1967) J. Neurophysiol. 30: 1263. neurons also synthesize a polypeptide, egg-laying hor­ Strumwasser, F. and Bahr, R. (1966) Fed. Proc. 25: 512. mone, in the intact cluster. Cell cultures have the potential for eventually producing a cell line of inverte­ brate peptidergic neurons since Coggeshall (1967) pre­ sented evidence that their numbers approximately triple 99

153. EFFECTS OF IONIZING RADIATION ON THE produce significant delays in the onset and offset of each CIRCADIAN OSCILLATOR IN APLYSIA EYES activity cycle. The magnitude of the delays are similar at Investigators: John C. Woolum, Felix Stnunwasser -3 the two phase points checked and at 5 x 10 M 2DG, a We are using ionizing radiation to dissect the 4 hr pulse produces an average delay of about 2.3 hr. We circadian oscillator mechanism in the eye of Aplysia. We tentatively conclude that depressing the ATP level tran­ have learned that 40,000 Rads of 50 KVP X-rays will stop siently (by 2DG) slows down the circadian oscillator during the circadian rhythm of compound action potentials such a perturbation. (CAPs) in the eye without changing significantly the size *CT (circadian time in hours) 0 ; onset of projected, i.e., or shape of an individual CAP or the bursting pattern of previous, light schedule; CT 12 ; time of projected dark schedule. the CAPs. This indicates that the circadian oscillator mechanism is distinct from the membrane mechanism References: responsible for the short-term (bursting) rhythm. Though Goldenberg, G. J. and Stein, W. D. (1978) Nature 274: 475-477. there have been reports of circadian rhythms that are Renner, E. D., Plagemann, P. G. W. and Bernlohr, R. W. radiation-sensitive (Rensing, 1968), there are no known (1972) J. Biol. Chem. 247: >765-5776. reports of the effects of radiation on a circadian oscillator. 155. CIRCADIAN RELEASE OF PRESUMED PEPTIDES FROM THE ISOLATED EYE OF APLYSIA We intend to extend these measurements to irradia­ CALIFORNICA tion of particular parts of the eye using lead apertures in Investigators: Dwican K. Stuart, Gerald Audesirk*, Felix Strumwasser the hope of determining if the oscillator is located in a particular region of the eye. We will also compare the A circadian rhythm (CR) of release of presumed proteins and small peptides synthesized by irradiated and peptides from the isolated eye of Aplysia californica is control eyes in order to correlate the rhythm of CAP demonstrated. Substances labeled with radioactive amino output and the rhythm of peptide synthesis. acids and released into the perfusate were separated on gel filtration columns and SDS polyacrylamide gels. In the Reference: Rensing, L. (1968) Z. vergl. Physiol. 62: 214. CR experiments, the perfusate of a single, labeled, dark­ maintained eye was collected every 3 hr for two days

154. PHASE SHIFTING THE CIRCADIAN RHYTHM IN while simultaneously recording the CR of compound THE EYE OF APLYSIA WITH PULSES OF action potentials (CAPs) from the optic nerve. Each 3-hr 2-DEOXY-D-GLUCOSE (2DG) perfusate was applied to a P-2 gel filtration column. Investigators: Felix Strumwasser, Daniel P. Viele Excluded substances (molecular weight >2000) and mate­ 2DG is taken up by cells by the same carrier­ rial fractionated in the region of molecular weight ..r1000 mediated transport system as glucose.- After phosphoryla­ showed a CR which was in phase with the CR of CAPs. tion, 2DG-PO is not recognized by the glycolytic Over half of these substances can be precipitated with 4 pathway and being impermeable accumulates within the trichloroacetic acid. Their release is stimulated by a high cell (Renner et al., 1972). 2DG can be used to set the potassium solution and inhibited by a low calcium solution ATP level within 3T3 cells due to this intracellular that also inhibits CAP activity. This and other previously accumulation. At 6 mM 2DG the ATP concentration could published evidence suggests that the CAPs and the peptide be reduced to 50% of normal within 15 min in 3T3 cells at release are directly produced by electrically coupled 37"C (Goldenberg and Stein, 1978). neurosecretory cells which may also contain the CR We decided to test the effects of 2DG pulses on the oscillator. One or more of these peptides may be a circadian rhythm (CR) of optic nerve impulses in the eye neurohormone and/or transmitter used for synchronizing, of Aplysia assuming, until direct measurements are made, entraining, and/or driving the rest of the animal's CRs. that ATP levels are depressed during the 2DG pulse. The *Present address: Friday Harbor Laboratories, University paradigm involved administering a 4 hr pulse of 2DG (5 x of Washington, Friday Harbor. 10-4 to 1 x 10-2 M) within 1 hr after dissecting eyes and replacing the 2DG with 10 mM glucose at the end of this pulse. To date 2DG pulses started at CT* 18.5 or CT 6.5 100

156. THE EFFECTS OF Ca++/Mg++ ON THE In conclusion burst rate in Rl5, at 14°C, is relatively ++ PROPERTIES OF THE BURST GENERATOR insensitive to [Ca J in the range 2 to 66 mM when the IN R15 total divalent concentration is maintained at 66 mM. On Investigators: Orland D. Mylan-, Felix Strumwasser the other hand the BPP is largest at low [Ca ++]-high Experiments were conducted on the neuron R15 to [Mg++] and declines monotonically at high [Ca ++]-low determine the sensitivity of the burst generator to [Mg++J. This decline in the BPP with increasing [Ca++} is manipulations of the Ca++ and Mg++ environment of the the likely explanation of the concurrent decline of the cell. Previous findings by Barker and Gainer (1975) spike rate. indicated that the amplitude of the slow pacemaker wave leading to burst generation (BPP [bursting pacemaker Reference: Barker, J. L. and Gainer, H. (1975) Brain Res. 84: 479-500. potential]) increased monotonically with increased Ca++ concentration (between O and 60 mM) at 21°C. PUBIJCATIONS Since it was claimed that the amplitude of the BPP depended on [Ca++] it seemed reasonable to expect some Audesirk, G., Kallo, J. and Strumwasser, F. (1978) ++ Humorally mediated influences of the eyes on the effects of [Ca ] on the rate of the burst generator. We activity of the neuron R15 of Aplysia californica. therefore examined the effects of varying [Ca ++]--from Brain Res. Submitted for publication. Audesirk, G. and Strumwasser, F. (1978) Free running 2 mM to 66 mM--on the burst rate, the spike rate, and the circadian rhythmicity of spiking in the neuron R15 BPP at 14°C. The divalent cation concentration was kept of Aplysia californica in the isolated, intact CNS with eyes. Brain Res. Submitted for publication. at 66 mM by adjusting [Mg++]. Kaczmarek, L. K., Jennings, K. and Strumwasser, F. In examining the effects of varying [Ca++] on the (1978) Neurotransmitter modulation and cAMP cor­ relates of afterdischarge in neuroendocrine bag cells BPP amplitude we could not verify the increase in the of Aplysia. Soc. for Neuroscience, Abstracts IV. In BPP with increasing [Ca++]. Our results (n;19 cells) show press. Kaczmarek, L. K., Jennings, K. and Strumwasser, F. an essentially monotonic decline of BPP amplitude with (1978) Neurotransmitter modulation, phosphodiester­ increasing [Ca ++1 with an anomalous extra decline at ase inhibitor effects and cAMP correlates of after­ discharge in peptidergic neurites. Proc. Nat. Acad. 7 mM Ca++. The BPP amplitude reaches 50% of its value Sci. USA. In press. at 2 mM Ca++ between 5 and 7 mM Ca++. Strumwasser, F., Alvarez, R. B., Viele, D. P. and Woolum, J. C. (1978) Structure and function of a neuronal The burst rate in R15 is quite variable from circadian oscillator system.. In: Biorhythm and its preparation to preparation and even within the same Central Mechanism, Naito International Symposium, Elsevier, North Holland. In press. preparation over 24 to 48 hr periods. 50% of the 19 R15s Strumwasser, F., Kaczmarek, L. K. and Viele, D. (1978) The peptidergic bag cell neurons of Apiysia: Mor­ had burst rates between 16 and 35/30 min when measured phological and eiectrophysiological studies of dis­ in artificial seawater (ASW) at 14°C on the first day of the sociated cells in tissue culture. Soc. for Neuro­ experiment. science, Abstracts IV. In press. ++ Stuart, D. K., Audesirk, G. and Strumwasser, F. (1978) To examine the effects of [Ca ] on burst rate, the Circadian release of presumed peptides from the following paradigm was used: the seawater solution isolated eye of Aplysia. californica. Brain Res. Submitted for publication. around the ganglion was removed and replaced with ASW Stuart, D. K., Chiu, A. Y. and Strumwasser, F. (1978) for 30 min. Seven Ca++ concentrations (2, 5, 7, 15, 22, 44, Neurosecretion of egg-laying hormone and other peptides from electrically active bag cell neurons of 66 mM) were tested in either ascending or descending Aplysia. J. Neurophysiol. Submitted for publica­ order, each test period being 30 min. An ASW test always tion. Stuart, D. K. and Strumwasser, F. (1978) Sites of action of followed each Ca++ test. the polypeptide egg-laying hormone (ELH) in the When burst rate was normalized to 100% in each head ganglia of Apiysia californica. Soc. for Neuroscience, Abstracts IV. In press. prior ASW test there was little effect due to changes in Stuart, D. K. and Strumwasser, F. (1978) Neuronal sites of [Ca++1 between 2 to 66 mM. The spike rate, however, action of a neurosecretory peptide, egg-laying hor­ mone, in Aplysia californica. J. Neurophysiol. showed a monotonic decline with increasing [Ca++ J. Submitted for publication. These results were the same when a synaptically cor­ Woolum, J. C. and Strumwasser, F. (1978) Membrane­ potential-sensitive dyes for optical monitoring of rected burst rate was computed. activity in Aplysia neurons. J. Neurobiol. 9: 185-193. 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

103

Associate Professor: John M. Allman Paula Berry from the Department of Cytogenetics of the Visiting Associate: C. B. G. Campbell City of Hope National Medical Center, we are mating our Speneer Research Fellow: Joel Myerson Weizmann Pellow: James F. Baker owl monkeys according to karyotype. Graduate Students: William T. Newsome III, Steven E. Petersen General Referenees: Visiting Graduate Student: EveLynn McGuinness• Elliot, M. W., Sehgal, P. K. and Chalifoux, L. V. (1976) Research Staff: Monica Cooper, Francis M. Miezin, Eric Lab. Anim. Sci. 26: 1037-1040. Saund, David W. Sivertsen Ma, N. S. F., Jones, T. c., Miller, A. C., Morgan, L. M. Laboratory Staff: Margaret M. Beloian, Wanda J. Dunn, and Adams, E. A. (1976) Lab. Anim. Sci. 26: Geri D. Joelson, Deborah P. V. Loeppky 1022-1036.

*Department of Psychology, Vanderbilt University, Nash­ ville, Tennessee. 157. THE PHYSIOLOGY OF PRIMATE VISUAL Support: The work described in the following research PERCKPTION reports has been supported by: Danforth Fellowship Investigators: John M. Allman, Joel Myerson, Mccallum Fund Francis M. Miezin National Institutes of Health, USPHS National Science Foundation Our goal has been to develop behavioral preparations Spencer Foundation Fund for the study of visual perception which will enable us to Weizmann Fellowship transcend the traditional neurophysiological approach that Summary: During the past year we have continued our treats the organism as a passive recipient of sensory investigation of the physiology of visual perception in information. To that end we are studying binocular primates. We have found that the macaque monkey can rivalry, in which the perceived stimulus changes although be trained to report the visual illusions experienced during the physical stimulus remains the same. We trained a perceptual rivalry and that the monkey's performance monkey, Macaca fascicularis, to report changes in the very closely parallels human performance under the same direction of movement of bar gratings by tapping one of conditions. This similarity in performance suggests an two keys. The monkey was then exposed to rivalry­ underlying similarity in physiological processes and en­ inducing stimuli (gratings moving in opposite directions ables us to explore the mechanisms of higher perceptual for each eye). This causes the perceived scene to phenomena with single neuron recording in monkeys. In alternate abruptly every few seconds from what one eye our continuing investigation of the functional division of sees to what the other eye sees so that the bars labor among the cortical visual areas in owl monkeys, we periodically appear to reverse their direction of move­ have found that neurons in the middle temporal (MT) and ment. The rate of perceived alternation in direction of the adjacent dorsolateral (DL) areas tend to be far more movement as reported by the monkey and by human directionally selective than neurons in the dorso-inter­ subjects tested with the same procedure was an increasing mediate (DI), dorsomedial (DM) and medial (M) areas. function of the velocity of the gratings (Figure 1). In Finally, our efforts to develop breeding colonies of several addition, gamma functions with nearly equivalent param­ primate species in order to establish a secure supply for eter values describe the distributions of rivalry phase future research with these increasingly rare animals have durations in both monkey and man (Allman et al., 1977). continued to be successful. Fourteen marmosets (Cal­ These results attest to the strong similarity between lithrix jacchus) and three lesser bushbabies (Galago human and macaque visual systems. senegalensis) have been born and reared in our laboratory. The fact that a monkey can be trained in this We also are making an important step toward the manner suggests that it will be possible to record from successful breeding of owl monkeys (Aotus trivirgatus). single neurons in visual cortex of monkeys who are Until recently owl monkeys have been very difficult to experiencing binocular rivalry and reporting their percep­ breed in captivity. However, it has been discovered that tions. The rivalry-inducing stimuli which we have used owl monkeys have at least 11 different karyotypes permit the testing of hypotheses concerning underlying (ranging from 46 to 54 chromosomes) (Ma et al., 1976), neural mechanisms. The waxing and waning of activity in and when owl monkeys are matched karyotypically, cortical neurons showing directional selectivity or differ­ breeding performance dramatically improves (Elliot et al., ential responsiveness to monocular stimulation, if cor­ 1976). In collaboration with Dr. Ray Teplitz and Ms. related with reported perceptual changes, would be 104

''° and the background activity vary from trial to trial, even when the same stimulus is used. Only through random presentation can one accurately determine whether changing the stimulus produces changes in the discharge rate. On-line data analysis provides, for each set of parameters, a post-stimulus histogram displayed on a storage scope, which is updated after each stimulus presentation. A maximum discharge rate for each set is tallied after the complete random presentation. -HUMANS --MACAQUE A second set of programs is used for off-line data 1 CYCLE PER DEGREE 4 DEGREE APERTURE analysis. Data summaries for each cell are produced with response rates corrected for spontaneous firing rate and various simple indices computed. The program next classifies cells into groups based upon anatomical location

VELOCITY of the cell (see Figure 2 for a summary diagram using DEGREES PER SECOND MEDIAL AREAS N=192 Figure 1. Rate of rivalry alternation as a function of grating velocity in macaque and human subjects. 30

"'::::w 20 evidence for selective facilite,tive and/or suppressive u mechanisms. d 10 Reference: z Allman, J. M., Myerson,. J. and Miezin, F. M. (1977) Soc. '--r-~~~~~~-+--~,_Q Neurosci. Abstracts 3: 551. 0 .2 ..4 .6 .8 1.0 1.2 1..4 >1..4

158. QUANTITATIVE ANALYSIS OF SINGLE UNIT DATA LATERAL AREAS N = 138 Investigators: Francis M. Miezin, Steven E. Petersen 30 Mapping studies have shown that several complete ::::I 20 "'w Visual field representations exist in extrastriate cortex u (Allman and Kaas, 1974, 1975, 1976), raising the question d 10 of how these areas differ in function. Examining the z L.....,--~~~~~~~~--1-<,..!=l discharge properties of single isolated neurons in a more 0 .2 ..4 .6 .8 w u 1..4 "..4 quantitative fashion may help answer this question. With '[)' INDEX this in mind, a series of minicomputer programs was Figure 2. Histograms of the directionality index for developed to (1) quantify unit discharge characteristics in neurons in the medial group (MT and DL) and lateral group each area to a variety of stimuli; (2) assess which of these (DI, DM, M) of cortical visual areas in the owl monkey. characteristics are common to an area; and (3) determine these data). Statistical tests (t-test, linear regression and which characteristics distinguish one area from another. Pearson's correlation coefficient) are performed on the The data acquisition program allows initial qualita­ grouped indices to determine (1) whether indices for tive characterization of the neuron by allowing the groups are significantly different, and (2) which indices stimulus to be manually joystick-controlled. The experi­ are correlated within a particular group and/or across menter can then specify which stimulus parameters to use several groups. (orientation, velocity, etc.) and turn stimulus presentation References: and data acquisition over to computer control. Addition­ Allman, J. M. and Kaas, J. H. (1974) Brain Res. 76: ally, one of the stimulus parameters can be varied in 247-265. random sequence using specified values. Random presen­ Allman, J. M. and Kaas, J. H. (1975) Brain Res. 100: 473-487. tation proves very important since the unit discharge rate Allman, J. M. and Kaas, J. H. (1976) Science 191: 572-575. 105

159. FUNCTIONAL LOCALIZATION OF NEURONAL species as presumably do the behavioral capacities of RESPONSE PROPERTIES IN EXTRASTRIATE different species. Our goal has been to determine which VlSUAL CORTEX OF THE OWL MONKEY of the set of visual maps found in different primate Investigators: William T. Newsome Ill, James F. Baker, Francis M. Miezin, Joel Myerson, species are characteristic of the order as a whole and Steven E. Petersen, John M. Allman which are specializations that have evolved only within We have quantitatively studied response properties certain groups of primates. Most of the information on of 330 single neurons in five extrastriate visual areas in the organization of the visual system in primates comes chronically prepared, sedated owl monkey (Aotus from New and Old World monkeys. Our goal has been to trivirgatus). The data thus far obtained show a marked compare the organization of the visual system in these difference in the responses to moving bar stimuli between better known species with a representative of the strep­ cells in the medial group of areas (DM, DI, and M) and sirhine primates, a group that has retained many charac­ cells in the lateral group of areas (MT and DL). During teristics of the ancestral primates. To this end we have experimental sessions computer-controlled visual stimuli been mapping the organization of the visual system in the were presented while recording from single prestriate lesser bushbaby, Galago senegalensis. The primary visual neurons, and cell responses were recorded and stored for cortex (V-1), the second visual area (V-JI), and the middle later analysis. Stimuli were moving bars of various temporal visual area (MT) in Galago are very similar to orientations and velocities presented in pseudorandom those found in monkeys. However the third tier of order. A directionality index (comparing the response in cortical visual areas appears to be composed of only two the best direction to the response in the opposite or possibly three areas in Galago as compared to five in direction) and a tuning index (comparing the response in New World monkeys. The pulvinar in monkeys contains at the best direction to responses in other directions within least two representations of the visual field; this is also

~0° of the best direction) were calculated for each cell. the case in Galago, but the second map in the superior Cells of DM, DJ, and M strongly tended toward low pulvinar in Galago is organized very differently from the directionality indices (indicating orientation selectivity or comparable region in monkeys. An important clue to the pandirectionality) while cells of MT-DL strongly tended functions performed by different neural maps of the visual toward high directionality indices (indicating direction field is provided by the relative magnification of certain selectivity) (see Figure 2). Of a total of 192 neurons parts of the map. The representation of the central visual recorded from the medial group of areas, 72% show field is enormously expanded in the primary visual cortex responses in the best direction less than twice the in Galago much as it is in monkeys. This magnification of response in the opposite direction, while 81 % of a total of central vision is much greater than would be expected 138 neurons recorded from the lateral areas show re­ simply on the basis of the density of input from the sponses in the best direction greater than twice the central retina to the brain. By contrast, the peripheral 2 response in the opposite direction (X , p< <.001). Cells of visual field is relatively much better represented in the DM, DI, and M also had significantly higher tuning indices superior pulvinar than in the cortex. (mean= 0.62) than cells of MT-DL (mean= 0.52, p«.001), indicating better orientation discrimination among cells of 161. THE PATTERN OF INTERHEMISPHERIC the medial areas than among cells of the lateral areas. CONNECTIONS IN VlSUAL CORTEX OF GALAGOSENEGALENSIS 160. ORGANIZATION OF THE VlSUAL SYSTEM IN Investigators: William T. Newsome III, John M. Allman A STREPSIRHINE PRIMATE In recent years physiological (topographic mapping Investigators: John M. Allman, C. B. G. Campbell, EveLynn MeGuinness, Joel Myerson, and single unit functional) and anatomical (architectonic Michael L. Cooper, Monica Cooper and connectional) techniques have been employed in The neural apparatus for the analysis and synthesis subdividing mammalian extrastriate cortex into several of visual information consists largely of a set of discrete discrete visual areas. Of particular interest is an topographic representations of the visual field. The anatomical technique used successfully in the macaque components of the set of visual maps, their internal monkey by Van Essen and Zeki (1978) which allows one to organization, and their interconnections differ among map onto the surface of a cerebral hemisphere the pattern 106 of termination of axons projecting interhemispherically primates, including humans, are unique in the extent to via the splenium of the corpus callosum. To the extent which facial expression is utilized in day to day inter­ that such patterns of axonal termination are congruent actions with conspecifics. Many basic expressions of with physiologically derived maps of the vertical meridian emotion are easily recognizable not only cross-cultllrally (midline) representation, the patterns may be used as in humans but also across species. To study the neural powerful anatomical landmarks for distinguishing borders mechanisms involved in the generation of patterned facial between visual areas. We have employed this technique in expressions is a formidable task. We are attempting' to do a prosimian primate--Galago senegalensis--so that we so by beginning with the lower order control of individual might (1) evaluate the effectiveness of the technique by muscle movements and eventually working back through comparing terminal patterns to the known vertical merid­ the neural hierarchy to more complex patterned expres­ ian representation in two anatomically distinctive areas, sions. V-1 and MT, and (2) gain information concerning the Our research on the face area of precentral :motor existence and organization of extrastriate visual areas not cortex has revealed two separate regions in which yet known in Galago or any other prosimian. The splenium microstimulation at current levels of less than 20 µa of the corpus callosum was surgically sectioned, and a five elicits movements of the mimetic muscles of the. face. day survival period was allowed before sacrificing the The larger representation extends along the anterior bank animal. Serial sections of visual cortex were stained using of the central sulcus; the second region was found ·in the standard techniques for (1) cell bodies, (2) myelinated posterior bank of the arcuate sulcus. The two regions are fibers, and (3) degenerating axonal terminals. Terminal separated on the surface of the precentral gyrus by a degeneration patterns were reconstructed over all of region devoted primarily to toq~'Ue muscles with some jaw visual cortex and compared to visual field representation and eyelid muscles. in areas V-1 and MT which were identified in cell body and In penetrations down the wall of either the central myelin stains respectively. Preliminary results indicate or arcuate sulcus we encountered a series of discrete that patches of terminal degeneration correlate well with zones each of which was related to the movement of a Vertical meridian representations in the two above men­ particular muscle in the face. The lowest threshold points tioned instances which can be assessed anatomically. A were found in the center of each zone and as the continuous band of moderate to heavy degeneration lies at microelectrode progressed toward the edge, threshold rose the V-l/V-11 boundary extending just onto the upper bank slightly until there was a shift to a new muscle movement; of the calcarine sulcus dorsally and just onto the lower the changes typically were quite abrupt and successive bank of the calcarine sulcus ventrally. Another discrete stimulation points separated by as little as 50 µ. could band of degeneration forms a belt around extrastriate yield different responses (see Figure 3). In the cowrse of area MT excepting the most anterior portion--a pattern many such penetrations, usually moving in 50 µ steps, we remarkably similar to the known area of vertical meridian rarely encountered simultaneous movements of mol"e than representation in MT (Allman et al., 1973). Terminal one muscle when stimulating at threshold level currents. degeneration does not strictly respect physiologically Our results support the view that precentral motor defined boundaries in the region of close apposition of the cortex is arranged in fine columnar-like zones, usually 200 central visual representations in V-1, V-11, and MT. to 800 µ across. The general organization of the representations of the mimetic muscles is roughly topo­ References: Allman, J., Kaas, J. and Lane, R. (1973) Brain Res. 57: graphical with adjacent muscles usually clustered tcr 197-202. gether. It appears, however, that a given muscle Van Essen, D. and Zeki, S. (1978) J. Physiol. 277: 193-226. movement may be represented in several noncontiguous 162. CORTICAL CONTROL OF FACIAL MUSCLPS IN zones within a region. MACAQUE MONKEYS *Undergraduate, California Institute of Technology. Investigators: llveLynn McGuinness, David W. Sivertsen•, John M. Allman Facial expression plays a major role in emotion and social communication in haplorine primates. Higher 107

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Figure 3. Microelectrode penetration down the anterior bank of the central sulcus. The location of the penetration is shown above. The star marks the point of entry of the electrode into the cortex. The lower graph illustrates the stimulus thresholds in microamper03 as the microelectrode was advanced. The transition between muscle movements coincided with elevations in threshold. 108

163. BEHAVIORAL SYSTEMS ANALYSIS References: Investigators: Joel Myerson, Francis M. Miezin Christiansen, B. and Myerson, J. (1978) Rate of periodic water reinforcement and the amount and temporal Killeen (1975) proposed a mathematical model of the pattern of eating. Paper presented at the May 1978 meeting of the Midwestern Association of Behavior temporal control of behavior that is remarkable in both its Analysis, Chicago. generality and precision. It describes the time course of Killeen, P. (1975) Psycho!. Rev. 82: 89-115. Myerson, J. and Miezin, F. M. (1978) Temporal control of instrumental and Pavlovian conditioned responses as well fixed-interval responding: a molecular analysis. as other behaviors entrained by periodic food reinforce­ Paper presented at the May 1978 meeting of the Midwestern Association of Behavior Analysis, ment. We have found this model to be applicable to Chicago. eating in rats entrained by periodic water reinforcement Myerson, J., Miezin, F. M. and Myerson, W. A. (1978a) Temporal control: growth, decay, and recovery of (Christiansen and Myerson, 1978) as well as to pecking in response strength. Paper presented at the August pigeons elicited by a signal for grain presentation 1978 meeting of the American Psychological Associ­ ation, Toronto. (Myerson et al., 1978c). Recently we have proposed Myerson, J., Myerson, W. A. and Miezin, F. M. (1978b) The modifications in Killeen's model which make it more theory of temporal control: behavioral clocks and stochastic processes. Paper presented at the May parsimonious while further extending the generality of the 1978 meeting of the Midwestern Association of mathematical theory of temporal control (Myerson et al., Behavior Analysis, Chicago. Myerson, J., Myerson, W. A. and Parker, B. K. (1978c) 1978a). Automaintenance without stimulus-change rein­ We started by formulating a system of differential forcement: temporal control of key-pecks. J. Exptl. Anal. Behav. In press. equations which described a process that could give rise to Killeen's fundamental equation and then we substituted a PUBLICATIONS different expression for the growth of response strength. Allman, J. (1977) Evolution of the visual system in the With this modified system, we were able to account for early primates. In: Progress in Psychobiology and the temporal control of those behaviors described by Physiological Psychology, Vol. 7, pp. 1-53, Academic Press, San Francisco. Killeen's fundamental equation as well as those for which Allman, J. M., Myerson, J. and Miezin, F. M. (1977) he found it necessary to invoke other mathematical Binocular rivalry: An experimental paradigm for the study of physiology of perception. Soc. Neurosci. equations. By a further modification, we were able to Abstracts 3: 551. incorporate feedback effects and account for behaviors Baker, J. F., Miezin, F. M., Myerson, J., Newsome, W. T., Petersen, S. E. and Allman, J. M. (1977) Quantita­ previously undescribed mathematically. tive response properties of neurons in the dorso­ Formulating the model in terms of differential medial area (DM), a third tier visual area in the owl monkey. Soc. Neurosci. Abstracts 3: 552. equations had several advantages: these equations explic­ McGuinness, E. and Allman, J. M. (1977) Organization of itly stated assumptions about the underlying process which the face area of motor cortex in macaque monkeys. Soc. Neurosci. Abstracts 3: 27 4. would not be apparent from the form of the solution; they Myerson, J., Manis, P. B., Miezin, F. M. and Allman, J.M. could be translated directly into a computer program for (1977) Convergence as a function of eccentricity in the retino-geniculo-striate system of the owl mon­ simulation of the system; and they permitted mathemat­ key. Soc. Neurosci. Abstracts 3: 571. ical description of feedback systems for which solutions Myerson, J., Manis, P. B., Miezin, F. M. and Allman, J.M. (1977) Magnification in striate cortex and retinal are impossible or unwieldy. Our model already describes ganglion cell layer of owl monkey: A quantitative the interaction of three simultaneous processes. Popula­ comparison. Science 198: 855-857. Myerson, J., Myerson, W. A. and Parker, B. K. ,(1978) tion biologists currently use similar, but more complex Automaintenance without stimulus-change rein­ models to simulate species interactions. Their success forcement: Temporal control of key-peeks. J. Exptl. Anal. Behav. In press. suggests that it may be possible to simulate several Newsome, W. T., Baker, J. F., Miezin, F. M., Myerson, J., behaviors simultaneously. We are currently employing Petersen, S. E. and Allman, J. M. (1978) Functional localization of neuronal response properties 'in ex­ this approach to attack problems such as choice and trastriate visual cortex of the owl monkey. Associ­ competitive interactions between responses. ation for Research in Vision and Ophthalmology (ARVO) Abstracts, p. 174. 109

