- <<•«;-,-- ,«y.iB- >t9ME±- "?'* X'M /77^t;f ORNL 5072
BIOLOGY DIVISION ANNUAL PROGRESS REPORT
J?eriod Ending £une JO, 1975 BLANK PAGE
' TM« upon lof «a*fc OOF wio Enfffr nMorcft •*•* OOf OW»/ Of OJjf OMOJ'VVOOJV OOT 0O*/ O* PJOaT or tho* omoJoyoMt mofcos owy wofrvify, moron 01* of ooy nwOfO'OwOO, its mv MMM not lOfViooj pfnofofy owttofj noJkn. vcm- WOLOGY DIVISION ANNUM. PROGRESS REPORT f«rNried( if | In 1130.1*75 J. B. Storer. Director SF. Carson. Deputy Director NOTICE This document contains information of preliminary nature and was prepwed primarily for internal use at the Oak Ridge National Laboratory. It issubiect to revision or correction and. therefore, does not represent a final report. Quotations from or reference to this report must not be made in open literature without written oermission from the individual authors. NOVEMBER 1975 OAK RIDGE NATIONAL LABORATORY Oak Ridge. Tennessee 37830 upsmatl by UNION CARBIDE CORPORATION tor fie ENERGY RESEARCH AND DEVELOPMENT ADMINISTRATION arsfaftm ORNL-13 Ptw^ Endwgrcawuaiy 2*. I*4» ORM.4.1 Penal r«—gMn 31. I*4» ORNLISC Period EndnvAugnat 31.I*4» OKNL-220 Nri tjMjnajNamnaVi 30.1*4* 0RNL-3I* PcriaaJ Eudngtilmary 2*. 1*4* ORNL-244 Period ra—gMij 15.1*4* ORNL-4S7 VvlMnfl DHMfA^pBl • J. IV^V ORNL-S37 OKNL444 Period buJagFcfcraanr 15.1*50 CMHL-727 Penod Cad—JMJJ 15.1*50 0RXL-W7 Period Ea*o«Ao«M 15. I*5C ORM.-M* Pcnad Enuoo«*4ooMbcT 10.1*50 ORNL-1** Period Ea*m fckrajry 10.1*51 ORNL-102* Period rndwjtaliy 10. I*5i ORM.40* Period Eadag Angus! 10.1*51 ORNLII67 Period CaawgMuitadjri 10.1*51 0RNII244 Period EriNlftkiwy 10.1*52 OWH 12*7 Period Endorg May 10.1*52 ORNLI3*3 Pcnod ElMbng Augnrt 10.1*52 0RNL-I4S* Period Ea*ng>iiii linen 10.1*52 0RNL-I497 Penod Eadwg February 10.1*53 OtWL-1614 Period bolMg August 15.1*53 0RNL-I693 Penod Ending February 15.1*54 ORNL-177* Period EnJng August 15.1*54 ORNLIM3 PenodEndingFcbruary 15 1*55 ORM.-I4S3 Penod Endmg August 15.1*55 ORN12060 Penod EtufeMg February 15. 1*56 ORNI.2IS5 Period EudMg August 15.1*56 ORNL-2267 Period Endmg February 15.1*57 O*NL23*0 Period Eudmg Angus! 15. IV57 0RNL-24Si Period Ending February 15. 1*5* 0*NL-25*3 Period Ennmg August 15.195S ORNL-2702 Period Ending February 15.1*5* ORNL-2SI3 Period Endmg August 15.195* ORNL-2913 Penod Endmg February 15. I**0 ORNL2**7 rVrud Ending August IS. I*M) ORNL-3095 PerioJ Endmg February IS. 1*61 ORNL320I Period Eiidmg August 15. 1*61 ORNL-3267 Period Lndmg February 15.1*62 ORMl-3352 Period Er.dmg August IS. 1*62 OHM. 3427 Period EuemgFebruary IS. I%! 3RNL-349S Period Endng August IS. I*h3 ORNL-3601 Period Endfcig February IS. 1*64 ORNL-3700 IVriodEnditg August I5.I9M ORML-37M PtnodEiidM* February IS. 1*65 ORNL3SS3 Period EndmtJuty 31.1*6$ ORNL-3922 Period Ending January 31. 1*66 0HNL3W Period Ending Jury 3!. !%6 ORNL4I00 Periiid Ending January 31 1*67 0RNL4240 Period Ending December 31. 1*67 ORNL-4412 Period Ending December 31. 1*6* ORNL-4535 Period Ending Dncmber 31. 1*6* OKNL4740 Period Ending Jvic 30.1*71 ORNL-4RI7 Period Ending June 30.1*72 0RNL49I5 Permd Ending June 30.1*73 ORNL4993 Period Ending Jute 30.1*74 CONTEXTS Introduction John 8. Surer flection spot resonance stud** of phofoiyzed peptide solutions - 0. G. Dohtrty. Ralph LhrmfRon. Manprct A. Turner, and Henry Zetdes 3 MecfiaOBmsuf cheinduinnescent oxidation-reduction reactions J. R. Toner and O. G. Ooheriy 4 Synthesis and evaluation of serine atianetabiifctes 0. G. Oofierty. Miniitt A. Turner. and James 0. Regan 4 Separation of base*, ribonucleotides, and deoxy ribonucleotides by aroon-exclusion and partition chromatincraphy on cation-exchange mm. Application to the assay of ribonucleotide reductase, deaminase, and nucleosidase Bwnal C. Pal. James 0. Reran, and F. 0. Hamilton 5 In ritrt transformation of 4-thioundine lo 4-selenoundine in F. cnU mixed tRNA Bunal C Pal and Diane G Schmidt 5 Regulation of wmethytsulfoxide-mduccd hemoglobin synthesis in Friend leukemia celts. I. Vncommittcd and committed slate* of hemoglobin synthesis during the induction period P R. McOintock and John Papaconstantinou 5 Regulation of dimeihylsulfoude-rnduced hemorlobin synthesis m Friend leukemia celb. II. Inhibition of hemoglobin synthesis by hydrocortisone P. R. McClintock and John Papeconstantinou 6 Regulation of mmethylsetfoxidc-induced hemooobin synthesis in Friend leukemia celb. III. Density •dependent induction of hemoglobin synthesis in the absence of inducer John Papaconstantinou 6 Regulation of albumin and a-fetoprotein synthesis in synchronized mouse hepatoma cells - C. L. Parker and John Papaconstantinou 6 K' filiation of dimethylsulfoxide-induced hemoglobin synthesis in Friend leukemia cells. IV. Effect of nroflavm on the induction of hemoglobin synthesis A. W. Wieru. > R. McOintock. and John Papaconstantinou 7 I notation iif albumin polysomes and albumin mRNA Peter Bro-an and John Papaconstantinou 7 rVigress in purification of tumor-specific transfer factor G. D. Griffin. Helen G. Stmn. DervMei Yana, and G. Damf Noaelli » TuncTvuhn transfer factor: a model for certain aspects of cell-mediated immunity 0.0. Griffin, Helen G. Sellin. and G. David Novcfli 8 Process on fractionation of transfer factor from beagles Helen G. Sellin. G D. Griffin, and G. David Novelli 9 D. .elopment of j generalized lymphocyte cytotoxicity assay using soluble antigens coupled to cell membranes - G. 0. Griffin, Helen G. Sellin, Sister Mary Paul Nevins, and G. Darid Novetli 9 in IV Transfer UNA alterations occwrnng in human leiutarac Krnphocvi.** G. O. Griffin. G. 0—id HottRi. and Wen-Kuang Yang Ill EjuMMMioa of urine from twnor-bearm*. rait foi mcmstd nuclei'intr content Audrey H. Bast and C. Dand NoveNi 10 SiiMJiesoadieilLNAofnetlnw-proaWnf baciera Audrey N. Best II Inlubition of DNA-uependent RNA polymerase off. o>* K phvpuoupids A salt-promoted uaubitor of RNA polymerase isolated front T4-phage-«i«rcicd A o>* Audrey I— Stevens and Joyce C. Photon 12 Abnormakoes in protein-syndiesbcornponenis as detected with an m ritn> protein-synthesis system - K. R. Hhavn and ML P. Sturberg 12 Transition of mRN As in an or rim? protein synthesis system M. P. Stutberg, K. R. Isham. Beth C.Munm. and L C. Waters 13 The content of modified bases in urafcniKthybied phem Uanine iRNA K. R. Isham. MWiunt Sutton, and M. P. Stutberg 13 Selective merJrytaiion of nrwfy synthesized tRNA from mnei' mutani of En Jurat/toe mK daring methionine starvation Lee R. Shugart 13 Purification of tRNA methytiransferases from Fschtnchm a*b Lee R. Shugart and Barbara H. Chastam 14 5-adenosyntomocysteine nucleosidase Lee R. Shugart 14 Tyrosy!-!RNA hgasc of DnnophUa: its ability lo discriminate iRNAT>r from a suppressor mutant and wild type K. Bruce Jacobion and J. B. Murphy 14 Cuanylalion ofDmsopluk tRNA and the guanylating enzyme ofDnmtphilt W. R. Farfcas and K. Bruce Jacobson 15 Quantitation of pteridines by direct measurements of fluorescence on thin-layer chromatography sheets Marilyn A. Sutton. T. G. Wilson, and K. Bruce Jacobson 15 Studies on the oiochcfimiry of the purple mutani in DrnsophUa metmnngasier - T. G. Wilson and K. 8ruce Jacofcson 16 Altered forms of phenylalanine tRNA in a yeast mutant K. Bruce Jacobion. J. B. Bell. Lee R. Shugart, and J. F. Lemontt 16 o-galactosidase in Mus muscuha - J. E. ToMer and K. Bruce Jacobion I7 Synthesis and degradation of specific tRNAs hi mammalian cells Michael Lin IX Automated sequential degradation of RNA Mayo UzieJ and A. J. Weinberger I* Analysis of modified bases in tRNA from eukaryote and prokaryote sources Mayo Uriel. L. H. Smith, and A. Jrannine Bandy IX High-resolution -high-speed separation of 35 modified nucleosides found in RNA Mayo Uriel and A, Jeannine Bandy . , l«» Affinity chromatography of RNA derivatives of dihydroxyboryl columns S. A. Taylor and Mayo Uriel 20 A novel ol%oribonuclease of Escherichia colt. I. Isolation and properties S. K. Niyogi, Brenda H. Underwood, and A. K. Dana 2(1 A novel oligoribonuclease of Escherichia coll. II. Mechanism of action A. K. Oatte and S. K. Niyogi 20 V Pr.mtxci ret»>a«%|olsinefe-*i!andedMI.?DNA S. K. Niyoai and Sankar Mitra The oilcol Kuktrukm o*dhwJ function and its integrative suppression *MI< aiapfc^ DNA synthesis Sankar Mrtra and 0. R. Stallions The n4e«»f f st-hrnHaieo* dbwf.' lunciNm m Ml»coiaptuge DNA synthesis Sanunu Dasfupta and Sankar Mitra Charactcri/atm* of hatiemyhay-T5 wluced DNA polymerase with DNA cuaiwiMf smgte-sirand breaks R. K. Fuemura. Barbara C. Roop. and D- P. AMson ExMWKienc associated ^nth bacteriophage T5 induced DNA polymerase S. K. Das and R. K. Fupnura A DNA^raumg protein from bactenupliage-TS-infected Eschtrichm rnfi S. K. Das and R. K. Fiajimura Intnctstronic mapping: «if bacteriophage T5 DNA put* merase mutant* W. M. Ardrey and R. K. Fupmura A nncroetifalmnary study on the «wipn of chJoroptastic amtnoacyi-iRNA synthetases J. G. FarreHy. L. I. Hacker, and W. E. Bamcft Purification and nucleotide cumposiiMm of phcnvlalanyl-tRNAs from tl e chloroplasis w.J cytoplasm of f'waViM - L. I. Hecker. Mayo Uwl, Arte* P. Teaskty. and W. E. Barnen The transcriptional origin of/iiqnVmr chioroplasi tRNA S. D. SchMrTzbech. L I Hecker. Artec P TeaUry. and W. E Bamrn Sequence homology studies with organelle and cytoplasmic tRNAs L. I. Hecker, S. D. Scbwartzbach. Artec P. Teastey. and W. E. Bameti The differential extraction and characterization of newly synthesized heterogeneous nuclear RNA from human neoplastic lymphocytes J. W. Bynum. M. Helen Jones, and Elliot Votkin The synthesis of three classes of pr4y( A Ron faming RNA in human (pydoma cells J. W. Bynum and Elliot Volkin RNA synthesis and proces-ing in neoplasma of slow and nondnridrng cells J. W. Bynum. Elliot Volkin, and James D. Regan Separation of nucleic acid components on polyacrylamide gel columns J. X. Khym An analytical system for thf rapid separation of tissue nucleotides at low pressures on conventional anion exchangers J. X. Khym hvahiai.">ns of mammalian ribonucleotide pools by combined polyacrylamide and anion-exchange chromatogiaphy J. X. Khym, Elliot Volkin. and J. W. Bynum Characterization of chromosomal proteins C 6. Mead and E. A. Hiss Cross-reaction of ttuoredoxin reductase and ribonucleotide reductase in Escherichia coH and HufjampacUu R. G. Hoft and F. D. Hamilton The ribonucleotide reductase system in Euffem pwafo S. Munwalli. Dorothea V. Parker. and F. D. Hamilton Isolation of ribonucleotide reductase in Pteudorrnxm Uuizeri - B. D. Mehrotra and F. D. Hamilton Studies on the function of ascrobalc sulfate - F. J. Finamont and Rose P. Fetdrnan Translatkmal control of protein synthesis in brine shrimp embryos A. H. Warner Lipid regulation of cell function Peter Pfuderer ill BIOPHYSICS AND CELL PHYSIOLOGY Mk.~roscoptc observation of intracellular ice formation in mouse ova j> a tunction «>t coulmg rate S. P. Leibo. J. J. McGrath. and E. G. Crawamo Pefraeabmty of mouse embryos 10 glycerol Peter Mazur. S. P. Leibo. and Nicholas Rifjnpoulus Survival of frozen-thawed mouse embryos as a function ot glycerol permeation S. P. Leibo Physiology and low-temperature biology ot bovine enihry •« Peter Mazur S. P. Leibo. B. H. Enckson. and J. C. Daniel. Jr Effects of subzero temperatures on eggs and embryos of .-tsnurar punciulaiti - Suzanne C. Jackowski and R. A. Wallace Phase diagrams of solutions of dimethyl sulfoxide in Hank\' balanced wit solution W. F. Rat! and Pe*r Mazur *4 Survival of slowly fro/en human red cells as a function of warnmte rate Peter Mazur and R.H.MMer »4 Survival of frozen-thawed human red cells as a function >>f mmimum temperature of exposure, elsceM conceitralion. and warming rate Hiroshi Souzu and Peter Mazur .»* Ctmpartson ol the response ol mammalian tissue-culture cells to freezing Nicholas Rifopoutos. S. P. Leibo. and Peter Mazur «•> Isolation and pri-peities of chromatin v bodies D. E. Obns. Ada L. Olins Marilyn 8. Senior. R. D. Carlson. Gwendolyn B. Howze. Ever line B. Wright and Mayphoon Hsw-Hsu '5 The formation of triple-stranded complexes between satellite DNAs and doublestiandeJ DNAs C. A. Chambers and 0. M. Skinner M* Moil-cycle-corrdaleii molecular chances in muscle and hemolymph in iKc land crab. Oeianinus lateralis ~ L. H. Yamaoka and D. M. Skinner " Interacting controls on mulling and regeneratnm in crusiacea C. A. HoNand and 0. M. Skinner Polyamino-acid stimulated uptake of extracellular fluid markers into Mela cells Emily Tate Brake and J. S.Cook >«> Turnover of He La membrane proteins J. S. Cook. P. C. Will, and Envly Tate Bn-ke 441 Radiosensiti/ation of Escherichia ofV by the free radical TMPN R. J. Brake 4(» The number and kinds of cyclohtityl pryiimdr dimers in human DNA W. L. Carrier. W.H.Lee, and James O.Regan .41 A sensitive method for the detection of ullravtolei phoinproducis mSauhcromycts cererisiae DNA - R. J. Reynolds 41 In vitro studies with bacteriophage T? W. E. Masker 42 Radktion-induced cessation of respiration in Escherichia a4i and its role in radiation cell death 9. A. Swenson 42 The role of adenosine cyclic X: 5 '-monophosphate in the ultraviolet-induced cessation of respiration in Escherichia <<* B/r 9. A. Swenson. R. L. Schenley.and J. G. Joshi 4.1 Changes in binding properties of pyridine nucleotide binding prote*ns in ultraviolet-irradiated Escherichia ciAi B/r cells J. G. Joshi and P. A. Swenson 4.1 VII Separation of viable and nonviable cefa from ultraviolet-inadiaied Ctikirkhm cot B/rcultum W. D. Fisher. R. L. Schentev. and P. A. Smnson 44 bxctmtn of pymNdme dimers fiom viable and nonviable crib in ultraviolet-irradiated ftrferftWhaoiftaVr cultures R. L. Schtntey. W. D. Fisher, and P. A. Sweraon 44 An Reproved procedure ot separation and purification of two principal toxBslridm V-3 and lllL and a hemagghiiinin from seeds of Maims amummr.. and crystallization of new lllL C H. Wei and OMnajCun Koh 44 Funnei purification of the must toxic ricnt V-3 from seeds of Rthmis commma - ChonaKsn Koh and C. H. Wei 45 Ammo-terminal sequences of two peptide chains for new V-3 S. S.-L. Li, C. H. Wei. J. Y. Lin. and T.-C. Tuna. 45 X-ray structural analysis of hycanthone mcthanesuitonaie. a highly active schistosonucidai agent C. H. Wei and J. R. Fintfein 45 Prehrnnury X-ray data for some compounds ot' bwlopcal interest C. H. Wei 46 The pk„ ot lie active-site cart»»xyl group of those phosphate isomense F.C. Hartman and G.M. LaMuraalia 47 Affmiiv labeling ot ribuhise brsphosphale carboxylase by 3-hfomo-l .4-dihydroxy-2-butan»ne 1.4-hisphosphale J. V. Schkns and F. C. Hartman 47 Evidence <>l a previous!} undetected lysyl residue a: the active site of aldolase J. P. Brown and F. C. Hartman 48 Affinity labeling of phosphogWcetate mutase ftom rabbit muscle I. Lidle Norton. C. D. Strirtyr. and F. C. Hartman 48 Dala-atquisifion >ystem Margarita K. Churchich. S. S. Stevens, and M. L. Randolph 48 Transfer of iripiet eiectionic energy in dinucleolides J. W. Lonoworth and S. K. Das 4° Dependence ol fluorescence and phospho.escence r>f Bence-Jones proteins on temperature J. W. Longworth and A. Solomon 49 Analysis of fluorescence lifetime histograms J. W. Lonoworth, S. S. Stevens. M. K. Churchich, and Jonas Hahteman 49 Fitting models to data - J. W. Longvronh and M. K. Churchich 5") D\A degradation kinetics after X rays M. L. Randolph 51 Energy deposition by neutrons M. L. Randolph 51 Adenine photochemistry R. 0. Rahn 51 Platinum binding to DNA Linda L. Munchausen and R. O. Rahn 52 Invito' labeling ol DNA with'"I R. S. Stafford and R. 0. Rahn 52 Photmy nibesis W. A. Arnold and J. R. A«i 52 Singlet excitation transfet from poly > A to iKtjnd dye at 77°K R. M. Pearlrteinand F. Van Nostrand 53 Theory of excited-stale interactions in Nomulecular aggregates R. P. Htmangtr K. Lindenberg. and R. M. Peavlstein 54 Analysis of fluorescence decay data R. P. Hemcngtr and T. J. Mitchell 55 Application* of multiple scattering methods to biology R. P. Hemenotr 55 Localized synthesis of /em in maize endosperm Benjamin Burr and Francis A. Burr 56 V»i Mutagenesis lor protein composition in soy heart* 0. E. Foard. Mfen-Kuang Yang. L. L. Triplen. and C. 0. Stringer *** Immunological studies of soybean lectin* O. E. Foard. Frances M. Tale. and Wen-Kuar* Yang *" Excision repair of LIT-induced p> rirmduie doner* in the DNA of pbni ccib G. P. Howiand ** Fiuorodeoxy undine inhibits thymidine degradation and stiniuLiie* it* aworporation mit> plant cell DNA G. P. Howiand and Margaret L. Yette 5" Preferential synthesis of cytoplasmic DNA in isolaied wid cari»i proiopb»t> G. P. Howiand and Margaret L. Yette v> Ethylene and CO; control of fern-spore germination M. E. Edwards (sponsored by G. P. Howiand) >*» Section III GENETICS AND DEVELOPMENTAL BIOLOGY Hydrazine mutagenesis in Haemophilus influenzae R. F. Kimball and Bermce F. Hirsch e»2 Development of an auxotrophic mutation .system fur Haemophilus influenzae and us use to compare the mutagenic effects of hydra/me ami .Ymeihyi-.Y'-niiro-.Y-nitri>M>guanidine R. F. Kimball <»: Mutagenesis with nitroso compounds Rosalie K. Elespuru. R. F. Kimball, and Jane K. Setlow «».; Studies on mutation indue lion in phages of Haemophilus influenzae - M. E.Boling and R.F.Kimball »>.' Repair and UV mutagenesis in Escherichia colt - Donna L. George M Mutagenic effects of (-is-dichlorodiammineplatmum in repair-deficient mutants oi Escherichia co!i Donna L. George and Linda L. Munchausen »>4 Autoradiographic studies of the cell cycle in confluent cultures of Chine* hamster ovary cells R. F. Kimball, Stella W. Perdue, Patricia A. Bnmer. andA.W. Hsie n* Combined tritium and carbon-14 autoradiography Stella W. Perdue «»5 Production by chemicals and transmission ol chromosomal aberration*, in mammalian germ cells H. E. Luippold, P. Carolyn Gooch. and J. G. Brewen <+> Cytogenetic analysis of dominant lethality induced in postmeiotic male germ cells J. G. Brewen, Helen S. Payne, and R. J. Preston i»~ Cytogenetic consequences of irradiation of diclyate oocytes J. G. Brewen, Helen S. Payne, and R. J. Preston o'» X-ray-induced translocations in mouse spermatogonia J. G. Brewen and R. J. Preston "I Cytogenetic effects of ozone: inhalation or in vitro exposures P. Carolyn Gooch, D. A. Creasia, and J. G. Brewen 7.* A'-methyl-/V-nit':KV-nitrosogtinidine-indiices reversion of gly D mutants in Chinese hamster ovary eel s E. A. Hiss and R. M. Wallace, Jr ~>(i X-ray-induced forward mutation rales in cultured mammalian cells E. A. Hiss and R. M. Wallace, Jr 76 (unitn.ir.iv -induced IA -like repair in normal cdh. and detective repair in xeroderma pnonenti^um I \Pt cdls detecteJ at Km* tunes alter uradiation Junes D. Regir. F M Fautcon. and R B Settowr Posireplication lepnii in normal human cdls and in xeroderma pigmentosum complementation cr»up> Raymond Wafers and James D. Regan "'H l-ack <>t phoi<«evciiai <•• IV -induced pyrinudinc dimers in normal human and xeroderma pigmentosum >km cdK W L Carrier. W H Lee. and James D. Regan "»» I dec! ••! putative repair inhirii<>r^ >ii UNA repair in norma! human skin cells J tier ultraviolet radiation James D. Regan and W C. Dunn. Jr. HO Vlrivmcarharyl and iuti.~icarKirv!-like compound-^ their effects on human DNA R. 0 Blevins. William Lipnsky. A. A. Francis, and James 0. Regan M) inhibition «>l serine iianstrvdroxymethyiase by a serine antimetahofcte A. A. Francis. D. G. Doherry. and James 0. Regan M < ompaiaiive mutagenesis J. L. Epler. Alice A. Hardigree. Ti Ho. Ruby D. Wilkersor.. and William Wmton >0 Mutagenicity ••! coal v>>R\er>i<>n tractions J. L. Epler. Alice A. Hardigree. and Ti Ho with M. R. Guerin. Hisashi Kubota. and I B. Ruben >: (iencik s^ieenine/ high - rev »hi turn .hrimiatocraphy and identification ot metaboiiie> J. L. Lple* C. O. Stringer, and William Wimon *: Prdiinirutv studio on the devdopment •>( j hoot-mediated mammalian cell mutational assay Jot \-.Yn:,ni ;H>tentul chemical mutagens A. W. Hs«, J. M. Holland. Richard ftfechanoff. and Jerry W. Hall *3 Dose teNponse <>t inuij|it>n> ji (he hy poxanthiiie-icuanine pho%phonhosy I transferase locus by alkylating agents in Chinese hamster ovatv cells A. W. Hsie. Patricia A. Brimer. J. P O'Neill, and D. B. Couch M Quantitative analyses nt mutations at the hvpoxartlhinc guanine phoiphorihosv I transferase locus by ph\Mcal agents in Chinese hamster ovarx cells A. W. Hs*. Patricia A Brimer. Richard Machanoff. J. C. Riddle, and A. P. Li H> Origin <>r taiaru> rcMsunt i<> purine araii^jes inChiivse hamster Coordinate aciivatit*i of a mullienzymc complex by the first substrate. Evidence for a novel regulatory mechanism in the polyaromatic pathway of Neuntspm cnaa - G- R. Welch and F. H. Gaertner 104 Influence of on aggregated multienzyme system on transient time kinetic evidence for compartmeniation by the aromatic complex of Seunnpun cnasa - G. R. Welch and F. H. Gaertner 104 PtuisphoceUulose. an affinity chromatographic system for chorismate synthase and the a- >matic complex of Xeurmpora crassa - K. W. Cote and F. H. Caertner 105 Scanning electron microscopy (SEM) of the oocyte follicle - J. N. Dumont I0S A culture medium for developing oocytes J. J. Eppig and J. N. Dumont 106 Inhibition of priHein incorporation in isolated amphibian oocytes - R. A. Wallace. J.N. Dumont. and A. W.Schuetz 106 Dieihylaminoethattol as an anlifertilily agent - Mary Lou Anderson and J. N. Dumont 106 DNA polymerase activity in Xemtpus kern oocytes during oogenesis maturation. and embryogenesis T. G. Hoflinger and Robin A. Wallace 10? Chromosomal-induced developmental abnormalities in embryos o( Xenopui hem — T. G. Hollingrr and Katherine Luby 107 Specificity for vitellogenin incorporation by isolated amphibian oocytes - R. A. Wallace art D.W.Jared 108 A kinetic model for determining amino acid pool size and rate of protein synthesis in rapidly synthesizing cells R. E. Ecker and R. A. Wallace 108 Membrane potentials in large oocytes of Xewtptn laevis R. A. Wallace and R. A. Steinhardt I (P Role of DNA ligase in DNA polymcrase-l-dependent repair synthesis in toluene-treated F.xherUhia coli - Daniel Billen, G. R. Hellermann, and D. R. Stallions 109 Roie of DNA polymerases I. II. and III in DNA repair synthesis in X-irradiated. toluene-treated bacteria Daniel Billen and G. R. HHIermanr- 110 Recovery from UV-light -induced damage in Bacillus suhtUis uvr I - C. T. Hadden and Daniel Billen 110 Defective excision repair in Bacillus subtilis uvr-l C. T. Hadden and Daniel Billen Ill DNA metabolism in Twcen 80 permeabilized Chinese hamster ovary cells - Daniel Billen and Ann C. 0!son .. Ill Section IV MAMMALIAN GENETICS Relative inducibility of heritable translocations and heritable inversions in postmeiotic germ cells of male mice - W. M. Generoso, Katherine T. Cain, and Sandra W. Huff 114 Ineffectiveness of alkylating chemicals in inducing heritable translocations in mouse spermatogonia W. M. Generoso. Katherine T. Cain, and Sandra W. Huff IIS Induction of herilaMe translocations in mouse oocytes with isopropyl methancsulfonate W. M. Generoso and Katherine T. Cain 116 Comparative inducibility by 6-mercaptopurine of dominant-lethal mutations and heritahle translocations in early tnciolic male germ cells and differentiating spermatogonia of mice W. M. Generoso, Sandra W. Huff, and Katfwrine T. Cain 117 Ill Sensitivity of different postcopubtion germ-ceil states of mice to induction of dominant leihab with three alkylating chemicals K. E. Sutar and W. M. Generoso II? Lack of duroirunt4ethal effects of ethyl alcohol in male mice - W. M. Gtntroso, Katharine T. Cam. Sandra W. Huff, and W. L. Russell 119 Results from a specific-locus test of the mutagenicity of sulfur dioxide in mice W. L. Russell and Elizabeth M. Ke»y 119 Specific-locus mutation frequencies induced in mouse spermatogonia at very low radiation dose rates W. L. Russell and Elizabeth M. Kerns 120 Criticism of a current model for estimating genetic risks of radiation - W.L. Russell 121 Effect ofhycantitone on X-chromosome loss in female mice W. L Russell. Patricia R. Hunsicfccr. Elizabeth M. Kern/. Carolyn M. Vaughan. and Georgia M. Guinn 123 Effect of route of administration of hycanlhone on liner size in Ihe offspring of treated female mice Patricia R. Hunsicfccr and W. I. Russell 125 Results from screening for radiation-induced hemoglobin variants in the mouse - W. L. Russell. Carolyn M. Vaughan. R. A. Popp. Lianc B. Russell, and K. Bruce Jacobson 126 An example of conditions that make the mouse specific-locus test highly efficient at low expense W. L. Russell and R. B- Cumming 126 Discovery of a tandem duplication in the mouse Liane B. Russell. W.'.. Russell.. N. L. A. Cachtiro. Carolyn M. Vaughan. and R. A. Pop* 127 Somalic-mutaiion method in chemical mutagenesis studies in the mouse Liane B. Russell. Clyde S. Montgomery, and Sandra W. Huff I2« Effect of age on induced and spontaneous nondisjunction and chromosome loss Liane B. Russell and Clyde S. Montgomery 12«> A case of nonrandom X-chromosome inactivalion explained by selection Liane B. Russell. N. L. A. Cacheiro. and Margaret S. Swartout 130 A new X-autosome translocation that may give nonrandom inaciivaiion Liane B. Russell. N. L. A. Cacheriro. Jean W. Bangham, and Margaret S. Swartout 131 A presumed pericentric inversion in the mouse - Liane B. Russell. N. L. A. Cacheiro. Clyde S. Montgomery, and Georgia M. Guinrt 132 Cytological studies of sterility in sons of mice treated at spermatogonia! or early spermatocyte Mages with mutagenic chemicals N. L. A. Cacheiro, W. M. Generoso, and Margaret S. Swartout 132 Cytological studies in sterile sons of IMS-ireaied females N. L. A. Cacheiro and Margaret S. Swartout 132 Attempts to find protein differences related lo genetic differences at single loci E. G. Bemstine and Lianc B. Russell 133 Eco RI digest: obtained from intact mouse liver nuclei - E. G. Bemstine 133 Energy transfer from aretophenone to DNA polylysine complexes - E. G. Bcrnstinc and R. 0. Rahn 133 A new experiment on the induction of specific-locus mutations in mouse spermatogonia by tiiliared water R. B. Cumming and W. L. Russell 133 Studies on potential genetic effects of 5-chlorouracil in mammals R. B. Cumming, B. C.PW, Marva F.Walton, andW. L. Russell 134 nn A test for 5 Section V PATHOLOGY AND IMMUNOLOGY Radioprotectron of mice by phetrymydrarine-danuged erythrocytes - L. H. Smith and T. W. McKinley. Jr. 146 Ability of thymocytes to increase proSifcratrte rate of transplanted marrow cens - Joan Wright Goodman, Sarah G. Shinpocfc, and Nancy L. Besford 147 Studies on control of the hemopoietic stem cell - Joan Wright Goodman and Sarah G. Srwnoock 147 Dissociation of age-related refractoriness of PHA-mduced lymphocyte transformation and lumorigcnesB - E. H. Perkins, Ching Yuan Hung, J. M. Holland, and Wan-Kuang Yang 148 A lower incidence r' pulmonary turners m Rude mice following intravenous injection fff varkMS syngenic tumors - C. B. Skov. J. M. HoNand, and F. H. Perkins 149 Agr-related changes in helper-suppressor ranciion of mouse thyrnus-derived splenic lymphocytes - R. L. Kruprud*i4 E. H. Ptrfcins 150 Age-related changes in spleen ceR responses to lipopolysaccharide - R. L. Krogwud 150 Failure \o implicate immunosuppression as a significanl inductive mechanism of radiation leukcmogencsis in the RFM mouse - E. H. Parkins and Lucia H. Cacheiro 151 XIV Recovery of imnrane competence funowing sublethal inadiation: therole of thyrnic funclion and agrcg - W.J. Peterson 152 A mkrotechnique for autogenic studies of mouse lymphoid tissues - C. F. Gottlieb. G.M.*1ernOT. and W..!. Pearson 152 Direct and indirect effects of fast neutrons or X rays on mouse embryos - Vfaftace Friedberg. G. 0. Hamwman. E. B. Darden. Jr.. D. N. FauNoMr. and W. R. Kirkham 153 Altered scram esterase and protein profits in cyclic neutropenic dogs - R. L. TyndM. Shirley P.Cotyer.and J. B. Jones 154 Effects of butytaled hydroxy toluene, dietrryirutroarnine. and X rays during lumorigenesis in mice- N. K. Chop. R. L. Tyndan. J.". Dougherty. Kuwtlha A. Davidson. C F. Gottlieb, and R. W.Tcmant 154 Effect of butytated hydroxy toluene on diethylratrosanune carcinogenesis in BALB/c mke - N. K. Clapp. R. L. TyndaH, UM C. Settvfnid.and W. C Kuma !54 Effect of whole-body X radiation upon buiytated hydroxy toluene or diethylnitrosamme ireatment in mice - N. K. Clapp, law C Setnu field, and W. C KKtna 155 Enzyme analysis of plasma from carcinogen- and noncarcdtogrn-ueaied mice - R. L. TyndaN. N. K. Clapp. Kowetha A. Omidwn. and C. A. Burtis 156 Plasma esterase alterations in mice fed carcinogens and/or the food additive bulytafed hydroxytoluene - R. L. Tyndaff and N. K. Clapp 156 Alterations in isoenzyme patterns following treatment with carcinogenic agents - J. P. Dougherty, N. K. Clapp, and R. 1. Tyndm' 156 Characterization of nonspeciik esterases in mouse serum - Kowetha A. Davidson, R.L.Tyndatl, and N.K. Clapp 157 Study of immune competence in mice treated with the potent carcinogen, dieihylnitrosamine. and/or butylated hydroxytoluene - C. F. Gottlieb.G. M.Peterman, and N. K. Clapp I5K Merits of histological comfinnalion of gross necropsy data N. K. Clapp. Lou C Satterfidd, and W. C Klima 158 Kinetics of appearance of covalenlly bound metnyl label (,4C> in DNA and RNA of mouse liver and lung following a single injection of ||4C|dimethylniirosamins J. P. Daughterly and N.K. Clapp 15« Subcellular distribution of r*Jioactr»ity in the acid-soluble and -insoluble components of mouse liver and king following a singfe injection of ('4 C| dimethyInitrosaminc J. P. Daugnerty and N. K. Clapp 15« Effect of neutron irradiation on Ihe incidence of various neoplastic diseases R. L. Ullrich. J. B. Storer, and M. C. Jirnigon 160 Effect of chloroquipe on recovery from carcinogenic injury to mouse King induced by split-dost radiation - R. L. 'Jtwich, N. H. Pazrnmb, and J. M. Yuhas 161 Influence of local radiotherapy on host-tumor interactions - R. L. Ullrich, J. M. Yuhas, and M. C. Jenrigan 162 DNA synthesis in megafcaryacyies of mHe after stimulation of megakaryocytopoiesis T. T. Odetl. T. P. McDonald, and Deborah A. Boran 162 Changes in platelet size after induced acute thrombocytopenia T. T. Ode* 162 Persistence of injected antiplatelet serum in the circulation of rats and mice Lucia L. Hodgrs. T. T. Odell, Diane K. Batman, and Deborah A. Boran 16.1 ^Mortality jnteiaction between singk-dose X ray and continuoustow-lewi dielhylnitini i nninf KiaafcC3Hmke - J. M. Hoaand and T. J. MrtdM* 164 Sex-and strain-dependent differences in the magnitude of the chroar- response to radiation - X ML HoMand, Mary S. Whitakcr, L. C Gttwn, and T. J. MrKhcN 165 Percutaneous toxicity of epoxy nam and amine hardeners in inbred C3H and C57BL/6 nice - J.M.Hotand.L.aGipHm.andMaryS.WMtakar 166 Traaspbatabaity of spontaneous hepatomas in C3H male mke - J. M. tloiand and Mary S-twutafcer 167 Activation of murine leukemia virus by gamma radiation - J. A. Cham. J. M. Ouarles. and R. W. Tcmant 168 Studies on the pathogenesis ofradiation-mduced leukemia in RF mke - J. A. Oram, Susan Coined Jonas. W. K. Ctopp. and R. W. Tenmnt 168 Replication of the DNA of Knhamrat virus - G.C LaiaHi and Anna Tai Li 169 Detection of murme tumor vims antigen m cultared cdb byflow microfluorometry - R. E. Hand. J. M. Ounrlas. and R. W. Termant 170 Use of flow microfluorometry for determination of DNA synthesis rates - J. W. Heidei, £M. Ovaries, and R.W.Tennant 171 Simulation of clinical radiotherapy with WR-2721 in the spontaneous dog-tumor system - J.M. YuhasandOarrylBiery 171 Antitumor properties ot-Gwynebmctmum pmnntm in senescent imrwnomcompeient mke - J. M. Yuhasand R. L. Uurich 172 Prediction of the effective dose of WR-2721 for humans from an interspecies distribution study - L. C. Washburn, J. J. Rafter, R. L. Hayes, and J. M. Yuhas 172 Dose-rate effects in murineradiation carcinogenesis- J. M. Yunas and Anita E-Wamer 173 Recovery from radiation and chemical carcinogenic injury to the mouse king - J. M. Yuhas 173 Section VI CARCINOGENESIS PROGRAM A sensitive assay for respiratory carcinogenesis, using transplanted trachea* R. A. Grimmer, Paul Neitesheim, P. H. Martin, and J. E. Caton, Jr 176 Comparative effects of 7.12-ojmethyibenz|d| anthracene and benzo|«|pyrene on tracheal grafts R. A. Gnestrner, Paul Netmheim. D. H. Martin. and J. E. Caton, Jr 176 DewJopmcnt of an epiihefel-cell culture system 'or detecting transformation m exposed trachea - Ann C. Merchok, Joyce C. Rhoton. and Rhonda F. Irwin 176 Effects of vitamin A or. proliferation and differentiation in tracheal organ cultures Arm C Marchofc. Joyce C. Rhoaon, Rhonda F. Irwin, and M. Vngjnm Cone 177 EfTecl of retinybceiale and retinok acid on development of carcinogen-induced metaplastic king nodules Paul Netafsfwim and Mary L. WWianw 177 Ehition of carcinogen from carrier particles in the respiratory tract of mice D. A. Crautw 17* Tobacco smoke hioassay project W. E. Damay. R. A. Griesemer. and Paul Nettatheim 179 Mechanisms of chemical carcinogenesis G. M. Soger 1*0 XVI Retabua between structure and carcinogenic activity of :Y-aMnmt cn—jnuueT - vrwkamUvmsky and ft W. Taylor IXI Siadawo«iMmoryiitittt^iWTOwm wrasat •*» - L. C Mn. •Hli CM-**. E. G. •**«. R.A.f)n^«NtfL.G.HMCN 193 Anatwnpiyaafhmyii nwdMiidcytotoninty assay ashaj laieted adenosine - AlleMStts to select l—or-spi*ifk lectins from kganats - VhM-KMnpj Yaagv M- MkjRjwit IMMamt. D.E. Foard. aadD.W.FovMain 194 r hwan ilngriahj - 0. W. Fnrnnani and Wan-Knaaa, Yana 195 Lrclai tnmrnl nf fonrtun IMWIMI of wybf D. W. Foantain, hi hnrfjnrt Iswnwat. MMteMYM»atfD.E.Fw4 195 bokctnn in Gfrrar- mmx - D. W. fi—iuiii aad Www Kina1| Yawn 1% Viral RNA snhasnts J. N. Me.XG. Fantty, aad F. T. Kaaney 196 Vlnlaatifens J.N. MKantfF.T.KcMwy 197 VknactnaiMM - J. N. Nrieand F. T. Kanwy 198 Attempts to identify specific IJIIUIWH —lull miffr nr pHyiihuu—i i - Kai-Lin Los and F.T. Kena-y 198 Preparation of anfi jodies ammst senjncaiial annam dcleiminants of hepatic tyrosme aminotransferase- Kai-Lin Ler and P. L. D»*e 199 Attempts m any tyrosine iminuiijnifiiaii iweimnmi UNA m cd-frce translation systems - Joanni ht Nicked. Kai-Lin Let, and F. T. Kanwey 199 Effect of Thiamin B» deficiency on the synthesis and dty titukm of hepatic tyrosine aminotransferase Kai-Lin lea, P. L. Darke, and F. T. Kanmy 199 Stadsrs on the degradation of hepatic tyroajne aminotransferase daring amnctton by hydrocortisone - Nicholas PomMo. Kai-Lin Lea, and F. T Kawwti 200 AMhor Index 202 MTRODUCTION THE PAST YEAR was one of transition for the Biology Division. The Atomic Energy Commission, our primary sponsor foi twenty-six yean, passed into history and its functions were incorporated into the broader responsi bilities of the new Energy Research and Development Administration. An early reflection of our response to this widened mission is the increased emphasis in this report on the biological effects of chemical agents. As usual, the individual reports consist of very brief summaries of principal research activities which will be reported in greater detail in the scientific literature. John B. Siorer Director. Biology Division I BLANK PAGE I ** SECTION I BIOCHEMISTRY EBwtVottw •JEaqmCatafjrsB ttmOac AaiOnmmlif DCDohrrty W.E.Coka J. R.Toller B.C. Pal Dont C. Sctunidt •rCdDMnnlMiM Mayolizid Ma Fapacou'CUnii C.LParker* NKfckAdi EKotVofcin W. E. Bametl G. David NoveOi J.W.Bymin/ Audrey N. Best A K Daila* JanDnk* R. K. FHJtnwn GD Griffin* J X Khym BrthCMuttn* C.CJtad E. F. Pfcarcs SMfcu Mitn Helen G.SeHn S.KNiyop Audrey L. Stevens Ann C. Obon' L. C. Waten S. D. Scfcwartzbactf Wen Knang Yang HNKIEVCMB* I F. D. Hamihan MP.SHJBJCTK L. R. Slwpn F.J.Fi K. Brace Jacotnon Peter Pradkrer •feted Litr* AMen Warner* J. E. Totter* W.F.Zapmkr 2 wrnmmw' *"SH t «*"-> - • case of ti-alanylsarc«Mne. where the peptide hydrogen is replaced by a methyl group, the radical attack is quite different; in this case the hydrogen is abstracted from the .V-methyl group of Ihe sarcosine residue, thereby giving rise to a mixture of tvo radicals which are ra and owns isomers. In the case of jJ-abnyl-t-valine and glycyl-L-valine, the -OH attack is not at the a carbon of ELECTRON SF1N RESONANCE the valine rcskhi? but ai the isopropyl side chain, giving STUDIES OF PHOTOLYZEO rise to a mixture of two radicals. rem DC SOLUTIONS Fiiur tripeptides have also been studied: 0-alanylgry- cyl-L-proline, ^-alanyl-l-prory(glycine. 0-alanylgrycyl- D. C. Duhcrty. Ralph Livingston.* Margaret A. Tumei. gjycine. and >3-alanylgjy< yi-L -alanine. The /talanyl resi and Henry Zeldes* due was used in the V-terminal position, since the work LmnptiMi and Zeldes found thai when two Bence- with the dipeptides indicated that more easily analyzed Jones proteins were photofyzed and the radkais formed spectra were obtained. Much to our surprise, essentially- were examined by microwave spectroscopy, charactcr- only one radical is formed in each case. Hydrogen istic ESR (electron spin resonance) signals were ob abstraction takes place almost exclusively from the a tained that could not be interpreted by direct analysis carbon o( the C-termmai amino acid. Results for two of of the spectra. With the expectation of discovering these tripeptides along with results from the corre systematic trends in Ihe spectra of simpler peptides that sponding dipepiides are given in Fig. I. The coupling would be applicable to more complex systems, we began a cooperative program involving the synthesis and •»».-0"S 1 S*-' exammation of specific «- and polypeptides. The initial study involved simple dipeptides of gly 0 cine, t -alanine and ^-alanine. Photulyses of an aqueous •HjK^CHfmKKpmKtoyca; s»4uinm of the prptides in the zwwtenon form contain a«* u* iso AM *» ing 1*7 hydrogen peroxide produced radicals by the abstraction of hydrogen by -OH radical attack. The results with the first five dipeptides were oven in the last annual report.' These dipepiides along with two additional ones. 4-alaml-t.-alanine and (J-alanyl-»J- o o alaniwc. have been reported elsewhere ~ in aH -ases the •iy»c^o^»^u»jc*crv nature of the -OH attack is highly systematic, hydrogen an *«t am MS TT.M a»T S.M aw IJO T7.«* •» abstracted from the o carbon between the peptide t • *.O0S«t »• LOOMS nitrogen and the carhoxylate group. When ^-alanine is in the ('-terminal position, a mixture of radicals is obtained by abstraction of H from both the a and »3 methylene groups. All these peptides were found to be m the transconfiguration. Hyperfine cowphngs and g constants and g are very little changed in going values were measured, and spin density for the carbon from dipeptides to iripeptides. Al present we do not from which abstraction took place was measured. know if the shupliicil y wM be exhibited by longer The study has been extended to dipepiides containing peptides with other side-chain R groups. If this is the more complex amino acids. Radicals from t -pv oh/hjly- case, the formation of the radical is not only significant cine and jtabnyl-i. -proline have been identified, and Hi for pinpointing the region of -OH attack but abo for both -ases hydrogen abstraction foUnws the previously labeling a peptide an unpaired electron, which is observed systematic trends sensitive (at least principle) to conformational In the former case the spectrum lines are quite broad, effects. presumably due to the presence of en and tmn Homers which have ''ighfly different (unresolved) values for the 1. R. Lmm$**cm. D. G. IWwir. M. A. Tamer, mi H. ZeMo. byperfme coupting constants In the latter caw the mm. Av. Amm. Ivwe. Hep. Mmt M IW4. ORNL-4993. p. 4. spectrum shows additional complexity which m thought 2. R. LmnaMnn. D. G. Dnfcrnv.aml H. ZeMn./ Am. Ckrm. to arise from a "ftp-flop" of the proline nng. In the Sn.91,im )HM||97S>. A MECHANISMS OF CHEMILUMINESCENT aqueous Mtlution. As with lumim»l. Iishi emission in the OXIDATION DEDUCTION REACTIONS absence of H<02 require* free 02. With fructiHe. I«ht emission I'ouWs the kinetics expected from a reaction J. R Toiler and O. G. Doheriy ot the type: The conversion of 5-aminophthalazine 1.4-dione (lummol) in aqueous solution lo 3-aminophlhalic acid A -* 2B -» end products . with the enussioa of light can be brought about by hydrogen peroxide and a catalyst, by peroxydisulfate The end products were .^and to he about '/ methyl- alone or in the presence of H 0>. by peroxidases and 2 : acridone and V dimethyl hiacridylideTte The oxygen H 0 . and by xanthine oxidase and its substrates or by 2 2 : uptake rate was found to he independent of the xanthine dehydrogenase, one of its substrates and a lucigenine concentniion above about 2 X 10 " .If. With reversibly reducible qumone. In the absence of H.O . ; hydioxylamine the oxygen uptake rate was dir-.jily free 0» is required for light emission, and the quantum related lo the >quare root of ".he lucmenine concer Ira- yield based on luminoi is much reduced. lion over a relatively large range 15 X lo * to 2 X It' * The peroxydisulfate chemiuminescence in the pres W) These data are presently interpreted to indicate '!.al ence and absence of HJOJ has been studied to elucidate the lucigenine undergoes a reversible decomposition to the mechanism si*p-by-siep. One current theory postu two identical radicals which then undergo disprtir**- lates a two-electron oxidation of luminol a-'-n by fionafion with unchanged Ju 1 OF SERINE ANTIMETABOLITES C»H.O:N, • S,0, -C»H,0;X, • SO, " • HS04 . D. G. Doheriy. Margaret A. Turner, and James D. Regan 1 c»H5o:N, • H2O2 -cmcuNj - • :H* . The initial discovery by J. D. Regan that human C,H,0,N,J-C,H,04X2 "••%, . granulocytic leukemia cells had an abnormal require ment for serine stimulated a search for serine anii- 2 ! mrtaboliies. A series of compounds selected to the C,H,04N " * -*C»H504N " • light . structure of serine was prepared, and one <»f them. HhydroxyaminoKyi'kihexane carboxylk acid, in This scheme requires the production of three H* per hibited DNA and RNA synthesis but had a minimal molecule of luminoi destroyed. Experiments in which effect on protein synthesis.1 The results suggested that production of H* was measured did not agree with this one carbon donation from serine lo purine biosynthesis stoichiometry. In fact, the H* production was quite via the enzyme serine iranshydroxymelhylase was variable: from zero to seven or eight H* per molecule of greatly reduced. The structures of those compounds luminoi in either the presence or absence of HJOJ a» already tested indicated fairly strict requirements for pH!2.0. inhibition. In consequence we have synthesized a series Experiments still in progress indicate that the 5-amino of compounds isosteric lo serine, e.g.. with a phos group may be oxidized to a radical which can combine phorus atom replacing the carhoxyl carbon, and various with itself in various ways fan "aniline Mack" type of combinations of amino and hydroxyl groups. At this reaction), but many of the combinations are again lime these compounds are awaiting testing in the tissue reduced to luminoi by any HJOJ present. This reversi culture system. To have an alternate means of testing ble system may then act as a catalyst for the decompo enzyme inhibitors we have isolated serine iranshydroxy sition of HjOj by peroxydisulfate. melhylase from rabbi! and rat liver and are currently Experiments have also been conducted on the chemi- developing a speclrophotometric assay. luminescent compound dimethylbiacridylium nitrate 'lucigenine). Kinetic studies have been made on the reaction between lucigenine and fructose, hydmxyl- I. N. H. fiimmn. D. G. Dnberi*. and J. D. Regan./ foil. amine, creatinine, or hydrogen peroxide in alkaline Curcrrfoil. 31.7*1 Hi ll*73>. -y-)«rj>sa» iLWiMiMnuowstq^^e^caiigigE^igg^^g^s^^-- SEPARATION OF BASES. RIBOMJCLEOSIDES. This led us to explore the possibility of chemical AND DtWYMBONUCLEOSIDES BY conversion of 4-lhiuundine to 4-scienoundine in tRNA. ANION-EXCLUSION AND PARTITION We have already reported the route to m rant labeling CHROMATOGRAPHY ON CATION-EXCHANGE of IRNA with "S: iRNAISHl'-!*-^-> tRNA- RESIN. AFFIXATION TO THE ASSAY OF tSCS>,5SH > iRNA("SH>.4 The same technique can RIBONUCLEOTIDE REDUCTASE. DEAMINASE. be used for partial conversion lev. t&H of 4-thioundme AND NUCLEOSIDASE to 4-selenouridine in #Y. cut mixed tRNA using SeH ' in the last step. Bmul C. Pal. James D. Regan, and F. D. Hamilton |5-'H|CDP (cytidine S'-diphusphaie) and CTPlcyti- dine 5' triphosphate) are used as substrates M the assay of I.C. ». MMm. G. C. Gia»*. R. S. Raps*"*. C. V. Tautvakan. K- A. Mrkfcuc*. aa< A. I. Dtfc*lara«. I**. Ak*L ribonucleotide reductase, deaminase, and nudcosidaHe .\tmt Az. SSK 29. IS H973I. activity in crude enzyme preparations After incubation 2. J. L. HuffuKM ami K- T. McKoaarS. «W*M. *np*i*. the nucleotides are hydrolyzed to nucleosides by At* 33*. IW11*741. sequential treatment with potato apyrase and a&aline J. Y. S. Pra*a*» Ran ami J. D. rfcerayit. lift So. 14. 2051 phosphatase. An aliquot is then chromatographcd on a >l*74>. 4. B. C. fat ami D. G. Sdmmmx.J. Am.Chnm.Sn. K.S493 cation-exchange column at 50°C with 0.1 V boric acid, 11974). adjusted to pH 7.4 with ammonia, used as eiucnl. The pyrrolidines Ura. L'rd. dUrd. Cyt. Cyd. and dCyd are separated and eluted in about 50 mm in small volumes. Assays by this procedure of CTP reductase activity in REGULATION OF MMETHYLSULFOXIDE- crude fractions of ribonucleotide reductase from INDUCED HEMOGLOBIN SYNTHESIS IN FRIEND kugjrm jcrufibs gave results comparable to those ob LEUKEMIA CELLS. I. NONCOMMTTTED AND tained by the standard method. The new procedure is COMMITTED STATES OF HEMOGLOBIN also applicable when adenine or guanine nucleotides are SYNTHESIS DURING THE INDUCTION PERIOD used as subs;rales. The adenine derivatives Ade. Ado. -JAdo. Hyp. I no. and dlno. as well as the guanine P. R. McCnnlock* and lohn Papaconstanlinou derivatives Gua. Guo. dGuo. Xan. and Xao. are sep arated Irom each other in this chromatographic system When Friend leukemia ceHs (FLC> are grown in 2% Me S0 mrdtum for only 24 hr. hemoglobin synthesis in about an hour. 2 begins on schedule at 48 hr. i>.. after two replications. The induction, however, is short-lived, and data indicate that synthesis soon stops. With a 4Jt-hr exposure to IX WTKO TRANSFORMATION OF Me:S0. the induction is initiated, but again synthesis 4-THrOURIDINE TO 4>SELENOURIMNE ceases within a short period after removal of the IN £ COL/MKED tRNA inducer. A 72-hr exposure to Me2SO is sufficient to give a rale of hemoglobin synthesis identical to the Bimal C. Pal and Diane G. Schmidt controls, where Me2S0 is present throughout the Selenium is present in iwr environment. The dement experiment. These results seem lo indicate that a is an essential micrnnutrient for man and other species. specific amount of exposure lime to MejSO is required On the other hand, many selenium compounds are before the FLC n committed or stabilized in the link at higher dose level. Sodium selenile has been pathway of differentiation characterized by the appear recently reported to retard the growth of various ance of hemoglobin. If the inducer is removed before experimental tumors in rats and mice.1 For these the stanihzaiion has taken place, then the reactions reasons the metabolism of selenium compounds is of which commit the cell lo this Rue of differentiation arc wide interest in nutrition and toxicology as well as in arrested. In addirion these experiments suggest thai the biochemisiry. It has recerlly been reported from two stabilization takes place between the second and third laboratories that £'. <>•#/ grown in a medium containing gencr?iktns(4ft 72 hr). 7 fSf-labeled sodium netenile or sodium sdenosulfafc incorporate sekiirum as 4-sftenouridinc in their *St«e«iH ai Iter VT Obk Rinw Graduate School of RiMwedi lRNAs.IaJ Biological studies on such selenafed tRNAs cal ScMncts. nesenr address: Frederick Cancer Research Cen arc hindered by poor yields from in rim experiments. ter. Frederick. Marybnd 21701. 6 REGULATION OF WMETHYLSULFOX1DE layers. (6) celb cannot be shaken loose from the INDUCED HEMOCLOMN SYNTHESIS IN FRIEND surface of the flask, (r) celb can be resuspended by LEUKEMA CELLS. a INHIBITION OF proteolytic enzyme treatment and wiR reattach to the HEMOGLOBW SYNTHESIS RY HYDROCORTISONE surface when subcultured. ( In the course of studies on the M*3SO-mediated synthesis at a specific phase of the cell cycle. The induction of hemoglobin synthesis in Friend leukemia critical point for albumin synthesis b at late Gj early cells, it was noticed that under certain culture condi mitosis: the critical point fot aFP synthesis is in tions a large number of (hex cells which normally grow mid-Gj. Pool analyses have shown that th? critical in suspension attach to the surface of the culture flask point cannot be due to an increase in the specific and form colonies. By selecting for these "attached activity of the intracellular leucine pool. Amino acid forms." cultures were established which have the analyses of immunopretipilaled \* II | albumin, isolated following characteristics: (a) cells form confluent, dense several hours after the critical point, hive shown that g-T. i !<• J,[*JHU* 99% «if ihc radioactivity is in leucine, indicating thai last mRNA templates. To expand our understanding of Ike increase m rale of synthesis cannul be attributed tt> the mechanism of induction of hemoglobin synthesis in a generauwd metabolic -.hsiribulion of |JHI leucine FIT. studies were done to determine whether proflavin NMO other amino acids. We conclude thai ihc critical win inhibit this induction. Our results have shown that pomi for albumin and oFP synthesis is due lo lue it) proflavine b a potent inhibilor of cellular repbea- aciivaiiun of new mRNA for these proteins. On the tkm. (0) induction of hemoglobin synthesis by Me2SO basis of these observations, we used the m/iraf paint as 0 MH nhibited by proflavine and the taie of accumula an assay to delect any interference with the transcrip tion of hemoglobin b not affected, and U} induction of tion of a specific mRNA. To determine whether the hemoglobin synthesis K delayed by proflavine. This transcription of albumin mRNA b disconiinuus and to delay b linked lo the effect of proflavine on the localize the phase in the cell cycle where transcription generation lime of these eels. Thus. I JIM proflavine occurs, we treated synchronized cells with inhibitors of wil extend the generation time of FIX* from 24 lo 60 transcription lactinomycin D. cordycepini at specific hr, and hemoglobin induction b delayed from 48 lo stages during the cell cycle. These experiments were 120 hr. In both cases, however, the induction and designed to localise an inhibitor-sensitive phase for synthesis of hemoglobin b the same. We conclude from albumin and ttfP. Our assay for the detection of an these studies thai («) the mechanism of induction of inhibiltir-sensiiive phase is the K of the critical point. hemoglobin synthesis by McjSO b basically different Treatment with aciinumycir showed that the critical from the mechanism of induction of ^utamine synthe point for albumin and o!T is abolished when the tase by HC thb conclusion b based on the fact that inhibilor is present during late S-phase and early G2. proflavine has a specific inhibitory effect on gwlamine One objection to the use of aclmomycin b its toxicity synthetase induction, and (b) proflavine, an intercalat and inhibit M m of milosb. Thb problem was solved by ing agent, has a potent inhibitory effect on the using cordycepin in cimcenlraiiuns that abolish the replication of FIT. Thus, thb potentially mutagenic critical point but have no effect on the cell replication. and carcinogenic compound shows a strong inhibitory Under these conditions, the drug-sensitive period fur effect on the replication of the cell but shows no effect albumin and aFP was found in bte S early G2 • We on the expression of a gene which b activated during a conclude from these studies that transcription of specific pathway of cellular differentiation. mRNA for albumin and oFP b discontinuous: the period of transcription is in late S and early G2. and the period of processing and transport of mRNA extends •Vnitinjt investiplnr. Department of HMocy, Kabmazob Crikyr. Kabmszon, Michigan 44003. from early Gi to the critical point (late G3): after the ^Student at the UT Oak Ride* Gradate School of Bio mRNA enters the cytoplasm because of its stability, it medical Sciences. Present address: Frederick Cancer Reararcb b traiiicribed continuously throughout the cell cycle. Center, Frederick. Maiybnd 21701. ISOLATION OF ALBUMIN POLYSOMES 'Postdoctoral investieator supported by subcontract 3322 AND ALBUMIN mRNA front the Biology Division. OttNL. lo the University of Tennessee, knmvinc. Peter Brown* and John Papaconstaptinou REGULATION OF MMETHYLSULFOXIDE- To study the regulation of transcription and transi INDUCED HEMOGLOBIN SYNTHESIS IN FRIEND tion of albumin mRNA in synchronized mouse hepa LEUKEMIA CELLS. IV. EFFECT OF PROFLAVIN toma cells, experiments were initiated to establish ON THE INDUCTION OF techniques for detection and isolation of albumin HEMOGLOBIN SYNTHESIS poiysomcs and foi detecticn and isolation of albumin mRNA. The detection and isolation of albumin poty- A. W. Wiens.' P. R. McClini.>ck.t tomet have been accomplished through the use of and John Papaconstantinou radioimmunoassay and iminunoprecipitaiion tech niques. Highly purified albumin was prepared and It has recently been shown by one of us (A.W.W.) coupled lo Sepharose (by CNBr treatment) for use in that proflavin b a potent inhibitor of the hydrocorlt- the affinity chromatographk preparation of mono sone-mcdialed induction rf glulamine synthesb in specific albumin antibody. Thb highly specific antibody embryonic chkk retina cells. Thb study indkaled that was rodirraier! with ' "Land the ('"Ijantrserum was & proflavin inhibits the transcription of giularnine synthc- reacted with liver poiysomcs. The polysomes arcre spun •y H s ia a sucrose gradient and the fractions were collected simply by sfparatMK the iahmiiory from the active aad counted. TWe radioactive profile showed a peak 01 curnponcutj. Stphadex C-25 i-hroatatography was used the heavy side of the polysome Mx*i-) profit. This, for this stpuratiaa. aad ciafci UV absorption peaks wew peak was sensitive to ribonuclease ireatmcui aad to resolved. Cytotoxicity assay of these components again competition with cold allwmia antibody. We now Jadkitfd varying levek of transfer (actor activity in al propose to use tins procedure to deteiniwt whethti peak components. Stphadex C-IO caroraategrapay latere b any change in size of jrbumin polysomes ia was also used to try to separate whmitory components synchronized hepatoma ceils. Thb infornutioc is im from the active material. Assay of pooled mssuul from portant for the distinction between a iranshtiunai this chromatography indicated that transfer factor stimulation of protein synthesis (more ribosomes/ activity was spread throughout the run aad that mRNA) or a transcriptional stmuaiian of albwrun lueuidtrabh. activity could be found in nou-UV-absorb- synthesis in synchronized ceils- In addition we propose ing material, indicting that transfer factor activity may to use the imimwoprecipiiation of albumrn polysomes not necessarily be associated with UV-absorbmg mute- for the isohlion of albumin mRNA. Prdirmnary expen- dies. ments on the mRNA isolation nave been done. Im- The separation of transfer factor into a number of munoprecipitaied polysomes have been phenol ex active components by two anile different processes tracted and the RNA fractionalcd on an otigo-dT (ion-exchange chromatography and motecuiar fihra- column. The poryadenytated RNA fraction shows a lion) may indicate thai the classic methods of transfer strong stimulation of m rfm> protein synthesis in a factor preparations produce various sized active compo wheat-germ system. The purity of the product of this *r nents of a larger molecule, and this brge molecule may ritn? synthesis is being determined by radioimmuno be resyntheseed by lymphocytes during the course of assay. the assay. In addition lo these chromatographic methods of purification we are also engaged actrvrfy in a study of •StancM at the VT -On* Riajr GotaK Sefcool of fco- the cell type from which transfer factor originates, since it might then be possible to separate tins particular cell type from other contamicalrng ecus h-rforc preparing PROGRESS IN PUStlFKTATtON OF transfer factor. The procedure could result in signifi TUMOR SPECIFIC TRANSFER FACTOR cantly less degradation of the active molecules in transfer factor. Preliminary experiments have indicated C. D. Griffin.* Helen G. Senrn. Den-Mei Yang.* Utaf a transfer factor preparation from "T* lympho and C. David NoveUi cytes is active, and tins activity resides in a larger The effectrves-ss of •nmunotherac.y of cancer by- molecular species than the activity of dialyzable trans transfer fo- 'or could p> ibably be greatly increased by fer factor. trsmg preparations of purified transfer factor which contain only the moleciilcs involved in transleriirj •USPHS tosMacionl FePvw. tumor immunity. Purified preprations would also allow quantification «if effectiveness of 7arious do>e I. V. Brers. A. Levin. A. tbvtoit.art II. Faarahcn:.-/. Cfci. schedules. We are currently attempting lo purify the /*rrt.'. SS.SO0 SI3H9TS). active components from transfer factors specific for oncogenic sarcomi and mammary carcinoma. TUIERCUJN TRANSFER FACTOR: Initial experiments with mammary carcinoma transfer A iMODEI. FOR CERTAIN ASPECTS factor used chromatography on Aminex A-28. Separa OF CELL4MEMATED IMMUNITY tion into IS UV absorption peaks occurred, and all of G. D. Griffin.* Helen G Sellin. and G. David Novell* these peaks were found to possess transfer factor activity when assayed in an in ritnt cytotoxicity auay.1 We are attempting to develop the tuberculin transfer The activity of some of the fractions could be demon factor system in animals and in human lymphocytes m strated only upon extensive dilution: this suggested the film to obtain information regarding bask aspects of prescncv of inhibitors of transfer factor retivily. tbi* immunological transfer phenomenon, such as which The presence of inhibitor* of transfer factor activity ceil type is the source of transfer factor, which cell type had gane unrecognized previously, and we felt that a responds i«» transfer factor, and lb? molecular species iignifkani purification step would be accomplished involved in transferring ability to react lo antigen by 9 btastogenests and/or ototoxicity. Current research ular-weigjn dniysate. catted transfer factor, from rup efforts have been directed at developing new methods tured. WBC from sensitized individuals. A practi lot preparation of molecules aMt to transfer innnunu- cal apphcaium of transfer factor has been found lupcal specificity, separation of these active nxdeculcs dinkslly in the treatment of various human diseases from endogenous mhmihir. and methods of assaying where a patient's cellular immune competence can be these activities which unainbiguously measure only one greatly improved by admmrst ration of appropriate aspect uf cellular immunity lie. ototoxicity. Mastu- transfer factor. Progress in the purification and study of gewesis.etc.i. the chemical nature of transfer factor has been s!ow due A methud for preparing tuberculin transfer tout to the lack of an adequate animal model and to the lack from guinea pip nTMnumzcd with lure BCG is being of a rapid, reproducible- m rirr» assay. developed which involves extraction of leukocytes from We are attempting to use beagles for this purpose ^nd Mood and spleens, homogenizaiion. and Sephadex G-25 would like to compare preparations from the same chmmatography. rr« •USms rVMUoctoraf tctfaw. DEVELOPMENT Of A CENfRAUZED I. <;. IV (icrffm. II. <;. Scthn. Sister M. t. Nevins. and <".. IV Xntefli. inn report. LYMPHOCYTE CYTOTOXICITY ASSAY USING SOLUBLE ANTIGENS COUPLED TO CEU MEMBRANES PROGRESS ON FRACTIONATION OF TRANSFER FACTOR FROM KACLES* G. D. Griffin* Helen G. Settm. Sister Mary Paul terms.* and G. David Noveii Helen G. Sellin. G. D. Griffin.* an J G. David Novdli The successful isolation nf the active components in Demonstration of in vm> transfer of cellular immune transfer factor a task in which we are currently mvohned. competence in humam can be shown hy inteclkm of requires a reliable method of measuring some aspect white bh>od cells (WBO or by injection of a Inw-molec- of transfer factor activity following various fraction* ••• *r<-. t ~.-~ -.<««r^VH^ 10 lion prucedwc*. aid dm MMWMKM mt>i be o( MWK aspect of actrrity which B Tinrminrocany mmortani m •rSfHS rv~a.o.i. *- .,- —a.jj-^*^nk--ivnajfl 12 mat Se due to a control mechanvjm preventing the loss promoter specificity, we have simhcd RNA poHmetasc or" needed mtermedMe* lur energy-generating metabo from K. am mfccled with a T4 phage gene 42 X 47 lism. Although INK has nut Bent tested, it » puraihfc mutant. exawmnug the activity of she susna subuuit of that. luW£/nvtnm% gram m die abseiice of folate.* the the enzyme. The mfcvied polymerase unuab about tRX***** of tneduwgemc bacteria B »<( lormylated 0.2 equivalent of the sterna twhunii. *wnficai»» fe* 4.wl V... poorly Mmwbior> toward core en/yme fractions ex IXt 72. 5»i 1*741 cept in the presence of Triton. With T4 ONA as a 4. H. H. VMU ml H. K«KKW. ««S f mm 5Ju i5* tOMpraie. these sigma fractiims nuubii the activity of • l»7<| core eiuyme phas host snjsna fractnats panievfarh in 5. H. R VsvU. «. SdMnft. mi H. K 1. I. R. Shurjrt. Mfl Pn. lima #** R Lee R. Shugart and Barbara II. fhastain I. I. .%. ftmmr.J *M. IVw JJ7. •'" sT4t«i•*:» :. I. R. yiMJU j** R. H. dknun. AW />.» Imm *»»c Became of the difficulties encountered during purifi Rep Jumt fit. I*~4. ORNl-IW. p If. cation, several different methods for the separation *t proteins from nucleic acids are being investigated in an effort to obtain a satisfactory procedure for the TYROSYHRNA UCASE OF JXftfJSMfflM.4 ITS tsobiron of the various iRNA methy transferases from 1 w ABUJTY TO MSCRIMRMATE iRNA FROM £ oil' Two techniques are currently bemg tried l*f A SUFPRESSOR MUTANT AND WHO TYPE precipitation of nucleic acids from microbial extracts with lysozyme. which involves removing nudek acids K. Bruce Jaci*son and J. B. Murphy and acidk pn>teu»s after complex formation with a haruer studies showed that the suppressor mutant, basic protein, and (el affinity chromatography, in nit-l". had only the first of two isujccepturs for which S-adenosylhomocysieine. an inhibit" of trans tRNA1 *' tret. I >. However. subse«|ueni stwU^s revealed methylation, is attached to agarose geb through a vHsf did indeed possess the wmd isoacceptor. hydrocarbon extension that terminates with an active tRNA,V/'/ n . but demonstrated that it was different carboxy .V-hydroxysuccMmide ester. Il is anticipated from that obtained from wdd-fype ffces. |RNA^*' „ that the tRNA metbyltransferases will form a protein- M (ret'. 2». The latter study showed further that the bgand complex with this material. Initial experiments variation in results between the two studies was m the indicate an increase in purity of the tRNA methyltrans method of preparaU-n of the tyrosyl-(RNA Ugase. ferases after treatment by either technique. Effons to define conditions that would allow repro In addition, the levels of these enzymes are being ducible production of the discriminating form of this studied and compared in an rrl' mutant of ¥.. cub enzyme were unsuccesfu.'.' Dtscnmnution n defined grown under different nutritional culture conditions. In as the oubdity to charge tRNA r' but ability to charge methionine starved cells fas compared to cells cultured R tRNA,,7'. We are continuing to study this problem in a complete growth medium I there is a twofold in through several approaches. crease in (he iRNAfadenosme-6 and •fguannsine- Knzyme preparations made m the post year have I hnethyltransferasc activities, hut a fourfold decrease produced discrimination in the majority of cases. in the tRNA(guanosine-7(methyltfansferate activity. whereas in the previous two years the majority was nondiscriminating. Storage of the discriminating en I. I.. R. Sfcvtan and ». H. Chwtain. §M. On Anm Pro* zyme at 20°C for I 3 weeks results in loss of Rrp. June JO. 1974. ORNL-499J. p. 16. discrimination, although activity remains: at I96°C the !> dtsctmnuitm isletamed lor t> ~ »e.-kv but. acan. thv |'*( (ruannte is ncufpiralew !•!•• : g»-W< jl S»,itm,tv ALTERED RaXMSOF PWEKVLU^VKCa tfttKA Pi A VE AST ULTANT SflMCS€N 1ME •OXMDNSnnr OF ItK k. Brave Jjcdro*. J B. Bel." 1 ee R Shweari. rVWIC HUT.4NT BM OKCIHVMLl ^ad J. K LewMHt Tnc yeaa .V* The aactnentr. i.mad at the 3-end p«ap -Me a» 1.4 *4CALACT0SMMS W bar* at aaa.«*»ict pet V.. peak A i !•* Ado O 41 MS HISC-III5 C>J. o io AUTOMATED SEQUENTIAL DEGRADATION •tWAvfctsI amrrivjaivf. (.'.irvWMxarw Icaaaat (ioat OF RNA May.. Ivirl and A. J. Weinberger* SYNTHESIS AND DEGRADATION OF SFECIFtC tRNAs IN MAMMALIAN CELLS The automated apparaius for sequential removal of auctevade residues from RVA is the fust operating Michael Ut* nsirument of its kind.1 lis major potential uses are la eufcary otic ceHs the lever* of UMH IRNAS reflect twofold: the preparation of truncated RVA molecules the ammo acid curnpustno* at the proteins King for further research m the relationship of RNA struc synihesued We are innc to answer rite questions ture to its lunclKM. ami the sequencing of tdigodwdeo- abuut the rjfceiMneuou: When crib differentiate, how twJe* larger than 10 to 15 residues. are specific iRNA levels adjusted? I* the adjust We have tested the KStrument wi;h four different mane by varying the rale of synthesis or the race of tRNA preparxtiuMs and several different preparations of degradation of specific iRKAs* What characteristics of potytAi I length ~JW or IUU0 residues per cbaml As the miracraubr eKvuonment utHuence rates of degrada- unhealed in the previnus report.' the removal of rw* and synthesis of specific iRYAs? Specifically, does uvcievissues ceases when about one-lourtb of the mole the extent of anwuuacylatiou of a tRNA species play a cule B removed. The is pronatriy due ti> the pecuhar role? Is the rale of synthesis or degradation of a iRNA structure of iRNA in this legmn of rfae molecule. m>t to specK* influenced by the mtnceRutar concentration of the chemistry of rmdroside removal. The three- that species'' dimensional structuie «J tRNA idrtermmeJ by A. Rich at MIT) is a specai sandwich-like arrangement with Using Fnend-virus-mfecfed mouse leukemia cells IFL hydrogen bonding between nonadjaccnt regows of the ceftsl as a model system. I have examHetl the degree of iRNA molecule: such a structure could stahiU/e the heterogeneity of turnover rates of 4S RNAs res«4ved by regnw !•• prevent further degradation. Pt4ylA)does not RPT-S chromatography. In exponeiKulry growing FL have tins structure, and we have gone through mine oris, the most laMe 45 RNAs turn over at least 1.75 than *0 cycles wrvhout .ibserving cessation of degrada times more rapidly than the most stable ones. tion such as is found in tRNA. In addition, the To measure rales uf synthesis and degradation of sequences in the tour tRNAs are quite different in thrs specific tRNAs. I have been attempting to purify region. The only common features f indicated below) tRNAs by a one-step mkromethod published by Ktyde arc the presence of pseudoundine and cy tidine. and Bernfieki.' The method involves specilk" amsno- acylaiion of a iRNA mixture with a single amino acid, followed by complex formairon with FIFTu-GTF and F.. n* iRvV,"^" T . ft; A A •eparation iif (he ternary complex of aatRNA-F.FTu- •fceal <"*rm inNA** '. . t" V. m.\ I GTf from uncharged iRNA «m I'K^ acrylarnide geK. A rwfc tnX\™" T . ( A \ A The ammo&cylaiion of tRNA by an amino acid other t m* rHNx'*' ?.r«;rr than the one added has been observed repeatedly and causes the production of complexes with unwanted iRNA. Apparently, proteases in the synthetase prepara *Analy:icai<"hrinHir> l>i»»«'»n tions generate unwanted amino acids. Sephadexing the I. M l/«rt. A. ). WeNikrreci. jnd A. ). namty. Bin* Orr synthetase preparations immediately before use re 4mm. th*. Rep. JmtrJa. I*U. OR\f-494? pr> M •!. duces, but does not eliminate, this problem. I am presently trying to overcome this difficulty by puri fying an aa-fRNA synthetase cimpiex from rciculo- ANALYSIS OF MODIFIED *A5U> IN IRNA FROM cyies. such as is described by Som and llardesty } EUKARYOTE' AND PROKARYOTE SOURCES Mayo U/iel. L. H. Smith, and A. Jeannine Bandy •On aMolKil leave from ibr Itmrbrmhlry DvwinmcM. I'nirmiiy of Oregon Medical Sthnnl. Portland 97IOI. The modified bases in RNA can be grouped into two I. n J. Klydc and M R. BernoVW. IHnrkrmatrv 12. .1752 categories: those involved in the recognition of messen M97J». ger codons and those involved in iRNA structure. This 19 oniA the pu*uMrt\ ft iiansLiiionai c »:c«4 ot proiem the rate ami senaiiiniy of the assay (K«s 2\. B ami synthesis ihixujfi the degree »l m«diricili»m I II llevker. M I-K-I and ». I. Karnrii. lb* r<-p.ti i. M I/-trl jml \. I Rjnd>. Ihr. t«T». HIGH-RESOLUTION HIGH-STEED SEPARATION OF 35 MODIFIED NUCLEOSIDES FOUND IN RNA Mayo U/wl and A. Jcanmne Randy fig. 1. I A) A pomp of standard iwclroiinu ha*r brcn Oiir earlier sy ems for separating ihc modilicd i-Nmmatnpaphrd with a snKrni rlanfr near MO mm. Ihc iirsr v»hi-nt is tr.im rrl I. jnil ihc Vk'>nd «><|seni i» 11 I .'/ tti-ulr. miclcoMtlcs' '" have Hcen lonsidcrahly improved, and 0 X M NIU r..rnut,- .pll 7 ID ,„,I (B) the aptfd of the anah-SB charge neutralization by pll -.-lunges and borate aihli- has brrn merrasrd bj> UST of a 9-v rrsin. rhr croup <>r tion' has been introduced, for the first time, to increase nmlciwdrt wpjrj'fd by vlvcnl I only are sh<«sn 20 at all pH values from 7 In 10.5. and that Ihe clTevtive 1. M. I'zirf. C\ K«*. ami ». K total. Anal. too. firm 25. 77 capacity Iequilibrium bindine. »f adenosine) is consid II96SI. 2. M. tziel and C. Koh. J (7v Time depending upon the pH and solvent. Subsume Subsunce Imin) (min) 25 V 175 m2(; •Oak Rider Associated Universities I acuity Research ftrlici- pant. Centenary CoHeec itl' Louisiana. Shreveport 71104. 29 4(SO,H 176 Cm 36 t' 259 rofcA I. ISOLATION AND PROPERTIES 50 (4ahu)Jl 272 C 56 V 2*3 AM S. K. Niyogi. Brenda H Underwood, t and A. K. Dalta* buffer chance 61 T 3IK Cm A new ribonuclease has been isolated from Exhe- 7t Vm JIB mY rivliia wli. The enzyme is present in Ihe 100.000 jr 75 X 346 nam' ** U supernatant fraction and has been purified over 200- HI *h 350 m2A t'old. Studies of tlie enzyme reveal that: 90 ln» 356 ml A ( (#) The enzyme prefers a free 3'-hydroxyl group for and weakly bound polymerase molecules we»e removed its action. by adding an excess of polyi. I). which has a high affinity (A) The enzyme attacks the oligoribonucleoiide sub for the enzyme. After digestion of the unbound DNA strate in a sequential manner from the 3'-end producing regions with pancreatic DNase and snake venom phos 5'-ribonucleoiides. phodiesterase, the protected DKA-RNA polymerase (c) The mode of attack appears to be processive: the complexes were isolated by Sephadex S-200 column enzyme acts by degrading one oligoriKmucleotide chain chromatography. The protected DNA sites were then to completion before proceeding to the hydrolysis of isolated by phenol extraction and alcohol precipitation. another chain. Studies of the DNA recognition regions reveal: (a) Very (J) The reaction rale is inversely proportional to the little binding is observed in the absence of the sigma chain length of the substrate: however, the enzyme has subunit the protein factor in £. Coli RNA polymerase a higher affinity for longer chains. that dictates specific binding and accunte initiation of (?) The enzyme activity is markedly inhibited by RNA synthesis. \b) The amount of protection varies secondary structure: oligoribonucleotides combined from 1.0 to 1.83 of single stranded MI3 DNA. In the with complementary polyribonucleotides are attacked absence of polyt I), substantially higher protections are poorly below the melting temperature of the complex observed. lc> The protected regions display two size and efficiently above the melting temperature. classes as determined by polyacrylamide gel electro {ft The enzyme is inhibited by 5'-nucleotides of phoresis (about 50 and 100 nucleotides in length*, id) adenine and guanine: those of cy loswe and uracil have a The two peaks have somewhat different base composi much smaller efTeci. The enzyme a not inhibited by tions, it) Hybridization experiments are in progress to 3'-nucleolides. determine whether these protected fragments are (r) Studies with dinudeoside monophosphates show "unique" and whether they arise from the same region highest reaction rales with pyrimidine sequences in the oi the M13 genome. order: CJJC > UpU > CpU > Upf. The presence of guanine al the 3'-end is strongly inhibitory, reaction rales being CpG > UpG 3 ApC > GpG. THE ROLE OF ESCHERICHIA COU 4mA FUNCTION AND ITS INTEGRATIVE SUPPRESSION IN MI3COUPHAGE •FUMAHIWOI invcMiplor sapportcd by subcontract 3322 DNA SYNTHESIS from Ibr Kftttocy Dmsioii. ORNL. lo the Unnwrwly of Trminscr. Kimsvinc. Sankar Mitra and D. R. Stallions I. S. k. Niy.ip. B. II. Underwood, and A. K. Dana, thb nrpnrl. F* derivatives of F. coli E508 ihermosensitive in dnaA locus (involved in DNA synthesis initiation function), its revertant. and an Hfr derivative of PROMOTER REGIONS* OF E508(ts) in which the temperature-sensitive phenoiypc SINGLE STRANDED M13 DNA b suppressed by integrative suppression have been compared for their ability lo support MI3 phage DNA S. K Niyogi and Sankar Mitra synthesis al the nonpermissive temperature. Upon Certain specific regions in the genome play a crucial infection at nonpermissive temperature (42°C). both role in biological regulatory mechanisms One such the temtant and the Hfr strain support normal phage region, called the "promoter." is involved in the specific replication, while the temperature-sensitive mutant does binding of RNA polymerase and subsequent accurate not. However, when infection is carried out at per initiation of RNA synthesis. Previous work (Arthur missive temperature and the temperature is shifted up Kornberg. Stanford University! suggests that there may late after infection, phage synthesis occurs in the be one unique site (promoter) on single-stranded Ml3 temperature-sensitive mutant also, although in lesser DNA thai is involved in the synthesis of the RNA quantity than in the reveriant strain. Ultracentrifugal primer needed for initial km of DNA replication. We are analysis of intracellular labeled phage DNA indicates (a) attempting lo isolate and characterize the promoter parental replicalive form (RF) synthesis is not de regionfs) with respect lo size, secondary siruciure. base pendent on dnaA function. (A) progeny RF synlheris is composition and sequence, and location in the Ml3 strongly inhibited in the temperature-sensitive dmA genome. RNA polymerase of E. coli was allowed to mutant but not in the other two. (r) progeny single- bind ti> labeled single-stranded M13 DNA. The unbound stranded (SS) DNA synthesis does not absolutely 22 require utanvl (unction, and <«/) progeny SS D the L'nr.rmir of Tcnnraec. Kaovriat. I S. MrtnindD. R Sulfcuit*. iinafcr.r52.4l7 424(19731. THE ROLE OF ESCHERICHIA COU dmC FUNCTION IN Mil COUPHAGE DNA SYNTHESIS Santanu Dasgupta* and Sankar Mitra CHARACTERIZATION OF MCTERIOPHACX-T54NDUCED Escherichia oli JnaG function is known to be DNA POLYMERASE WITH DNA involved in the initiation of formation of Okazaki DNA CONTAINING SINCLF STRAND BREAKS fragments during DNA synthesis. Romberg and rrs R. K. Fujimura. Barbara C. Roop. and D. P. Aluson collaborators have shown thai Jnmii protein is a new UNA polymerase that synthesizes the primer RNA Earlier studies by Steuart et at.' have shown that. like during replication of single-stranded (SS) DNA of other bacteriophage induced DNA polymerases. TS coliphages OXI74 and G4 to the corresponding replica- DNA polymerase prefers the templates that are single- live iorms (RF). It was also shown by workers in the stranded with either 3'-OH ends looped back to form laboratories of Romberg and Hurv.il* that in viim primer* or oligonucleotides annealed to the denatured systems for replication of M13 phage SS DNA to RF do DNA to serve as primers It was shown clearly with T4 not require Jnaii protein. polymerase that the polymerase by itself is not capable Miira and Stallions1 have shown, using a tempera of synthesis from a single-strand break in a duplex ture-sensitive ikiaii mutant of E. <"//. that JmKi molecule accompanied by strand displacement.2 function is required for M13 phage synthesis. In this Our studies with temperature-sensitive DNA polym report we summarize our results on the role of Juaii erase induced by bacteriophage T5 revealed that DNA function in various stages of M13 DNA synthesis: synthesis with nicked DNA as a template is more (a) In confirmation of the in vitn> experiments. M13 temperature stable than the synthesis with denatured parental RF synthesis does not require Jna(> function. DNA.* This observation induced us to investigate more |r» MI3 progeny RF synthesis is strongly inhibited in rigorously the nature xtf DNA synthesis from single- the dnaG mutant at the nonpermissive temperature as strand breaks using wild-type T5 DNA polymerase. compared with the synthesis at the permissive tempera Bacteriophage T5-induced DNA polymerase was char ture or when compared with the synthesis in the acterized using mainly circular duplex DNA of bacte temperature-resistant wild-type E. utli. Similar results riophage PM2 with single-strand breaks formed by were obtained both with wild-lype phage, where prog DNase I action. The purified polymerase preparation eny SS DNA synthesis occurs, and with MI3 gene 5 had synthetic activities with both denatured DNA and mutant, where SS DNA synthesis is absent. nicked DNA- In polyacrylamide gel electrophoresis of (c) MI3 progeny SS DNA synthesis does not require native protein, the polymerase activities with denatured tbuG function. The reduced synthesis of Mi3 DNA at DNA and nicked DNA superimposed on each other. In 42°C in the mutant host is presumably due to inherent SDS gel electrophoresis there was only one band. Both temperature sensitivity of the MI3 SS DNA synthesis polymerase activities were shown to be phage-induced. itself. The intracellular SS DNA synthesized at the Thus both polymerase activities are very likely due to nonpermissive temperature is circular, and the pulse one phage-induced protein. label can be chased into mature extracellular phage at DNA synthesis with nicked DNA as a primer template the nonpermissiv; temperature. increased with increase in number of single-strand 23 break*. Mu*t .it the characterization was earned uut The exonncteasc actrvny associated with the porym- with fHl DNA with one ur lea than one single-strand cases has oeen implioted in the editing fraction by break pfr si rand Essentially ail such breaks were various investigators. Therefore it was of interest to see repairable by '~pat Alkaline sucrose gr. iienl centn- what happens to the template and the newty synthe fugation showed that synthesis IKVUO with the strand sized strand under synthetic conditions We followed with a single-strand break as a primer yielding DNA the degradation of the newty synthesized strand by longer than une phage DNA unit long. Newty synthe following template-dependent conversion of dNTP to sized DNA t» cuvakntly linked to the proper strand. dNMP. Under synthetic conditions, Lc in the presence Thus the synthesis is occurring very likely by strand of -WNTP. the template was totally protected when displacement. This is supported by electron micro nicked double-stranded DNA was >jscd as the template graphs. However, the n-wty syn'hesized strand was degraded, as indicated by dNMP production. These results indicate the TS-polymerase-associatcd exonuclease might be 1. ('. O. Swiurr fitL. J. BkJ. Ota* 243.5319 • I9M>. crucial for a faithful replication of TS DNA. 2. Y. Mramutw aiid C. C. R.Kltudw3. J. Bwl Cham. 244. 2WI11*7It. J. R. K. I uruaou and B. R. Biractl. Biol. Orr. Anrnt. P*>%. Krp. Ju.tr 30. 1974. OUNL-IW. p. 24. •Student at the UT Ouk Ra%c Gmfralc Scfcool of Bio- madkal Scimcrs. EXONUCLEASE ASSOCIATED WITH BACTER10PHAGET5-INDUCED A DNA-MNDQ4G PROTEIN FROM DNA POLYMERASE BACTERIOPHAGE TSJNFECTED S. K. Das* and R. K Fujimura ESCHEtJCHlA COU Upon infec:iur. uf its host Escherichia cvti. bacte S. K. Das* and R. K Fujimura riophage T5 induces synthesis of i DNA polymerase. Even in the caie of simple bacteriophages, current The DNA polymerase is presumably involved in T5 evidence indicates that the replication might be a DNA replication. Studies with is mutants which map in complex process revolving many components. In the the structural gene of this protein, indicate that this T4-infected E. coli system, gene 32 protein has been en/yme might be an essential component of phage shown io stimulate the rale of DNA synthesis in rim>. replication machinery. This enzyme has been purified A DNA-binding protein, which is presumably coded by to homogeneity, in our laboratory, as indicated by the T5. has been detected and purified from T5-infected A. presence of a single band in native and SDS gel cn/i cells. DNA-cellulose affinity column and DEAE- electrophoresis. Sephadex columns were used in the purification pro Other groups have shown thai bacterial- and phage- cedure, lis effect on TS DNA polymerase has been •nduced polymerases also t.jve exonuclease activity. We studied. Our preliminary- observations indicate that, have found an exonucieas* activity in the purified DNA unlike the gene 32 ptotein. this DNA-binding protevn polymerase preparations. The activity was shown to be inhibits DNA polymerase even at low concentrations. associated with the same polypeptide which has the The inhibition was seen wMh nicked DNA as template. polymerase activity. The en/yme degrades both single- The inhflritory capacity of the protein was found to be and double-stranded DNA. Single-stranded DNA is very strong in that only microgram quantities were degraded at a faster rate. The enzyme attacks the DNA at needed to produce $0% inhibition under our assay the 3-Oil end and produces S'-deoxynudeotidev The conditions. rale of degradation was found lo depend on the number This protein may hare some role in recombination of available J'-Oil ends when excess enzyme was used. and probably protects the 3'-OH at the nicks and gaps The pH optima of the en/yme coincide with the pH on DNA template. Currently we are investigating a optinu of the polymerase activity. The enzyme showed number of DNA synthesis mutants to learn more about an essential requirement of Mg]* for its activity. the protein. jV-Ethylmaleimide inactivated the enzyme, which indi cated the presence of an SH group crucial for its activity. For optimal activity during all purification •Student at the UT Oak fMcc Graduate School of Bio- steps the presence of DTT was found to be desirable. imdpcal Sciences. 24 MnuasmoMC MAifmc OF the tRNA h—ting sue) wtlhtn the three enzymes Both RACTEUOnUCETSDNA approaches require that a substantial quantity of all POLYMERASE MUTANTS three enzymes be purified to homogeneity. We love recently apphed three techniques which are W M Antrey* and R K. FwMnmra new to tins laboratory toward the purification of these Time amber mutants of structural pent for T5 DNA three enzymes: preparative acrylamide electrophoresis, putymerase. desnpated dm/. «n». and *nJ*». were acrytamade-agarose gel filtration, and affinity vhmena obtained from Y. Lamu Two factor owats were tography. The tlrsl two have been incorporated into our carried out between these and one additional mutant, scheme for purification of the t'lgkm chlon'olasiic designated «MD/S t obtained from KcCorqnodaler. en/yme t'smg these methods n conjunction wi«h known to be adjacent to the polymerase grne Nrt hydroxy apatite and DtfAF chromatography, small-scale outside ot" the polymerase ctstron. To our surprise, the preparations mdicale that homogeneous en/yme will he map was avaname soon. We are presently purifying a large preparation of this en/yme. Since the rViigft-rw cyto ,imA tan/ *niDI5 PURIFICATION AND NUCLEOTIDE •OHM mi An analytical anum-exchange procedure ha* been dcithiped for the ranW «eparati«ii of acid-soluble nucleotides (the so-caded "free" or tissue nucleotide*) CHAIUCTEIUZAT10N OF It permits assay at low pressures (40 oO psii in less OUtOMOSOMAL PROTEINS than I hr on 10-vm cohnrms of Anunex resins 'con C. G. Mead and E. A. Hiss ventional iiyrene-iype amor exchanger*) with alkaline citrate solutions as the eiuenl. Separation parameters Chromosomal pr.tteins have been isolated and puri have been investigated to determine optimum con- fied from Chinese hamster ovarian tissue culture cells dwinn* for the routine analysis of samples cnnlaininf blocked at rnefaprusr with cokimid. The isolated Issue nucleotide*. A simple solvent extraction pro protein* were found to be in such small quantities thai cedure has also been developed to remove rldO* or it was necessary to label them with l25l in order to CCIjCOj H quantitatively from cell extracts confainin|[ demonstrate different classes of separable proteins. acid-soluble nucleotides. This procedure involves Ihe Gradient sirvnrpfive chromatography has been success removal of aqueous acid sortitions with a water-in- fully used to separate the chromosomal proteins from snhiMe amine dissotred in a waler-inirnisciMc solveni. each other and from the chromosomal nucleic acids. 2S Pn*ens fro* uaJthrlrd dHumosumal preparation contains two subumu. and does not appear to contam a have b en separated and assayed for DNA-exmmcteue prosthetic group- In addition, our studies haw shown and DrfcA-cadonikieast activity m the presence of that the ribonucleotide reductase that is active in the either big1* or Caz*. No nodes* activity n ihe«e Emgkim NADfH-thiorcdoxm reductase system H more separated protest fractions has yet been detected Other complex than the protein reported m a previous DNA-mvurved enzymatic activities are bene exanuned. pubfccatiun.' The mure complex eusyinr contains four Some effort has hem spent on increasing the amount of different types of polypeptide chains. metaphase eels nsed as starting material and the yield of metaphase chromosomes from these cefc. biw 'CmmegK C**rmnom Fanny Rnanii fjiiwjnfi buai father nwiiivemfnts are needed. ihr CnrnBiln DeaomaaM. Unmpmmc CoBcnr. SJobary. 9bnliCarann2tl44. I. V D. Ilimniia./ mot Chrmt. M9.442S 44j4il*74>. CMSMEACTMIN OF TWOREDOUN REDUCTASE AND RtftWUCLEOTIDE ISOLATION OF RluttNUCXEOTlDE MMBUCTASBmaCWOUCmACOUJOtD REDUCTASE M KEVDOMONAS STITZEU EUGUNA GMACIUS B. D. Mehrotra* and F. D. Hamiton R C Holt* and F D Hamilton /* uuizeri contains a rihonucleosidr triphosphate TtnoredoxM rednctase and thioredoxin. proteins that reductase which requires added dilhwl and adenosylco- couple NADPH oxidation to ribonucleotide reduction balamm for activity. The enzyme has been purified by ribonucleotide, have been identified in a number of 40-fohJ by acid pH preciprtaiiofl. ammonium sulfate prokaryolk organisms- The thioredoxin reductase fractionalion. and Bruget chromatography. CTP re described for fnafcrnt1 is the first such system reported duction is activated only by dATP. CTP is not reduced for a eufcaryotic ceil. To determine if the prokaryolk. to a stgnificaui extent if other dNTPs are used. No and eukaryotic systems would function together, the activity has been obtained with ATP. GTP. or IJTP as t'ygkm thioredoxm reductase system was tested with substrates. We have not determined if the lack of £ cnK ribonucleotide reductase. Tests were aho made activity with these triphosphates results from the lack of the reaction of the lu&m ribonucleotide reductase of an essential ingredient for the assay of the en/yme or with partially purified £. cnU thioredoxin and thiore- the need of another enzyme for these compounds. doxin reductase. In both of the heterologous reactions we observed 45 73T of the activity obtained with the homologous system. 'Carnegie Corporation Vacuity Research Participant from the ChcMisiry DrpanineM.TouralooColkltc. T«»ga*m>. Mioinippi 37*14. *Sra*nrr at the 11 Oak Riajr Cradmir School of •*>- I. 5 NnraK. D. V. Farter, and V. D. Hamiitoa. Mmiifica- STUDIES ON THE FUNCTION OF IIM of the NADfH-lkiorcrJoxin redacts* sjrstCM in Kmgkim nwraw. Ivor Nfl. Atmd. Set USA. smmrttcd. ASCORtATE SULFATE F. J. Frnamore and Rose P. Feldman THE RIlOfWCLEOTIDE REDUCTASE The natural occurrence of a sulfated derivative of SYSTEM IN EVCLEHA GRACILIS vitamin C was first demonstrated by this laboratory in S. MunavaJli.* Dorothea V. Parker, and F. D. Hamilton extracts of Artemm smHm embryos; more recently it has been identified as a metabolite in rats, guinea pigs. fish, Studies have shown that Eugkm contain a protein rabbits, and man. At present we are actively engaged in system which can utilize the reducing power of NADPH studies of the function of this compound and its in the ribonudeolide-reduclase-caialyzed reduciion of relation to vitamin C. CTP. The proteins required for this reaction are To determine whether ascorbate sulfate is a storage thioredoxin reductase, a flavoprolein of approximately form of vitamin C. we have embarked on a search for 185.000 mol. wt. and a second protein (Protein I) whose the existence of an ascorbate sulfohydrolase that has function in the reaction sequence is unknown. This new the ability to convert the sulfated derivative to the protein has a molecular weight of 240.000 daltons. active vitamin. We have succeeded in isolating and 29 parttaty purifying a* enzyme with thrs property from 2. A. J. VobapcrixJkR.O. Mwi.,Jl*oui»fci»i• 17.37 extracts of digestive (buds of /Mix pummtm. The ll»7J» purification procedure involves gel filtration. DEAE- ). V. F. Zaiistv a*4 L A. MyasafeM.Car Vamfhrnmrn^ fc. 19 t!9*4». ccamuse column Lhrumatography. and isucieclric focus 4. B. SaMorr. M. Han. C. C. SacaW. T. Una**, aai T. ing. The enzyme extubrts arytsulfaiase activity when baai./ Ami Hrrmtr Sue. 14. I2» When ttsh are «weicrowded. a* a survival mecrumsm Oxidized lung od ha* been used by several worker* in they secrete lipids which depress their growth, repro the past to treat patients with renal hypertension, duction, jnd heart rate and increase their •aortauty.' because of its sanaanty to a natural lipid antmyperten- We haw wed a heart rate depress* assay to isolate the «on agent i ANRLl secreted by the manaaahan kidney. apid respoasMe for crowding M goktfrsh. and found it The active animyprrtension fractitm isobted by us to ham the chromatographic behavior of a digjyceride from rat kidney aad assayed on a reaaHy hypertense rat and the spectrum of a conjugated irieaoic fatty acid.1 showed activity separating on the trailing edge of the It exerted its effect through the central nervous triglyceride peak from a silica gel column. Again, this system1 and was found MI brain and spaial cord i issue fraction had the UV spectrum of a conjugated trienoic as well as m the fish water fatly acid and also gave a positive thioharbituric acid We have also Mutated an analogue to the heart rate lest depressor froM commercial lung seed oil. Tung seed oil contains one of the few Rataral conjugated trieaoic ois. It appears that lipids containing these unusual conju Tm* aaalogne *s ptty^otorxaRy active and is chromat gated fatty acids are prevent in mammalian tissues and ographicaly smvibr to aad has a similar spectrum to the may be physiofegicaRy active m mampiab. A novel goldfish factor. This active aaalogat is a minor constit Kpoxidase has recently been found m human blood uent of the lung oil preparation, probably an oxidation platelets capable of forming cuapgatatf fatty acids from product, and appears to rearrange on prolonged cuniaci inethylene-inierrupted unsaturated fatly acids through with protonating solvents such as ethanol. One of the an eudoperoxide compound.' rearranged products is a potent growth mbJbitor to Xennpm Item tadpoles, which led us to investigate crowding in Xenvpta. We have found thai Xenopus tadpoles abo secrete a 1. A A tn«B. K Smmh. amt f>. rYanerci. Flag, fnfc Cmlt. npid when crowded, which inhibit> iheir growth. This Ji.l% SECTION II BIOPHYSICS AND CELL PHYSIOLOGY Peter Ma/ur Cefl TwysUhtgf X*ay Peter Mazur J. R. Einstein J. F. Brandts* C.H Wei S.P.Lcibo Nicholas Rifopouios Krushi Souzu* L.E.Towiir F. C. Harlman ChnMriiy J.P.Brown' I. LiKile Norton D. t CMins R DoueJas Carlson' Gwendolyn B. H«>wzfr Excilcd-State BwfJryjks Ada LOIins* M. L. Randolph Marilyn B. Senior' J.W.Lofigworth Growth and RcfranatiMi Linda L. Munchausen' R. O. Rahn D. M. Skinner S. S. Stevens Kiah Edwards III*" P.F.Sanders' r L. H. Yamaoka riSoU-State Biology and Cell Division R. M. Pearistein W. D. Fisher W. A. Arnold* H.I.Adlcr J. R. Azzi D. P. Allison S. F. Carson D. R. Stallions R. P. Hemenpr* Francis Van Nostrand' Radiation Biophysics J.S.Cook Plant Sciences W. L. Carrier J.E.Donneiian.Jr.rf 0. E. Foard J. G. Jiwhi* Benjamin Burr W. E. Masker Frances A. Burr* P. A. Swenson G. P. Howland G. L. Vaughan' 0. J. Schwarz' PC.Wiir L. L. Triplet! "Comultjni. Vi*ilinjt invctlfcalor from abroad. *^»'dociorjl feivcstiiciior. "LtawofatwfiU-e. rVmlin|c invc«fipif»r. 31 32 damage without permeating then: prs* :» freezing. OK the other hand, there is evidence IHHII rabbti embryos that survivals are superior it" protective additives ate allowed to permeate.1 However, when permeation occurs, it can cause osmotic problems during thawing and subsequent dilution. Furthermore, injury during freezing appears in some instances to be related to the MICROSCOPICOBSERVATION or volume of cells prior to freezing.* INTRACELLULAR KE FORMATION IN An understanding of the contributions of these MOUSE OVA AS A FUNCTION OF COOLING RATE various factors can be greatly facilitated by knowing the S. P. Leibo.* J. J. "WcGrathJ and b C. CravauW permeability coefficients of the solute in question and A physicai PHYSIOLOGY AND LOW-TEMPERATURE BIOLOGY EFFECTS OF SUBZERO TEMPERATURES ON OF BOVINE EMBRYOS EGGS AND EMBRYOS OF ARBAC1A MJNCTVLATA Peier Mazur. S. P. Leibo. B. II. I tick son.* Suzanne C. Jackowski* and R. A. Wallace and J. I. Daniel. Jr.T Tliere is increasing evidence thai a physical-chemical To be agriculturally significant, the procedure of model1 of the response of a ceil lo freezing is both superovulation and transfer of bovine embryos requires qualiraiKely and quantitatively correct. For example, if the development of five techniques: Ui) endocrino has been shown that several cell types freeze intracel- logical techniques for superovulaiion. in) techniques for lularly at those cooling rates predicted by ihe model.1"* the recovery of embryos. (<( in rr'rr-> techniques for Since the original model included an analysis of the determining Ihe viability of treated embryos. i«/> response of sea urchin eggs lo freezing, we have begun freezing procedures that will yield a high proportion of an experimental test of the model with these cells. Eggs embryos that arc viable on the basis of the in vitro and early embryos of Arbaua punctulaia were exposed assay, and (<•> surgical techniques for reiniplanling to subzero temperatures and assayed for normal devel embryos into foster mothers. The weak link in the opment by examination under the light microscope. sequence is the inability so far lo perform Mcp li/i. the Samples were suspended in sea water and cooled lo freezing. -5.8'C. then seeded with ice. Ten minutes later We have developed competence in steps a. b. ind i samples were cooled lo temperatures ranging from As an example. Ihe n>rmal cow ovulates a single egg per 10.5 to 27.0T at a rile of 0.7°C<;min and were reproductive cycle and produces about six calves in her immediately warmed ai a rate of 2 iT'min. Survival lifetime. We have succeeded in developing reliable was assayed by fertilizing the eggs and culturing them as procedures for the superoviilalion and collection of well as Ihe treated embryos in seawater at 22°C. The embryos, obtaining an average of IS. and as many as fertilized eggs were scored for viability ai ihe four-cell 40. fertilized ova from one cow at a single cstnnis cycle. stage, and Ihe embryos ai the blastula stage Only 70^ We have also modified Iwo published procedures lo of Ihe unfertilized eggs survived cooling alone to assay Ihc viability of treated bovine embryos by in vitro 5.8 f, whereas °5^ of Ihe fertilized ejgs (5 lo 115 culture and by fluorescence. The culture procedure is min after fertilization) survived thai treatment. After usable for embryos between the two- and eight-cell seeding and cooling lo -10.5T. only 2r'r of the stage, but promotes cleavage of only about 50^ of unfertilized eggs survived. The survival of fertilized eggs 34 and embryos was greater, depending on embryo stage DMSO to salts of 3.61. 6.46, and 9.43 respectively. and stale. That is. survival of fertilized one-cell eggs, The phase-diagram data obtained for these solutions two-cell embryos, and four-ccU embryos was 57, 24. have been found to be virtually identical to previously and 71K. respectively, after cooling to -I5.0*C, published phase-diagram dura obtained for DMSO- whereas survival of embryos during first and second NaCI-HiO solutions of the same weight ratios. Since cleavage division was only 15 and 107 respectively. NaCI comprises -Wl by weight of the total sails Similar patterns of susceptibility were obtained aftn present in HBSS. the data indicate thai the other minor cooling to -I7.0*C. Regardless of embryo stage cr salts have utile or no effect on the solution's phase state, no more than 5% of the embryos survived cooling properties. Preliminary data analysis suggests that the lo -27.0"C. These preliminary studies demonstrate that mechanism of DMSO's cryoproteclive ability cannot be there a a cell-cycle dependency of sensitivity to explained entirely by its colligalivc properties. freezing damage. But they abo suggest that the quanti tative model can be tested using this cell system. •Student at the I'T Oak RidfC Graduate School .>f Bio medical Sciences. •Student at the ».T Oak Ridce Gradual* School or Bio medical Science*. 1. ?. Mamr. J. On. fkystol. 47. 34? ,»3>. SURVIVAL OF SLOWLY FROZEN HUMAN RED 2. M. Usteyama. E. C. Cravafco. K. R. OUkr. and C. E. Hugpat. Ojrotiologr 10.517(1973). CELLS AS A FUNCTION OF WARMING RATE 3. M S. Mandel and K. R. DMer. OroHotot? II. 540 Peter Mazur and R. H. Miller 0»74». 4 J. J. McGralh. S.M.M.E. Thesis. Massachusetts Insiiiuic of We reported last year that when human red cells Tcchnolocy (1974). equilibrated in 2 M glycerol were frozen slowly to - I96°C. survivals were considerably higher when warm ing was slow (0-5 or I'C/min) than when it was 24. PHASE DIAGRAMS OF SOLUTIONS OF 160. or 52S*C/min. This response is analogous lo that 1 2 DIMETHYL SULFOXIDE IN HANKS- observed recently in mouse embryos * and in higher plant tissue culture cells3 and to that observed for BALANCED SALT SOLUTION many years in higher plants. It confirms previous W. F. Rail* and Peter Mazur observations by Meryman on human red cells.4 It may reflect osmotic shock from r./id dilution, but if so, the One theory of freezing damage suggests that slowly basis of the osmotic shock is uncertain. We have since cooled cells are killed by being exposed to increasing determined (a) that the damage from rapid thawing concentrations of electrolytes as the suspending me occurs chiefly between -30 and -20"C, and (b) that dium freezes. A corollary lo this view is that cryopro- the differential effect of wanning rate is maximum in 2 teciive additives such as dimethyl sulfoxide (DMSO) M glycerol. In concentrations of 1.7 M glycerol or less, protec* cells by acting colugathrery to reduce the few cells survive regardless of warming rate. In 2.5 M electrolyte concentration at any subzero temperature. glycerol or higher, most cells survive regardless of Previous data fnm this laboratory suggest that this warming rate. Cells frozen slowly in 1 or 2 M solutions interpretation may not be correct. However, to test this of the rapidly permeating solute, dimethyl sulfoxide, interpretation rigorously requires precise information as behave entirely differently. Slow warming is somewhat to the electrolyte concentration in partially frozen more damaging than rapid. The explanation of these solutions previously used for cell survival experiments. effects of warming rate and its highly nonlinear To do this, phase diagrams of these solutions are being dependence on glycerol concentration remains obscure. constructed. Two methods have been used: (a) a temperature-rebound nrthod to calculate the freezing point of solutions of high weight-percent water, and (b) 1. D. G. Whittingham,S. P. Lcibo. and P. Mazw. Science differential thermal analysis lo measure the freezing 181.288 M 973). point an J other phase properties for solutions of lower 2. S. P. Leibo. P. Malur. and S. ('. Jack<'W*ki.£xp. rellRr*. weight-percent water. 89,79(1974). 3. L E. Tow ill and P. Mazur. *fenr Fhytiol.. in press. The three series of solutions observed were 0.4, 0.7, 4. H. T. Meryman. in Cellukr Injury and Restsiance in and 1.0 M DMSO in isotonic Hanks' balanced salt FrtrJing Orgtnisitu. institute of Low Temperature Science, solution (HBSS). which correspond to weight ratios of Hokkaido University. Sapporo. Japan. 1967, pp. 237 238. tscta&r* mmmtm g 35 SURVIVAL OF FROZEN-THAWED HUMAN With stiB higher concentrations (2.5 and 3.0*). there RED CELLS AS A FUNCTION OF MINIMUM is no LT,#; that is. more than 50% of the cells survive TEMPER ATURE OF EXPOSURE. GLYCEROL freezing to - I00*C. and survival again becomes inde CONCENTRATION. AND WARMING RATE pendent of warming rale. The next step will be to compare the percentage survivals with the concentra Hiroshi Souzu* and tVter Mazur tions of sodium chloride present at each temperature. One widely accepted explanation of slow freezing These concentrations will be obtained from published injury is that damage results when the concentration of pfrase diagrams for glyceroi-NaCI-water. ekctrotyie readies a critical level in portly frozen solutions during freezing. We have conducted experi •Vniiin • ut.^vinplui from iht Ituiiluic of Lo* Tempnaiurc ments on human red cells to test this hypothesis. Cells Sotntt. Hakkanto L'nrnrroly. Sapporo. Japan. were suspended in saline or phosphate-buffered saline containing 0 lo 3 M glycerol, held for 30 min al 20°C COMPARISON OF THE RESrONSE OF to permit solute permeation, and cooled at a rale of 0.5 MAMMALIAN TISSUE-CULTURE or l.7*C/min to various temperatures between 0 and CELLS TO FREEZING - I00"C. Upon reaching the desired minimum tempera Nicholas Rigopouios. S. P. Letbo. and Peter Mazur ture, the samples were warmed at rates ranging from I lo 550*C/min and the percent hemolysis was deter This laboratory was the first to recognize and to offer mined. The results for a cooling rate of l.7*C/min (Fig. a rational explanation of the fad that the response of a 3> indicate the following: wide variety of cell types to freezing was similar in that (a) Between 0.5 .If and 1.8 M glycerol the tempera- their survival is a Diphasic function of cooling rate.1 2 lure yielding 50** hemolysis (LT*0) drops slowly from Those analysts also led to the concept of an optimum 20 to 35J C. cooling rate for maximum survival. To collect further (A) The .-fjeS oter this range of concentrations are examples of this phenomenon as well as to develop relatively independent of warming rale. practical procedures for tow-temperature preservation (<•) With glycerol concentrations of 1.95 M and 2.0 of cell lines, we have been conducting a survey of the M. the LT,o drops abruptly from 55 to -lOO'C. response of a variety of mammaian tissue-culture ceil and becomes dependent on wanning rale. The LT5« is types to freezing and thawing. The procedure is lower with slow warming than with rapid. straightforward. Small samples of cells are suspended in dimethyl sulfoxide, seeded with ice to induce crystal lization, cooled to - I96°C at rates from about I to 0ftm-9l*$ 75-t3090 800cC7min. and then warmed rapidly. Survival is assayed by procedures appropriate for a particular cell §-«» type. Thus far, the following cell lines have been tested WR and found to exhibit maximum survival at an optimum • SS0*C/«i.« cooling rate or range of rates: two mouse lymphoma o»30*C/mm »1«C/nw> lines. L5I78Y/TK' and L5I78Y/ASN'. and a mouse t hepatoma line. BW7756. Non- of these cells survive cooling at rates in excess of 60°C/min. I. P. Maiur. AV«/. /foe. 24. S-175 11965). 2 P. Mazur. Stitntr IM. 9391 i97(->. ISOLATION AND PROPERTIES OF CHROMATIN •> BODIES* D. E. Otitis. Ada L Olins.* Marilyn B. Senior.* R. D. Carbon/ Gwendolyn B. Ilowze,* 0.5 »0 «5 fiverline B. Wright, and Mayphoon tlsk-tfsu GCTCCR0L CONCCHTftfttlON 14T) Fig. i. Rctolfomhip between glycerol concentration and There is now considerable electron microscopic and htm&tjm femperatwre in 0.IS #f 51 J. Cooling rai< ITY/min. biochemical evidence that the first level of DNA folding 3b in eukaryotk chromosomes is the spheroid chromatin r 'RfMTjKh tptmwccd w pari by HI DA and in part by Mil body. It appears that the association of histories with •^search Gnat CM •'•334. DNA results in a six- to sevenfold compacting of DNA t within the » body. MH p>Ml*Klufai amnplor I t-rikmhip HD MM3t». 5SK;MS pushfcHtural Ntvnt«alu( it-VUuwdup C. A. Chambers and D. M. Skinner DNA kttfth (bar pan» -2U0 -125 llmyc vMimi *• pmaa Mt pwnw The interaction of purines or synthetic simple se FJcvtrua mKiameaff w kadm witi) taih w budwj WHIMM tab Luo-jaftc X-fajr Ftcmt PmcM quence ribo- or deoxyribopolymers with comple rcdcvliMn mentary polynucleotides to form double- (ds) or triple-strand (ts) helices was reported some years ago (reviewed in ref. I). We find a similar formation of is From these and other data it appears that sonication complexes by the G-rich (50Tc (.) or C-rich (50% C> produces double-strand breaks on the connecting DNA single strands of two satellite DNAs with ds main strands between the v bodies: the resulting monomer component DNA from the same organism as she particles represent the upper limit of the fundamental satellite or with DNA from heterologous sources {it.. repeat unit in chromatin {i.e., ~200 np). Nuclease ihe main component DNA from other organisms digestion appears to result in the rapid destruction of including bacteria). The satellites studied were one of 2 the connecting strand followed by slower degradation very simple sequence Satellite I of ihe hermh crab. of internal DNA. Preliminary studies reveal that the tkgurus pvilicmm whose sequence is pure ( T A early stage of nuclesse cleavage produces v bodies wi'h G G)„ l A T C C >„ (ref. 3) and ihe more com tails, suggesting that early digestion resembles breakage plex asatdlite of ihe guinea pig. Caret ponettus. 5(Tf by sonication. of whose residues are ( TTAGGG)„:iAA High-resolution microscopy studies now in piugress T C C C )n. with ihe remaining sequences variations suggest that " bodies may have a characteristic internal theron. The ss DNAs have been labeled in vitro by 1J 1 structure, possibly possessing a hole or crevice in the ' iodinalion or by H-5ubstraies using DNA poly middle of the particle. merase I. Control experiments were performed wilh J J Computations of the low-angle X-ray scattering prop [ H|Thd or H-C-labeled guinea org a-satcIRr i*oiate4 erties of various close-packed arrays of spherical parti from tissue cuhure cells (GP80- The satellite DNAs 3 cles, assuming different models for the internal electron have been purified by entri'ugafion in Hg * • Ag* Cs S0 (wf. 4) or Ag* Cs}SO. gradients. Their single density distribution, have shown that: (a) the ~ I10-A 2 4 : reflection could arise from the particle lattice; (b) the strands have been recovered from alkaline CsCI gra- reflections at ~55. 37. 