Professor: Derek H. Fender of the pattern can be less than a millimeter spot on the Sherman Fairchild Distinguished Scholar: Bela Julesz* retina. We are injecting blood into the vitreous of monkey Visiting Associates: Raymond P. Briggs, Patrice L. French, Stanley Klein, Thomas E. Ogden*, John eyes, extracting the opaque vitreous, and testing with Trimble laser light. The simplicity of this approach makes it Research Fellows: James P. Ary, Mark C. Citron, Michael T. Hyson potentially valuable in the clinic. Graduate Students: Jiu Wei M. Chen*, Terrance M. Other approaches under consideration include ultra­ Darcey*, Daniel B. Diner*, Ross M. Larkin* Research Staff: Cornelis M. Dekker* sound, X-rays, and focused magnetic fields to stimulate known parts of the retina without light. *Division of Engineering and Applied Science, California Institute of Technology. This is a preliminary report of work in progress and as yet no written reports are available for distribution. Support: The work described in the following research reports has been supported by: National Institutes of Health, USPHS 165. SPATIO-TEMPORAL COURSE OF EXCITATION Alfred P. Sloan Foundation IN mE HUMAN BRAIN Investigators: Terrance M. Darcey, Derek H. Fender 164. ASSE!SING MACULAR FUNCTION IN VITRECTOMY CANDIDATES A system has been developed which allows us to Investigators: James P. Ary, Terrance M. Darcey, follow human sensory information processing via multi­ Thomas E. Ogden, Derek H. Fender channel scalp potential measurements and the determina­ A common symptom of diabetes is hemorrhage of tion of corresponding electrical cortical sources. Scalp retinal blood vessels and consequent contamination of the potential distributions resulting from certain sensory vitreous with blood. As the blood cells break down the stimuli are analyzed using a field theoretical approach in normally clear vitreous turns opaque resembling chocolate combination with realistic assumptions regarding the milk. Normal vision can often be restored by surgically electrical and geometrical composition of the head. removing the blood vitreous and replacing it with saline Electrical sources are specified in terms of their position, provided the retina has not detached nor atrophied. The orientation, and extent. surgical procedure is called a vitrectomy and presents This scheme has been applied successfully to a study some risk to the patient. Hence diagnostic techniques of the mapping between the visual field and visual cortex which can assess the condition of the retina, especially in humans. This was done by presenting a patterned the macular part, help the physician judge the likelihood stimulus to various areas of the visual field and determin­ of recovering normal vision and help him decide if surgery ing the cortical sources which gave rise to the scalp is appropriate. potential responses. Sources were found to depend on We are pursuing muitiple approaches to this prob­ stimulus locus in a manner which matched the topography lem. First we are looking at the scalp distribution of the and functional anatomy of visual cortex. As a result, it visually evoked potentials (YEP). The YEP is dominated has been established that evoked scalp potentials may be by macular inputs, and its scalp distribution depends on used as a crude tool for the definition of the anatomical the part of the visual field stimulated. We are testing projection of localized sensory fields onto the cortical patients who have one normal eye and one eye with a surface. known macular degeneration. By using as many as 96 We are now commencing experiments which apply simultaneous scalp electrodes we are able to measure these techniques to other forms of sensory processes. precisely the scalp distribution of the VEP and compare These include stereoscopic depth and auditory stimuli. the distributions for the two eyes. Preliminary results show clearly identifiable differences. 166. IDENTIFYING BRAIN FUNCTION mROUGH EVOKED SCALP POTENTIALS Our second approach uses a safe level of coherent Investigators: James P. Ary, John Trimble light from a laser to set up an interference fringe pattern on the retina. As little as one millionth of the input light In identifying the function of the human brain, there must pass through the vitreal opacity without divergent is an enormous gap between the understanding that can be scattering in order to produce a pattern that becomes achieved by single-cell studies on lower animals and the identifiable on phase reversal of the fringes. The extent understanding that derives from behavioral or psycho- 110

physical studies on the intact human. Probably the only used as the stimulus and a cross-correlation technique is realistic way to bridge this gap is by studying evoked scalp used for analysis in a laboratory situation. Our priesent potentials. This approach is clouded by the fact that project involves the combination of the conventional sensory inputs pass through many stages of processing, technique with the techniques we have developed for each of which generates a signature in the scalp poten­ laboratory use into a more powerful clinical tool. A real­ tials. The goals of this research are to disentangle these time computer has been developed to perform the cross­ signatures, locate their sources, and identify them with correlation analysis since the results are needed while the function. patient is still in the hospital. We have begun the process Powerful tools are required for extracting definitive of classifying these ERGs in terms of the retinal pathol­ relations from the hodgepodge of scalp potentials. We ogies of the patients. have applied two complementary techniques for this This is a preliminary report of work in progress and purpose: white-noise nonlinear analysis and computer­ as yet no written reports are available for distribution. fitted source localization. Time-invariant, finite memory systems can be fully characterized by a set of kernels 168. SYSTEM MODEIJNG OF HUMAN OCULOMOTOR SYSTEM derived by cross-correlating the output with a white-noise Investigators: Derek H. Fender, JiuWei M. Chen input. We have measured scalp potentials while white­ noise modulating the luminance of a 6° square presented in The function, control, and information processing of the right half of the visual field and computed first-order human eye movements have been of central interest to kernels which resemble the classic flash response. We are researchers from different fields for many years. The determining the optimal stimulus conditions for minimiz­ purpose of this research is to modify existing system ing the variance of the estimates of higher-order kernels. models for rapid saccadic eye movements and smooth The sources for prominent features of these kernels can pursuit movements from the viewpoint of control engi­ be located by applying volume conduction theory and least neering and system theory. squares fitting to equivalent model sources. Having The control system approach was first employed by identified the cortical regions that contribute to the Fender and Nye (1961). The original model has been response, it may then be possible to sense the activity in modified several times. This research may bring about a one region relatively independently of others. We are better understanding of different stages of control 111echa­ exploring electrode weighting schemes and correlational nisms and information processing by the brain and clarify algorithm for extracting the general input-output relation existing ambiguities by using different stimuli target of cortical regions of known activity. movements and complex stimuli patterns. This is a preliminary report of work in progress and Because of intrinsic nonlinearities in the system as yet no written reports are available for distribution. model, white-noise types of nonlinear analysis techniques will be employed. Optimal control theory will be ,applied 167. CIJNICAL STUDIES OF THE HUMAN ERG USING to typical saccadic strajectories. NONLINEAR ANALYSIS This is a preliminary report of work in prog'I'iesS and

Investigators: Derek H. Fender, Thomas E. Ogden, as yet no written reports are available for distribut~on. Ross M. Larkin Reference: The electroretinogram (ERG) is a measure of the Fender, D. H. and Nye, P. W. (1961) Kybernetik 1: 81-88. activity of the retina in response to stimulation by light. 169. HYSTERESIS IN HUMAN BINOCULAR FUSION The conventional method for recording this activity from the human retina is to stimulate it with a bright flash of Investigators: Derek H. Fender, Daniel B. Diner light directed into the eye and to record the ensuing In 1967, Fender and Julesz measured hysteresis in electrical response with an electrode embedded in a the temporalward limits of Panum's fusional area. In contact lens placed on the cornea of the eye. This 1977, we measured the nasalward fusional limits and found measures the impulse response of the retina. Previous them to demonstrate hysteresis not significantly different studies by members of our group have established a from the hysteresis found in the temporalward fusional procedure whereby a randomly modulated light intensity is limits. 111

The question arose of whether the hysteresis was its moving components then the phase of the flickering due to a shifting or a stretching of Panumts fusional area. uniform background should not affect the detectability of In the past year, we tested two subjects with retinally the grating. We found however, that for test patterns of stabilized images and found both to demonstrate highly 0.8 cycle/deg flickering at 20 Hz there was a strong phase significant shifting of Panum 1s fusional area. Only one dependence. There was considerable masking when the subject demonstrated a corresponding stretch of Panum's uniform field flicker was in phase with the grating and fusional area, and this stretch was only mildly significant. there was no masking when the uniform field was 90° out The shifting was so prounounced that, under certain of phase. These results are not compatible with inde­ conditions, the zero disparity retinal location was not in pendent motion mechanisms. Panum's fusional area. (b) Counterphase grating mask. In this series of experiments the background was composed of superthresh­ Reference: Fender, D. H. and Julesz, B. (1967) J. Optical Soc. Amer. old levels of equal rightward and leftward moving grat­ 6: 57. ings. An increment of the rightward component sum­ mated totally with a decrement of the leftward grating. 170. NONLINEAR ANALYSIS OF CAT ERG The test patterns are thus not detected by independent Investigators: Ross M. Larkin, Stanley Klein motion-selective mechanisms. A series of experiments has been carried out on the (2) Direction-selective adaptation with very slow cat's ERG. The stimulus was a randomly flashing light. motion. This experiment indicates that there may be Every 7 msec there was a 50% chance of a flash. Neutral some mechanisms which are very sharply tuned to motion. density filters allowed exploration of 5 decades of light A moving sinusoidal adapting grating was found to produce intensity. The data was analyzed by time-locking the a considerably greater threshold rise for a subsequently response to a test flash accompanied by a particular viewed test grating than a test grating which moved very pattern of flashes called the conditioning flash. A slowly in the opposite direction. This suggests that such conditioning flash might for example be three succeeding very slowly moving test patterns (as slow as 0.12 Hz for a time intervals (21 msec) with no flash. The data are 0.4 cycle/deg grating) are detected by direction-selective displayed as the response to test plus conditioning flashes motion mechanisms and not by sustained mechanisms that minus the response to the conditioning stimulus. A family do not analyze motion. of graphs are generated for different time intervals between conditioning and test flashes. Our results show 172. BINOCULAR HYSTERESIS AND FUSION WITH that the conditioning stimulus has a strong effect on the VOLUNTARY EYE MOTIONS response magnitude and a weaker effect on the response Investigators: Michael T. Hyson, Bela Julesz latency. The dynamics of ERG adaptation is revealed. One function of the eye movement control system is to point both eyes at the same point in space. This allows 171. MOTION MECHANISMS IN HUMAN VISION the extraction of binocular depth information by the Investigator: Stanley Klein visual system. (1) Nonindependence of motion mechanisms. A sine Fender and Julesz (1967) found that if the images to wave grating flickering in sinusoidal counterphase (the the two eyes are stabilized on the retinas (thus canceling light bars become dark bars and vice versa) consists of the effects of voluntary eye motions) the left eye and identical right- and left-moving gratings. There have right eye images can be misaligned horizontally up to 2° recently been several experiments which indicate that the before the sensation of depth in a random dot stereogram two components of the collllterphase grating are detected is lost. To regain the depth, the images must be almost by independent, direction-selective motion channels. realigned, thus showing that the detection of depth and Using background masking patterns, we show that the the tolerance of the system for alignment errors depends motion channels are not independent. on its history of stimulation and thus shows hysteresis. (a) Spatially uni.form flickering mask. Consider a We have extended this idea to normal vision with counterphase grating in the presence of a uniform field voluntary eye motions. We misalign the images while flickering at the same rate. If the grating is detected by monitoring the vertical and horizontal motions of the 112 eyes. The subject maintains his depth percepts during this 174. AUDITORY EVOKED RESPONSE! TO SPEECH misalignment, even though the images now fall on new Investigators: Michael T. Hyson, James P. Ary, parts of the retina. This suggests that the brain is Derek H. Fender dynamically altering its internal frame of reference The goal of this project is to find auditory evoked during this process. We call this a 0 cortical shift" response dipole localizations for various speech sounds representing cooperative processing among different areas such as /pa/ or /ba/. Since the localizations to visual of the brain. We have found that with voluntary eye checkerboards correspond to expected anatomic locations, motions, the subject can tolerate misalignment of up to one expects the same to occur in other modalities, such as 3°, somewhat more than under stabilized conditions. Also, audition. we have found that the motions of the two eyes are We use a speech synthesizer and have developed different; they do not simply duplicate each other. Thus, preliminary programs to output simple speech sounds. The experiments in which the motion of one eye is inferred work will proceed by doing evoked response audiometry to from measurements of the other must be seriously calibrate the system, followed by tonotopic mapping of questioned. dipoles. Then we will use the synthesizer to present This is a preliminary report of work in progress and sounds whose percepts are different to check if the as yet no written reports are available for distribution. dipoles also differ. Reference: This is a preliminary report of work in progress and Fender, D. H. and Julesz, B. (1967) J. Optical Soc. Amer. no written reports are available as yet. 6: 57.

175. A COMPONENT ANALYSIS OF THE ERG USING 173. EYE MOVEMENT IN READING NONLINEAR ANALYSIS Investigators: Michael T. Hyson, Raymond P. Briggs, Investigators: Derek H. Fender, Stanley Klein, Patrice L. French, Derek H. Fender Ross M. Larkin

The goal of this project is to investigate the role of In parallel with efforts in the clinic to collect and eye movements in reading. We are developing a multi­ analyze real patient data, we are also undertaking a more processor, real-time computer system and a speeded-up theoretical approach leading to a better understanding of plasma display in order to change text on the display in the ERG. This is being done with nonlinear analysis where response to a subject's eye motions. The processors should the second-order kernel represents the activity of inter­ allow the prediction of saccadic motions and alterations acting elements in the retina. To understand where these of text in 9 msec. components originate and what conditions modify their The reading studies will proceed by studying recog­ characteristics, we have embarked on a companion study nition of different letters to select a font for the display. in a more controllable laboratory situation.. Our present We will then collect descriptive statistics of eye motions efforts are directed toward experiments with stimuli in reading. We will then predict points of regard in order designed to elicit the dynamic interactions between to place a "window" of text at the point where the subject elements of the retina. is looking. Varying the size of the window, we will This is a preliminary report of work in progress and determine the role of peripheral vision and other cues in as yet no written reports are available for distribution. reading. These studies of normals will eventually encompass PUBLICATIONS dyslexic children in order to determine if poor eye motions are related to poor comprehension. Darcey, T. M. and Fender, D. H. (1978) Partial field studies of human visual cortex via multichannel This is a preliminary report of work in progress and evoked potential source determination. In prepara­ as yet no written reports are available for distribution. tion. Diner, D. B. (1978) Hysteresis in human binocUlar fusion: A second look. Ph.D. Thesis, California Institute of Technology, Pasadena, California. Diner, D. B. and Fender, D. H. (1978) Hysteresis in human binocular fusion: I. Temporalward and nasalward ranges. In preparation. ll3

Diner, D. B. and Fender, D. H. (1978) Hysteresis in human Larkin, R. and Klein, S. (1978) Nonlinear analysis of cat binocular fusion: II. A stretch or a shift? In ERG. Presented to the Association for Research in preparation. Vision and Ophthalmology (ARVO), Sarasota, Flor­ Hou, L. R. and Fender, D. H. (1978) Programming strategy ida. of saccadic eye movements. Vision Res. In press. Stromeyer, C. F., Kronaver, R. E., Madsen, J. C. and Kavanagh, R. N., Darcey, T. M., Lehmann, D. and Fender, Klein, S. (1977) Bidirectional motion mechanisms in D. H. (1978) Evaluation of methods for three­ man. Presented to the Association for Research in dimensional localization of electrical sources in the Vision and Ophthalmology (ARVO), Sarasota, Flor­ human brain. IEEE Trans. Biomed. Engrg. In press. ida. Klein, S. (1977) Unifying white-noise methodologies. Pre­ Stromeyer, C. F., Madsen, J. C. and Klein, S. (1978) sented to Quantitative Analysis Workshop of the Direction-selective adaptation with very slow mo­ 1977 Neuroscience Meeting in Anaheim, California. tion. J. Optical Soc. Amer. Submitted for publica­ Klein, S., Kronaver, R. E. and Stromeyer, C. F. III (1978) tion. Checkerboards and edge mechanisms. Perception. In press. Klein, S., Stromeyer, C. F. and Madsen, J. C. (1977) Counterphase flickering and moving gratings masked with spatially uniform, flickering fields. Presented to the Association for Research in Vision and Ophthalmology (ARVO), Sarasota, Florida.

acoustical cues in the song. Our strategy in search for the Professor: Masakazu Konishi Visiting Associates: Hans-J. Bischof, Hans-J. Leppelsack clues has been to use computer synthesized songs in Research Fellow: Eric I. Knudsen tutoring white-crowns. If they learn a synthetic song, this Graduate Students: Mark Gurney, Alvin J. Hill Jr., Daniel Margoliash*, James S. McCasland must contain the necessary clues. Such a song was Research Staff: Eugene Akutagawa produced and the next step was to modify the song Laboratory Staff: Cynthia Akutagawa, Christopher M. Ingram systematically until it became unacceptable to white­ crowns. Judging from the results obtained so far the clues *Division of Engineering and Applied Science, California Institute of Technology. must be very simple, because birds accepted radically simplified versions of the original computer song. The Support: The work described in the following research reports has been supported by: absence of these clues, as well as the presence of certain National Institutes of Health, USPHS sound patterns, may inhibit learning of alien songs. National Science Foundation Spencer Foundation Fund 1?7. THE OJLBIRD: HEARING AND ECHOLOCA'nON Sununary: The main efforts of our group were spent on lnvestigators: Masakazu Konishi, Eric L Knudsen three areas of investigation: single neuron correlates of sound localization in the owl, specificity of bird song The oilbird derives its name from the fact that learning by means of computer music technology, and natives used to extract oil from young birds which are study of brain sex differences in the zebra finch. We laden with fat during a certain period of their develop­ carried out two expeditions to Trinidad to study the ment. This bird navigates by sonar in dark caves where it auditory and visual systems of the oilbird. lives. Neither the auditory range of the oilbird nor the smallest object that can be detected by its sonar was 176. SPECIBS SPECIFICITY OF SONG LEARNING IN known. We studied oilbirds in the field and the laboratory THE WHITE-CROWNED SPARROW in Trinidad to investigate these aspects of their acoustic Investigator: Masakazu Konishi behavior. The oilbird's ear is most sensitive to 2 KHz Bird voice, a seemingly esoteric topic for scientific beyond which its sensitivity declines by about 20 dB per inquiry, is known to exhibit some of the most interesting octave. 2 KHz is also the most dominant frequency in the phenomena of behavioral development such as the effects oilbird's sonar _signal. The use of low frequencies by the of sensory exposure, deprivation and isolation, inborn oilbird implies that its sonar system is designed to resolve perceptual preference, and critical impressionable period. relatively large objects. We confirmed this prediction by Young white-crowned sparrows learn the song of their own subjecting oilbirds to obstacle avoidance tests in their species in preference to alien songs. How do they know home cave. The results show that a disc of 20 cm in which song to copy? They must recognize certain diameter is barely resolvable to the oilbircPs sonar system. 114

178. AUDITORY RECEPTIVE FIELDS: CENTER­ 179. DEVELOPMENT AND SEXUAL DIFFERENTIATION SURROUND ORGANIZATION OF THE SONG SYSTEM IN THE ZEBRA FINCH Investigators: Eric L Knuci!en, Masakazu Konishi Investigator: Marl< Gurney

In the course of our studies on how auditory neurons Unique to the songbird brain is a robust neural in the barn owl respond to spatial aspects of a sound system which controls the frequency patterning of song. stimulus, we have found that the receptive fields of In the adult zebra finch, the individual song system nuclei specialized auditory neurons are subdivided into separate are conspicuously sexually dimorphic in volume excitatory and inhibitory areas similar to the center­ (Nottebohm and Arnold, 1976). We have examined the surround receptive field organization described for other ontogeny and sexual differentiation of this system. sensory systems. These specialized units were located in The birthdates of nuclei within the song system have the lateral and anterior portions of the midbrain auditory been determined using 3H-thymidine autoradiography. nucleus (mesencephalicus lateralis dorsalis, MLD), the The internal and external striata comprise separate avian homologue of the inferior colliculus. Units in this proliferative zones. Proliferation in the dorsal ventricular region respond only to sounds originating from a restricted ridge produces a spatiotemporal gradient of time of origin area of space (receptive field), and are arranged according with first born neurons lying deep in the matrix of the to the location of their receptive fields so as to form a external striatum. At hatching, little cytoarchitectonic physiological map of auditory space. The inhibitory areas differentiation has occurred within the telencephalon. In of these units were mapped under free-field conditions by the peri-hatching period rearrangement of neurons using two movable sound sources; one source positioned through migration, and glial proliferation characterize inside a unit's receptive field to drive the unit; while a telencephalic matlll'ation. As the telencephalon matures, second source, positioned at various locations outside its the individual song nuclei coalesce and increase in volume, field, tested for inhibitory effects. yet as late as 20 to 25 days post-hatching the sexes are Noise bursts presented outside of a unit's receptive equivalent. During days 25 to 35, the song system nuclei, field inhibited these units. The strength of the inhibition RA and HVc, rapidly expand in volume in the male. The depended upon the location of the source, and the programmed sexual development of the song system intensity and spectral content of the sound stimulus. occurs independently of either song vocalizations or song Inhibition increased as the source moved in from the crystallization. Injections of estradiol benzoate (1 µg/g periphery and approached the borders of a unit's receptive body weight) in oil on day 2 post-hatching suppresses song field. As the source entered the unit's field, the effect of vocalizations and courtship behavior in the male, while the noise changed from inhibitory to neutral or excitatory the nuclei of the song system achieve normal volumes. within a few degrees. Increasing noise levels resulted in Deafening on day 30 post-hatching prevents song crystal­ stronger inhibition and an expansion of the unit's inhibi­ lization through interruption of auditory feedback yet tory area toward the periphery. Tone bursts also inhibited allows song vocalizations. Again, no effect on song these units, but the effect was weaker. The range of system development is observed sound frequencies that contributed to inhibition was wide, Reference: and changed with sound source location. Thus frequency­ Nottebohm, F. and Arnold, A. (1976) Science 194: 211. response curves measured using different speaker loca­ tions were often qualitatively different: inhibitory fre­ PUBLICATIONS quencies at one location being neutral or excitatory at Knudsen, E. and Konishi, M. (1978) A neural map of another. auditory space in the owl. Science 200: 795-797. The fact that the auditory system has created Konishi, M. (1978) Auditory environment and vocal devel­ opment in birds. In: Perception and Experience, R. center-stll'round receptive fields based on functional prop­ D. Walk and H. L. Pick (Eds.), Plenum Publishing erties of the input, and independent of the topography of Co., New York and London. Konishi, M. and Bentley, D. (1978) Neural control of the sensory surface, argues strongly for the importance of behavior. Ann. Rev. Neurosci. 1: 35-59. cente~urround organization in spatial analysis of sensory input. 115

Research Associate: Marianne E. Olds has been proposed that neurons containing the mono­ Senior Research Fellow: Dorwin L. Birt Graduate Student: Gary S. Jones* amines and neurons containing the endogenous opiate-like Research Staff: K. Jeffrey Eriksen, Dana B. Nelson, substances constitute, at least in part, the neuroana­ Robert Nienhuis Laboratory Staff: Donald D. Morris, Robert F. Nickerson tomical substrate for a central motivational mechanism. The experiments this past year have aimed to determine *The Salk Institute, La Jolla, California. (1) whether these substances applied intracerebrally in the Support: The work described in the following research immediate vicinity of neurons participating in neural reports has been supported by: National Institutes of Health, USPHS transmission correlated with ICSS or escape behavior National Science Foundation modify in a specific manner the neural transmission at