27, and 22A arise principally as dienis. a consequence of the spherical particle shape: and (c) We find that: (0) Centrifuge '•» C$C1 gradients the relative peak intensities are sensitive to the exact without ds DNAs. either single strand from either choice of internal electron density distribution. Meas satellite forms a very broad band (width at half Height urements are in progress to determine the proper choice extends ant a 50-mg density range), (b) In contrast, of a radial electron-density distribution. each ss DNA forms a narrow band (width at half height The universality of " body occurrence is now well extends over a 15-mg density range) with any on* of documented; evidence demonstrates their presence in ihe different carrier DNAs, either mam component plants and fungi and in various animal tissues. Prelimi DNA (homologous cr heterologous) or DNA from nary studies from our group indicate that v bodies can cither of two bacterial sources \M. kit fa (7 IT ii * C. be visualized in synchronized CHO cells sampled at p • 1.727 g/cmJ) or E. coV (50% G • C. r « 1.7035 different stages of the cell cycle. g/cm*)). The ts complexes form bands at characteristic 37 positions in a CsCI density gradient 5 to 7 rtsg/cra1 4. D. M. SkiMMT and W. G. Betttie. fWc. .Vtit Atm*. Sri. more dense than the peak of the carrier DNAs. Thus the USA 79. HM iW3). density of the is complex is 1.732 g/cmJ with M. 5. A. R. Muqpa and R. U. wdb./ .MM *rf- 37.43 epidermis, fain of animals were joined and sealed with POLYAJMNOACIO^TIMUIATED UPTAKE dental cement. To test the effectiveness of this co- OF EXTRACELLULAR FLUID MARKERS joming. (3 H11 ^K! was injected into one animal and the INTO HrU CELLS hemolymph of it\ partner sampled. Within I hr. Emily Tate Brake and J. S. Cook the >pecifk activity of the hemolymph of the non- injected animal equaled that of the injected animal. As part of our program to investigate factors affecting Experiments with such animals should indicate whether membrane turnover, we have begun a study of pmocy- the factortsi cuntroUing growth of regenerates and tosis as induced by polyamino acids' in HeLa ceils. molting is hemoiymph borne. Apart from observations with an electron microscope, The data described thus far could possibly be ac our quantitative measurement is the uptake of counted for by the minimal hypothesis that the |l4C|sucrose as a marker for the volume of mednim mhibiiion of growth and DNA synthesis after autotoray imbibed by the cells- We find that under all conditions, of partial regenerates is induced by secretion of MIH. there is a fast component of uptake completed in about To test this hy^HhesB. experiments were performed on I hr. followed by a stow uptake that is the same for animab wttose eyestalks had been removed. In such both control and stimulated cells. We therefore use the anwnais. presumably unable to produce MIH. growth 1-hr uptake as a measure of the effectiveness of any and ONA synthesis of the regenerates remaining after compound. Following uptake, if the cells are returned autotomy of their partners showed stage-dependm to normal growth medium, a fraction of the radio- inhibiiiun cumparabk with that observed in intact activity is rapidly «*st. while Ihe remainder leaves the animals still capable of producing the hoinone. As in cells very slowly. As a preliminary conclusion we aavnals with eyesialks. during the early stages of assume that the fast component is weakly bound while regeneration, autotomy results in an induced premolt ihe slow component represents medium that has been pause accompanied by a decrease in DNA synthesis in taken into ihe cell. the regenerates remaining while re-regeneraiUm of the Controls take up a volume of medium equal to 3 5"* missing limbs occurs, bedysr* is delayed, making the of th* cell volume per hour. Poly-D-lysine is more premolt period in animals that regenerate two seit of ettective than poly-t-ornithine in stimulating uptake, limbs in sequence as much as. twice as long »tn animals the maximum enhancemcit of the rate being upwards that regenerate ord> ant vz\ Since moiling can be of sixfold. The cells grow at their normal rate in either significantly relayed in animals without eyesulks. we compound a' 2 X 10 * M. which is an effective conclude that either another s»!e of synthesis and/or stimulating concentration, but are growth-inhibited at 6 Mocage of lh em 40 TURNOVER OF HrLa MEMMUNE MtOTONS ceRs to ionumg radratiun at otherwise nontoxic concentrations.1 •* TMFS competes with J. S Cook. P. C Will • and tmily Tale Brake oxygen for transient, radimmn mdwncd radicais in Continuing with the double-isotope, two-pulse tech DNA. leading to covalent TMPN-DNA adducts or > nique.' the use of which in our membrane preparations oxidation of DNA ndicais. In cuntrast to the oxygen we validated last year, we haw measured turnover of effect. TMPN sensitization occurs with fcttie if any 4 membrane proteins m logarithmically (rowing HeLa increase in the yield of DNA jingtr-strandbreaks The ceils. On SDS-gel electrophoresc of purified membranes radiochemical basis of sensitization is not known for we find, confirming similar observations on other either oxygen or TMFN. but probably mvotves both the systems from Schknkc's laboratory, that, in general, the number and repairabmiy of damaged nucleotides m the larger molecular weight proteins turn over faster than ceUnbr DNA. I have examined the sensuimion to the smaller proteins. There are approximately 30 gamma rays of strains of E. tuB deficient m the identifiable bands per gel The range for all proteins is prereplicativc excision (uvMl and pourephcatrve 0.5 to 1.5 turnovers per generation. In the vicinity of tmv4) padtways for repair of l/'V-induced pyracidHU 125000 mot wt. which is the approximate size of the dimers. and a doubly deficient fuvM nrAl mutant. known ouabain-binding submits of Na*-K*-ATPases. Dose reduction factors for pmmi inadtaUon of bac the turnover is about 1.5 per generation. This value is terial suspensions under air or anoxicatty m I MN not greatly different from the value of 2 per generation TMPN have been calculated, with irradiation under derived from ouabain-binding studies. Thus the ATPase nitrogen as the standard m both cases (Table 2h These appears to turn over more rapidly than the mean value data agree with earner results.* for membrane proteins I I.I turnovers per generation i The sensitivity of the strains to gamma rays increases but not significantly more rapidly than proteins of noticeably as repair functions are removed (reading similar molecular weight down the commits), with the greatest effect at removal of the ncA-dependent pathway. Sensitization with TMPN emphasizes this effect, inducing a maximal 'rVHtdoctonl mmnpiof vpfattti by Sobvoniract No. 50-fold greater sensitivity of the double mutant as 3322 from Ihr uwlofy Division. ORNL. to (he t-mveniiy of compared with (he wild type. The cxci*iou-fepa>r Tcaarwr. Knm«m>. 1. I M. Ariss, D. Doyle, an* R. T. Sviwakt./ JM Ckrm. function, absent in the mtrA mutants, appears to be 244.3303 TaNrZ. uy TauTrt sun oxyuni of c cwi Comma-fay dn*r f kutaano at g#f*yt R^RvnfmjtVMBnH I0t wiiiin traction (aclnf* f. eoK tinm Ovywm N, • TMTNf OsynnV* TMTN Oxypm »TMTN ABIl57wml-fyair 40 20 12 12 2.0 3.3 AtlfMnvM 27 4.5 12 12 2.4 2.3 AB2443rvr>4 4.4 0.7 IS 2J 44 IX AB24aOovMmr/4 2.7 0.4 14 1.4 4.7 1.7 'Dow momncatiwi factor* are Dtt)«rmiiue4¥D|«(iMrocni) ^Osynrmmnn natd by bvbbtwM, am. aonxia by babbJmg mange*. rTMPN cMicaMref i»N wg* I mM. 41 note loimpiii cuitumm 2 am) 4) rhat oxygen competes TaMr 3. Cyclnw^pfwmmwtdnnM^mlMmmiDNA against the ttwatumg eflect uf TMPN and essentially -protects" the rcpau-defkvtut mutants. Time data are ImmmWO^mWVmml •MMX 100 consistent with a model where TMPN enhances the Fhow llmzt non-srraed-break. ~UV4dce" faction of pnwn-ray C<>C CoT TOT TiHal damage f subject to nr and rer repair), wbticas oxygen SO OOM 0.052 Oil 0.27 enhances a ~aun-UV-hfce." perhaps strand-brcsk- too 00* J 0.11 0.24 0.41 amociatcd. fraction, at least partly repairabk. inde 200 0J0M 0.15 0.42 044 pendent «t" the mtrA and nerA functions changes with mcreasmg doses of UV. The production of tyiosote-cuniammg dimers levels off before thymine- •Sla*** HlklTOnt *afer feain* SA—t «f •*»- aaAial Scieant*. couumiag dimers due to the difference in absorption 1. r. T t—itm aai r NmWbwn. .tear Ml. characteristics of these bases. We can now make a l«*5il«Ml reasonable estimate of the total number of dimers 2. f O. tVfwr«m.R OlKbi».)*4T.»ra«j4.Atr./feriBr. simply by knowing the percent radioactivity m the «W M. 52) • I974» thymine -corn jmmg doners. These numbers provide a J K W*M mt T Orattal. /ar / «•**. AW 25. 225 ll»74i refinement in the estimation of the amount of radiation 4. I Mum. Fint Em. maatri. C—p. Vol. U. p. M9 damage and repair. For example, we can now correlate «l«7|» the nurobti of dmm-specific endonuclease-sensrtive 5. W. D Rap*, t ZMW.C waEnra.D Rfaa.L IWiaMr. sites m irradiated DNA with the actual number of mi f. II— JI4-Flaaicn. Caa/iiumi «t four «** Owr pyrioudme dimers present. IUimmm*mt a> JUawww JW**r. ONL 5920*C-S7» 1. P. limp. R. Whou-nrfi. jmt 0. S. Cox. Btodnm. JUNJMV*. .4r»Jl2.62*ll«73». THE NUMtElt AND KINDS OF CYCUOMJTYL PYROMDfNE DMERS W HUMAN DMA ». L. Carrier. W. H. Lee. and James D Regan A SENSmVE METHOD FOR THE DETECTION OF ULTRAVIOLET PHOTOntODUCTS IN Dmwu are prudnced in DNA between adpeent SACCHAKOMYCES CEKEVISlAt DNA pyrnmdmes by (JV light- The ami couvemrul way in estimate the amount of doner* » to incorpi»raie R. J. Reynolds* radioactive thymidine mto the DNA. irradiate, hydro- h/» with formic acid, separate the acid-stable thymme- UV irradiation of 5. cemame n known both to viiBljmmf photoprnducts from the thymine by paper induce mutations and to reduce viability. Ulti chromatography, and finally compare the amount of mately, the lesions most probably responsible for label in the photoprodacts (TOT. TOO with the these effects, either directly or indirectly, are amotmt m thymine. Recently Unrau ft a*".' wed cydobutyt pyhmidine dimers. We have now de |'*f IBHKII as a general precursor of pyrimidme* in veloped a technique which is capable of detecting DNA an I UNA They were able to determine Ihe dimers m yeast DNA after ftuences of less than 5 J'm1. number and kmd of doners in the DNA -M several This is 10- to 100-fold more sensitive than methods rnvroorpnmm irradiated with UV h|hl. We used this normally used for the detection of dimers in yeast. The method to measure dimers formed in cultured human increase m sensitivity rs made possible by the use of a fibroblast cehs exposed to :S4-nm UV Injhf. The dimer-specific endonuclease found in extracts of number and kmd v( dimer formed are shown in Table Mkmencoa luton, an endonuclease previoudy shown }. Tlryimnt-containing doners are more readily formed to incise near wyimndiwe doners.1 Measuremeni of the at this wavelength of UV light than cyfowne-containing resulting smgle-sirand breaks may be used to quantitate 1 dnwrrs After 50 Jm the ratio of COC. COT. the number of doners. TOT is 14.20:66. In contrast, this ratio is I6:.*a:.'? Briefly, the DNA to be assayed, prelabdcd with m A. culi. At lower doses of UV. ihyinine makes In-1 H| uridine, is released from yeast spheroplasts and up about 75** of the bases involved in dimer for gently extrxicd with phenol. The latter step dirnmates mation: this frjire for £'. o*H n about bOTi. The ratio nonspecific nurkascs found in yeast lysaies The DNA 42 is treated with the M. farms extract and sedtmented un source of this nuclease activity and. in particular, to calibrated .ilkaline sucrose gradients, from which the determine whether the activity persists in extracts read* number of single-strand breaks can be determined. We from hosts lacking the K-12 restriction enzyme have shown that the number of breaks is linear with We have abo begun to examine Ihe response of this m fluence and that no breaks are induced by the enzy- nrrv system to UV radiation. In rnv T7 is quite i>.-«tic treatment of DNA from unirradiated celts. sensitive to UV and has a biphasic survival curve which Th:< technique has been used to follow the loss of shows dependence upm the h. coU MTTA utrB genes. UV-enoonuclease-sensitive sites in the wiM-type haploid However, the rate of m ritrv DNA synthesis seems to be strain s288c. a strain from which a number of UV- relatively unaffected by UV. Doses of SO J/m2 applied sensitive mutants have been derived. After 30 J/m1. a to the exogenous template reduce the rate and extent fluence resulting in about 70** survival. 80*? of the sites of m wkrv DNA synthesis by only 50*. Also, we have are removed within 2 hr at 28*C in potassium phos- as ye. found no evidence for m riirv excision of phaie buffer. pH 7.0. After 24 hr. about ?S5 of the pyrinudine darters in this system. Moreover, although sites are gone. Studies are now being extended to the extensive serniconservalive replication synthesis takes radiation-sensitive mutants. place, we have thus far been unable to detect any UV-sumulated nonconservaiive DNA synthesis charac teristic of repair resynthesis These observations suggest •SiaJnu >i ihr LT Oak krifr GraAntc School of Bk>- that T7 may deal «vrth UV damage primarily through —dk jl Scicmxv posfrepbcational repair rather dun by a "cut and I. V. L. Carrier and R. ». Sctfaw. J. MmrtthoL l«. I7S «I970>. patch" excision scheme. Further experiments will be needed to test this interesting possmatfy. IS »771t0 STUDIES WITH MCTHUOntAGE T7 1. D. C. MM* MMI C. C. Rkfcw4w«. J. Mini Otrm 249. 2914 2W4liy?4>. W. t. Masker 2. W. F.. Maker aari C. C. RkfcaiUna. uchmilcrf to J. . for separation of viable and nonviable celk from thermal treatment acts on the control system. Our imdiaicd cultures, and the repair capabilities of the hypothesis rs that the cAMP buadiug protein is thermo- i wo classes of ceBs. labne at that its binding constant is altered. An alteration m the binding constant would prevent tran scription and subsequent respiratory failure in UV- 1. P. A. Sweat** at(L Sttralty. far. / Jtarfwr fas*. IS. mdweed ceRs, but exogenous cAMP would enhance the Slll»74» 2. I- Wirkm.Aar. Mart. Atmd Sri USA 71.I9XHI974I. possmmty of binding and favor transcription. i. P. A. Smcwmm ami R. L. Sdwuiry.J. atonriat III. *S» We have detected significant differences in eJution 11972). patterns of radioactive cAMP binding proteins from unirradiated and irradiated eels using agarose geh to which cAha* b covafcutty bound (affinity chromatog raphy). We are now in a position to test our hypothesb TNE ROLE Of ADENOSINE CYCLIC of altered cAMP binding properties by thermal (42*C) J r MONOTHOSTHATE IN THE ULTRAVIOLET treatment with m rnw and m nuo experiments. INOUCED CESSATION OF RESPIRATION IN FSCHEKHHU COU m* 'taint* marry Depar mem. Untaunty of Tc—met. Kaax- P. A. Swcnsou. R. L. Schenley. and J. C. Josbj* vaV J7»l». 1. P. A. Saw man ana! It. L. Sckcnby. 4iawuro Am. Sor An important piece of evidence that cessation of /a»wan<.a.»t«W74». respiration after UV irradialion of E. cum' fcV'r rs an 2. A. D. Mn». C. itemcs*. and G. Zatay.fVar. Mot Atmd. induced phenomenon is that adenosine cyclic aYifOvi*. 1222 • 1971». 3^5'-naonophosphate icAMP) causes more ghjcose- J. P. A. SWOMMI. I. M. norlr. and R. L. Scfccmnr. / mtcunot. 19.111*741 gmwn cds to shut off respiration and lo become nonviable than in its absence.' The intracenuJar concen tration of cAMP in ffucose-frown ceis is quite low. and our interpretation is that the increased level of this cyclic nucleotide stimulates the induced synthesis of CHANCES IN RaMMNG PROPERTIES OF the respiratory control protein in the same manner that PYRIDaHE NUCLEOTIDE IVONNC PROTEINS it stimulates the synthesis cr ^gaiactosadasc in glucose- IN ULTRAVrOLET4RRADIATED grown cefls that are induced by a d-gabcloside such as ESCHERICHIA COU R/r CELLS lactase. For maximum induced formation of (tfatacto- J. G. Joshi* and P. A. Swcnson snbse. a complex of cAMP and a cAlR*bmdmg protein must be present, presumably lo enable the RNA Associated with the cessation of respiration in UV- polymerase to transcribe the £gatactosidasc gene.2 We irradiaied E. cwdj B/r ceRs b the selective loss of infer that cAMP exerts its tfnbitory effect on respira pyridine nucleotides. Thb loss might be caused by tion by combining with a cAMP-bmdmg protein and changes in the bmdmg ptMpcrties of pyridine nuckoride alowing transcription of a gene coding for a respiratory binding proteins- In affinity chromatography experi conirol protem that b involved in the cessation of ments, we have used agarose columns to which NAD respiraiion. was bound covaleutSy and have found very different During the past year. cAMP has been used by us in ehKion patterns for radioactive proteins from unirradi tombiiMliiin with two treatments. S-Auorouracil IFUral ated and irradiated ecus. Certain peak activities in and elevated temperature (42*C). each of which mam- extracts from unirradiated crib are absent in irradiated tains respiration m many of the cdb where it would ones and vice vena. We have begun the process of normaly cease after UV.' The cAMP has only a smaH nofartmg. pnrrfymg. and analyzing the composition of effect am the respiration maintained by FL'ra but binding proteins to determine how radiation causes ahnosl completely prevents respiraiion in thermally these changes. One possaMc way thai the changes occur treated ceRs. Our mterpretalion is that FUra treatment b by selective protenrytk cleavage of amino acids or causes production of a fraudulent messenger RNA so short peptide chains from pyridine nucleotide binding thai, even though cAMP causes more mRNA to be proteins. We arc conducting a search for such a formed, this mRNA is not active. Thermal treatment ndot ton-induced enzyme. was formerly thought by us to inactivate the respiratory Our plans arc to study the biochemistry of the control protein, but the above results suggest that this irradiated ceR. and large Quantities of cens in which 44 f3BI3xWmBB*JCCtt &&0SCS R9¥t tSKCH 0BCC WtH v£ EXCISION OF rYMMOINE MMERS FROM needed. We have designed and constructed a quartz VIABLE AND NONV1ARLE CELLS IN flow cd for large-scale irradiations. Two flow ceRs are IJLTRAVIOLET-f RRADLATED tSCNUUCHIA M series and saMtwiched between UV prima, id J! OMibVr CULTURES lamps. IK conaboralioa with E. F. flares of the •kochciantry Section we have pumped 2% b^ers/min of R. L SchenJcy. W. D. Fisher, and l\ A. Swenson log-phase eels front a fetmentet through the irradiation We have measured the pyrinadhw dimer excision in system and have been able to dnpbcaie respiratory and separated viable and nonviable cefc from UV-irradiaied lethal response! obtained in onr imaU-scalr experi gfyecrof-grown cultures. Withm 120 nun after UV 120 ments. We expect to accumulate a Uugram 01 more of J/m*). in the presence or absence of detergent. 60 70% irradiated cefc for fcwcfccmkai studies. of the timers were excised from an irradiated culture: hide or no excision occurred dvruu; an additional 2 hr of mcubatioa n. growth medium. When the viable and Kannvar 37916. nonviable eels were separated at 120 mm. the number of doners in each population was identical to that of •he whole cakare. No further excision was seen in the viable culture when it ws incubated in growiM ajeannu SEPARATION OF VIABLE AND fvOJIVlAJLE for an jawthinjl 3 br. Rcirradtatioo of viable cefc CELLS FROM UL1KAVMMXT«KADUTED crmtammg residual doners showed two nNcwstmg ESOIEIUCMACOUViCVUVKS features. Doners were once more excised, and the cefc W. D. Fisher. R LSchcntey.and t. A.Swcnsoo were no awe sensitive than lug phase cefc that had not been pievioasty irradiated. These results show that Aliboafh Ihe repair of DNA in UV-irradiatcd bac- survival is not wcR correlated with the extent of dimer lerial eels has been extenswety stndied. no method has removal ami suggest the pniamnily of selective repair of existed which conM dcstrnmndi between Ihe events diners in LV-kradiated cultures. the catture. We have developed a ccntrifnpi method for the pveearatae sepjuthm of viable and mmviahtc cefc from a UVHrrawBUd caviare When a UV4rndteicd<20 AN anVfX>VFJ>ritOCEDt*E OF SEPARATION J/m*. approximately 10% sarvival) culture of E. n* AtvDPtJMritailONOFiTOnWCT incubated m the presence of the nomouk dewintnt nomsamonucunscxmmnm.Am> Triton X-IOO. the nonviable cefc. whkh have stopped CinrSTALLIZATtON OF RKCM BJL nVj*Vj7JnTBBBB? IxVa* ttMA ^mmf faTJbntafnnl ^Ranl RRgT^juTmn* ^untdmCT within 120 nwaafwn irradiation. Vnbte.rcifinng cefc C. H. Wei and OtongKan Koh are not vaJnenMe lo the detergent. To separate the Two print naal toxins and a lectin present in the seeds dates of cdb. the enknre B mixed into a 0 to S* of Rfcuues enrnwams have previously been separated nential sucrose gradient on a 5% cushion and subjected and purified to homogeneity by a ptmedare. the final to dvftcreatiai lemiiragation. After three cycles of stage of winch r CM from UV-hradialtd cultures grown on ghjeme and from rather than fonowed nontoxic anjtutmiu IHH. Fanner- gammjiiiiiijied cultures. more, the 111^, fraction was resolved into rwo com- 45 punent protems. which have the tame uoekvtrk puint. AMuTvOTTJrJR^LSEQ^TCNCESOFTWO 7.4. and are also mdraiuguishable with respect to SOS rEFTlOE CHAWS FOR MCVIV-J" ckcirophoresis patterns and tu ettctrophuret*: inobm- S. S.-L. Li.» C. H. Wei. J.-Y. Liu/ and TXT. Tut** ties at various pH values f4.5.7.0. aad *»-5i. RcsutuiSm of a preceduu} dtuuMct from the MUM peak of riciu After careful screening of five difTeretit protein 1 V-3 was nut achieved. crystals fir obtained m out laboratory frwr seeds of After intensive iriab. tuxm lllL has finally been Akmt nrcvMonuB and /teams wiwimwu. preliminary cryiiJh«d in the form of fine needles. A modified X-ray data fur four of which bane been reported.1'4 we nuciodiffusiuo eel* was used foe slow eipindinum chose to concentrate our X-ray structural studies on darns apiusi 0.05 M Tm4l,P0. buffet tpH 7.2) to ricin V-3. :he most toxk protein present in the tatter which anal increments d 3 N ,—mm n* sattat* ;n seeds. Knowkdfe of the ammo acid sequence would be the same buffer were added daily. The crystals started of utmost importance in the X-ray sinrctura! detetmi- to appear at a* MUMMM sulfate concentration of nation. The toxm consists of two peptide chants (A *ni 1.25 M. and their growth appealed ciimplrte after one B) of sfcfbtly different molecular sues. CoRaburatn* at 1.41 M ammoauum sulfate concentration, research has so far rewind in the aenmo-tenuma! r of the hunted sue and unfavorable shape of the scwaeucc of 2t residues of the A chant and 14 residues lllL crystals, they do not appear to be useful for X-ray of the Bet structural mvestiptiuu. I 5 10 12 A-dM Hr«W4W4.]rv«kf.Trf^k»llc«c4lc VJ-TW 13 15 » 24 C. H Wo. J. DtMeftcrrjr.T nVaul V.-K. Ymr.nwf Dm Hr-Ab43r-Ab-Thi-Val4«»Sct-TTi-A«-Vjl41lr- 1. ft H «cUn adM. ti ••*»«. *r* J»«+tm Jtau*n 25 2* MI.4C*tl*10». I 5 10 •-chaw .4b-Aif%jl-TMr- of scMstowaiastt. a parasitic-worm disease estimated iu nsethanesuifonat* anion has a tetrahedral configuration affect more than 200 mnhuii people in the tropin. of D,j symmetry. Its charge is deJocaheed. and all S-O Although this drag is a potent framrthtft mutagen, so bunds are espial to within ±0.01 A. that hssaf.ty asa drjg rs questionable, it was estimated (as of July 1974) that at least 700.000 people infected with SCMMMMW hamtuhmm and S. mmaom have been treated with hycanthone during the past six yean I. f. *. twmcri. t- mntac- «*»*- *- lb""*". Vmv IS*. • •raal. Africa, and the MhMh? East.' The structural MT119741. invesligatiuu of hycanthone was undertaken as port of the ninor portion of our work devoted to the structure deirrmm ithm of ~smau~ molecules of biological m- terest an* as a contribution to the eventual explanation PREUMNARY X-RAY DATA FOR of hycanihone activity. SOME COMPOUNDS OF MOLOGICAL INTEREST The compound Isuppued by Dr. F. J. dtSencs of C.H.Wei NIEHS. Research Triangle Park. S.C. I crystamtes with SnJktm snV Jrrtrmrire ,tf tkyimlrtyH f-5' h? Jn>xrtkrimknr Ca» 5TpT-2lljO. generously 0 HNCH2NH(C2H5J2 suppbed to us by Dr. ". \. Ibyes of Lis Alamos Scientific Laboratory. w*. further purified and con vened snto a sodhim salt by passing the aqueous [ I |T II | JO j • CMsSOs I sowtion through a sodium-converted Dowex 50 cul- umn. Single crystab were obtained from water eth- anol solution at 4*C over a period of months. The CHjOH crystab seem to be extremely sensitive to X radiation. Pkriimmary Werssenberg and precession photographs mdkate that the cry stab possess the following data:« - 12.70. * * !!.**. €- = 13 47 A. *} = 122.5*: space group four mutecuks m a monoenwe unit ceH of symmetry C2 r or CV: Z = » Or 4; V = 12*1 A\ As judged from tl,* and parameters* « 2«».i«»35*l3i.ft » X 7W7|4». the sue of the unit eel*. ?h? d*n*ed compound could be r « I0.6573|S) A. and d * 116.42017)*. The structure a nucleoside degradation product, rather than a dmu- was solved by the heavy-atom method and was refined cleowde monophosphate. by ftnl-matm least squares to a discrepancy index. Dmwfrm nf JtAiDf if.F*-Ji^miiniiK^5'-ifir Cll:Nt(";ll,»j group and that this proton ami the and evaporated to dry new. Fr»m Ihe llrk-voniatmnc hydroxy! hydrogen atom are hydrogeiHbonded to aqueous solution, pbielnte crystals were obtained. This oxygen atoms of two neighbors*!, methanesulfonaie material crystalli/es in a monoclintc unil ceil of •on*, thus fornnng an infinite chain of hydrogen-bonded symmetry f2,r and parameter* n ' 4.63. b - nwdecules around the ctystaftographic two-fold screw 10.01. r - l«» II A. ami *i«'W.4' A c<«mpari*m of the axes. The carbon atoms nf rings I and III are planar to crystal data with those for guanine and pianme IITI wiihrn 0.02 md 0.01 A. respectively and these planes denvatrves suggests that the derived comp-mnd is likely intersect along the C|«»)... St 10) bne to Unm a slightly guanine hydroNormJe m>«<4iiydrate. the strucliire of bent configuration with a duVural angle of 167.7". The which has not yet been reported. 47 THE pA. OF THE ACTIVE-SITE AFFINITY LA1FJJNC. OF (ARBOXYL GROUT OF RIMJLOSE MSIIIOSriLATE CARBOXYLASE TRIOSE PHOSTHATEISOMERASE BY J-WvOMO-l.4-WHYDItOXY-2-RJTA.NONE 1.4-MSrHOSrHATE F. T. Hartntan and <. M. IjMuraeha* J. V. ScluVvs* and K ( Ibrlnun „»-ltaloaceiol phitsphaies and ah iJ*>l phosphate in- actrvate uu**e phosphate i*omerase hy 3 hiehly selective esieuficatiiHi i>r an essential glutamyl residue.'* .< Brimio-1.4-dihvdroxv -J-butanone 1.4-brsphosphate. Attempts n> determine the pA,, of 'he active-site a reactive ana'-ieue >*( the sub>trale rihu'oie I /4*i*ph«>s- carboxvl irmip inmi the pll dependence oi I he phale. has previously he>*n >vnthesi/ed as a potential inavtivalion rale have been ciMiiplicatcd by j variable affinity label f.« ribulose bisphosphate carboxvlase.' affinity of the reasenls for the en/ynie as a result of This reasent inaclnrates ibc carh»oi\|a>e from spinach as changes in the •••nvaiini' stales of 'he reagents <>vei the a consequence of alkylaiixn i>f two essential lysvl pll ranee examined. T" circumvent this problem, we residues." If these residues play a crucial r<4e in have synthesized chlocoacetol sulfate. 3 nuwopion-; catalysis. a.-ialoeous residues sh-mld be preseri in acid ihat exists i^ilv as a monoanion i»ver the entire pll rthulose biNpho>piiate carb«i\yla>es from diverse species, ranee at which protein carhoxyl sroup* i.in;/e. This new and therefore tiie sank.* reaeent should also inactrvale reagent estenlies the same glutamyl residue «>! triose these eiuyiiKs by aikylatnm of lysyl residues. Thus, we phosphate isomerase as do the previmsly described have inspected the effects of hromi4*utanone htsphos- r aelive-siie-specific reagents. at»J therefore the pll de piute on nbulose hispho->phate ca b>>xybse from pendence of the inactivation rate by chlor»aceti>i Kb\k'\pmUnm n.hnm!. a purple nonsulfur Hacteruun. fins orsanism i- auiont: the :110s; prinniive kmmn to sulfate provides a measurement of the pka "t the essential caihoxyl sroup. For the yeast enzyme, the conijin the carbtOiVb*'. In ci>niia>f !•» the enzyme apparent pA., calculated from the pll dependence o( from spiriach (which has a moiccular weishi ,ti 5ftO fMKf the maciivation rate is .».«> 1 Ml. I 1. We were unable to and contains eiehi prohnneric units, each with a 56.000- and M.IXKr-JjIion Mihuniti. the enzyme from K determine the pKa o|' ihe corresponding group in the taNni muscle enzyme because of lis instability a! low rtihnmi has a molecular weidit of ! 14.000 and is nil. However, since the maclivalion rale wa. virtual!} ciHiipose-J of two itlentical Mibunits of 5".000 daltons.' the same at pll 5.0 as al pll VO. the pA,, must be well The punfie-J carboxylase fr->ni R rubrum is inacti below 5.(1. vated bv hroni EVIDENCE OF A PREVIOUSLY . strate protection. The kinetics of insctivafion are UNDETECTED LYSYL RESIDUE AT THE consistent with the initial formation of a reversible ACTIVE SITE OF ALDOLASE en/vnie-re^genl complex preceding the irreversible in- activalion step. Abo. from kinetic studies in the J. P. Brown* and F. C Hartman presence of substrate, we conclude that the initial His-359. adjacent to the penultimate residue, of binding of reagrul " at the substrate binding site. An rabbit muscle aldolase has b-en implicated as a com additional observation consistent with a highly selective ponent of the active site by its preferential alkylation at modification of an essential residue is that one mole of pH 6.5 with the affinity label iV-bromoacetyteihanol- reagent is covafeiilty incorporated per mole of catalytic amme phosphate.1 We now find that die specificity of subunit inactivated. Further studies are in progress to the reagent is pHdependent. At pH 8.S. alkylation with elucidate the kind of amino acid residue alkylated by the reagent. pHIBrAcNHEtOr* abolishes both Fru-l,^P2 cleavage activity and trmsaktolase activity. The stoictuometry o( incorporation, kinetics of inaclWation (pseudo first- order and a rate-saturation effect), and the protection DATA-ACQUISITION SYSTEM against inactivation afforded by competitive inhibitors are consistent with the involvement of an active-site Margarita K. Cnurchsch. S. S. Stevens, residue. A comparison of *H profiles obtained from and M L. Randolph chromatography on the amino acid analyzer of acid hydrolysates of inactivated and protected samples Since the automated data acquisition and processing reveals that inactivafion results from the alkylation of system of the Biology Division was operating at near lysyl residues. The major labeled peptide in tryptic capacity, more processing capability has been added to digests of the inactivated enzyme has been isolated. meet user requirements expected over the next several Based on the amino acid composition of this peptide years. New equipment is now in final stages of testing: fur measurements of energy depusniun by ioniiing •uVwirti mf+int* m part by Wl f AM (A I5» 7). photons, utile care need be taken to match exactly the demencary composition of the detector to mat of biological tissues of interest. However, for neutron nadtaiions the energy deposition b entirely by a UNA DEGRADATION KINETICS variety of umJeat interactions and b strongly de AFTER X RAYS pendent on the target nuclei and neutron energy. Hence II. L- Rand ilph for neutron irradiation work, calculation of kemta factors (cssentiaBy dose per unit neutron flueuce) b tVrriuus work on DNA defndMion in Him ipunVi necessary. Results of such calculations (done at the uuwarurur cdh which have been X irradiated has been National Bureau of Standards by R. S. CaswcS and J. J. extended.'-2 The uncertainty which we find in statisti Coyne with cotaboralion from here) based on 17 nuclei cal resolution between models depending on unilateral most important m tissue composMior and the latest degradation vs modeb depending on bialcnl degrada nuclear cross-section compilation E>dJF/B-4 and cover tion at eqbaf rales is a^taKtalivcty utyhuMc it one ing the energy range from thermal neutrons to 20 NeV jiinwnfT, that degradation is geueraBy buafcral iwbkh 1 have been reported. Separately a survey of variations implies at least two exonucieases) acting at unequal in kerma factors at various neutron energies with rales in the two directions. In this framework onr variations m elemental composition of mamnmhan results zre consisteni with die conclusions that: (*i al tissues tcveab thai the hydrogeu concentration b the degradation is buateral: In) in log-phase DBl 17 crib, dominant considrraiiun at neutron energies greater iun degradation is mnch faster in one direction than the about I keV and nitrogen lor perhaps **•> at energies other, but in log-phase DBl 12 the two rates are nearly less than about 5 eV. In the higher energy domain the equal: and (ri in stationary-phase crib die degradation kerma factor differs by less than 10% from that for rale in the slower direction b greater than m log-phase standard tissue, hut at low energies die factors may be crib, so that die rates become nearly equal for considerably greater (3.5 limes for kidney and 1.6 times wild-type crib. Since the-interruption rales are nearly for ox lens i or considerably less (0.35 times for fever or equal for log- and stationary-phase crib, the measwed ovaries) dun that for standard tissue At least by degradation b faster and greater in stationary phase. implication, kerma factors are for ionizing radiation, The similarity of results of measurements of degrada yet al neutron energies o. less than perhaps 30 keV. tion with a temperature-sensitive strain, tint 9. vs wild elastic scattering, which accounts for most of the total type provides evidence that the bulk of what we kerma. results in such low-energy recoil nuclei that they measure b truly remaining DNA and not a reflection of cannot directly cause ionization. In such cases only an mlracenuiar pool of DNA precursor or reincorpora l4 , exothermic interactions, such as N(jr./>) *C. tion of degtjded radioacfively labeled fragments. Pre- l 1 *Be(n.a) Li. and rffn.yM). can produce ionizing sec linwnary results mdicaie that there b an X-ray-inducible ondaries. In these cases, calculation of an "ionizing inhibitor of degradation. If so. what we have called an kerna factor" rather than the conventional "total "interruption (of DNA degradation) constant" prob kerma factor" may be the meaningful calculation for ably represents the growth rale of inhibitor during radmbiotogy. incubation and simultaneous, in our work, with degra dation. I. It. S. CnwvU. J. J. Coyne.awl M. L. hjaJulpk.Stunk* in cosffQr depositwo o by neutrons, ffffc. 29Ml SJWIQ. on NtutFOR 1. M. I„ tUwiityti. mM. Ph. Anim. Jtagr. RfP J*'* SO. Ommrlty m Hahgy mid Hfdkinr (Mmwrb, 1974). to be 1974. OftNL-m3. p. 50. prbfeMd. 2. M. L. RawJnipb ani I. K. Scrkvw. JNnpftn. J. M. 441 ADENINE rHOTOCHEMtSTRY ENERGY DEPOSITION BY NEUTRONS R. 0. Rahn M. L. Randolph UV irradiation of poly dA results in the formation of When materials arc irradiated by ionizing photons, one or more phoioproducts.' It is of interest as to each electron looks pretty much like the others. Hence whether similar products are formed in DNA The 52 pb»(opioduct» can ho followed m p»4\ J.A by measur- piled with phtinuttl b lowered below Jul '4 me, ibe mcreasc in ahsorimtce JI .»IO nm a» well ac*topheiiowc l"he viscosity of DNA decreases sharply a> by measuring lite mcrcase m fluorescence al 4.»0 m. noon billing pbt>num. »uh halt the chance uccurmg Acid hydrolysis of irradiated pot) J A erne* JaMe •hen less chat t* •' t4" ifce hates are complcxed. From products as unhealed hy an aiHarf m absorhance at the rate of reaclmn with formaldehyde, w* conclude .»IO nm and a new tluoKtccwcc rruxnmwn ai *V» oat. lhal haulm ••< ibe piiiMtun compuMnd «•• DNA Paper dwornarocram* •* the tiydrolysatcs wm a indlKex sniaR derulMieU repi>ns thai unwind m the Htunul waler acetic acid »J*U Mi !2l solvent showed presence «4 i<4fiuMeh\de wilh a rale about 40 •unes pruuWb mumme with an R, .4 0 21 0.2"». The »»iwet rlur; ihjc «4 a s«i$le-«traaii cham hreai. fluorescence associated with these product was KI- *ired dwevth on the cbromat<>crafn. The pririilc in lawed agreed wei with thai •feained u«rt radioactive •XIII f\«ti4>i"l<>Ml !<*•«. fK«lM J!.<«: IVftntOHlM -4 labetmg procedures A cotaparrson ol ruine and de- fhauin. L-antm State Itwl) jjtwn tUmet >«»*. natuced DNA irraduted with 50.UW J m: ai 254 nm showed lhal «mK the latter had an appreciable flw>- rcsveuce resernhiim: ihac ol poly d.A. T*H result is tx rmu; LAKUNC cmtststeni with I!M observation lhal hydrogen bonding OF DNA WITH ,I5I tit' poly dA to puty rl quenches pnotoproduct formation. R. S. Sialfotti anJ It. O. Rah* bark difiicullR-s asaviat.-d with ': * I reaction with tiller paper discs seem no longrr ; pruNem. -Some material of a nonspevilW natu.e seems lo he miroduceJ into the DNA-lahehn* rcaciimi ahxu; wich the ad-Jiimn of '**l. Much of the ni*is,)ecilic lahelnw; dn he PLATINUM M.NOIISC TO DMA reduced I without reducmc the specific DNA bMn;) hy p*nc lo Ions reaction trmes near 24 hr. It is now Linda L Munchausen* and R O. Rahn possible lo label ~25 mt of DNA and detect il with a The amount «»l «»»-dichl«>ri>diammineplaiinumllli tolerable "sisul-io-notse ratio.' S SINGLET EXCITATION TRANSFER FROM POLY Pidy rA fluorescence is half-quenched by bound rA TO IOUND DYE AT 77 K proflavine or ethidiura bromide at a fractional dye concentration of about 0.01 smaller by an order of R. M. Pcaristcm* and F. Van Nostrand' magnitude than that required to sensitize the room- temperature fluorescence of dyes bound to nucleic acid •uth stngkf and triplet electronic excitation energy put} mere. No quenching occurs of the fluorescence of can be transferred from nucleic acids to bound moie similarly fro/en adenosine solutions having the same ties. The most efficient transfer reported is that of absolute concentrations of base and dye. triplet excitation in adenosine systems at 77* K or less. From these results we draw the folowing conclu The phosphorescence of putt nhoadrmlie acid (pufy sions: rA) in gh col-water glasses is half-quenched by para to I The fluorescence of adenosine is not quenched by- magnetic mriaS ions at fractional concentrations (metal dyes in glucose-acetate solution at 77° K. thus ion/adenosineK'i/2 ** OJOI. Similar low values of r,,> vcrifyins '•»' adenosine does not form aggregates arc obtained for metaMon intending of the phospho to which dyes can bind in that frozen solution. rescence of stacked adenosine aggregates in frozen aqueous solutions. Significantly smafter values of r,/2 (A) Poly rA fluorescence in frozen aqueous solution at are observed for such aggregates when the phospho 77 K is half-quenched by either proflavine or rescence is quenched by the dye proflavine. ethidium bromide at 0.1 0.2 the r value required Sinffci excitation transfer from nucleic acids to to sensitize the fluorescence of dyes bound lo bound moieties appears to be less efficient. Studies nucleic acids at room temperature, and less than 0.5 done with nucleic acid-bound dye systems at room the r value required lo half-quench die fluorescence of stacked adenosine aggregates at 77°K. This temperature suggest values of rl/2 ~* 0.1. Metal-ion quenching of nucleic acid fluorescence at 77* K also has represents the most efficient transfer of nucleic acid been observed, the most efficient case again being smgkt excitation yet demonstrated. J stacked adenosine in ice. for which quenching by- Cu " gives rtlJ — 0.02. No report of fluorescence quenching in frozen glucose-acetate solution is facilitated by of covairntry linked nucleic acid polymers by dyes at the presence of phosphodiester bonds (an unlikely 77* K has yet been published, although it has been possibility I. the transfer of singlet excitation from recently noted that the increased (unquenched) lifetime poly r A lo dye is intrmtmlecukr. of the nuctek acid singWl at low temperatures should greatly increase the efficiency of singlet transfer at Although we have shown thai poly rA can efficiently 77"K as compared with room temperature. transfer its singlet excitation lo bound dye and thai the Using a frequency-quadrupled Nd.YAG laser as exci transfer is most likely intramolecular, we have not tation source (X,x * 266 nml. we have observed at shown thai the transfer is entirely, or even mostly, 77* K the quenching by bound dye of the fluorescence in-stack. tc. essentially one-dimensional. If it is in- of poly rA fro/en in an aqueous medium containing slack, this certainly proves the existence of singlet sugar. Previous experiments on excitation transfer from excilon transfer in poly rA. because a one-dimensional nucleic acids lo bound dyes, done at room temperature, transfer range of about 50 bases of approximately 150 used aqueous solvents. Nucleic acids frown in most A is far too great lo be attributed lo direct Forster 54 transfer from adenosine to dye. |We calculated A. ol 1. In chtonuipnute aggregates «»l btulopcal iwetesJ. about 2D A lor transfer to either proflavine or ethftuum the tndrndual chromopnorc spectra display no sharp bromide, using the poly rA fluorescence spectrum J**! features. wy taking advantage ot the contnwum nature (be measured ab&Kbance spectra ot the bound dyes.) of these broad structureless spectra, and by nuliaUy We have not. however. :iiW owl the possibuity that neglecting Mferactiuus with (he heat bath, we have puty rA is highly iokled. ir.. has relatively short constructed a model Ifamiltoncau fur a dmter. Such a stacked report*, in fro/en ghtcose-aceta We have developed a fairly general dtcBnvamo.un t~kncntu> Umwi.Mwih ; Tmdim. ImnWPtJCBnfcOt vSBCmW 4MB wamwCml Owl fiT9nm£HwS OV •JmOwOmBCSnw tfcauiraei wttc—my PVHWIML Eq. (I) m a hi iqniii sense, subject to a system of precisely dn? set of potable mluiiiini (delta function ANALYSIS OF FUUORE&XENCE DECAY DATA rapwuarsL This set is jnst the set of al f fin** or R. P. Htmeugcr* and T.J. MfcMT cueflkitars. This das of fknetions oontami a wide A fimdamental probhm in extracting useful informa tion Iran measwjrmtnts of me time coarse of molecu- deiaikd shape of ike excamgaghl- These quantities are daUcwcctty toaspedrknvodel: connected hy the couvohrimn mHgijI W) k is apphcabk to situations Cor whkh no simple model barwl Mr. * of the system without any distortion «W to results of our deconvohition method using real data detail of the actual exciting flash function. have shown immediately m each case whether a Formal solutions of tq. Ill for the delta function one- or two-decaying exponential model is appro response /If I. using raw data for pit) and «1f I. are priate, or whether a more compbeated model is readily found. However, these solutions haw not been required- Moreover, our method has been useful m useful, became it is in the nature of fcq. 11) that smaB finding mitial model parameters. Le.. hfetimes. to distortions m »Wi and iflt) lie. experimental error! be used in further attempts to fit the data to some lead to enormous distortions in /Hit Therefore, a large defmite model. number of morc-oe-kss indirect methods of extracting information about the delta function response of fluorescent systems haw; been proposed. *CknnMfy DKIMMI: fonacriy o>itM>'u*i. In most of ihese methods fit) is taken to be an ' ConoiKf SoriK** Dmwow. explicit function of a small number of parameters of snorc-or4ess owect physical interest. The problem re mains of using f-q. 11). with pit) and «(M Hetennined experimentally, to find the best values of the parame AlTUCATrOfvS OF MULThTLE SCATTERING ters. Thus, for example, it b usually assumed for METHODS TO MOLOCY fluorescence decay experiments 'hat /If) is a sum of s, R. P. Iltmenger* small number of decaying exponentials. The coeffi cients and decay constants of these exponentials arc the Several results have been obtained in the general parameters of physical interest. theory of multiple light scattering which have potential Several more general deconvoluiion methods have usefulness in biology: been proposed. In every case restrictions of some sort, in) The leaf is an example of an intact biological such as smoothness conditions, must be placed on structure in which structural irregularities strongly allowed sohitions. The difficulty with these melhods is affect optical properties. The primary observation is that the class of allowed solutions is not well defined. thai an intact leaf is a much more efficient absorber of St amtejfc! das 3 sapessws *ri its «xmcied funnnii ' LOCALIZED SVMTHESS OF 2FJN Tar absorption spectrum of a leal, at **• as its other m MAIZE ENDOSPERM opticai properties, can be esataaaed very wel by a irmanun Ban and Fiances A km* maple two4ayet anM. 11k upper layer of tins auM contam* MOM ot the pannrrts. has an win of tlectron microgranV* show du; protean hudae*. accurately ihe concentration of bacterial eels in sus < iwirWtjill. pension over a aery wide range of optical densities.2 (r l Measurements of light transmitted through a slab of intensely scattering material may also yield useful MUTAGENESIS FOR PROTEIN COMPOSITION results. For example, using Beer's law to find the IN SOYBEANS absorption spectrum of pigment-containing cells in dilute suspension leads to results severely distorted by D. h. Foard. Wen-Kuang Yang. L. I. Triplet I. scattering by the cells. However, if the absorbing cells and C. D. Stringer are mixed with a suspension of norobsorbing but Methionine is present in soybean slor.ee proteins in intensely scattering particles, then the transmitlancc of limiling amounts for human nutrition: fr.-e methionine ibis mixture can be used, al least in principle, to obtain is absent in mature seeds. We are trying lo induce, by the undniorted absorption spectrum of the cells. radiation and chemical treatment, mutants which have higher levels of protein-bound methionine. The greatest Chcmiury Diriwm; forme;ly cnirailrwil. problem continues to be Ihe lack of a reliable and rapid 1. R. A. Mim jnd W. V. Lmiimt. HIM FhyikH. 27. J7<» 91 tmu. quantitative screening assay for methionine. Since other 2. (. W. Ibnthcr. I. II. Trucker, jnd V. V. Plurct. Bm- methods have thus far been of limited usefulness, we Itxhnol. Binrng. 16.475 M«I974>. are trying to develop an immunological assay. This 57 any omcr. B aprotcMducs iMcanbei has been the notation and character- iiation >if sack a prosea ftun amis of defatted soybean meaL Fwc peaks were resolved by wns uatrhnd. The pooled fractious of each peak went dtnH *ed. ty iphniiiif J. and IrydroJyttd • preparation for tauvc anwno achl analysis. Mrrjninhw the first three peaks only and in ihr anwant inrite third of these peaks. as a from only wwh extracts of cafyic- UVMUNOLJOGICAI. SfUDKS OF SOVMEAM nl bynrwtjrl cutsets i LECIVS A D.F. Fuani, Frances M. Taw.* acfjmy hi cotyledons occurred; MM) wcn-Kuang Vang aged. Twelve days after the cucu. II » hewiagfbjinusaon actiriiy Neither rite role of lectins in legwne seeds nor their extracts of cotyledons', however, the root extract. (ale during seed gcroiiatiun is known. I sute soybeans. which detected at low tilers in wk carry stages of we have beenn a study of these penbhnu. Oar anus arc 10 develop immanuligic methods to detect lectins in The of lectin fractions IV VIII in soybean seeds and scedkngsaad to me tins knowledge has been assayed using the to dciernunt the rule and fate of lectins. Sach dnTasi tarctni sera arc htcotpo- •nfttrwtaliun najbt be nsed to test the postulate' •' thai rated arc punched to bold lechus are the hinJwf mrvtunrsa tor the nitrogen- A circular precipitin spot forms fume bx-icnum Mmzfbmm. whicn m«*J^ jnJ forms the wen where antigen and antmody combtne. rtnuute* on the an heap* mots. The area of the spot is proportional to the concentra The two lectin fractions used in this study are peaks tion of the antigen. Comparing the areas of these spots IV and Vlll isolated on a Df Af.-ctOutosc cotautn. as with those formed by known concentrations of lectins, has been previously de*-iibcd.J Two different crude a quantitative measure of lectin concentrations in the extracts of lectins from seeds were obtained one with extracts was obtained, "bathe results were found only a pll 4.7 buffer, the other with a pfl 7.5 buffer. in extracts of cotyledons. Fraction IV lectin was Extracts of lectins were made from terminating seed present at a concentration of 0.2IS mg per 100 mg of lings I to 12 days after moistening the seeds. wet weight on the first day after soaking the seed, and Solutions of lectin fractions were injected sub- at 0.200 mg per 100 mg of wet weight on the third day. culaneously into rabbits al intervals of two weeks over It was undetectable thereafter. Fraction Vlll lectin was a period of eight weeks: final injections were intrave present in a concentration of 1.383. 2.050. 0.966. nous. Antiserum was prepared from Mood taken period 0.533. and 0.266 mgper lOOmgof wet weight on 1.3. ically from the rabbits' cars. 6. 9. and 12 days, respectively, after soaking the seed. Double diffusion on Ouchlcrlony plates was used to Immunofluorescence has been employed in a prelimi characterize the lectin antigens and antibodies. The nary manner using linn sections of different organs of antigen-antibody interaction is specific, i.e.. there was the seedlings. After adding fraction IV antiserum to the no cross-reaction between anlilectin IV and lectin Vlll sections, coat antirabbit immunoglobulin labeled with or IIS globulin. The appearance of diffuse precipitin fluorescein was applied. Sections examined under the bands indicated that the lectin preparation either was UV microscope showed especially «rorg fluorescence not completely pure or was a mixture of mote than one m the cotyledons and the epidermis of the roots. We component. Although the double diffusion test is need to refine our methods before drawing firm highly sensitive, il is not aMc to resolve a mixture of conclusions from our initial observations. r^-tSOt* "OftAt 1. I. «W7Jt 2. •, k.i»UMlj^t.USriaM*.Jnnr<-l».>«ll«74l. F«M4. AM AT 4m /*» A*p JW >»- #•**. tl«SI.-«W»J. EXOSON REFAaK OF UV-MWCED •mSWIMEMU OF IUWT CEILS UMfMM Last year we repotted that isolated protoplasts of cultured wild carrot eels could tttiatmdf repair DNA strand breaks induced by iasaunag radiation.' We also reported the first evidence of DNA repair wphcjliun m ptaM crib fotooring UV imdrHiou; however, direct *• K 4« to T2 assays of pyiiiiadiae dimcr excision gave equivocal rant orrc* uv inri results.2 Difficulties with the dimcr analyses were overcome with a mure effective DNA labeling pnice- bi the wild carrot rwtoplasls. thyi doners are nMtaly induced at a rate of 0.002'.; for a ? UV *nc cf I J/m . The 24-hr excision data fit a curve ratfftwimf v M lh> • showing 100^ dimer removal after low dines trjr. 14 * I0O xurr rjw*wfmry m ihv« J/m2 >. but the proportion of dinners excised is reduced 4 lo about 2^ at the higher UV doses. The efficiency nf the prrtopbsls continue to give a positive vital slain dimer excision after low UV doses is comparable to that reaction. observed by Regan and Carrier' in cultured human A wide range in excision repair capacity has been crib. Previous failures lo detect dimer excision in pbnl observed among different anuria! species.*' so a similar eds possibly resulted from the high UV doses em variation might be expected among plant species. We ployed in those studies (cf. ref. 4>. have begun lo examine a variety of cultured pbnl cells Although the disappearance in the dark of dimers for the ability lo excise UV-mduced dimers. Substantial from the acid-insoluble cefl fraction is generally ac dimcr excision has been found in celb of Hapttpappus cepted as evidence of excision repair, the dearest proof and tobacco, and in isolated Petunia protoplasts. bes in the simultaneous demonstration of excised dimers appearing in the acid-soluble eel fraction (Fig. 4). These data also indicate that, after a UV dose of 42 1. G. P. Ihmbwrf. R. W. Ifarl. Md M. L. Vrtic. Mum. *«•*. J/m1. dimer excision in these crib begins with a high 27, III (1975). 2. G. P. liowbnd jnd M. L. Yrllc. Btnt. On. Anmt. Phtg initial rale and proceeds with decelerating kinetics. Krp. Mmr JO. 1974. ORNL-4993. p. 64. Under the conditions of our experiments, no further 3. G. P. Hnwbnd and M. L. Ycllc. immediately inHowin^ excision is detected after 24 hr. even though many report. dimers remain in the cells' DNA (Fig. 4 and ref. 4). and 4. (V. P. Howbml. Malurr 354.1 W> I1975). 9» I'HilllJIIliaWI • II ||fal to « k of wii Aw jm_ if 74. u«M-tr»i. r- »- ami (AMI «*MV». I> M wed tana fc. «. m. NM( M1I.IL SC«WB. f»ur .1M .*** 5m 1X4 these fffwilL I'HIdTM —porj-w into aadtar DNA ns iwili aianj wcajat-aatfaae and wt ot I50X 10* MCCV0RA1NM iWO PLANT CELL BNA |JHldTfcdbjbihi DNA «.a • ihiii sacme avadwats. Idlwil. IFwUidl ow OC1 gradmts. and by auiiiianiytpaj laMo- t by Sirib A- FeiwBe)- dTM •to DNA-* fast vcar. wr awe < et at' law* aba neawncd thai a variety of •owed tar FdL'id wan by polyacrr get dMoanN«e*apay. U X RNAoraroMi Klana arlard lo *rt op sesj After a 20-fc* of iMumplw UNA ntMiM m the presence of 10'* M FaUrd and •r^aobyraaMagiacMawi |2**C|dTnd. W of ihe "C acuvny iwn« DNA. lotwMt ecu fraction oi cvrotard wild carrot oHk b We wish to we this ledwaaae ia arobiagtac fate of cbrauatoejaenjtaay tinned nth |: '*r|4TM «fc* (DNA. M tar afecinY of added FdLtd. lest dm IO? is. Ilftnr iriwh* are dtscwnrd mow rafty cbewhew J 1. I. WLI—r. V. R. FkckMcr. arf R. i*wwH". .wflRf. Rutfiar yiUkHMi of 10"* Jf FdTrd dun*; a 4 •MtOHk pctcMoraie or IC- w itm— hydroudr acav -w«!sp»!tfB. T-- ds'e. read's hsa bees ins^al »::h * be set nssade a desKcator to remove ataajsphKric smgfr ethylene coakVMraiioa. i ppm. which I icportcd ethyteae or carboa dtocude respectively. GnwiiJw is earber iaMbrts spore -yt mauttwt ivt 50 •. Ftekmmaiy scored atwr 50 br with ik Jkirfu aceuicaraane SECTION III GENETICS AND DEVELOPMENTAL BIOLOGY W E. Baraett and Cytochemistry I Genetics R F. Kimball R. A. Popp M E. BHirw; B. S. Bradshaw* Dimna L. Genrpr* J P DaueJierty* C. P. Hwsch Diana M. Popp Cytogenetics J.C>. Brcwen Yeast Genetics P. Carolyn Goivh J. F. Lem«Hilt F. A. Hiss R. J. Prestim Drosophih Biochemical Genetics E. H. Grell Medical and Mokcnbr Genetics James D. Regan DrosopMa Recombination and A. A. Francis Chromosomal Behavior Raymond Waters' Rhoda F. tircil Comparative Mutagenesis Structure and Function of Aromatic J. L. Fplcr Mnftienzyme Systems TiHo F. H. Gacrincr Mokcnbr BMogy Electron Microscopy and Cell Biology W. F. Barneit J. N. Dum rrwfdoctoral invcMipitor. 61 62 DEVELOPMENT OF AN AUXOTROPHIC MUTATION SYSTEM FOR HAEWtHHHS IXFLIEXZAE AND ITS USE TO COMPARE THE MUTAGENIC EFFECTS OF HYDRAZINE AND .V-METHYL-A'-NITRO-.V-NITROSOGUANIDINE R. F. Kimball HYDRAZINE MUTAGENESIS IN Last year1 it was reported that two proline auxo- HAEMOPHILIAS ISFLI 'ESZAE trophs had been isolated, both of which were clo ely linked t>> a locus for thymine requirement. It has now R. F. Kimball and Bernke F. Hirsch been shown that the two mutants are very closely Our studies on hydrazine mutagenesis, part of which linked to each other, pw a delectable but very low rate were reported last year.' have been continued. Further of proline independent recombinants, and are pruhahK work on mutation "'fixation." using lysates made at mutants at different sites in a proline locus. various times at'ler mutagen treatment and measuring A;- reported previously, one of the two auxotrophs mutation in bacteria transformed with these lysates. has has a low iprnBI) and one a high [prnB2\ rale of hoth failed to confirm the existence of the mutatiim maxi spontaneous and induced reven.ion. It has now been mum at intermediate 'in--, mentioned in the previous shown that pruBJ is a suppressible mutant and that report. Instead, mutati-m in the transformants increases about lHy- of the rt%-ersions are unlinked suppressors. to a maximum as the time bei»ven treatment and lysis The frequency of true reverlants is. however, quite high increases jus; as in the eariier work with nitroso- enough to allow detection by linkage to thy' in a carbaryl.: transformation assay. Thus the v. stem allows simul Almost all the fixation seeins to he associated DNA taneous ar.u separ jte assay ft r forward imitations at replication, since in a icmperati're-sensitive DNA syn suppressor loci anJ revertants at the pn>B2 site. The thesis mutant very little fixation occurs at the restric pr»BI mutant gives a low but usable frequency of both tive temperature. However, in experiments designed to spontaneous and induced reversion entirely by rever avoid any residual replicarive synthesis, no fixation was sion at the locus. found. Thus the only fixation is at replication. Comparison of the induction of mutation by MNN(J We have been unable to detect any single-strand (.V-meth>T-.V'-nitro-.V-nitrosoguanidiTe> and HZ (hydra breaks or alkaline labile sites in the DNA of bacteria zine! shows that the pmBZ systems, both true re- treated with hydrazine. We have also been unable to vertanls and suppressors, give very similar kinetics to find any evidence for post replication gaps. In both the previously studied mutations to novobiocin resist respects, hydrazine differ* markedly from alkylating ance. With MNMS. the mu'alitins increase as a power of agen's such as nitroguanidine and nitrosocarbaryl. We the dose between two an J iree ami reach J high have J!SO i>cen unsuccessful in finding any evidence for maximum at a f MUTAGENESIS WITH NTTROSO COMrOUNDS lUmtH Lsekgmund. The role of genetic back ground in mutagenesis by nitroso compounds is being Rosalie K. Elespuru.