Summary: We continue to study the neural correlates of these sites, and (2) whether these substances infused learning and motivation. The aim of the learning study is intracerebrally at sites supporting ICSS have positive to identify the sites in the central nervous system of reinforcing properties as shown in self-administration tests. mammals where specific changes in responsiveness to a stimulus occur as it is repeatedly paired with a reward and therefore comes to signify the association between the 180. ASSOCIATIVE NEURAL CHANGES IN MEDIAL GENICULATE AND INFERIOR COLLICULUS OF two stimuli. The aim of the motivation study is to use THE RAT DURING CONDrnONING direct electrical stimulation of the brain at sites where Investigators: Dorwin L. Birt, Robert Nienhuis, such stimulation has been shown to be rewarding or Marianne E. Olds punishing to identify and characterize the pathways Results obtained in a recent experiment in this comprising these functional systems. laboratory drew attention to the possible contribution of In the learning study previous findings indicate that subtle nonassociative processes to changes in the early changes take place in the early component of the neural latency unit responses obtained in the medial geniculate response to an auditory signal used as the conditioned body (MGB) and the inferior colliculus (IC) of the rat stimulus during the acquisition of a differential classical during the learning of a differential appetitive condition­ conditioning task. Experiments during the past year had ing task. An important source of such nonassociative two main goals: (1) to determine, by a set of additional effects was shown to arise from differences in the control procedures, whether changes in these short laten­ interval between a food pellet presentation and the cy evoked responses are specifically related to the pairing following presentation of positive and negative condi­ of the conditioned stimulus with the presentation of a tioned stimuli, CS+ or CS-. To determine whether food pellet or to a variety of nonspecific changes; (2) to previously observed changes in MGB and IC were due to characterize, anatomically and physiologically, the neu­ associative processes or to nonassociative ones, experi­ rons and neural circuits that demonstrate learning through ments were carried out using a counterbalanced stimulus specific changes. presentation schedule which placed the CS+ and CS- at In the motivation study the findings in this and other the same average time following a previous food pellet. laboratories have implicated ventral diencephalic struc­ Criteria adopted for characterizing a change as associa­ tures such as the hypothalamus in intracranial self­ tive required that the response to the CS+ be enhanced stimulation (ICSS) and medial brain stern structures such relative to that to the CS- throughout repeated reversal as the central gray in the escape behavior produced by sessions. brain stimulation. These structures contain high concen­ The results confirm previous findings of early trations of rnonoamines, opiate receptors, and the en­ latency associative changes in MGB, and are consistent dorphins. Some of the amines, some drugs which modify with the idea that such changes are most likely to occur in amine metabolism, morphine, and the endorphins have the medial division of this nucleus. The findings in the IC been shown to facilitate ICSS, and under different test thus far do not support the notion that this nucleus conditions, to modify the responses of an animal to participates in the process. However, in view of the punishment. Also, it has been shown that these substances complex structure of the IC and of the fact that only can function as positive reinforcers in their own right as neurons in its central nucleus were sampled, these shown in self-administration tests. On these grounds it negative findings must be viewed with caution. Experi- 116 ments are presently being conducted in which neurons in 182. POSITIVE REINFORCEMENT OF RAT BEHAVIOR PRODUCED BY INTRAHYPOTHALAMIC INFUSION the external nucleus of the IC are being sampled, since it OF MORPHINE is reported that this nucleus projects heavily into the Investigators: Marianne E. Olds, K. Jeffrey Eriksen medial division of the medial geniculate. The self-administration method is often used to 181. SINGLE UNIT ANALYSIS OF LEARNlNG-SPECIFIC demonstrate in animals the positive reinforcing properties CHANGES IN RAT MEDIAL GENICULATE of substances known to have euphoric or addictive Investigator: Dorwin L. Birt properties in humans. In these tests, the substances are Recent and current experiments in this laboratory made available orally, systemically, or intravenously, and indicate that although changes in unit response to auditory more recently intraventricularly. Although much infor­ stimuli occur in many neurons throughout the mammalian mation is thereby obtained about the comparative rein­ brain during conditioning, neurons with a much more forcing effects of these substances, the findings reveal restricted anatomical distribution show changes that meet little of the site(s) in the brain that mediate these effects. criteria for being specifically related to the pairing of The reports that most of these substances, at appropriate conditioned and unconditioned stimuli. It is now desirable doses, facilitate ICSS, and the more recent report that to study isolated single neurons, in order more fully to receptors for opiates and endogenous opiate-like sub­ differentiate neurons that show learning-specific changes stances are present in high density in regions known to from those that do not. It is usually not possible, support ICSS, has suggested a possible relation between however, to hold isolated neurons reliably for the ex­ the endogenous substances and ICSS. According to this tended periods of time required for the acquisition of a view, morphine and related compounds have their euphoric differential conditioned response. Furthermore, the time effect in humans and positive reinforcing properties in necessary to acquire a reasonably large sample size would animals because they have an action at ICSS sites. be prohibitively long. Therefore rats have been highly Rats implanted with combination cannula-stimulat­ overtrained in a cued successive reversal paradigm in ing probes were pretested for rewarding effects of which one of two tones is paired with food pellet hypothalamic brain-stimulation and then tested for self­ presentations under one reversal condition, while the administration of morphine infused at these same sites. other tone is paired with the food pellet under the other Two experiments were carried out. In the first, the reversal condition. Rats quickly learn to respond appro­ effects on bar-pressing behavior to obtain the substance priately to the changed conditions. A chronic microdrive of two doses were compared with the effects of infusing system has been developed to permit systematically artificial cerebrospinal fluid on the rate of responding related penetrations of a brain region with small-tipped when two pedals were placed at opposite sides of a test electrodes. It will now be possible to study, within a short chamber, one pedal delivering the tested substance and period of time, differences in the response of isolated the other yielding nothing. Analysis of the rates of neurons to the same physical stimulus under conditions in responding of the group yielding moderate to low ICSS which the stimulus is paired and unpaired with food rates in the preinjection tests shows that the rats pellets. These experiments will first concentrate on the invariably responded more frequently on the pedal yielding medial portion of the rat medial geniculate nucleus. It the morphine than on the negative pedal and the rate of will also be possible to determine a variety of physio­ responding for morphine on the "positive pedal" was logical characteristics of each neuron, with the aim of significantly higher than it was for the pedal in tests discovering whether a discrete category of neurons may yielding the vehicle. The larger of the two doses was the be involved in learning-specific changes. more effective one. In the second experiments high-rate ICSS rats successfully learned to reverse when the positive pedal was changed from one side to the other in successive morphine sessions. Finally, results with the antagonist naloxone indicated that the reinforcing effects of morphine at ICSS sites were specific. We are presently conducting similar tests with the enkephalins. 117

183. ACTION OF HEDONIC SUBSTANCES ON NEURAL regulatory functions among which is consummatory be­ ACTIVITY IN STRUCTURES SUPPORTING BRAIN­ havior, and also it is a structure known to support STIMULATION REINFORCED BEHAVIOR behavior positively reinforced with brain stimulation. The Investigators: Marianne E. Olds, Dana B. Nelson experiments were carried out on rats prepared with In the present set of experiments the behavior of multiple small-tipped electrodes for recording single unit neurons in structures Im.own to support brain-reinforced activity. Methods allowed prolonged periods of recording behavior was studied as a function of chronic injections of in the freely-behaving rat working to obtain brain rein­ morphine in the lateral ventricle of the rat. The aim was forcing stimulation. The unit activity selected for study to study the changes in neural responsiveness during required a fairly high rate of spontaneous firing in the dependence, as shown by autonomic and stimulant effects intervals between brain stimulation, and the suppression on behavior. The latter consisted in an impairment of the of this activity for a brief period following each electrical discrimination between two acoustic stimuli, one having pulse of the train constituting the reinforcing brain significance and the other not, in a classical conditioning stimulus. The selection was based on earlier observations test rewarded with food. The preliminary findings that this type of neural response in hypothalamus to indicate a selective action on neural activity which reinforcing brain stimulation seemed to be specific and correlated with the behavioral indices of dependence. The might therefore represent response patterns in a posi­ normal difference in the amplitude of the neural responses tively reinforcing system, which if this were the case, to the two acoustic stimuli was reduced but not obliter­ would be the target of compounds producing reinforcing ated, and the early component of the conditioned neural effects on behavior. The application of morphine reduced response was followed by a longer-lasting depressant the spontaneous activity and potentiated the inhibitory phase. These patterns of neural Ch&ll5es occurred within responses. At the same time, the behavior of the animal the first 40 msec of the presentation of the two acoustic was either not affected or gave evidence of behavioral stimuli, clearly anteceding the stimulant effects of stimulation. Effects on different types of neural re­ chronic morphine administration on behavior observed in sponses in this structure are presently being investigated. the interval of 200-1000 msec following the acoustic The results obtained thus far are confirming the findings stimuli. These changes characterized neural activity in obtained in the anesthetized or immobilized animal with the ventral tegmental region and in the raphe nuclei, two morphine, and are consistent with the notion that a structures known to support high rates of ICSS; no obvious specific site of action of morphine is in the hypothalamus changes were found in the region of the locus coeruleus, in test situations involving neither sedation, nor analgesia, another structure known to support ICSS. The analysis of but instead positive reinforcement. these data is presently continuing. 185. NEURAL SUBSTRATE FOR CONDmONED 184. ACTION OF HEDONIC SUBSTANCES ON BRAIN­ EMOTIONAL BEHAVIOR IN THE RAT STIMULATION EVOKED NEURAL RESPONSES IN HYPOTHALAMUS Investigators: Marianne E. Olds, Dana B. Nelson Investigators: Marianne E.. Olds, Robert Nienhuis The relation between structures in the mammalian Studies using the iontophoretic method to apply brain that mediate positive reinforcement to brain stimu­ substances in the immediate vicinity of brain cells to lation and those that mediate negative reinforcement to determine receptor sensitivity have shown that opiates brain stimulation is at present not clear. The anatomical generally have a depressant effect. An important evidence from mapping studies of both effects suggests a question in understanding the action of these substances close relationship between the structures mediating both on behavior concerns the site of this depressant action effects, though one set of structures lies in ventral that correlates with the well-known analgesic effect and diencephalon and the other in thalamic nuclei and the the less often studied positive reinforcing effect. The aim brain stem midline region. The present study is addressed of these experiments was to investigate the hypothalamus to the question of whether these two sets of structures as a possible site mediating the positive reinforcing might be part of one general system mediating emotions effects of morphine. The basis for selecting the hypo­ in which case changes in one set might be accompanied by thalamus was that it is implicated in a variety of changes in the other set. To achieve this goal we have 118 selected a paradigm that is a model for 11conflict11 rates over the next 200 to 1000 msec. LC cells showed no behavior through the establishment of a conditioned obvious correlations with overt motor behavior. Qualita­ emotional reaction. We study the changes produced in tive observations indicate these responses do habituate, conditioned neural activity responsive to acoustic stimuli usually beginning within five presentations of the same during a classical conditioning task rewarded with food stimulus. when the animal learns that the consummatory act will During recording which persisted throughout sleep­ result in punishment in the form of a footshock. At the waking cycles, discharge rates were highest during the behavioral level, this results in the suppression of the transitions between either slow wave sleep (SWS) and behavior leading to the retrieval of the food reward, the waking (W) or between rapid eye movement sleep (REM) degree of suppression being a function of the intensity of and W, with rate increases preceding the SWS to W the punishment, the motivation to obtain the reward, and transition, but not the REM to W transition. Spontaneous the training of the animal. We are interested in rates in waking rats otherwise were highest, with only correlating changes in neural responsiveness to the acous­ occasional spikes during SWS and virtually no firing during tic stimuli, one of which has been paired with the REM. presentation of the food reward and the other not, with These preliminary observations indicate the feasi­ the establishment of the conditioned emotional reaction. bility of testing hypotheses of LC function based upon In the past year, we have studied neural responsiveness studies of discharge frequency during manipulations of the under these conditions in structures supporting positive freely-moving rat. and negative reinforcement and in structures which are *A.V. Davis Center for Behavioral Neurobiology, The Salk reported to integrate information of an 11 emotiona111 Institute, La Jolla, California. **Weizmann Institute, Rehovot, IsraeL nature. We are presently analyzing the data. ***Deceased Professor of Biology, California Institute of Technology. 186. LOCUS COERULEUS NEURONAL ACTIVITY IN FREELY-BEHAVING RATS Investigators: Gary s. Jones•, Stephen L. Foote", Menahem Segal**, Floyd E. Bloom*, PUBLICATIONS James Olds*** Birt, D. (1978) Separation of associative from nonassocia­ The nucleus locus coeruleus (LC) in the rat is a tive short latency changes in medial geniculate and inferior colliculus during differential conditioning highly compact group of noradrenergic neurons from and reversal in rats. Soc. Neurosci. Abstracts. In which a uniquely divergent efferent system of axons press. Birt, D., Nienhuis, R. and Olds, J. (1977) Effects of arises. Pharmacological, physiological, and behavioral bilateral auditory cortex ablation on behavior and observations have generated many hypothetical functions unit activity in rat inferior colliculus during differ­ ential conditioning. Soc. Neurosci. Abstracts, Vol. in of these neurons which can best be evaluated the III, p. 231. freely-behaving animal. We are currently investigating Birt, D. L., Nienhuis, R. and Olds, J. (1978) Effects of bilateral auditory cortex ablation on behavior and the changes in discharge rate and pattern in neurons of unit activity in rat inferior colliculus during differ­ the unrestrained albino rat LC in relation to three ential conditioning. J. Neurophysiol. 41: 705-715. Birt, D., Nienhuis, R. and Olds, M. E. (1978) Separation of variables: (1) stimulation of specific sensory modalities; associative from nonassociative short latency (2) spontaneous sleep-wake cycle alterations; and (3) changes in medial geniculate and inferior colliculus during differential conditioning and reversal in rats. freely-occurr~ behaviors. Brain Res. Submitted for publication. Both multiunit and single unit data indicate that Brauth, S. E. and Olds, J. (1977) Midbrain unit activity during classical conditioning using food and electri­ histologically confirmed LC neurons exhibit pronounced cal brain stimulation reward. Brain Res. 134: 73-82. increases in spike rate from basal resting levels of about 1 Le Moal, M. and Olds, M. E. (1978) Peripheral auditory input to the midbrain limbic area and related to 10 Hz, with a latency usually between 20 and 50 msec, structures. Brain Res. In press. in response to gross auditory or visual stimulation; Le Moal, M. and Olds, M. E. (1978) Unit responses to auditory input in the dorsal and median raphe nuclei qualitative tactile stimulation experiments give similar in the rat. Physiol. Behav. In press. results. Most LC cells showed multimodal responsivity. Nienhuis, R. and Olds, J. (1978) Changes in unit responses to tones after food reinforcement in the auditory The sensory evoked activation was usually followed by a pathway of the rat: Intertrial arousal. Exptl. transient absence of spikes with a gradual return to basal NeuroL 59: 229-242. 119

Olds, J. and Nienhuis, R. (1977) Associative and nonas­ Olds, M. E. (1978) Hypothalamic substrate for the positive sociative changes in auditory system unit responses reinforcing properties of morphine in the rat. Brain during classical conditioning in the rat. Soc. Res. Submitted for publication. Neurosci. Abstracts, Vol. III, p. 237. Stein, E. A. and Olds, J. (1977) Direct, intracerebral self­ Olds, J., Nienhuis, R. and Olds, M. E. (1978) Patterns of administration of opiates in the rat. Soc. Neurosci. conditioned unit responses in the auditory system of Abstracts, Vol. III, p. 302. the rat. Exptl. Neurol. 59: 209-228. Olds, M. E. (1978) Inhibitory evoked unit activity in the medial forebrain bundle in the rat and self-stimula­ tion behavior. Neuropharmacology. In press.

Associate Professor: John D. Pettigrew 187. NOREPINEPHRINE MAINTAINS VISUAL Visiting Associate: Toru Itakura CORTICAL PLASTICITY IN KrITENS Senior Research Fellow: Takuji Kasamatsu AND CATS Weizmann Fellow: Gary G. Blasdel Investigators: Takuji Kasamatsu, John D. Pettigrew, Research Fellow: Marylouise Ary Marylouise Ary Graduate Students: Eileen A. Bagdonas, Michael L. Cooper, Kenneth L. Marton Further experiments were carried out to complete Research Staff: Sarah E. Kennedy, Phyllis F. Knudsen, Robert W. Tajima the study described briefly last year (Biology 1977, No. Laboratory Staff: Christopher D. Vestuto, John C. 182). In kitten visual cortex which had been depleted of Wathey catecholamine (CA) by local pecfusion with 6-hydroxy­ Support: The work described in the following research dopamine (6-0HDA) (4.0 mM), the usual shift of ocular reports has been supported by: Fight for Sight, Inc. dominance did not take place following a week-long National Eye Institute, USPHS monocular lid suture. Norepinephrine (NE) (48.6 µM) National Institutes of Health, USPHS National Science Foundation perfused simultaneously with monocular deprivation re­ Spencer Foundation Fund stored the plasticity in the visual cortex depleted of CA Walter and Sylvia Treadway Fund Whitehall Foundation by 6-0HDA which had either been injected intraventric­ Robert E. and May R. Wright Foundation ularly or perfused locally. Since the microperfusion of NE Summary: This group studies the visual pathway as a started after the end of prior treatment with 6-0HDA, it model system of central nervous system organization. is unlikely that NE could have prevented 6-0HDA from Particular stress is laid upon the study of early postnatal inducing its CA depletion effects. The predominance of development of the visual system and the marked sensitiv­ monocular cells in favor of the undeprived eye therefore ity that the visual cortex of some species shows to cannot be due to the competitive interaction of the two environmental changes during development. chemical agents used. Rather, plasticity appears to be Most work is carried out on the domestic cat, but a restored in some way by exogenous NE. new line of investigation has been opened up by the Microperfusion of NE in older kittens and adult cats discovery that a part of the avian brain is convergent in also restored the susceptibility of cortical cells to function with mammalian visual cortex, even to the point monocular deprivation to such an extent that following 1 of showing a similar degree of developmental plasticity. to 3 week-long eyelid suture the proportion of normal Preliminary studies on various avian species are helping to binocular cells decreased significantly but the complete provide an evolutionary perspective upon some of the shift of ocular dominance was not observed. more puzzling aspects of developmental plasticity. These plastic changes were found only in the local The interplay between genetic and environmental area of the visual cortex perfused with NE or 6-0HDA. factors during development of the visual system is also Since no essential changes in binocuiarity were noted in studied in the Siamese cat, a single locus mutant with the visual cortex perfused with NE or 6-0HDA without widespread changes throughout its visual pathway. simultaneous monocular deprivation, these results strongly Further progress has been made in the past year in suggest a specific role of NE in maintaining cortical the delineation of the nonvisual inputs, such as eye­ plasticity (Pettigrew and Kasamatsu, 1978; Kasamatsu, position information and monoaminergic fibers, which Pettigrew and Ary, in preparation). appear to be necessary to account for the cortical effects Reference: of visual deprivation. Pettigrew, J. D. and Kasamatsu, T. (1978) Nature 271: 761-763. 120

188. THE RECOVERY FROM ABNORMAL VISUAL CA terminal fibers (with varicosities) seems to be highest EXPERIENCE: EFFECTS OF NE AND &-OHDA in the superficial layers (II + III) but not in the molecular Investigators: Marylouise Ary, Takuji Kasamatsu, layer as reported in the rat (Levitt and Moore, 1978). The John D. Pettigrew dense plexus formation in layers II and UI is the most Previous work has established that the catechol­ characteristic feature of CA innervation in kitten visual amine (CA)-depleting agent 6-hydroxydopamine (6-0HDA) cortex (Itakura et al., 1978). prevents the shift in ocular dominance that results from monocular lid suture in young kittens. Cortical perfusion References: Bloom, F. E. and Battenberg, E. L. F. (1976) J. Histochem. of norepinephrine (NE) reverses this effect and also Cytochem. 24: 561-571. restores susceptibility to monocular occlusion in adults. ltakura, T., Kasamatsu, T. and Pettigrew, J. (1978) Neuroscl. Abstracts. In press. This study tested whether CAs also influence the Levitt, P. and Moore, R. Y. (1978) Brain Res. 139: process of recovery--the restoration of functioning binoc­ 219-231. Loren, 1., Bjorklund, A., Falck, B. and Lindvall, o. (1976) ular connections. Four-week-old kittens had one eye Histochemistry 49: 177-192. sutured closed for 1 week. The eye was then reopened for 12 days, during which time 6-0HDA (10-3 M) and NE 190. DIFFUSION OF NOREPINEPHRINE AND &-OHDA PERFUSED LOCALLY IN CAT VISUAL CORTEX (10-3 M) were continuously perfused into the right and Investigators: Takuji Kesamatsu, Toro ltakura, left visual cortices, respectively. Single-unit recordings William Shoemaker* were then done, and the percent of visual cortical neurons In order to delineate the extent of diffusion of showing recovery of binocularity were compared for the norepinephrine (NE) and 6-hydroxydopamine (6-0HDA) two hemispheres. The side treated with 6-0HDA showed delivered by a continuous microperfusion technique, three no recovery: the binocularity (25%) and ocular dominance approaches have been taken. pattern were the same as that found in control kittens (1) The spatial distribution of tritium in the brain immediately after 1 week of eye closure. However, the was studied following a week-long microperfusion with side treated with NE showed almost complete recovery 48.6 µM "cold" NE which had been mixed with a tracer and yielded a normal ocular dominance pattern with 70% quantity of 3H-NE. The radioactivity was measured from binocularity. These results suggest that CAs are involved tissue samples taken from each of many sites at various in visual cortical plasticity in general and affect changes distances from the perfusion site. Counts were maximal in response to manipulation of visual input in both at the site of perfusion and decreased sharply in the directions, whether the process involves an increase or a neighboring area in both antero-posterior and medio­ decrease in binocularity. lateral axes. The counts leveled off at a distance 10 mm from the perfusion site in the antero-posterior axis in the 189. CATECHOLAMINERGIC TERMINAl.S IN KITTEN parasagittal plane and 8.5 mm in the medic-lateral axis VISUALCORTEX: FLUORESCENCE HISTOCHEMISTRY (Kasamatsu, Pettigrew and Ary, in preparation). Investigators: Toro ltakura, Takuji Kasamatsu, (2) The cerebral cortices of kittens were locally John D. Pettigrew perfused with 4.0 mM 6-0HDA for one week. Catechol­ Using a modified glyoxylic acid histofluorescence amine (CA)-containing terminals and fibers were visual­ technique (Bloom and Battenberg, 1976; Loren et al., ized by a modified glyoxylic acid histofluorescence tech­ 1976) the normal distribution pattern of CA fibers and nique (Bloom and Battenberg, 1976). In the area close to their terminals was studied in kitten visual cortex. the perfusion site the usual yellowish green fluorescence The preterminal CA fibers are parallel to the of CA terminal fibers disappeared completely. This cortical surface in the white matter. They give off primary CA-depleted area extended about 4.5 mm in half perpendicularly collateral branches toward the deep layers diameter centering at the perfusion site. Next to this in the gray matter. These stem fibers run, perpendicu­ primary affected area we started to see fragmented CA larly or obliquely, toward the cortical surface sending off terminal fibers which had brighter fluorescence and were many short and long collaterals in various directions. thicker in size than usual. This seemed to be due to the Finally the stem fibers reach the molecular layer where accumulation of CA at the proximal end of partially the fine terminal fibers run horizontally. The density of denervated axons. When we moved further away from the 121 perfusion site, we observed a relatively normal pattern of sutured eye and after 16 weeks this percentage had risen distribution of CA terminal fibers. However, the intensity to 27. The percentage of binocularly driven cells stayed of these fibers was still lower than that at the correspond­ about the same. Some plasticity is apparently present in ing site of the opposite hemisphere (ltakura et al., 1978). the owl even at these older ages. (3) Either NE or 6-0HDA was perfused locally in the visual cortex of kittens. The animals were anesthe­ 192. THE EFFECTS OF ALTERNATE MONOCULAR DEPRIVATION IN THE OWL AND CAT tized deeply with the excess dose of barbiturate and killed Investigators: Eileen A. Bagdonas, John D. Pettigrew by transection at the medulla. A small amount of tissue was punched out from each of many sites at various During the period of alternate monocular depriva­ distances from the perfusion site. The radioenzymatic tion, the animal does not receive simultaneous binocular assay was carried out for NE and dopamine (Coyle and input. When this occlll's during the critical period, Henry, 1973). A preliminary result showed a significant physiological experiments reveal a severe deficit of decrease of the content of NE and dopamine in the area binocularly driven nelll'ons. This effect is seen even if the near the 6-0HDA (4.0 mM) perfusion site. Work is in animal has had normal binocular vision up to the time of progress to delineate the extent of diffusion of 6-0HDA deprivation and continues to experience periods of binoc­ in the neocortex. ular vision during the course of the deprivation. One cat, alternately occluded between eight and eleven weeks References: Bloom, F. E. and Battenberg, E. L. F. (1976) J. Histochem. received over 100 hr of binocular experience during this Cytochem. 24: 561-571. period. Only 2196 of units recorded from its visual cortex Coyle, J. T. and Henry, D. (1973) J. Neurochem. 21: 61-67. could be binocularly driven. Itakura, T., Kasamatsu, T. and Pettigrew, J. D. (1978) Two owls were alternately occluded between eight Neurosci. Abstracts. In press. and twelve weeks of age using fabric hoods which only *Arthur v. Davis Center for Behavioral Neurobiology, The allowed vision through one eye. As the hoods sometimes Salk Institute, San Diego, California. fell off, the owls did receive an amount of binocular 191. A DETERMINATION OF TIIE EXTENT OF THE experience during this period which did not exceed 100 hr. CRITICAL P~IOD IN TIIE OWL Recordings from the visual Wulst showed a normal ocular Investigators: Eileen A. Bagdonas, John D. Pettigrew dominance distribution; i.e., a majority (81 and 9096, The visual system of owls, like that of cats, has been respectively) of units could be driven through either eye. shown to be sensitive to perturbations of the early visual This result is quite different from that seen in cats. environment. Monocular deprivation during the critical period in both animals results in a loss of vlsual neurons 193. THE CORTICO-GENICULATE PATHWAY which can be driven by the deprived eye. If a cat is INCATS Investigators: Kenneth L. Marton, John D. Pettigrew reverse-sutured early in its critical period, a recapture of uni ts by the originally deprived eye is seen. A loss of At least half of the neurons in layer VI of the cat units responsive to visual input through the reverse­ visual cortex (VC) send axons to the lower visual center sutured eye also occurs. called the lateral geniculate nucleus (LGN) (Gilbert and An owl was monocularly deprived between 4 and 9 Kelly, 1975). The functional nature of these neurons is weeks of age and then reverse-sutured for 23 weeks. The not understood as it is difficult to isolate and identify ocular dominance distribution after this period was very these cells physiologically. We have developed a tech­ abnormal: only 3696 of the visually responsive units were nique to do so and have begun to study the properties of binocular and only 4% could be driven solely by the these nelll'ons. reverse-sutlll'ed eye. This result indicates that the owl's An array of 12 insulated metal electrodes designed sensitive period extends to at least 9 weeks of age. to cover at least half of the LGN is implanted in that Recordings were also made after 1 and 16 weeks of nucleus. Each electrodets position can be identified by binocular experience to determine the extent of recovery. finding in visual space the position of a flashing spot that After one week, 1396 of the visually responsive units optimally stimulates the neurons near the electrode tip. responded to visual input solely through the reverse- F.ach electrode can be used to stimulate that part of the 122

LGN. Individual neurons in the deeper layers of the VC tical comparisons can be made. are recorded from, and in particular, the response of a To date, a few LGN cells have been studied cell to LGN stimulation can be used to confirm that the extensively and strong influences probably from the VC cell sends an axon directly to the LGN. If the VC cell have been found. In particular some LGN cells show up to responds reliably at high stimulation frequencies and if a 100% facilitation of normal response by stimulation of the spontaneous spike from the cell blocks the response, then normally nondominant eye (for that cell) when the visual the cell is classified cortico-geniculate. stimulus presented to that eye is an oriented contour Once a neuron is identified as cortico-geniculate, its moving at a particular velocity, and in particular when the properties, including orientation, velocity, and binocular motion of that stimulus starts or stops in a location in the preference, as well as others, are studied in detail nondominant eye equivalent to that of the receptive field Although a comprehensive study is not complete, we have in the dominant eye. Studies are in progress to clarify indications that these neurons tend to be of the "complex" these effects. category, with strong binocularity, and possibly a prefer­ Reference: ence for slower velocity of visual motion (perhaps 2 to Schmielau, F. and Singer, W. (1977) Brain Res. 120: 16°/sec. 354-361.