* R. F Kimball, studied in various repair-deficient mutants of F aili. No an J Jane K. Set low' mutant has been tested in which nitroso compounds are Mutagenesis with nilri>so compounds is heiiig studied not mutagenic, in contrast to other mutagenic chemi as a tunctKHI of chemical structure, experimental cals and radiations. Some evidence has been obtained conditions, and genetic background in two species of that two porymerase-defeclive mutants are less mutable bacteria, Fuherkhia ».••# and Haemophilia influenzae. with nitroso compounds and that the dose response Snueture-acririiy n-kttiimdiips. (a) Binding experi follows different kinetics. Experiments with a ments: Three nitroso compounds of differing biological temperature-sensitive DNA synthesis mutant have not activity (nitrosomethylurea. NMU: nitrosomethyl- provided evidence of repair under the conditions used. nitroguanidine. MNNG; nitrosocarbaryl. NO have been found to bind differentially to both species of bacteria. 'Student ai the IT Oak Ridye Graduate School of Bio The order of binding of radioactive compound follows medical Science*. the order of mutagenicity in both species, but only in Trrornt address: Biology Department. Bcookfcmen National F. a a does the relative binding correlate well with the Laboratory. Upton. Ne» York 11973. biological results. In H. influenzae the differences in binding are IHM great enough to account for the differences in mutagenicity, r..* do the binding results STUDIES ON MUTATION INDUCTION IN HI AGES reflect the differences in mutability between the tv. ~ OF HAEMOnaLLS INFLUENZAE bacteria. For example, whereas the binding of nitnnto- M. E. Boling and R. F. Kimball methylurea to the two species of bacteria is very similar, F. ciAi is mulagenrzed at a concentration During the past year, mutation experiments were tenfold lower than that required to imitate //. influ initialed with two Haemophilus phages. HPIcl and S2. enzae. From these experiments we conclude that the The basic goal was to compare results with the phages structure-activity relationships seen for mutagenesis by with those obtained in the whole cells. Both phages are these compounds are due only i<< part to differential lysogenic: consequently it is. in principle, possible to uptake of the compounds. Other factors such as compare the results when the phages are treated outside metabolism and reactivity of the compounds at dif the cell and th: n replicate vegefatively inside the cell ferent genetic loci must also be important after infection, when they are treated inside the cell and ih) Isotope effect Ihe mutagenicity of deuterium' multiply vegeiaiivcly. and when they are treated as substituted 'PtaMncluni Mwntqaii* «ypi»rwd hy Sabcitatnn 3322 The distinction between 3H and l4C radioisotopes in fruM ifcr Biiilory Oman*. OHM. i<> the Inntraiy of Tnwnm. KnoxvnV. autoradiographs would aflow a wide range of applica rNIH ftMtnoctmal Ken»w. VYacM i TsMt 4. HMWM Rwkocyw TF.M Time of Total TFM Normal Intercnaium treatment crfh fl> lw> Intrachanre* Triradial Shattered lame.) crib Sym. A«ym. (hr) Knred Control 200 19ft 2 0 0 0 0 0 0 5* I0"1 M 34 36 ion 96 3 1 0 0 t x 10"* V 34 36 200 179 15 9 0 0 2.5 * 10"* M 34 36 200 180 16 9 2 0 0 5 / 10"* W 34 36 200 140 49 29 3 5 3 0 1 < 10s M 34 36 200 82 194 35 22 29 5 7 10 1 x 10 *' V* (1 2 200 169 23 9 3 1 0 *PNA ttimiibted after TKM treatment. 67 '""* SfrrauitiftMU Ik •enrnn: lawnr.il Ihti N».of Urfe. S\m. A«B. Intta. No..* Deb. Sym A oi m* ** rt.v 6 .Wo 2.0 a 0 0 300 4.0 0 0 0 12 300 X.33 ii o 0 300 6.33 0.33 0.66 0 IS 3nn 14.3 066 ion 0.33 3on 10.33 0.33 0 0 24 3«» 4.0 0.33 0.33 0 300 9.66 0.33 0 0 30 3on 3.33 0.66 o 0 300 1.66 o 0 0 36 3no 6.66 0.33 0.33 0 0 2 mf kg TfV b Sin !.nn 0 0 o 100 3.0 it 0 0 i: 3on 7.0 0.33 o 0 300 6.66 0 0.33 0 IS 2oo I6.no 3.5 0 0 300 10.66 0.33 0.66 0 24 JOO 6.33 1.33 0.33 0.33 300 II.O 0 0.66 0 3M 30ft 4.66 H.33 0 o 300 4.66 0.33 0 0 lOmgkgtr.M 6 3W 3.0 0 0 0 300 S.O 0.66 0 0 \2 3on IS.33 1.33 0 0 300 36.33 3.66 2.0 0 IX 2on SO 1.0 1.5 o 24 N.. dmwofn" 300 62.66 12.0 16.0 0 30 No dnreom 44 No dnNoit* 3on 25.0 6.33 S.33 I.O y (fmfkgTfM 6 3«» 2.33 0 II 0 100 6.0 0 1.0 0 12 IX jUNt 4.o 0.33 0 0 3O0 20.6 2.0 2.0 0 24 V» dmfeXK .*» No dnrruon* 300 48.66 31.66 44.66 1.33 3* No dnmnxn Thi* part M ibr experiment wan frne twitx. TaMe6. Reciprocal we have undertaken the study of the cytogenefk after TtM consequence* of treating poslmeiotic male germ cells. The experiments are done by treating young adult male No. of OIMT imp/kc) No. <>r rrib mice with the mutagen of choice and then mating them, lran ajtfUrfr^rH-*^.*-'. 6S studied. The precbe inlenrab used ai each dose are of a visMe piece of chromosome material wovkl cause a summarized in Table 7. The interval* studied eno—.-pass lethal event. Thus total dominant lethality is thai the spermalogenic slants of early-spermalid t:.rough proportion of cells that contained at least one aberra- spermatozoa. ti«Hi. The level of predicted preimplaniaiion los» was Two basic types of chromosomal damage were ob- arbitrarily adjudged to be that proportion of cells with senred in these studies. The one type was simpk more than five aberrations, and the predicted level «»f chromatid aberrations, usually isnehromatid deletions dead implanis was calculated by taking the proportion and chromatid interchanges (Figs. 51 and Sift. The of cells with I 5 aberrations of the total with 0 5 second predominant cytogenetic effect observed was aherraikms. the shattering of the male complement I Fig. 50. and The levels of predicted dominant leth-,:i\ for the 50- this occurred most frequently ai the highest MMS dose and 100-mg/kg doses as a function of time are pittfted and the period of peak sensitivity. The complete data m Fig. ft. Actual dominant lethal data from a study are summarized in Table 7. with F.MS by (ieneroso and Russell' are also presented. The aberration data were used to calculate "pre These daU were chosen because of then completeness dicted" levels of lethality. In making these calculations and the similarity of actum of MMS and F.MS. An it was assumed that anv aberration that resulted in loss interesting aspect of the present data is the very high Tabic 7. Distribution of >wi*MMS Frnlvtcil ftcihttetl Pmlmol Pine Interval Crib with abemlinns Tout Cimirol 99 1 O r. II 0 0 IIHI 1 n 1 ion 2'/, 2J 6 10 3 2 3 «i 511 52 IV 49 1 5 /. •*'/, 6 0 1 1 1 4 45 61 •hi So 5»» fi 0 i> 2 H 5 h 27 5« |l Ml M> |im ll'/Uu'A 19 7 II 5 1 5 9 Ml «t 23 5X H'A 45 4 11 1 n (I 1) 5H III II lo I5'4 45 3 1 li » II II 5«» III o In 17'/, 45 4 1 <» » 11 II 5» IM II Ii' l*'A 49 1 •• 0 0 ll II 50 •» II * 22'/, 50 0 0 0 0 0 il 50 II II II 50 2'A 25 o n 0 II 0 0 25 0 0 ll J'.'. 54 9 0 0 II (I ft fci 14 II 14 5'A 27 »4 5 1 0 0 2 5«> 4* 4 M 9'A ;t 12 12 1 0 0 1 M! 54 I 52 in'A 29 II X t 0 0 0 50 42 II 42 II'AMJ'A 32 12 7 2 1 1 2 5* 45 5 42 »% 41 6 1 0 0 0 1 50 IX 2 16 l?'A ft9 5 1 0 0 0 II n 75 9 il <» I*'/, 49 1 0 0 0 0 0 n 50 2 II 2 2*'4 50 0 0 0 o 0 0 a 50 n 0 0 25 *% 44 5 1 0 0 II 0 0 50 12 n 12 A 4* 3 0 » 1 0 0 0 50 X 0 X 8'A i 9'/] Ml * 1 0 •• 0 0 0 H9 10 0 in "Total percentage nf cefc cany in? ai leaM ime i-hronwnomal aberration. Proportion of cefls with more than fiv* chromnwHiu! abrrrafionv ' Proportion of crif* with five PHOTO 2CI7-7S -S05S« IOC ' . 1»X\ \ ^ w> »•- w* *7« t ,Vt INTERVAL (dOfSi AFTER TREATMENT *r F*.*. YicMof (#> EMS a* MMS. SuM Imr » 3 ftncnlacd rrprcicnuiiua «»l the 34)0 rag kg IMS dau of brneiuw and Ruuril. *—-*. cakubicd tront abcnatiiMi data alter IIKJ mf. kr: •—• calculated 1 r-.»m abeiratiiM data alte." 50 mg kg MM5 CYTOGENETIC CONSEQUENCES OF IRRADIATION Of DKTYATE OOCYTES J. ft. Brewen. Helen S. Payne, and R. J. Preston ', In the past leu years a great deal of information has become available on the cytogenetic effects of irradia tion of male germ cells. This advance in knowledge is \ due principally to the simple techniques developed to in make preparations of ihe rneiotk chromosomes. Until recently, however, similarly si.nple techniques were not ?%.$. tyimplt 1 of lint efcavaff nufcuu •» lipfci ir ftfara ill available for studying the meiotic chromosomes of eoKvwd OTowr ova after wcafamt of ^cnMMM wrali MMS. oocytes. We have recently begun stvdying the types and (/!» Smgtr-doabir fragment urrowi. 1S1 (r Radial chromatid frequencies of chromosome aberrations produced in mfercbangr with accompanying fragment larritw*). fO diat- dictyate mouse oocytes by X ray*. l frequency of shattered chromosomes on days 5'/2 tion. Young adult female mice were irradiated with 200 through *>'/, after a dine of 100 mg/kg. This time R at various iniervab before they were induced to corresponds 10 when a high frequency of "infertile supetovulate. The intervals used were 1.3.5. 7.14. and maiings" occur. We propose that me "infertile mating*" 21 days. Metaphase I oocytes were analyzed for result from my early death of the hlasiomercs as a chromosome aberrations. Since the dkiyale oocyte* consequence of the massive chromosome damage. have completed DNA synthesis, the aberraliom ob served were chromatid-lype. The results of the experi ment are presented in Table 8. Once it was established that ar interval of 14 days I. T'. M. Gtmtmn and W. L. RMI. Nirut Ra ». SOT M between irradiation resulted in the highest yield of aberrations, a dose-response curve was done using this 70 interval. The doses employed and the aberration fre where the frequency o. translocations seen at Ml quencies obtained are presented in Fig. 7. which is a increases approximately linearly with dose. plot of log aberrations vs lug dose. The slope of the In early studies1" on the recovery of reciprocal curve for translocations is 1.7. indicating the presence translocations in the F, of irradiated mice, it was noted of a significant two-track component. This is unlike the that very lew translocations were recovered after doses data reported by several investigators for the male. of 300 and 400 R. This seems surprising in light of the fact that we observe l>r*- and ><»'< interchanges at these doses respectively. The actual cylological data were of mW oocytes used to calculate an expected recovery frequency of at after ac—c X-ray dnar of 200 R reciprocal translocations. In making these calculations, Interval No. of crib Dctetkia* InlCTcfcHipM four assumptions were made: la I based on ("-band •days! wuml IT I staining, symmetrical and asymmetrical interchanges occur at approximately equal frequencies: !/>> there is 1 100 1.0 • 1.0 1.0 • 1.0 no preferential segregation of exchange, or nonex- i 100 4.0 • 2.i» l.llr I.I) 5 125 4.S • 2.0 0.8 • 0.R changr. chromatids into polar nuclei. (<) inclusion of 7 100 6.0 * 2.5 6.0 • 2.5 any deleted chromosome, dicentric chromosome, or 14 125 10.4 ? 2.9 12.* - 3.2 duplication deficiency in the mature ova will result in 21 100 6.0 • 2.5 6.0 - 2.5 dominant lethality: (. possible segregation patterns is diagrammed in Fig. X. The possible segrt- gants conform to the ratio '» normal 6 duplication deficiencies: I balanced translocation. In the case of a symmetrical exchange plus a deletion, the probability of both exchange chromosomes and the undeleted chromosome going to the same pole t-> 'A X 'A: this '0 2C 50 '00 400 «000 then must be multiplied by the probability (V: lot"that pole not forming the polar nucleus, at All the proba Fig. 7. Vnaal log yield vs log dose plot of abcrratm* bility of the two exchange chromatids segregating oteiwr in •maphijt I oocyte*. i I I inter..lunprs. iBt p.n>lcd together is 'A multiplied by the probability I'A) that drielHtnv TaMr 9. Expected fmyortiuns of ova gmorvpn retuvertd 'run the data at 400 R 14 days before kxpectrd propof i N>n-. of: Aberration No. of Duphealion 1 t-abnerd 2babnced cbw cclH Deleted and dcfkieiHy fran%loc.iiion Iramloutiom None 36 1 deletion IX 1/2 1/2 1 "Presumed to be dominant relhah. 71 BIO-30672 / A ft OR »n »n A5 AC 99 99 tt fff Vf f ft OR OR OR OR OHX R RDr» ROHK ROHK OrW Dp W » T Fig. a. Scknmk RBfesnMatiM of the awHMr i the>' will not form the pola' nucleus. Thus the chance of approximately 43%. This agrees well with Searle and of recovery of the iranshKalior. in a balanced form is Beechy's frequency of 3S7 dominant lethality at 400 R given 21 days prior to conception. The calculated frequency of expected translocation heterozygolcs in the live F( of femaks irradiated with 1. I. B. Rracfl and L. Wickhair.. fknelia 143. 392 93 400 R of X rays 14 days prior to ovulation is IJMF (1957). (0.01/0.566. Table *>). If the same calculation is made 2. A. G. Scarlr and C. V. Bccchy. MuHi Res. 24. 17! M for our 300-R data, the predicted recovery rate is 067 (1974). compared with Seatle and Beechy's confirmed fre quency of 0.4*7 at the same dose.2 Searle and Beechy X-RA Y-INDUCED TRANSLOCATIONS reported eight semisterile offspring in 680 tested F, IN MOUSE SPERMATOGONIA from females given 300 R from I to 42 days prior to conception. All of the semisterile were females, and i G. Brewen and R. J Preston only three of them were cytologically confirmed as In previous reports on this study1-1 we presented translocation heierozygoles. The chromosome analysis data on tne yields of reciprocal translocations induced was done on somatic cells, however, and it is possible in spermatogonia! «tem cells of mice and observed in that some of the other five were indeed translocation primary spermatocytes following single or split doses of helero/ygotes. X rays. It was clear that the dose-response curve One other interesting aspect of our data is the following single acute exposures was hump-shaped, with calculated frequency of presumed lethality in Table 9 a maximum at about 600 R It was argued that this was li due to a differential sensitivity of cells in the different If doses which gave a yield on the ascending pari M stages of the cell cycle to both ceil killing; and the dose-response curve were split into two fractions, chromosome abenatii-n induction. The split dose ex the yield decreased when computed with the yield from periments described below were designed to determine the same dose given in a single exposure for intervals of whether or not this was a feasible suggestion. The °0 mm or more between the tractions as is observed in rationale behind the experiments was that cells should many other cell systems from TnJncantia to mamma show a cyclic response to translocation induction as the lian cells Howevei. for total doses which gave a yield cells surviving the first dose, that is. the ones more on the descending part of the dose-response curve, the resistant to killing, pass into sensitive or resistant stages splitnlose response was somewhat different I Fig '». of the cell cycle at the time the second exposure is Table 10). A dose of 1000 R gave a yield of 5 4 • given. translocations, and a single dose of 500 K gave about ownc-we TVSCM 0 3 6 18 24 48 72 3 4 ~"v V HOJRS WEEKS •MTERvA^ BETWEEN DOSES Fig. 9. The yield of trandocatioMs foHowmg 1000 R or 500 R * 500 R separated by variovs lime mierrab. Table 10. VteWs of reciprocal Iremlnoliom iadaccd by a 1000-R dose, a 500-R dose, or 500-R • 50D-R doses separated by nrino* lime interval* Dow Time interval No. t>l iTlls TMnshn.jth>ns • Ri bciwrcn di»e\ soircd r } 1000 500 5.4 • 1.0 SMI xno 17.0. 1.5 500 • 500 30 mm MM) 4.J- III 500 • 500 3hr 70O 17.1 • 16 500 • 500 6 hi 650 15 7 • 15 500 • 500 18 hr MM) 40 7 • 2.6 501) • 500 48 hr 600 23 8 • 2.o 50O • 500 72 hr MM) 19.5 • 18 50O • 500 1 week 600 23 5 • 2.0 500 • 500 2 week* MM» 27.0 • 2.1 5o« • 5oo .1 week* MMI 25 .o • 2 0 5»W> • 500 4 wo;k« 800 2o.5 > 1.6 Soo • 500 * wicks MM) J2.0 • 2.3 rmMHH 17 0T :.unsloc.»Kim The translocation yield increased chance hav- needed to be in stages of the cycle with as the interval between the Joses was increased, up to a similar sensitivities, because of the relative uniformity maximum of 40.2*? for an interval of IH In The yield of response of cells over this range of fractionation decreased l«> 2-vH*"? for a 4K-hr interval and still lower intervals. to \**.5'~, for a 72-hr interval, which i> close to ihe yield for a single -Jose of 5U0 R. The translocation yields for I R J Prm»n ind I G. Brcwm.lfc.Mf Rn 19. 2IS 23 fractionation intervals of I. 2. 3. and 4 weeks weie r 74 TaMel I jhuntiim Iicqacacin m Chianr twiwt hoae aanam afm unmr wlntiiiun nmexposufr Avcip Length >t ChrorejtiJ jberrjiHHi< r > vnrrifkr No. of edh 0, exposure exposure I line jiuly/>il Achromatic tppmi lht» l>eteik>R> tlwiunpri t.Kl ksjont Control 400 t HI •. "50 O.50-0 35 «•<»» 0.2 J 5 300 0.67 r 0 4? I 33-O 67 I1JM 11.15 ppm hr> 6 3w» o.33 • o.33 0.33 • 0.33 o.t»» 12 300 133-0*7 0.67 - 0.4? 0.0M 52 6 300 100-058 1.33-0 67 0 00 1312 ppm hrl 300 2 00-082 0 67-0.47 0.33 -0 33" 300 I.00 • OSS 1.67 • 0.75 0.00 Two dK-ermis-v I chromatid exckuiiee in the same cell TaMc 12. ChfoMinoMal afccmiioa (rcqaencits hi MOUC kafcocy les afttr ozone inhdition Average Postexposure Chro malid aberration* l':> No. of celk Oj exposure blood with- Achromatic analyzed Deletions hxt-hanee* ippm hrl dnw.il lime Ic-.tons Control 400 1.75 0 66 1 .00 * 0.50 o.no 075 Immediate 250 4.40; 1.33 0.4(1 • 0 40 n.oo 12 hr 200 3.00 - 1.22 2.00 • 1 «» o.on 1 week 300 2.00 o 82 5.33 - 1.05 n.jt} >o )j 2 weeks 300 2 67 0.94 1.00 • 0 58 O.tHI I..I5 Immedule 105 0.9* • 0.95 3.81 • 1.90 0.00 2 week* 200 1.50-0.87 3.50 - 1.32 n.oo 1.98 Immediate 30t> 3.33- 1 05 1.00 - 0.58 o.on 1 week Mm 1 67 -0.75 2 (SO t 0 82 (M*> 2 weeks 300 2.33 • 0.88 2.33 - 0.88 o.oo again it can be seen that there is no increase in through leukocyte cultures, which had been stimulated aberration frequency in the treated group compared with PHA 12 or 36 hr previously. Aliquots were with the controls. Spermatocyte preparations were also lemoved at different limes, so that a rart^e of exposure made from these mice eight weeks after exposure to levels was given Thf complete results for the cultures determine if there was any increase in reciprocal exposed 12 or 36 hr after PHA stimulation arc given in translocation frequency. No reciprocal translocations Table 13. There appears l:> be a significant difference in were observed in the 1500 cells analyzed, indicating no the frequency of chromatid deletions between treated effect of ozone at the exposures used. and control groups for exposures above 7.J* ppm hr in In addition to these inhalation experiments, several in the cultures exposed 36 hr aiter stimulation. vitnt exposures of human leukocytes were performed. The second method of exposure was to put leuko There were basically two methods of exposure. The cytes, which had been stimulated 12 or 36 hr earlier, first was to bubble ozone, of a known concentration. into ozone-saturated solutions The exposure times. 7S together with the aberration data, are given in Tabi> 14. exposure level dun IHM significantly increase the There was mi difference between the treated and frequency of chmcioviinc aberrrtHins. except in the control {(roups for cells in different stages of the cell one instance Thh is the case whether exposure is by cycle TJMTIJ. kaJfcorylr MI2i IJtfcrjfterniA Chromatid aberration* I ~> Oznne txposute No of crib Achromatic tppm hrl analyzed Deklum rxchamw* tnium 12 hr Control 900 4.0 ±0.67 3.44 . 0.62 0.00 13 300 1.67 • 0.75 2.00 • 0.82 O.OO 2 4 300 2*7 • 0.94 4.00 • 1.15 o.no 2.6 300 3.0© • 1.00 3 00; 1 OO 0.00 4.8 300 2.67 • 0.94 3.30- 1.05 000 7.5 250 5.60? 1.50 5.20 • 1.44 0.40 * 0.40 3* hr Control 900 3.78 • 0.65 3.67 f 0.64 0.22 • 0.16 165 300 1.67*0.75 3 67-1.11 0.00 250 300 4.00? 1.15 5.33- 1.33 0.33 i 0.33 '40* 300 1 67 r 0.75 4.0O- 1.15 O.0O 300 520 3.t>7 till 7.00 : 1.53 o.on 5.73 231 0.87 • o.ftl 7.36- 1.7* O.O" 7.00 300 4.33 - 1.20 4.67 • 1.25 o.l? 7.23 120 10.00 • 2*9 14.17 • 3.44 0.00 7 95 250 5.20- 1.44 12.00 • 2.19 0.00 14.2 ISO 4.no ? 1.63 800 • 2.31 o.no TaWe 14. HWUM knkocyie culfwcs exposed to an ozoiie-nfanled snkitiM at 12 and 36 hr after PHA sfimnblimi Kxposure Chromatid aberration* IT) No of cell* lime Achromatic analyzed Dektkm* Fxchanjrs (mini lesion* 12!- Control 900 4 00 • rt.67 3.44 • 0.62 ono 30 30u 3.00 • 1.00 4.00- 1.15 o.no 60 250 3.60? 1.20 6.80 t 1.65 o.no 90 160 5.63- I.M 5.00? 1.77 0.00 36 hr Conirol 900 3.78 • 0.65 3.67« 0.64 0.22 • 0.16 5 300 8.67 • 1.70 4.33' 1.20 o.no 10 300 7.00 • 1.53 7.00' 1.33 o.oo 15 300 6.00' 1.41 5.67- 1 37 n.no 30 300 6.00- !4I 5.67 • 1.15 0.67 • 0.47 60 250 2.40 • 0.9* 2.JMI • 1.06 0.00 90 150 2.00- 1.15 6.00 • 2.00 o.no 76 •or exposures above 7.J3 ppm nt to culture* 3b hr after an asynchronous population. However, the number of PHA itunulatioa. when there was no itmhr increase m mitotic figures was reduced severalfold m TO' . At snnibt eels exposed to saturated suiwions of UAW present we ate investigating irw effect of TG' on V» realty satisfactory expbnati.* can he offered for reversion of gfy D locus, using a spontaneous TC the one Mgnirkant increase in aberration frequency. nwtant derived from K, I* It is possible that the TC These results are very different from those of Zelac et mutation alien the metabolism of the cell in such a way tL, and somewhat different from those of Men et at that a defective repair process m MNNC-treated cells Ozone is death. from the description here, a very weak results in increased lethality and possibly an increased clasiogcn. iv-tut -mutation me. 1. It •.;. ZrUr. H L Cronos. W E Mck.B.C DWOM. jnd B A k BIO- 27991 t.5kr* aOfcran, N « MCUBATiON t 20 hr •.».<*.• NORMAL CELLS XP CELLS OS-iilO"* 12 *»109 0 4 6 16 *J05 313 nm FLUENCE (trgs/nwn*) Fig. 10. tUkHomMp of the difference in the reciprocalo f the wwjnn wrap mokeuar weip>i of trthtr nuimul cefc or XT crib irtuMnicd vritfc JO kntfs of ""Co Trays wdw wWtopm ana wenbtwl cither at long times or moel time* before awry. The different *ymbpl» refer to different experiments. w lnKKWIPIMV •*•£••> «** «•> •* «MWM*«>r-41 r;j«f»»HWIW t,/e^s*#ii&*i*mm*«'i<*> 71 BIO- 30463-1 patients with XP exhibit varying abibties IO perform this process.'-1 Cotnf4en»rniation tests have revealed ETHYL HETHANESULFONATE: poops A. B. f. D. E. and the XP variants, these being 2 K)~ M 2hr, INCUBATE l8-20hr able to excise <2. .17 25 55. 60. and I0V7 of fBrdUrd • HO-UREA) ,., UV-mduced pyrl km respectively .3 INK _ V.S'Snm IdTW • HO-UREA J* The existence of a postrepticalior repair process m hmmn celb has been shown more recently.* UV 1.0-i«10_t irradiation retails m DNA synthesis being Mocked by a pyrt Ipyr. leaving a gap of ~I0J nwcleoiides This gap rs then fnVd in to join the shorter segments, resulting in h%h-nnjlrctilar-we^bi DNA. Mil ef nt* found no difference with regard to this ability in normal cetts. in XP cells unable lo excise pyrt Ipyr. or in XP variant cetts. More recent studies by Lchmamt el «t* on similar cell lines at earner times after UV irradiation resulted in the statement that XP variants are considerably deficient in posirepncaium repair, and XP A-group cells are inexplicably intermedi 12 * 10' ate between normal and variant cells with regard to this 313nmFLUENCE (ergs/mm2) ability. Studies in our laboratory have shown that XP B and D-group cells are also intermediate, resembling Fn> II. K*a*$ at a* amtfant *immmI ami XT cent after group A. However, a closer evaluation of both our data s MUM wn* 2 x 19 Mtmmi* WHUMptmMimwmtf and Lehmann's reveals that the XP variant, and lo a av«* at 29 hr after iman. N»M ibat ike XT crib nan ihetc lesser degree XPs of groups A. B. and D. although conditions apncar to display detective repair of fcwon* induced by thb chemical agent when auayed at long times after the synthesizing DNA of a smaller si/e than normal cells at drfevery of the original injnlt. and noraal cent appear to exhibit a set time after UV irradiation, increase the molecular an UV-type repair response, contrasting with the mail pre- weight of their DNA/unit lime after UV. the same as in vioady found al ifcori limes after treatment with this chemical normals. XPs. and variants (see the slopes of Fig. 3 in agent. | J. D. Rcpnand R. B. Sctiow. Cancer Ret J4. 33!* 25 ref. 6). Secondly, although Lehmann rial, slate that in ll»74M all strains the DNA becomes "big" after 5 8 hr. their gradients have a limit of resolution at a molecular and whkh may appear l» be rather differently repaired weight of 150 X 10*. thus nuking difficult accurate in normal and XP cells. DN A-damaging agents may estimates of their si/e of "big" DNA. We have shown induce several types of damages, these damages will he that such "big" DNA is larger at 5 X hr and longer in repaired according to the structural configuration of the normal cells as compared with XP and variant cell lines. damage and the suites of enzymes available and evolved Control experiments are presently being conducted to for dealing with these damages in human cells. determine whether the rates of DNA replication, both with and without UV. vary considerably in all the cell lines. If variations occur, both pulse and chase limes 'Prevent addict*: Boito^y Department. Rrookhjven National will require adjustments for accurate comparison. Laboratory. AvMmjtcd Unncrtiiies. Inc.. Upton. New York 11973 Finally, some differences in Ihc sizes of newly replicated, chased DNA may tentatively be attiibuiable to a phenomenon reported by Buhl and Regan.7 These POSTREPUCATION REPAIR IN NORMAL authors found that in an XP line unable to excise HUMAN CELLS AND IN XERODERMA pyrf Ipyr. for I in 30 of the pyrt Ipyr "by-passed " the PIGMENTOSUM COMPLEMENTATION CROUPS newly replicated DNA contained an endonuclease-sensi- tive site. No such site was observed in Ihe "by-passed" Raymond Waters and James D. Regan regions opposite pyr()pyr induced in normal cells. An Initial studies on the prereplication repair of UV-in- extension of this study lo include groups A. B. C. D. t. duced pyrimidine dimers |pyr()pyr| have shown that in and XP variants has been undertaken. It is possible lhal normal human skin cells these phot^producis are although posireplicalion repair makes ihe DNA larger, removed via excision-resynthesis. whereas cells from attempts (defective attempts?) to repair endonuckase- •Ml 79 sensitive sues in ike newly synthesized DNA opposite We subsequently showed that XP cefc were pyrtlpyi make the DNA sughiry smaller in the XP and essentiaty incanable of excision of these dnaers.1 variant eel nan. Recently we showed that, wane in earner reports we Considerable work wdl be required to clarify the found only 50TJ excision, with impiuwjd methods and rcfaiiondiip between pisircphcaiion repair and the XP lower doses we could not demonstrate complete exci phenotype both m XP and variants. It is hoped that sion of dhntrs in normal human eels.1 these experiments wdi etnwnate some of the possible Sutherland ef nt* haw recently reported tbjl human artifacts and clarify the significance of what b inserted ecus are capable of the pfaotoreversal of UV-mduted opposite the pyi< myr in these various XPceU lines. pyrimidine diners and possess pnotoreactivinj enzyme activity Sutherland etal* further showed that XPceRs have lowered levels of photr.reacthatmg enzyme. We *r\m*Wto»l JwcoUfaio sapewioi Hr Suacuwiract 3372 have therefore investigaied (using Sutherland's experi from ibe •**•*) Dmuua.ORNL. to rfcr Iweraty trfTemev mr. Katoivinc mental conditions) the photorevenal of UV-mduced 1. J-E.Ckawr../vaf»v2lS.tS2li9tt). pyrimidine dimers in normal human ecus and two 2. R. B. Srtfcnr. J. D. ReganRegan. JJ. Genua*, and W. L. Carrier. different XP fibroblast strains. The data of these Aw. XMI. AtwJ. SciVSAtt. 10*5119*9). experiments are shown m Table IS. As can be seen 3. J. II. Rattan. K. H. Kranuct. M. A. Lain** >. W. from these data, there rt no photoreversal of UV-m I-VMMIY. and H. li CtMm.j4m. Intern. MeJ. Su. 22 I 1974). duced pyrimidine dmers after 60 mm of white-light 4. S- N. MM. R. M. SlitaKM. R. B. Scnw» and 1. D. RcfM. mk>pky* J. 12.1IS3 < 1972). treatment under Sutherland's experimental conditions. 5. S. N. BaM. R. B. Sctlo*. and J. D. RVpn Hopkyv J. 13. We therefore concluded that neither norma! cefts nor 12*511973) xeroderma «.cRs are capable of m mo photoreversal of *. A. R. LehnMM.S. Kirk-Rdl.C. F. Arlrtt. M. C. falenon. dimers. Although, it has been clearly shown4 that these P. M. Lohman. V A. drWcrrd-Katiciem. and D. Buofxwa. *>ur. cells possess an activity which appears to be pbotore- Xrtl Acml Sri ISA 72,21911975). 7. S. N. BaM. and J. D Regan.Smtwrt 24*. 4S4H973) acirvatfcm enzyme, this enzyme apparently does not perform its proposed function m mo. LACK OF PHOTOREVERSAL OF UV-INDUCED PY1UM0INE DIMERS IN NORMAL HUMAN 1. J. D. Regan. J. fc. Trosko. IW. L. Carter. *mpmjn. J.«. AND XERODERMA PIGMENTOSUM SKIN CEU5 3:9 2S«l9*t) 2. R. B. Serinw. James D. Regan. J. German, and W. L. W.L. Carrier. W.H.Lee, and Carrier, hoc. Stil Aamt. Sti USA *4. 1035 41119*9). James D. Regan 3. W. L. Carrier. W. H. Let. ami i. D. Regan, iinwnrtied lo Btophyt. J. We first reported in 1968 that normal human cells are 4. B. M. Sutherland. M Rkr. and E. K. Wagaei. five. XttL capable of the excision of UV-induced pyrimidine And Sri. VSA 72.103 711975). TahV 15. Lack of pbnfnrcwnaj of IV-maWed •jihnrdwidunewin •cima! tmmm and %cvodcrma amutiifojw n dua cans Dimm rjmivT* I'VoWai 254 mn < Norm?. 3 0.055 0.05S irBBT) 75 0.270 0 320 XTI2BE hi 0.041 0.043 (lay Tim) 75 0 34 0 30 100 0.43 0.42 XT5GOR (SnGre) 10 0045 0.045 EFFECT OF PUTATIVE REFMR WUMI with .*l3-nm bjhf. produce* a photodynaanc effect ON DNA RETAK M NORMAL HUMAN SUN resnkmg m fnrther breakage of the DNA. However, by CELLS AFTER ULTRAVIOLET RADIATION the methods of onr analysis this photodynanMc effect B subtracted DM from the repair inhdiiiory effect (Table lames D. Regan and W. C. DWM. Jr. 16). Hydrazine snlfate at IO'1 Jf was also fonnd lobe Using the BrdUrd phototysB away fin DNA iepair. we an effective mhdMor uf repair bni had no photo- hair evtmmti a number of pussdde hnnbitorsofDNA dynamic action. Three other omnjunndf I l-naphmyl- repair. SUNK of these chnwi.att ate prodnced by coal anuue. fnryifnrjnndr. and nalidixic acid) had significant conversion processes. Cerua others of these agmts pboiodyuanMc effects, in combnuinn with the 313-nm have beta saggesied at pussaMe •nrpair inhljiiors or are hghi. on the cens. but did not inhabit DNA repair. mlubitors of semKonsenrati*e syMbesb. The data from Further studies of the mechanism of the cumpuimdi. these experiment* are «nanu»ued in Table 16. foundtobemMbtory to DNA repair are in progress. Ojmacrine hydrochloride is an effective inhibitor of DNA SMgk-siraiMi-break rr^inmg after jinwni radia- MTROSOCARRARYL AND NTTKOSOCAttAlYL lion in human ceMs. We fownd it had no effect on repair UUCOInVOUNDS: eels aftei ohravtolet radiation. S-ARHKO- I EFFECTS ON HUMAN DNA at I0'3 M is a my effective inhibitor of R D repair, inhibitint by approximately 50T. Loner -or.ccn- ky. A. A. Francis. tratiotts aho inhibit to a lesser degree. 2.-»-DKtitro- i D.Regan phenylrydrazine at 5 X I0~* M » abo an effective Hnman skin vets (normal snd xeroderma pin inhibiiur of repair. It also, upon radbtion of the crib mentosumi were treated with carbaryl I.V-meihyl-l- TaMrM. ENactafl i duncvm DNA UVvaau ll/i . vrl * 10 " nn>:oity AfTM Dow* 0*fr.4 **p*.1 OMMTak byaVutMnoaV 2x !©"*.¥ y» J42 N.-K- in"*.*/ J.5JI it>2 1 X J.53 155 S*me ""-"*-*" 5x lll"*.V • i-«w;» 2.21 2* IO"*Jvf • 2.51 Mtydroxyqamohac 1 X IO"*Jf Hi 4 Ml \.me l-NapfcihytMninr $A IO"*.W 2.V.1 242 Pfcrtt^yiUNMV iw4 wl rcaWluMi t>y .111 mn ctpul m JThd jMi HfdVid mtnbui--a1 »•*•» 244ethyku91k*te»c 5* W*M 2*3 24K N.<»c 2.4-DmMr»pfccnyuiyd»/mr 5 ' •• 275 1 J* Ph«i<«iyiij«Wi n!"l wi nrdihtmn 1 t. lrt"*,W i.im In .11 i MI MMra|ajl m «Thd jnd Rrdtrd mtwhifni or** Hydra/me %«N*ic \ it IO"'.¥ ••* 1.54 Konr t-oryK.rf.iHM*> «.\i--2» 1* n»>t<»?yiumk Mill wl rrdattmn 1 ' 2 V* by .11 i nm Dr»>\yihotj«r 5* in"5.*? 4 04 V7ft PBinr HjhdwR JCM 1 X 257 rtmlitdyiMmfc' H»I| wl rrdavimn 1 ' 2*7 by 31 .t .-MB n|iul m aTfbP and ifdl'ral HHnlMWd ,rn> "I xpmnrc IIMC -- up** pvnud (20 hr» Un (iV-rcpan HMfcrt -• 90 mm fin DNA Atmaire Madim fti l/rnol. wim mrjMMCof itee macniivdr nf rrpotr A bmer & l/mol. wl mraiK k%i Rfdl'iri mvrnow. hrihT Irw rrpuit. •1 •TM"1* «10* feW—im lhw>wa y nan: Znvmac 2hr 20 hr Tamel Ttcarn* Tnatctf TlOMi TICMOI wMh .V-Mtruw •MM Atnmncaf •BOtn-wfc •uttlit'wtr •BKCfSCHOf Ahfcoffc 294 US 272 122 207 135 BsypM 270 III 220 •0 203 90 •VX 200 151 279 ISS 274 149 CalM'Baa 2S9 I0S 270 172 270 170 1 IBOIW 2M ISO 310 210 2K ISO •Vrhuarrl 250 139 200 170 J22 270 Owirot -jno -300 -300 napihyl-tarbjRuiel. a ommun agricultural pesticide, ur from ihe culture, the DNA-repairing events normally Ms A'-nrtrderi*aiive. mtrosocarbaryl. and the DNA of occurring in human ceBs after UV- or ionizing-typc rhe cefe was stdimentcd in alkaline sucrose gradients at damage did not occur. These observations suggest thai several times after treatment. Numerous smgk strand these six nitroso derivatives of AT-nwmy1 J. L. r-pkt. Alice V Ibrdrgrer. and Ti Ho 1. J. D. Rcpn. H. Vndupkfc. S. Takctb.». H. I **. Mil XI with Kabm,SrMiKr 1*3.14521 !**»». 2. N. r»jnwni>. D. G. Dofcrm. awl J. D. Kcjaa. J \a* M. K. Guerm.* HrsasJu Kubota.* and I. B Rubtn* Ctmrrrlna. SI. 7*1 H»7Jl. 3. R. T. Taylor and H. Wcnsfc**. Am* B**h.m 13. *> In a prehrnnury effort t» examine energy c»nvenion ll**5>. processes, a coRahoralion with the Analytical Chem istry Division was begun. The chemists furnished partially purified and identified fractions from the COMPARATIVE MUTAGENESIS crude product of ibe coal conversion proves*. Ptelnni- J. L. Epler. Alice A. Hartbpee. Ti Ho. nary screening for mutation with "Ame>" >min\ Ruby D. Wilkerson. and William Winion (Sefmiwreiat) was carried out. A numbei ol the iracthins produced genetic damage. A tew were extended t<> the The major coal of the comparative mutagenesis group human leukocy te sy stem I«H C> logenctic -inalyses. B to provide a means of mutagenicity lesting of those The microbial assays m%<4ved the determiruiM>n ol a compounds produced by various existing or proposed crude nuclrvalion curve and. whsequenily. reversmn of methods of energy generation. These compounds in four different hr-ndme-requi'ine. mutants The tour clude the primary- effluents of existing fossil fuel "Ames" strains utili/ed differ m their mutational sources such as sulfur dioxide, the oxides «»f nitrogen, origin: TA 1535. a base substitution, and T\ l>3*». ozone, hydrocarb-ms. ami heavy metals, as well as 1537. and I5JW frame stuffs. products of newly propiMcd methodology such as cool Results are preliminary and are ifoioBsiy obtained liquefaction and of auxiliary methodology such as ustng rmpure fractions. Attempts wiR be made (•> cooiing'iower additives. ascertain the sprcirk compound!*! responsible '••» the The work is divided into two pluses: one dealing with mutagenic action. The assays will he exiended ••• known compounds expected to occur in the environ fractions obtamed by hqwJ-hqnid chfmaiiyapfcy ment through energy production, conversion, or use. These mvcslrgaiions. especially the mcNtence <4 and another dealing with actual samples from existing frame-dufl mutagenesis and its owietattmi wnh carenvo- or experimental processes. genesrs. are the mmai attempfs i«» •*•-*•!•* nmr»n- To approach the proteins of dealing with and the riea laity imporiani processes fur genetic damage testing of large numbers of compound*, we set up a form of the "tier system." Operating units uftli/ing Salmnncfh. /:". c<4i. yeasi. human leukocytes, mam * Iwryrirel < fcnmunv l)»»wa malian celb. and DrimtphUa have been initialed. Coor- dinaied work has begun in whole-mammal systems. GENETIC SCREENING: MGH-RESOUfTION Compounds giving positive results in the "lower tier" CHROMATOGRAniY AND IDENTIFICATION (microbial) assays will he tested further in the higher OF METAIOUTES organisms. J. L. Kpter. C. D. Stringer, and William Winion During the post year the hulk of the experimental results was obtained with the StUmmdh reversion The research of the Mochcrmcal Analysis .»f tienefic system. Approximately 150 compounds were prrlimi- Vanants group consists of iwo general efforts liii ihe S3 tcreenmg and rhromatiieTaphtc analyses of selected Recently, we have developed aCHO mMattonat assay genetic vanantv and I»| the isolation and ideniificalion suitable lor unaniitating inducrw mutations at the of me»aholtte% presumably unrobed n genetic lesions. HGPRT locus by various ptr>steal and chemical agents.' The primary materials mvestigaied hate been himun Recause of their nhrubtasi ongwi. the cefts lack the body thud* and pbnl extracts None of the human activation systems for converting some mutagen* to sample* imeMigaied thus tai has pointed to a metabolic then active forms To broaden the applicability of lesion under genetic control >iui »a> mil suspect or mutagenicity testing of our CHO cell systen*.. we have a!ready known to exist. With resoeci to plant material*, undertaken an approach thai combines many advan a preimwury coUahontrve effort was wrt up with Dr. tages of the bust-mediated assay hut without the Peter Carlson* in order i<> look at an array of i<*n uwcertamiies concerning qualitative dissimilarity be mutants from the point of VMW of detecting bio tween the bust and indicator genomes. In these prelim chemical parameter* that might be indicative of the inary studies, genetically defined CHO ceHs. clone baar* for the genetic lesion. K.-RIU (generaly referred to as CHO-K.RM* cells), In addition, (he collaborative effort with the Rant which h cetb could be etmunaied because mednm F12FCM|IU> DOSE RESPONSE OF CITATIONS AT THE dfci not favor the growth «»f primary mouse ceH* On HYrOX ANTMNE-GliANINF ntOSTNORtBOSYL recul'ure. the mdrcaior CHO cek denw-J from the TRANSFERASE LOOTS BY ALKYLATING tumor had smite ceil morphology i» ihe original cell AGENTS IN CHINESE HAMSTER line ami karyoiyrMcaM* were exclusively ("HO cells. OVARY CELLS' HrstologKal section* of a series «4" THO-tumors' growing in nude imce revealed a tumor thai was well A w llstc.Patiicu A Burner. J. T. 0"Vill.» circarrtscrihed but ntmencapsuiaied. The tumor celb and I) B » « •5 experiment* Me being earned oui mm? V-metfcyl-.\ - frequency per unit of lunlmrp dose demonstrated that niir<»*>ur«M JO«J V-ethylA-Mtr>«w«nea Result* from I mm of iwntamp exposare yielded an "eqssivjknt"* I'V these *iudic> w* etiibfcsh ike rule «•! ihc chemical dose of 70 ergs/mm'. X ray at doses up to 100 rads did Mructure and tin: reactariy <4 jtkyljlme a*enl> <>n two nut significantly affect me survival of CHO-K, BH* b»&>*jcal cttcctN cell tunrnal and induced ••ulaiioiiv cefc. However, doses of 200 X00 rads produced an exponential low of cellular reproductive capacity. X ray I In |>u«i m V,»FW.I. <.-0<~-«nfNt I.J47 Mil**^" ai doses lower dun 200 rads dtd not appear to induce •VIM tW.I.*t..r.ri fdhm. mutation* to (vttHoguamne resistance appreciably. A r»M4>H.l BY rtfYSITAL AGENTS IN CHINESE t In pari in SUMMIT ft* fewnrs i.Z*l *l.-iv75». HAMSTER OVARY CELLS' 'Slaarai at Ifcr 11 Oak Ryjtr (*jafe:t« Scfcml at B»>- A.W. MM:. Patricia A Brimtr. Richard Machanoff. J. (.Riddle.* and A. Mi* ORIGIN OF VARIANTS RESISTANT TO Kxposure .4 CHO cert* i<> I'V dose* up !•> "^r> PURINE ANALOGUES IN CHINESE flpnwi: did n«n significantly reduce cell Mirvnai. IV HAMSTER OVARY CELLS dose* •>! H«» MK crgv'mnr produced an exponential A. W. HSK. Patricia A. Braner. Richard Machanoff. ceH kdhng. The observed mutation frequency !•> b. P. Kirsch. L.-urse B. Ewing.* and Linda S. Borman* n-ihmguancnc resistance mduced by (*V increases in proportion "* increasing dines up (•> 2oO ergs'mm1 m Studies from many laboratories have established that the ranee of 5 MN ergs mm2 examined. The pooled the enzyme HGPRT is responsible for the sensitivity of data >>l mutation frequency. fl.X). as a tunvtion of dose mammalian ceRs to purine analogues such as X-a/a- X (urn* U ZUi ergs'mnr is adequately described by guanine and MhJoguanme. Whether variants resist an J to /f.Vl - 10 * 113* • 2.04*1 One interpretation of the purine analogues arise as the result of gene mutation or Imea: dose response is lhat the llr'-induced premula- of epigenetic changes in diploid I IS) and tetraphud |2S» liimal lesions, presumably the unrepaired pyriimdme CHO cell lines was examined by investigating tat the dimers and other photoproducis. increase with increas difference of the dose-response relationship for EMS- ing l!V irradiation, which is directly responsible tor the mduced cell survival and mutations to Mhioguanine induced mutations. The possible cell-cycle phase speci resistance, which are known to affect primarily if not ficity of CV-mduced mutations to tvthioguaninc resist - exclusively the HGPRT locus: (M the differential ance was studied using synchronr/ed cells obtained by effects of H-azaguanine vs Mhioguanine on the spon the mitotic shake-off procedure The results indicate taneous and EMS-mduced mutation frequency. •<» the the highest increase >.A mutation frequency in early S physiologic-biodiemical analysis of the HGPRT activity over that observed in G(. with a subsequent drop again of ihe druiE-resisiani clones. We observed that EMS as the cdK progress through S and G2. Late mitotic induced higher cell kilting in IS than in 2S cells at every ceHs appear In yield a relatively low mutation fre dose examined. Both DK and D„ ate higher in 25 than quency, l.'sing an exposure distance of .1* in., as m IS celts. In the IS ceHs EMS at 25 ug/rrrt induced recummended by the manufacturer sunlamp txposvre mutations to r>ihioguanine at a frequency S times the of up in I mm did not affect cell survival appreciably, spontaneous frequency. The induced mutation fre an exponential cell killing was observed wtien cells were quency increases with increasing doses up to 200«g/ml. exposed to (he sunlamp from 1.5 6 mm. Sunlamp- whereas in the 25 ceffs. EMS is ineffective in inducing mduccd mutations to Mhioguanine resiMance could be any appreciable increase in mutation frec/iency. The observed for an exposure time as low as 15 sec. and virtual absence of induced mutation at this recessive increased proportionally with exposure limes up to 4.5 locus in the 2S ceHs is consistent with the notion lhat mm. The shapes of sunlamp-mduced cell survivals and ihe Unear dose response to 6-lhioguanine resistance mutations to 6-thtoguanme were similar to those induced in 'he IS cells arose as a result of mutation at produced by UV. Analyses of the rate of mutation the gene or chromosome level. Furthermore, the spon- m both HCPRT and CwPOK negai* wonhl be classified eel type* WJ* conquued by varying the MUMOIW at targe detenu**. of »ni| i 11.2. SO. 7.5. and 300 Mj/ndt ani > nniigniniur 11.7 and 6.0 ng/ud). to bulk eel types, both tb* spontaneous and the nuluced mutation tre- CEIXCYCLE PHASE SPEOHnTY Of nmntiti decrease with iucreanng concentration of UV4JCHT4MRJCED MUTATIONS TO punnr mJkiipui. When S^opHnt at 3Q M£ mi was •^TlflOGUAJvJNE RESISTANCE IN 1MKLM. •acd. no reiiinui cniimiri were utwwJ in spuntane- HAMSTER OAA*T¥ CELLS ons or EMS-treated adman of etmer eel type. rYtnni- J.C. RnUkr.* Richard Mactsanoff and A « Hsur nary phy nulugn. bjuthtuuijl dMacteri/anou u» nV HCPRT activity of !fce resistant clones uuncaies thai Studies from nwciohnl >\>iern> have *h»wu thai wntc anjph proportion of nVwarrsinf at lower ft-aognamur cbenucal muiinrai. act pmnarny on repncaiMC DNA In cunccnhattons (1.2 and 3.0 ng/nril couiani lug* levels order to Jcttinmn, whtthei manHwdun eel* rmgtti a#-»» of HCPRT activity. suggesting that variants unaffected dhow a preferential S phase seummts i«> mntagem we ai the HCPRT loon were selected under less string* ut are investigating the mutant me eftexts of IV rrradiainw selective conditions. at different tune ponu* durmg the eel cycle «•• CHO eels n culture. The genetic marker fctuWed r* o-rhmpuninc resistance, which » known t» rout! from •SrwJrw jl iWll (Mi ftufer (MBK Scthi-I .«f Bw- alterations m the HCPRT bicus m C1K) veih. The pmcedure used involve* nhiamm *> nchf A V AMANT OF CMMEK aVUsSTEK OYAftY he swuaMated foe bysjoKanaaue ant ajycaar at the COlSmraALTBtEPTKAVBOE OFCELL CYCLE The observed ahdity of fohak acid to uplau hypuunaine and f)ycaK in ate calawr natdnan J. P 0~NcdL* LaaJa S l«n: i^AWIfat saajnts aaa das vanaai may he dtlkieM ia the fW> eels M cahare base a ceft cycle «f 12 13 he fwnaanua «rf Ktiahydnduliv and iTHFl aad its dema- wmch can he divided apfXwiwwleK arto C.14.5 5.0 Mes. alach play arale m iht ftaaialiua of ffyciat frum xnae aad m the cumctswa ot deoxyaridne najao- brl.St4 4 5btl.O:l2 23 lal. aad Ml I hr». Variants with a kawri cei cycle laae were IMIIJUJ by waif phoaahaie lo deoxyihynMdnK- nsuanphiaphan. as actt eels with EMS fobowed by iacabaliag satire presence s a at hwayaihiMu of panaes. Mchjdiaf hypo- ol 54* CATAMMJ9I OF THE V.O1 -UnUTYKYL derivatives. prcstHnahty at different rates, |r) rapid CYCLIC ADENOSINE 3' S'-PHOSfHATI AND nbotphudicster bund hydrulysb as wed as deavybtiun THE MOItrNC«X)CICALT1LANSFO«MAT10N OF of mttatePwtM O2 -nwuobulyryt cAMP; < dcacybrwn of J.P.O'Neill.*C.H Schroder/ KohtaroKawadunu.* , •nracenubr Bt cAMP, ptbwruy to Bt}cAMP is raphty deacylated lo iV'-monobatyry! cAMP ami more slowly to 0* -tnunobutyryl cAMP. The CEU-CYCLE PHASC-SffCIFIC hNMPHOtOCICAL O'-ntonobutyryl cAMP b farther rapbty ungraded TitAN>yortauTioNorci>maciiAMsrat through two pathways: deacyiafion to cAMP and OVARY CtiAS VY S+.O1 OIlVTYltYL CYCLIC hydrolysis lo S'-AMP as wen as hydrotysb to O1 • ADENOSINE 3"5 4H08PMATE' monobutyryl 5'-AMP and deacyiation to 5'-AMP. The A*-monobutyry! cAMP b slightly dcacytated to cAMP J. P. 0"Neil.» C. H. Schroder .• J. C Wddle.* (and then hydroiy/ed to S'-AMP) and also hydrorywd andAW.Hsk lo /VNrwnobwtyryl S'-AMP. In amera) ihe /V*. We haw wctnonslraied previously tha* m CHO celb a monobutyryl sub- chain b fairly stable. These ceil hbjh concentration of Bt:cAMP alone or a low cuncen- extract studies are confirmed by the whole-cell uptake tralion of Btlv' AMP phis hormones such as testosterone studies, in which /V'-monohulyryl cAMP and /V*- or rmnfaelandin ¥., has a (emarkaMe rm. phogenelic monobutyryl S'-AMP were found to accumulate within effect in converting celb from a compact cpHherraMike the cells. morphulofy to the spindle-shaoed fibroblast ftwrn. This study demomirates that the butyryl derivatives Under our standard growth conditions, the feneration of cAMP are degraded per se hy CHO ceN cAMP lime of these cells is approximately 13 hr. Bt:cAMP phosphodbsierase. This observation, enmbined with the treatment nxraases Ihe generation lime slightly. The uptake studies reported previously, point out the possible cell-cycle phase-specific susccptiMily of Ihe problems whkh may complicate the inierpreiation of celb to morphtdogical transformalkm by these agents experiments employing BtjcAMP as "second messen was yludied using a synchronized cell popubiMm ger." Intracellular melaholism b not usually considered, prepared by Ihe conventional mittfic shake-off tech although we have demonstrated such metabolism in nique, which yields am °5% mitotic celb. Bt2cAMP at CHO celb resulting in the accumulation of /V*-munr> I m/tf was added every hour for various lime intervab butyryl 5 AMP as well as 5-AMP. ADP. and ATP. iwer iwo cell cycles. Thb prolocol permits anarysb of There b also an accumulation of /V*-monobuiyryl ihe responsive phase ($). since ihe motptwgefieiic effect cAMP as would be expected from earlier studies. The of BtjcAMP b dearly recognizable after I hr. Only ce!h effect of incubation of CHO cells in culture with in the first or second early 0( phases converted lo the BtjcAMP is envisioned as follows: (e) partial hydrolysb fibroblast-like form within 2 hr after BtjcAMP addi of dibutyryl cAMP lo both /V*. and f^'-monohutyryl tion. Cells in other phases were not affected morpho cAMP in trw medium; (A) uptake of all three-bulyryl logically, even aflcr lorn* treatment periods. It can f» RuUBaWBa)g^naaa> Baam aTUnjaaaP^BBBaBBBaBM' IBBBBBR aL^^jata nSBWlBBBBBBBBaBBBBBBBB> aalWaT m nro ACTIVATION a CYCLIC APENOVC the eel doafptim process are icsfvom lo BttcAaaP 3'S-PNOSPIUTE^fPB«OENT PKOTcTN UNASE only during ike carry C( phase of growth, Sluusrr tm INCMNESC eUMSTER OVARY CEUS TREATED the possMe changes of cetmtar cychc aaeeromle wlTNPUuWtEDCMOLIRATOXM systems anon fc,cAMT exposure and of cvdk AMP receptors daring the eel cycle we m process. A. P. Li.* KnhMeo Kiwwlnmit id A. W. Hsie Enierotoxius am known to stinmlate ait ay I He I. J. P. OThlC. a ftteUir. J.C. Mfc.wI A. W.Hae, fsniCWVAk*. JOB***. been denionrtraled that anon exposure of CHO eels to *Su*M * ear irr-Okk Rafer CuaiH Scaaal «T Ma» j>u|L^lLJBL« aaaaaaaaX^g^aaajae to 9JB adftamfaajft-naj fgRftTaaBi RRfja_^naj to that prgduced by me exogenous adiiriaa of •tscAMP. Our dot nipunii study shows that a ntmi- ACHY ATION OF CYCLIC ADENOSINE »v rrro mum of I ag of protein per ml of pwriwtd ftakftwrin y: V-rWVHATE-f»ENDENT PROVE** KINASE cholera toxaa causes ecu efounaUon wahm 3 hr when M CIsWESE HAMCTBt OVARY CELLS TREATED the oBt are grown in mi mum FI2 luaalfmrnlid with mm *\o* •omrfYRYL CYCLIC ADENOSWE 10» ictaJ caV strum. Tawt-coum stames nubltoh a 3^-PNOSPNATE1 A.P. Li," KohtaroKim nfcam.t and A. W. K*e cAMP level (uwectohj over the control), activation of protean kinase at nro (dtreetokj over me control), and Treatment of CHO cens with Bt The rrrorphotogkaJ response of CHO-K, calls to I. A. t. Li, K. IbwasMms, ami A. W. Hsie. ttocfiem. lljcAMP treatmenl involves polymerizalior of micro Mnutys. ftn Cnmmn. M, 307-13 (1975). tubules and their alignment into arrays parallel to the *SlwJrnl al Ihe UT -Oak RMs* Graduate School of Mo- new long axil of the cefl.' Using several morphological nuuKal ScifnceS' variants,' isolated from CHO-K ceils after ethyl tftnioncioni invcsiitalor wpporied by subcontract 3322 f from the RMotv DnMoo, ORNL, lo the Untovraty of rnethancsulfonaie treatment, the cotchkine-Wnding TennvMce, KnoAviui. capacity of cell fractions has been studied » a probe for ' ' iaamamu«..«w. •ndetstanding the stroctorc of twaofca and its altera- I. L S. nniii. J. N. DMMU. ami A. ». Mm. *JOX Ct0 Kn n|.«i S| !*?$». kai variants have been examined: an epithelial like earitnt oj7 vthicn polymerizes its mefruttebolcs in tLAOAJmUNDUCtDHOtOGLtmr* nupunit 10 •t2cAI«r tagamet, but dues ml align them, and variant If*, watch is alwaysfibroblastic, eve n VARIANTS M THE MOUSE wtthoat BitcAMP treatment. Since alkmnuit of incro- R. A. Fupp. Carolyn M. Vaaghan, Lost B. Russet!. Ijhnkj may involve the cen membrane, crib for assay W. L. RasteR. and K. oracc Jacobsoa were humogeniud and separated by centrifngation into a membrane faction and a saprrnatanl fraction, both One newt. SPRR, 472. with a radiatioit-mdaced of which were assaycu for caUncine-ninding activity. hemoglobin difference.1 was sterile, and its anomaly In fcneial. tnboan comprises approximately 1% of coahJ net be sterncd by breeding tests. The riectrupho- total eel protein of all the cd types. Upon treatment, retk and solnbcafy properties of the hemoglobin from wan* dots not appear lo be a marked increase in the 472 inggCTtcd that its a chains were JifTcient from the amjowH of tuneJm present. aMioegft mere B an increase m chains of its stbhags. Hemoglobin from 472 was of abort 20%l*btam in the jnpernaunl fraction. In al andyied thcmkjBy to determine the natmc of the eel types, tokhkiat hiadmg in the presence or absence of CTP imreasts wgnoJdiwy with respect to protein chains were separated by dnomatogfaphy over car- concentration. The effect of innejjing colchicine con boxymethyl ccnatme. Each char* was djgfiiid by centration on colchicine binding activity revcab a dif trypsin, and anenwit of me tryptk digests were ference between CHOJti crib and the morpnoloeical awsyzea ny tvrO'dnaenstonal chromatograpny and elcc- variants when memVrim fractions are assayed. In al tropnoresrs frmgcrprmtmg). Sohdne peptides m the casts, supernatant fractions from nnlreated or treated o-cham tryptk digest wese separated imtiaRy by chro crib show a linear increase in amoont of cokhicmc matography over uwu 50-X2 and farther panned by boond as the colchkme cancenlratMm mcrcascs. How paper onomatogmphy and/or paper etKiropnonEas. ever, in the immbunt fraction of CHO-Ki crib the effect of colchicine mntmlralmn on cotcbkmt landing liyptk peptide. Alpha iham tryptk peptide • |erT-») changes from a sigmoids! owe in nmscatcd ceB extracts M maant crvjtaBa^nBai BBa^afBaamanm* farnaaVan am naBawafiamaf SaT nanwatanTaamai to a linear one m treated cefl extracts. Tins change b M in one of the two • chains (the other dram hat dependent cm the presence of CTr MM Itomopmnrmg striae at poafion «•> m the himsglubm of d72*s sire. •offer. There n no change in colchicine bmdmg of me Horn ciimpbti jwalyni of nT»revealed that jiparjjiat mrmbunt fraction of M7 variant with or without was the only amino acid at posrtnm tm^ tnm. nerener 01 oticAMP treatment: the binding Kinetics remain rig- Ihe dnpneeted and adjacent e>cham genes of me ntomal with respect to colchicine concentration. This irradialed strain SEC d parent of d73 were expressed. observation is consistent with the hypothesis that whenrer the deficiency of the expression of the SEC tubulin associated with the ineinbram b involved in ocham genes resnfled from X-ray-mdoced gene macli- microtubule alignment. The change in binding proper- vation wtthoot absowle loss of Ihe gene or through lies observed in the CHO-K» crib' membrane fractiom deletion of a region of the chromosome bearing the does not occur in the M7 variant which does not align dnpikated oxham genes was not established. i*> mhTrotubtdes as CHO-K, crib do. Variant M6. which Similar metrtods were wed to analyze the hemogiobm fe always fibroblastic, shows a linear cokhirim-bnidmg in another variant, SPBo* o*352. Chemical analyses profile with icspeci lo colchicine concentration with or mowed that Ihb mouse abo has an o-chain deficiency without BtjcAMP ireaimcni. In untreated crib this indistinguishable from that in 472. proTilc is dependent on GTP, but m treated crib il K Mice identified as HGBF*., eft I and SFM» 4122 not. The presence of a linear response to increasing each were anemic when originally tested, and they colchicine concentrations in a cell line which always has continued to be anenric when tested many months polymerized and aligned microtiibules n consistent with bier. Although a few progeny of 4*1 had high lite possible involvement of ihb membrane-associated, reticulocyte counts, most of its offspring had pbeno- cokhkinc-binding activity with microtubule alignment typkaRy normal blood. The hemoglobin in all progeny and cell shape. of 4122 appeared lo be normal. Hemoglobin from each of these anemic mke was analyzed following pro *Srae«M at the UT-Oak KMfe Gcaewte School of Wo- cedures outlined for 472. Fingerprints of the iryplk anwcal Science*. peptides of both the o and fi chains appeared normal. 91 NtwuhthM. many of iht larger iryptk peptides «m • these erythrocytes as K r • for neutraly charged afrngbfeull mbslMiitkMs. but mm* was famd. Tims, the dtl and dl22 docs not appear to be caused by I alterations of of the bfupjabio from a vanmtt nJcntilied as 300 FHCB» 986 aw ir. havr aoi begun on ihe hiuniuJnhuM from SFBnV 927 9732. I (tram Dr. GmoZanoBi)ii s mixture of |nMH rays to refuue the mmilimuBi of a hajh Y-12 accident. Tbe sy nthesu. To estimate the McKty of hemoglobin of cacb of these four samples was less man •i riwo, the level of incorporation of me upper control ranee of S parts of ujokwcmr per iiyrk acid (iAbu) 100,000 amino acid residnes. The ammo acyl tRNA Tbe higher Bolcucme content nt bcmogJobm of persons exposed to radiation could nave avowing its mcorporation only 80 limes per base-subst HnthM point motalions tfcst s an awiage substitution frenjnency for other leucine into nolcocme codons in crythroid sleni ecus. i adds in the hemoalabia of mouse wfiiyhium Efforts are being made to prow this m an uperunrnlsl To control for potential differences m protein synthesis annuel. Another possible came for an elevation in the rates and pool sizes, the incorporation of die ansJogne Quantity of isuleinine inrise hhjMy purified henuujiobm was compared with the incorporation of leucine, of persons exposed to irradiation imghf be Ihe pre since botopk iAbu was recovered, after protein hydrolysis of of a protein that contains isotencine and is not Hver and kidney tissues, representing 30% of the total completely resolved by chromatography from hemo radioactivity. Separation of tissue proteins by molecular globin A. Fctd hemoglobin is such a protein, and it weight revealed that Ihe analogue was incorporated in couM be elevated in exposed individuals owing to proteins of all sizes. radiation-induced hwclivation of the f-cham gene in a In mouse strain C57BL/6Cum, analogue incorpora few erythroid stem eels; as a consequence. HbF might tion rate (iAbu/leu) was 25% higher in kidney tissue 92 which kdb nomnl ccRs in ca thesana •art not Ui cehs which fack ehr tide ceayae.ni m»«. mbra».The level of the dreg kwb aunaal itiidiagcfnsin the pit and anas. at24hr except Hot and it canacsareduction i n thenaaaher of cohmy-farm ate kidney ing anils in hone amrrow. Female offspring from males of these tea act of SOftfm the error twice with 6-dnagaanme wear chaneaged after he same conauaond. Heterocygoas (r- C3H lo uuac carriers Car mis X-hnked locws aneuhl expacas me was 47% at beer of enzyme activity at only half of mew somatic ccRs and ofaje.ObJ we thearfare expected twee resistant tohajh levels of the drag. None of 124 female offspring has as yet 20 mg pet kg per Coy of incahyf nanamntmafeenac-and «Mna9lMl9tra Hat CXpfCwCS pintnwVlyp?. A luTnTMuatuaMt OB da of X ray at tut tW lEVd of i ANASMYSYSHMFORSTDIXILL SDCUIIVIIV TO DRUB wan being 35. I*. and 2IShidM hr. Ai4» and 72 hr *e MtSOweated i Ifo r at Natcd cefc may mnlinmfc lo a OTfPftVlH RJf iPn? SMVC ftHBPf M IWO VffPCWlH ujapounr wwhoof affecting stem cans. The Kline, a study was initialed to lampw the effect of earinas an stem ecus n dnscrcuwatcd cens. When ' stem ecus from none marrow are mfused . EMUCMNENT FOR THE into an hraamtcd monse. some of the cent settle m the mooucnoNOF uscmmuif spleen and in nine days develop into discrete clones of fHVraMNTMNE«UANOSME cens described as spleen connect. The nuubbcr of stem ntOSTNOIUiOSYLTIUWr^lUSEDCFICIETCr) ccHs in a bone marrow pool can be detcmnned by MmrAirriKE anaying the number of spleen colonics dtiUuping from a prescribed inoculum of bone marrow cens. The effect C. P. Hhach, Diana M. Popp. of a ding on stem ceHs can be determined by swaying and R. A. fvpp the nantber of colony-forming ecus m On? marrow of FwlitniMiy cxpcrinicnhi went ondcrfafcen IO crahnte Heated and untreated amtnoh. The effect of a drag on ihr potential prodection of umlaut offspring of ex the nasre-aWrerenfiatcd cam can be defcrnetned front pected genotype by enriching the population for of bone marrow in treated n spermatogonia of the selected phmotypc prior lo metmg. Spcrmatogonial enrichment woaM reduce the cost of gewiiMl mutagen screening in the male since butyl nitrosourea, a carennnjen; the frequency ofrecovery of mutant offspring would be nnntonjppvcmsui and potent cell increased. Mice wen? treated with the sdcciivc Man aard to sdecf for HCfRT" ecus m tun*e culture: 93 cortisone acetate, an mrnnniosnppressaal that destroys wul be extended to mcf'ide other en lymphocytes: and HlrydroxyUmmoKyduaexanrcar- broader treatments. This preliminary stndy already boxylic acid, an ammo acid analogue synlhesued by demonstrates the potential vaase of the colony-forminf- D G. IXmerty m this laboratory and suspected of beme wnit assay a* frtrrmming the effectiveness of a drag on a serine antimetabolite. the stem-cell pool, it shows that 0.1 mM6-thii The drugs were nlmmiileied in regime ni which the in the drinking water for five days hteratwre suggested would furnish a mixiawm nun- hnnatopoietk SICHKCU pool to 4% of that of i lethal dust or yidd a nunim—i effect either by bone marrow. kahug cehs or iuduciag mutations, femuri and tibias were icmmed from the mice after treatment: die EFFECTS OF MHYWBOXiXAIvWOK was flashed from the marrow cavity and CYCLOHEXANECAfttCKYUCACIDON in Tyrone's solution. Nat, hated eel counts IMMUNERESFONSE TO SHEET I 10* and 10* hone marrow eels were RED BLOOD CELLS (SMC) iotogaas. Icdofy jrradwied mice. Nine days after bone marrow mjecuoa the mice MM krikd. KTC removed and placed m Bourns n at that a cychc the nmnher of spleen colanir restricts growth of bmnan cytic eels in caftan?. These eels be seen in Table I*. nVe drags can be that affect dnffercntiaied eels UhydroxylaannotcycluhiJt awn nbmyhi acid, only f cortisone acetate), affect differentmied eels and to fmutiua as a serine anthnetahohte. It UNA stem eels ahkc Dnt CA 23 a* I V IIS 23 a* i 1 St « err 3 n* 1 Jn ss 5 a* 1 1* IS «TC ft.lmV 1 <* IS •J mm i 15 4 Mil* to a* 1 SI 22 >0 an; I 49 31 1-HMT 03mV 1 102 I2S 03 mM i •» 133 (•uuV 1 110 127 lOmM > 127 132 *CA •- CmMM jcrUM-. «••prain l in same . mjrmd in: Cff = Ordophn inV. onoMd M « anr. unActcu MK CTG**-fl uoaauunr. awatoi m NjO. ft* MMWa > M water bofdr: WHV * bntytouo- Mwica. * alia Vr* ctkyl Urunid ««J pWianaMtanVr»N4S . If* • t jb»A _ .Mi_^J*-t.^ .1 •PU*YIW mbtot a 1 •bnaai n mwn»:mx * * %^mfwHnkyWmWmf99€f€ti UMVUT jtMvd n«t««dinlM>a mUtdamUmmm \f M ttjIvT wMltf The cuaawaad was syaifcesized by aad obtajaed fiua effect OR the steal c«Ss of the bone aormw txe -Aa D. C- Dubrrty Mkc wear fed 2- sc - *»—«!0 r 20 i Irk'*' •m («• 4 •0 4l(¥l N»ll4 4m» 20 *o 4m» 20** 4«t 20me 3*5 Fia.ll. tat iiriawu«t UKiaiar ajlwamtjmaawd»!• wiaiiaiiaf IIM 5RlyaafcaatrlyMaknryNsIrma (4 aMNb* aai aad 19 aaaabl dnaars 1.4. art 5 days altar iajmbai (A - cntianwr acciaic-trcatcd *mmr*. N anfmal *>nti«» X»ab«rtM?..enihcvc\ ' Miabrr SEPARATION OF H-2 ANTtCEN *f\Mie *fvoCvbcvvH( annaaafor ivpawltvl fc* mannxi JM2 ahmmg at me aamf opmnai J?C. Smce mtrate at mc ferns mr Mm Dmm «ttn_ •» w* Vmnumt ** sole mlruacn somce. cd dr«Moa is not pmiwhlt became of the doabty Mocked pathway of jiinmliipun. It jbuabJ be noted that mm' tewenmt* do not grow UX*Z»tCr9JiBlAllUi4MD*KED camer. becamv they sail carry me nonkaky. nonvevcrti- MirTACENCStt: HmWTriON AM» DCPKESMOW BMVMRLE ANDNOrWIABLECELLS vmr ptmmsd and fmcied m Mmajiit GS nammraar I0J2 ma) discs, and the dear medmm was assayed for J. F. Lemonti nstrite cnaxenfratioa aamj prcviima> tanbuttd S and The m rmt enzymatic assay jinainc to mdaced NED reagents. One MM of m rnv NR enzymaiK icrtrsmm of imr/-m m VtHmmt mavmr. dmribed m last acwrKy is defined as I aannmnfc of mtnte primnrcd by year's leport.' has hem vtiimd to study aamma-fjy- 10* ceffs per hnm. Since me acovity is a faactina of abe proportvon of eels mat have reverted and pvnaWed mhuMe mKlrmtmn of cohmy formation. The remits active «n/ym». cazymaticaly determinedreversion far - dearly indkJlr mat nmtauon MKMM am) eipwimi avjencats wear cakalaied by drvabrnj this jcimty by occur in nonviable eels with ihe same frequency as in mc average im rm> activity eaJabiicd by a comxtmn of the snrvmng eels. •aogeme ntvcrlanls. Tms aaammiam ami al revenants Smce SARI is the structural pmc for miratc reductase mad lo a smfV constant actrrwy may not be mc cam. (NR) enzyme, levtnions of a mnaml bnt imiimh For example if base sabstMatams are mmrved. a aide, imwl-m. leading to an actmt enzyme arc detected ajaaajber <•« difrcnmi base parr changes may prove by shaming nwremtd pniaWiioa of nitriic. me en- acceptable for fal or partial fancinm Mnreovcr. zyutatic prodnci. from nitrate present at very large arvcrsioa .mammae* so caambwed nmsi he cunts led cimccntraiiom. Tms is achieved in a mr/-/ bactmnmnd. by a factor mat mmcalcs boar nmch direct radnjNon that is. neuclREaay defective nnritc icduciaae. sntn mat damage has been mffered by the .SAM ttractwral gene njtrrfe arcurmmtcs in cdb and in ihc mcdmni. Under ExpoMHV of wad-type or rcverlant SARI ccfk lo these cnndNium the rate of fummfiun of nitrite is raamaon cames a dim drpendrni bg in die fmt ourpurtnmal in me ciiuccntratiou of active enzyme. amammtcc of NR. math the same as prcvMimly #7 UV ifcaMKM. The fiml kid of eazyne. aboat padraays of iadaccd an the ooar a loo peal- lai a iiaajj htfber daa* aavest dm. ifga—aa-ray iaiaccd revcrtaats efmrl-m inflict iacreauagry imt DN A daanf* to art ftaeraied by an error-proae repair systea,riant ibi s NAM, aaateag • ami taadj baa m wmo actaary. system is prababty aot itself iooodMe by the raaaattaa sarnots fat bettci thai the caiac eel todf. vbidi • •>. - -. ... . I. J. r LnaMKi.aa* Dm Ammm. ftm* Mip. Jm*r ». I9M. m ww^_. ... laaac aaw w» warn o»!tt«»m.pp.*: ••. *•* *al ** •ol» caa ami do 2. U HiWt i.*—"****-l 2n.2»fl«7l». For exaaate. a lakes 500 bam to am.twju cdb of das very ^to2*sarn«d.ahie*dasdiae CBKIK ANALYSIS OF aar MUTANTS IN TEAST rCKartaityaicaacediooalyXrSofdgii I til J. F. IjemoMt aai Efaabem I. feryaa* had. The panaamy AM the samriag 2% of da? cdb MatatKM fieaataLJej esiianted aawag lanhaa, UV-i ctaan after raaaaa-ray tieanarai ofrias mrl-m tml-t comer* rcsbtaacc to da-; strata coanore teauauMy wxR asdi daae obtained «inae of a faaky fmrsunfy m the original mrl-m no* noble.' Eary- rhese bad aMKaiy ami—aid matatinn frtaarncifs. corrected were p IpcMep: l» were for siraciaral grae iaactnatioa fir., act raaciwaal pcMe; aai 3 aanaats from each of I SAM cStroat. ate airy close m dnnc obtained aaaaac mwati not mate. Of UK IO.OI aaoie laravmg cdb The aaat HJJI amb ancrpreia- ixed variants. 2 ant any UV \ rioo «/ ibrse reiafcj is that exprtudar iadaced reier- sina B occarriag, aai wah the same freaatacy m boch vnMe and noanaMe Jab-a inoljiiiinj of cdb. If. oa rhe AI | odMr hand, mB awamtd dial — tjgratnj. any omy MM ooanakmeatatam wait ml. ml. mi nwX •icenr m cdb dm aai form cotnairs fsarvnorsl. dnr aflsacbaan?ivrliybrab aim carraddw scry UV-anen- caryaMic iwtmmi ficanimiti srodd be expected to Me mgd/71 rcamawMaaaftmi carat panted as remnants per cdl lini. Thea. a pbted Tan latter kmi of trad hat beca nataard atainid ia daw iim am i hj awmd IfY i pronauh by rlohafay.2 trim fast eaadoyed the of amd-17. dot «*. a XAMI-Sml peme-tmnrmr ir»ana to awaator eaaaao-n>- mm dot expected fran • •. aaF**. or »»'• ca praed that rev« wtbautiuaal reyait earyaan are tana- irr/.rerZorarvJfoataleleaicachlecail. *cbe» RKhK-dde. «» dut iec«a«Maaiii«) coaM nccar The icataaaaj tea iadam acre each croaed to a #iarr m crib that hoi • worry*» lacccwfal repair. aatabty aarlwd aat/ ttraat lo yadi o^floab of die tht «anrmir* Ahanajh an> btiie a kaoaa fnikariag type U** arm 3 CmVt Hi$5-2 /ysf-f M*$ The peine tirarm were am oianderei at ihb ia*r fuNnwaaj aKabatma aai priaanjiaai. pare chaMi of After ateiiBK aai l«) Siagk aadear aaaaiMas are respoasdde for de- iwa thai UV aaHafcaciH natjht be retried iw dark (Kline UV aailjhdiiy 10 «•>/ in ame OM of ten repair of letbai daanet. la «mae of ihevt I'V-seKmive CfWatS. straias. the fre«ps p»M pathway*, dm duwMe awtants EFFECT OF rerJ ON DAMt-NOLUNG RECOVERY sfcadd be WK seasHire diaa eidwr sa»jle«aKaai AND«OTOR£ACTI>ATiW IN YEAST sirjM. The MMeraciMM coaM be addane or syacrgtsik- Mataats aw jiraawd lu be amdtaky. Keadra L. CaMwei* aad J. F. Leasuirti Tbe mmr-130 mm-339 doable mmam was foaad to j^ ^ ^.uiiwi h bettered 10 be anohed at aa be aw Wscasttiredtaa cither ttfdwsaajkasataats. (nwfMK ^^ pathway actiag oa DN'A daaufe This safjrsts dot each MUM B - a diffcreM »ftct«| by UY hahi. Haptuab The data do Vnd fc.^c to dw hypodwiii dm tr*3 blacks a* crror- r of IJVdaaKaK. dfflcrent front dwewinoa 1974. a w&3 aad anr-JJf Mack step* in a I to dm Mocked by saW39. Fail nrter awr saaj i a place for J tacVT OS* aaajrCijaiiirfcliaal «t»i—» The twst step is a •.-** Somen Dlsaats. For das 100 names vf Boyd and Mucarif1 tor detecting DNase i»n either side uf the htstuae genes. dossing-twer in the activity in puryacrybande gets wfck.lt haw been eieciro- demarcated repua measared O.IM l'~ (7 c«» 1722** lliesp pfcoresed. Must of «mr success has been with the DKx>e* in the annealed coated 125 * l"0 Heal ueaiaKal |.»5 ik3i are actiw at a pH of 4.5. In brat and adui(> there t |*C> t»f *2 hi. grwa before, daring. >H hdhiaiag S. are two coaipoacats which aagrate ai different rates to gawa peak respuase latent Sot I.43T IISOco I2JM>3 Ihc amide and there b considerable activity at ihc taesl. < tMrespnadna* in a 36-fold increase in lecuadma- origin, la papae oat ur stnaetaacs iwtt more compw- tiuc I Fie. 131. This peak is m sharp contrast to ihji lor amis migrate to the aaude oa polyacrybande gel. the eatire granna,. which ocvim — 12 hr earher. A aWaniajfivr is potyiawptac l«w JI least three t die ling, studies haw shown that ihe proximal reaua aWs of ibe loots that we caked DNase I. Ii appears of 2L is bte bbefmf .s If labebag of Ihe bbtoae bjads thai al forms of the activity that we observe are ci*tfc UTE CROSSOVER RBTONSE Of TK€ MTSTONE REGION RhodaF Crel Thnmal stadies of the ftiiaiahiai «»«cyie haw demuasiraied a cniat.ideai.-e beiawa the Sphase 124 30 hr m daratinnr aad the stasitiw period km recombmatina Qa^iaajMiwl Ihe anaaat. the scaatiw period, dnlcvcai remnas aDpby chsnctcristic peaks of response, pjmibly correbted with the iimpnijl stoaeace of DNA repbcaiioa/ We dwahJ hkc to know if Ike cinncaVnce coabJ be olradcd lo a swaW reajna hnaiawj nary a lew ftnes. An approach lo tlw oatstina teeaMd panibai wwh ihe la aaother syuem the iramcriptinaal activity of the histoae fears has been shown to he chaety cnaaled lo DNA uphution.* Tumiajtiun of hatowe iranscnptmn ictwrmatinn of S by several hinjn. and it b daring the bte. aacnaaled phant that MHOVIC yencs fCpiicsnC. in §M9f9n^mJintw inc Mfifmc fevm hawf been >iw.jii^c?i lo rtpim .^D.E in ike salivary pBNM ClHWIIlOWtlWC IVyVVf(lllMiy 3 m33UHMMI of I * PMOI 1*1 Ifcf pffOXIIWmi pUflMlff Of CRfOlflOSOflll1 2 • To irttnwiw»f if rtW (nwf-tT rAfonsc lo heat r* blc in I hi* oTff"*}. dnsdy naiiliitif niawkcr* were iMmdaced 101 Her MUTANTS IN MtOSOPMLA Rather than us»m; numtnmiKiiiNi as an indicafoi of Esteb t Gcnetuwi. Rfcoda F. Grdi. reduced lecunriMiation. our system measures crossover ami J W Day* frequency mrtvily ami selects for mnunts winch ehmiaaie or drastically reduce exchange. Attention thus A program t» recover and duiactcrue EUS-mdwced far has focused urn a cfnenocomal region of 1$ bamfc fcr' omtants H intHnwmir.. S«ch Minn, mclwdmf, the within which the m- gene c/J/C hes. Newly induced temperai«e-seiHMr«« variety. sbuaM serve as useful mutants in thH segment are characteriz** by gmeiic loob for analyrmg the tctumbmafiu* process m At>- studan including complmtniation tests, temperature AvadaMity of a tempeiatwre-seiMHwc rrc scmiirniy. am) segregation behavior, as we* as by M shuaU permit selective control of rcttmmma- elect run microscopic examination - IHIM Ivf regions whose responses aw lemponBy distinct A total of 20000 EMS-twaicd tmrd chromosome* from one another, and ai the sane lime ihunld twrnish haw MOW been screwed, ami 17 new mutants haw been a* independent criteria* for the lime of mtftrtic recovered. Amung these. 14 are recessive jvMts of exdi tfSfC which completely etmunaf e meiotic exct »-.v C-• - : t omv« ?t . -J **-•'-- w- >.•* •-*•*'• Pb>M. PwM^rmmpimmirtcMmtameiWflHmarrrJ^maimi. -« 102 are recessive alkies at a new |: H /DROXY AKTHRANILATF. AND RELATED KYNURENINASETYK ENZYMES OF TROTOTMAN METAMUTES AND KSKILUUM ROQVEFORT1. ASKHGILLVS THEIR ROLL IN CARCINOGENESIS MGER. MMBOftSSTOLOMFOt. AND ANDM;TACEN«SB KEVDOMOXAS FLVOHESCENS: FURTHER EVIDENCE FOR MSTWCT KYlWRFJWiASE AS. Shelly.* J L. lip!-r. and F.H.Gaertner ANDHYOROXYKYNl-RENWASF ACTTVmES Chromatographic, metabolic, and kinetic studies re A. S. Shetty* and F. H. Caertner vealed two distinct kymueninase-type enzymes in many micr organisms. In these studies'~2 a single constitutive The kyrwreninase-iype enzymes of three r%np and hydroxykynurcninase was observed in Strtkanmivm one bacterium were isolated and examined kmeiicaf y arrrrism- Due to the activity of this enzyme. .V. for their abwty to catalyze the hydrolysis of L-kynure- (TrrroMr excretes hydroxyanthrandaie (HA). The lat nine and i.-i-hydroxykynureninc. The phycou.ycete ter observation ted to an analysis for the presence of Rhtzofms xri4n*tfrr was found to contain a single. HA la putative Madder carcinogen; ••» fermentaiiun constitutive eszyne with km for L-3-hydrox}- products such as wine and beer. Al'hough HA was no! kynuvenine and L-kynurenine of 6.67 X 10"* and 2.5 detected by paper or gas chromatography, some aro X 10 "* M respectively. The ascotnycetes jisaMgaaVj matic compounds with ifcorcsccnt and chromato mgrr and ftiminViiwt mqmrfimi each contain an en- graphic characteristics similar to HA were four d. zymr. induced by L-tryptophan, with sismtar Km far Since most carcinogens are known to be mutagens, we t -3-hydroxylcyntireiane and L-kynurenine ranging from air testing the mutagenicity of several tryptophan 5.9 X I0'5 to I4J X I0~' Af.asweUasaccmtifutCee metabolites, as well as ethyl acetate extracts of beer and enzyme with A'*, for the two substrates of 4 X 10"* wine, with the Stlmmtelh strains of H. Ames et nf.J and 10 ~* M. The bacterium PstuJcmoma flaumerta Our preliminary result* indicate that hydroxykyn- has a single, inducible enzyme with Km for L-J-hy- ureinne. or an oxidized form of it. may be a frame-shift droxykynurenine and i-kynurenine of 5 X 10"* and 7 mutagen oi strain TA r537. We arc also interested in X 10"' iV. In addition. sigKtfkanl differences in the mechanism of detoxification of HA. Our experi maximal velocities (!'„„) were observed in two cases ments with mouse-liver preparations show the presence The Vm„ of the inducible activity from P. fhtonxxm of a highly active hydroxyanthranilatc oxygenase was 4.5 times greater for L-kynurenine than for iHAOh Rapid metabolism of HA is further evidenced L-3-hydroxykynureninc. wkereas the Vman of the by Che lack of HA in urine of mice, collected after constitutive ac'ivity from R. stotimiftr was 2.5 times injections of this compound either singly or in combina greater for i.-3-hydroxykynnrenine. It is concluded U) tion with reported inhibitors of HAO such as salicylate thai the constitutive acthriiies are hydroxy- or/>-aminohenzok avid. kynureninases involved hi the biosynthesis of •ucoiina- nvde adenine dinuctrolide from L-tryptophan. (o) that Ihe induenme activities are kynureninases involved in ihe catabolism of i_-tryptophan lo anlhranibic.and (r) 'Supported by a Carncpr Corporation Grant lo the Uni- that R. stohnifer and P. fhnwnctrs. respectively, cany icraly of Tennewe. an Nlll Grant RR mil 11 in Florida A * M the most specific examples of each type of enzyme. L'nrmtily. and ikr U.S. Fncrey Research and Itevctopment AdmmKlration. Permanent addmv Florida A * M University. TaRakanc* 32307. 1. A. S. Shelly wd F. II. Gacrfnrr. / Ihctrriri. II). i 127 11973). *S»ppofie4 by a Carncpe Corporation CtaM to the Uni 2. A. S. Shelly and I II. Gaeilner. / Htctrrinl. 122, 23S versity of Tenhcxwc. an NIH Grant RR 08111 to Florida AIM 11*75). University, and the t.'.S. Fncrfjr Research and Development 3. R. Ames rt •*.. hnr toll. And. Sri. LISA 70. 2MI Adminrttnimi. Permtaent address: Florida A A M University. 11973). TaOahassre 32307. •'<»**»*£**•• '"^fSf? 104 COORDINATE ACTIVATION OF A INFLUENCE OF AN AGGREGATED MtJLTIFNZYME COMPLEX BY THE FIRST MULT1ENZYME SYSTEM ON TRANSIENT SUBSTRAIN. EVIDENCE FOR A NOVEL TIME: KMETIC EVIDENCE FOR REGULATORY MECHANBM M THE COIR>AltTMENTATION BY THE AROMATIC POLYAROMATIC PATHWAY OF C0MTLEX OF \H.KOSfO*A CK.4SS.4 SEVKOSHML4 CKASSA C. R. Welch* ami F. H. Gaertner G R Welch* and F H Caerinet The catalytic constants of four of ihc five engines of The aromatic complex of .Vrams/*** mss* is an the aromatic complex of .\nmnpmm amaa were aggregated muhien/ymc system which catalyzes live enhanced significantly when incubated with the fmt consecutive reactions in the central pathway leading to 7 substrate. *-deoxy-i> «nsfes»i>-hepiut-»st>naie -phos- the biosynthesis of the aromatic ammo acids. In an phat* iDAHPi. Activation with OAHF was ancom- attempt to understand the physiological importance of pkshed independent!)- of catalysis by incubating the this complex in particular, » well as the importance of purified enzyme «ystem in a mixture devoid of re«|uisiie ceRubr organisation uf enzyme systems in genera! . we cofactors and uMtrmediaie substrate*. The activity of have isolated the complex and have begun to character each enzyme in the complex was subsequently assayed ize its catalytic properties. Optimum conditions for the in appropriate complete reac:iuu mixtures. Double assay uf the overall five-step reaction catalyzed by the reciprocal plots «f the kinetic data •vat used to partially purified complex have been determined. An determine the effect of DAHf on the catalytic con analogue computer was programmed to represent an stants of each enzyme. The results for the live cn/yme> um^grcgated system of live enzymes w«th rate con- dehydroqjwnaie synthase. •cfvydroujumasr. Oehydro- slant* identical to those found for the constituent shtfumaie reductase. shHuotaic kinase, and *m>t- enzyme* of the complex- By direct comparison it was pyruvytshikiinaie synthase were as follows. Incubated shown that the lags I transient I Rites) oh! anted for the in the absence of DAKPlie., unacirvated) the maximal overat* reaction were 10 to 15 limes longer for ihe velocities f H in relative unirs were I. 20.4. 2. and 5. hypothetical unaggregaicd system than for the com respectively, and the km 's were 0.06.0.1.0.04.0.1. and plex. We conclude from these data thai the unac- 0.1 IIIW In dirr.i comparison, tvben the complex was gregated nwiiienzynie >ysiem compartmentalizes inter incubated with DAHP tf.e.. activated), the I' values mediate substrates during ihe course «>l the overall were 2. 20.4. 2. and 5. and the km\ wrrt 0.01.0.02. reaction. We suggeM that, in addition to "channeling" 0.02. 0.1. and 0.01 uW. This suggests but does not intermediates of competing pathway v reduction ol ihc pmve thai a singie site, dbtinc! fi.nn the catalytic sife. transient lime may be an important consequence of the is responsible fn» the coor&natc activation. We propose containment of intermediates within a physic ally as that the physiological importance of the activation sociated enzyme sequence. The fact thai the aromaiic invuKes a wvei regulatory device which provides a compkx exhibits a se.-i»nd catalytic property unique i<> means for directing the flow of aromatic mtermcotalcs aggregated enzyme systems, "coordinate activation."' from the anabolL- polyaromalic routs to a catabolic one indicates thai 'he physical association of these enzymes in response re thv energy charat of the ceil. In support may have more than one physiological function. of 'hrs view are the facts that shikimate kinase was found lo be i.ihibiled by ADP and that, as a result of the acthratc-.m of the other four enzymes in the complex, shikimate kinase catalyzes a rale-limiting aonequilibrium reaction. •PredfKttwal I««..» tuppmlcJ by CMIM <.M 1974. MUMS Tmcw jddrro: Scrvior dr Oiiimc Ptiy«)uc 2. I/HOCTMW Librr 'Prcd«v(i>m i-cllim tuppnrkJ by (J.jnt UM 1974. NIGM5. de BluscTVv Awnw I- D. Rninevcll. 5)1. RrmwK. IW-lpmm Ptvrm jddr(«: Scrvkr * Chimic Phytmiie 2. LJIIWTMU' Lwre I. (tit vVclcn and I H. lixnncf. Arch Hnirhrm. Mnphri. dr Bruxrllcv Avrnue!: D iSmMrvrli. 50. Hf«wc'.«. Reljriwn. inpre«. 105 rmsniocELUJLosc. AN AFFINITY SCAMHNC ELECTRON MKROSCOPY CttROMATOGRATtfiC SYSTEM FOR (SEN) OF THE OOCYTE FOLLICLE CHORBMATE SYNTHASE AND TK AROMATIC COMPLEX OF J. N. Dumont SEinOSPOtU CKASS* SEM provides a new dimension to the study of the K. V. Cole and F. H. Grtrtner morphology of eels and tissues. In our studies of developing amphrbbn oocytes it has provided new data In previous studies i* the aromatic whir *tyme regarding the relationships of rj»e ceRs and tissues system iH,V nm, we have unserved thai II of 13 comprising the oocyte foKde. Such hsformaliou is enzymes catalyzing the of erytnrose-4- important if we are to understand, for example, the phuspbaie and phujphofwufpyi urate to L-tryptophan influence of certain cets lie., foabclr cefls) on gamete bind to DEAEvcHufase and elate in approximately the development and maturation or tut transport of mate same concentration range (OJOS 0.12 M\ with a linear rial to the oocyte surface. The fouWtewhic h surrounds phosphate gradient. In paciicnbr. the five-enzyme dtwhiumg oocytes is composed of several discrete aromatic complex and the three-enzyme anthramtate tissue components. Covering the external surface of the synthase complex etate nearly in coincidence. fouVte ii a continuum of extremely flat squamous ceRs Recently, a very active and highly stable protease referred to as the superficial tpnhilium. The margmi of activity was demonstrated in N. emtm which, comci- idjjicnt ceRs form dose junctinns with each other so denrty. hat some of the same molecular propcr*ies as that mdiiiduJ ceRs are ssmcsi md^mguishable from the aromatic enzymes. Fortunately, this protea-ie is each other. The surface of rbr* epithehal ceRs is inactive dwing early stages of purification because of a studded with short nucrovwV Icncaih the superficial highly efficient and specific uuturai inhibitor. cpitlithum is a layer of connective tissue composed of In our own stndies on this protease and its udubuor canape* fibers, a few fibroblast ecus, and capdbnes. •unpublished data), we have fovnd thai the protease When examined with SEM it is revealed that nV dees am bind to DtAE-ceiMuse. and the ndwtilor cohagcw fibers are aggregated into rather discrete binds only sligHry (which again fortuitously protects loosely packed bundles and that these bundles are the aromatic enzymes from proteolytic ailacKi. How randomly oriented throughout tins stratum. The capd- ever, some proteolytic activity may be unmasked at bries ate rmhtdded in ihe connective tissue Underlying bier stages m purification which conM lead to arti- the connective tissue byer rs anottwr stratum of ecus, factua! resnlts in studies concerning the strnctwe and the fonVi? ceRs. These are rebtrvety brge steflate function of the aromatic enzymes. imiamouj hkr celb. but. anNke the ceRs of the super As a consequence «>f the jbove observations and ficial epithelium, they are not as dosety associated with conchnions. w have attempted to eliminale ihe pro ewh other. Although tbey form a continuous byer. tease from our preparalioKs at an early pnrificatrm assoriatrons befwecn adjacent cefls are made via ra stage by taking advantage of Ihe wide difference in the diating pseudopodial processes There are. ;herefor*. affinity of the aromatic enzymes and the protease in mwry spices between adjacent cHlt. This feature is ion-exchange chromatography. Since the protease does important when one considers that oocyte nutrients not bind to DEAE-cetlutose fan amon-exchange col and yolk protein precursor (which n synthesized in the umn), we anticipated thai it would bind lightly to a liver* must be transported from the .apwlaries through cat ion-exchange column such as phosphocellwlose. On this layer of cefls in order to reach the developing the other band, since Ihe aromatic enzymes bind to oocyte. Separating the follicle cefls from the oocyte is DEAE-cellulose. we expected them to simply wash an aceflular. finery (XamenKMS layer referred to as the through the phosphocellulose. free of the protease. vitelline envelope. Here we report the surprising res'ri: that two of the SEM has revealed features of this envelope which aromatic enzyme components, chorismaie synthase and were previously undetected. Randomly dispersed the aromatic complex, bind tightly to phosphocellulose. throughout the vitelline envelope arc relatively large Our results indicate tha' this cation exchanger may pores or channels. Macrovilbr processes from the serve as an affinity chromatographic system for these foKkie cells traverse some of these channels to make enzymes as well as other similar types of enzymes. contact with the membrane of 'he developing oocy.e. if* Oaatr cuaaaets appear amply The porosity «•» the WMMIIOIf OF IMTEJN nitflif envelope aahcates rhat this CMHWKM •««" ih* inONMBOLATta) tottWie doe* not provide a *ub*uairjl harm i.» the passage of aairieats and vol; ptecarwiv*ft UM the Muod lo :he devetuaiag oocyte. The tartace of we «>ocyie R A Vabx.J.V I A. ». Schart/' itself H studded uiih aacrovah vrfach mead upward /it rtlr.' effect* ••) sienna* ldeo\yo«lK»*tfVi*c contact villi the vaeaaie envelope. pvojtrtetoat. estrone) •« aptair .4 radaoactiteh labeled Mwd provem uaehoginaO In wdatrd AM /upanv* oocytes were waited. Oocyte* wear pwm- A CUL1UK MEMUM RNt steroid tot umiywmMth 3* at 4' «uc>ie* were exposed to *an«u» of ifcKuds tOJOl IA af/adt. oocyte* afte» 12 lw H Fiwone al al •flair. Drony- osahncctaie x> aa of normal lo «auu>of of the oocytes after varioa* kayths «f Electron M a sapptrnacated oocyte •tweaked a chae retatauulup be In al cspcriareats oocytes ta' airimmtam nnotawainni and foKctes were wed. First. *e abdrty of the uf raiCTopaavytasu and other stractaral oocytes to mpnad to jNujtnau at the oocyte cortex. The roan* sapjpra * by nmswiag die abany of thn> bnrraoac to unji degree of steroid structural specific"* for _ iraaaat vesicle breahdowc. Oocytes e mm of mtlofitam UKorporatiua i for 15 day* aw jpaMe of thisresponse. Second, it that UNA and prorcat syntheiii coatinaes i die aaratioa of cakare. TMrd. the aMhty of * Pvn r». J *jo II if*ii 1 m- the oocytes to ccdncytote bbekd yofc protcias was 219W. Incorporation is reduced 50? ia the first days of canarc bat iberesfter Kiiiaai at a Hauvefy constant rale. Oocytes canared ia a pre- MtJWVLAmmHOEmAMOLXmAS vroady developed saaae medium du not maart-ati meir ANTIFBTnUTY AGENT abarty to incorporate proteins beyond aboat three days. FmaRy. the morphological integrity of cultured Mary L««a Aadmon* and J. S. Oumnai obcyics was exaaunrd by electro* rnicr'jscopy. The KAE has been shoaa to be an effective iwal remits indkate rkai ceHabr organeRc* appear norma) aaiifertdHy apntt in trace at a conccntratim of OXft W. and that endocytork activity al ihe oocyie surface is The ctimpiHind x'l« » a pmtcoital cn^traccptne. which nuaiiainrd. The development of a nippkaiental oocyte b effectne during the prcimpbnuiion ttages oi rnaintcaance medium wal aid oar studies of hormonal pregnancy (days I through $)• AR eviariK-e mdicates control of membrane activity, the rote of mr 'Vane in that DEAF, preventsearry embrytmic development with thr adnrptiaa of macromolccules at the ce.i sarface no obvious effects on Ihe mother. Cleavage of embryos and iheir entry and transport aithin the ceR. and the from DEAE mothers is repressed. These embryos never «aerer and syntheticrequirements o f the cell during consUi of more than four or five Mssinrneres at day 3. development. uhaV those from control mothers are undergom; Mastulatiim Culture of embryos in media containing DEAF, at a concentration M 10"'' M also rrvolied in *Jack«m Laanrainr*. bar Harfcnr. Maiw OMn*. inhibition of cleavage of the embryos. Marw'iri ii in 107 to OKA pury—wat-fl *•"• *yMtjrwt may he atfeied by OEAE: OEAf Ji 10 : J» owMnM had «k * abeam « Mfft-IA «e» » by VJtf nM>WmaValnm Of I IM^waCjnr. tfcfc fttM BMA NiLviauK Acmtnr w MEwnmuem IQOCYIB andfanVrmeUby' .bMTUUTtOft. T.G. fi b» been known for tome tane that jbnmmihnr* j*e caned by wwnaammnal jbeimnm* T.G. Severn! wearknown examples ase Donm s. Uajseftcftet s. ^^M Y^Mff'a w^^bt^M* ffl^M^fe^M M^^^MM* *^B fe# made to mow thai chrumraomal jin najjmiri may arise nfeoagh ovcfriaewm of the oocyte That is io my. oocytes semammy loo hmj m earner war ovary or vtrras to may develop shnanoiinail or cytoplasmic finely ties. cawtrawed mtcracinms tsccjessary tor emtayapeneajs. Inacfc work has been done on overripeneas natare of ihr oocyte's vhwapr These catty sfwJhts pstdontmanity mvohed two it essential lor cfniirymr, ideas oa die control of prneuime, overripe oocytes First, oocytes m of deithipmuiljl procesar*. Xtmtpmt Jarrnr were aboard to remain m the ovary for We foand ranapfc DNA pory mowing activity in j mundu or even years prior lo ovalaiiun and feruhza- mglr oocyte to incorporate 100 300 pmulri of lion. Second, in Aew /ajarm it is mmrible to ovatatc I'lijdAMP when assayed villi potylrA • potyldTi females and alow amtare (it-., second metaphase) primrr-tempbic. Incorporation was NKM for at least oocytes to remain in the ateras for a nmnber of days JO MM M the praeiK-e of artificial icmpfctie. ibowjfc prior tofciiduane, m rirrn. In both cases it IOMT deriaiiun was seen when activated DNA was awn. Nkcry thai acme oocytes bad chromo* The addition nf equal amoants of cold rATF to the ties and "typical overripmess syndromes. However. I'HjdAT? in the amy Mix did not redact radioactive ajmg of oocytes either in the ovary or in rhe ateras incorporation No incorporation was obtained in Ihr don not providr exposarc of the oocytes to experi absence of primer-template or when poryfrAI was used mental mampatiiion. Dae to the hardmtni and large in place of the polyfrAI • potyldTr. This activity is ntee of the amphJbvjn oocyte, many uatM expertntental imiibi to that reported for DNA porymemse-a from manipnbtions are poamne. A method for ajmg oocytes chickens. in a manner men that experimental access is possible pnwnftr a tool far OUt.lt a aarnM for ajaK .T tenriab observed by other workers for dissected oocytes mmm/2 nminiiihiimCunjiiiiih'i nmmtyll,C»0). thus appear to be a preparation artifact Instead, one of me terminal events of oogenesis rs a suppression of the of labeled amim- acid amt — aiming f*at various times. tennrnc-y to generate an Na'Jt* transport process when /, ami I'can be evaluated. FunnY. since oocytes are ovulated artificarty (by dissection t or •aturaty twhrch occurs approximately at the same time as gr mmal vesicle breakdown I Apparently, little in the way of membrane potential changes occurs during the where I» B the pm4 site whew me external concen normal process of oocyte maturation tration of ammo acid is lero. vamei wt lean bepkmed as a function of C to obtain both »'• and a measure «f pool espandabvfcty. Tms model has wow bee* tested *ncvarttncfj( of Zminff. tnwTnTy of CMUvmu. *tAttc% with tmet actively synthesizing systems: U» tea urchin btastutas. f 0) mouse cleavage embryos, ami (r> amphuV ran oocytes. Vabses for rates of protein syntbesb bate been derived which agree reasonably wel with pub- ROLE OF DNA UCASE IN DNA bshed data obtained by more cumbtnuHU methods. rOLYMERASE-l-DEPENDENT RETAIR SYNTHESB M TOLUENE TREATED ESCHERICHIA COU Wjw«ml Ubmtunf. Afpnmr. Wmm MM)*. Darnel Bdlen. C R. HePerntarin • and D R. StaRiom In lohieHe-lreaied bacteria, both replicalive and repair MEMMANE rOTENTULS IN LARGE synthesis occur in the presence of all four deoxyribonu- OOCYTES OF XEXOn>S LAEVtS deotide triphosphaies. In such bacteria many small R. A. WaSace and R. A. Sfcmhardt* precursor molecules are lust from within the cells, and therefore their role in DNA metabolism may be Large oocytes (1.2 IJ mm) wvhro their fcmcles measured. have an averafr resting potential >f 25 mV. When In toluene-treated Bacillus subiitis and A'. «*' possess marwally dissected out of their fani-les. these oocytes ing normal DNA lipase activity, nkolinamide adenine undttgii a hyperpabrizalion over the next 30 min in dinudeoiidr (NAD) reduces and nicotinamide mono- values around 70 mV. Small increases in external nudeoiidc (NMN) stimulates a DNA porytnerasc-l- |K*| hyperpolarize further. The hyperpobruation is depmdent DNA repair synthesis following X-ray ex prevented by mainiaining oocytes in the follicle at posure.1 Since NAD is the cofaclor for DNA ligase in 20*C. keeping disseclcd oocytes at 5*C. or incubating bacteria and NMN is an end-product inhibitor of DNA dissected oocytes at 20*C in 10 ' M ouabain or K'-free ligase. Ihe tentative conclusion was drawn that DNA medium. Dissected oocytes at 20*C also accumulate K* ligase is involved in a coordinated control of DNA and lose Na* over an *.-hr period. The evidence thus poiymcrase-l-directed repair synthesis. IM For direct evidence of aYe rale of DNA bfase m syutaeiuv. OTK DVA pJyumase-l-duecied icpan r» •jHHtoMaf RV extent *4" Kadeotide uHcrtMi during cuorduuted «*a OKA bgase actmty. \> xtnemrJ M B repair syMhesr*. we constructed a duubtt MBM «f*' satndn. the-e K an exagpnaled lepau %ynuVrn> at ..ehV o* K-12 with a condrtMuOy lethal HMtatM a *r ctmtaunnc polymerase I when DNA ugasr r» MUCUCC. structural gene foe DNA ugase cmfuiuu; nunifiHuii whetc;t m rautaah tacfcmg DNA p>4yw«n«c I bet imwriimg ptdjrmerase N and or M actraly. aW extent If NAD acts sotek raruugh n» rule a» cutactot for of tcpau wrihcM » will udtatneed by DNA hgase Ihc OKA ngase. *n at the • infuinniuni' Hftufit of same Mfati mdunu k*f tor X-ray mduccd atand break. 4. c use presence or absence of NAD should have hrde effect on 4K nkwK repair mAns observed m I'smg i—tan i> m winch dtt p»4ymci.nc I detect irradiated tohreue-treaied celK from which NAD ba> resales m Ms >'-*••* eAouucfcasc actwity. »c hoe been removed by in dung liwrntenri drat at JOT asccrtamed that iw*> acttnty Imcfc itansLitk*! DSA.«N 4K donate mutant eJuniied an NAD wuiirint X-ray- MTV Urn the exaggerated repaw \\Mhor> •IT (MlMrcGalBMS ROLE Of DNA POLYMERASES I. N. AND HI K DNA REPARt SYNIMESB M RECOVERY FROM UV4JCWT*»DUrED X-HUtADIATED. TOLUENE-TREATED DAMAGE M BACILUSSlBTIUSmr-t BACTERIA C. 1. Madden* and Darnel Bdkn Daniel Mien and (.. R. IMIennann* A mutant tirrr-/1 ot llndhti nronfh which t* detKieni in escrstiin of l"V-induced pyruntaW dhners fr««n We have previously reported that in BmiKus uthritis DNA shmvs a marked increase m abdity to survive l"V made permeable to nucleotides by toluene ireaimeni. a inadblinn when Dialed on annni^acid-supplemenlcd DNA polymerase I repair activity was markedly stim- agar medmm compared with survival Jnliry when ulaicd by X-ray exposure of the permeaMued ceUs.' In pbted on nulrieni agar. The extent of kdkng depends a mufanl lacking DNA polymerase I activity, repair on the richness of the plating medium 'Dn - 2.2 J m" synthesis was observed, but on a more limned scale. for nulrieni agar, and 5 b J m" for «y»ihetk- medium». DNA hgase activity was found to reduce the DNA so (he effect is considered t>> be one i>f grow in porymerase-l-direcied repair synthesis, hut had little dependent lethality. Irradiated stationary-phase iirr-l influence on DNA synthesis in poll ~ mutants. Because cells incubated in liquid medimn lackmg amino acids re many more mutants of DNA metabolism have been quired for growih recover Inm: this sensitivity to rich isolated and characterized in ¥.. tvdi. extensive study of medium within J 4 hr after irradiation. Recovery is DNA repair and replkative synthesis in f.'. o* is greatly reduced in the absence of glucose or in the underway. presence of NaCN. although not completely eliminated. The results to date show that all three DNA polym Exponentially growing cells have only a Irmiied ability erases, namely I. II. and III. are capable of carrying to recover from sensilnily to rich medium. out X-ray-induced repair synthesis in toluene-treated Growth-dependent (elhahly can also occur in liquid cells. However, there are several striking differences of medium: in nulrieni broth the ability of irradiated action among the ihree DNA polymerases. X-ray- stationary-phase uwr-l ceHs lo form colonies on defined induced repair synthesis carried out by DNA polym agar medium decreases during poslirradiaiion incuba erase II or III is totally dependent on Ihe presence of tion, but ireaimeni with chloramphenicol inhibits the AT?, unlike that of DNA polymerase-l-directed repair loss of this ability. Recovery from sensitivity lo neb Ill nolo t» aaMMied by calfeaar hat »4 In Mf*-a>dn>\\ • 22* J *-. and K>t ar>-/J = I > J m" >. ihew itfJin MVattwuHM^i aa adaa** «4 DVA Kpacattoa * 4K ancn*Mdec«bf lewe* ate drffnoii At low l"V AaValaK-^acTinc-eradKai prordet itmm that coadi- tweMcei. mw-l cefK cam •*• fCfaw AMkeai JI aua» aaVntaa; Kv>« •II tint Mtrntt t«3*ma*c Van.4 M fc wn*kJ Stawor*. •I I (ft* Utic* I^JAUI.- V*-4 .<• Rk-OTofcul S.vn.i* DEFECTIVE EXCISION REPAM IN DNA METADOUM IN IWEEKtf MILUS StVTIUS mw-l PTElUaTr^MJZEDC1faT«SEIUM»TER OVARY CELLS C IMIadden* and Darnel Mm DaaieJ Men and Ana t Olson* Excision repair of p>nrnNline dimers. J mator leston rn DNA following IA irradiation, can be stanaiari/ed We have deterrruaed that \" Tween W pfetreaimeai by the following steps 1*1 endonucleolyiic mcrsion of wiN alow nacieoiides to penetrate Ounese hamster DNA at the site of the lesion Irwckmgi. IM exonudei*- ovary ceRs. As with puiiKjNIi/ed bacteria, this system lyiK: removal Imccn S«l . ***> IV MAMMALIAN GENETICS % I Rwd E«Kto«fl Uncft.1 Id BmufMe VI Vtaffcmo R B (ummmc • . M. Cirn«« Vwim imr«tiiaft>r tr»m jhrojd 113 •'«•—JB.PU- ~*m 114 I aartvth. suprv have been mdstd ah* tot B^IM M hfnrjbif aneinwu am nanaai prrnwel scapes by Under let, and limn.' C nannti • «l ihr •etatwe iana •.*•!> ut Ik n*» bentaK. .-rJ rwnsftvan be made liable 1*1 thee ant *w«t dnterenevs aa the cuada- rht •esteem* Itoffcir* saer* •BLAim eowriiif\ OF MUTABLE > DBA 29 reewsed tV i nUNnOTATIOM AND InHttTABLE hybnJ (•01 (TLLSOFMALfMKl* CSHafi male* wear nsed. Second, m tbr ttaabes ifce aniaaak towed wear conceited 1L M. U«RMI. kaFhetanr T. Cam. first raw weeks atarr Haram. In the >«Hntf stcjsrs ihe X-ray data were afcw hased « h a that the «an»«* afcYtstant concerned dntinf ihr first row week* alter i effective at indncing chumnnuaat breakage m hm fo» EMS and TkM the nuyem tested e germ otfc MMC their highest effect Ihe few days shew d» nnnant-lethat. Maps Isprnnahiew and spunaandsi b» tratusiw. effects ate Lawn to that (he mum sensitire period any vary with the he nwiiiiiain. And third. whie ihe TIM dose n the hi addnraai to minimi, a number id" afcyt- sane in the m» stndars. hsjher average «W* ai rMS atiag chemicals thai am effect** mdncers of" dtiwmjat- and X Mrs near nsed ni the nr«rrssi« stady. TCwtv lethal naitafnnri haw also teen fawnd to nwhsce aacher fre^sencirs «>f tranaW-afn-ns o«nld be e\«evted faenixUc transiocatiom and htirubk amiMt m ihe at ihe dnses Ml rMS and X rays used Un tut aa«vn»Mt same male pminwiiMk germ-celi stages at those in ttadses. which dun—Jin Inhab are indnced. Thus, it is now k* ihe iranaV^atkin Mperiaarnts. irandiicatkK: punMt io compare ihr relative nsefwmess of 4» innuni heteiw/yetMMy aaannf note nmpniy nss deternnned hy lefhab. hrrifaUe translocation*, and heriiabk mcersnins IcTtnaTy test and cy ttdi*»icaf exananatmn vt dsatinesrt- J» nrakators of induced chromosome breakage ai these meiaahaar I spermrt«>cyles.* The mrthnd med ny germ*.*! stages. We nave already established that, for RMderkk and Hasan for detcciinf m»er»>»n hner»- Y.MS and TFM. heriiabir iraasiucaiiuns are a nmch «y*nMs is esam«ially a scarcn for high frc«aaencies «•« more sensitive and drfinitrte end point of chromosvtne bridfrs in ihr first meiufic anaphase at mate pniymy. breakage induced in ihe male potfmrkHk stages than One of Ihe testes is temmed and nuit+»p. jfiy prepared «>minani4eihal mutations.'": f«K hndpr analysis. The aniiml is kept j»ve and bred f«r r.MS. Tt-M. and X rays, winch are effective in further sindirs if ihe incidence of anapf a* bridprs is at inducing beriiahie translocations in the mate pos:- leasi 15'; IIIK *bacier«Hii»d"" fre«nw»cy Bahrnii * \ TaMrl*. TminKDI Dw»tr X.1. <»f ttfttftmr letted V«. M ftox**y *Mb jhrmlkHi n 7no lonoR 1557 I5i».% » fMS 200 400mp'kf M*. I I0A4.I TFM r>.2 mic/ke •7 Cnnirt*! 2MJ JH0.II -\ 7«wm ISI 4l!27.l5;» IMS 2nr» mp/ke 24A 791.12.1 r;» n.2 ms/kj: 204 5«»»2*.W.» *AII Ikaw da dateteac^s m exartaaraul >---vda» •« "ll'TH -Tmnjirxn i fi"~n"ki"riTTH ~tfi—"""*" '*" ' r r f" "#"•;?*-•- «J>^»*v^ajg*-r-Tv«-->iwy-.'-B.^-.«-^r-ywi.'My»- lib induced transmissible translocate ns in mouse sperinau*- the early stage, treated females were caged individually gonia. I)n the other hand. X rays, when given at with untreated males carrying (IK- sex-linked gene optimum conditions, induced uncquiv.ical increases in (tn-asy {Hit immediately it'ict injection. A total of heritable translocations at this ceil stage.* * although I4f»4 male progen) were tested for translocation the rates are lower than would be expected on the basis heterosy gosity. Ot' these. 54u were conceived within 24 ol the cytoiogical frequency determined ai diakmesis days after treatment ?nd 915 at later periods. It should metaphase t. be noted that there is considerable dominant-lethal effect on oocytes ovulated within 24 days after treatment. Two sterile and no partially sterile males Rd'atcd *rvn«*iv b» 'Ik: Y>lk»njl ( enlcr !>»» were found among progeny conceived at the early I.'XK»I.-.-KJI Kcwjich ami h> ihv tS I IK-ISV Ri-varvh and period, and one sterile and one partially sterile male lA-icli-pim-m \diranKralk>n under contrail «iih the I ro»n were found among progeny conceived in the later I arhtdc I »rp»tjit»n. period. The partially sterile male was confirmed cyt>»- I. V I. \. I jck'Ho. I. B. Ku««-ll. ami M. V S«-jri.«u. (ogically as a translocation heiero/ygote. One ^i the („'/•//.v7K?.; «M.i«m(. sterile males die<> before cytologicai analysis was done, :. N. I. \. 1 achrnv. M. S. Svjrt.mi. ami L. B. KuwH. Bf'l Ifcv. Inmi ft»»« R.p him <>t l*~4. t»RM-WI pp arid the other two that were analyzed (testes weights HZ J5. were normal) did not show cvtologjcal evidence of .'. t. I. I .>rj. A «.. ScirtV. I. P. I »anv and B Jean WOM. translocation hetero/ygosity. Thus, the results indicate (1/..-1 ••!,•!!, v n. 44? '.» i itvtt. that IMS. which is an effective inducer of dominant- 4. O. S. RoJUi. Vtiur K.> l.*i ilftoi. lethal mutations in diclyate oocytes, does not induce a 5. ». M. Uncr.-... K. I. < am. ami S. *. Hurt. «/«/. Ihi tmiu. /*>•* K'p Jum JO. I1J4. ORXI.-4W3.p- 13*. significant increase in the incidence of translocations in those cells that were transmissible to male offspring. Our results are similar to those of Kussell and INDUCTION OF HERITABLE WickhanV' and <.illuvod and ieonard5 with X ra> in TRANSLOCATIONS IN MOUSE OOCYTES that no translocations were recovered when male WITH ISOPROTYL METHANESULFONATE* progeny were scored. Searle and Beechey.*' on ihe other hand, claimed that while they also did not detect *A. M. deneroso and katherine T. Cain transmissible translocations among note progeny from A number of chemicals that are known to be effective X-irradiated oocytes, induced translocations were re dominant-lethal inducers in male postmeiotic germ cells covered in female progenv (three confirmed transloca have been found 'o induce dominant-lethal effects in tions in 2'M female progeny tested). This finding awaits diet> ate oocytes of mice. While it is generally known verification because the number «>f female progeny (hat one of the consequences of chromosome breakage scored is ratl»er small and because unlike with X ray. in male pos!meio:ic stages is the formation of reciprocal they did not find such an effect with fission neutrons. translocations, no information for such chemical effects on oocytes has been available until now. We studied the inducibility by isopropyl inethanesulfonate (IMS) of Rt'«:.ir>'b >r*>n*w.l h>inil> In :1K VI1IOTI.II I i-tm-r i«>r r<>M,.i>li>t:K.ii Ri-M-.itvii .in,l In l\w IS I n,Ti> Row.irih.iml heritable translocations in dictyatc oocytes of mice that Itetrlopmenl \,lmin»tratk>n iimk-r ton'rati nirli ihv I nun were in various stages in follicular development. IMS < .irhntc (i>rp»uii..n. was chosen rrecause it is u. effective inducer of I I . II. IhlinL-. I>. ti. ItotK-m. .111,1 II. V. Millim:. HUM. dominant-lethal mutations both in iru'.- pi>stmeiotic Kr>. 15. 175 X4il'»7:». cells and in diclyate i>ocytes.' "' : ft. \l. ««TKT.W.. S. ft. Hull. .m,l S. k. S: MIL Mutal. N.s 11.411 :iul'»7|, (SIX x. C57BI.IF, female mice ,10 12 weeks old) .!. U M. <«ih-r..vr. S. K. Ni.»iti. .m,l S. ft. Hull. \lnui. Rry were injected intraperitoneal!) with a dose of 75 mg kg IJ. 171 *4il>»7li. of IMS. This dose, in addition to dominant-lethal 4. I . K Kii.^ll .im! I. ftKkh.ii!) („n COMPARATIVE INDUCIB1UTY BY mutations and heritable translocations are produced. t-MERCAPTOPURINE OF DOMINANT-LETHAL While heritable translocations are a more sensitive end MUTATIONS AND HERITABLE point in postmeiotic stages, the reverse seems to be true TRANSLOCATIONS IN EARLY MEIOTIC in the early meiotic and differentiating gonial stages. MALE GERM CELLS AND DIFFERENTIATING However, il should be noted that the comparative SPERMATOGONIA OF MICE* inducibility of dominant lethals and heritable transloca tions in the early meiotic and differentiating gonial W. M. Generoso. Sandra w. Huff, stages was studied only for the compound 6-MP. More and Kaiherine T. Cain information on these ceil stages is certainly needed. Recent studies in male mice with two mutagenic cltemicak. mitomycin C and 6-mercaptopurine (6-MP». have contributed significantly to our present knowledge Rocmh \p»n-»red f>n(l> b\ the Njfiuiul (Vnicr Un JuxkuhrsKjl Roc-jfch jnd bv the IS Litest t Reicinh and of the overall picture of chromosome breakage effects Dtnektpmriit Admmi%lrjtinn under cntrjit »ilh the Inmn of chemicals in male germ cells. I nlU results with these t arbrfe < orpmM*m. two compounds became available. the chemicals which 1. V. \. RJ> jnd M. L. Il>ruck, /.nrvwi. H.allh /Vryx1 *. clearly induced chromosome breakage in male germ 27 Jt6il973>. cells either were alkylating agents or were known to be 2. *. M. ricnenxK. R. J. fte»t»»n.jn d J. li. Bremen. Vutat Rrv 2H.437 47119751. transformed in >n<< into alkylating forms, and clear-cut 3. Y I.. ( jvhe»«> jikt W. M. IU.-ner»«>. W>WMP .Yt-wsl.m..in chromosome breakage effects of such chemicals in preiv males were found only when posimesotic germ cells or hte spermatocytes were treated. The antileukemic SENSITIVITY OF DIFFERENT purine analogue 6-MP is an exception in that it is not an POSTC0PULATI0N GERM4TELL STAGES alkylating chemical and is not known to be transformed OF MICE TO INDUCTION OF DOMINANT into one and in that the chromosome breakage effects LETHALS WITH THREE ALKYLATING were induced only in germ ceils thai were presumably CHEMICALS* in late differentiating spermatogonia! and early meiolic K. t. Suter" and W. M Generoso spermatocyte stages. The chromosome breakage effect of this compound was first delected as clear-cut In an eariier study' we found thai IMS treatment of increases in induced dominant-lethai mutations.,l male and female germ cells after mating but prior to Subsequent cytok>gical analysis of ?he germ cells in 'he sperm entry yielded frequencies of presumed dominant diakinesis metaphase I stage revealed that chromatid lethals thai were much higher than the aJded effects on deletions were the likely cause of dominant lethality ' iiiosl sensitive dictyate stage and sperm in vas and The cytologic?! study also revealed, unexpectedly, that epididymis. In this study, the i>ocyies were predomi the rebiive yield of chromatid interchanges was very nantly at metaphase II at the lime of treatment. This low. posf dating precleavage stage is still several hours prior Results on induction of heritable translocations with lo fironuclear formation and DNA synthesis and may 6-MP al the same germ-cell stages are as follows. Out of no! be the most sensitive period. There are already good 215 male progeny tested from male parents treated indications from wo k on mammalian cells in ram that with 196 mg/kg of 6-MP. I partially sterile transloca certain alkylating chemicals are most effective during tion was recovered. Four hundred additional male S-phase.' And studies in mice with X rays3'4 and progeny from male parents treated with ISO mg/kg fission neutrons5 showed that early pronuclear stages weie tested for translocation heterozygosity. Of these, were most sensitive lo induction of dominant-lethal no translocation hetero/ygotes were observed, but there effects and sex-chromosome loss. Thus, the present were four sterile male progeny. Cytological analysis of report is ar investigation of the relative sensitivity of these sterile males1 revealed the presence of XYY postulating precleavage germ cells when treated before condition in three of them. The fourth one was normal. feilili/ation. al early pronuclear stage, or during DNA All three XYY males had small testes and spermato- synthesis. In addition to IMS. this study included I MS genic arresi. and TEM. These findings clearly demonstrate a marked differ Temales used were either from IC3II X 101 |f-( or ence between postmeiotic stages on one hand and the from a mixed slock obtained by crossing Gs/Y maks early mciotic and differentiating gonial stages on the with (SEC X C57BDF, females. These two slinks will other in the relative rales by v.iuch dominant-lethal be referred to as Jlf and SBGS respective!;.. In all •.