Reference: 195. A STUDY OF DEVELOPMENT IN AREA 18 OF Gilbert, c. D. and Kelly, J. P. (1975) J. Comp. Neuro!. THE VISUAL CORTEX OF mE CAT 163: 81-106. Investigator: Eileen A. Bagdonlls

194. filNOCULAR INFLUENCES ON SINGLE CELL It has been shown by both morphological and RESPONSES IN THE LATERAL GENICULATE NUCLEUS electrophysiological methods that area 18 in the visual Investigator: Kenneth L. Marton cortex of the cat receives a direct thalamic input. Stone and Dreher (1973) have shown that all the LGN cells which A large projection of axons comes to the lateral project to area 18 are driven from retinal fibers of the geniculate nucleus (LGN) from layer VI of the visual Y-type (while area 17 receives both X- and Y-cell input). cortex (VC). The neurons of the LGN respond optimally to Some of the characteristics which distinguish Y-type cells monocular stimuli; however both inhibitory and facilitory from X-cells are faster conduction times, larger receptive influences from stimulation of the other eye have been fields, and their ability to respond to extremely rapid found, and in particular it is known that cooling the VC motion. These properties are reflected in area 18 visual eliminates the facilitation and decrements the binocular cortical neurons. inhibition (Schmiel.au and Singer, 1977). Work is in Norman et al. (1977) demonstrated that X-cells in progress here to characterize the nature of these cortical the LGN develop earlier than Y-cells. X-cells with influences. mature latencies are found as early as 21 days while Single cells in the VC respond to more specific Y-cells did not have adult latencies at 40 days. We plan "trigger features" than do cells in the LGN. It is to record from cortical cells while stimulating electrically hypothesized that influences from the cortex back onto in the LGN to see if this difference in maturation rates is the LGN can be mapped by varying visual parameters that also reflected at the cortical level. normally only cortical cells are directly "tuned" to and A determination of the critical period (i.e., that studying the "average" effect on LGN cells. In particular, period when cortical cell properties are susceptible to LGN cells receive mainly monocular excitatory input change by environmental alterations) will be made through responsive only to contrast, whereas many VC cells are monocular and other forms of visual deprivation. The excited binocularly by contours of particular orientations normal development of cortical cell properties such as moving at particular velocities. The effect of monocular ocular dominance and orientation selectivity will also be versus binocular oriented stimuli on LGN response is being investigated. studied. Due to the normally high variation in LGN response, special computer-driven shutters, prisms, and References: Norman, J., Pettigrew, J. and Daniels, J. (1977) Science optical stimuli are needed to interleaf alterations in the 198: 202-204. cat's visual environment so that optimized paired statis- Stone, J. and Dreher, B. (1973) J. Neurophysiol. 36: 551-567. 123

196. A NATURAL NATURE-NURTURE EXPERIMENT binocular interaction nor very sensitive to early visual OK THE OILBffiD deprivation. Investigators: John D. Pettigrew, Masakazu Konishi PUBLICATIONS Since the oilbird, Steatornis caripensis, nests deep in Ary, M., Kasamatsu, T. and Pettigrew, J. D. (1978) Local neotropical caves, the protracted development of most perfusion of 6-0HDA prevents recovery of visual young birds takes place in total darkness. The role of cortical cells from the effects of monocular depri­ vation. AR YO Abstracts, p. 294. experience in visual development can therefore be studied Blasdel, G. G., Cooper, M. L. and Pettigrew, J. D. (1978) in the natural situation. Evidence in the Siamese for the simultaneous ex­ pression of Boston and Midwestern patterns. ARVO In this investigation the aim was to study the visual Abstracts, p. 216. properties of single nel.ll'ons in the visual Wulst of oilbirds Blasdel, G. G. and Pettigrew, J. D. (1978) Effect of prior visual experience on cortical recovery from depri­ of different ages, raised with and without visual experi­ vation-induced amblyopia in kittens. ARVO Ab­ ence. In owls, the visual Wulst functions in an analogous stracts, p. 269. Blasdel, G. and Pettigrew, J. D. (1978) Effect of prior manner to mammalian visual cortex and is dependent upon visual experience on recovery from monocular depri­ visual experience. It was therefore of some interest to vation. J. Physiol. 274: 601-619. Cooper, M. L. (1978) Studies in the visual system of the examine, in a putative, close relative of the owl, the cat. I. Retinothalamic pathway in normal and response of the Wulst to natural conditions which are Siamese cats. II. The vertical horopter. Ph.D. Thesis, California Institute of Technology, Pasadena, tantamount to severe visual deprivation. California. A single netn'on recording laboratory was success­ Cooper, M. L. and Pettigrew, J. D. (1977) Naso-temporal division of retinothalamic pathway in normal and fully set up at the Simla field station in Trinidad, with Siamese cats. Soc. Neurosci. Abstracts, p. 556. portable equipment brought from California. The visual Cooper, M. and Pettigrew, J. D. (1978) A neurophysiolog­ ical determination of the vertical horopter in cat response properties of over 300 neurons were studied in and owl. J. Comp. Neurol. Submitted for publica­ six oilbirds netted in the wild at Oropuche cave. Two tion. Cooper, M. L. and Pettigrew, J. D. (1978) The vertical birds were normal adults who had had visual experience horopter in the cat. ARVO Abstracts, p.173. outside of the cave, two birds were visually naive Cooper, M. L. and Pettigrew, J. D. (1978) The retino­ thalamic pathway of the Siamese cat (Manuscript). juveniles taken from nests deep in the cave where no light Cooper, M. L. and Pettigrew, J. D. (1978) The naso­ penetrated, and two birds were taken from a nest at the temporal division of ganglion cells of the normal cat's retina, with a note on the major meridians of mouth of the cave where visual experience was possible the cat's eye (Manuscript). during the day. Crewther, D. P., Gillard-Crewther, S. and Pettigrew J. D. (1978) A role for extraocular afferents in post­ All birds were comparable in terms of their visual critical period reversal of monocular deprivation. capabilities, as revealed by the response properties of AR YO Abstracts, p. 294. Crewther, D., Gillard-Crewther, S. and Pettigrew, J. D. Wulst neurons. The visually naive juveniles had no obvious (1978) A role for extraocular afferents in post­ deficit of responsive netn'ons nor of specific response critical period reversal of monocular deprivation. J. Physiol. In press. types when compared with the adults or the visually Daniels, J. D., Norman, J. L. and Pettigrew, J. D. (1977) experienced juveniles. All three pairs of birds had roughly Biases for oriented moving bars in lateral geniculate neurons of normal and stripe-reared cats. Exptl. comparable numbers of orientation-selective and direc­ Brain Res. 29: 155-172. tion-selective neurons, with the orientation-selective neu­ Daniels, J. D., Pettigrew, J. D. and Norman, J. L. (1978) Development of single neuron responses in the rons tending to prefer orientations clustered around kitten's lateral geniculate nucleus. J. Neurophysiol. horizontal or vertical. In press. Gillard-Crewther, S. (1978) Plasticity in the cat visual A surprising finding was the total absence of any system. Ph.D. Thesis, California Institute of Tech­ binocular interaction in any of the netn'ons studied in any nology, Pasadena, California. Gillard-Crewther, S., Crewther, D. P. and Peck, C. K. oilbird. This stands in marked contrast to the owl, where (1978) Maintained binocularity in kittens raised with the vast majority of neurons are binocular, and is both eyes rotated. AR YO Abstracts, p. 269. Gillard-Crewther, S., Crewther, D. and Pettigrew, J. D. particularly notable in view of the large degree of (1978) Maintenance of cortical binocularity in kit­ binocular overlap exhibited by the oilbird. tens reared with bilateral rotation of the eyes (Manuscript). We conclude that, despite its frontally-placed eyes, Itakura, T., Kasamatsu, T. and Pettigrew, J. D. (1978) the oilbird is like the rabbit, and unlike the cat and owl, in Catecholaminergic terminals in kitten visual cortex: the normal distribution and its changes following the that its visual "cortex" is neither primarily involved in local perfusion of 6-0HDA. Soc. Neurosci. Ab­ stracts. In press. 124

Karten, H., Pettigrew, J. D. and Konishi, M. (1978) Pettigrew, J. D. (1978) A comparison of the visuotopic Telencephalic representation of the owl's claw. organisation of the visual Wulst in diurnal raptors, Proc. Neurosci. Soc.. In press. with a note on the evolution of frontal vision. In: Kasamatsu, T., Ary, M. and Pettigrew, J. D. (1978) Frontiers of Visual Science, S. J. Cool and E. L. Recovery from effects of monocular deprivation: Smith (Eds.), Springer-Verlag Series in Optical Sci­ acceleration with norepinephrine and suppression ences. In press. with 6-hydroxydopamine (Manuscript). Pettigrew, J. D. (1978) Binocular visual processing in the Kasamatsu, T., Pettigrew, J. D. and Ary, M. (1978) owl's telencephalon. Proc. Roy Soc. B (Lond.). In Restoration of visual cortical plasticity by local press. perfusion of norepinephrine (Manuscript). Pettigrew, J. D. (1978) Vision and birds of the night. Knudsen, E. I., Konishi, M. and Pettigrew, J. D. (1977) Engineering and Science (Caltech) 41, No. 2, pp. Receptive fields of auditory neurons in the owl. 8-13. Science 198: 1278-1280. Pettigrew, J. D. (1978) Is the locus coeruleus involved in Norman, J. L., Pettigrew, J. D. and Daniels, J. D. (1977) cortical plasticity? Trends in Neurosciences. In Ea·rly development of X-cells in kitten lateral press. geniculate nucleus. Science 198: 202-204. Pettigrew, J. D., Cooper, M. L. and Blasdel, G. G. (1978) Pettigrew, J. D. (1978) The paradox of the critical period Improved use of tapetal reflection for eye position for striate cortex. In: Neural Plasticity, Carl monitoring (Manuscript). Cotman (Ed.), pp. 311-330, Raven Press, New York. Pettigrew, J. D. and Kasamatsu, T. (1977) Local norepi­ Pettigrew, J. D. (1978) Stereoscopic visual processing. nephrine perfusion and effects of monocular depri­ Nature 273: 9-11. vation in 6-0HDA-treated kittens. Soc. Neurosci. Pettigrew, J. D. (1978) A role for the avian pecten oculi in Abstracts, p. 572. orientation to the sun? In: Animal Migration Pettigrew, J. D. and Kasamatsu, T. (1978) Local perfusion Navigation and Homing, pp. 42-54, K. Schmidt­ of noradrenaline maintains visual cortical plasticity. Koenig and W. Keeton (Eds.), Proc. in Life Sci., Nature 271: 761-763. Springer-Verlag, Heidelberg and New York.

Professor: R. W. Sperry in split-brain monkeys; (3) neural and behavioral plasticity Research Associate: Charles R. Hamilton Visiting .Associates: Felicia A. Huppert, Evelyn Lee-Teng, after neonate surgery on oculomotor muscles in kittens; Margaret Y. Scott (4) aspects of memory processing, learning, and engram Senior Research Fellows: Ronald L. Meyer, Eran Zaidel Spencer Research Fellow: Timothy D. Field localization in brain-operated, newly hatched chicks; (5) Graduate Students: Karen E. Gaston, Sheila GillaN:l­ retinotectal growth, plasticity, and engram formation in Crewther, Karen F. Greif, Larry E. Johnson, Betty A. Vermeire goldfish. Visiting Investigators: Carol K. Peck, Helga Schiff­ Sertorio 197. VERBAL IDENTIFICATION OF LEFT VISUAL Research Staff: Leah Ellenberg, Eef Goedemans, Susan FIELD NUMBERS IN COMMISSUROTOMIZED E. Kass, Lois E. MacBird, Josephine Macenka, HUMANS Steven D. Mueller, Edward Ogawa, Leslie L. Wolcott Investigator: Larry E. Johnson

Support: The work described in the following research During tachistoscopic presentation of visual stimuli reports has been supported by: American-Israeli Binational Science Foundation (that is, very brief flashes of 100 to 150 ms) those stimuli Frank P. Hixon Fund to the left of a fixation point (or left visual field = LVF) National Institute of Mental Health, USPHS National Institutes of Health, USPHS pass to the right cerebral hemisphere, while those to the National Science Foundation right of the fixation point (right visual field = RVF) go to The President's Fund The Stone Foundation the left hemisphere. In most right-handed people verbal University of Cambridge speech appears to be localized in the left cerebral Summary: Research in psychobiology continues to deal hemisphere although many nonverbal linguistic abilities with a range of problems concerned with the higher may also co-exist in the right hemisphere (Zaidel, 1978). functions of the nervous system and neural mechanisms of When the major forebrain pathways between the left and behavior. Work in different laboratory sections has been right hemispheres have been cut (commissurotomy), many focused on five problem areas: (1) hemispheric specializa­ reseachers have concluded that verbal identification of tion and functional organization in human commissur­ LVF stimuli (passing to the right hemisphere) occurs very otomy, hemispherectomy, other neurosurgical and normal rarely and may usually be explained by peripheral cross­ subjects; (2) mechanisms and pathways for the processing, cueing or other poorly controlled factors. interhemispheric transfer, and recall of visual information In the following experiment, two "split-brain" sub- 125 jects (NG and LB) were tachistoscopically presented single the same or different, or to identify verbally the two digit numbers to their LVF, RVF, or at a fixation point numbers presented. All stimuli, viewed through a and were asked to verbally name the number flashed. The Gerbrands two-channel tachistoscope, were 2° in size and numbers were 1° or 2° in size, and black on a constant flashed as black digits on a constant white background. white background. It was found that when the patients are required to The results are as follows: (1) LB can accurately make go-no go manual responses (that is, move a lever name numbers in both the LVF and RVF, up to 8° with the right hand if the two numbers are thought to be horizontally from the fixation point. NG can correctly the same or make no response if they are thought to be identify single numbers in the RVF but cannot name different), subject NG is able to make correct choices at a numbers in the LVF. (2) Naming stimuli in the LVF, highly significant level when the numbers are presented '!!' whether correct (LB) or incorrect (NG) requires twice as and 3° to each side of a fixation point, while LB can do it much time (about 650 ms vs. 1300 ms) as naming stimuli in only rarely. Each subject performs equally poorly when the RVF, and the variability in reaction time (RT) is much stimuli are flashed 4° from the fixation point (i.e., 8° greater. (3) The ability of either subject to identify these apart) and not above chance. stimuli is independent of the number flashed, its size (1° NG can verbally say "same" or "different" to the or 2°), or its distance (up to 8°) from the fixation point. bilateral stimuli with an accuracy approximately equal to (4) LB can verbally identify single numbers perfectly at her manual response. LB cannot acclll'ately say 11same"- the fixation point with RTs equal to his responses to RVF 11different" to the stimuli, and also performs more poorly stimuli. NG can identify numbers at the fixation point at than he did when using a manual response. a greater than chance level (61 % vs. 14% chance) with no However, when requested to name the bilateral errors consistent with hemifield masking. Correct re­ stimuli, NG can only repeat twice the number presented in sponses by NG have reaction times about equal to her the RVF. LB, in contrast, can accurately name (left to usual RVF responses while incorrect responses have RTs right) the two numbers presented. about equal to her usual LVF responses. In summary, one of two split-brain subjects studied In summary, subject LB is easily able to name can consistently cross-compare numbers flashed simulta­ numbers flashed in the LVF. And although NG cannot, her neously to the two visual fields, and manually or verbally reaction times are almost identical to LB's, which may indicate whether the two numbers are the same or not. It imply that some underlying mechanisms are the same in remains unclear not only why the two subjects differ in both subjects. Further experiments are in progress to try their ability to visually cross-integrate, but also what to determine how the split-brain subject can verbalize neural strategies and structures may be involved. Further LVF events, whether information must be ultimately experiments are in progress to try to answer these passed to the "verbal" left hemisphere or whether the questions. right hemisphere has verbal abilities not previously recog­ nized. 199. VISUAL CROSS-INTEGRATION OF PATTERNS IN COMMISSUROTOMIZED HUMANS Reference: Investigator: Larry E. Johnson Zaidel, E. (1978) Cerebral correlates of conscious experi­ ence. INSERM Symposium No. 6, 117-197. Forebrain commissurotomy (including the corpus callosum, anterior commissure, hippocampal commissure, 198. VISUAL CRQSS-INTEGRATION OP NUMBERS IN and massa intermedia) eliminates a broad communication COMMISSUROTOMIZED HUMANS system between the two cerebral hemispheres. Although, Investigator: Larry E. Johnson in fact, the two halves of the brain are not completely Two patients (NG and LB) who have undergone disconnected, the kinds and amounts of information which forebrain commissurotomy were tested for visual cross­ can continue to be intercommunicated remain unclear. integration by tachistoscopically (150 ms) presenting two Experimental results have been previously reported single digit numbers simultaneously in the LVF and RVF. (Biology 1977, No. 210) concerning the capabilities of two The subjects were required to make either a manual or split-brain subjects (LB and NG) to cross-integrate simple verbal response indicating whether the two numbers were line figures flashed tachistoscopically (150 ms) to the two 126 visual fields. These experiments have been continued and extension of the method developed by Huppert and Piercy expanded, and the following results are now available. (1977, 1978). Retention of pictorial information was Using a manual go-no go response mode (moving a tested by presenting slides of scenery, people, etc., to the lever if the two stimuli are identical or doing nothing if left or right visual field of split-brain subjects and testing they are not), subject NG can consistently and accurately yes-no recognition (using a manual response) 10 min, 2 indicate whether simple figures (X and O, or + and 0) are days, and 7 days later. The results indicate that although the same or different. She can do this whether the figures information was better retained by the right hemisphere are 2° or 5° in size, ·whether they are 1° to 4° to each side (left visual field) at the shortest retention interval, it was of a fixation point, and whether they are white figures on also more rapidly forgotten. In contrast, performance of a black background or black figures on white. There is no the left hemisphere (right visual field) was more constant significant difference in acctll'acy with +O stimuli com­ over time. pared with XO stimuli, implying that NG is comparing One interpretation of these data is that the left and more than just the most medial portions of each stimulus. right hemispheres initially encoded different information In contrast, LB almost never attains a significantly about the pictures, which in the case of the right acclll'ate score, although he usually performs higher than hemisphere, was well suited to short-term but not long­ chance 50%. With enough trials it is likely that LB's term retention. Accordingly, in the second experiment, scores might reach significance, which may explain the simple pictures (line drawings of familiar objects) were results reported last year. employed, and their features experimentally manipulated When the subjects are requested to say "same" or in an effort to determine which features were remem­ "different" after each presentation, NG continues to bered by each hemisphere. The features selected for perform very well while LB remains near chance level. study were color, shape, detail, and orientation. One However, when LB is asked to name the stimuli flashed he version of an object was flashed to the left or right visual can do this very well If he is requested both to indicate field and the split-brain subject required to point to the if the two stimuli are the same or not and then to name correct alternative after 0 or 10 sec delay. For both the stimuli, he becomes confused and his performance fields, performance deteriorated when the delay was breaks down. introduced, but there was no difference between fields at Mar_iy questions remain to be answered as to why one either delay. For left-field stimuli at both delays, the patient is able to easily cross-integrate these patterns correct color was almost always selected, while the other while the other cannot. It is also possible that LB's verbal three features were each correctly selected only 70% of identifications may involve right hemispheric speech. the time. The pattern of responses for right-field stimuli Further experiments examining the reaction times re­ was quite different from this. Moreover, for right-field quired for responding to various tasks may provide some stimuli the pattern was quite different at the two test insight into how split-brain humans cross-integrate visual intervals. At 0 delay, color and shape were almost always information. correct and detail and orientation rarely correct; at 10 sec delay the most striking change was that color was 200. AN ANALYSIS OF MEMORY IN LEFT AND RIGHT rarely correct while orientation was almost always cor­ CEREBRAL HEMISPHERES rect. Taken as a whole, the results indicate that the left Investigator: Felicia A. Huppert hemisphere initially encoded more features than the right Investigations by Sperry and his colleagues of the (which appeared to concentrate on color) but that the left capabilities of the separated cerebral hemispheres of hemisphere failed to utilize all the information which was split-brain subjects have yielded valuable information available to it. It is at present unclear why this should be about specialization or relative superiority of one hemi­ the case. However, the results of these two rather sphere over the other on a variety of cognitive tasks. different studies clearly demonstrate the existence of a However, one question which has so far received little qualitative difference in retention in the two cerebral attention is whether the hemispheres differ in their ability hemispheres. This finding could have profound implica­ to store and retain information. Two studies were tions for theories of normal as well as abnormal memory, undertaken to answer this question. The first was an and further investigations appear to be well worth 127 pursuing. and nonsense syllables in Israelis suggests that the two tests tap multiple and diversely lateralized component References: Huppert, F. A. and Piercy, M. (1977) Neuropsychologia 15: linguistic processes. Although the nonsense syllables seem 643-652. neutral with respect to English and Hebrew, it remains to Huppert, F. A. and Piercy, M. (1978) Nature. In press. be found whether the differences in laterality effects on 201. CROSIH;ULTURAL DIFFERENCES IN BRAIN the dichotic CV task between Americans and Israelis can ORGANIZATION? serve as indices of cortical differences due to the Investigators: Eran Zaidel, Harold W. Gordon* contrasting scanning habits in English and Hebrew (read A dichotic tape containing simultaneous natural from left to right and from right to left, respectively). nonsense Consonant-Vowel (CV) syllables from the set Ba, *Department of Behavioral Biology, The Medical School Da, Ga, Pa, Ta, Ka, of the kind used for laterality The Technion, Haifa, Israel. experiments on many American subjects, has been admin­ istered to about 55 native adult Israelis. Subjects were 202. SYNTAX IN TIIE RIGHT HEMISPHERE required to write down both of the sounds they had heard. Investigator: Eran Zaidel The common results with American subjects include (1) a A long-term study of the syntactic structures large and significant right ear advantage which is usually available to auditory language comprehension in the interpreted as left hemisphere specialization for process­ disconnected right hemisphere continues to highlight a ing the speech sounds, (2) a larger laterality effect for substantial grammatical competence and large subject-to­ men than for women, (3) a shared feature advantage subject differences. Poor right hemisphere performance yielding lower performance (and sometimes a higher right was more likely to occur in sentences involving word ear advantage) for dichotic pairs differing in two phonetic order, complex negation, wh-questions, embedding, and features than for pairs differing in one feature only. subject pronouns than ones involving simple negation, Other observed effects with American subjects include (4) tense and aspect, and object pronouns. the following order of decreasing laterality effects for Attempts to characterize the syntactic competence individual stop consonants: B>G>P>K>D>T, and (5) of the right hemisphere in developmental or pathological better performance with unvoiced than with voiced terms were generally unsuccessful. Although the right syllables. hemispheres obtained equivalent mental ages of between 4 In contrast the same stimuli with the Israeli popula­ and 8 years, they revealed different error patterns from tion (1) failed to show a right ear advantage, (2) failed to normal children of the same age. There was a higher show a sex difference in laterality effect, (3) did show a correlation of right hemisphere errors with those of small shared feature advantage in performance level aphasics than with those of children, but here, too, the though not an associated drop in the laterality index, and right hemispheres were relatively less sensitive than (4) yielded a different order of laterality effects for aphasic patients to linguistic complexity proper and more individual stops: G>P>T>D>K>B. However, (5) unvoiced affected by difficulty of vocabulary, by memory, and by syllables still yielded better performance than voiced redundancy. ones. The disconnected right hemispheres also seem to Dichotic control tests with triplets of simultaneous exhibit modality-specific part-of-speech effects in recep­ Hebrew or English monosyllabic words did show compar­ tive language tests, and their syntactic sophistication able and significant right ear advantages in the native seems independent of their general stage of cognitive Israeli and native American subjects, respectively, as well development. Further experiments are planned for study­ as similar sex differences in laterality effects for both ing the effect of response mode on right hemisphere populations (H. W. Gordon). In view of the strong left competence: can the right hemisphere act out an hemisphere specialization for processing the stop-conso­ instruction as well as it can point to a picture correspond­ nant syllables indicated by split-brain patients (E. Zaidel), ing to it? the data suggest possible differences in cortical organiza­ tion of language between Americans and Israelis. The dissociation between laterality effects on word triplets 128

203. LONG-TERM PSYCHOLINGUISTIC CHANGES e.g., Bee with "B," Tee with "T,11 etc. In the "Picture" IN THE ISOLATED HEMISPHERES condition each sound was associated with the name of a Investigator: Eran Zaidel simple line drawing, e.g.; Bee with the picture of a bee, The psycholinguistic profiles of three hemi­ Tee with the picture of a tea cup, etc. F.ach dichotic pair spherectomy (one dominant) and six commissurotomy was followed immediately by a letter (or a picture) patients were compared during a three- to five-year flashed for 0.1 sec randomly either to the left or to the followup study in order to detect possible learning trends right of a central fixation dot. The subject then had to or other systematic changes. indicate with the hand ipsilateral to the stimulated half­ Where clinical status remained stable, one of several field whether this letter (picture) did or did not match a distinct patterns occurred. First, hemispheric deficits sound he had just heard. Both accuracy and latency were remained generally uncha~ed. There were subtle trends measured. in the mature disconnected left hemispheres towards Analysis of variance of the acc\ll'acy data revealed a improved performance and regression towards the norma­ significant right ear advantage, indicating left hemisphere tive mean. In other words, there is, as in the free-vision dominance. Surprisingly, there was a trend for the ITPA profile of six commissurotomy patients, a small pictures to be better recognized in the right visual half­ change towards reduced variability across tests and field and for the letters to be better recognized in the left towards normalization of scores. visual half-field. A second pattern discernible in the younger patients Analysis of variance of the latency data showed that shows development of abilities that are consonant with letters were recognized significantly faster than pictures, the native competence of the growing hemisphere. Thus that responses in the left visual half-field were signifi­ the right hemisphere of a girl who had dominant hemi­ cantly faster than responses to right visual half-field spherectomy at age 10 showed impressive growth of stimuli, and that correct responses were significantly auditory vocabulary but no change in a severe inability to longer than incorrect responses. There was also a decode long, ordered context-free sentences. Where significant ear x response interaction with latency of linguistic development occ\ll'red in either isolated hemi­ correct responses far exceeding incorrect responses for sphere it tended to do so at the expense of visuo-spatial left ear stimuli but not for right ear stimuli. abilities; the converse was not observed. When either the The results show that different laterality effects isolated left or right hemisphere showed deterioration of a can be obtained for the same dichotic stimuli when they skill with time they showed this on different skills and are interpreted differently by the subject. Quite different apparently as a consequence of cognitive readjustment laterality effects were also obtained depending on the rather than natural growth. feature contrasts of the dichotic pairs. Accuracy tends to Experiments are now under way to determine more emphasize left hemisphere advantage for processing the precisely how the two hemispheres vary in their respec­ auditory component of the task, whereas latency seems to tive learning strategies. It is already known that the right emphasize right hemisphere superiority in processing the hemisphere is not responsive to step-by-step trial and visual component. error learning whereas the left hemisphere is. *Undergraduate, Department of Psychology, Dartmouth College, Hanover, New Hampshire. 204. MEANINGFULNESS IN DICHOTIC LISTENING Investigators: Eran Zaidel, Bennette Kashdan* 205. PREFERENCES FOR COMPLEX STIMULI IN A dichotic tape containing random simultaneous SPLIT-BRAIN MONKEYS pairs of Consonant-Vowel (CV) syllables from the set Bee, Investigators: Betty A. Vermeire, Charles R~ Hamilton Dee, Gee, Pee, Tee, Kee, was constructed at Haskins Labs Studies of the visual preferences of split-brain in New Haven and administered individually using stereo monkeys (Biology 1977, No. 209) were undertaken to see headphones to 16 Caltech undergraduate students in whether there are differences in the ways the two Psychology and Linguistics classes. The experiment independent cerebral hemispheres discriminate, recognize, included two conditions. In the "Letter" condition each or categorize various types of complex stimuli. Split­ syllable was associated with the name of a stop consonant, brain monkeys were trained to hold down a lever to obtain 129