—ry*mi,-m»iim'.< TaMeZO. Stage xmtuhtf of WEVMCCft* llourt after No. of No. of total Dvmy implant* Dead Dominant Treatment midpoint of copulated fertile implant* per per t'erlik- implant* lethal* dark period females Icrrukt fertile female fcmale il • »• Control 4 5 44 42 7.2 6.8 6 IMS • 25 reek* > 4.5 42 38 7.2 5.8 19 14 11.5 46 44 7.4 5.3 27 22 IT* 45 40 7.1 4.1 42 40 Control 4 5 46 45 7.6 7.3 4 f MS 13lN» me ksi 4 5 43 39 4.7 1.6 65 78 l!5 ii 29 5.0 2.2 56 70 17 42 39 6.1 3.0 50 58 TIM (0.6 mt ky» 4.5 44 43 6-0 4.2 30 43 115 46 41) 4.3 2.7 37 63 17 44 39 6.6 4.1 39 44 'Only rewlt* with ill ferrule*are included in thi* la We. "The preponderant yerm-cell *tape* lot 4.5. 11.5. and 17 hr after the midpiiint uV dark period are. rctnectivcly. ovulated egp in melaphaw II with *pm.i in oviduct, mak and female proni'viei formatkrfi. and male and female pronuclei undergoing I)Y\ *ynihe*iv Living implant* per preplan! female in experimental group I m.' i x 100. Living implant* per pregnant female in control group 1 119 *ftotoWturjl ianmtaaiur wpfurlrd parlt> by tabcuauact days alter the last intubation). With these numbers of N... 3)22 irwK the Rvlufv UXMW. (WML. lu the Lwrermy mated females, high dominant-lethal effects such as that of Tcaacucc jad rainy b> ibr p«eian»c»l ol the Caalun of observed by Badr and Badr should have been easily Zarna iSaii/rrtaail). detected. Because we folowed their procedure dosdy. 1. K. I. Saict. .VW«f. jt<-i.. in prro. 2. II. J. tnat ami D. St»ti. Ivwr. »>i S*-. h>mium. Str B the only explanation possible for the remarkably lli.*H 5l2U9Mi. different results is that the strain of mice tkey used y I. H Raivil aad «". S. **MU*umrr>. Inf. / Rmh*. AW (CBA) b highly sensitive to dominant-knlal induction I*. 151 Mtl W. M. Generoso. Kjtherme T. Cain. Live impbalt afcxpcrnanital grovp/femate x 100. Sandra W. Huff, and W. L Russcl live inpbatiot' control groap/lraolr A recent report in Xtturt by F. M. Ibdr and R. S. Badr' compefied us to conduct our own investigation RESULTS ROM A SPECIFIC-LOCUS TEST on the mutagenic effectiveness of ethyl alcohol in male OF THE MUTAGENICITY OF SULFUR mice. In this report they presented a seemingly un DIOXIDE IN MKE equivocal, high dominant-lethal effect of ethyl alcohol W. L Rinsed and Elizabeth M. Kelly in spermatozoa and spermatids of CBA mice when 0.1 ml of either 40 or 60* w/w alcohol was given once a Since large human populations are exposed to ap day for three consecutive days. Effects ranging from 3 I preciable concentrations of sulfur dioxide from energy to 67*7 dominant kthals were observed in their study.2 production and other sources and since sodium bisulfite We conducted our study on two hybrid stocks of has shown mutagenic properties in rnkroorganisms. it male nice. (101 X C3H)F, and (SEC X C57BL>F, seemed desirable to test whether any mutagenic effect These mice were 3 to 4 months old at the lime of of sulfur dioxide, or, more specifically, of sodium treatment. Sixteen males of each stock weighing 22 to bisulfite, could be detected in mammalian systems. 27 g were given, by intubation. 0.1 ml of 557 w/w Human exposure to bisulfite and sulfite as food ethyl alcohol once a day for three consecutive days. additives was an additional reason for mammalian This dose is equivalent to 156-191 mi of pure alcohol mutagenicity testing. given all at once to a 70-kg person each day for three Still another argument for conducting this investi consecutive days. Twelve control mice from each slock gation appeared after the work had started. Shapiro and were handled similarly and given the same volume of Louis: presented new evidence on the nature of the distilled water. Each experimental and control mouse deamination action of bisulfite which led them to was caged with two virgin (I0IXC3H)F, females conclude that bisulfite might be mutagenic at concen immediately after administering the last dose. Females trations much lower than ha** previously been sup were examined for presence of vaginal plugs every posed. They argued that "the presence of sulfite morning (for 21 days thereafter), and plugged females oxidase is not a sufficient guarantee against the possi were replaced by fresh ones. The procedures for our bility of bisulfite mutagenesis." and they recommended study are generally similar to those used by Badr and extensive testing in mammalian systems. Badr. After a dominant-lethal test in mice by Generoso1 Contrary to the clear-cut dominant-lethal effects had shown no mutagenic effect of injected sodium found by Budr and B?dr. ethyl alcohol did not induce a bisulfite, we proceeded to a specific-locus test, using detectable increase in dominant-lethal mutations in the some of the same males that had been employed in the two slocks of mice in our study. We killed for uterine dominant-lethal study. These had been injected pen- analysis a total of 73 experimental and 53 control toncaUy with 400 mg/kg of sodium bisulfite daily, 5 females that were mated at the time when Badr and days/week, for a total of 20 treatments. At 7'/, weeks Badr found dominant-lethal effects of elhanol (4-13 after the completion of the treatments, these wild-type 120 males were mated to females homozygous for the seven dose rate of ON R mm. induced mutjium tiequency in marker genes use J in our standard specific-Incus rest. mouse spermatogonia is independent of dose rale. Ilus The offspring were scored tor any imitations ai the Mas proposed as (he most tcasonaMe hypothesis to til seven I«KI (hat could have occurred as J result oi the observed lack ot statistically significant change in treatment ut spermatogonia! stages. mutation frequency as dose rate was hnvered ••ver an lo date, in a total <»l l.».5hK offspring scored, no NOO-toUl range, tiom ON K mm tor-High 001>» K nun mutations have been observed. I nirl further intorma- to 0.001 K mm tion becomes available >HI the metabolic late undei In our now ••xpeniueniv the !<>»exi do-*- :JI- used was varhHts conditions, it i> illlicull I > nuke J quantitative 0.000" R nun. The •cumulated dose ot MMi R used in comparison between the exposure level in the testes ot this experiment required a continuous exposure ot over mice that had received a total of XO0O rug kg >>t sodium 10 months to out low-level ,J7Cs source. The results sulfite, imected over 20 days, and the exposure level in ohtained at this dose rale are being compared with the gonads \'\ humans breathing sulfur dioxide-polluted those trom mice exposed t- (lie sariK* total dose air llf a generation. However, we have attempted an accumulated in >x days at a dose rate i»l 0.005 R mm. estimate of the possible upper limit \ti the genetic risk Two factors that were not considered in earlier m man from sulfur dioxide in the atmosphere, lakmi: experiments have been controlled in the current series. the upper (/> However, ihe effect dritps lo «me-ienih of ihe The results untamed are shown in Table 22. Although above when the du«e b halved, and. in offspring Intra the number of litters scored is not huge, il K dear thai, conceptions occurring in all bier weeks alter the first, of the routes of adnunisirauuu tested, intraperitoneal •at effect al al has been detected. injection b the only one that has a dear-cut etftct on (rj There wiU ab» probably be a large drop m XO litter size. It is questionable whether any effect al all incidence when injection is intramuscular instead of vnH be detected with the other routes of admuusira intraperitoneal, judging by Ihe drop in effect on litter tion. size. It is obviuudy important lo test whether ihe effect of I*) In spile of Ihe large effect with 150 mg/k«. the hycanlhooe on X-chromusome loss wiN also be de human risk is estimated lo be very small indeed, if not pendent on Ihe route of administration- Up lo now. we zero, from ihe therapeutic dose Thus ihe test has have estimated genetic risks from treatment of human turned out lo have a fantastically fine resolution. Once females on the basis of results of intraperitoneal one knew where to look, ii was easy lo get not only a injections in mice. The findings reported here indicate delectable effect, but a quantitatively measurable one thai such calculations may greatly overestimate ihe risk which permitted ihe estimation of ihe human risk lo be from intramuscular injection, the procedure used in probatory negligible. humans. It should be pointed out that even the human risks estimated on the basis of mouse intraperitoneal injection are minimal at the therapeutic dote. So the 1. W. I.. Rvnril. P. R. HamKtrr. I. M. Kctv. C. M. Vaarfean. and G. M. Gains. Art. Mr. Amm. /W*. Rep. hmt practical result of applying the new finding, if it abo 30.1974. ORNL-4993.p. 11*. proves valid for X-chromosome-low induction, may 2. W. M. tintrr.n... V. J. «e Series. S. W. HutT. and K. T. simply be the addition of an extra safely factor. Cain, AM. Arr. Amm. Hog. Rrp. hmt 30. 1972. ORNI-4W7. The present results could have an important influence pp.125 2». on Ihe methodology of mammalian chemical muta 3. P. R. HwtncfccT and W. L. RuratN. thin report. genicity testing. If mature oocytes on the surface of the ovary can be exposed, via intraperitoneal injection, lo a much higher concentration of a chemical than can any EFFECT OF ROUTE OF ADMINISTRATION OF other germ-cell stage in either sex. then screening for HVCANTHONE ON LITTER SIZE IN THE possible mutagenicity by litter-size reduction in early OFFSPRING OF TREATED FEMALE MCE mating* and confirmation by ihe X-chromosome-kiss lest may prove to be a sensitive, as well as relatively Patricia R. Hunsicfcer and W. L. Russell easy and quick, way of delecting some classes of Since a transmitted genetic effect of hycanlhone in mutagens. mice had been detected only in treated females, despite extensive testing on males, and only in litters conceived in Ihe first week after treatment.1** the question arose 1. W. I.. RimrU. P. R. Hamiffcer. r. M. Kcty. C\ M. Vauthan. and G. M. I'mnn. Ihn report. as lo whether the route of administration might be 2. W. M. Gcnrow. F. 1. dr Serre*. S. V. Iftrfr. and K. T responsible, or partly responsible, for the sex difference Cain. *W. Mr. Anna. Phy Rep June 30. 1072. ORNI -4817. in response and possibly also for the limitation of the pp.125 26. effect to early matings. Intraperitoneal injection had been used throughout all experiments, and il seemed conceivable that the surface of the ovary, where mature T«*Mr 22. Effect of vjfiovf nates of srioitMMrafioii of foMcles arc located, might he exposed to a higher 150 MB/ fcfo f fcycsBflfcoiito n toUtr sfet in ofnpfwig concentration of an intraperiloneally injected chemical CflMCCMVo in uflN? twSf WtVut 4WPW than would Ihe testis, or perhaps immaiure follicles in WEMMMttf Of PtfMnVC MKC deeper regions of the ovary. Accordingly, a test has been run on different routes of administration. The genetic effects thai have been detected in our earlier studies on treated females are filler size reduc Control 17 6.1 tion.1 increase in dead implantations, and X-chromo Intorliofl CM»V 13 3.0 some loss.' For a preliminary screening of the possible InjKimntimr I* 6.3 effect of different routes of administration, litter size Injri-liiHHw) 14 6.1 Goner 12 6.5 was chosen as the easiest end point to measure. 12b •BUMS neoM sntttN«G FOB are heme tested. At Wast lour of these are iransmHitag tAMAIHHMNDUCED NBIOGLOMN the effect. VAMANTS M THE MOUSE 1. a. I. rwd. t. M. V••>»*•• •- A. r\f*>. at K. a. W. L. RMSMJI. Carolyn hJ. Vaughn*. R- A. fupn. JbcafcM. aW Mr taw Itu*. * EFFECT OF ACE ON INDUCED AND of age to X*c/Y units. Pari of each group of I SrOOTANEOUSNONOBJUNCTIONANO is irradiated with a sunk dose of 200 R X rays I CHROMOSOME LOSS week prior to mating (I day prior lo matmg in the early part of the experiment). AI four groups are luadfed B. Russet and Clyde S. Montgomery concurrendy. This procedure is lepbcaicd cutty 2 The wen-ki weeks as mmrih beccrne available. The i of Down's syndrome and of certain piimiii the phenatypk detection of both i X-chrnnuiinmr anmnabci in nan' Ins long led to the pnernd sexihiumoinnw losses, and of both i thai oocyte agog predisposes lonri spon- and paternal first-dmawa •uniirjunitinn (or second- . In the nsonse, mcftased age has diiiiiun aiwliijuai linn of crossove with duleased frequency of Certain monks and trisomies may also be < mcrcascd frequency of detectable, Exixptroaal animals arc tested oocytes at meiosis-l.z and with and analyzed cytohjeJeaHy by means of tissue cultures plohty in fetuses conceived. A effect of from the puma. The phenotypc by watch unly 5 R of irradiation on aged finale J has also been •ondtsjunciion would be detected m sons. chawed on the basis offetal chromosome analysis* but XT»/X*/Y. is unequivocal. However. XM km (from any die reams are somewhat open to doubt. cause) would yield 0/X'*. the phrwotype of which is We have undertaken a cytogenetic study on the effect iiumitfctd by one extreme of the range of expression of 1 of nuHmjl *f on spontancons and indnced sex- the normaty segregating X7X *. Therefore, moil of w cfcromosome nundrsjvnciiun and loss in the mouse. the "possible 0fX " animals that are saved nun out. on 1 Maternal X-chromojome nnndJijunction has not been testing, to be m fact X7X *. reported in the past, and the upper 9S% confidence No irradiaied female had more than two litters or liniit of the zero spontaneous frequency is 0.04%.* The produced a niter more than 3 months after treatment, spontaneous frequency of XMfos> averages only 0.05%. while controls have yielded up to * litters (even m the and the effects of acute irradiation are readily meas- old group) and are stW producing litters 10 months urabk.* after inrlntion of the experiment. For more exact In an experiment still in progress, genetically uniform comparison, we have listed separately the first two XT»/X* females (F, between the inbred TABR and litters of controls, provided tbne are bom within 3 C3Hf strains) are mated at either ca 3 or«. *> months months after mating (Table 23). It may be seen that old Aft at IV« Komber «rf Xmubrr of „ lemurs offinriiie Mien per ^ j KTIT Xwrwal (R) fcrlincT M M r P cbiMfieJ X X Y 0X X"X Y %% Other Hktl fewob Mile Femur 9 2»» MM) 0.91 *6 402 3W 0 2* r 4? y 2M» 541 1.00 4.9 271 25» n 1*5? r n 55J" I.«I 25* 2*> 0»J? 0 o jnrr* i.*o 5* IJ5 IM 0 .! n 1035 5ot 52? 0**? 2*1? n 40J 424 n* j? o 3? *A ? lymknl faNnwMe * wwlm mitc*tc% iNai nn ymrtk <--r cylflfoeiol Mniitbi area carriemld . namficafin* by snmvHrac «inrr i* unceitaiii i*ee tciil. fvrtt iwo ktfrrv fttnittd that ilmr arc hrvm wifMn } mnoth* after fMiwg. jtt near rkr md «f ffcrtr rcfri*Jw».i*»? fcfr^futv vMIr offcvrs owry recnwfy started tTiTVVMlC. IwRR?ftafftPS Wt*fJ M iRf r*Mo*mH^lrH jrf Hill fH""*?. 130 females produced beiler than young, both in the we decided to carry out autoradiographic studies in irradiated and control croups. ar» J thai, while ther? was fetal nonkidney and kidney tissue. J an obvious effect of radiation on number of litters, One T(X:4) and one T|X:7) were chosen lor this \ there was (title or no effect on average litter size. study, namely T|X:4)IRtz and T(X:7>hR« ' * These jj The results with respect to nondisjunction and loss will be referred to as RI and Rb respectively. Fetuses 1 ate as yet quite preliminary. Because of phenotypic were removed from the uterus I* days after discovery 4 uncerlainlies (see above), most of the possible excep of a vaginal plug. At that age. sex can he recognized: i tional animals listed in Table 23 must be viewed as very and. when appropriate markers have been used in the j tentative. No unequivocal instances of maternal nondis mating*, translocation fetuses can be recognized by eye | junction have so far been observed, although there has color (except for rate cases of crossing-over). Tissue been one case of paternal (spontaneous) nondis cultures were initialed either from the kidneys of these * junction. We are continuing with the present croups and fetuses or from their rcmaming tissues. Chromosome '-\ adding others at older ages and higher doses. preparations were made by a technique combining j fluorescence bunding (for identification) and early- \ bbeting autoradiography.* 4 1. V. M. Comn Brm*. P. law. ami .. Smith. .4**. Www. Table 24 compares the earlier results for Rl and R6 Cowr. 33.1 I4IIH*). (first 2 lines) with the present una from I It-day fetuses. 2. S A. Ilmfcn— and R. G. Mvnnb, tonvr 21*. 22 2» It may «e noted that Rl shows random maclrvahon tmder both circumstances. Ro. which gave extreme 3. M. Vjnawolo. A. Kn*». ami U. Wxanabc. ,\«IMY Xrw nonrandomness in adult kidney cultures, behaves ran aW 241. 141 42 11*7J». domly in fetal nonkidney cultures and gives iuler- 4. M. YJSMMH. T. SkaKMb, A. tmtn. ami G. Wilambc. v«wr Srw Hot 244.2INVIH 1*73). mediale results in fetal kidney cultures. 5. I •. Rvnril. m Cmrmkml N**wntj Vol. 4 1(4. by A. Since considerabte portions of the autosome arc tnNntiMCT). ill seen. subject to inaclivalHW in T(X;A)s.4 functional hemi- zygosity for a large number of autosomal loci exists in about 50% of all cells in cases of random inaclrvation.* A CASE OF N0NRAND0M X-CHROMOSOME It b concehrabie that such a situation may not be INACTTVATION EXPLAINED BY SELECTION tolerable in tissues where the loci involved play a | Liane B. Russell. N. L. A. Cacheiro. critical role, and an extension of this explanation has. in | and Margaret 5. Swarlinil fact, been invoked to account for the fact that T(X.A)s j are recovered with a frequency considerably below that Wc have earlier reported1 the result of autoradio which would be expected from the incidence of graphic studies tm seven X-autosome translocations T(A^h.J |T(X;A)"sJ that involve either chromosome 7 m 4. Tne present results suggest that in R6 |and possibly in Using an early-labeling mrthod in cell cultures derived some of the other T(X;7)s also) one or more autosomal from adult kidney tissue, we found that either the genes are inactivated that are critical for survival of intact X chromosome IX") or the long translocation adult kidney tissue. Selection therefore occurs for those product (X*) was differentially unlabeled (presumed cells in which X" is inactive, i.e.. in which the critical inactive) while the short translocation product in each genefs) on X1 is (are) active and thus functionally case was labeled indislinguishably from autosomes. diploid. The intermediate result obtained for fetal However, mmrandom inactivation was found in three of kidney indicates that the critical period of activity for the four T Tafck-24. toXt Ftomtat of faWltfcstialy Ageirf Kaabctaf NMBBCTOT Ti- Crank x„~~ iranlis cent x".«r x'.rt* AMI Knfcaty Rl 2 172 45.4 54 j» Aaa* K«fc«jr R4 * 54 9t_2 IJI It-feyfrtei Ki*n R4 2 TT TO.I 29.9 IBtfajrlctas T-tal* HI 2 47 55.5 443 It-** fan Total M 3 IJ7 55.5 44.5 "X* - 9: A than Jcramowter ifc* matt X: X* "* upiwMiiX'gipiMMlf -rx«. HK CMM ik* iMiK «*«**. after rn»irj|#j nV kafcarjrv lucahxc critical genes fine such in specific strains titer sate of 2.7 is is show* in the table. About a third of (he but only 2% of die males, are sssaH. A i of nonwd jiqd Bales were tested for fertibiy: 8 1. H t_ A. (xftriro. M. S. JMM. L. B. Ra round to be ferine and IS sterile. Three smut Gmran 74. *3411973). 2. I_ B. Rml ami 1. W. •jtkiw. Crarlnr 4ft. 509-25 currently being tested ave apparently sterile. We shaft llfftl*. analyze these cyiotogkaliy to check the J. I_ B. Rnrl ami C. S. Miinmnw»i>. Grarrin43.103-20 that they may be RI3 males with an extra X. II949>. 4. L. B. knarii atal C. S. Mwwn—tcty. lirmnks 44. Se* 2*1 31211970). 5. M. N. N**MI an* S. M. Garth*. Crfoj.-nrrtrj 9. 212 21 22 1019 IS I0S9 H97©>. 273 5«5 24 M2 4* L. B. MM. •§ 71MF ROTT cy CMnviivpfOHiey cpt utwtwp' mmt (ca°. by M. Locket. Anorawt Prea. New Ycifc. 1944. pp. Cyiological analysis (by quinacrmr-mnsiard banding) 153-il. of kidney cultures from small females indicates a reciprocal translocation, with on; break near the A NEW X AUTOSOME TRANSLOCATION THAT centromere of chromosome 12. the other in the X MAY GIVE N0NRAND0M INACTTVATION chromoscme. at about one-third of its length from the Liane B. Russell. N. L. A. Cacheiro. Jean W. Bangham, centromere. The two translocation products designated 12 and Margaret S. Swartout 12* and X (with centromeres of chromosomes 12 and X respectively), arc of medium size. Early pube Information derived from the study of X-autosome labeling with ,3H|diymidine was carried out in kidney translorations |T(X;A)s) has contributed heavily to our cultures from two adult RI3 females. In this procedure, existing knowledge of gene action, gene inactnation, an asynchronously late-replicating (presumed inactive) and gene-dosage effects in mammals. Of ten mouse chromosome wiU be selectively unlabeled. Among 25 T(X;A)s known until recently, nine have involved either ceils in which asynchrony was observed, there were 18 chromosome 7 or 4, and have shown random inacf hra- in which the 12* chromosome was the unlabeled one. tion, excepi in special circumstances.1 We have this In four cells the single unlabeled chromosome was year obtained information on a new T(X;A) which probably the normal X (although mffustness of X- involves neither of these autosomes and may produce chromosome fluorescence presents occasional diffi nonrandom inactivaiion of a different sort from that culties of identification). In the remaining three cells, observed in the past. This translocation. RI3. was two chromosomes - the normal X and one or the other briefly reported last year1 (when it was erroneously of the translocated chromosomes were lightly named R9). labeled, while the rest of the complement was heavily RI3 was found in a slock set up from a small labeled. daughter of an irradiated male (300 R of X ray to If the preliminary indications from die labeling postspcrmalogoniai stages). In propagating the slock studies prove to be correct, they would indicate from small females outcrossed to normal males of other nonrandomncss Hi an opposite direction fiom that 132 observed in T(X;i»)i6H. where buth tamsiocated CTIT)U)ClCALSTL'0i«0rSTr3»AiTriN90r« chromosomes are almost always active whie the normal OF MICE TREATED AT STEM4 ATOGONUL X is inactive in at cew*. Crosses that introduced OK EARLY SffERMATOCYTE STAGES X-chromosume markers (sgfl or /r) and chromufiinvt-12 «OTMMUTAGCNrCHE3w)CALS markers (m*. ft\ have »v far not yielded results N. L A. Caritriro. W. M. Generosu. •MbVaune nonrandoninrss. However. dtrse luci may not s and Margaret S. Swatawt be situated in 12 . We have rtudkd cylutuckaaH and hwiokfjcatly 14 sterile sons derived from iptrmMinywii treated with I. L. n. HTU. V t. A- (jcwrim. ami M. SBaflMM. Pm •CVMtt. cyd iph iiBhwnsi. TBI. »«r TWA. The 14sterilemice >. L. n. Ummtt. K l. \. (idknf.. J. n. •iwn.ii.anJ M were found among 3X12 F, mats tested rot fertility. SnavtMN. JW Mr 1mm IVwy AVsi ibr Mt. t9U. As was the case after X-ray treatment of spermato (MtNL-r**).rr. in 22. gonia.1 only a ssnaat wnrwutian 13 of 141 have chrontoaimtt astdtnahrs. One maV each was XYY: XX/XXY nmsaic: asm 40 chnmmumwi XX. TlY.I?). AfKstma>nMKOOmK9waaiott AM three had stnaM testes and ipermatiigrntr, arrest. MTNE MOUSE In addition, we have tfudtrd four stride suns (out of 6IS F| tested for teriifctyi derived tram differentiating Liane B. RusteR. N. L. A- Cachem». spermatogonia and very early spermatocytes treated Clyde S. Montgomery. and Georgia M. Cninn with 6-nwrcaptopufinc. Chromosome abnormalities In me course of an experiment designed to measure were observed in three males. aH of which were XYY sex-chrumosome loss after irradiation of spermato and had mtafl testes and sfermatogenk arrest. Although gonia.1 we found, within the presumed X*0 (i.e.. this number is still small, the frequent XYY condition paternal-loss) class, a female that had a submetacentric in sterile males from the 6-MP study may be attrib chromosome in a complement of 39. Farther geneik utable to induced nondisjunction. Tfus prublem is now and cytoiogkal work showed that the submetacentric understudy. segregated independently from the 39-chromosume None of these 18 sterile mates derived from chemi (XO) condition, indicating that the abnormal chromo cally treated spermatogonia or very early spermatocytes some was wholly autosomal. Mriotk preparations from had the type of aberration most frequently found in animals heterozygous for the submetacentric failed to sterile sons derived from irradiaied pustspemtatogomal yield multivalent configurations and had only 20 stages namety. reciprocal translocations in whkh at btvaknts. Since the shorter arm of the submetacentric is least one break h near the centromere w telomere.2 of considerable length, multivalent configurations would be expected if the abnormal chromosome were 1. N. L. A. CacfcewD ins M. S. SwartoM. Kmlmt A>i. 59.45 the result of a reciprocal translocation. A pericentric 119741. inversion was. therefore, suggested by this finding. The 2. N. L A. Catfcriro. L. ». Ran*. Mm M. S. Swanoal. suggestion has been tentatively confirmed by the results GntrririTt. 73 91(1974). of cytoiogkal banding studies, whkh indicate that the affected chromosome is 9. A breeding stock has now been successfully established, and homozygotcs have recently proved to be viable and ferine. We shall CYTOLOGIC AL STUDIES IN STERILE SONS attempt to introduce chromosome-8 markers in order OF IMS-TREATED FEMALES to determine the effects of the presumed pericentric N. L. A. Cacheiro and Margaret S. Swartout inversion on recombination. The submetacentric may also prove to be a useful cytoiogkal marker In earlier work we studied sterile sons of males chromosome. treated in postspcrmaiogonial' or spermalogDnial1 stages. We have now cylotogkally examined seven I. L. B. fteorfl and C. 5. Montgomery. Mum. Res. 25, sterile sons of females treated with IMS in experiments J*7 76(1974). ofW. M.Generoso. 133 ChMMuauMe aanurnabtie* were observed KI five of complexes. When intact nuclei from the liven of C3H the males. Finvmgs of the seven sierilr sous may be mke were incubated m me presence of the Ecu Rl sununavBrd as blows. restriction endonuckase. 15 JOS of the total nswfcaf DMA was recovered at soluble form as high molm-mar- wewjM mattiMl. The dkjesl contaiks DNA. RXA. and protein. Over *tfl of the digest it excludrd from targe-pore cufcimni «f agarose gel. DNA purified from J. 41 ifcniwn HIn, XYY. the dkjett is til" lnjhin»lecularwe«^a»*vfrnan*dby 4. MaairXY/X*. agamr «r> thiiiophnreiu. Protrini from «V agarose- column cmate exiractabk with SD5 have been anaKied by elecWiiphaKsts m SPS pofracolannue gets. The resuhs show thai most if the protein is htstone. the rest Anarnamv m bfyMryjr. being a compk^ array of other proteins. A variety «f hattwisutiun procedures wM be used m It showid be noted that the types of jnnmatiti found attempts to obtain humujanuui preparations of arcra the whoh dvfErrcnt from those m storm; sons of uiadUitd or EM5-treated postspennafofaniaf stages. should provide a hasn fa the detection of proteins dutl moat of which cany uiipn*»l tnmlocTltotii hairing a** not uniformly duiribuled over use genome as wel one or both breaks mar the endf s> of dtromocomcfs).1 as for the study of a wrk variety of fundamental processes in the expression of genetic information. 1. N. L A. Cxfccim. L. U. Kmmdt. and M. S. SwM. <«**rars *•, 73 -91 (1*74). ENERGY TRANSFER FROM ACETOPHENONE 2. N. L. A. facte*,, awJ M. S. SwMtart. JfadfiM. *«L 59,45 11974). TO DNA-rOLYLYSINE COMPLEXES E. G. Bernstme and R. O. Rahn ATTEMTTC TO ITN» PROTEIN DIFFERENCES RELATED TO GENETIC The bwchenucal assessment of damage to the genome DIFFERENCES AT SINGLE LOO by a variety of agents is a logical extension of work m mammalian genetics in progress at ORNL. Prettimaary E G. Brrrtstine and Liane B. Russell experiments have shown that the number of thymine The availability of a delated complemrniaiiori map dimers introduced into DNA by energy transfer from of the duuie (J) sborl ear (te) region of chromosome 9 acefopfrtone molecules excited by irradiation at 310 of the mouse' makes Ihe biochemical identification of nm decreases linearly, after a brief hg. as the DNA » the product of the dilute gene of interest. The mutation titrated with poryiysine. The project was begun in order if'* affect) pigMeniatkut by catping clumping of to detemune whether an estimate of exposed and meianosomes and opblhot<*k: seizures believed to be covered regions of DNA in chromatin and nudei might due lo myelin degeneration.3 Since both of these be made by comparing the extent of acetopbenone- defects may result from an altered membrane com mediated dirner formation in thae samples to that in ponent, we are comparing myelin proteins from mutant pure DNA. The sensitivity of the method is not and wild-type animals by analytical SDS-polyacryl- sufficient. Dimers were quantitated m unlabeled mate amrde gel electrophoresis. rial by hydrolysis of irradiated DNA, chromatography on km exchange paper, ehition with HCI. photoreversal at pH > 11, and analysis by difference spectroscopy at 1. L u. Rn«ll.«bM/ Rrs. II, 107 23(1971). 275 nm. 2. D. V. Ketlon anJ H. Ranch. Kxp. Ntwn*. «, 252 62 U9»2). A NEW EXPERIMENT ON THE INDUCTION ECO Rl DIGESTS OBTAINED FROM OF SPEttFIC-LOCUS MUTATIONS IN MOUSE INTACT MOUSE LIVER NUCLEI SPERMATOGONIA BY TRJ71ATED WATER E. C Btmsti,-ie R. B. Cumming and W. L RusseU The current goal of Iras program is the isolation of In experiments previously completed' in this labora defined regions of the mouse genome as DNA-proiein tory on trilmnvmduced specific-locus mutations in the 134 MVK, ^SCStwOwtt WCIC IwBKVl WMCll HMt K MwpOftflM chlorine are ats> used in jadutfijjf praccsses mvwrring Cnkrme in water wqy react with any organic mate need ofctanficaUMu We previemsty fwnvd that theME rials it encounters t» form stable cMuriue -. Jtjhucg tmwuwfjTj jVB^ffm^PHBaBBBBBBjajfwawTwwnnawMnwBWBwffjiB0 ^BBBwaTawMvAVmSanVJl Innay* tnamW; nwawawnflaajnnmnjB>ur. twaBuw•JPaaH. I• wananlwavnwk* a* tfraanks.' many «rf which ntty hare potcw- bw4>*gKal •MM ruinmr HM n shjMy abmc 2. MM* dab are effects.1 h is cstuvntrd that, hum sewage cMurmauun weeded t» detenu** if tnuum dues indeed unidnce ahme. www than 1000 tuns «rf such stahtr ddurmr- nwre gcactk damage in Sfjetmatunmna per wait of abaarbud dose than X or gamma raws. We abw fM an ;nfa» surface waters each year.1 JutVy' he demwn- utwfcatmu in out first series of experawrutt that the itnted the presence vf mare than #0 chfaanr iiw- dhmumtmu of wmiuni amuug the amen loci scwrcd wught be Afferent than for gamma rays. These two sewage efrureuts. and 17 of these uumpu—df hare been pwiats need to be clarified by iddHainjd data. •cVutifted. The most abundant ot the cnmpmi idr We hare started a new experiment dir%md to identified to dale is 5-ddurouraci |5-C1U>. a thymine auu«wuuatrty double the number of data from cews analogue whkh may be mcurpuratcd into USA and treated at spenuatofmua. The logistics of this experi whkh is mutagenic in some bacterial systems.' This ment ant greatly reduced by not coucctmg any data contpound. therefore, may serve as a good model to test from eds uradnted in pcntspcrmatogomal stages. Thus whether a known maty dumped into natural waters the males aw not mated at the isolators, and more may pose a genetic hazard to man. It is of great interest notes can be treated at one time. As before, ore nudes to know whether mimmah which ingest 5-CIU in thru are given a single ip injection of tritaied water at 0.5 drinking water actually incorporate this base analogue mCi per gram of body weight. They are placed in into DNA of their germ cets. It is also important to isolators with five treated males per cage or 72 treated know whtthti it is k^wkaUy possible to sit up a animals per isolator. This compares with 18 treated meaningful genetic test in mammals exposed to 5-CKJ animals per isolator in our previous experiments. The in their drinking water. ansmak arc held m isoblnrs for 4 weeks and then Male (C3H X 101 )f, mice were given water contain removed and placed in a special room. At 10 weeks ing cither 100 mg/Kter ot 1 g/litcr of 5-CIU for eight puttmjettion they arc mdrridualiy mated to catch the weeks. Water consumption was normal, and there wet' first poststerik offspring, and they wwl remain mated no easily observable nl effects. Weight remained normal. for life The higher of these two exposure levels is at least To date, two groups have been mated and one has 1,000.000-fik'd as high as man can be expected to produced offspring. There are presently 492 offspring receive in Ins water supply. At the end of 8 weeks these and one mutation at the ft locus. The data from this mates were individually mated to T-stock females, and experiment should be much more extensive by next the pairs were mamtamed on the same levels of 5-CIL' as year. previously given to the respective mates. Offspring were collected from this small number of matmgi (12 and 11, respectively, for the lower and higher doses) and I. R. B. C«MNM«. W. L. lUwH. an* G. A. Sept. Hal. Mr were scored for specific-locus mutations. Fertility and Amu. hvt Ktp hmt JO. 1974. ORNL-49*). p. 12*. litter size were normal in both groups, but there appears to be a smaH reduction in the percentage of offspring STUDIES OH rOTENTIAL GENETIC raised to weaning at the higher dose level which, if it is EFFECTS OF 5-CMUHtOUItACIL IN MAMMALS real, is probably due to a slight toxic effect to young exposed at this high dose. To date. 393 offspring ha-e It K. Cumnunf, t. C. Pal. Marva F. Walton, been scored in the lower-dose group and 314 in the and W.L Russell higher-dose group. No specific-locus mutations have CTdorinafion is the most common method of dis been seen in these 697 offspring. The use of this very infecting water wed by humans in tins country. Most small sample for risk estimation is dealt with sepa municipal water supplies are chlorinated heavily enough rately.4 to provide a specified residual chlorine content during Male offspring from this specific-locus test were dntribution. Most sewage effluents are required to be saved, when weaned, and maintained on 5-CIU water at chlorinated, and more than 100.000 tons of chlorine the same concentration as their parents had received. As are wed annually for this purpose. Large amounts of young adults these mates were kitted, their testes and I3S knees MM rcntMcd. and UNA wancsiijitcd from these VkCVMorjr The OKA was hydMvied by •w* DKaari. 'snauui •f d—syeh iiudn" MIL' rwed by chr imn niuhr «f the DNA tl.li was S€K. Ahuwi 1.49 «4~ the me level. Tins mi mill to aawat I S-CIU for every 250 wdcurtdri (averaged tlvowganwt the ge- t). or ahem 2.2 X I0T S-CHJs per I fuur 5-C.KTs per structural atne. •feat 1. R. L. i*»*y. ORNL-TM «»•- 342 pp. I If??). 2. C W. Gcun. L. D. EyaM. R. L. Mb}. J.E. Tlw ••«• •..*•«»«• 24*. S45»U974>. »n 3. R. B. C ii|. D. Ccwtc. M.F. WakjM. R.J EtaJum. ika> nrpuri. 4. W. L RBMC* aatf R. B. Cmmmmf. this report There n a slight and probably not lignifk it reduction 5. C. A. Sep. R. R. < •»•»•». aau" M- F. Wan—, mm. Mn in total impbnts in the S-CIU-trcated group. There is no 24.317(19741 bud dominant lethal is not significant and is hailed to A TEST FOR S41fU»*OUtAClL4NDUCED apparent implantation loss. Thus there is no evidence DOMtiANT-UTHAL MUTATtONS IN THE MOUSE for a genetic effect. When the present experiment is R. B. CuMtning and Barbara J. Ehnhorst* complete, approximately twice as many females will have been scored. At that time these results can be S-CIU is a compound which is produced by cMorina- reevaluated. lion of sewage effluent,' is incorporated into the DNA of mammals when it n consumed in the drinking water.2 and is mutagenic in bacteria.1 While no *ORAU aattfgntfvatc rcxaick pankipnf. JUWUI 1975. specific-focus mutations have yet been detected in the Vmtnrmy of Wncemia-Strveits Point. Stcmw POM. S44SI. movse. it is of interest to know whether this com 1. R. L. Mky. ORNL-TM 4290. 342 pp. 11973). 2. R. *.Cmmmmt.t.C.rj.M.r.W*o*.*m4W.L-Km*ta, pound is capable of producing other classes of genetic tin report. damage in mammals. A dominant-lethal protocol has 3. It ». CtoHNvm;, D. L. George. M. F. Write*, ami ft. J. bfen devised which allows mating of males under rHMloril, IRBS report. continuous chronic exposure to an agent with females which are not exposed- Using tins protocol a dominant- lethal mutation experiment to test potential muta MUTATIONS PRODUCED BY S4m0R0URAC1L genicity of 5-CIU is now in progress, and about half of AND S-BROMOUIUaL IN ESCHERICHIA COU the data we anticipate are already available. R- B. Camming, Donna L. George,* Male offspring from the specific-locus mutation ex Marva F. Walton, and Barbara J. Elmhorstt periment1 had been exposed to I g/liter 5-CIU in drinking water since before their conception. Under 5-OU is amMg the most abundant cMorine-contanv these conditions, 5-CIU is incorporated into their ing organic compounds resulting from the chlorination DNA.2 Twelve of these animals were caged mdrviiually of sewage effluents: consequently, it is an environ and supplied with 5-ClU-cofilaining drinking water mental contaminant for which there is some human during the work day. Twelve matching pens, contammg exposure.1 Its analogue 5-brmnouraci (5-BrU) has been three unmsted females each, were supplied with regular shown to be mutagenic in bacteria.1 Both compounds drinking water during the day. At 4:00 p.m. the 5-CIU arc incorporated into DNA and replace thymine, and males were transferred to the female cages, and the we have shown* that 5-CIU is incorporated into the 1* »ffkMMWiwjl ••rNvakW ffuiinl »• ^kwM« I a*; •MB *r M« PkwM. OftM.. t» tfer li•KM I «f T«BOW. KOIIMSJI *«KAl' HillUJJ II NKKk fMIMfOM. «•w t 1975. I'm JIIIOJ •» Tn.ni Jm« FfcaM. AHM> fWmt.544l | 1 «- 1 Mk«.«Ml!«.-T1l49*. J4IfVLil*TS* iL» Ma *ri f. t. Km. JVMM In 0.4*7 9 U974*. *. «. & mi. a. < ML Nanar". **••IJ^<1 . after JO MHI or 5-OrU. Him aas aa NIDI *© *0 MM W#ltf Jhf. Ifct—llllBIW IL0C- 5-OU B dearly system far 25 lo 100 F.*fcni«rM 1 F^nimil wrm/lo* warm 1 ftvforfiMfl' wmt/lo mtim Iteimtimm NT* »J» t 0.99 13.53 t ».97 BHT S.9S • OJI I3.7J t 1.34 rwnMtojrbJtal I2.3S '. 1.24 l« St • 1.17 - RMS NT- 4*5.5* • 55 *04_5*»2 •HT JMA'22 21 411.9(29 35 fmwifcjiaiU 34IJIi27 29 407.5'30 3* MMS Ht 9*2.1 • 12 20*5.9'254 urn *59.* • 45 32 M3.2 • 42 *5 rWCTVnWIVItM 935.5 • 75 3 I54A.* • 172 25 Ho ffcQCMnMiil. 137 tar al «vf m* ma* points. The rlndor DKA •o ng|i«imii I'HIdThd activity m the «as 27 days pv—mimrm wbjk use X-vajr- «CJH X IOIIF, 10 i: (rood animak ahmved the pveseace of |'H|dThd- WH11*.79tm*n)fm2 kvd *f I'HldTlHl uptake into she *as aveem of i lac a i iltdayi The perm erf stanes shvMuat repair «f X-ray. of of mm- of |*K)dThd after X-ray teas. This is m spite of me net mat H2»i _ UPWVT •wapnuaYnYfuS •*• upvavgvjapu*#pswvl aVvAmamw mV wavauujr* B^0"J^nl wry FJMHF RafXtays.*-* The vJOMt X-ray dear is not iaaaaMag notes hecanse we mad an elevated level of iJOORiiaaed. DNA by X rays Roberts* uiimaiti that for every lesion m DNA. ippmiMalili 1001 •col Scot ***. iter repavjcdsvvr.il is. I I. *. ft. CMM« ariilF Ifclinl. mwer level of DNA i 11.547 53IIW3IL using 600 R of X rays compared with 250 mg/kg of in.* CMM. M. F EMS is a nomvestahlon of the mmmer of bases inserted J. C A. Scfa. *mr. Xrtt *arf Jri CXI 71. ««5S 59 Mo the repaired teams. II974H Work has also been started relating the level of DNA 4. G. A. Stjra. J. C Owm». a4R. I. CMMM*. taw icfMl- repair m the germ ccOs of Mate mice to the dose of X rays received. For example, the level of DNA repeat UNSCHEDULED DNA SYNTHESIS M UK oceanmg m early iptrmatidi has been foand to increase GERM CELLS OF MALE MCE AFTER linearly with X-ray dose from 50 R to 600 R. Higher IN WW EXFOSURE TO X RAYS doses are currently being stadted. It shoatd also be G.A.Stga noted that the detection of mtschednkd DNA synthesis m the germ effc of mate mtee at X-ray doses astow a s Unacheduted DNA synthesis in poitnviiuiic fjnaxcu 50 R attests to the sensitivity of these experiments of nnk mice was fiisi demonstrated using die since Fnater. for example, has nvavcated that a dose of muapm EMS.1 N was also of Merest lo at least 1000 R was needed to detect DNA repair in the if X rays cowM induce wsstkcavlcd DNA synthesis in male perm ccRs. Therefore, two groups of Another aspect of DNA repair m mouse germ ccfisis (C3NT X 101 )F, nates were both given testicular the yVjMiiianatiun of how the rave of repair in a given of ('HlthynwJne (('HldThd). with one germ-cell stage changes with time after treatment. Thai of notes abo receiving a 600-R dose of X rays can be atvestigated by giving the lestkular injections of over a I0>mm time period. I'HIdThd at various times after the end of the X-ray Three animals were kmed from both the control treatment. Since we have found that the labeled (I'HIoTM only) ami the X-ray-treaied (f'HIdTM • thyntidme is omV aiiHibtf for incorporation Mo •00 R of X rays) groups at 2- to 3-day intervals 3-27 germ-eel DNA for approximately I hr after it is •ays after treatment. The fHldThd activity contained injected.' it is possible to measure da level* of DNA in several mvmon purified sperm heads taken from the repair occurring at different times after the end of the vast deferenta ef both the control and X-ray-treated X-ray exposure. The results for early spermatids ex > of animals nvs measured ht a Kojvid scMnBtion posed to 500 R of X rays so far indicate that die repair IJt i a. K »t in *. •.» «I«W|L .itf« T^Livrrpr n av S. I. f. vbtom. J. K rw*c. B. A. Sank. ** *.BX lai maul Co*Mt.aniJMhMM X«) Mil*?!*. afteri *. C A. &•>. ftor. W* .%** **. (» 71. «*» *» ARfewrofl «T |*K|dThd into cany at me same (mar mr f'HfdThd of HUB ON 1ml RELATIVE EFFECTS DNAofeariyi of METMYL. ETHYL, PROPYL, AND I ht after utMma*. The MinbarYLI*TTfUI«£SUI^ONATEvN effects wf al tour thsnmafa m dw CAISNCCV ITIVUVKKD1IUDDNA of ptiwhceR DNA repair appear to be I SYMIMBB M TBI GERM OEUS oner the dose ranges studkd. The greatest DNA i OFMALEMMT* response was seen using MMS. fohwwed by EMS. IMS. G.A.! . J. C Owens, and R. ft. C« and finally PMS. The same relative order for MMS. LMS. and PMS appbes m their effectiveness m the induction ot donunanl-kthal mulJiiwm in spermatozoa cd stagrs of nob unor dm undergo DNA Kpnir < anal late spernunms. However. HD tans between exposed lo EMS.' we are now looking at four« MMS and EMS in this case.* Comparmg MMS and EMS. MMS. EMS. PMS. and IMS todetc for exanvpb. it was found that at rquimwljr admin i to induce DNA repair in monse germ cells, istered concentrations of the two chrinh-jli. MMS was m particular, sperm from candal epididymides are being about 5 times as effective as EMS in inducing DNA 16 days after treatment. (Sperm from die repair m early spermatids. A similar relatjuwihip has I epididymides are used because there are about been found between these two chemicals m the twice as many of them per animal as there are of sperm induction of DNA repair in cultured injiiamhjn in the caput spimdymis or in UK vas.) This meant that cHb." although in that case the difference was at the time of treatment these caudal sperm were early tenfold. Roberts ei of.'' have found that m their cultured For each of these chemicals a dosc-responsc curve has mammaban ceHs repair syndiesis is directly related to been obtained which measures die relative unscheduled die ovenl level of DNA aUcytation m die cdb. If the uptake of |JH|dThd ialoearty spermatids as a function same result bobb true for the mouse germ cefb treated of admmssttrtd concentration of die chemical mutagen. m vnv. then measurement of DNA repair in these germ Abo. the lebfrre amount of repair occurring over a eds could become a fast and economical way of 3-day time period fobowmg aduamstratiou of each measuring chemical dose in the genetic material. The chemical ha* been studied, ftefore die duration of re technique would be economical, since expensive iso- could be investigated it was necessary to estabJisI topkaly labeled chernkal mutagens could be replaced long labeled drymhfine remained avanabb for by much bat expensive labeled thynudme and un poration into the grrnweH DNA m die testes after labeled mutagens to assay for the molecular dose injection. We have found that after testicular injections received by dw DNA. of the |*H|dThd. its removal from die thymidlnt pool The measurement of DNA repair m die mouse germ of the testes is quite rapid, with most of the free ecus abo appears to be a considerably more sensitive |' H| dThd disappearing from the testes within about an biological end point than measurement of dominant hour after injection.' This result is in agreement with lednb or translocations in the germ cefb. The measure- uo I2> in L9» 9» •f OKA 5 tw 10 i 261 1. C. A. S«js». M«r -W .*atf Jri 1X1 71. 49J5-S9 Il974n 2. E F Oja.a«F-.4ai.,'/tar 99. 391 4I3U95** 3. F * OafcKg.,!* / Amt 99.597 I9H954+- 4. LOOW(«4f B. UMKt.GMnrln tt. 1*17-22 |I9W». forme 5. $- OwnnH«b. L Eaacaacic. arfC.A. *«dK«d- « CIWII U* J arf ifer 11 Ear** noted that when SO id of | ll|rilhd Mm MM *» the testes, the iwaica encap- 1 C. A. Sc**. r B. (MM*, ant M. F. of the |JH|dThd sohNioa would leak oat. Therefore, a ta>. Aw* AVp. *mr J*. f »7*. OKXL-M93. » 12* smaBev. 35*1 volume of |*H|dThd vkiMraMcJ 2 r. D. Lrrto mi t. terutrv fcitw V Mi. 433 47 lc try to prevent this ruftwmg. There was no dsf- 1 C V feci. 1W ffcr AM*. fta*. Jtro. Jhnr A l»74. faeuce m the level of wnachedated mcosuoranon of J OKXL L 141 it»heafeaw>Ktwa«05 a* 115 aftf «f the bhM aVnaasnc daecwy •*» cack 6m keen aMkraait ky a 4rW Sra f XI 71. «f>5 9* larCyckiakos- DSA tor Stga's |'H|dqraHanr) • early ipnnuiidi. la untreated aace DMA ao —Kfctuwhd DMA svntkesn was observed, (a) The i n rclaled to DMA raw. An i anaauM of •an.fccaaWa DNA svnaVws ia treated germ ike MBI ikM bit ecus varied accordmg lo ike laae aroanMy rcujarfcd for the capacity far DMA jtlwatiag rke nuMageus. Warn very bilk- or ao aclwa- tkrir DNA • cdr/bsed. Farther- M conM ke achieved at ike Mat of iacorporafmi of it is known thai EMSi ,JH|dTkd. unsckedakd DNA synthesis was consider- saectfk-vocos mac akiy lower ikaa a* later nan when activation kad pcrmitR sbsjr*. wktrc no I'Wiaawbiy occurred. Unscheduled DNA synthesis in DNA syrHhesis was delected in Sep'* experiments. creased with time ia both cycle ahojpfcamidf- and It is interesting, livrrcforc. to del amonvn-trcaiew frrra eels, reached a anaaa. and r a srmtar pattern of DMA *kta detuned, kkknaea mdaccd a rmxanaai response il woakJ ke wry attractive lo postatate akoai 0.5 hi afier keiag injected, white cyctophos- IkM ikt genetic davnape MNN ky EMS an* outer pfcaaaat drd ike same skoal I kr after treatment. aftytating agents may roan front failure of DNA reaair Unscheduled DNA synthesis remained at significant lo occur. !*»•* several hoars after treatment with both chemicals. To mvestigate this possikdity a aaiiaw of chemical These mate are interesting for a nnnajcr of reason*, najtagens arc ai present btmg tin died in oar laboratory. h a jppiient that cyclophosphamide or its active •7 BwjanBanjamjnaj w |a>^paHjaj9 v^awa. w Y w>ww*vnnaannBmawja»mjavana> aaaaaji BBBjaa^paflB>v« netatotoe irigpri a DNA repair response in those wnl he skoam hrre as examples of tan different fcraxel stages wtach are the mast sensitive to the matagens that produce Afferent patlerai of frnn-ceil aW-elron of genetic damage ky ike compoand. la other stajt stwrilivity to tkt amaction of feaetk damage. wwe.. a maximum matagenkity coexists at timr and QrdophospMrnidc induces chrumjsvmi breakage in gcmvcel stage, with an active repair process. The an postmcicHic germ-cell stages of PJBNC mice, die most puwikility. mentioned before, that FJB-iwdaced sensitive being thai of carry spermatids.1 Mitomrn, on genetic rUmege may orifjnite from failure of DNA 142 The tan of F, wu* EMS -t 200 nag/kg. Mf/fcg. or sahne ICUMNOL 5.5 from e>«.hiphus- 17.5 to 20.5 days Control F,*» are conceived 5.5 to*.5wr 17.5 to 20L5 days puetnonwrnt. Thus the F, s art • Jnccd by the particular stitcud because it is I I Yefganean s onpnal report. us setecseul uccause tuent rss dm there is a ffcujncncy in •EJUTAOLE ntBtUrA1MPML LESMNS treated with tins agent. F, MTNEMOUK at approxunalciy sewM weeks of age. and cytefogicai preparations 120 skies RIC< Ea»ill.E.&rt yor each) are made uang a nweufiratmn of the air-dry A project has been Itihniipn of Evans rt •*..* which has been used in our between the Food and Drag laboratory.1 Fonowing a .-ccovery period, the F, oaks ERDA to kmsficMe the are mated to produce F: mates fat least 12 F, mates for each F, V The F, nates are then treated with urelbane. the DNA of nanMaaban germ ccHs. Thcst prcmuta- and at various limes bier (from 5 to 15 days) they are tional lesions could then be iraasnwttaj to < kdted and cytatopkal preparations arc made from the uniam u% testis. In the event that chromosome abnor frequency, or they nugbt bt developed into malities occur in Ihe spermatocytes of the second testis, expressed mutations by exposure to a second agent preparations from the first testis wnl be examined in winch is not nwmufy mntantnic. In 1971 Lavappa and drtai to see if the same abnormalities were present YcrgaMan' reported such a situation in the Armenian prior to urcthane treatment. If such an abnormality is hamster, in which uKtnane caned chromosome aberra observed in F, mates. Fj animals from Ihe same F, will tions in certain mate frrm-crll stages in animals whose be examined. grandfathers h?d been exposed lo EMS, white no effect To date we have produced, from FMS-treaicd fathers. was seen in urclhane-trcated animals whose ancestors 141 nonsterik F( mates which have had one testis had received no treatment. If this is a real effect, it removed and from which good cytotogical preparations constitutes a very important class of emetic damage have been made. Of these. M have been mated to because it provides a mechanism for interaction be produce Fj's. and 29 have produced 12 or more F, tween afrnts which arc not by themselves mutagenic - male offspring. From cyrlophosphamkte-treatcd mates a class of genetic damage which would rot be detected we have, to date. 145 nonsterite F, 's which have given in any mutational test system currently in use. Our good cytologies! preparations. project is an attempt to repeat the Lavappa and A prcurmnary examination of slides from the EMS- Yerganiafl experiment in mice, using a much more treatcd F, 's showed 9 clear translocation heterozygotes 1« •mtui the 141 evamiwd. ami there were. m iddiriiin. 4 The mmw frequency at 000066 R/mm rheretoee sterile ammab in mis group- Thus the freuuency of pvesumed heterurygotes 113 145» is ham than would itself at nurmai levels. The pummmty of setectM he expected for this dote of fctJS. Ve dunk, however, tannin bt ruled out. biriri appears ummeK. The rale «f that Ihe n'liimrr wdi mceeast as the aides ate examinrd OJ0O56 R/mm. which reduces me A, ipiimit ujinrii lo • gamer dead, wet thus fat only a few ceH* ham 379 of control, gives about dur same freuufucy of hem analysed for each F,. No cytulugtcal data are yet nmuriim. ami 300 R. whew oft survival is «mty I6S. avaiaHe far F(*s from the crctoptrjsplMmde-ucaied gircs me highrst mutation rales UaL This rcsuk is the groups: however there were 5 sunk F,'som of ISO reverse of mot expected if thrre is a pwsiuve onrdmMi between sutsstwiry ID ceH kmmg and anuitiniij to IILS. LHjffb ari t. \€tpmom. Sntmcr I7J. |7I 74 JndJraird as the primary factors in the reduced fre- uuincy of multlhmi ubse.ved at low dose rales. > R. I . S«MM»«C Mil.1. IMM|. JfcM. *V*. 27. $75 asu*T*» J t. P. tcun. li. Imlwa. ja4 C. t. t/Mtf. (Vfwf>ri*rr*-> X EFFECTS OF *>MERCAPTOffJUNE ON *T» **«l*4l. SPERMATOCONUL SURVIVAL AND DURATION OF SffERMATOCENESB ft* TRE MOUSE SflRttATOGOrOAL STEWCELL SURVIVAL t. F. Oafcberg and Deborah T. Pa!afim» AFTER HUUDUnON AT LOW DOSE RATES E. F. Oakbergand Deborah T. ralatima This experiment was prompted by me uuMrvjinm of Gencroso et d.' that a high freiaurncy of dominant These data were obtained to evaluate the possdde nde iethab was observed 32 J- 35.5 days after mjecliM of of cci survival «u mutation freouencies at different mate rruce with 6-MI\' 'J Chromosome breakage was dose rales. 101 X Oil hybrid males were given 100 R elevated at days 14 and 15.' It was important to of cesium gamma rays at 0.0056 and 0.00066 R/mm determine if rate of speriuatogenesis had been affected, 156 and 6.7 R/wfc) continuous exposure and -.wnpsicd or if chromosome breaks were being recovered from with maks given 300 R X rays at 94 R/m*>. spermatogonia. One group of mice was lulled immediately after Five groups of 12-week-old 101 X C3H hybrid male removal from the gamma-ray field: a second group was mice were used. Group I was used as control for cell luhVd 24 w later in order to observe miration of cell counts. Group 2 was given IS *rCi [JH|inyinJJim. n-poputatnm. Cell counts for these intervals were the fohowed 30 mm later by an injection of 0.25 ml 0.03 S same for pmnu-irradiased mice, and ihe data were NaOH. Group 3 was given IS *rCi |3Hlthynudine. pooled. Mice of comparable age exposed to 500 R Group 4 was given 15 pCi of |'H| thymidine, followed X rays at *M R. min were kdled 120 hr alter irradiation, 30 mm later by 150 mg/kg of 6-MP in 0.03 S NaOH. when ceH survival is minimal. Group 5 was injected with 150 mg/kg of 6-MP only. Since the exposure to low dose rales involved all Animab were killed at intervals ranging from 72 hr lo stages up lo the lime of kiWng. daia were presented as 17 days after treatment. cci type scored. The most drastic effect was observed Testes were fixed m Zenker-formol. embedded in after 300 R acute X rays, where only \tT< of the stem paraffin, sectioned at 5 sim. and stained by the periodic cdb and only a few differentiating spermatogonia acid Schiff technique with Fhrlich's hematoxylin as a survived. Al 0.0056 R/mm. stem-cell survival was 37^ counterstain. AutoradiTgraphs were prepared of sec for A, and 24^ for A, cells. The remaining sper tions of testes receiving |JH| thymidine. Surviving matogonia! types showed depletion lo about 5** of spermatogonia were scored al 3 jnd 10 days. Progres control, indicating a marked increase m sensiiiviiy for sion of celb labeled as prelcplotene spermatocytes was these stages. At 0.00066 R/min. the number »t stem fnttowed lo 17 days, or for two cycles of Ihe ecus was unaffected, with survival values for A,-A« wrnmiferous epithelium. spnmalogonia ranging from HI to °7">. Thus, the Number of A, (stem I spermatogonia was not affected. equdrbrnim al lower cell numbers which occurs al this Al 3 days, frequency of A2-A«. In, and B sper dose rale results from sensiiiviiy of At-A« sper matogonia was reduced, respectively, to 80. 