food while simultaneously viewing one stimulus at a time or at different speeds. By decreasing the rate of from selected pairs of photographic slides varying in movement, detection thresholds for differential move­ content. Which of the two available stimuli was viewed ment may be studied in addition to learning ability for all and the duration of viewing were Wlder the monkeys' these discriminations. control. It is presumed that since the monkeys must look Three monkeys have been operated on to date, but at something in order to obtain food, they will elect to only one has been tested extensively. This monkey has spend their time with the more favored of the two stimuli. shown a clear deficit in the ability to learn movement but It was found that picture content can differentially not pattern discriminations. At least 12 monkeys will influence the direction and magnitude of preference eventually be studied as they become available from other exhibited by each hemisphere. Although each individual experiments. monkey may show a systematic difference in the perfor­ mance of his two hemispheres, seven split-brain monkeys 207. INTEROCULAR EQUIVALENCE IN SPLIT-BRAIN MONKEYS have spown no evidence of an overall trend towards Investigator. Charles R. Hamilton hemispheric specialization on this task. However, these experiments are subject to the Interocular equivalence refers to the ability to methodological criticism that the monkeys may have interpret with one eye what was originally perceived, worked solely for the food and thus their behavior was not recognized, or learned with the other eye. For normal guided by the content of the photographs, Further studies animals this ability almost certainly results from the of visual preferences will employ two different appa­ convergence of inputs from the two eyes onto populations ratuses that do not involve food-motivated looking, but of binocular neurons in the striate cortex. If the optic rather tap the natural curiosity and investigative charac­ chiasm is divided in the midline then each eye directly teristics of monkeys. projects only to the hemisphere on its side and interocular equivalence depends on commissural connections between 206. BEHAVIORAL FUNCTIONS OF EXTRASTRIATE the hemispheres. The specific site of initial binocular VISUAL AREAS interaction may be manipulated by selective partial Investigator: Charles R. Hamilton section of the commissures, and the remaining degree of The numerous visual areas in the primate brain (e.g., interocular equivalence then may be determined behavior­ Allman, Biology 1975, pp. 37-39; Van Essen, Biology 1977, ally for a particular type of task. In this way the pp. 144-145) presumably represent regions for differential functional level for processing different types of informa­ analysis of visual information, the selective removal of tion in the hierarchically organized visual system can be which should produce differential deficits in behavior. studied. For example, it can be shown by selective While behavioral determinations of fWlction are notori­ disconnection that the perception of tilt aftereffects ously difficult, they are essential if functional interpreta­ occurs at a lower level than discrimination learning which tions of physiologically and anatomically defined areas are in turn occurs before interocular equivalence for visually to be made. By studying the effects of unilateral lesions directed movements. Anatomical studies (Biology 1977, in split-brain monkeys on visual discriminations selected No. 205) have provided sufficient detail about the locali­ on the basis of the physiological properties of cells in zation in the commissures of interhemispheric connections these areas, it is hoped that even subtle behavioral between visual areas to permit more precise tests of deficits may be observable. As a first attempt, lesions interocular equivalence at different levels in the hier­ have been made in the posterior bank and depths of the archy that should reveal what kind of processing occurs at superior temporal sulcus in rhesus monkeys, a visual area each level. notable for its cells that are particularly sensitive to One experiment now in progress should help decide direction of movement of stimuli. Stimuli to be discrimi­ which of two mechanisms, convergence or transfer, best nated include large rotating spirals that appear to expand interprets the phenomena of interoc_ular equivalence in or contract, radial lines that rotate either clockwise or animals with partially split brains. The convergence counterclockwise or in one direction but at different model, usually favored by physiologists, attributes equiva­ rates, and line gratings that move in different directions lence to simple, bilateral convergence of inputs from the 130

two eyes to produce binocular cells at any level left mechanisms for pattern or focal vision versus spatial or interconnected by commissural connections. Areas nearer ambient vision, we found that, as with patterns, inter­ the eye, such as striate cortex, would have binocular cells hemispheric transfer in cats of discriminations of move­ only near the vertical midline of the visual field while, ment and brightness is largely dependent on the splenium because of continuing convergence, areas further into the of the corpus callosum. A small but sometimes significant system would have binocular cells with larger receptive savings of about 20% on the transfer tests, however, may fields that extend farther from the midline. The transfer be attributable to a proportionally small role of the model, usually favored by psychologists, interprets equiva­ midbrain roof in the discrimination of these stimuli. lence as a result of transfer of information processed in one hemisphere to the other hemisphere by commissural 209. INTEROCULAR TRANSFER OF A CONDmONIID connections specialized for this purpose. It predicts that FOOD AVERSION IN CIDCKS Investigator: Karen &. Gaston information derived from the entire visual hemifield should be transferred. Therefore the two models may be Last year I reported preliminary findings which differentiated by observing how far off the midline suggested failure of interocular transfer for a visually­ interocular equivalence is preserved at different levels in mediated conditioned food aversion in chicks (Biology the system. We have found that both the anterior 1977, No. 204). Further investigation, however, has commissure, which interconnects areas deep in the sys­ demonstrated good interocular transfer of this learning. tem, and the splenium of the corpus callosum, which Young chicks with one eye occluded were subjected interconnects areas at many levels, allow information to a food aversion conditioning procedure (see Biology from at least 25° off the midline to be used by the 1977, No. 203) in which ingestion of an unfamiliar colored hemisphere indirectly receiving input from the commis­ liquid (green sucrose solution, GSS) was paired with sures. Further surgical division of the splenium to leave delayed LiCl-induced illness. In a subsequent preference intact only the restricted vertical midline connections for test (GSS vs. uncolored water) chicks displayed marked areas near the striate cortex is now in progress. avoidance of the GSS whether tested with the trained or the Wltrained eye open. Substantial interocular transfer 208. EXTENT OF MIDBRAIN VISION IN CATS of the monocularly-acquired visual food aversion was

Investigators: Carol K. Peck, Sheila Gillar~wtber, therefore found to occur after a single conditioning Charles R. Hamilton session, despite an interstimulus interval of at least During the last decade the pretectum and superior 10 min. colliculi of mammals have become increasingly implicated The factors which govern whether or not interocular in visual perception and spatial orientation. We have now transfer of avoidance learning occurs in birds are not completed a previously described study (Biology 1976, clear. Cherkin (1970) and Benowitz (1974) found that Nos. 150 and 151) of the role of the midbrain areas of the chicks that learned in one trial to inhibit pecking of a bad­ cat in discriminating two types of stimuli frequently tasting object demonstrated good interocular transfer of associated with midbrain function--direction of move­ the passive avoidance task. In a series of active ment and brightness. Rather than using the traditional avoidance experiments with adult birds, Stevens and method of testing the midbrain for surviving capabilities Klopfer (1977) found good interocular transfer of con­ after removal of the primary visual areas, which alters ditioned withdrawal from a visual stimulus when the the ~unctioning of the isolated midbrain system, we tested unconditioned stimulus was painful shock to the wings, but the ability of the midbrain commissural connections to failure of transfer when the unconditioned stimulus was a support interhemispheric transfer, and compared this loud noise or a monocularly-presented looming object. ability to that of the forebrain commissures. It is Thus, research to date on avoidance learning in birds has assumed that the functions of different areas can be failed to find interocular transfer for responses estab­ inferred from the type of information transferred by their lished through distance receptors, i.e., visual or auditory, interhemispheric connections. whereas positive transfer has been found for responses Contrary to expectations based on current theories established through somesthetic or visceral receptors. It that postulate largely independent fore- and midbrain may be that a crucial factor involved in determining 131 interocular transfer of avoidance learning in birds is the type of learning in chicks might be expected to vary organism's perception of the unconditioned stimulus as an directly with age and the functional maturity of the internal, rather than external, event. participating interhemispheric connections. The present experiment is the first stage of a developmental study Refereneei: Benowitz, L. (1974) Brain Res. 65: 203-213. designed to establish the degree of interocular transfer of Gherkin, A. (1970) Nature 227: 1153. pattern discrimination learning in newly-hatched chicks Stevens, V. J. and Klopfer, F. D. (1977) J. Comp. Physiol. Psycho!. 91: 1074-1081. and to determine whether the extent of transfer varies with age, possibly as a result of progressive myelination of 210. NONVJSUAL CUES MAY MEDlATE INTER­ a forebrain fiber system such as the DSO. OCULAR TRANSFER OF A VISUAL FOOD AVERSION IN CHICKS In the first phase of the investigation, 24 3- to Investigator: Karen E. Gaston 6-day-old domestic chicks were trained ,monocularly to perform a simultaneous two-choice visual pattern dis­ While binocularly-trained chicks do not exhibit any crimination (+, ti) for heat reward and then were tested evidence of an acquired taste aversion under the experi­ for retention or interocular transfer of the discrimination mental conditions described in the preceding abstract learning. After each animal was shaped to peck in an (Gaston, 1977), it remains possible that the novel taste operant chamber, one of its eyes was covered with an and/or viscosity of the colored sucrose solution has a role occluder and it was trained on the discrimination to a in mediating acquisition and/or interocular transfer of the criterion of 36/40 correct responses. The discrimination visual aversion. was readily learned, with nearly all animals reaching To examine this possibility, chicks were adapted to criterion in fewer than 300 trials. Immediately following drinking uncolored sucrose solution (USS) prior to the the acquisition session the chicks were retrained to monocular conditioning procedure which paired green criterion on the discrimination problem with either the sucrose solution (GSS) with delayed LiCl-induced illness. same eye (monocular retention, n=12) or the other eye The results of a subsequent preference test (GSS vs. (interocular transfer, n=12). The chicks retested with the uncolored water) indicated a dissociation between factors trained eye demonstrated excellent retention of the dis­ influencing acquisition of the aversion and those influ­ crimination task. In marked contrast, animals tested for encing interocular transfer. While adaptation to USS prior interocular transfer showed no significant savings. Initial to conditioning did not decrement the degree of condi­ performance (errors in the first 40 trials) and total trials tioned aversion to ass displayed by chicks tested with the and errors to criterion with the second eye were not trained eye, apparent interocular transfer of the aversion appreciably different from those of the earlier acquisition was much less than that observed in the previous session with the first eye. These results indicate that, experiment in which the sucrose solution was completely under the present experimental conditions, pattern dis­ unfamiliar. Investigations in progress are aimed at crimination learning fails to transfer from the trained to determining the significance of this finding. the untrained eye in very young chicks, in contrast to the Reference: findings in adult birds and to the good transfer reported Gaston, K. E. (1977) Behav. Biol. 20: 441-453. for one-trial passive avoidance (Cherkin, 1970) and for an object discrimination (Bondy and Harrington, 1978) in 211. TESTS FOR INTEROCULAR TRANSFER OF A HEAT-REWARDED PATTERN DISCRIMINATION newly-hatched chicks. Progressively older chicks are INCWCKS presently being tested on the pattern discrimination in Investigator: Karen E. Gaston order to determine whether there is a positive correlation Complete optic decussation in birds restricts direct between behavioral transfer and the maturation of inter­ visual input from each eye to the contralateral hemi­ hemispheric connections. sphere, making interocular transfer of visual learning References: dependent on interhemispheric connections. An intact Bondy, S. C. and Harrington, M. E. (1978) Science 199: 318-319. supraoptic decussa.tion (DSO) is apparently necessary for Gherkin, A. (1970) Nature 227: 1153. transfer of pattern discrimination learning in adult birds Cuenod, M. (1974) In: The Neurosciences, Third Study Program, pp. 21-30. (cuenod, 1974). The extent of interocular transfer of this 132

212. HORSERADISH PEROXIDASE STUDY OF eye is only to the contrelateral midbrain, the optic OPTIC FIBERS IN GOWPISH* tectum. However, it is possible to misdirect surgically Investigator: Ronald L. Meyer one optic nerve into the ipsilateral side so that both eyes In goldfish and other lower vertebrates, severed innervate a single tectum. This technique was used in optic fibers have the capability of regrowing back to their conjunction with eye lesions wherein half of the retina main target neurons of the midbrain tectum. This was removed. Which half-retina was removed, whether regenerative capacity has made the retinotectal projec­ the lesioned eye projected contralaterally or was forced tion a popular model system for studying the development ipsilaterally, or whether one or both eyes were lesioned of selective axonal connections. In numerous previous were systematically varied In this manner the avail­ experiments the location and function of the terminals of ability of appropriate target sites and the kind and number these regenerated fibers have been determined after a of ingrowing and preexisting optic fibers could be altered. variety of experimental manipulations. However, we are The growth of fibers was analyzed anatomically by still quite ignorant about the general morphology of these labeling fibers with tritiated proline for autoradiography fibers including the pathways through which they have on serial sections. The results so far have been complex. grown. On the presumption that such information will Fibers show some growth preference toward their normal provide important clues about the mechanisms underlying targets regardless of preexisting optic fibers but at the growth of selective connections, an anatomical study of same time this growth preference is also modifiable by optic axons in goldfish was attempted. The approach was the presence of other optic fibers. In some cases the to apply the marker enzyme horseradish peroxidase (HRP) tectal innervation was uniform but under other conditions to the retina or optic nerve and at the same time cut a the fibers from the two eyes interdigitated in patchwork few optic axons by various microsurgical methods. Since fashion. An anomalous double lamination was also the HRP is taken up primarily by cut fibers which occasionally found. This suggests that the factors transport the enzyme down towards its terminal, the hope controlling fiber growth are multidimensional and inter­ was to selectively stain a small number of fibers. So far active. it has been possible to stain reliably large groups of fibers PUBLICATIONS originating from selected regions of retina and to stain occasionally isolated fibers apparently including the ter­ Gaston, K. (1977) Visual mediation of one-trial condi­ tioned aversion learning in chicks. Soc. Neurosci. minal arborization. Efforts continue to improve the Abstracts 3: 727. reliability and resolution of the technique so that it can be Gaston, K. (1978) Interocular transfer of pattern dis­ crimination learning as a function of age in the extended to fish with regenerated optic nerves. developing chick. Soc Neurosci. Abstracts. In press. Gaston, K. (1978) Interocular transfer of a visually­ *Part of this work was carried out while the author was on mediated conditioned food aversion in chicks. Be­ summer leave of absence at Cold Spring Harbor Labora­ hav. Biol. In press. tories. Gaston, K. (1978) Brain mechanisms of conditioned taste aversion learning: A review of the literature. Physiol. Psycho!. In press. 213. SURGICALLY UNCROSSING THE OPTIC NERVE Greif, K. F. and Scott, M. Y. (1977) Localization and IN GOLDFISH generalization of visual memory after tectal lesions Investigator: Ronald L. Meyer in goldfish. Soc. Neurosci. Abstracts 731, p. 234. Hamilton, C. R. (1977) An assessment of hemispheric specialization in monkeys. Ann. N. Y. Acad. Sci. Since the 1940s, it has been known in lower 299: 222-232. vertebrates that when severed optic fibers regrow back Meyer, R. L. (1978) Evidence from thymidine labeling for continuing growth of retina and tecturn in juvenile into the brain, the fibers differentially interact with goldfish. Exptl. Neural. 59: 99-111. · various brain cells in the course of forming selective Meyer, R. L. (1978) Deflection of selected optic fibers into a denervated tectum in goldfish. Brain Res. In connections. Recent work in this and other laboratories press. has now indicated that interactions between the optic Meyer, R. L. (1978) "Elctra" optic fibers exclude normal fibers themselves may also importantly determine the fibers from tectal regions in goldfish. J. Comp. Neurol. In press. selectivity of fiber growth. The retinotectal system of Meyer, R. L. (1978) Autoradiographic mapping of retino­ goldfish has been used to explore further these interfiber tectal fibers in normal goldfish and after regenera­ tion of following nerve crush. Soc. Neurosci. interactions. Normally the main optic connection of each Abstracts. In press. 133

Peck, C. K., Crewther, s. G. and Hamilton, c. R. (1978) Zaidel, E. (1978) Concepts of cerebral dominance in the Partial interocular transfer of brightness and move­ split brain. In: Cerebral Correlates of Conscious ment discriminations by split-brain cats. Brain Res. Experience, P. Buser and A. Rougeul-Buser (Eds.), In press. pp. 263-284, Elsevier, Amsterdam. Sperry, R. W. (1977) Absolute values: Problem of the Zaidel, E. (1978) Man's elusive right hemisphere. Scien­ ultimate frame of reference. In: The Search for tific American. In press. Absolute Value: Harmony Among the Sciences, Vol. Zaidel, E. (1978) The split and half brains as models of II, pp. 689-694, Proc. Fifth Jnternat. Conf. on the congenital language disability. Jn: The Neurological Unity of the Sciences, The International Cultural Bases of Language Disorders in Children: Methods Foundation Press, New York. and Directions for Research, C. L. Ludlow and M. E. Sperry, R. W. (1978) Consciousness as causal. In: Oxford Doran-Quine (Eds.), NINCDS Monograph, Govern­ Companion to the Mind, Richard L. Gregory (Ed.), ment Printing Office, Washington, D.C. In press. University of Oxford Press. In press. Zaidel, E. (1978) Long-term stability of hemispheric Token Sperry, R. W. (1978) The human mind. In: Consciousness, Test scores following brain bisection and hemide­ Personal Identity, and the Divided Brain, The cortication. In: The Token Test: History, Clinical, 1977-1978 Frank Nelson Doubleday Lectures. In and Experimental Applications, Future Directions, press. F. Boller and M. Dennis (Eds.), Academic Press, New Sperry, R. w., Zaidel, E. and Zaidel, D. (1978) Self­ York. In press. recognition and social awareness in the deconnected Zaidel, E. (1978) On measuring hemispheric specialization minor hemisphere. Neuropsychologia, Vol. 16, in man. Jn: Advanced Technobiology, B. Rybak Pergamon Press. In press. (Ed.), Sijthoff and Noordhoff, Alphen aan den Rijn. Zaidel, E. (1978) Auditory language comprehension in the In press. · right hemisphere following cerebral commis­ Zaidel, E. (1978) Performance on the ITPA following surotomy and hemispherectomy: A comparison with cerebral commissurotomy and hemispherectomy. child language and aphasia. Jn: Language Acquisi­ Submitted for publication. tion and Language Breakdown: Parallels and Diver­ Zaidel, E., Zaidel, D. and Sperry, R. W. (1978) Left and gencies, A. Caramazza and E. B. Zurif (Eds.), pp. right intelligence: Unilateral performance on the 229-275, Johns Hopkins University Press, Baltimore. book and board forms of Raven's Progressive Zaidel, E. (1978) Lexical structure in the right hemi­ Matrices following brain bisection and hemidecorti­ sphere. In: Cerebral Correlates of Conscious cation. Submitted for publication. Experience, P. Buser and A. Rougeul-Buser (Eds.), pp. 177-197, Elsevier, Amsterdam.

are applying several other approaches to the problem of Assistant Professor: David C. Van Essen Graduate Students: John L. Bixby, Karl J. Fryxell, John visual cortical organization. By selectively labeling the H. R. Maunsell cells of origin of the projection across the corpus Research Staff; Phyllis F. Knu

218. THE TIMING OF SYNAPSE ELIMINA'DON IN of multiple innervation. Differences in these two param­ SKELETAL MUSCLES OF NEONATAL RABBITS eters did not appear to be related to position in the body, Investigators: John L. Bixby, David C. Van J!ssen nor was there any obvious correlation between the Mammalian skeletal muscles are known to undergo contractile properties of muscles and the time course of striking changes in their pattern of innervation during synapse elimination. These findings indicate that the early postnatal development. The most prominent events timing of this important phase of neuromuscular matur­ are an initial polyneuronal innervation of individual ation is not regulated by simple body-wide signals; rather, muscle fibers and a subsequent period of synapse elimin­ it is likely that the process depends upon specific ation that leaves each fiber innervated by one and only interactions between each muscle and its motor supply. one motor axon. We are investigating the elimination of synapses in the rabbit because it is much larger than the rat, the most extensively studied animal in this regard, PUBLICA 'DONS and so is more amenable to certain experimental manipu­ lations designed to explore the mechanism underlying this Bixby, J. L. and Van Essen, D. C. (1978) Suppression of original nerve inputs to a mammalian skeletal phenomenon. In contrast to the results found in studies on muscle by a foreign motor nerve. Soc. Neurosci. Abstracts 4. In press. the rat, we have found regional differences in the time of Bixby, J. L. and Van Essen, D. C. (1978) Regional onset of synapse elimination in various skeletal muscles of differences in the timing of synapse elimination in skeletal muscles of the neonatal rabbit. Brain Res. the rabbit. In particular, muscles situated more anteriorly Submitted for publication. in the body tend to lose their "extra" synapses earlier than Van Essen, D. C. (1977) Neural signaling in vertebrate and invertebrate visual systems. In: Function and muscles situated more posteriorly. The largest difference Formation of Neural Systems, G. S. Stent (Ed.), pp. we found was between the diaphragm, which begins losing 253-283, Dahlem Konferenzen, Berlin. synapses several days before birth, and the soleus muscle, Van Essen, D. C. (1979) Visual areas of the mammalian cerebral cortex. Ann. Rev. Neurosci. 2. In press. which shows no detectable synapse loss until several days Van Essen, D. C. and Bixby, J. L. (1978) The distribution postnatally; in the rat these same muscles differ by no of cells projecting interhemispherically in extra­ striate visual cortex of the macaque. Soc. Neurosci. more than a day in the time course of synapse elimination. Abstracts 4. In press. Van Essen, D. C. and Zeki, S. M. (1978) The topographic We have also found differences among muscles in organization of rhesus monkey prestriate cortex. J. the rate of loss of synapses and possibly in the peak level Physiol. 277: 193-226. NEUROGENETICS

Seymour Benzer

Ronald J. Konopka

139

Professor: Seymour Benzer 219. STUDIES ON THE PHYSIOLOGY OF napts Spencer Research Fellow: Chun-Fang Wu Investigators: Chtm-Fllll! Wu, Barry S. Ganetzky Research Fellows: Barry S. Ganetzky, Lawrence M. Kauvar A second chromosome, temperature-sensitive para­ Graduate Student: Duncan Byers Research Staff: Alice I. Cox, Eveline Eichenberger, K. lytic mutation, napts (no action potential, temperature­ Elaine Miller sensitive), isolated in this laboratory, causes the normal Support: The work described in the following research excitatory junctional potential (ejp) recorded from the reports has been supported by: larval neuromuscular junction to disappear at restrictive The James G. Boswell Foundation for Virus Research temperatures (above 35°C), even though graded ejps can Jane Coffin Childs Memorial Fund for still be evoked at these temperatures by depolarizing the Medical Research National Science Foundation nerve terminal electrotonically. Gordon Ross Medical Foundation We have demonstrated directly that this is due to a Spencer Foundation Fund mutant defect in axonal conduction (Wu et al., 1978). A Summary: .our activities are directed at the mechanisms fine bore (tip diameter 5 to 8 µ) suction electrode was underlying behavior, making use of genetic methods and used to record en passant from the larval nerves. Using the fruit fly, Drosophila. Work is in progress on three this technique, we found that for individual units, and for main levels--developmental, physiological, and behav­ the compound action potential, axonal conduction is ioral. normal in the mutant at 22°C but completely blocked The larval form of Drosophila is an excellent above 35°C. preparation for neurophysiology, since it contains large The effect of temperature on muscle action poten­ muscle fibers that are easily impaled with microelec­ tials was also investigated. Under physiological condi­ trodes. The properties of the neuromuscular junction have tions, the larval muscles do not fire action potentials, but ++ ++ been studied in detail, and glutamate has been identified can be made to do so by the presence of Ba , Sr , as the transmitter. Certain mutants, detected by behav­ tetraethylammonium ions, or very high. [Ca++] to over- ioral abnormalities in the adult form, have physiological come the rectification by potassium current (Suzuki and abnormalities demonstrable in this larval system. Some Kano, 1977). The inward current for the muscle action show temperature-reversible blocks in axonal impulse potential is carried by Ca++. To determine the effect of propagation. Others show abnormal transmission at the napts on muscle membrane excitability we recorded neuromuscular junction. Another has abnormal facilita­ intracellularly from larval muscles in the presence of tion at the junction. This larval system thus makes it 24 mM Sr++, with direct current injection through a possible to use behavioral mutants to dissect physiological second intracellular electrode. Both normal and mutant mechanisms and work toward the identification of the larvae give overshooting action potentials under these molecular components of neural structures. conditions at 22°C and retain this ability up to 37"C or On the behavioral level, we have concentrated on more. Thus, nap ts appears to affect a product whose learning and memory in Drosophila. Normal flies can normal function is essential for the sodium action poten­ learn to avoid selectively a specific odor that has been tial in nerves but not the calcium action potential in associated with electric shock. A mutant, called dunce, muscle. does not learn this task, in spite of having essentially References: normal ability to sense the odorants and the shock. The Suzuki, N. and Kano, M. (1977) J. Cell Physiol. 93: defect is due to change of a single gene. The search for 383-388. Wu, C.-F., Ganetzky, B., Jan, L. Y., Jan, Y. N. and additional mutants continues to be fruitful and we will Benzer, s. (1978) Proc. Nat. Acad. Sci. USA 75: attempt to use these in dissecting the steps involved in 4047-4051. memory formation. 220. CYTOGENETIC STUDIES OF napts Investigators: Barry S. Ganetzky, Chtm-Fllll! Wu

nap ts (no action potential, temperature-sensitive) is a recessive, second chromosome, temperature-sensitive paralytic mutation that blocks axonal conduction at 140 restrictive temperatures (Biology 1977, No. 227). To approach to this question is to construct double mutants. identify the altered gene product by biochemical tech­ If the mutant defects are independent, the phenotype of niques, electrophoretic and other variants would be useful. the double mutant should simply be the sum of the Thus, we are attempting to isolate additional nap alleles. individual phenotypes. Where the two gene products This project hes been simplified by the cytological interact, one may expect two main types of result--a sup­ localization of the nap ts mutation and the identification pressive interaction, or a synergistic one. We have of a chromosome deleted for this region. We found that initiated studies of this type using nap ts combined with flies carrying a second chromosome deleted for salivary other mutations. region 41B-42A, heterozygous over nap ts, are identical in The Shaker (Sh) mutants show ether-induced leg­ phenotype to nap ts homozygotes. Thus, nap ts is located shaking behavior and are associated with a prolonged cytologically between 41B and 42A and the deleted excitatory junctional potential (ejp) at the larval neuro­ chromosome lacks the normal nap gene function. This muscular junction, believed to be the consequence of a deletion can be used in a simple screen for new nap alleles defect in potassium channels (Jan et al., 1977). Flies in the first generation after mutagenesis. Normal doubly mutant for Sh and nap ts do not display leg-shaking mutagen-treated males are crossed to deletion-bearing behavior (even at 22°C, at which temperature napts flies females and offspring carrying the deletion and a treated are not paralyzed). In other words, nap ts behaves as a homolog are tested for temperature-sensitive paralysis. suppressor of the Sh phenotype. Preliminary experiments Several thousand newly mutagenized second chromosomes suggest that this suppression is true at the physiological are now being examined in this way for new nap alleles. level also, since the time course and [Ca++] dependency of Another area of study with the nap ts mutation is the the ejp in the double mutant is more nearly normal than identification of the primary anatomical site of action, or that of Sh alone. foets, of the mutant gene (Hotta and Benzer, 1972) which A synergistic interaction was found between parats requires the construction of sexual mosaics, or gynandro­ and nap ts. The parats mutation is X-linked and causes a morphs. For an autosomal mutation like nap ts the method temperature-sensitive paralytic phenotype similar to that is normally not applicable. However, we have identified of nap ts. Although each of the single mutants is viable at an insertional translocation in which the normal nap allele 22°C, the double mutant is completely lethal. Experi­ has been inserted into the Y chromosome. From the Y ments are in progress to determine the precise time of chromosome, it is an easy matter to move the gene to the lethality. X chromosome. The crosses to do so are in progress. Other double mutant combinations among the known Once the appropriate X chromosome is constructed we temperature-sensitive paralytic mutations--para ts, shits, will be able to make flies mosaic for nap ts in order to com, and nap ts__ have been examined and only the fate-map the mutant focus and to study the behavior of parats nap ts combination shows this synergistic effect. flies in which only specific identifiable parts of the This suggests that there may be something special about nervous system are blocked in axonal conduction. the function or interaction of their gene products. For the Sh nap ts and parats nap ts double mutants, the nature Reference: Hotta, Y. and Benzer, S. (1972) Nature 240: 527-535. of the interaction remains to be determined but may eventually be accounted for by the physiological proper­ 221. GENETIC INTERACTIONS AMONG MUTANTS ties and molecular structure of membranes. wrrH NEUROPHYSIOLOGICAL DEFECTS Reference: Investigators: Barry S. Ganetzky, Chwi-Fang Wu Jan, Y.-N., Jan, L. Y. and Dennis, M. J. (1977) Proc. Roy. Soc. Lond. B 198: 87-108. Recent studies in this laboratory have been con­ cerned with mutations causing defects in some part of the nervous system. Some of the mutants appear to involve 222. SEARCH FOR MUTANT GENE PRODUCTS different components of the nerve membrane, and we Investigator: Lawrence M. Kauvar would like to know whether the products specified by The high resolution two-dimensional protein electro­ these genes physically interact with one another or share phoresis system described by O'Farrell (1975), which hes related functions in the nerve membrane. One genetic been successfully used to identify mutant gene products in 141 phage, E. coli, and mammalian tissue culture systems, is individual. Half the flies were trained to avoid 3-octanol being used to compare extracts of brains from wild-type and half to avoid 4-methylcyclohexanol, in alternating Drosophila with comparable fractions from mutant series. Summing all trials, the learning score for normal 35 strains. In vitro labeling with s-methionine of hand­ flies was 0.32 (n = 600 trials, 29 flies) and for dunce 0.05 dissected larval brains provides sufficient activity to (n = 660 trials, 19 flies). These are very similar to the produce a fluorographic image in a two-week exposure. scores observed for populations. Learning performance Two fractions from a total Dounce homogenate, 100,000 g for the ensemble of individually tested flies does not pellet and supernatant, are being examined. Treatment of change even after repeated trials (40 trials each for wild the homogenate with DNase before centrifugation con­ type and dunce). Also, the flies did not appear to be siderably reduces streaking of the pattern. fatigued by an hour of continuous testing. Therefore, repeated testing can be successful. From the results, it is Reference: O'Farrell, P.H. (1975) J. Biol. Chem. 250: 4007-4021. estimated that 100 trials are required for reliable pheno­ typic classification of an individual fly as normal or 223. Na+-CHANNElrSPBCIFIC TOXIN BINDING dtmce. STUDIES Investigator: Lawrence M .. .Kauvar Reference: Quinn, W. G., Harris, W. A. and Benzer, S. (1974) Proc. In collaboration with M. Raftery's group in the Nat. Acad. Sci. USA 71: 708-712. Division of Chemistry and Chemical Engineering, we are attempting to detect specific binding of various Na+ - 225. A SECOND ALLELE AT THE DUNCE WCUS IN DROSOPHILA channel inactivating toxins to homogenates of adult fly Investigator: Duncan Byers heads. By freezing whole flies with liquid nitrogen and shaking them vigorously on a vibrator, the heads can be The dunce mutation lowers olfactory learning per­ broken off and collected in high yield and purity with formance almost to zero (Dudai et al., 1976). It also nylon mesh screens. Initial attempts to detect binding slightly reduces female fertility. A second mutant has a 3 using H-labeled tetrodotoxin on 1250 homogenized heads similar effect on learning and strongly reduces fertility of were unsuccessful. We plan to try again using saxitoxin females (Biology 1977, No. 230). Both mutants map to the and scorpion toxin, each of which can be labeled to about same chromosomal vicinity. In complementation tests, 100 times the specific activity attainable with tetrodo­ new mutant/dunce trans heterozygous females have the toxin. If binding studies prove feasible, we plan to screen learning and fertility of dunce. Therefore, the mutants 2 mutants, particularly temperature-sensitive paralytics are allelic. The new allele has been named dunce . The 2 such as nap ts, for inability to bind the toxin in an attempt two phenotypes of dunce are fully recessive. to identify the gene(s) coding for components of the Na+_ Referenee: channel. Dudai, Y., Jan, Y.-N., Byers, D., Quinn, W. G. and Benzer, S. (1976) Proc. Nat. Acad. Sci. USA 73: 1684-1688.