54. and matogonia. The possibility thai stem-cell kinetics is 507 of control. Number of preleptotenc spermatocytes affected is being invcstipled by 1*111 thymidine label- (treated as bit* A« early In spermatogonia) was at the •I control level. Recovery was slower than expected, with 144 numbers of A|-A« goaia and prekptotcnt sper Male 101 X C3H hybrid mice. 12 weeks oht were matocytes depressed to 84-9)% of control at 10 days. given a smgk ip mjectiua of 20 mg of wjethaae per Progresnoa of labeled cefc through spermitonraeiii mease (22-23 f vesrfMX Aaimalr were knkd over a was identical for groins icceivmg (*H|tlrjrmidme ami 12-hr to 12-day iaietval. aad Sim paraffia sectioas of |JH|ilryn*oWpktt64ir the testis were stained by the periodic acM-Sdnff The results on spermatogonia! kimag are mtcrcstiag m tcrfmafae with EMich's hemaloxylm as a amatcrstaia. that no afepktioa of stem ccUs occurred, ami that late Spnaancgpari were eaaarnHd m 99 tubular cross sectioas distribaiedoa the basis of freqweacy of me 12 sanative cd type, showed ao effect as measured by stages of the ryck of the aaaaifriuuj ipiihilmm •amber of prdeptotciie spermatocytes at stage VII72 The aamber of A, (stem) spermatogonia was depressed fer after treatment. Abo, recovery was slower than we only at721n.Ai-lnccns>liowevet.aVmcdadtdme by have observed for other rhrmkali, with a samiffcaat P hr. and prekptotcae spermatocytes were aot deptessioa m dnTercatiatiag sperantogoaia aad prctep- rcdaccd anlil 72 hr (ie. aald cans treated as A»-la loteae spermatocytes 10 days after irramatioa. 6-HF spermatogoma were sampled). Thereafter, afl otsl types owl aot affect the rate of spermatocyte ami spermatid from A| spcrmatoaoaia to arckptoteac spermatocytes ambpmrat. Therefore, it cm be concluded tha * LHSwih T.TOM J M Hottwd E.H.Itofcim R-W.Tc C.F.Gottlkb C.C.Uvdte W. J. Peterson J. A. Otttn C. n. Sfaw* J. If. Qwrks. Jr.* R.J.kacati" H-3B. G.E.Cosgnwe W.D.Cwde N. K. »\w%r J. P. Daufheny* JMYuhat Effect* •flftfi-LETRatirtiMs RE.Toya.Sr. J. B. Sioier K. W. Christenberry* Kvwetfu A. Davidson* ft L Ullnch *KM(4>K-IOTJI mvetfjgtfof. 14S ., I MR 146 conclude that enhanced activity of the RES is not a major factor in radioprotection by PhRBC. In another series of experiments, we tested the possmwty that PhRBC could create an environment in hemopoietic tissue that was conducive i» recovery of radiation-damaged stem eels. To explore this possibil ity, we measured the spieen-colony-forannf capacity of RAMontorocnoN OF MKE BY bone marrow m several types of recipients. Injection of H»iYUiYIIIUZWE4UMACEOEftYTHtOCYTES unirradiated marrow ceus produced about the same numhtr of colonics in mice that received PhRBC as in L H. Smith and T. W. McKmley. Jr. mice that received saline If > 0.2). This observation appears to exclude enhanced trapping or growth of r'beayBrydrazine (PhNHNH]) is radioprotective if marrow stem ccfts in spleens of PhRBC-trcated mke. Of given to Mice about I week before exposure to X rays.1 greater interest, however, n tne fact that the number of In experiments d* signed to characterise cwnoprotectiou spleen colonics arising from irradiated marrow cdb was by this drug, prehnanary resuErt showed that erythro- greater (F * 0.05» in PhRBC-treafcd mice than in cytes (RIO damaged by IWHNtH (PhRBC, were also saane-treated controls. We tentatively conclude, there radioprotective.1 SpedfkaBy, die injection of PhRBC I fore, that PhRBC may create a splenic environm-wt that day before irradiation resulted in enhanced 30-day promotes repair of radiation-damaged hemopoietic stem survival and splenic hemopoiesis. Apparently, RBC that celts. are altered by PhNHNH, initiate a chain of events that The results to date show that PhRBC provide a establishes a preirradiation condition which renders the soluble or particulate (actor that acts directly or animal mote radioresistanl. In a previous progress indirectly on hemopoietic tissue of mice. As a result of report .* we presented evidence showing that thb this action, hemopoietic tissue has the capacity to enhanced radioresistance of mice was not the result of recover following lethal whole-body irradiation. The an increase either in the D0 li.e. radioresistance) of fact that PhRBC are radioprotective when given before hemopoietic stem celb or in the number of stem ccUs at bat not after irradiation indicates that the factor is not risk at the time of radiation. effective after injury has occurred. Our data abo appear Since there n histologic evidence that PhNHNHj to rule out both of the following stem-cell factors thai increases reticuloendothelial system (RES) activity.' would enhance survival: increased radioresistance of injection of PhRBC could also possibly increase RES stem ceHs and increased number of stem cetts at risk. activity, which in turn might provide the irradiated The only observation winch suggests a basis for radio- animal with a better defense and consequently improve protection by PhRBC is that irradiated marrow grows survival. Therefore, we have continued our studies of better m mke treated with PhRBC than in mice treated radioprotection by PhRBC by examining the ,'unctional with saline. Enhanced marrow growth could indicate slate of the RES in the treated mice. As a reflection of the presence of a hemopoietic mkroenvironmeni more RES activity, we measured the 7*^ for circulation and conducive lo repair of sublethal radiation injury to the liver accumulation of injected '' Cr-labeled sheep hemopoietic stem celb. Accordingly, the factor pro RBC. Enlianced RES activity would he expected lo vided by PhRBC would be involved in creating this reduce the T>fj and increase accumulation of sheep mkroerwironrneni in hemopoietic tissue rather than RBC in the liver. The results show that, compared with acting directly on stem cells. controls, neither expectation was observed for any of the groups on days I. 3, or A after treatment. Liver accumulation was slightly, but not significantly, in creased above normal by each of the three treatments, and PhRBC alone or in combination with irradiation did not significantly affect accumulation compared with X-ray control values. Accumulation data for lung 1. L. H. Smith and T. W. McKMey. Jr.. *«*»/. Ka. 50.611 and spleen were also obtained. Lung values were M972). consistently low in all groups «2%). and the spleen 2. L. If. Smith. KM. Drr Axmi Prog. Rep. hmt JO. 1974. ORNL-4993.P. 145. values were no higher in the PhRBC-treated irradiated 3. J. It. Jamil. N. M. Fifes. S. H. narmrtl. and K. A. mice than in X-ray control mice. From these results we MKDTMM./ Kxp. Med. 122.299 f 1965) 147 AMUTY OF TtmMCYTESTO ducing the same kmd of data which permst a smmar MC1UASEKOLIFEKAT1VERATEOF TftANSnJUiTEDMARiWWCaiS ervthroBoietic colonies Thm ejovenmem had been Joan Wright Goodman. Sank C Shinpock. attempted on earner occasions with Utile success and Nancy L.ftasford because of the dnuKuky of finding a Awe of marrow Our nasi work using a l*-F, poor-fniwth combmatMMi ecus whoat progeny would produce enough crythro- has indicated that thymocytes brio control division and poiese! within a iiuunauh time fo be meaiurablt by eaffcreutiaiioa of irawylanled nonbumnk bone mar the **Fe uptake method without, at BVs same time. row stem cds. Data from Zipori and Traimu.1 who studied nude and thymecumnaed mice, and from Lord number of splenic colonies- We juccemh! m the and Schofieid. who transplanted oogenic marrow damaged by asmal expomre to Xrays, inggiitid that cefts (2.5 X 10s). <•) giving 80 times as many the thymus pfays a amtroamg role in hemopoiesis in thymocytes. fiom the iMMMilogists' Irisiiitf Jhjf of various subsets aged take exhibit dramatical* depressed responses of of dryatocyies. we a*e Jstvnrigniug specific characteris- DNA synthesis ||sH|thynmbac uttorporaiioa) when tks of different eel pufwsWtiuau. Cefc ubt.imeJ bom stimulated with the T-oeft-specific ptant lectin. phytolKssiagtmtmm (PHA). This loss uf activity is mm* elective m augmenting growth of marrow eels thought to be related to the decrease in w rriv tsasnUBuned iMo urtdjatrd reetpwats than ruuriahni >,el m/diiird muatsne response seen with advancing doses of rhymk lymphocyte i. We as* exploring the age. Rrhaaajij efforts have demonstrated that the origin of the augmenting cents) few* die peritoneum. PHA response can he restored at older adult mke. and Results to sate show that they aw astsfftectcd by to some extent m aged mice, by removal of a auti-theta serum putt oiatpltntrat. We has* found that plastic-adherent cd population before citlturing spleen m antigen jbuurplsua slashes the sssitss—Istesl peri eels with PHA. It has also been reported that separation toneal easily of the mouse yields leUtrvery few theta- of spleen eels unuipunirw to PHA produced a beanoccdk pupviaiioa that suppressed the PHA response of normal hi a kss-owect appiuuth to this ulemifkanon prob- spleen eels. KH. we are asking expcrimentaly whether there aught Spleen eds from virus-infected mke and animals with also he a man lejiiar T eel mtitn.4 m coatiwthag either a variety of progressively growing tumors respond the stem eel i'self or the lymphocyte stcm-cel inter poorly to PHA. Furthermore, suppression of ur wUro action. Despi.e ate negative awswer that cast be interred autumn induced lymphocyte stimulation by tumor-eel from studies asmg thyineituntiied donors and/or recip- supemalants has been noted. Therefore, it was im iente m the parent into F( IrybridfP^F,) pour growth portant to see if related mechanisms ought account for consbmatiou. we have transplanted a msal amount of the dmtmiihed age-related spleen eel response of isogenic mamiw fie.. P -* irradiated F) with or without BC3F| mke. where tumorigenesis is marked during F or F| thymocytes. After S days we found there was senescence (neoplasia is present in 80% of our BC3F| no significant difference m sptenk cokmy rwrnber as a mice at the time of death) but restricted primarily to resort of transplanted thymocytes. Another series of Type A reticulum cell sarcoma. experiments used doubty thymos4eprived F(*s Accordingly, we have determined if the decreased (thymectomized mice that some weeks earlier had been PHA activity we see in spleens of normal size from irradiated and given marrow from donors that had pathology-free aged mice could be related to suppressor earlier aho been thymectomized. irradiated, and given eels arising in response to beginning tumorigenesis. but isogenic marrow transplants). Had a host suppressor cell before hhlopathology is present. Hence, celb from both been an important factor in the growth of P marrow normal and palhologk old spleens have been tested for transplanted to the "normal" irradiate J F, hybrid, suppressor activity. We first measured the decline in doubly thyreus-deptived hosts should have permitted PHA responsiveness of spleen celb from old animals development of many more splenic colonies. This was free of pathology and from old tumor-bearing mke. It not the case, and we must conclude with the evidence was found that activity of spleen celb from old animals so far obtained that there is no -suppressor T cell free of pathology was sevenfold less when compared to operating in hemopoiesis. young adult animals, whereas the PHA responshvness of spleen celb from old animals with enlarged spleens due to reticulum cell sarcoma was •*» 100-fold less, dramatizing ihe marked additional depression occurring DISSOCIATION OF ACKMKELATCO with pathology. REFtMCTOMNESS OF FHA4NDUCED If the decreased PHA responsiveness is due primarily LYIvFHOCYiXimANSrORMATlON to an active suppressor cell population present in ANDTONMUGENESB pathologk spleens then these spleen celb should exhibit regulatory activity when added to young-adult spleen F H. Perkins. Citing. Yuan Hung.* J. M. Holland, and Wen-Kuang Yang cells and dramatically suppress the response of the latter to teveb approaching that seen with palhologk The concept that lymphocytes with specific suppres oM spleen eels. It was found that celb from normal old sor activity function as an essential part of the spleens exhibit some suppressor activity, on the average regulatory mechanisms of the immune response has 33%. This suppressor activity KlwofoM) can be con attracted much attention. Our recent studies demon trasted to the ~ sevenfold decrease observed when the strate that splenic lymphocytes from pathology-free activities of spleen cells from normal young and old 149 animals are cultured separately and compared. Simi 1. S. GHENrt mi J. C. Saark. Jr.. Scitmr 17*. J$7~*l larly, only sbgfctty more suppressor activity KtwofoM) U972). was seen when ccns from enlarged palhologa. spleens 2. S. GCKMM mt C. L. Craw, m Thttnm* 4UMS «/ Agmt f* kr M. naitrtiinr. Acadonk toss. Mew York. 1974. fmn individual old animals were cultured with young -»I05 17 adMk spleen cefk. This finding b in contrast to the marfcedry reduced activity of mdmdnal old patlwtogk snteens when cnltnred separately (~-IOO-fold). The AumoLOKmrnncEwnjiMOHAKY observed decreased activity in these mixtnre experi TvmmsmMmmwctFOLLomwG ments is T-cef specific, becansr B-cefl activity, as •fTKAVINOUS MBCTBONOF measured by LfS sthnnhtion. is not snppiessed. VARIOUS SVNGEMC TUMQSS The decreased IHA rcsBomtvcness of T cdb in the ipk cm of aftd animals can he acconnted for by at least C. •. Skov • J. M. Hoftmd. and E. H. PMdns four mrchauumi: (*) duuliou of responding lympho Developing tumors, whose antigenic chancier makes cytes by proKrcraiion of MA nonrcsponsne ecus;!*) them susceptible to immuniiliigii d luugnwiiw and absolute decrease in the nnmbiti. of lespunsiie cds;(r) response, may be eliminated or their growth may be demand reactivity of indhidual T lymphocytes rcsuh- ing from an age-dependent alteration in a pathway wC«MBVtHmTmT1> BMnWumwCfl vy ••vmnVIHaV JHVwCnVmwvjCv UVECUTMV" nisms. On the other hand, initiatron and growth of essential to FHA responsiveness. DNA replication, or weakly antigenic or nonantjgcnk tumors may be little cdnbr proliferation: and id) decreased reactivity dne alTecled or actually rnhmrrd in their initial develop to inert astd suppressor cell activity or manbers. ment by means of mmmnmtmwfation. Their is evi While duuliou of PHA-respnosive crib by PHA-non- dence which suggests that the thymus dependent responsive neoplastic reticnlnm cefb offers the most branch of the immune system may be primarily acceptable explanation for the minimal activity of aged responsMe for both of the above aspects of the mice bearing enlarged (I -3 g) pathologic spleens when tumor-host relationship. We therefore are mvestigatiur collared separately, this fans to account for the the growth of tumors m athymic nude mice to further decrease seen in spleens of normal size and free of elucidate the role played by the thymus-dependent pathology. Our earlier studies have demonstrated com immune system in tumor development. parable numbers of T cdk in the spleens of young and aged animari, similar scnsiliviiy to m vitro cultural The growth of several transplantable syngenk tumors conditions, and equal abilities to bind PHA. The present was compared in athymic nude and normal hetero results clearly demonstrate that neoplastic processes zygous liitermales that had been back-crossed to three inbred strains. BALnVc C57BI/6. and OH. Five that lead to splenic pathology in senescing BC3F( mice do not give rise to specific suppressor ceHs that exert different spontaneous or induced tumors have been regulatory control over T-ceB responsiveness. Therefore, used. The tumors, including two sarcomas, two carci the decrease in PHA responsiveness observed in old nomas, and a melanoma, were passaged subcutmcously mice appears to be related to yet unidentified defects, or in culture. A constant number of indrridual tumor perhaps at the intracellular level. involving processes cells were injected into age-, sex-, and weight-matched essential for DNA replication and cellular proliferation. syngenk nude and litleimatc mice. The cell dose was Collectively our finding can be interpreted as support established in preliminary experiments to produce for Gelfant's model of cellular aging,' •* described as a discrete lung colonies without confluence- The lung progressive conversion of cycling to noncyciing cdb colony assay was used to assess tumor growth after with increasing age. In other words, immunocompetent permitting tumors to grow for 2 to 3 weeks. T cens become blocked m either the G, (prior to DNA Five experiments have been carried out. In each experiment, the mean number of lung colonies was synthesis) or G2 (following DNA synthesis) period of the cell cycle, and the capability of reversible transition lower in nude mke than in httcmutcs or inbred to the cycling stage (cellular proliferation in response to conirob. Litlermalc and inbred mice developed IJ to stimuli. It., release from G| or Gj block) is lost or 4.4 times more colonies than did nude mice. The diminishes with chronological age. development of fewer tumors in the lungs of nude nrke was consistent with each of the five tumors tested. However, once the tumor became established, or •Postdoctoral inveMigMM' waponsd by a Research in Agmj following subcutaneous injection, the rale of tumor TraMRf Gram; PHS Grant Ho. HP 0929* from NICHHD. rVYMMf JMWVfSr mtOtOfff HVpVftllVVflf, IHROB Sfttff uMWIlify, growth apparently did not differ I.I athymic nude and Normal ftl7*l. normal animals. Experiments with radiolabeled tumor 19 I •NTibeB wMTcdb bamfct ONMNI lewief B cdb A* we I all «rfT cdb was winced, they lth*| * appean n%eb/ mat dstsmiKcs in occwwd at; of tnw»r cdb and cdl grow* raaedt* not 3XW spleen cdh. A fat war ubatried dwlfcience » cwhmy uctwned ai abm* I X 10* «U spleen cdb I I. We omcmewa n •utrpmaaay awajcca cc ^v ^Mfeitf^tf^M ^^ M^KAHM^M af~ wW T-^tfbwr Tcdbfn we activity wf the hapien-pnmed aWd activity. •CMMtrf 1*1 Ik* mjrttian of the carrier a hcsprr and a snppwssa* cwwajfwncM wfT cdb. l*)T-hdp*» ay naiwrawy uccvirrwg nnaprcsaHf cdb mat been atpumd tw he present in the ipleeni wC «ht * Irl the jfpi'JMi cuwpjitwrni. rather the hriper cMnpwrtment. was expanded by the mprctinn of ICC. or |«Tt neither helper m* suppressor ceSs pruMeraied. The existence uf >«ppcui*r activity dem onstrated in this tfady must be cwwidered when iMcrprctmg Pie wecsmc m unmnne responses or • *. L. Krogsmd* IE. H. Pnkiw *Sf*M M rfcr UT Cfak RaJcr Coda*** Sritaml M few- response ID many antigens has been tfHwMCiM ScfVIKVV. decrease dratwatkaly with apt. The deii- I IF A. P. Mmrr ani «. F. WhM. timifltitl Ktv I. .1 dency canrrot be completely explained by dvTIerenccs in thefrequency o f thymus derived (T» lymphucytes or 2. N. A. SwKhiM*. Km J. tmuminl. I. in • 1*711. rMue-marTow-dcriwd IB) lymphocytes or macrophages. J. N. Grrknr DrlMb. P. McceMi. J*J R. I. WaWMW. ¥*4 the three eel types which are known to cooperate in M.IS9M97S). these repomes.' We haw measured ihe abmiy of the T cetb to partkipatc m ihe mwwne response by making we of their "earner-specificity" m a hapteircarrier AGE4IBLATEO CHANCES W SPLEEN CELL system where diniirophenyl fDNP) groups are Ihe RESPONSESTO PJPrjrOLYSACCMAatl DC hapten*: determinants while bovine gamma frobdin R. L Kmgsrnd* (KG) or human pmni globidin (HOC) serws as "carrier"3 A haptcn-specilk B-ccf population was The age-relaied decrease in hnmond rmmwi activity. fmeraied in young mice by injeciing DNP-HGG and. 2 as ^fggested by investigations wing T 153 suppkmentcu with 9% approved fetal calf serum and gestation day 16. Our working hypothesis was that antibiotics ISO USP units pein^dmi and 50a* strepto lethal injuij and subsequent punwd death resulted mycin per ml), villi incubation at 37"C in a I—nidified froui passage of a proton through a sensitive region in 5%COj atmosphere. The pHwas varied from 6.7 to 7.S each of the two bbstomerev The first data obtained by varying the concentration of COz in the atmosphere, supported this two-hit kating nuxhanrun: however. and pH 7J (5* COx) was found to be the cenier of a subsequent experiments at higher radiation doses re broad plateau. The mitogen is added to each culture in vealed BKonsistencies. Mote embryos died than bad 0.01 ml: optimum amounts are: phyiohtiiiigfljlinin been predicted on the basis of results at the lower (PHA). 0.2S and 1.25 #rg per culture for spree* and doses, and also a greater proportion of the deaths peripheral Mood respectively; cuncanavahn A (Con A), occurred at later stages of gestation. The fatter finding 0.60 jig pn culture; and £ ew*" 055:15 bpoporysac- suggested radiation injury of the mother as the cause of charide. 1.25 fig and about 5 ag per cnitnre for spleen the unprcdicted increase in mortality, since it seemed and peripheral Hood leukocytes. The higher optirrmm unlikely that an increase m radiation dose would result conccntraiions for peripheral blood leukocytes may be m a greater proportion of later deaths as a direct effect accounted for by the presence of residual erythrocytes. of radiation. surviving a brief rypotouic shock treatment of whole- To test the indirect lethal-injury concept, we irradi Mood samples. To measure the DNA synthetic re ated two-cell embryos m «n»v with an absorbed dose sponse, a ptuse of tritiated thymidine | initial concentra of 500 rads of X rays and examined stained tissue tion 10"* Jlf. speciric activity 5 Ci/mrrraie) is added the sections of implantation sites on gestation days 7.5 and last 24 hr of culture. Triliaied thymidine incorporation 11.5. In a previous experiment we found that 95% of was highly dependent on the concentration of thymi the embryos given this dose at the two-cell stage were dine for concentrations less don 10"* M, while dead when examined on gestation day 16. The 75-day increasing the concentration above 10"* Af made little implantation sites were mainty of two general types: difference in the measured response. The response was one type contained a normal-appearing embryo, and the directly proportional to the length of the pulse, with 30 other had no identifiable embryonic tissue. Both hr the longest puhc tested. The 24-hr purse was chosen showed a decidual reaction. On day 11.5. the implanta for convenience and to maximize the measured re tion sites were also mainly of two types. In one type a sponse of a small number of ceRs. The optimum time of decidual reaction but no embryonic tissue was seen. We harvest with all mitogens is after 4» i»r of culture, are tentatively assuming that the absent embryos of although because the responses to PHA and Con A are both these and the 7.5-day implantation sites died at sustained, the magnitude of those responses at 72 hr is implantation, or within a few days thereafter, from similar. The response to LPS declines after 48 hr. as do direct effects of radiation. The second type of 11.5-day the PHA and Con A responses after 72 hr. Several implantation sites had embryos and placental tissues mouse strains have been tested, including BALB/c. that showed varying degrees of degenerative changes. C57BL/6. and C3H. Variation among these strains AH the embryos had developed at least a day or more approaches tenfold in some cases', with the BALB/c beyond gestation day 7.5. A characteristic finding was tending to be the best responder. hemorrhage of maternal Mood in the uterine lumen. The results seem to be in accord with the view that at the higher doses of radiation, some embryos that would otherwise have survived, died some time after gestation DIRECT AND INDIRECT EFFECTS OF day 7.5 because of radiation injury of the mother. To FAST NEUTRONS OR X RAYS ON avoid such indirect effects, we are routinely examining MOUSE EMBRYOS implantation sites on gestation day 7.5. Results to date Wallace Friedberg* C. D. Haimeman ,• E. B. Darden, Jr. * are consistent with the two-event, direct-effect model D. N. Faulkner,* and W. R. Kirkham* of our working hypotheses. A major tod of this study is to obtain risk estimates of lethal injury lo preimptanUiion embryos for radia tion levels below those at which an increase in mortality can be detected experimentally. In initial experiments *C«H Aeromedkal Imtitvte, Federal Aviation AdmMstra- on two-cell embryos irradiated in utero with fast (ton.Okbhoma City.Oklahoma 79100. neutrons, we examined the conccpiuses grossly on *UT-ErU>A Comparative Animal Research Laboratory. 154 ALTERED SERUM ESTERASE AND PROTEIN We have now expanded tins preliminary investigation PROFILES IN CYCUC NEUTROKNIC DOGS to determine whether RHT and DEN show an inter action: whether Riff interacts with ionizing radiatiun: R. L. Tyndal .• Shirley P. Cofryer* and J. R. Junes' whether the two known carciwugens. DEN and radia Esterase analysis of doc scram was perfumed by tion, interact; and what the effects of each agent are grattstnt aciytanwde gel clxiropboresis. and protein mdmdualy. Male and female RALR'c profiles were determined by cethdose acetate electro used. Doses of the agents are («» 0.75^ BUT in the teed phoresis. Cyclic esterase activity was detected in sera of throughout life. IB) DEN in the drinking water for 7 adult and prepubertal dogs suffering from cyclic neu weeks for an average cumulative dost of 300 mg/kg. tropenia. Activity of two scram esterase bands was and (r) 250 rads of X rays. Suitable control groups are correlated with the level of peripheral neutrophils. One also mamtainrd. The mice will be kuVd at either 12 or band of esterase activity was maximal prior to and 18 months for patholugkal examination. In addition to dnring the increase in number of peiiphcul neutrophils, obtaining pathology data iCIappl. we are making serial whereat activity of a second esterase band was maximal observations in individual mice on plasma esterase when numbers of peripheral neutrophils were mammal. changes (Tyndafl). metabolic fate of the chemicals Activity of both these esterase bands was higher m fDaugherty). hormonal dependence and source of es normal dogs than in dogs with cyclic neutropenia and terases (Davidson), immune status (Gottlieb), and viral did not appreciably vary with time. Serum from dogs activation (Tennant). with cyclic neutropenia had increased levels of alpha 2 Some of our preliminary results are reported in protein which varied appreciably with time but ap companion papers. We have observed: («) interactions parently did not correlate with the peripheral neutro of BHT upon DEN carcinogenesis, most of which phil count. Since esterase changes occur prior lo the appear beneficial and arc sex dependent; lo) esterase precipitous diminution of circulating neutrophils, anal changes thai are detectable after I week of treatment ysis of mammalian plasma esterases after total-body and that vary with different compounds (these altera irradiation might be a useful physiologic indicator of tions are most pronounced in females): (c) tumor- radiation dose or impending marrow dysfunction. susceptible organs that have different mefabdtic pat terns within the same mouse as do the various subcellular components; (d) virus 6-8 months after •Cownrftam. +Mr*cal Dmnon.ORAi;. ionizing radiation which may be associated with a lAufcrntv of TciMvwfv .McmnnaJ Rcsordi Center ana reduced viral antibody tiler during the leukemogenic Hospital, Knoxvmr 37914. period; (e) no dramatic changes in immune status during the later stages (12 and 18 months) when mice are i DEN; rOEN. Fcarfe TM> oe«E» TfcaCmWiH Naamcr " pmrtrcom against PFN effects P-. Rctfaon MMWCI of M Vmti Uu* LW*I 4tpce at I DEN iMKMqprans f McnAcactl CMMUI 37 0 0 3 BHT pttoHirtKM .TDENcflcct* •NT SO 0 n 4 GKaicraqpcrulia OUT *X ray* 4S 0 0 25 Xrav* 47 0 0 25 DEN 49 0 31 77 liniment with BHT.1 We expanded Ike mvesligalMMi lo Xnys*DEN 43 9 S3 72 mcmJe larger numbers of mice of both sexes, with ibe MIT* DEN 4* 0 2* 54 same kftmg lines of 12 and 18 Months of age; these times conebrtcd with the first deaths and with a time Control So 0 0 20 when a majority of the mice were sick or moribund •NT SO 0 0 * BHT*Xrays 49 respectively. Mice leeched cither fc) 0.757 BUT in the 0 0 1* Xray* 4t 0 0 19 feed throughout life. (•) DEN in the drinking water for DEN 47 0 34 J* 7 weeks (mean cumulative dose of ~330 mg per Xrayi+DEN 43 0 M 70 kilogram of body wcighD. <0 DEN phis BHT. or \J) BHT* DEN 45 0 •W 47 no carcinogen (controls). Other studies are being conducted to determine enzyme changes and alterations 'KIM at 12 h»; pou atcropsy only. in immune competency in these mice. reported a potentiation in C3IF, male mice, and we The pathology data from mice killed at 12 months are have observed a slight protection m BALB/c males and completed and are being analyzed. Table 27 demon 2 females. The interactions of BHT upon DEN carcino strates the complexity of BHT-DEN interactions as they 3 genesis led us to investigate a further concern - are affected by sex. The mechanism whereby BHT the effect that a leukemogenic dose of X rays might modifies the DEN effect on the foreslomach is not clear have on treatment by BHT (a free-radical scavenger and at this time: however. DEN carcinogenesis is signifi an enzyme inducer) or DEN (a known carcinogen which cantly affected by hormonal environment, as is Ihe preMunably requires intracellular enzyme activation BHT modification of the DEN process. The 18-monfh before it is effective within Ihe organism). mice have been killed, and the histological evaluation is Male and female BALB/c mice were given either (#) now being completed. The paihokigy data and (he 0.757 BHT throughout life, (ft) DEN (330 mg per kilo results from the other investigations will then be gram of body weight) for 7 weeks in drinking water, (r) correlated for inferences regarding mechanism of 250 R of X rays at II weeks of age. or (rf)X rays plus action. DEN or BHT. Appropriate controls were maintained. Mice were killed at 12 or 18 mouths for comparison *C«NMltant. with previous BHT-DEN studies. HtslologicJ examina I. N. K. Clapp. ft. L. TymfaR. K. 0. ('•mining, and J. A. Olttn, Pond Cosmrl. Ttaxkvl. 12.3*7 71 U974). tion is not complete, but the preliminary information based on gross necropsy suggests modifications in both sexes (Table 28). Radiation plus DEN appears to cause higher stomach tumor incidences than DEN alone or EFFECT OF WrOLE-BODV BHT plus DEN. For rung tumors, radiation alone X RADIATION UPON BUTYLATED increases the incidence in females but not in males. HYDROXYTOLUENE OR Radiation plus DEN increases Ihe incidences in males METHYUHmtOSAMINE TREATMENT but not in females. There was no difference in mortality IN MICE between the treatment groups al 12 months. Informa N. K. Clapp.* Lou C. Safierfield. and W. C. Klima tion regarding changes in esterases is now being eval uated, as are Ihe pathology data at 18 months. The effect of BHT upon radiation lethality has been reported to vary with strain, dimming and Walton' •Consultant. IS* 1. R. U- CMMI an* M. V. WAoa. Aaat Cacairf. Foamf. r^ASaUESIERASEALTEKATIONSm 11.547-$3 We undertook efectroBhorctk analysis of plasma ctferases m mice fed DEN and MfT. singly or in ENZYME ANALYSS Or riASMAFMM combination, to detect their early physiologic effects. CAMONOGBt- AND One group of BALB/c mke was continuously fed chow NOKAKMOOEN-nEAlED MKE wintahnng 0.7S* BUT. A second was fed BUT and R. L Tyvial * N. K. Chum• Kowctha A. Davidson* given DEN (total dose -330 mg per knofram of body andC A Boms' weight) m drinking water for 7 weeks. A third group was given DEN alone, and a fourth was untreated, hnce Esterase profits of plasma from BALB/c nice treated were necropittd after 10.20. and 4t weeks. with a variety *»f carcmofjrmc and wMCMciaafjnNc Flassu esterase changes were apparent in BALB/c chemicals were aujatyznd. nasnia levels of gamana mice after feeding them laboratory chow with 0.75% of •Jutamyt traiispeptidase and ejutwir-oxatacetic trans- the antioxidant BUT added. Other esterase changes, anuuasc were atso determined. Esterase analysis was different from those in BHT-treated anrnuds. were also carried oat by gradient acrytantfde gel electrophoresis, apparent in plasma of mke during exposure to the while levels of the othet plana enzymes were de caninuntn DEN. Interference with these DEN esterase termined with the mini-GeMSAEC system. Mice ex alterations was apparent in plasma of mice treated posed to the carcinogens diethyliwtiunnnnr. concomitanih/ with both DEN and BHT. Esterase dujsropyliMrosannne, dmUrosupiptMzmc, dunethyl- changes resulting from either the carcinogen or antiox hydrazine. dmifthyldinmosopiperaziuc. and urethane idant exposure precede the overt histologically detected had idcntkaBy altered piaswu esterase profiles after 7 changes induced by these compounds. The early days* exponsre to the chemicals. Exposure to the esterase changes and subsequent lutnorigenesis resulting noncardnogens nitrosohydroxyproune. nitroso-2j6- from DEN exposure were more severe in female mice. diwelhylpiperidiiie. nitrosometboxymethybmii^. Some of the esterases altered by DEN or BHT exposure I -wttroso 4 inethylpiperazine. and ethyl methane- were testosterone-related. sulfonate caused no obvious plasma esterase attentions. The interference of BHT feeding on DEN-induced Ingestion of carbon tetrachloride resetted in a plasma esterase changes was reflected in the subsequent altera esterase Jteralkm different from that seen in car tion m type and severity of tumors in mice fed both cinogen-treated mice. Lewis of the plasma enzymes DEN and BHT as compared with mke fed only DEN. gamma glutamyl transpeptidase and glutamic oxalacctic We are currently anaryzhw eslerwe alterations in mice transaminase did not distinguish the carcinogen-Heated exposed to total-body irradiation with or without from the carbon-tetrachloride-treated mice. Thus, concomitant BHT treatment lo determine whether within this group of 12 chemicals, esterase analysis of esterase changes are predictive of pathologic sequelae in mouse plasma distinguished the carcinogens from the such mice. noncarcinogens I week after beginning treatment. We are currently testing a variety of additional carcinogenic and noncarcinogenk chemicals to determine the effi "Consultant. cacy of plasma esterase analysis as a potential screen for environmental carcinogens. ALTERATIONS IN ISOENZYME PATTERNS FOLLOWING TREATMENT WITH CARCINOGENIC AGENTS f t*M4ocioral iftmtimtor supported ky subcontract 3322 J. P. Daugherty.* N K Clapp,* and R. L. Tyndall from the gMoey DMmm. ORNL, to the Uasvcnny of Tennessee, Knoxvtnt. Numerous studies have shown that many classes of Cncmicsl Techaoloey DrfUkMt. enzymes in plasma and tissues are changed during 157 duTcreat pathological processes. Plasma esterases were Cd*\ Ca1*. and Zm1' had essemiaty no effect om aketcd when BALB/c nice were givea DEN.BHT.or a esterase activity. The tnvaarat caiioa La9* enhanced conhiaatiun of both.' To detriauat if sach attentions actmty ia one enzyme aad adaTiitrd activity ia ate arc common among dnTerenl (maps of eniymes. we are fVPJSanlarnTaTl. ^wRu aanur#3%V*Iw^R» Pa^^CluT^"* aaaawwaauwlJwj 9 •^••Tlawa*. investigating the clertropnoretic ambatiy of several showed some degree of inhibition ia aft* bat three ester- enzymes under diffevcu* conditions. Tumorous aad asc bands: only one band was completely adubtied. anffdtoriag nontmnorous tissues (fever, lung, stomach, IHunjiani ia the presence of eserne abolished the in ovary) aad pltian lone heca obtained from take hibitory effect of eseriae on three esterases aad soma- litaied with BUT. DEN. aad X rays, alone ot m bled or enhanced effect oa two enzymes. aawbamtioa. aad aril be aaalyzcd fot vtutujes in Studies to analyze sabstratc preference revealed on* different enzymes by patyacrylaaade art electro anaphthyl batyrate snowed a lesser preference ia mar phoresis. We are screening several cazyaKs. including cazynws aad a greater prefcience ia 2. Napthol AS-D acid phosphatase, alkaline phosphatase, esterases. glu acetate and AS-D ctdoroacetatc revealed a very low cose 6-phosphale dehydroftaase. lactic dchydfofraase. prefcience. and only II of the It enzymes were pbosphoglucaRHitase. aldolase, aad ^ucuioaidase. fot dtavnajtrated with the forme? aad 6 with the totter. attentions aad conetatioas with known carcinogenic Thirteen esterases appeared to c-ntaia eery smaR activity of (reaiment. amoaats of lipase activity when assayed with nnaphthyl capryble ia the presence of rorwun cftobte; a-naphnyl laarale and myristaie were not hydrotyzed. 'lusakai-tani umiwjjior twppwnti ajr mamniuii 3322 Heat-siabany tests showed marked variations ia the fam the BJofaey Dmaoa. ORNL. *• UK Uanvniiy of sensitivity of various bands depeuding in part oa tConwtaM. whether the senun was heated prior to or after I. R. L. Tyiabtt. S. Carver, and N. Oar*. fur. J. Cmnr. m electrophoresis. Plasma from mice fed DEN was sciaJyzed using the parameters described above. Al estcrases had character- istics identical to those in control plasma with the CHARACTERIZATION OF NONSPECIFIC exception of one. band 10. This esterase activity in 1 ESTERASES M MOUSE SERUM normal (control) mouse plasma was enhanced by Mn * aad resistant to eserine; however, in plasma from Kowetha A. Davidson* R. LTyndaH.* DEN-treated mke it was not affected by Mn1* aad was and N K C1appt sensitive to inhibition by eserine. Band 10 is also one of the enzymes which shows a quantitative increase during Mouse serum and tissues contain numerous non DEN treatment. These results reveal that it is also specific esterase enzymes, many of which are altered qualitatively different. under different physiological and pathological condi Experiments arc rntr planned to: («} examine plasma tions of the animal. At least 18 plasma esterases have from mice treated wi'h other carcinogens to determine been demonstrated by polyacrylamide gel electro if band 10 is qualitatively different, (0) measure the phoresis, eight of which are quantitatively altered in level of circulating testosterone to determine if the mke fed a chemical carcinogen. DEN.' The tissue quantitative alteration in irMtnierfwe-dependent ester sources of these esterases are yet unknown, and their ases may be mediated by an effect of the carcinogen on identification will depend in part on characterization of testosterone production, and STUDY OF MMUNE COMPETENCE IN MKE individually and in combination Although the results TREATED WITH THE POTENT CARCINOGEN. are not yet complete, no dramatic changes were METHYLMTROSAMNE. AND/OR observed in the immune competence of DEN-ueated BUTYLATED HYDROXYTOLUENE mice compared with BHT-treated mice or untreated controls, at either 12 or 18 months after treatment. C. F. Gottlieb. C. M. Pelentan, and N. iC. Clapp* Preliminary analysis suggests a slight increase in re The involvement of immunosuppression in carcino sponse with the MtsheO and Dulion assa> in DEN- genesis has been suggested as a partial explanation for trealed mice, as well as a slightly reduced PHA response the carcinogenicity of ionizr. g radiation. If immune in those mice with lung tumors. Further analysis of the changes are consistently associated with oncogenesis, data B being made, and correlations are being at the immune depression would be expected to occur tempted with tumor types and organ sites. with other carcinogenic agents besides radiation. Within ibis context, we have been evaluating hnmu'tocompe- *C TaMr 29. Cumanriiun nf wmOt VMjT WNM MMMI a* affecting tonr* madmen m BALB/c UNCIt after BHT ami/m DEN T Mnor mcnJenct W) Treatment Stomach LMK ttowtf HM§ ,»g m ^^ Mag Gross HiMofofjiul Gross nawOMRpCM Control 0 n 20 20.0 BHT 0 0 • 14.0 BHT*DF.N 49 91 •7 •8.9 DEN 34 93 3* •1.7 Control 0 ft 3 10.8 BHT 0 0 4 6 BHT + DEN 2* 70 54 84.8 DfN 31 *3 77 91 .• ''Mk* killed at 12 month* of apt. 19 in the iqiiajanns lawag of the forestoaaach is difficult: various inbtiluhr fractions of both Bver and lung did thus the anticipated increase in tumor incidence with not incorporate appreciable radioactivity. histological confirmation was not swprianf. Pwknoury DMN s raawRy metabolized withm the body and tumors were recorded frosty after inflation of the shows rcmufcjbk affinity for tRNA m two organs laags and were then cleared and sabstqueaify ex- susceptible to DMN's carciaogeak activity, but has aaaned under a ditsectiag aacioscope. Histological Stile affinity for DNA or for ana tunanr-susceptiMe sections were node frequently to differentiate between tissues. The sigaificaace of these observatioas to the tumorous and aoatmaorous nodules. With gross obser carcinogenic process h uncertain at this time, bat is vation, tumors >03 mm can be detected. whMe with bring explored expernaentaBy. In addition, each of the da* dissecting scope we can detect nodoks 20.1 nan. subceaafar fractions b being analyzed ft? radioactivity Onr observations show that the data from gross associated with hptd and protein compopenrs. aad necropsy alone are valuable, but some conclusions analysis of the methybied bases of tRNA fractions is ia wonbj ham been different withont histological exami- progress. nations. These remits reinforce the desirability of coamaaag pathological changes by histological observa tion, and guarding interpretations whkh do not include •UMIOMIUHI m****** IMP mud by snbcMtnct 3322 (nm tar anJagv OMN, ORNL. to the Uwjmutj «r histology. Tcaacwce.KnMvaV. «ConanaM. 1. N. K. Chpn.lt L.Tvnajl.awJJ. A. Onca. Oncer ftet •CMWKUM. 3I.IW-M(I97I>. 2. J. P. Dwajniii awi N. K. Chap.das report. KINETICS OF APPEARANCE OF COVALENTLY wOUNDMETHYL LABEL(MC) SUBCELLULAR DISTRIBUTION Of RADIO IN DNA AND UNA OF MOUSE LIVE* AND ACTIVITY IN llsE AODWLIJBLE AND LUNG FOLLOWING A SINGLE INJECTION 4NSOUIaUCOMrXMENTS OF MOUSE UYER OF | • *C) 0l»KlliYLNiTRO£AMlNE AND LUNG FOLLOWING A SINGLE INJECTION Or |' 4C)DIMETimjviTROSAJ«NE J. P Daugherty* and N. K Clapp* J. P. Daugherty* and N. K.Cbppt The reactions of dimethylnitrosamine (DMN) with A large amount of literature has accumulated on the cell«lar nucleic acids are of interest in determining the diversity of the toxic, mutagenic, and carcinogenic possible cellular targets that are responsible for the activity of numerous mtrosamme compounds. The observed organotropism of DMN awl DEN.' The incidence of primary tumors following DMN and DEN differences in the reactions with DNA and RNA may administration in two strains of nice (BALB/c and RF) help define the cellular reactions which ate significant has been described by Clapp et d.' Differences were in the carcinogenic process. The experimental protocol noted between DMN and DEN tumor induction within is described in an accompanying report .2 strains and between strains for each carcinogen. The The greatest amount of methyl label, in bc«ti lung and differences in tumor types suggest that differences may liver, was associated with the RNA components of thr exist at the cellular level in the affinity, transport, cytosol fraction, the I RNA In the case of the liver and/or metabolism of these compounds. cytosol fraction, tlte radioauivily bound to RNA The present study was undertaken to investigate the increased rapidly during the first 2 hr. reached a distribution of DMN in various tissues of RF mice. It is maximum at 8 hr. and declined thereafter. The distribu hoped that these data will provide information to tion of radioactivity in the cytosol fracti'm of the lung elucidate the observed differences in cellular and organ increased rapidly through 4 hr and thei declined, 'he susceptibility between BALB/c and RF mice and level at 16 hr being equal to the level at 15 mm. 'be ultimately between DMN and DEN. radioactivity in the nuclear, mitochondrial-lysoiorr.il. Radioactive DMN was r -.ninistered (10 mg per ribosomal. and membrane-microsomai subcellular frac kilogram of body weight) by gastric intubation to three tions reached maxima at 16.4.4. and 8 hr. respectively, replicates of 8-week-old male RF mice. The animals for (he liver, and at 8.4.0 (no accumulation), and 4 hr. were killed at timed intervals (15 mm-24 hr). and respectively, for the lung. The DNA components of the tissues were removed. The tissues were homogenized ife fractionated by atflcrcntiai ccatrifugation into five of taaaor incidences hi the RFM groups ' fractious: nuclear, uuftocboudrial-tysososnal. which received 0 SO ram of Tisfaoa neutrons. Details of aatmbrane-nuciososttal. and cylosol. Each the experimental protocol, as wcN as the effect of these fraction was incubated m trkMofoacetk doses on lift ruan. haue been reponed previously.' aria* and filtered to separate the and-soluMe and Table 30 indicates the age-adjusted incidences of theradioactivity o f each com- •did tuaMMS in the various ircatnKni nd expressed as cpm/aag of An increased incidence of thynric lympkuna DNAmthehomogenate. over controls was observed at al doses except S rads. Rmtioacthity was detected m the tnmw-susceptmle When these dose-incidence data for the induction of tissus (Imt aai lung) following the administration of thymic rjaajihuaai are conspired with those of smmaity OMN. white radioactivity was atar burrgrnund ia mamlamed RFM females exposed to X uradntion usccptMe times (heart). The acid-somble \IMS., nniiabliihfd data), an RBE of sjipmijnmety 3 i of the laaf incorporated about sixtimes as is obtained. Althungh there was some flactuation in radioactivity as the liver at IS aria after aemmrs- of reticulum eel tratioa; at 16 hr theradioactivity in both tissues had was appfoxmsatcty cojual. The acid- Tins is smmar to results ot both Macs incorporated obtained after X-ray exposure. much lessradioactivity than the acid-soJuble comno- Sand tumors were pooled into three categories: (a) acats. nuJkatmg that oafy a sssal aaioaat of mrthyl endocrine tumors. (»> lung adenomas, and (r| other label was covakntfy bound to ululat ancroeaolrcattj. tumo.*, bexauje of sample sizes. The increased inci The icsalts suggest that differences do exist ia the dence of endocrine tumors is primarily a reflection of metabolic fate of DUN ia Km and hang tissaes. A ovarian tumors, although shght increases in pituitary liiilir stady is tmder way using ,4C-DEN and iadad- and mammary tumors were observed. The most inter ing BALB/c mace. esting observation was the apparent increased incidence of lung adenomas at SO rads. To our knowledge, such an increase has not been reported previously using . br sabMMfact 9322 either neutrons or X rays except after relatively high Drriaoa. ORNL. to the Vtmtimr of Tcauessae.bftMrim. doses localized to the thorax. tCvasaRaat. As a result of these data indicating that neutrons may I. N. K. Clapp. R. L. Trratala , and J. A. Olf«a. Cmtttr Met. be significantly more effective in the induction of rung 3l.l9t-9S(l97|). adenomas than low-LET radiation, we have initiated a more detailed, mechanistic examination of the response of the rung to neutrons and X rays. Results of these studies are too preliminary to report at this time. EFFECT OF NEUTRON IRRADIATION ON THE INCIDENCE OF VAJUOUS NEOPLASTIC DISEASES R. L Unrich. J B. Storer. and M. C. Jernigan I J. a. Storrr. G. I Coserove. E. B. Daraea. Jr.. L J. Serrano. M. C. Jenupa. W. C. Kama. L. C. SaiterfirM. H. C Our studies on the late effects of neutrons in femaie Swam, and F. S. Mania. WW Mr. Amm. frog. Hep. JmmrJO. RFM and BALB/c mice are sufficiently complete to /•74,OftNL-4993.pf> 149 SO. TaakSvX I in RFM ladatace (% * SX.) for coat of 0 (control) 5 ram lOraes 20 ram 50 ram IpJfHuW lyM^IMptvvB 10.0*3.0 IS.7*5.9 24.5 **\5 3U t S.3 Retkawm can aarcorM 42.3 * S.3 43.6 t a.5 53 J * 7.7 30.3*7.3 47.$ * 4.1 Ouarkakcana 11.3*3.7 23.7 * S.9 9.3 * 3.4 12.4 *«.3 101*4.1 Endocrine tamors 7.0*2.9 S.2 * 3.1 10.7 * 5.9 50.4 * 0.9 54.2 * 4.2 Lane adenomas 23.9*4.7 10.11 3.7 23 J * M 30.11 SJ 45 J * 5.3 Otter ramors 4.2 * 2.2 9.5 * 5.0 4A*2J 25.6*7.0 47.2*4* Ml EFFECT OF CHLOKOQUNE ON tEOOVEKY FftOM CAJbtMOGEMC MRJKY TO MOUSE UMC»Mir^DBYSnJT-DO5EtAI)UTI0N R. L. IMrkh. N. H. r>nwni.* and J. M. Yete Nwniw studies have shown that fractantation of a radiation exposure B less effective in proofing a exposure. Thn> decreased effectiveness win fractiona tion is apparently due to movers between fractions. Recovery aaay be s function of JntimMnlw repair and/or mtenrBulir recovery of target anal uniiMiug cd pwpulatiuau. Intracellular repair is rcbiivery fast and is essentialy conwJete in a nutter of hours, lutmclulai recovery, on the other band, is a dower process. Some aatevccflulBr recovery any occni wvdun 24 hr. kwi most occurs over a period of days and in Akhoaajh the icdnced effectiveness of tow-dose-ratc or exteaded fiauioaatiou icguaens in tumor indnciion snajeststhat recovery ocean, the role of this phenome- C tOOO 2000 3000 oose of the importance of lias phenonrenon in carcmogtuesis as well as an undent arnmig of the relative Nnponance • UMvausauuwjaaaui panes • •anajreaat.a—°ajti* of iufracegubr and iniercefhdar recovery in carcino «hne. A——'- ^M • cMomcauK. genesis is essential for the imdersiaudmg of the mecha- nnms mvolvcd in the c'rirnogtnk process. iRtraceMar of body weight ntaurdijiery after the first exposure. repair ntecruniinp may act on two t)p?» of injury: Animals gwen spht exposnies received the same total injury which would nlliniately lead to either neoplastic dose as the single-exposure group. Appropriate non- transformation or potentiauy lethal damage. In both treated, sahne-treated. and ihloro quia*-treated conttob instances recovery could ultimately influence the carci were included. Since there were no differences in the nogenic effect either by affecting the number of tumor incidences among the control groups, they were transformed ceHs (initiating event) or by influencing the pooled for analysis. The 5-hr fraction interval was activities of the target- and mteracling 162 UNA SYNTHE5B M MGCAKAIYOCYTfS Of rmBtncK v>MSf Rcscatdi CCMR, FWPEI* kt.lfafytMMt 21701. AFTEft SIMULATION I. N. N. ! 1. M. Y« ftcs.wH,54-«l (1974). T. T. Odel.T. I*. McDoaaM.* and Deborah A. Bona Tfce iArhag index of ananfcaryocytes 30 nan after injection of tritiated thymidinr (ftijTdR) indicates •HFLUDKE OF LOCAL RADWTBWATY thr proaortioa of the naaafcaryocyte popabtioa that » m ONA syanVesii at the time of jajevlina. beca R. L. Ufckh. J. M. Yuhas. and M. C. I'HITdR labels DNA and has a very short half-ta the body. Other work had shown that the tsatr ncrcases in ammab that have others. nas the host i to increase their platelet production. It Atteaapts to one this therefore predicted that an mcrease in DNA therapy often fa!, in ; from a pioidy increase in stimabtcd control, because the saftastavrs nace coaM be seen as an metcast in the bbehug mdex In fact, it appears in this system of the megakaryocytes after |'H)TdR injection. Tins thciapy can at la any accelerate raeasare naght serve s a method to assay the staauta- h injury to the tion of awgjlrai jwyiopow. iii. control asechanrans which help to C3H aace were injected with rabbit aMi-moase-plate- front the tr let atnan and then injected at intervals ap to %hr later ale. Asa with I'HITdR. Mice wm Intel 30 nan after |JH|T«R (local control only at the expense of accelerated adanairfratMn. and bone marrow smears w«ie prepared. s), we hat* gpvca the mice a radioprotective Aatoradiograms were made, and lab fling index was WR-2721, jwst prior to their localized radiation determined. The antiserum reduced the platelet counts . Previous experience indicated that tins drag to about 4% of the normal level. The normal level was tanms. hot did protect the retained at approximately 96 hr. The labeling index was the radiation field. In greater than that of controls Co < 0.05»at 24 hr and dwg-tiwated aaintals. the sanse radiation dost was less (• < 0025) at 72 hr after injection of antiplatelet to achieve local control of the tumor, but serum. These remits indicate a stimulation of nvegakar- or mese wcany careu ananas survive* yocytopoiesb followed by a depression It may be of accelerated snetastasB. possible to use either of these ctonges fr JVH normal as progress to drtrtamsf the an end point for studying stimulation of rnegakaryo- natwe of this regional inrubrtron of metastases, and its cytopoiesB. Additional experiments wW be needed to response to irrahitiun in the presence and absence of determine whether a more moderate stimulation of WR-2721. megakaryocytopoiesis wal produce a measurable effect A related series of experiments haw began involving by this method. the combination of local radiotherapy with C mrvwm in the tteatment of the Line I aheobr eel carcinoma. UmveoHy or TCMKMCI MtmorM Rcwardi CffUMf «UMI The objective of the stody is to identify the radmbio- Kamvmt 3791*. factors of importance in I the efficacy of this mode of cancer therapy. This type of cumbbud therapy wonU alow us to more effectively control distant metastases while achieving OIANCES IN PLATELET SHE AFTER local tumor control. Althoagh these experiments are INDUCED ACUTE m.OaJWCYT0rENIA sill in progress, it is apparent that fractionation of the T T.Odefl total radiation dose m combhwtion with C fmmm treatment is agnifkarwly more effective than a similar Earlier studies in several laboratories have indicated smgW acute dote phn> C. ptmm in increasing the that there is an increase in the size of some of the survival times of tumor-bearing mice. circulating blood ptatetets in thrombocytopenic stales. 