224. LEARNING IN INDIVIDUAL DROSOPHILA Investigator: Dmlcan Byers PUBLICATIONS For mosaic fate mapping of the dunce mutation, the Jan, Y.-N. and Jan, L. Y. (1978) Genetic dissection of olfactory learning performance of normal and dunce short-term and long-term facilitation at the Dro­ sophila neuromuscular junction. Proc. Nat. Acad. individuals must be measurable. The procedtn'e used is the Sci. USA 75: 515-519. same as that used for populations (Quinn et al., 1974) Jan, Y.-N., Jan, L. Y. and Dennis, M. J. (1977) Two mutations of synaptic transmission in Drosophila. except that an individual fly is tested repeatedly. Be­ Proc. Roy. Soc. B 198: 87-108. cause the trained avoidance extinguishes within seven Wu, C.-F., Ganetzky, B., Jan, L. Y., Jan, Y.-N. and Benzer, S. (1978) A Drosophila mutant with a unreinforced trials, pairs of training trials were alternated temperature-sensitive block in nerve conduction. with pairs of test trials, up to as many as 40 tests for an Proc. Nat. Acad. Sci. USA 75: 4047-4051. 142

Assistant Professor: Ronald J. Konopka This result reaffirms the conclusion that the oscil­ Gosney Senior Research Pellow: Nancy S. Petersen lator controlling locomotor activity is located in the Research Pellow: Alfred M. Handler Graduate Students: Steven H. Green, Dominic Orr, brain, and raises the possibility that the action of the Randall F. Smith clock is mediated by a diffusible, possibly neurosecretory, Research Staff: Steven M. Wells Laboratory Staff: Rebecca F. Bodor, Caroline Vermaes substance. Since short-period rhythms were not super­ imposed upon the host arrhythmic activity, it is possible Support: The work described in the following research reports has been supported by: that the humoral factor acts by gating activity, rather Gosney Fund National Institutes of Health, USPHS than stimulating it. This factor is presumably lacking in President's Venture Fund per0 animals. The fact that the arrhythmic host exhibited Spencer Foundation Fund the genetically mutant short-period rhythm of the donor Summary: Our group is continuing the study of nervous brain precludes the possibility that the transplanted brain, system function and complex behavior by means of single or the operation itself, activated a normal oscillatory gene mutations in Drosophila. One specific approach has mechanism in the arrhythmic host brain. been the analysis of mutant brains by light microscopy and histochemistry in order to correlate behavioral abnormali­ 227. Sl!ARCH FOR NEURAL CORRELATl!S OP THE ties with nervous system defects. We are also investigat­ LOCOMOTOR ACTIVITY RHYTHM IN ADULT D. MELANOGASTER ing the effects of behavioral mutations in cell lines in Investigator: Dominic Orr order to learn the action of the mutation on the molecular level. Finally, we have spent much effort recently in It is well known that circadian systems with differ­ developing in vivo and in vitro organ cultures of brain and ent free-running periods and/or different sensitivities to a thoracic ganglion in order to study the detailed physiology phase resetting agent will entrain to this agent with and biochemistry of wild-type and mutant nervous sys­ different phases. We have found that, in the locomotor tems. One interesting offshoot of this program hes been rhythm of adult Drosophila, a drastic phase difference can the successful transplantation of a mutant circadian be induced between the wild-type strain (24 hr free­ pacemaker into a genetically arrhythmic host. running period) and the short-period mutant strain (19 hr free-running period) by imposing a 6 hr light-on and 6 hr 226. TRANSPLANTATION OP A CffiCADIAN light-off schedule. Under this condition, wild-type flies OSCILLATOR IN DROSOPHILA become inactive only in alternate dark periods while the Investigator: Alfred M. Handler mutant becomes inactive iri all dark periods; the transi­ Mosaic analysis of the adult locomotor activity tions from active to inactive phases being very abrupt in rhythm of Drosophila suggests that the neural oscillatory both cases. system resides in the brain. To test whether the oscillator We wish to know whether neural signals from the functions in the isolated brain, and to determine whether brain of the fly are responsible, at least in part, for a diffusible substance produced by the brain governs the inducing these active and inactive phases of its activity rhythm, we have transplanted brains from short-period cycle. Accordingly, a preparation has been developed in 8 (per ) flies into the abdomens of genetically arrhythmic which the anterior part of the fly, with the ventral 0 0 (per ) hosts. The locomotor activity of the per hosts and thoracic and neck cuticle open, is bathed in saline, and the control animals was monitored during entrainment and a posterior part of the fly, with the major spiracles for subsequent free-run period. Out of 59 surviving per0 hosts breathing, is exposed to ventilating air. We have been implanted with a pers brain, 6 showed short-period able to keep flies neurally active under this condition for activity as shown by digital activity records and periodo­ up to 36 hr. Neural signals to and from the brain have gram analysis. One individual in particular exhibited an been monitored with suction electrodes placed on the 18 hr period for 6 cycles, with an additional activity cervical connective. Units may be identified from fly to component introduced after the fourth cycle, and persist­ fly on the basis of relative spike amplitude and firing ing for an additional cycle apparently induced by a food pattern. So far, we have found that neural activities at charge. The remaining host animals showed no clear the cervical connectives of wild-type flies are much rhythms, as did per0 flies implanted with per0 brains, and higher and orderly patterned in the dark phases of a 6:6 unoperated per0 flies. 143 light-dark cycle. We are proceeding with further charac­ References: terization of the firing pattern of these physiologically Konopka, R. J. (1972) Ph.D. Thesis, California Institute of Technology, Pasadena, California. identified units and to correlate them with the activities Konopka, R. J. and Benzer, s. (1971) Proc. Nat. Acad. Sci. of the leg nerves and the behavior of the fly under similar USA 68: 2112-2116. Young, M. w. and Judd, B. H. (1978) Genetics 88: 723-742. conditions.

228. PER LOCUS GENETICS I: CLOCK PHENOTYPES 229. PER LOCUS GENETICS JI: GENE DOSAGE OF CHROMOSOME BREAKS IN THE PER REGION ANALYSIS Investigators: Randall F. Smith, Ronllld J. Konopka, Investigator: Randall F. Smith Dominic Orr Another method for studying the role of per in the Three circadian rhythm mutants (arrhythmic, long­ genetic organization of the Drosophila circadian system period, and short-period) of Drosophila melanogaster have involves manipulating the number of doses of the per been localized to a single locus (called per) within the gene. Doses of per+ can be added or subtracted using 3Bl-2 region of the X chromosome (Konopka and Benzer, duplications and deficiencies for the entire per region. 1971; Konopka, 1972). Chromosome rearrangements that We have found that for the eclosion rhythm, the deletion have a breakpoint in the 3Bl -2 region have also been of one of the two doses of per+ normally carried by a shown to produce arrhythmic and long-period rhythms wild-type female increases the period by about 1/2 hr, (Young and Judd, 1978). We have been reexamining the while the addition of an extra dose of per+ decreases the clock phenotypes of six 3Bl-2 rearrangements (five period by about the same amount. The addition of the + deficiencies and one translocation) by assaying both the equivalent of two doses of per to a wild-type male eclosion and adult locomotor activity rhythms of flies decreases the eclosion period by about 1 hr. The carrying heterozygous combinations of per alleles and locomotor activity rhythm shows similar, but not identical these rearrangements. per+ dosage sensitivity. Our analysis of eclosion rhythms shows that each These data are consistent with the hypothesis that 3Bl -2 rearrangement produces either a nearly normal the role of the per gene is to shorten the period of an + .. (approximately 24 hr) rhythm or an arrhythmic pattern. underlying infradian oscillation. The per dosage sens1t1v- Altered period lengths, such as the long-periodicity ity also raises the interesting possibility that the short­ observed by Young and Judd (1978) for one combination of period and long-period per alleles may overproduce or 3Bl -2 rearrangements, were not observed in our eclosion underproduce per+ gene product. However, complementa- studies. Our analysis of locomotor activity rhythms shows tion analysis indicates that these two alleles produce that the activity phenotype of each 3Bl-2 rearrangement qualitative, rather than quantitative, alterations in per matches its eclosion phenotype. However, some individ­ gene expression. uals from usually arrhythmic combinations, especially Dosage sensitivity has other functional significance. those including the translocation T(1;4)JC43, expressed The observation that all three per mutants alter the long-period rhythmicity. periods of both the eclosion and locomotor activity Both the eclosion and locomotor activity data rhythms in a similar manner suggests that both rhythms indicate that arrhythmicity results from the absence of are controlled by a single oscillatory mechanism (Konopka per gene function. One interpretation of the locomotor and Benzer, 1971). However, these two rhythms show activity data is that the per gene may be partially marked differences in their sensitivity to changes in the expressed in certain "arrhythmic11 rearrangements, pro- ' dosage of the short-period allele. This difference indi­ ducing weak rhythmicity that can be detected in loco­ cates that the oscillatory systems controlling these two motor activity, but not eclosion, records. An alternate rhythms are not identical. interpretation is that the per gene functions to shorten Reference: the period of an underlying infradian (long-period) oscilla­ Konopka, R. J. and Benzer, S. (1971) Proc. Nat. Acad. Sci. tion which is sometimes behaviorally expressed in the USA 68: 2112-2116. total absence of per activity. This latter hypothesis is supported by extrapolation of per gene dosage analysis (see following abstract). 144

230. ACTIVITY RHYTHMS OF ADULT DROSOPHILA close to 2 hr, with periodic intervals of inactivity. Since MOSAICS circadian rhythms should ideally be observed over a Investigators: Ronald J. Konopka, Steven M. Wells several day period under free-run conditions, it is our hope The cells controlling the locomotor activity rhythm eventually to keep isolated brains viable for extended of adult Drosophila have previously been localized to the periods under in vitro conditions. brain. Since the brain shows bilateral symmetry, it is reasonable to expect that two oscillators exist in the 232. SEROTONIN N-ACRTYLTRANSFERASE IN brain, one on each side .. We have constructed mosaic flies DROSOPHILA which have a 19 hr oscillator on one side of the brain and Investigators: Josef Zwass*, Ronald J. Konopka a 22 hr oscillator on the other. Some of these mosaics The enzyme serotonin N-acetyltransferase exhibits show arrhythmic activity in constant conditions, suggest­ a pronounced circadian rhythm in vertebrate pineals. We ing that each oscillator interferes with the other's undertook an investigation of the level of this enzyme in operation. Other mosaics show activity rhythms which, adult Drosophila as a function of time of day, as well as when subjected to periodogram analysis, show components genetic constitution. Adult flies kept in a 12-hr at both periods, indicating that each oscillator is capable light/12-hr dark cycle, which normally induces a strong of operating independently under certain conditions. rhythmicity in locomotor activity, showed no evidence of a rhythm in serotonin N-acetyltransferase. In addition, no 231. IN VIVO AND IN VITRO DROSOPHILA difference in enzyme level was found in strains carrying BRAIN CULTURE 5 certain body color (tan, tan , yellow, ebony11), eye color Investigator: Alfred M. Bandier (chocolate), or behavioral mutations (parats, Shaker5, 2 0 Preliminary genetic mosaic analysis indicates that a Hyperkinetic , per , pers, and sine oculis). However, the circadian oscillator in Drosophila is located in the brain. mutation black body consistently showed a 60 to 80% Little is known, however, about how this oscillator is decrease in total enzyme activity as compared with wild entrained and how it exerts its influence on periodic type and the above strains. This result suggests a behavior. One approach towards answering these ques­ correlation between decreased enzyme activity and dark tions involves studying biochemical, nervous, and hormon­ body color. Since the enzyme also appears able to utilize al periodicities in isolated brains kept in culture. Certain dopamine as a substrate, the dark body color observed in rhythmic activities emanating from the brain can easily black may be due to a decreased amount of acetylation of be studied in this way, and experimental manipulations dopamine and other components of the chitin polymer. can be more carefully controlled than when the brain is *Undergraduate, California Institute of Technology. left in situ. We have therefore begun to study brains kept under in vivo and in vitro culture conditions. In vivo 233. CHARACTERIZATION OF A TEMPRRATURR­ culture involves dissecting out a brain and transplanting it SRNSITIVR CELL LINE DERIVED FROM into the abdomen of a host individual. Brains kept under SHIBIRR EMBRYOS Investigators: Nancy S. Petersen, Ronald J. Konopka in vivo culture for as long as a month maintain much of their morphological integrity as shown by histological A continuous cell line has been obtained from examination of silver-stained brains. The ability of dissociated embryos of the Drosophila mutant shibire, a transplanted brains to effect activity rhythms in arrhyth­ temperature-sensitive lethal. The mutant cell line fails to mic hosts is discussed in another abstract. grow at the restrictive temperature of 29°C, unlike cells In addition, we have begun to keep brains alive under obtained from wild-type embryos which grow well at this in vitro culture conditions, using an enriched insect organ temperature. The shibire cells have an increased level of culture media. Brains kept in culture for 24 hr are able to acetylcholinesterase at 29°C and some cells show morpho­ synthesize a normal pattern of proteins as shown by logical changes which resemble axonal outgrowth. We are 35s-methionine incorporation on SDS polyacrylamide slab currently isolating cloned cell lines from the initial shibire gel electrophoresis. Electrical responses have also been line in order to further investigate the neuron-like recorded from the cervical connectives of freshly isolated properties of these cells. brains. In one instance burster activity was recorded for 145

234. EFPl!CTS ON CNS OP HOMEOTIC MUTATIONS 235. ANALYSIS OP THE SYNDROME OP THE MUTANT APPJ!CTING THE ANTENNAE IN DROSOPHILA DROSOPHILA "DROP-DEAD" Investigator: Steven H. Green Investigator: Dominic Orr

A fundamental question of developmental neuro­ Hotta and Benzer (1972) reported on a "drop-dead" biology is what cues are used by afferent neurons in mutant, which initially develop and behave normally but finding their CNS targets. Some information pertinent to early in adult life become uncoordinated in movement and this question can be obtained by studying homeotic then die prematurely. The same study also provided mutations in Drosophila. These mutations transform one mosaic mapping data that indicate the brain of the fly as appendage into another. Specifically, I have been studying the primary site of the defect. Antennapedia (Antp Yu), Nasobemia (Ns) and spineless This study was aimed at clarifying the syndrome of aristapedia (ssa) which transform the antenna wholly or this mutant from the onset of abnormality to its death. partially into leg. Mutant and control flies were individually placed in glass The tarsus (the most distal segment) of normal legs tubes with normal Drosophila medium and inspected contains taste receptors which project to the thoracic visually at intervals for morphological and behavioral ganglion. When touched with sugar on the tarsus a fly abnormalities. The locomotor activity of the flies was extends its proboscis. This response is not seen if a wild­ also continuously monitored by infrared sensors. The type fly is touched on the distal segment of its antenna in survival curve obtained with isolated flies is very similar accord with the fact that the wild-type antenna has only to that obtained by Hotta and Benzer (1972). Briefly, 56% olfactory receptors. However, proboscis extension can be of the flies survive through the third day and 18% through elicited by touching the antennal "tarsus" of ss8 flies but the fifth day (control values are 86% and 54%, respec­ not Ns or Antp Yu flies. This correspqnds to presence of tively). At or around the moment of death, 21 % of the tarsi only in the antennal "legs" of ssa but not Ns or mutant flies are morphologically normal at light micro­ Antp Yu in which only proximal segments are transformed. scopic level, 19% have grossly swollen abdomens, and 60% These experiments, done first by Deak (1976) and have completely collapsed abdomens. Flies in this third repeated in this lab, suggest that afferent sensory class appeared very similar to control flies under extreme projections find their functionally appropriate targets starvation, even though plenty of fresh food had been regardless of their origin. Nevertheless I could find no available to them at all times. The behavior of difference in the pattern of projection of antenna! sensory "staggering,11 first reported by Hatta and Benzer, is highly cells in silver-stained preparations of AntpYu, Ns, ssa, and (but not absolutely) correlated with the appearance of wild-type flies. Apparently the fibers find their place of abdominal collapse. A fly in a fairly advanced stage of origin. Presumably the proboscis extension response in ss8 staggering and abdominal collapse could be cured of both results from a very small population of fibers which symptoms by feeding with 1 M sucrose solution (less con­ terminate somewhere other than the olfactory glomerulus, centrated solutions give much less striking results). the place of termination of most sensory fibers. However, recovery from these symptoms did not seem to A mutation, antennaless (ant), exists which entirely extend survival: some recovered animals died as soon as a eliminates one or both antennae in some homozygous flies. few hours afterwards. The hypothesis currently being This mutation also eliminates antenna! 111egs" in the ant; investigated is that an onset of sensory failure prevents ss8 double mutant. The brains of ant flies show this mutant fly from feeding on normal food and that the apparently normal olfactory glomeruli whether or not the resulting long-term starvation leads to the observed olfactory projection from the antenna exists. degeneration of the nervous system.

References: Reference: Deak, I. (1976) Nature 260: 252-254. Hotta, Y. and Benzer, S. (1972) Nature 240: 527. Stocker, R. F., Edwards, J. S., Palka, J. and Schubiger, G. (1976) Devel. Biol. 52: 210-220. 146

236. ACTIN MUTANTS IN DROSOPHILA ingly are screening each stock at 22" and 29°C. Over 1000 Investigator: Steven H. Green stocks have been screened thus far and some lethals have

Interest in the role of contractile proteins in been picked up in one region of interest. In order to structure and function of cells, particularly neurons, has determine whether these lethals are truly affecting actin prompted us to screen for mutants in actin. This protein, rather than adjacent genes, we are extracting actin from in the form of microfilaments, has been thought to be different larval tissues (brain, gut, body wall muscle, involved in a number of processes, especially those related imaginal disc) and obtaining peptide maps by a modifica­ to the motion of a cell and the organelles within. tion of the method of Bray and Brownlee (1973) to check Comparing the biochemistry and physiology of mutant for abnormalities in the actin of mutant candidates. cells with wild-type cells may yield considerable informa­ References: tion on the molecular mechanism of actin's function. Bray, D. and Brownlee, S. (1973) Anal. Biochem. 55: 213-221. Recent work by Sally Tobin in mapping putative actin Tobin, S. (1977) Ph.D. Thesis, University of Washington. genes cytogenetically has made a mutant hunt feasible. Of some 29 regions of the Drosophila genome that bind PUBLICATIONS cDNA made from actin message, 3 are on the X chromosome. We have crossed males of stocks mutagen­ Handler, A. M. and Konopka, R. J. (1978) Transplantation of a circadian oscillator in Drosophila melanogaster. ized with EMS (ethyl methane sulfonate) to females with American Physiological Society Meeting, Fall 1978 deletions for one of these regions to isolate lethal (Abstract). Konopka, R. and Wells, S. (1978) Circadian rhythms in mutations specific to those regions. We are particularly Drosophila cell culture. XIVth International Con­ interested in temperature-sensitive mutations and accord- gress of Genetics, Moscow, August 1978 (Abstract). DEVELOPMENTAL GENETICS AND IMMUNOGENETICS

Edward B. Lewis

Ray D. Owen

149

Professor: Edward B. Lewis 237. A NEW CIS-REGULATORY MUTANT OF THE Visiting Gosney Professors: Berti! Rasmuson, Marianne BITHORAX COMPLEX T. Rasmuson Investigators: Madeline A. Crosby, Edward B. Lewis Research Fellow: Loring G. Craymer III Research Staff: Madeline A. Crosby, Nancy Hwang, We have found a new cis-regulatory mutant within Eileen P. L. Roy, Amelia M. Smit, Gudrun Vener Laboratory Staff: Francisca A. Castorena, Ana L. the bithorax gene complex, Miscadestral pigmentation Gharzeddine, Nancy Harris, Gertrude Jordan, Eva (Mcp), which causes the fourth abdominal segment of the Westmorland fly to acquire characteristics of the fifth. This transfor­ Support: The work described in the following research mation is especially easy to see in Mcp males, in which reports has been supported by: National Institutes of Health, USPHS the solid pigmentation normally characteristic only of the National Science Foundation fifth, sixth, and seventh abdominal tergites extends Summary: We are studying a cluster of genes in Dro­ anteriorly to the fourth tergite. The mutant is dominant sophila that plays a major role in controlling the develop­ with complete penetrance, but shows some variability in ment of the fly's thoracic and abdominal segments. This phenotype. cluster, known as the bithorax gene complex (BX-C), is Mcp maps between Hyperabdominal and Micro­ composed of at least twelve genetic elements. Eight of cephalus, which places it within the distal region of the these are defined as genes, since they act as if they code bithorax complex. This location is consistent with other for specific substances. The. other four elements act like evidence indicating that the distal region controls abdom­ cis-regulatory regions which govern whether a given gene inal differentiation. is in a repressed or derepressed state. Three of the As a dominant mutant which effects the transforma­ twelve units have been discovered within the last year and tion of a given segment to a level characteristic of a more form the basis of the first two of the following reports. posterior segment, Mcp is comparable to Contrabithorax, In the head and first two thoracic segments the which has been shown to be a cis-acting regulatory genes of the bithorax complex are normally repressed. element. Mcp acts as if it turns on (or derepresses) a Commencing with the third thoracic segment the genes hypothetical structural gene, tentatively designated infra­ become derepressed, the more proximally located ones abdominal-5 (iab-5) which is normally repressed except in being the first to do so. Finally, all of the genes of the the fifth, sixth, and presumably re_maining abdominal cluster appear to be derepressed in the eighth abdominal segments. segment. The working hypothesis we have for interpreting The impairment of such an Mcp-controlled struc­ these results is that the complex is regulated by means- of tural gene, or the deletion of Mcp, shoUld result in a loss two types of gradients. An antero-posterior gradient in of the dominant Mcp phenotype. Acting on this hypothe­ concentration of a repressor substance within the early sis, we have mutagenized (with X-rays and EMS) homozy­ embryo is invoked to explain the gradual derepression of gous Mcp males and screened their progeny for Mcp­ the genes starting with the third thoracic segment and revertants. Five such revertants have already been proceeding posteriorly. A proximo-distal gradient along isolated and characterized. Two are lethal when over a the chromosome in the relative affinity for repressor of deficiency for the entire bithorax complex (Df-P9); one of the various regulatory elements adjacent to each gene is these has been characterized and is a deletion of the invoked to explain the derepression of the genes in a distal-most part of the bithorax region. sequence that corresponds approximately to their order in When the remaining three Mcp revertants are tested the chromosome. over a chromosome carrying Df-P9, the resultant males In the third report we describe evidence for the completely lack normal male-type abdominal pigmenta­ existence of a separate regulatory gene that makes a tion, as if the iab-5 structural gene (and possibly Mcp as repressor of the bithorax complex. well) has indeed been damaged. Such animals can be said to be pseudo-hermaphrodites: although they have male genitalia and gonads, the abdomen closely resembles that of normal females in its pigmentation pattern. These results lend further support to a model of segmental differentiation achieved by sequential dere­ pression of individually regulated structural genes. 150

238. AN ULTRA-ABDOMINAL MUTANT IN Uab 4 hemizygotes lack gonads, structures of mesodermal DROSOPHILA PRODUCING GONADAL DYSGENl!SIS origin which are believed to be derived embryologically Investigators: Nancy HwllJlt, Edward B. Lewis from the third through the sixth abdominal segments. Thus, the genes of the bithorax complex appear to act in We are studying the control of abdominal differenti­ widely different types of tissue in effecting specific ation by genes of the bithorax complex in the larval and intersegmental transformations. adult stages of the fly. Each of the eight abdominal segments of a fly is uniquely characterized by its pattern of cuticular structures. The second abdominal segment 239. POLYCOMB: A MAJOR REGULATOR GENE OF for example has a unique structure of uncertain function mE BITHORAX COMPLEX which we designate Wheeler's organ, after M. Wheeler who Investigators: Gudnm Vener, Edward B. Lewis first described it. It is a raised circular area on the We have obtained evidence that the bithorax gene ventral surface of that segment. The second abdominal complex (BX-C) is under negative control. That is, the segment also has a unique distribution of ventral or BX-C genes which normally are repressed in the head and sternital bristles. The third through the sixth segments first two thoracic segments become derepressed in those differ from the second in lacking a Wheeler's organ: and in segments when another gene outside the complex, Poly­ having more elaborate patterns of sternital bristles. comb (discovered by P. H. Lewis), is inactivated by Within the bithorax complex a dominant regulatory mutation. Animals homozygous or hemizygous for an mutant Ultra-abdominal-5 (Uab5) is known to transform extreme mutant allele, Pc3, die in the late embryonic the first and second abdominal segments towards the third stage, have their thoracic regions partially converted into abdominal segment. Such an effect is understandable if abdominal regions, and have extensive modifications of the rearrangement associated with Uab5 (an X-3 trans­ the head suggestive of an abdominal modification of that location) damages a regulatory element that derepresses a region. By varying the number of doses of the BX-C gene responsible for promoting a third versus second region from 0 to 4 in the presence of homozygous Pc3, it abdominal level of segmental development. We call this has been found that no transformation of any segment gene infra-abdominal-3 (iab-3). The prediction is that from a thoracic to an abdominal state is achieved even when iab-3 +, the wild-type allele, is inactivated the third among the abdominal segments themselves unless at least abdominal segment should transform into a segment one dose of the BX-C region is present, and that the most resembling the second abdominal one. We have found two extreme transformation to abdominal states occurs when instances of this type of change associated with chromo­ the animal has four doses of BX-C. In the latter cases, a somal rearrangements of the bithorax complex; namely portion of the head and all thoracic segments strongly Jn(3LR)Uab4 and T(2;3)P10. These rearrangements have transform to an abdominal state. This dependence of the breakage points near the middle of the bithorax complex action of Polycomb on the number of doses of the BX-C and evidently they induce a position effect of the iab-3 + genes suggests that the wild-type allele of Polycomb that weakens its dominance. The effect is especially produces a specific repressor of the bithorax complex. 4 marked in the Uab hemizygote (an individual which Presumably an antero-posterior gradi~nt in concentration carries Uab4 in one third chromosome and a deficiency for of this repressor controls the degree of repression of the bithorax complex in the other). The recessive loss of BX-C during normal development. function in this case is manifested by a transformation of not only the third but the fourth, fifth, and sixth abdominal segments towards the second abdominal state. PUBLICATIONS Not only are cuticular tissues affected, but a Lewis, E. B. (1978) A gene complex controlling segmenta­ strik_ing effect on mesodermal tissues is also evident. The tion in Drosophila. Submitted for publication. 151

Professor: Ray D. Owen We have successfully isolated the MHC transplanta­ Gosney Research FellDw: Richard M. Maizels tion antigens from spleen cells of four strains of rats, by Graduate Student: Elizabeth P. Blankenhorn Research Staff: Debra Dison Hall precipitation with specific rat alloantisera. These mem­ Laboratory Staff: Dennis R. Shelby, Carol Shotwell brane molecules (called 11 AgB" antigens), labeled biosyn­ Support: The work described in the following research thetically with tritiated amino acids, have been charac­ reports has been supported by: terized by microsequencing techniques and by two­ Charles B. Corser Fund for Biological Research Gosney Fund dimensional gel analysis. Our findings are consistent with National Institutes of Health, USPHS U.S. Department of Energy the following conclusions about these rat antigens: (1) The behavior of AgB antigens on SDS polyacryl­ Summary: Most remarkable during the year has been the amide gels, both in the original preparations and in the emergence of our laboratory at the leading edge of the fraction retained on and eluted from insolubilized lectin soft-pellet world. Suggested originally by Watership affinity columns, indicates that they are approximately Down, this excursion into the fascinating domain of 45,000 molecular weight glycoproteins. In each strain, coprophagy has sniffed out immunological territory still to there are multiple species of AgB antigens, revealed on be charted, and we have moved into the gap between basic the two-dimensional gels, that differ by weight and and applied soft-pellet research. Over the same interval, charge. These antigens differ among rat strains as well as our studies of antigens associated with the main histo­ being different from each other within a strain. compatibility complexes _of the rat and the mouse, done in (2) The N-terminal partial amino acid sequences of association with Lee Hood's laboratory, have come to the AgB antigens also show that in three strains of rats at satisfying fruition. The work on a carcinoembryonic least two molecules are present in each preparation. antigen, alpha-fetoprotein, has retained its provocative These two molecules differ from each other by only one of character. Our studies of animal models of two human the twelve residues identified. The sequences show strong autoimmune diseases, myasthenia gravis (done in part in homology to those of the MHC transplantation antigens of association with Michael Raftery's laboratory) and sys­ guinea pig, mouse (H-2), and human (HL-A). temic lupus erythematosus, have progressed. And the (3) There are haplotype-associated residues found in integrating thread that has run through our rather diverse the sequences of four rat AgB antigens by which we can program over the years, an interest in development and its distinguish the antigens from each other. These residues genetic regulation, mainly using the immune system as a occur at positions 9 and 22. model for developmental analysis or as a tool for We are currently devising strategies that will enable following the development of other systems, now knits up us to identify which of these sequences corresponds with the raveled sleeve of care. the different spots found on the two-dimensional gels of the AgB molecules. 240. CHARACTERIZATION OF TRANSPLANTATION ANTIGENS ASSOCIATED WITH THE MHC OF THE RAT 241. ANTIGENS CONTROLLED BY THE Ir REGION Investigators: Elizabeth P. Blankenhorn, J. Michael OF THE RAT Cecka Investigators: Elizabeth P. Blankenhorn, J. Michael Cecka The major histocompatibility complex (MHC) is a genetic region in animals that encodes the structural or Our work on the immune response (Ir) region regulatory information responsible for a number of im­ alloantigens (Ia molecules) of the rat has been extended to munologically important functions. Two functions of the the analysis of four strains of rats. Like the transplanta­ MHC are separable: the production of antigenic proteins tion antigens, these Ia molecules are also glycoproteins, that are recognized as foreign in the transplant rejection and like their counterparts in other species (mouse and process, and the regulation of the level of the immune human in particular), they are found to be composed of response by the animal to certain antigenic stimuli. The two polypeptide chains of 33,000 molecular weight (alpha) transplantation antigen molecules of the rat are the and 28,000 molecular weight (beta). The number and subject of this study. Our work on antigens coded for by nature of rat Ia molecules has been studied using the immune response (Ir) region is reported in the microsequencing and electrophoretic methods. The defi- following abstract. 152 nition of rat Ia-like molecules rests on their isolation by An earlier investigation at Caltech (Biology 1975) specific alloantisera directed to the whole MHC in the rat had found remarkable variation between alleles of the two target strain, along with their behavior in electrophoretic H-2 loci, K and D, as judged by partial amino acid separation. By comparison of the N-terminal partial sequence analysis of the N-terminal portion of the amino acid sequences, we have confirmed that the molecule. We were interested in extending these results, molecules isolated under our conditions are indeed closely and carried out a similar analysis on four more H-2 homologous to the genetically defined mouse Ia antigens, molecules, the K and D products of H-2q and H-2s and to their human counterparts. In addition, two general haplotype mice. points were established. From these analyses, we were able to confirm that (1) The rat alpha molecules resemble both the antigens coded for by the K and D loci are extremely human and the mouse I-EC subregion alpha sequences. In similar even though they are separated in the mouse the first 30 residues, there are no differences between the genome by 0.5 cM. The products of each locus vary alpha polypeptides of different rat strains, and only one considerably (up to 30%) among alleles, but both loci sequence was found in each alpha preparation. In showed parallel polymorphisms, such that Ks, for example, contrast, the beta polypeptides of each strain were is more similar to the Db molecule (which had been revealed to have at least two sequences (equivalent to at analyzed previously) than to any other K molecule. least two molecules), one of which is comparable to the Taken as a whole, the accumulated data from these I-EC beta mouse sequence and one homologous to another and other studies on N-terminal sequences of H-2 mouse I subregion (I-A) beta polypeptide. These amino molecules show a number of other unexpected results. acid sequence residues in the beta molecules from The frequency of amino acid substitutions is among the different rat strains. These amino acid sequence results highest of any allelic system so far characterized. This, were confirmed by two-dimensional gel analysis of the BN together with the pattern of similar substitutions found rat Ia molecules. We made use of a strong cross­ among products of either end, could suggest that the reactivity found between rat Ia antigens and highly evolution and organization of the H-2 loci may be more specific serological reagents detecting either the mouse complex than previously supposed. I-EC or I-A subregion molecules, and found that the two This work was carried out in collaboration with Dr. cross-reacting species are distinct in both molecular L. Hood and with Dr. J. A. Prelinger of the University of weight and charge. Southern California School of Medicine. (2) There are also distinguishing haplotype-associ­ ated residues in -the beta molecules from different rat 243. STUDD!S OF ALPHA-PETOPROTEIN IN THE MOUSE strains. Thus we are able to conclude that the rat MHC Investigator: Elizabeth P. Blankenhom encodes or regulates the expression of at least two Ia antigens, at a point when the genetic analysis of the MHC I have continued efforts to identify the genetic region has not yet revealed the subregion structure of the elements controlling the synthesis of AFP (alpha-feto­ complex. protein) in mice (Biology 1977, No. 243). This secreted liver protein is found in embryonic mammals, in primary 242. ANALYSIS OP mE DIVERSITY OP MOUSE carcinomas of the liver, and, in smaller quantity, in adult TRANSPLANTATION ANTIGENS serum. I have established matings between mice which Investigator: Richard M. Maizels differ in the adult level of AFP, and recenUy have The major histocompatibility complex (MHC) of analyzed the first backcross generation for APP levels and many species of mammals has elicited much interest for their albumin types. We had suspected that the AFP because of observations that genes encoded within this regulator might be linked to tt1e structural locus for region are involved in the control of immune responses. albumin. I did not observe any such co-segregation of Perhaps most conspicuous of these gene products are the these two markers. The AFP serum concentration of 15 transplantation antigens, in mice the H-2 antigens, which mice was determined individually at two different ages. appear to determine not only graft rejection but also a In each age group, the AFP levels were found to fall in range of cell interactions in the immune system. one continuous range. The average concentration of APP 153

in 30-day-old mice is 6425 :!: 2500 ng/ml, and at 150 days, 245. IMMUNOCHEMICAL STUDIES ON THE INDUCTION OF EXPERIMENTAL AUTOIMMUNE MYASTHENIA the level drops to 3280 2160 ng/mL While the decrease :!: GRAVIS AFP is in concentration expected, these values are higher Investigator: Tooi R. Claudio* than those reported in the literature (Olsson et al., 1977). Several laboratories have shown that a disease with When two classes of high and low AFP mice are the characteristics of the human autoimmune disease constructed by including those animals above and below myasthenia gravis can be induced in experimental animals. the mean value, average concentrations of APP are This disease, known as experimental autoimmune myas­ twofold higher in the high category, but individual mice thenia gravis (EAMG), has been induced in animals ranging may be in one category in the 30-day sample and in the from mice to monkeys by injecting them with purified, other 15 weeks later. There was no parental-type detergent-solubilized acetylcholine receptor (AChR). We association of albumin and AFP levels within these too have induced EAMG in rabbits with purified deter­ classes. Further work of this nature is being conducted on gent-solubilized AChR but were unable to induce the more recent litters. disease with any of the four AChR subunits or SDS Reference: denatured AChR (Claudio and Raftery, 1977). The disease Olsson, M., Lindahl, G. and Ruoslahti, E. (1977) J. Exptl. Med. 145: 819-827. could be induced, however, with membrane fragments containing AChR. Studies are currently under way to try 244.. THE IMMUNE RESPONSE IN EXPERIMENTAL to induce EAMG with membrane proteins located at MYASTHENIA GRAVIS neuromuscular junctions which do not contain AChR. Investigator: Debra Dison Hall Such studies will ascertain whether the syndrome is There is evidence that myasthenia gravis is an characteristic only of immune effects on the AChR, or autoimmune disease in which spontaneously occurring involves other molecules at the junctions as well. This antibodies to the acetylcholine receptor cause severe would be very valuable information for the better under­ neuromuscular weakness. Model systems for the human standing of human MG. disease have been established in various animals by There is now one report in the literature (Lindstrom immunizing with purified acetylcholine receptor (AChR) et al., 1978) of inducing EAMG in one strain of rat using protein; the molecular changes and physical symptoms in individual AChR subunits. We are trying to confirm this experimental autoimmune myasthenia gravis (EAMG) are report using the same strain of rat plus two mouse strains. very similar to those observed in the human disease. If the disease can be induced with isolated subunits, we The role of cellular immunity, distinguished from will attempt then to look at the antigenic determinants of humoral antibody, in the disease has not been defined. We the isolated subunits. It may be possible to look for are attempting to induce EAMG in rats, and are following common antigenic determinants between human AChR the cellular response to AChR over the course of the and the AChR isolated from Torpedo californica which is development of the disease by measuring the ability of used in these studies. Attempts will also be made to lymph node cells taken from immunized rats to kill induce EAMG with fragments of subunits. Such studies thymus cells which have AChR on their surface. We are can eventually tell us how many regions of the AChR are also checking the assertion (Zurn and Fulpius, 1977) that it capable of inducing EAMG. is the subpopulation of antibodies directed to the alpha­ References: bungarotoxin binding site of the receptor that is involved Claudio, T. and Raftery, M. A. (1977) Arch. Biochem. Biophys. 181: 484-48 9. in the development of paralysis. Lindstrom, J., Einarson, B. and Merlie, J. (1978) Proc. Nat. Acad. Sci. USA 75: 769-773. Reference: Zurn, A. D. and Fulpius, B. W. (1977) Eur. J. Immunol. 8: 529-532. *Graduate student working with Professor Michael Raftery, Division of Chemistry and Chemical Engineering, California Institute of Technology. 154

246. DEVELOPMENTAL STUDIES OF THE HUMORAL The contents of the soft pellets are rich in protein, ASPECTS OF MORINE AUTOIMMUNE DISEASE assumed to be mainly bacterial in origin. No one seems to Investigators: David J. Asai, Lori E. Winkelstein*, Alana M. Wallace have considered that secretory immunoglobulin may be included in the soft pellets, and may play a critical role in We are continuing our studies on the humoral maintaining and using the internal ecological system. We aspects of the murine parallel to human systemic lupus have found that JgA is indeed present, in substantial erythematosus autoimmune disease, using NZB x NZW Fl amount, in association with the pellet membrane. We are mice (see Biology 1977, No. 239). Our preliminary most interested in the functions of this immunoglobulin. evidence is that the drug Ievamisole (1-2,3,5,6-tetra­ Does it serve in a nutritional role, such as aiding in the hydro-6-phenylimidazo [2,1-b]thiazole hydrochloride) is absorption of peptides, as IgA is thought by some to do in able to prevent or delay the development of antinuclear human digestion? Is it specific for particular bacteria, antibody in this autoimmune system. We are pursuing this perhaps regulating the bacterial population by selecting observation. against potentially harmful inhabitants of this small *Undergraduate, California Institute of Technology. universe, and promoting the welfare of beneficial ones? There are many possibilities, offering much opportunity 247. NATURE AND FUNCTIONS OF lMMUNO­ for rumination. We will hopefully eliminate some hypoth­ GLOBULINS IN THE SOFT-PELLET COPROPHAGOUS SYSTEM OF THE RABBIT eses, and it will all come out in the end. Investigators: David J. Asai, Lori E. Winkelstein*, References: RayD. OWen Freed, D. L. J. and Green, F. H. Y. (1977) Early Human Devel. 1: 107-1!2. Rabbits produce two kinds of fecal pellets, the Thacker, E. J. and Brandt, c. S. (1955) J. Nutrition 55: 375-385. familiar 11 hard pellets" and special "soft pellets," which they reingest. The soft pellets have a defined struc­ *Undergraduate, California Institute of Technology. ture--a membrane bag containing partially digested food material, many bacteria selected to serve in a symbiotic capacity with their host, and well-buffered enzyme PUBLICATIONS systems. The reingestion cycle creates a remarkable Blankenhorn, E. P., Cecka, J.M., Goetze, D. and Hood, L. internal ecological system adapted to the rabbit's nutri­ (1978) The partial N-terminal amino acid sequence tional and behavioral world of rat transplantation antigens. Nature. In press. 155

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

248. IN VITRO STUDIES OF PLASMID DNA this effect has allowed us to predict when membrane REPLICATION proteins precipitate. Experimental studies have shown Investigators: Susan E. Conrad, Judith L. Campbell* this theory to be at least qualitatively accurate. Support: National Institutes of Health, USPHS When studying the proteins, it was expedient to use We have developed a cell-free system to study a thermodynamic model of the lipids. We have more replication of the plasmid RSF1030, which is 8.3 kb in size recently studied their microscopic features, and found and carries genes for ampicillin resistance. Replication that our halo model coincides with previous theories of occurs in extracts of both cells that carry endogenous membrane thickness elasticity. This suggests that protein plasmid and cells that do not, and is stimulated by effects on lipids depend on thickness changes. Statistical treatment of the cells with chloramphenicol before mechanical studies of the lipids have shown these changes harvesting. The preparation of extracts involves removal to reflect lipid tilt, rather than conformation. of endogenous DNA, so that even in extracts prepared I have also used the model to study lipid phase from cells carrying endogenous RSF1030, replication in transitions. It correctly predicts the phase behavior of vitro requires exogenous template. Both sucrose gradient protein-lipid systems. It further reveals that the usual and gel electrophoresis analyses indicate that the products analysis underestimates the amount of lipid perturbed by synthesized in vitro 1 contain both partially replicated each protein particle. I have also found that those lipids intermediates and fully replicated, supercoiled molecules. are usually constrained between the disordered and or­ Preliminary experiments examining mutants of E. dered states, closer to the former. coli suggest that the proteins encoded by dnaB, dnaC,D, *Working with Professor Sunney Chan, Division of Chem­ ~' dnal, dnaP, dnaF, dnaG, dnaZ, and~ are required istry and Chemical Engineering, California Institute of Technology. for in vitro synthesis. In addition, rifampicin, novobiocin, nalidixic acid, and phenethyl alcohol inhibit synthesis, indicating that RNA polymerase, DNA gyrase, Pnal and 250. STRUCTURAL STUDIES OF AN ACETYlr CHOLINE RECEPTOR FROM TORPEDO DnaP protein are required for plasmid replication. CALIFORNICA A partial restriction enzyme map of RSF1030 has Investigators: Michael W. Klymkowsky, Robert M. been constructed and is being used to map the origin of Stroud* replication used in vitro. We are also using the in vitro Support: California Foundation for Biochemical Research system to study bacterial plasmids with altered replica­ National Institutes of Health, USPHS tion properties. National Science Foundation

*Division of Chemistry and Chemical Engineering, Cali­ Using antibodies raised against purified acetyl­ fornia Institute of Technology. choline receptor, adsorbed either onto colloidal gold beads or recognized by a second antibody (ferritin or colloidal 249. LIPID ORDERING AND MEMBRANE PROTmN gold) conjugate, we have demonstrated that the receptor 0 AGGREGATION corresponds to the 85 A diameter rosette seen in negative Investigator: Jay Edelman* stain electron micrographs of electrocyte-derived mem­ Support: Lucy Mason Clark Fund branes. Direct electron microscopic observations confirm My main research interest has been the theoretical and extend our previous conclusions, based on X-ray study of protein-lipid interactions. Previous work has diffraction analysis (Figure 1), that the molecule extends 0 shown that membrane proteins are surrounded by halos of above the membrane surface by about 60 A (Figure 2). perturbed lipids. My goal has been to study their Calculated molecular volumes (Figure lB,D) indicate that structure. the receptor has a molecular weight of between 250,000 For instance, when two proteins approach one to 310,000 daltons, a figure consistent with current another, their halos merge. This produces a force estimates for the monomer molecular weight (250,000 to between the proteins. A statistical mechanical analysis of 280,000). 156

Korenbrot at the University of California, San Francisco, A we have shown that it is possible to form membrane sheets on clean water surfaces by osmotic shock, and experiments using this technique are in progress. (2) We are continuing our image analysis work (Biology 1975, No. 262; Biology 1976, No. 290) on ordered receptor areas with the hope of determining the maximum molecular sym­ metry consistent with the observed data and locating ligand binding sites. The identity and characterization of the large two-dimensional lattices found in these mem­ brane fractions are still being investigated. Images of the internal membrane surface should resolve the question of ,300- their identity. References: Figure 1. Electron density profiles at 11 (Al and 6.5 0 Klymkowsky, M. W. and Stroud, R. M. (1978) Manuscript in (Cl A resolution derived from X-ray diffraction analysis preparation. of receptor-rich membranes. B and D represent the Ross, M. J., Klymkowsky, M. Agard, D. A. and Stroud, corresponding molecular volumes. The circles in B and D w., represent hydrated sodium ions drawn to scale. R. M. (1977) J. Mo!. Biol. 116: 635-659. *Department of Biochemistry and Biophysics, University of California at San Francisco, San Francisco, California.

251. SYNAPTIC VS. EXTRASYNAPTIC ACETYir CHOLINE Rl!Cl!Pl'ORS Investigators: Wilson C.-6. Wu, Michael A. Raftery* Support: llarle C. Anthony Fellowship

There are two forms of acetylcholine receptors (AcChR) on the skeletal muscles of rat and adult frog, namely junctional receptors, and extrajunctional receptors that appear following denervation. Studies have revealed significant differences between the two forms of recep­ tors. Extrajunctional receptors appear to have an acetylcholine-activated channel open time that is 3 to 5 times longer than· that of the junctional receptors (Katz and Miledi, 1972). Biochemical characterization has shown that the two forms of receptors differ in their isoelectric points (Brockes and Hall, 1975), suggesting that a basic difference in the primary structure exists between the two proteins that gives rise to a change in the total Figure 2. (Al The edge of a receptor containing net charge. vesicle is shown tagged by anti-native receptor gold. The It is possible that junctional and extrajunctional projecting receptor molecule and halo of antibody sur­ rounding the gold beads are visible and in most cases the receptors differ in the extent of phosphorylation, which connection can be seen. (B) The arrow points out a clear image of a single receptor molecule seen in cross-section. would account for their difference in net charge. In this regard, phosphorylation could be the basis for the forma­ Our work centers on (1) the visualization of the tion of junctional receptors in myotubes that is believed intracellular surface of the receptor membranes. Covered to occur through aggregation of extrajunctional receptors with postsynaptic proteins in the intact cell, this surface to form dense patches of junctional receptors following is hidden in membrane fragments which have their innervation. extracellular sides facing out. Working with Dr. Juan Phosphorylation of the acetylcholine receptor has 157 been shown to occur in vitro in Torpedo and the electric the binding component for cholinergic ligands. However, eel. Recent study of the AcChR from Torpedo in our it is not yet well established that the molecular apparatus laboratory (Vandlen et al.. , 1978) has demonstrated in vivo responsible for ion translocation copurifies with this phosphorylation and identified the phosphorylated amino receptor complex. acid residues. In the same study the numbers of these The best and perhaps the only way to answer this modified residues in the whole AcChR and in its constit­ question unequivocally is to reincorporate the purified uent polypeptides were determined. By applying similar AcChR in a membrane system. Attempts to reconstitute techniques we hope to determine whether junctional and the AcChR have been carried out with sealed vesicles extrajunctional receptors differ in the number of phos­ formed from membrane fragments (Hazelbauer and phoryl linkages. Changeux, 197 4) and protein-free lipids (Michaelson and References: Raftery, 1974). Functionality of the reconstituted AcChR Brockes, J. P. and Hall, Z. W. (1975) Biochemistry 14: was assayed by monitoring ligand-induced efflux of pre­ 2100-2106. 22 Katz, B. and Miledi, R. (1972) J. Physiol. 224: 665-699. viously accumulated Na from these vesicles. Unfortu­ Vandlen, R. L., Wu, W. C.-S., Eisenach, J.C. and Raftery, nately a number of basic problems associated with M. A. (1978) Biochemistry. Submitted for publica­ tion. reconstituted vesicle preparation makes it difficult to correlate the results obtained from these studies with the *Division of Chemistry and Chemical Engineering, Cali­ physiological function of the AcChR. For instance, the fornia Institute of Technology. 22 kinetics of Na efflux follows a time course which is 252. RECONSTITUTION OF AN ACl!TYLCHOLINE orders of magnitude slower than that of physiological. RECEPTOR Reconstituted vesicles are often leaky to ions and Investigators: Wilson C.-S. Wu, Michael A. Raftery* contribute to high background noise. Contamination of Support: Earle C. Anthony Fellowship the preparation with a large proportion of nonvesicular Postsynaptic depolarization at the neuromuscular membrane fragments also contributes a significant 22 junction or in electric organs is a result of two basic amount of background Na reading. steps: (1) binding of acetylcholine to its membrane-bound We are currently working to overcome some of these receptor and (2) translocation of cations across the problems. postsynaptic membrane. The receptor protein for acetyl­ References: choline has been purified and characterized in a number of Hazelbauer, G. L. and Changeux, J.-P. (1974) Proc. Nat. Acad. Sci. USA 71: 1479-1483. systems. The structure and properties of the acetyl­ Michaelson, D. M. and Raftery, M. A. (1974) Proc. Nat. choline receptor (AcChR) purified from electric tissues Acad. Sci. USA 71: 4768-4772. have been extensively studied. It has been clearly *Division of Chemistry and Chemical Engineering, Cali­ demonstrated that the purified AcChR complex contains fornia Institute of Technology.

VISITING LECTURERS

GRADUATES

FINANCIAL SUPPORT

161

VISlTING LECTURERS

Richard Flavell, University of Amsterdam: Physical Ted Begenisich, University of Rochester: Conditioning mapping of sequences flanking the rabbit beta globin hyperpolarization affects kinetics in K channels of gene. myelinated nerve. Peter Gage, University of New South Wales: End-plate Gordon Fain, University of California, Los Angeles: channels. Synaptology of the vertebrate retina. Daniel Hansburg, Washington University Medical School, Francois Michel, Lyon Neurological Hospital: Hemi­ St. Louis: Anti-1,3 dextran antibodies as a model anacousia: Hemispheric cortical deafness. system for the investigation of the generation of Rolf Zinkernagel, Scripps Clinic and Research Foundation: diversity. The thymus and H-2 restriction. Otakar Stark, Charles University, Prague: The fine Robert T. Schimke, Stanford University: Amplification of structure of the rat MHC. dihydrofolic acid reductase genes in methotrexate­ Geoffrey Wilson, University of Edinburgh: Molecular resistant cultured animal cells. cloning of fragments of bacteriophage T4 DNA. Gregory Mears, Columbia University: The molecular basis Amar jit Singh Klar, University of California, Berkeley: of genetic deficiencies in hemoglobin expression: Evidence for jumping genes: Switching of the a-thalassemias. . mating type. Phil Leder, National Institutes of Health: The structure William Hayes, University of Sussex: Effect on ribosomal of mouse a-globin genes. behavior of an E.coli mutation (tif-1) which induces Arthur P. Arnold, University of California, Los Angeles: DNA repair functions. The neural and hormonal bases of bird song. Russell Devalois, University of California, Berkeley: Fred Sigworth, Yale Medical School: What does noise Responses of cortical units to one- and two­ analysis tell us about Na channels in nerve? dimensional spatial patterns. Herb Kallickey, University of California, Irvine: Whisk­ Leslie E. Orgel, The Sall< Institute: Origins of the genetic ers, barrels, and brains. code. James Sprague, University of Pennsylvania Medical James Wittliff, University of Louisville Medical Center: School: What is the role of the striate cortex in Steroid-protein interactions and tumor cell response. vision? Paul Barstad, University of Alabama Medical School: A Craig J. Benham, Lawrence University: Elastic models of mass tissue culture facility for the large scale supercoiling. preparation and characterization of membrane pro­ Stanley Schein, Massachusetts Institute of Technology: teins. Colicin K: A physiological voltage dependence ion Robert Zucker, University of California, Berkeley: Intra­ channel protein studied in a planar artificial mem­ cellular Ca release at fertilization and partheno­ brane. genetic activation in the sea urchin egg. Richard Campbell, University of California, Irvine: De­ Richard Flavell, Plant Breeding Institute, Cambridge, velopmental and behavioral biology of nerve-free England: NuC?leotide sequence organization in re­ hydra. lated cereal genomes. Burt Dorman, Yale University: Human chromosome Richard Axel, Columbia University: Isolation, transfer, specific cell surface antigen on human x mouse and integration of the thymidine kinase gene into hybrid cells. mutant mouse cells. Tom Hall, The Pasadena Foundation for Medical Research: Morton Bradbury, Portsmouth Polytechnic, England: Molecular biological approaches to cancer therapy. Physical studies on the structure of chromatin. Joanna Olmsted, University of Rochester: Cellular G. D. Fischbach, Harvard Medical School: Early events in control of microtubule assembly. the formation of neuromuscular junctions. Norman H. Giles, University of Georgia: The organiza­ Oscar Miller, University of Virginia: Ultrastructure of tion, function, and evolution of gene clusters in genetic activity. eukaryotes. Robert Tjian, Cold Spring Harbor Laboratory: The binding Jennifer Lund, University of Washington: Architecture of an SV40 T antigen related protein to a specific and development of cat and monkey visual cortex. site on SV40 DNA. Harvey Karten, State University of New York, Stony Kunito Yoshiike, National Institute of Health, Tokyo: Brook: Evolutionary origin of the mammalian Recombinant DNA in SV40 productive infection. cortex: Cellular hypothesis. Peter Sterling, University of Pennsylvania: Microcircuitry James Lake, University of California, Los Angeles: Ribo­ of the visual system. somal proteins_ and functional sites mapped by Dietmar G. Braun, Basel Institute for Immunology, Swit- immunoelectron microscopy. zerland: Rabbit anti-polyse.ccharide antibodies: Bernard F. Erlanger, Columbia University: Application of Structure and genetics. nucleotide-specific antibodies to the study of DNA Rita G. Rudel, Columbia University: Neural plasticity in and chromosome structure~ CNS of man. Mark Ptashne, Harvard University: Gene regulation in Steven Green, Rockefeller University: Sociobiology of the bacteriophage lambda. lion-tailed macaque monkey. Maureen A. Gilmore-Hebert: University of California, Edward Evarts, National Institutes of Health: Control of Los Angeles: The structure of immunogloblllin genes movement by the brain: In.sights gained from studies as revealed by analysis of hnRNA. of the activity of single nerve cells in the monkey. Arnold Berk, Massachusetts Institute of Technology: Bio­ Matthew Scharff, Albert Einstein Medical School: Anti­ synthesis of spliced messenger RN As of gen binding and constant region mutants of mouse adenovirus-2 and SV40. myeloma cell lines. Larry Sandler, University of Washington: A set of closely Robert Campenot, Harvard Medical School: Local control linked autosomal genes that interact with sex­ of axonal development in cultured sympathetic chromosome heterochromatin in Drosophila melano­ neurons by nerve growth factor. gaster. 162

Gunter Blobel, Rockefeller University: Mechanisms for Charles F. Stevens, Yale Medical School: Molecular basis the intracellular compartmentation of newly synthe­ of transmitted dependent permeability changes at sized proteins. synapses. Dean Hamer, National Institutes of Health: Animal John Abelson, University of California, San Diego: Tran­ transducing viruses. scription and processing of intervening sequences in N. Jerne, Basel Institute for Immunology, Switzerland: A yeast tRNA genes. description of the immune system. Jonathan Howard, Agricultural Research Council, Cam­ R. K. Hunt, The Johns Hopkins University: Starting points bridge: Monoclonal antibodies to major histocom­ for a developmental genetics of the nervous system. patibility antigens of the rat. Robert Wurtz, National Institutes of Health: Vision during Richard Goldsby, NASA-Ames Research Center and Stan­ eye movements. ford University: Specific antibodies without specific Bernard Forget, Yale Medical School: Genetics of human immunization. hemoglobin. Michael Waterfield, Imperial Cancer Research, London: Haim Manor, The Technion, Israel: Polyoma virus-specific Structure and function of membrane proteins of RNA synthesis in a productive infection and in an influenza and Sendai viruses. inducible line of polyoma transformed cells. Fred Sanger, MRC Laboratory of Molecular Biology, Jean-Pierre Changeux, College de France and Institut Cambridge: The nucleotide sequence of bacterio­ Pasteur: Selective stabilization of developing syn­ phage 0X174 DNA. apses as a mechanism for the specification of Hideo Mohri, University of Tokyo: The role of dynein in neuronal networks. flagellar movement and cell division. Nurit Kalderon, Rockefeller University: Gap junctional Irving Weissman, Stanford University Medical School: T communication and myoblast fusion during striated cell lymphomas and T cell receptors. muscle development. Fritz Bach, University of Wisconsin: Immune response to Michael Ashburner, University of Cambridge: Genes, major histocompatibility antigens. bands, and puffs in Drosophila. Allen Lehmann, University of Sussex: DNA repair and Terrone Rosenberry, Columbia University: Acetylcholin­ human genetic disorders. esterase is attached to collagen-like subunits at Charles Heidelberger, LAC-USC Comprehensive Cancer synapses. Center: Cell culture studies on the cellular mecha­ Corey Goodman, University of California, San Diego: nisms of chemical carcinogenesis. Neuronal differentiation during embryogenesis in John E. Desmedt, University of Brussels: Event-related grasshoppers. cerebral potentials and hemispheric specialization in Peter Bryant, University of California, Irvine: Pattern man. formation and symmetry in imaginal discs of Dro­ Uno Lindberg, University of Uppsala: The regulation of sophila. polymerization of actin in nonmuscle cells. Pamela Dunsmuir, Harvard University: Evolutionary Kenneth Jones, Jet Propulsion Laboratory: One Mars year change in the Drosophila genome studied with cloned as seen by the Viking landers. DNA fragments. Darwin Berg, University of California, San Diego: De­ Richard Firtel, University of California, San Diego: The velopment of cholinergic neurons and the formation structure of actin and other developmentally regu­ of neuromuscular synapses in vitro. lated genes in Dictyostelium. Steve Heineman, The Salk Institute: Study of synapse Nina Fedoroff, Carnegie Institution of Washington: The formation and receptor metabolism with clonal cell oocyte SS ribosomal RN A gene family in Xenopus lines. laevis: Its primary structure and transcription. Sally Krasne, University of California Medical School, Los Stephen Hughes, University of California School of Medi­ Angeles: Interactions between potential-sensing cine, San Francisco: Mapping the integration sites dyes and membranes. for avian sarcoma virus DNA. Edward A. Clark, University College, London: The John Smart, Imperial Cancer Research, London: Biolog­ immune response to the Thy-1 alloantigen. ical implications of comparative tryptic peptide Siegfried Weiss, Basel Institute for Immunology, Switzer­ analysis of tumor antigens of polyoma virus. land: Immunoglobulin genetics: Rabbits lacking Michelle Fluck, Harvard Medical School: The role of two kappa light chains. early genes of polyoma and SV40 in transformation. Birute Galdikas, University of California, Los Angeles: Roger Jeanloz, Harvard Medical School: Isoprenoid sugar Orangutans. phosphates in the biosynthesis of glycoproteins. Ann Graybiel, Massachusetts Institute of Technology: The Walter Stewart, National Institutes of Health: Intra­ brain stem oculomotor system. cellular staining and dye coupling between cells seen David Farb, Harvard Medical School: GABA as a neuro­ with a highly fluorescent dye. transmitter in spinal cord cell cultures. Stephen Arch, Reed College: Biochemistry and physio­ David W. Weiss, Mt. Sinai Medical Center: Passive logical effects of neuroactive peptides. immunotherapy of neoplastic disease with effector Rolf NOthiger, Anatomisches Institut, Ziirich: Develop­ cells sensitized in vitro. mental genetics of sexual differentiation in Dro­ Arturo Falaschi, University Pavia, Italy: Enzymes in­ sophila. volved in DNA synthesis and repair in human cells. Edwin Lewis, University of California, Berkeley: Func­ Stuart Rudikoff, National Cancer Institute: Structure of tional and phylogenetic correlates of morphology in antigen-binding myeloma proteins and idiotypes. the frog's inner ear. Lionel Jaffe, Purdue University: Control of development by steady ionic currents. 163

GRADUATES

Twenty-six students in Biology were awarded the B.S., M.S. or Ph.D. degree in June, 1978. Names, degrees conferred, titles of doctoral theses (with plans of recipients for future work), are as follows:

Bachelor of Science

Marc Stuart Berger, B.S. with honor. Margaret Ann Marshall, B.S. with honor. Lois Freeman Brubaker, B.S. with honor. Ray Bowen Morris, B.S.

Richard Brownley Gayle III, B.S. with honor. Lawrence Ira Mortin, B.S. with honor.

Joel Brent Gunter, B.S. with honor. Eileen Patricia Leslie Roy, B.S.

Lisa Cox Heinz, B.S. with honor. John Charles Wathey, B.S. with honor. Sandra Jean Koch, B.S. with honor. Josef Benjamin Zwass, B.S. with honor. Eric Kurt Lucha, B.S. Master of Science Janet Roberta Kallo, M.S. Doctor of Philosophy

Antony Clifford Bakke, Ph.D. Thesis: Studies on the Henry Vincent Huang, Ph.D. Thesis: I. The ontogenetic chromatin of the cellular slime mold, DictyoStelium expression of antibody variable-region genes in the discoideum. Postdoctoral research, Scripps Clinic chicken. II. Methods for fractionation of plasma and Research Foundation. membranes and membrane proteins. Postdoctoral research, California Institute of Technology. Welcome Bender, Ph.D. Thesis: Electron microscopic studies of sequence arrangement: poly(A) mapping, Robert Edward Sheridan Jr., Ph.D. Thesis: Kinetics and RNA tumor viruses, and slime mold actin genes. equilibria at nicotinic receptor in Electrophorus Postdoctoral research, Stanford Medical School. electricus and Raia erinacea electroplaques. Post­ doctoral research, St. George's Hospital Medical Jay Barry Edelman, Ph.D. Thesis: Statistical mechanics School, London. of biological membranes: Protein aggregation and lipid ordering. Postdoctoral research, University of Barbara Landale Stitt, Ph.D. Thesis: Role of the host cell California, Irvine. in bacteriophage T4 development. Postdoctoral research, Public Health Research Institute of the Teryl Kenneth Frey, Ph.D. Thesis: Biochemical and City of New York. biophysical studies on the RNA species of Sindbis virus. Postdoctoral research, University of Pitts­ Duncan Knight Stuart, Ph.D. Thesis: The neurosecretion burgh School of Medicine. of the polypeptide egg-laying hormone (ELH) from the bag cells, neuronal sites of action of ELH, and Sheila Gillard-Crewther, Ph.D. Thesis: Plasticity in the circadian release of polypeptides from the eye of cat visual system. Postdoctoral research, National Aplysia californica. Vision Research Institute of Australia. David Tang, Ph.D. Thesis: A DNA nicking-closing Karen Faye Greif, Ph.D. Thesis: Memory processing in enzyme from mouse L cells. chickens and goldfish. Postdoctoral research, Uni­ versity of California School of Medicine, San Fran­ cisco. Stanley Hoffman, Ph.D. Thesis: The role of the plasma membrane in the development of the cellular slime mold, Dictyostelium discoideum. Postdoctoral re­ search, Rockefeller University. 164

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 agencies for specific projects; from unrestricted annual gifts; from fellow­ ships for the support of individual scholars; and from contributions to general funds provided by lndi.Jstrial Associates and Institute Associates, as follows:

Fund--Research Support Purpose

American Cancer Society Cancer research

American Heart Association Greater Los Angeles Cardiovascular research Affiliate

Anonymous Gift Fund Research in educational programs John E. Barber Fund Biological research, particulary 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

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 Bruce Research in biology

B. W. Foundation Research in immunology

Louisa Jane ChLU'ch 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 Mrs. Mary Bruce Delbr'Uck Research in biology

Josephine V. Dumke Fund Cancer research Energy Research and Development Administration Research in developmental immunogenetics Fairchild Foundation Fairchild scholars program The Max C. Fleischmann Foundation Research in molecular biology

Foundation for Cardiovascular Research Dr. F. Noble lectureship

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 re­ search Irma Hoef!y Fund Cancer research Hoffman-LaRoche, Inc. Research On barbiturate and minor tranquilizers

Arnold R. Jones Cancer research 165

Lasker Award Fund Chemical Genetics

Litton Bionetics, Inc. Instrumentation grant

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 ali.alysis, biophysics, developmental biology, genetics, neurobiology, plant and cell biology, psychobiology, and virology; graduate re­ search 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 pro­ gram

Northwest Area Foundation Medical science program in biology

Ann Peppers Foundation Research in auditory physiology and anatomy 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

The Spencer Foundation Behavioral biology research

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 Sta tion

Joseph G. Venable Fund Arthritis research 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 166

Fund--Fellowship SUpport

American Cancer Society Grass Foundation

Earle C. Anthony Fellowship Lawrence A. Hanson Foundation

The James G. Boswell Foundation Mackenzie Foundation

California Foundation for Biochemical Research The Helen G. and Arthur Mccallum Fund

California State Graduate Fellowship Medical Research Council, Canada

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

Carnegie Institution of Washington National Academy of Sciences, Interacademy Exchange Program Centre National de la Recherche Scientifique, France National Aeronautics and Space Administration Claremont College National lnstitutes of Health, USPHS Lucy Mason Clark Fund National Science Foundation The Jane Coffin Childs Memorial Fund for Medical Research Gordon Ross Medical Foundation Consiglio Nazionale delle Ricerche Damon Runyon Memorial Fund Danforth Foundation Evelyn Sharp Fellowship Deutsche Forschungsgemeinschaft Smith, Kline and French Laboratories Deutscher Akademischer Austauschdienst The Spencer Foundation The Camille and Henry Dreyfus Foundation, Inc. Walter and Sylvia Treadway Fund European Molecular Biology Organization Vern Underwood Undergraduate Scholarship Fairchild Scholars Program USA-USSR Health Exchange Program Federal College Work-Study Program Veterans Administration Fight for Sight, Inc. Chaim Weizmann Foundation Foundation for Cardiovascular Research Helen Hay Whitney Foundation Gloria Gartz Fund

E. S. Gosney Fund

168

AUTHOR INDEX (by page number)

Allman, J. M., 103, 105, 106 Ellison, J. W., 36, 37 Kaczmarek, L. K., 97, 98 Anderson, D. M., 37 Eriksen, K. J., 116 Kasamatsu, T., 119, 120 Angerer, R. C., 34 Erlanger, B. F ., 92, 93 Kashdan, B., 128 Armstroqr, D. L., 92, 94 Ernst, S. G., 33, 35 Kauvar, L. M., 140, 141 Ary, J.P., 109, 112 Ewald, S. J., 48, 49 Kehry, M. R., 47, 48 Ary, M., 119, 120 Klein, S., 111, 112 Asai, D. J., 71, 154 Fender, D. H., 109, 110, 112 Klein, W. H., 36 Attardi, G., 17, 19, 20 Finbow' M. E., 80 Klymkowsky, M. W., 155 Audesirk, G., 99 Foote, S. L., 118 Knudsen, E. I., 113, 114 Frelinger, J., 50 Koblin, D. D., 91 Bagdonas, E. A., 121, 122 French, P. L., 112 Konishi, M .. , 113, 114, 123 Baker, J. F., 105 Frey, T. K., 65, 66 Konopka, R. J., 142-144 Bakke, A. C., 30 Fritsch, E. F ., 55, 57 Kronenberg, M., 48 Balzer, D. R. Jr., 75, 79 Fuhrman, J. S., 47 Krouse, M. E., 91-93 Bell, J. R., 62-65 Kuppermann, A., 42 Bell, P. B. Jr., 83 Galau, G. A., 32 Benzer, S., 139 Ganetzky, B. s., 139, 140 Lacy, E. H., 55, 56 Birt, D. L., 115, 116 Gard, D. L., 65, 66, 77 Lafay, F., 49 Bixby, J. L., 134-136 Gaston, K. E., 130, 131 Lafay, J.-F., 73, 74 Blake, G., 57 Giffin, C. E., 42 Larkin, R. M., 110-112 Blankenhorn, E. P., 151, 152 Gillard-Crewther, S., 130 Lauer, J. E., 55, 57 Bloom, F. E., 118 Gimlich, R. L., 34 Lawn, R. M., 55, 57 Boettger, H. G., 42 Goldberg, D. A., 57 Lazarides, E., 75, 78 Bonner, J., 22, 25, 28-30 Goldstein, S. F ., 70 Lee, A. S., 34 Briggs, R. P ., 112 Gomer, R.H., 80 Lenches, E. M., 63 Britten, R. J., 32, 38, 39 Gordon, H. W., 127 Lester, H. A., 91-94 Brockes, J. P., 87 Granger, B. L., 77, 78 Lev, z., 34, 35 Brokaw, c. J., 69, 70 Green, S. H., 145, 146 Lewis, E. B., 32, 149, 150 Butler, E. T. III, 56 Greif, K. F ., 79 Lipps, L. S., 58 Byers, D., 141 Griepp, E. P., 81, 82 Lowy, P.H., 60 Gurney, M., 114 Lyle, H. L., 72 Campbell, C. B. G., 105 Campbell, J. L., 155 Hall, D. D., 153 Machray, G. c., 29 Cantatore, P., 19 Hall, T. J., 39 Maizels, R. M., 152 Carraway, K. L., 83 Hamilton, C. R., 128-130 Maniatis, T ., 54 Cartier, P. K. III, 5 2 Hamilton, R. M., 73 Marder, E., 92 Cecka, J. M., 50, 151 Handler, A. M., 142, 144 Martin, M. s., 63 Chamberlin, M. E., 32 Hardison, R. C., 55, 56 Marton, K. L., 121, 122 Charlang, G., 53, 54 Harshey, R. M., 73 Marty, A., 92 Chen, J. W. M., 110 Heller, E., 96, 97 Maunsell, J. H. R., 90, 134 Ching, E. P ., 19, 20 Helphrey, D. B., 42 Mayne, J. T., 64 Chiu, A. Y ., 96 Hirose, T., 29 McGuinness, E., 105, 106 Chomyn, A., 59 Hobby, G. L., 52 McMillan, M., 50 Claudio, T. R., 153 Hood, L. E., 41, 42, 44, 62 Merkel, C. G., 19 Clegg, K. B., 33 Horn, G., 54 Meyer, P., 74 Conrad, S. E., 155 Horowitz, N. H., 52-54 Meyer, R. L., 132 Cooper, M. B. W., 105 Horvath, S. J., 42 Miezin, F. M., 103-105, 108 Cooper, M. L., 105 Hough-Evans, 8. R., 33, 36 Miller, M. M., 83 Corey, D. P., 89 Huang, H. V., 43, 49 Mitchell, H. K., 58-61 Costantini, F ., 34 Hubbard, B. D., 76 Moller, G., 59, 60 Crews, s. T., 17 Hudspeth, A. J., 82, 88-90 Moore, G. P., 38 Crosby, M. A., 149 Hunkapiller, M. W., 41, 42, 49, 51, 62, 96 Murphy, R. F ., 29, 30 Huppert, F. A., 126 Myerson, J., 103, 105, 108 Darcey, T. M., 109 Hwang, N., 150 Mylander, O. D., 100 Davidson, E. H., 32-37 Hyson, M. T., 111, 112 Davis, M. M., 47 Nass, M. M., 91-93 De Francesco, L., 20, 21 Itakura, K., 29 Nelson, D. B., 117 Delbriick, M., 71, 73 Itakura, T ., 120 Newsome, W. T. III, 105 DeOgny, B. L., 41 Nicholson, B. J., 82 Dever, J. E. Jr., 26, 27 Jacobs, R. A., 89 Nienhuis, R., 115, 117 Diner, D. 8., 110 Jacobs-Lorena, M., 33 Nishiguchi, D. J., 17 Dreyer, W. J., 40, 42, 43 Jayaram, M., 73 Norris, D. D., 42 Jennings, K. R., 97 Early, P. W., 47 Johnson, L. E., 124, 125 O'Connell, c. D., 55, 57 Easter, D. w., 80 Johnson, N. D., 42, 46, 48 Ogden, T. E., 109, 110 Edelman, J., 155 Johnson, R., 69 Ojala, D. K .. , 17, 19 Edens, J., 80 Joho, R. H., 47 A., 55, 56 Okuno, M., 69 Efstratiadis, Jones, G. s., 118 Olds, J., 118 Julesz, B., 111 169

Olds, M. E., 115-117 Sargent, T. D., 25, 26 Van Essen, D. C., 133-136 Orr, D., 142, 143, 145 Scheller, R. H., 34, 36, 37 Van Harreveld, A., 83 Otto, M. K., 72 Schilling, J. W., 45, 46 Vass, S. I., 42 Ou, J.-H. J., 66 Segal, M., 118 Vener, G., 150 Owen, R. D., 151, 154 Shaffer, G. D., 29 Vermeire, B. A., 128 Shen, J., 56 Viele, D. P ., 98, 99 Parker, R. C., 57 Sheridan, R. E. Jr., 91 Patalano, J.P., 41 Shinowara, N. L., 82 Wallace, A. M., 154 Pearson, W. R., 39 Shipley, S., 69 Wallace, R. B., 25, 29 Peck, C. K., 130 Shoemaker, W., 120 Wang, C., 82 Petersen, N. S., 60, 144 Shotwell, S. L., 88 Wassermann, N. H., 92, 93 Petersen, S. E., 104, 105 Shupak, P ., 72 Wathey, J. c., 91 Pettigrew, J. D., 119-121, 123 Sim, G. K., 55 Wells, S. M., 144 Pike, L., 33 Sivertsen, D. w., 106 Wiktorowicz, J. E., 24 Posakony, J. W., 17, 37 Smith, R. F ., 143 Winkelstein, L. E., 154 Potter, E., 28 Sperry, R. w., 124 Wiseman, A., 20 Presti, D. E., 72 Stanton, T. H., 51 Wold, B. J., 36 Strauss, E.G., 62, 63 Woolum, J. C., 99 Quon, D. H., 55, 56 Strauss, J. H. Jr., 61, 62 Wu, c.-F., 139, 140 Stroud, R. M., 155 Wu, J.-R., 27, 28 Raftery, M. A., 156, 157 Strumwasser, F., 95-100 Wu, W. C.-S., 156, 157 Readhead, C., 41 Stuart, D. K., 96, 99 Revel, J.-P., 79-83 Stumph, w. E., 27, 28 Yancey, B., 80, 81 Reyes, A. A., 25 Rice, c. M. 111, 64, 65 Zaidel, E., 127, 128 Rohan, R. M., 38 Tanguy, J ., 92 Zimmerman, E. R., 41 Teitell, M. F ., 27 Zwass, J., 144 Sabatini, D., 81 Thomas, T. L., 34 Sala-Trepat, J. M., 26, 27 Trimble, J., 109 170

FINANCIAL SUPPORT INDEX (by page number)

The Ahmanson Foundation, 40 National Aeronautics and Space Administration, 52, 95 Rita Allen Foundation, 54 National Eye Institute, USPHS, 119 American Cancer Society, 17, 22, 32, 44, 54, 75 National Institute of Mental Health, USPHS, 124 American Heart Association, 95 National Institutes of Health, USPHS, 17, 22, 32, 40, 44, American-Israeli Binational Science Foundation, 124 54, 58, 61, 69, 71, 75, 79, 88, 91, 95, 103, 109, 113, Earle C. Anthony Fellowship, 32, 44, 61, 79, 156, 157 115, 119, 124, 133, 142, 149, 151, 155 National Science Foundation, 17, 22, 32, 40, 44,. 54, 58, 61, Biomedical Research Support Grant, 133 71, 75, 103, 113, 115, 119, 124, 139, 149, 155 The James G. Boswell Foundation for Virus Research, 139 Northwest Area Foundation, 54, 79 Ethel Wilson Bowles and Robert Bowles Professorship, 44 B. W. Foundation, 44 Ann Peppers Foundation, 88 The President's Fund, 124 California Foundation for Biochemical Research, 155 President•s Venture Fund, 54, 142 California State University, Los Angeles, 95 Cancer Research Institute, 44 Research in Biology, 71 Carnegie Institution of Washington, 32 Research Corporation, 61 Centre National de la Recherche Scientifique, 22 Rockefeller Foundation, 58 Jane Coffin Childs Memorial Fund for Biological Re- Gordon Ross Medical Foundation, 44, 58, 139 search, 54, 139 Ruddock Fund, 7 9 Lucy Mason Clark Fund, 71, 155 Damon Runyon-Walter Winchell Cancer Fund, 22, 54 Consiglio Nazionale delle Ricerche, 17 Charles B. Corser Fund for Biological Research, 151 Alfred P. Sloan Foundation, 75, 88, 109 Smith, Kline and French, 44 Danforth Fellowship, 32, 103 Spencer Foundation Fund, 58, 91, 95, 103, 113, 119, 139, Deutsche Forschungsgemeinschaft, 71 142 Divisional Funds, 83 Stanford University, 44 The Camille and Henry Dreyfus Foundation, Inc., 44 The Stone Foundation, 124 Josephine V. Dumke Fund, 58 Walter and Sylvia Treadway Fund, 119 Fight for Sight, Inc., 119 Leland Fikes Foundation, Inc., 40 Universit8 de Paris-Sud, 44 The Max C. Fleischmann Foundation, 40, 44 University of Bari, 17 Fort Lewis College, 22 University of California, Los Angeles, 79 University of Cambridge, 12~ Gosney Fund, 22, 54, 58, 71, 142, 151 U.S. Department of Energy, 151

Lawrence A. Hanson Foundation, 95 Veterans Administration, 32 The Hearst Foundation, 88 Frank P. Hixon Fund, 124 Weizmann Fellowship, 32, 103 Whitehall Foundation, 119 Litton Bionetics, Inc., 40 Helen Hay Whitney Foundation, 17 Robert E. and May R. Wright Foundation, 119 March of Dimes, 32 Mccallum Fund, 32, 79, 95, 103 John A. Mccarthey Foundation, 40 Medical Research Council of Canada, 22 Medical Research Council (U.K.), 87 The Merck Company Foundation, 61 Muscular Dystrophy Association of America, 75, 91 •

A phase contrast light micrograph of a face-on view of a Z disc sheet from chicken skeletal muscle. When a muscle fiber is extracted first with glycerol and with potassium iodide and then cleaved in a mini-blender it shears on both sides of a Z line rroducing individual Z line striations. These striations readily adhere onto a glass coverslip and can be viewed face on. Such a face-on view of the striations reveals that each Z line is composed of numerous interconnected Z discs; each Z disc is approximately 1 µm in diameter. The dark phase dense objects in the plane are remnants of mitochondria which are tenaciously adherent to the Z planes.

A portion of a record of the swimming movements of a sea urchin spermatozoon after removal of the flagellar membrane with Triton X-100 and reactivation of move­ ment in a solution containing MgATP. Flash photography using a continuously moving film in a specially modified 35 mm camera provides more detailed information about the development and propagation of bends on the flagellum than has been available previously.