163 It has been suggested by many authors thai yonng deprivation By 24 hr the frequency distribution of platelets are larger than average, which would accawnt pbitcsn size had began to shift back toward the control for increased ate rant pbtdet size in mdividuals recover condition, suggesting A*1 ** period of delivery of ing from thiumbocytopeaia. and that platelet site these large platelets to the circulation is quite short. decreases with age. On the other hand, it has abo been Morco-.er. these large platelets may possibly have a suggested that large platelets are produced as a conse shorter than normal survival time, because they seem to quent t of the stress of acme throatbocytopenia and be csseatiany gone by 60 hr. whereas it has been that these large "stress" pbtctets may not be the same reported ami the average lifetime of ral platelets is 4 as the large yonng pbtdets in normal indiiidudi. In to S days. Alternately, these large platelets may undir! of inegakaryocytopoiesis and platelet prodac- decrease in size with age. as has been iiau/itid for tion after induced acute miombocyiGpcaii. we had nonnaHy prodaccd pbtriets. noted the presence of large platelets m platelet-counting In addition, the data collected m these experiments hemacytometer chambers at abent IS hr after impac are consistent with a shift m the proportion of platelets tion of thrombocytopenia with antiplatelet senna. We in the circulating popabtion toward the upper (larger) wondered wheihei these large platelets were normally end of the normal size distribution about the tine when produced yowag platelets present m high propoctioa the most rapid platelet production begins (sample taken soon after ihronmocyiopenia induction or whether at42hr). they were produced specifkany m response to the stress These remits suggest that "stress" platelets that are of acute thrombocytopenia To learn more about these larger 'han any found in normal individuals can be platelets, we made measurements of the size of huh- produced m response to acme, severe thrombo «idaal ptatekts at intervals daring 60 hr after infection cytopenia. The results do not rale out the posseMty of antiplatelet serum into rats. The dose of antiserum thai newly produced platelets in normal or treated used was adjusted to reduce the peripheral platelet individuals are greater in size than die average of the coant to about y< of the pretreatment count. In such circulating population rats there is a slow increase in the number of circulating platelets for about \% to 2 days and then a very rapid increase between 48 and 96 hr. Platelet size measure POttSTENCE OF INJECTED ArfTuff. JtTELET ments were made on platelets of a control rat and of S«IMM TIC CIRCULATION rats killed at 12. 18. 24. 30. 42. and 60 hr after OF RATS AND MCE injection of APS. Relative sizes were obtained by taking JS-mm photomicrographs of platelets in hemacytome Luda L Hodge*-,* T. T. Oddl ter counting chambers, placing the photomicrograph* m Diane K. Beeman. and Deborah A. Bonn an enbrger and tracing the platelet perimeter on paper Antiplatelet serum (AP5) has been used to induce cutting out the tracing, and weighing it. throanwcyiopema m experimental animals in order to Average platelet size was larger statistically \p < study platelet production and megakaryocytopoiesis. It 0.001) than in the control at 12. 18. 24. 30. and 42 has been shown that the degree of thi umfc ucytopema b hours. By 60 hr. average platelet size and size distribu related to the dose of APS injected. Results have abo tion had returned to a pattern very similar to that of suggested that the duration of thrornbocytopenia nay the control. In the 18-hr sample the frequency distribu be related to the dose of APS. The latter observation tion appeared to be bimodal, and nearly half of the raises the question whether larger doses of APS persist platelets were larger than any observed in controls. in the circulation and produce a cottfmuing effect of Because the new large platelets at 18 hr were bigger remaining APS on newly produced platelets or whether than any present in controls, they apparently do not a loafer-lasting depression of platelet production after correspond to normally occurring large young platelets, bran doses of APS is due to an effect of the APS on but rather represent ? specific response lo the acute megakaryocytes, the precursors of pbtetett. The pres thrnmbocytopenc condition. The early appearance of ent investigation was begun to exaanne Hit length of these extra-large platelets suggests that they were lime APS remains in the circulation after intravenous derived from megakaryocytes that were already under- injection. going the later stages of their maturation process at the Rats or mice were injected mtravewoi^'y with APS time of antiserum injection. If so. the message to that was made in rabbits against rat platelets. The serum produce these large platelets was received by potirepli- was absorbed with rat red blood cells. The doses of cation megakaryocytes, and the stimuhn was pblckt antiserum were sufficient to reduce the circmattag 144 platelet count to about aero. At intervals duriag the 24 the br after AfS injection. Mood niapln were taken and inn the to dm. The serum was then couectcd. Rat The concept of wdds « to tested tor its anmty to ajghjiiaar rat farvival data, nay be at foMows. For each or to take part in ronaation of a of two popabtioasof S. the lite-span is dnided /.-#,./,. ieagrh is arbitrary bat is aw dry choata to reflect ihe senan was tested only aril paliera of anrtaliiy pecuniar to the species The marts ijiafclirhtd that anweran specific to rat coaaderatioa. For aace a 50-day jatcncal isc« pbtetets can be detected in serant of At5 injected rats For each proap the odds for dyiag III*} ia a by an at mao pbtetrt wjlaiantiiia ledanoae. The AfS interval are given by the pjiuhaliiliry of dyiag in the of recipients can abo be detected iarerval divided by the probably of sarnviag the lo interval. For each iaterval. then, it* can be estaaawd i of the AR. AR was by the ratio: of rtripwaU for 3 hr after in rats and far 24 hr in ana K*DfA. n» the i of AfS haecMd and its persatencc in the where P H Ihe rannbet which die in the intenal and A is the aaraber which survive to Ihe end of ihe interval. If this ptucedate a applied lo an intervals, an array b obtained that represents the changing force of naxtahty 1*74. •OftAU in each groap. As a ateasare of the degree of differ Tree tfcc UmnuKr ofPjnii.ii Tout 770*9 between two groans, the odds ratio y'a defined ia each interval to be: MORTALITY WISUCnON IETWEEN 7 »*,•/*,* I2> SMCLe>M»CXRAYAND GONmiUOW UW-LEVEL *, * and it]* are rhe odds far dying ia that InTRtYUOTnOSAMrnllN interval cormpuawag K> groans I and 2 respectively, IULE OB MCE la the present context, where groap I isa reatedgroop and grant 2 is aa untreated control group, we thai J. M. Hovtaad and T. J Hitched* refer to y as the wjortality tailor liaCSH* •• X nws. DCN.or bam r»Mt Mean varvoai Groap T ~- sx. Xrar* DKNWoJr* wMe'aavsr m 1 Anna 0 V 139 «M la 2 Xnv 300 0 147 7*0 W i OEff 0 1.} IS* •32 14 4 DEN j M 22S «M 7 S DEN+ X far 3S» U IM S32 IS « mn*%m 3ft> M m m 7 •XrarMBfcVp. I. HVl 0.5 mm Ca. *m me -130 RVaaa. 5 « of aw. *DtN: 1.2 at *J mfrmfcan want lor We nwonniwm III) I MS Aa for das iaieractioa by analysis of tisaaes i a coaaatcr program lo dtieimint the from necropsy km of al latctmood cstiaBic of y. together with dyiag in these experiments • 95% confidence raaits.' The pragma abo exact lest of the hypothesis thai 7 * Lie.. *C«aawcr Sciences I that there is ao difference bet««ea the treated aad I. MM*. 5NK Jin. Drpt Ammm- hwg. Ktp. Awr M. 1973. coatrol groap. IXXMCDCSDII The resahs of these cakaianoas are 1a— iiiird ia Table 32. Ii is of iawrcst to evjaaae the rcsaks for cvaJeace of iateraclioa between the radiatiaa aad DEN SEX- AND SnAOlDBENIKNT WfTIKEJCES treatments, la the preseai coaiext we shal say there is WTIKMAGNiTIJDEOFTHiClvKONK ao mfcrai-tioa if the effects of these treataieats (as USKNSETOItADlATiaN •JKasared by the MF) are aadtiptimwe.ie.. the MF for tar coariMted trotatent is approxanately eqaal lo the J. M. rttdkmd. Mary S. Watakcr. ict of the HFs for iadividaal treatments. L. C. Gipaoa. aad T. J. Mmchel* As part of a luariaaan, program to identify, ferae, and asssaa relative important to those genetic factors which mflaeace effects of radratioa. a series of 95%. aadtiple straia aad hybrid sarvival experiments were Mt- Mtnted Foar inbred strains (C3H. BALaVc. BFM. C57BL/6). two F, hybrids (C3CF,. B6KFMF, >. ana a 2 1.44 1 19.1.91 O.O09 four-way cross (C3CB6RFM) were exposed to 300 P. of 3 2.1* IM.2J* TaMtJl Tkri ••fani Mortality factor It** Stow Ratio i(tm*c!**ct r Rank MA 1 C3N 4,31 1.44 2.9> <0.0»l 2 CJCF, 4.23 2-1* 1.93 <0.0I 3 MRFNFi 3.44 I.S4 I.M <0.0I 4 *FM fcuJ 3.70 1.S5 *NS-nM B that the response of C3CF, hybrids is so similar to fikety to be observed in a random breeding population? each parentaJ strain that we cannot make genetic We only raise these questions now. EventnaJly we hope inferences. Female B6RFMF| mice have a response to have some of the answers. nHermedate between the two parents, althongh there is a tendency for the hybrids to more dosery restmWe Computer Scicacc* DTHMM. C57H/6. Mak B6RFMF, respond as the more re I. J. M-r!otaarfairiT-J.MMchra.ilHifcport. sistant CS7BL/6. Both sexes pooled foRow this same trend, that the F, of this cross between resistant C57BL/6 and sensitive RFM resembles the resistant parental strain. Female four-way-cross mke also re PERCUTANEOUS TOXICITY OF EPOXY RESINS semble the resistant B6RFMF, male parent. Male AND AMINE HARDENERS IN INBRED four-way-cross mice have a response that cannot be C3H AND CS7BL/* MICE djitmyiuhid from either parental response. To sum J. M. Holland. L.C.Cipson. marize the remits of the hybrid analysis, if we exclude and Mar*- s. Whitaker the C3CF|. which is a cross between two related strains that show very similar responses to radiation, inherit The epoxy resins bb[2J-epoxy-cyclopeniyl} ether ance of resistance appears dominant over sensitivity in (Chem. Abstr. Serv. Reg. No. 2386-90-5) and diglycidal F| mates of a cross between the divergent C57BL/6 and ether of bisphenol A < 1675-54-3) are representative of a RFM strains. Female hybrids show a slightly different large class of materials that are used extensively pattern in that dveir response b clearly mtermediahr. An throughout industry. An amine hardener, m-phenyi- intermediate response is consistent with the hypothesis enediamine (I0M5-2), is used with various resins and that factors which control the somatic response to resin mixtures as a polymerizing agent. Given the radiation arc multiple and that these factors manifest extennve use. economic importance, and opportunity additive dominant afteractions. for skin contact associated with tpoxy resin compo Genetic factors are obviously important m defer- nents, a long-term study was initialed to evaluate the the majnitnde and direction of the radiation cumulative effects of these materials applied to the skin these differences doe solely to the of mice. Preliminary toxidty-ranm-frndmg experiments differential induction of tenhrmw and ovarian tumors were performed in order to establish the maximum dose in females as ha* been nigpmid, or are there additional that would be tolerated without inducing acute toxic factors involved m the mate-female difference? Are the ity. i responsible for beiween-strain differ- Fifty microliters of a 50% v/v acetone solution of i anea, or matt we assign certain effects each ream and a 1:1 mixture of both resins were applied to sex and others to strain? How representative of the to the shaved mterscapular skin of male and female "avenue mobse** is the nan of icsaonaes obaerved in inbred C3H and C57BL/6 mice. The amine hardener wvwvvjpmw> pvwwinmr aw VJUVJW rwrnaar ^rw nwjwvnrwftaapvav ^^vjvvaw wva^a* awa this series of inbred monse experiments? Are observa was also diluted in acetone, but to a concentration of tions obtained from genetically divergent but homoro- 10% v/v. Treatments were given daily 5 lima/week for strahw representative of the range of responses 2 weeks or until death. Survivors at the end of the 167 2-week treatment period were kdkd and examined for CS7M/6 nuce are being exposed to the same spectrum gross or microscopic evidence of toxicity. of matciiah but at a lower (25%) concentration of resin C3H mkc tolerated the resin exposures without 2386 Under these conditions no toxic deaths have mortality and with no chnkai evidence of overt occurred in C37BL/6 mice. The amine is being appned toxicity. The amine hardener did. however, induce to both strauss at a 2* concentration vritfmu* signs of 100% mortality m females and 60% mortaKty in makes toxicity. It is concluded, bused on owe experience over the period of exposure. The obseivcd difference between sexes B of Questionable significance, because each material are being apphed which was be tolerated survivors had gross and microscopic lesions indicative of without evidence of cumulative toxicity and shortened systemic toxicity. survival time. It is under these conditions that those C57BL/6 mitt exhibited SOX mortality after ex advene biological luponau which are Kkery only after posure to resin 2386. No mortaKiy or signs of toxicity prneungid exposure, such as tumor induction or auto- were observed with either of the other resin treatments. anergk phenomena, arc likely to become manifest. As was the case with C3H mice. CS7BL/6 mice White the long-turn study has only just begun, after exhibited 100% mortality after exposure to die amine over 5 months of continuous exposure, no evidence of hardener. any deleterious effect has been observed in either sex or In both strains the clinical response to the amine strain. hardener was the same. After one or two treatments, signs of toxicity were obstived as dehydration, leth argy, and brown, discolored urine. Win continued treatment the animals became depressed and comatose TRANSYLANTAMUTY OF SPONTANEOUS before death. It was origami) assumed that the BETATOM AS IN CM MALE MKX discolored urine wn die result of massive intravascular J. M. HcCand and Mary S. Whitaker erylhrdysis jnd ex.-reiion of hemoglobin. That this was not the case was concluded by failure * delect Spontaneous or induced hepatocellular tumors are hemoglobin in urinr using the benzidine kst. Acetone difficult to classify as benign or malignant on a purely solutions of commercial-grade nr-phenyknediamine are morphologic basis. The liver, due to its practically dark brown. If the compound was absorbed through the unlimited growth potential, typically responds to intact skin in sufficient quantity, it or its calabohtes chronic injury, and in some mome strains to senes could have caused urine discoloration. A UV chro cence, by irregular compensatory hyperplasia. These matographic profile of individual urine samples revealed compensatory hyperplastic changes grade continually that the material was absorbed. Some of the material into frank autonomous neoplasia. The C3H male mouse was excreted apparently unchanged, but several new very commonly develops hepatoma with an average peaks were observed, suggesting that metabolism of the latency of 14 months and an incidence between 40 and material had taken place. 50%. These tumors vary greatly in gross form and Toxicity of resin 2386 in C57BL/6 mice was un location. Most are well circumscribed, pedunculated, expected, since the same material had no conical effect and resemble normal Kver in color and texture. They in C3H mice. Death in C57BL/6 mice was the result of rarefy exhibit malignant behavior such as local invasion a Schwartzmann-like phenomenon with massive renal or systemic metastasis. Usually they km the host by cortical and hepatic necrosis, suggesting an angio- rupture and hemorrhage into the abdominal cavity, by vascubr or allergic pathogenesis. This conjecture is infarction, or by obstruction of portal and post caval supported by the observation that death was very rapid bkwd flow which leads to circulatory coHapte. and occurred after only one or two applications. If the In an effort to better characterize the biological animal survived the initial anergic syndrome, gradual behavior of this common tumor, we have initiated a recovery ensued even though treatment continued. TMs transplantation experiment to determine (#) whether tolerance or resistant state persisted throughout the there it any correlation between gross and microscopic remainder of the treatment period. characteristics of the primary and growth of the rumor On the basis of these preliminary experiments. 50% following trantphmtation. (*) the avenge latency and acetone sohinons of resin 2386. the resin mixture, and growth rate, (r) how the morphology of the transplant resin I67S have been applied three times weekly in mrmbks or differs from the primary, and ( The procedure we haw adopted involves deierminit ACTIVATION OF MURINE LEUKEMIA VIRUS the characteristics of the primary which include gross BY GAMMA RADIATION form, location, color, texture, size, and evidence of J.A.Otten.J.M.Quaries.* metastasis. Sections of the primary, residual normal andRW.Tennanl Seer, and representative other names are taken. The tumor is mmced in Hanks' buffered salt solution, and Oncornavirus genetic information is present in all soul fragments of tumor tissue are grafted mouse ecus m an unexpressed stale. These endogenous mbcutaneousry mtc five or ten 10- to 12-week-old OH viruses may be activated by chemicals such as halo- recipients of the same sex as the donor. Transplant genaied pyrhuidmes or protein synthesis inhibitors. recipients are examined at 2-week intervals for evidence Large acute doses of X irradiation also induce the of tumor growth. Latency is established as the elapsed expression of endogenous virus, ahhough X irradiation time from tumor implantation to the first palpable is idatively inefficient compared with hamgenated evidence of tumor growth. Tumors are allowed to pyrinudmes. Since the activation of virus by such progress for 30 days after first observation. The hosts chemicals has been shown to be dependent on cell are then kitted, and the tumor is dissected and weighed division, we devised an experimental procedure for to obtain an estimate of growth rate. Cross and low-dose-rate, long-term irradiation of actively dividing microscopic appearances of transplants are compared ecus. Non-vmis-producing AKR ce»b were grown in with the original primary to determine if one or more flasks fitted with medmm and mo bated at 37*C with cell components have become predominant. While the the cd monolayer peiptudkutar to a ,,7Cs source experiment has only just begun, preliminary observa providing I7.S rads/hr of gamma rays. Total doses of tions are interesting. Of 19 hepatocelhnar tumors which 350 rads (20 hr) and 700 rads {40 hr) wire applied. have been transplanted. 13 grew successfufly to re Activation and expression of virus was Jetermined by semble the original primary closely. AH of these tumors, conducting XC-pbque assays on the subcultured irradi both primary and transplant, were classified as minimal ated cells. The minimum total dose of 350 rads resulted deviation hepatoma. A variable small cell -omponcnt. in virus activation, and 700 rads induced a 40-fold resembling the "fetal hepatocyte." was observed, but increase in plaque-forming units. Serum deprivation for the presence cf this cell did not correlate with 18 hr prior to and during irradiation inhibited cell iransplaniability of the tumors. Only I of 19 primaries division and prevented virus activation. These results had undergone spontaneous metastasis. In this case, show that low-dose-rate gamma radiation activates multiple pulmonary nodules resembling normal liver endogenous leukemia virus from AKR mouse cells m tissue with sinusoids and hepatic prate architecture were vitro and that cell division during irradiation is required observed. for virus activation. latency varied from 27 to 267 days, with an average around 180 days. For this entire period, tumor cells remain viable but presumably do not divide. Eventually, *NC1 rVMtdoctorai Fdtow. however, the tumor cells begin to divide and give rise to large subcutaneous lumor masses. Data on differential growth rate and correlation of growth rate with latency STUDIES ON THE PATHOGENESIS OF r.i incomplete. These observations arc consistent with RAMATION-fNDUCED LEUKEMIA IN RF MICE early reports' which ais> encountered long latency in the first-gtTetJitiop transplant. Our current working J. A. Otten, Susan Custead Jones,* hypothesis to rxptoin prolonged latency is that host N. K. Clapp.t and R. W. Tennanl factors are involved in maintaining the dormant stale in The RF-strain mouse b sensitive to radiation-induced transplanted syngenk hepatoma. These same host neoplasms, and an RNA tumor virus has been associated factors may be responsible for controlling the time of with r^iation-induced lymphomas and myeloid leuke appearance and rale of growth of the primary. For this mia in the RF-strain mouse. However, the relationship reason, the identification and characterization of these of irradiation to activation of endogenous RNA tumor putative host factors and the extension of our data on viruses and effects on the autogenous immune response the biology of the primary are in progress. of mice to these viruses is not understood. We have examined RF mice for endogenous type C I. H. I- Awdmmut and T. 9. Dwm.I. N*H. Cmetrlmi. 13, virusca»and naturally occurring antibody to these viruses 455(1952). following irradiation. The study is designed to follow 169 iudwidujl aaimah throughout their life to determine experiments, designed to identify formation of a it*e lebtmrdup between expression of endogenous double-stranded intermediate, mlraccthdar forms of hreb of virus-specific antibody to tfr vims. These it input KRV DNA were examined after infection of ceBs nr«> studies arc paralleled by nt Mm actrratioR studies with virus containing radioactivefy labeled DNA. Be described in this report. RF sake were irradiated at 2 tween 6 and 10 hr after infection of actively growing months of ace with 250 rads of whole-body X NRKcei^virWDNAilKmrdasluTtmd^itatybyCsa radiation; at 2-month intervals foBowmg irradtstiou, isopyenic cewtrifugatioa. DNA beading at the new seram samples for antibody titration and a small density was primarily of viral unit length in -sfrdinr portion of tad for isolation of viras were taken from sucrose velocity gradients, which suggested con mdmdual mice. The individual mice were then held and servation, rather than degradation and reutmzation, of examined at further 2-month intervals. The radio- viral DNA. In agreement with use appearance of viral immune assay was used for determining viral antibody DNA in new density species, hydroxy apatite chroma levels, and the XC-ptaquc assay was used for detection tography showed an mciease m the douhle-strandcdness of infectious viru>. Prdiminary results juggtil that vims of viral DNA between 6 and 10 hr after infection. New appears by 6-8 months following irradiation and that species of input DNA were also prominent m neutral with the appearance of virus there is a concomitant sucrose gradients by 10 hr after infection, tn aftahwe drop in antibody titer to the endogenous leukemia gradients, DNA from the broad I4-20S (neutral virus. These studies are being continued in order to sucrose) fraction was homogeneous in size, but ap further understand host regulation of endogenous leu peared to be snghtty less than unit length. Analysis of kemia viruses and the role of irradiation in the loss of resrtance to single-strand spume nuclease, SI, before dus regulation. and after denaturaticei/rcannealing showed that 14-18% of 14-20S DNA was double-stranded. Addi tion of excess virion DNA to annealing reactions DepMtntvjt of McroatotoKV. Ifruvciiitji of Tcnpjcncc. completely inhibited ^association of 14-205 viral Kamvine 3791*). DNA, confirming the presence of complementary viral tOmsaKaM. sequences in I4-20S nohxules. AD of these results support the conclusion that parental viral DNA B present in cells in virus-specific molecules which arc at least partly doubte-stranded. REPLICATION OF THE DNA OF KILHAM The replication of the RF DNA was also studied by RAT VIRUS infecting synchronized NRK ecus with virus and pube- C. C. LaveDe and Amu Tai Li" labHing with |'H] thymidine. DNA obtained at differ ent limes contained labeled complementary strands Kftham rat virus (KRV) is a member of the parvovirus which could be identified by hybridization to unlabeled group of small (18-26 nm) isometric viruses containing virion DNA fixed to nrtroccflulose filters. Ptelirninary linear, single-stranded DNA. KRV has the important results revealed that maximum RF replication occurred capacity to induce developmental defects in the rat and from IS to 20 hr postinfection and always after cellular hamster. Our interest in parvovirus multiplication has DNA synthesis was initiated. In cells which were been further stimulated by recent evidence that a blocked continuously m Gl,little RF could be detected paivuviius-like agent is the cause of acute gastroenteritis by hybridization. Thus the replication of viral RF m humans. appears to be cell-dependent. The time required before As shown earlier by R. W. Tennant in this laboratory, the detection of progeny RF agrees with the time of KRV production requires cellular functions found only formation of parental RF. Whether the formation of in actively dividing ceils and associated with DNA parental RF is also ccU-cycle dependent is not yet synthesis. Specific events in the replication cycle of the known. virus which are dependent upon cellular function, Finely, hydroxyapalile chromatography of virion however, have not been denned. DNA revealed sdf nical bmrtalions of this method ahow examination of at me UT-Ojfc only relatively low nwnbers of cdb (on the order of 500-1000 per experimental sample fur a typical assay), with a resulting wide statistical variation. Recently flow nwcrofhjorometric rechmques for measuring selected DrreCTKMOFmJBJNETUnXlRVIItlJS parameters of single cefts have become available. These ANTIGEN IN CULTURED CELLS BY techniques provide rapid examination of large numbers FLOW ItKROFLUOROMETKY of ccBs (10,000 cells/sample for a typical assay) and thus yield very precise statistical results. R. E.Hand, J. M. Quarks* We have developed a method for preparing cdb for andR.W.Tentunt flow mkrofreoromelric measurement of specific stain ing by mutMoftuorescent reagents reactive Inmiunofluorcscenl tests aw widely used to detect murine RNA tumor vires antigen and an tke urest nee of viral antigens in infected eels. The quantitative parameters of the technique. CeBs. cither standard procedure involves infecting cells growing on uninfected or infected with Moloney leuhrmia vims, are glass coveislips and fixing and Hawing the cells for fixed in ace lone, washed in buffered saline, incubated antibody, re nt. 16. Sratwr partem and wmpim of a aniMatUm M* mprtw (vmwecwd) cats (A and •> tar (warnil hafcrmh »bw-rnfctl»d) ecu (C and D). Foi in* nailer mnerm, lb* Y axis represents light scalier and the X ass fluorescence. For the hJstocrarm. in* Y axis represents number of cells and lb* X axis fluorescence. 171 washed, and examined by flow inicrotriMrometry. The for defined conditions, we conducted a mithcmiricil number of fluorescent cds as a percentage of the total anaryiit of the synthetic phase of several types of cd population is dritnwined for each sample nsing a normal ecus using data obtained from she Los Alamos CytoflnorogfaT, Model 4801 (Ko/Physks Systems. Scientific Laboratory. A formula was drrived giving rate Inc.). Tins instrument, which has the capacity of of DNA increase as a function of the distribution of nmwllininuirj measuring two optical properties of ceMs flow HHcronworometric data. The resulting differential at a nmimunt rate of SQOu ecus/sec. is used to equation was integrated and graphed to ytsld the determine light scalier (representing ceM size) vs flaorcs- amount of DNA and synthesis rate as a function of cence (representing the specific antigen-antibody re time. This provides a theoretical model which we are action); these properties ant dnpliyed on an oscSo- currently testing to determine how these curves vary as scopc with a grid overlay. Typical dnplayi of negative a function of altered growth properties, and whether and positive ceMs are shown in Fig. 16. A scatter pattern external perturbations of the eels (*$.. infection with (4) and histogram (**) of a negative test arc shown in viruses or treatment with caicmugrnic chemicals) can the left panels and the corresponding displays for a be detscted as alterations in DNA synthesis. The positive test in the right panels (C and £>). This Mttrubwn and applicability of the mathematical model wt> be evaluated experimentany. other virus-cell systems, often the advantages of modi mate rapid detection of specific viral antigen on a VOMPMCf SCttncd tfffHMO. prr-cd basis and a larger nomber of ceRs than can be obtained with microscopic examination and thus yields a better statistical analysis of the results. SIMULATION OF CLINICAL RADIOTHERAPY NCI nMMoclofiH Fcflvw. WITH WR-2721 IN THE SPONTANEOUS DOG-TUMOR SYSTEM J. M. Yuhasand Darryl Kery* USE OF FLOW MICROFLUOROMETRY FOR DETERMINATION OF DNA SYNTHESIS RATES AD of the information which suggests that S-2-[3- annnofKopyUnwnoletlrylpliosprtOiotNotc acid (WR- J. W. Heidri.* J. M. Quarto/' 2721) offers differential protection to normal and and R. W. Tennanl malignant tissues has been obtained in rodent systems. Flow imcrofhiorornelric techniques provide rapid Wide such studies are of value for preliminary work, measurements of selected parameters of single cells in they do not lake into account the gross differences suspension. The use of large numbers of celts, approxi between the mouse and man. and the tumors each mately 100,000/min, provides high statistical precision. bears. Since the dog represents a closer approximation Fluorescent dyes specific for DNA are available and of man, both physiologicaly and anatomically, and have been used to stain ceStubr DNA for flow mkro- certain canine tumors arc essentially the same as their flnorometric determinations of the DNA content of human counterparts, studies on the effectiveness of single cells. The life cycle of mammalian cells in culture WR-2721 in increasing the efficiency of radiotherapy can be divided into four main period* C( (gap), S of spontaneous canine tumors were originated. (synthesis), G», and M (mitosis). Ideally, celts in Gt The Department of Radiology. University of Penn (Le.. prior to synthesis) have one unit of DNA. cells in sylvania, b a referral center for the East Coast for the Gs and M have two units, and cefls in S have between radiation treatment of spontaneous tumors in canine one and two units. Histograms of the number of cells vs pets. The two tumors selected for this study were fluorescence (i.e., the number of cells vs the amount or squamous cell carcinomas and fibrosarcomas of the units of DNA) yield peaks at G( and ai Gi • M, with head and neck. The overall plan calls for the admini ceils in the synthetic phase between the two peaks. stration of 0, SO, or 75 mg of WR-2721 per kilogram of Working on the assumption that the rale of DNA body weight before each of four to eight localized replication during the synthetic phase is an important exposures totaling up to 4000 tads. Full clinical and distinguishing characteristic of cells and thai the work-up and follow-up arc made on each dog. shape of the histogram described above is related lo the The first study, whkh is now Hearing completion, rate of DNA synthesis if the generation time is known involved the exposure of non-tumor-bearing dogs lo 172 Or WR-2721 RK MMAHS ROM AN LC. J J. tWtrr.» *. L Hwes.' UWMrifr rf dJ.M.Yahas of the 5-2-| J ianiaiiaiiipylnnmii|eiliylB»waj^^ (WR-2721) has potential vaJat in the ijdiothtrapy of ANIITIJMOIt IWDtTIES OP solid tasnors. swee the drug Mtotaaes Hit COHYNEBACTaUlMfAKVlMWi of uumal ta to the tumor, at least in I.M.YuhasanuR.L.URrich I system*. Deficient attocntma of the drug by a variety of sand tumors has been avmonstratcdi this Most cxecrinvrntal studies on specific and nonspecific deficiency appears to be the major factor mponiibk unnnmothcrapy ate conducted in young adult Mice lor the observed dMTcreafial protection of wound and beating strongly immunogenic iraaaeaHtcd tumors. In das respect, they bear Mlk similarity to the cUnkal The* i in that the Miochthonrus twnots ate weakly of two parts: How mach WR-2721 isogenic and the immune responsiveness of the t*red to a human m order to obtai host it rtdated due to age, tumor growth, sidr effects concentrations which offer cx^tUcnf protection lo of therapy, or their combination, we have therefore lower species, and does tne same tissue concentration initiated stodits which attempt to circumvent these offer similar protection to all species? Thefirst portion problems: both young and senescent Hnnmnolj|icaRy of the oacstion is covered in this leport. incompetent mice are employed, strongly ami weakly The tissue concentration of an mjtctcd drug can range immunogenic tumors are studied, and, in certain cir- from being a simple function of ike dose per unit body curretaaccs. spontaneous tumors growing ir old animals weight through the move complex relation of dose per arcstudkrd. unit surface area. This tatter reblkmaWp is a reflection The nonspecific irnmurostimulant being studied is of the fad that the surface area is a good correlal? of CufyntttcKriutti p&ttwn, which has been shown by the metabolic rate. others to be a potent inhibitor of tumor jrowlh m the In the hope of being abfc to predict the drag dose aforemenlioncd systems. In our laboratory, the use of required to achieve excellent protection of human C pmmn docs inhibit tumor growth, but no cures has* normal tissues, we have studied the distribution of yet been obtained, ettn in young ink*. Further, the "S-hbekd WR-2721 in mice. nts. rabbits, and dog/. overall effectiveness b far lower in weakly immunogenic These sludtes have demonstrated that the tissue distri tumor systems, such as the Line I king carcinoma. bution in these species is not a strict function of cither Senescent mice arc less responsive to the antitumor body weight or surface area. Extrapolating this inter- in • ari/LE.) OWL OlOJ. apt l»~%*. ; oflinliHiioMy.fi,, l«.«aal«J2 BAU*c ajanae. Of ******* rasfhaatva •uunriOTCi muE URIC J.MYohu lo «tof*t wids da? by-pevdects of each a wjefety of dOSJOf P^ammmf aflMLW An exception 10 AH nk • *« efficiency when they arc given noaojnant long carcaaornss. Instead of a dnoct toacatdy. la brief, it apoanbte stop between doer nlc and aamiyswm nviehtbra>teio"rca>vcr,> r taaatii by one, bat effective, ie., in the range of 7-14 rash/day. to recover from the mjary indand by dK other. rrctbninary studies on Ika tarprtad The poaaonjty daft each a pattani enisled ins type II ahcohf call of ibt now* am, by oar iwlbaJriiiry stadias on the acate psraboocresafJonsnip rttan a from tkt drlhate I for of waMpaiM trenafonneUBn and call cytotoxicity. lion. Both carcinogens yielded a Abo iacloard in dam ttoaUtt were matym of da* wiponss carta for the prodactioa of rang tamers hi die rote of apt at cxaoaare in (klcmiioJiig stsattvHy to moans (Fig. 17). In the low one range the yieM of indociion. As a genera) rate, M appears thai ccl tamon was Mnearly proportional to doer (kttog whose faction asthnsi with apt (««:, die *>pr-I.O), wttte ia the Mghtr doss raapt die yieM ovary) show a marked deck** in sensitivity lo twmor was proportional tc a ajreatar than IJO power of die indnction with Mhandnf apt, whereas cat systems carcinogen dose. Thaae data sapjest dart m die low dose whose integrity remains ctatnf taby coaaiaM duoMghont ranpt • stank event arespmssbk for die production of lite (*#., alveolar taring cells of the rang) snow no tamors, wMe in die higher dost range, not only single i i 174 125 290 900 1000 290 900 MOO fJOO UPCTHMCDOSEfjaa/af) X-IUY OOSC frwJs) » -" Aat IZSwt/kgofi B 1230 raw. oil lakes iato accoaat Ae fact Aat traajva of the* Ant of artAaac woaM afco ywM of one awMoan act tratkm of 1250 rait woahJ yieaJ tar fewer bat eaeal iaieractioa B coatribatiaf. Carryiwj tamon. ii CM WO toagrr be apca AM Aese Awn of a step farther, oae coaM araae Aal Am Ae two caaciaoytas are cojaaby haiawjuas. IMea be ao recovery fram total Aws whkh fch oa wformatioa sach asAabobtaiaea'for Ae by-aroaactt aortam of Ae can* bat Alt sajnificaat of caerfjr techaotoajes. cosi-bcacfit aaaly«es wal be Mcoaerv AoaU be observed ia Ae i wal jwnrn law •"•wawwai; aaaawaaaw* w/w* wwaaivwa) aw# wawa> »»»wtT»'"»a»mw^rww VI CARCINOGENESIS PROGRAM F.T. J S Cook WHATmmmVbm cmwuhtr R.W.TCMMM G.C.Lavde J. A.Oitcn J. M. Quito. Jr.' fntdactonli tiptoi 175 ™J«. ra COmTAJUTIVr. fFFCCTS OF 7J2 IWMT1MILMHH|»|4W1WMCIHC AND R- A. CnonKi. Pad Nftlnarim. D. H. 3MU.ECaMi.Jr.* ASNSTIVEASSAYFOR :rats or I of 7.i: •cnoU|pyrene (a?) m HtiKf grafts 1 w pnaecraan and Away of priku for nac ami of i*iwjw myxoid dip minion of laatiin taae. Onr miath me ill «m MW perminrw bkr me lapupfaak iiiihihaiii wa repriced try a uf low dose «RM to the graft and a>^c cd aycr of di irrnpta aW awahic ipithinnm the do* ratt. Analysis of the dbrs maM a ott of «« tW«iia.ijlfc cenanr stypm. The M. dystnipak first MMT. TW a*MM of cactMjtaj arkara wa m,,. Eajki Mamtaftcr J* suriof arnpcnmcwt concentration dependent pkes 0.1 ng of insafn and 0.1 ng of I per ni. or m Wjymoum's i secwm 12%) tot 57 days. Vitamin A < 0J02 pg or 10 ng/ml were mew added, h rem HfECIS OF VITAMN A ON ftOUFEMATION AND DvFFEKENTIAHON IX HUCMEAL found on expfauts cultured in strum-free i ONCANCUUUKS and/or wwenatr hypersecretory activity can be i m cxpbaMs cuttuwd in me low/ Joyce f. Rhoton F.li nd M. Vhfinii Cot I. AM r. Last year we deniaWd a tracheal orgm-cohere {#*. /or*, i kn die purpww of studying ivmchai nunc diftcreiiHatMu m Our way we are Mdirawc wws system » to AOomoevtiJonKNxofCAMamicBi- rnrtber me effect* of vitamin A lal-Awas- mOKUtmjMLmSnCUJMCHOOOLa reumd) on ceR prnhtcialinK. secretory ceN activity, and ram Ncttemcmt and maty L. Winams mbmiliuu nf squamous iwrtapbm. We have reported thai 0.2 and 2.0 ug per ml of We have previously reported that rats mamtamed on mcdmm umwnates |JH|iliywiidme incorporation, cel different levels of rctmytaceiaie (RA) show different lular byprrpbsa. and hypersecretory activity m tra scnsiimlies to hmg cancer induction. Animals on low cheal exports cuilured m Waymooth's MR 752/1 RA intake mow an mcreased sensitivity when compared mcdnm supplemented with I0T hone serum.1 As a with rats on high levels of RA. However, it was not .Hmtmwatini of mere studies, the foRowing series of dear from the previous study whether this was indica utptiimnHs ate neiug carried oat. f«)The effects of 0,2 tive of a stale of immicd susccptibMity in deficiency and 2 pg ot rctimd per ml are being tested on tracheal and its mere correction by addition of vitamin A. or expvaMs cm tared m Waymoulh s medim whether a general anticareinogemc effect of vitamin A meMcd with low serum (2>) or in the defined i alone. In both cases a marked increase in In a new study, three groups of rats were maintained | H|thymidmc-bbelcd epithelial cells if found on (beginning at 12 weeks of age) on a diet providing autoradiographs. and ccwubr hyperplasia and hyper 250 300 ag of RA per week. In addition, two of the secretory activity are stmmbted. <») Wc arc carrying three groups received daily doses of either 1500 mj of oat mese kinds of analyses osmg tracheal expbnls from RA or 1000 «g of l3 UM K*ub ccrtaai I have dnnvn Otai Boa does MM auenatny Ky B IBM MMKT CCfUM « m mx rehaaid from ihr earner particle • is not watahlr to interact withtesae. W e Hofaaarmudro r clearance of the the ihmmitiiin of the ca to the carrier narttdtetnam i carbua particles (to I) warn '•**•. a | KUIT10NOFCAKlNOC»ntOMOUrJtin Tha neihoaotogy was ased to PAKTICLCSM TIK tesrnuTOrrr of Wt (tarn two different TtACTOFMCX (05-1.0 « and IS 30 a) was vastly different. When IP adsorbed to 0.5- to IJ0* D AC carbon particles was adanniiicred anratrachealy to Effect of cmritr mnkk stu. - Several stadia from mice. $07 of die iniria! lam; harden was ehmnated at other laboratories have reported an increased retention aboat 36 hr. and *0¥ was ewmnated at abost four MML-M« TS-iS0«C too 01 2349*70 0 1 2 3 4 5*7 rmt uritn INSTILLATION V 0.7 u ••-CMrtOM 7HME Af7E»WJ5T»LLATl0N0r 20ut»-CMr»0N MftTlClES (•**•) •AffTlCLCS «*•»») Pig, IS. Phniairtini of S* now a* wpbennj met of ma*. Mice were Mfatracnofty mjrciaJ with •» crr*iats abmc or with •* crystals aowrbnl to M> 0.1-u^riB) Vh, carbon mrfkws. Data »tsarv*Ma at acrccM of the iftrini rona bmreit. •. M> crytMh atnnc: . RPcluted rmm earn* particle*; •'•. carbon partkles. 179 •ay*. Ai sevca days. 50% of Bar carbon parades woe ato-soa*c ian 11 (see Fig. IMi. Has yum imkV« being ebttd from me carboa wale • me bag. The IMHI me B aparuxiatairty !6%/-by for at least low days. Tmt means dm there B always a steady. relatively iWHUl m—^H>- of fieci-afcangeaata persists maar bag tot low days, la contrast, when me same aaaatwy of free m* crystals tahn 03 1.0 *) » Mimnchaly immiimiii. 50* B ilaiimit d wmw* 15 hr aad >90K ehaaaaied wvriaa tine say. These bets are aaponant with regard lo naciwuatB activity, ma Btinrtew cannaogm contact B probably meir coa- •MK to lamurigrak artcvity axm a rapid aewte m contrast lo the ctmaaatHa rate of fate IPciystab ami IPaeaoibed to 0.5- to IJOB particles, me ehmma- itoa from me bag of Wt adiurhtd to 15- to 30* aartkles B aaKh mmer. la nm cast <50% of oVe Vis t hamuli J at f«m days (see Fig. IM). This B ahao* the same paha unary clearance rate as for the carboa aartirlf i. mdkjtinv, thai rhel*BiK»rdimociariagfroin the carbon ami therefore letitwcly hide is free to interact with tisme. It is noteworthy that with taniuriatak stames. both free If crystab aad Wt adsorbed to 15- lo 3©# carboa have a low carcmogjemc activity, whie adsorption of Wt to 05- to 1.0* carboa particles prodaces aa enhanced catcmogeak activity. rtm. mw> mwo mttxitmmmi Effeci nf mpimttwy mfecikm. In the past wr haw? rn> IT. KWHWWWMI wt WW MB WW WBWWWSfy BVJC1 Wt shown that severe viral paewatomtis oases a Ian jag WwOwmttcttm awl aswaawcvMl wast* Tat wsct WCVE MCIS- tndMaay mjMtd «nk IP oysub akmt or widi BF oysub defect ia the respiratory Jcarjact of JaiohiMe particles. atuibed to 0.1-m cwfeo* awnktr*. Dbu are csarcacd at Utasriag oar' #>IU-Wwnng tcchaaaje described above, niuM of iaicwl nwa wiwt- o. wT«ryMjbalowt.aowiwfccitd a* stated the effect of viral arfection oa the rate of WJWC; . wf cfyajb alowr. awfded WKC; *. cwaoa wjftKtrt. ihaanaiimi of Wt from nwase bam. When die 0.5- to wamtcewt an: •••. caraoa awtidn. iaSccttd wicc: •. Bf cwMd from ofton awlidn. —aiwfuiid tmt*. -. Bf dated I JO* Wt carrier particle is iairatrachealfy jdaianitiuil fioai carbM asttKfcs. iafocKd Mkc. two days prior u> the peak paeaatoawai mdand by die atfeclMia. there appears to be aa cananccd chnaaatioa of Wt from the bag (ate Fig. !•>>. Wirhia 24 hr.75* of T0IACCD SMOKE MO ASSAY PROJECT die awiiial dose B elaiaajifd. Uaiafecied coatrob W. E. Dtmcy, R. A. Cnetemer. and ram Nettcmetm cfntanate 40% of the IP wtriaa 24 hr. aad 75% it cantmiltd by three days after iasfabtwa. A mam Therehmbtenaaeedforabkmwayofdwimaactof ptotemaccoas exadate it prodaccd at da? bag at a chroak tobacco^atoke expomve dbecdy on respiratory remit of the viral iafeciioa. It it cnaceivaMe that dm tract tBBjes- The proMem hm heen approached hew in pmnfBtJteiaH exudate enhances the daaocieiioa of If two ways: Bdadatioa expoean? of cats aad dehveriay from the carboa carrier particle. sntoke to atbcataneoasiy tranaabMed tracheat. There am no appttuu effect from die vims infection Saasl bboralory anhmm are to be pwfened for mch on the pahnoaary dearaace of carboa particles daring bioatsays becaase of economical aad logittical consid the seven-day observation period, Based oa oar ptevhau erations. The only smaR laboratory anjawj that had clearance studies with nanmMe particles, seven days for bant sysfcsMf arsHy explored for iaJaaatioa mpomrct carbon-particle clearance it loo ahuri an inierval for aa was da? hamster. The amy dpiiricant Itajont avdaced in effect to be noted. these ananab wtn bryngtal aBjkopbdm ami carci- IK Uf rat* chrtmicaly exposed to seven and ten ctga- •f M^. I UEUJSCai CaffCUMUeueWS SvHMSes nSBHTSUCU IIKN reurs/day. 5 and 207 respectively have dwd »pou- the at might be significantly mote sensitive to imn lanrouly after 15 muvMhs. At anion*. lesions have mdnctsviu m lb* mpimavy iracl dun tbe hamster. been observed twK m the sespuaiory tract. ltrsti4ogi- Howevti. the nl hat not been used exninwvcly m caSy. dm lessons included occassuwai mrid local smoke iuMation studies because of its greater suscepli- hypernbssas of she upper airways. brondNubtis of the bavry to irspwasory tract mtecinms and acute tigarette- termmal brtmcnaoles. and aheubtb. unokt toxicity. Tracfceas transplanted MsbcuimtnuiK beiween mbred A series of acute experiments led to a feuiNbly study rats show pronMse for auayme. caicif* The NMR arectrometcr wil be astd * 4K n favare 10 stwdt hydroy ataiiiwii exchange kiaetics of the beta aoamoa}. al poaM to activation of bydromaa aanau aanpnaads to amstigaK the pusaaatiiy of alpha to the aitraao groap as a raae-hnarjag step ia cartaaam «ot ticipatHja in the iwnhaaiwi of carciao- The increased carcawanadtjr of artrosureliaaydro- Hgh-prcssare aamd cmortattograahic tcctaaaars are anVnvaamm* ^njWjnmmafJwi vammfmi gmntaBnUBmmma*rRjmamB*/ ^mmi tmm*. ImaVfV being developed for the aaatyas of cuaavkx ihermaty nannrs • dated in rats by the former bat not me latter, aaaaUe. aoavabtde .V-nitroio coaaviamas and/or lam together with me aoacarnamtakity of the doacty paecarsors. which are often aapovtant reiinwln aad • • - - • W«^J . . .. — •• -' « > HTraCWJCS. VUPWJ. HCBU1C SeanraUDn KCMinaVS I0VC hydtnayridme». are diflkalt tocxpram in this way aad beta worked oat. More importantly, a mem. wjuitiw.. •adoabtedry mvutu othti factors. anroaB^onmonan actectov B oemg aevetopca. aan •atal remits are very eacoaragiag. Has rfowirg wet- snjmaoHivmmKMHBmwt chenastry detector wil detect raicrogram onantiaes of. far example, minmicarbamam which cannot be icaday analyzed by ps4M|uid ctamvMofjaphy. Reimeracnts of KW Taylor rhf detector are at progress. In previews sladks. aMroioaathvtdodecylamiae ia daced tunjiiionji cca carciaomas of the arinary RELATION •CTWEEN STWCTUtt AND bladder ia Spragar-Dawley rats. It has aho been shown cAKONOCENK: ACTIVITY OF mat ariaary Madder tpwmlmm ia rats caa be traas- VMUMBO comouKn pkmted to a waVaianiiwi poaiion aad wal develop iaio "pvmdo bladdtu" hard by a typical traasitional WMham LpHky and H. W. Taylof bliimn are bemg fed rutrosoaKthylaodecylawme to li has become uovtuus iba! small changes in chemical detrmaar if these ectopic bladderi wal be affected Uraclure of nrtrosaminrs and other .V-naroio com- limiarry to the nataral blahtei. Systemic effects of the ponads can make a large difference in caicrnogenk compoand can thas be duiingaished from the local acidity, either in potency or m target organ of the action of some puarbfc metabolite in the wine of caKiaogen. By comparing lac potencies of several treated anaaak. ratios** coramamds of aaatar sfractarc. some mdica- tkms can he gained of the ihtmkjl characteristics rcbled to csKiaogenicity and hence inferences aboat SYSTEMIC EFFECT OF the mcihaniaii of their action. Several nrtroso com- WlllOSONKTrAltETrlYUIwQMfMEON goandt have been given to rats at *e sane mobr TIUNSnANTEDAKOIKIST concentration for thr same lime period, and the sarvrvai TKAOvEASOFRATS and lamor mcidcncc in the ammab haw been mens- H. W. Taylor. R. A. Gnearmef. ared. DictaVwonn'rosopipendmc it mack more potent aadPmn Ncttcsnemi than aMrasopiprridmc. and drbioaiuniiojupvptridme a a mde irm poteni than the dkhtoro compoand. aH r^ruaalwpumeiirylearimiae (NHMI). which is three giving me to esophageal lamors m high incidence known to in date neopiaia ia the trachea and hmg of Smmarty. dkhluruniirosopyirolrdme a mndt more rats, was given by gmge to F-344 rau bearing potent than nrtrostipyrrokdine. cauamg cwphagtal mbcntaneom trwipmnted irachea*. The effects of das lamors rather than fiver lamors. and nnrosonornico- naroso cumpwind on me host tracheal and on the line, another derivative of rdtroauay i lumliM present in trantpoated trachea) acre corwpared at Ave-week inter- tobacco smoke, canes natal cavity tumors, again with tab wp to 30 weeks. The initial effects on host trachea* Meher pofency than mlrosopynohmne. Nitroeo-2.5- ware hyperplasia and mrfaplaiu to stratified Mvoamoas dnnewrylpynofnmic was aoncarcmogenic. showing that epfdvelmm. accompanied by a lymphocytic mfihratc. blockage of the alpha carbon atoms reduced cjreino- Later, hyperplastic marocmary epi'Jvtliwn relumed to gmk activity. The redoced carcinogenicity of alpha- most of the tracheal Nning. except 'u rsotated foci. acnrerrafirtaoeica nnrofomorpaonne compares wnn Fmaty, pofyps, papmomai. and tamwiotii cell card- me anftrbefed compomd. together wilh the above nonm> devekyed. The effect* on the tnnepfaritcd revjlts and lire greafjy increaaed carcmoaii.'cily of tracheal were those of atrophy and dytfrophy, with no 132 neoplastic changes. These studies strongly suggest thai induced by DMN While iboc studio arc not yei Ihe effect of NHMI on ihe trcchea involves more than a cKvnptrted. the iouli\ xtgpot thai lurnormmiciiy e» i*4 simple organotrophic action. Some rnetabohte may be enhanced by a popniati»-n .«! dnndine hepatocyio formed in the tunc, or may be lorn ."d elsewhere and HMoecncsis Miwhev in mhfc.li aamul'v were kmed at removed by way of the lung and trachea, and exert its four-week mtervals after toHulmt *-t ti«. -intent wiifc action !ocalry on these ononis. DMN. show thai tht» compound r» I»\IC i» bcpai«cy to and that tumors pr«*aW\ JIIMT from tmal I«H.I ••» undnYcrcnuaied celh thni JOMT m ihc peripiial • „•- STUDIES ON METABOLISM OF pons. Tboe celK seem to hate a plcunpoicni potential DtMETHYUOTKOSAMUa IN RATS Mime I'IHTI later resemble lanall ••sal hcpaiocyio. ««•*• H. W. Taylor. Catherine Snyder, and Wtfkam Lnmsky areas proliferate as endothelial cells rh-i! arc cjpahk- ••! phag>H."> ti/mg carbon parttclo and ccH> in \>«nc l»*i We have mriBied metabolic studies with tntmrn- acuuwe cdia and re-emMe primitive cholaneitiLu cclh. labeled dmtmylnrtrosarnine {DMNr. The distribution TranspbntalkHi UudW* with thoc lu:m»tN ace undci of DMN among the five Brer lobes at various tune way. as are htstoensy me %lu«iiev intervals after oral feeding of the niirosamine has been determined, as well as its distribution in four hvcr tissue fractions: acid-soluble, lipid, protein. 2nd nucleic acid. FURTHER CLASSIFICATION OF CHEMICAL Distribution in the lobes is approximately equal after CARCINOGENS:UXORWNC TO THE MODE OF recirculation time. Although most of the liver radW DNA REPAIR THEY INDUCE IN HUMAN CELLS acliviry remains in ihe acid-soluble fraction throughout the first 24 hr after oral administration of iritisicd Jamo D. Regan -*ni A A Francis DMN. a significant portion of label IXW^I is m (be Using Ihe bromodeoxyundine photulysr* *>j\. »c lipid fraction after the 1-hr lime point. Work is under have previously described in deiad the two modo <-t way to deterrnme the location of the label within the DNA repair winch occur m human cellv' They are various lipid classes. Methodology is also being de short finni/me-iypc) repair and long tUV-type) repair veloped for studying ihe morphologic distribution of These two modo aie observed in human cells alter the label through autoradiography. Because DMN is treatment with a variety t»f chemical carcmogen> and soluble both in water and in nonpotar solvents, frozen mutagens. We have extended these studio l<> include a tissues must be cut in a cryostafk microtome and number of other .-ftemkal caicmogens representative <>l freeze-dried under vacuum before they can be used lo certain classes of chemKals having similar ttructures Nil expose photographic film in a meaningful manner. We varying in their ability i<> induce cancer. Results of are. in addition, studying techniques for separating these studies (along with our previous result* for Kupffer cells and hepaiocytes and quaniitaiing ihe comparison) are shown in Table *4. 7-Bromometh\l- labeled DMN hVeach cell type at periods after carcino ben/anthracene at 10 5 M inducts abnut fine breaks gen administration. per 10" daltims alter Brdt'rd incubation jrnl HI* ergs/mm1 of .U.i-nm radiation. The activity • MSTOCENESIS AND BIOLOGY OF HEPATIC compound in regard to repair in xeroderma cells has not TUMORS INDUCED BY DIMETHYLNITROSAMINE yet been tested. It is conceivably a complex agent IN RATS similar to 4-nilroquinoline-l-oxide |4NQO» which pro duces more than one type of damage. The K-rcgion H. W.Taylor epoxide. BP 3.4 epoxide appears to **e a classical Several studies were performed in an effort to help short-type agent similar to previous short-type agents clarify ihe cellular origin of tumors induced in the liver we have described. The non-K-rcgion epoxide of BP. the by DMN. In metabolic studies of DMN. ii is imperative 7.8 epoxide, has a complex interaction with DNA in the to know what cell type actually gives rise lo tumotj. so presence of the 313-nm light and appear, to induce thai relevant ceOular metabolism can be elucidated. The DNA cross-H.iks. The dimethyfben/anihracene 5.6 influence of concurrent administration ofCCI« and ihe epoxide appears lo be a long-type agent, and iis repair influence of partial hepaleclor y on DMN tumori- in xeroderma cells is defective. Dr. James A. Miller supplied l.l-bisfphenyl)-2-propynyl-A -;yclohexykarha- genkity have been studied. Il a|ipears that CCI4 had link influence on the action of DMN. while partial mate. whkh we find lo be a classical long-repair- liepalectomy increased Ihe time-io-dcafh with tumors inducing agent with defective repair in xeroderma cells. 183 M. 11 V>. turw >;rj«! bfcjfc.^ p" Rrpuu IJutjlK-n :•• AJ-,.n» jitcr BrdlrJ l»pc-«t term lA> 1 h«j\».k-i hrfal • ->4 nm> -•"•cr*vmm* ••4 2" 1" *•« Intra* 4 'i_5 1 "A short I«> !••"'.¥ I2ii 2 1.5 Sk*t i»i 1.5 L.-IW mis 5 • l«~* X 5 1 »>4 Sh.t: V>rlo>.ijid Pf-fMOC Ulll.ifK' 2 • In "* V 12" •» • 14 Sh.rt KK-iri. lit * V Mi 2«i I «VJO 5 • |i»*'.If *• 1.5 Sh»rt IM ^^ l-tte 7-fc..nvmctln lfeciujathi*.cnc l"» ' V *< IS -4 5 UP ;4 rp>\«k ik rrxkini : • H» w *• IK '1.6 Sfc-rt •f ''.Hcpi.xnk- monk retain! 5 • to ' V S»i IH ( r.—.-ImkxIlNA Uancrliv INrn/jnllajvcnc 1.15W *: •->! v_.ii w„ .i v„.B H uwU A similar compound, but having one less hen/ene ring. attacked.1 A tautomeric form of deoxyguanosine that B I-acetoxysafnile. This agent, by ctmirasi. is a wil pair with deoxythymidinc is produced. Hence the short-rcpair-iype agent which produces repair patterns G-T gives rise to t GC and an erroneous A-T at the next identical m normal and xeroderma cells, even at long replication.1 In both bacterial4"'' and human'"'1 cell>. a times. /.<-.. IH hr. jfter damage. We are continuing our considerable amount «il this damage rs removed via the studies with these agents and wiM further characterize excision-resynlhesb mechanism. them, particularly with regard to the number of Rodent crib, unlike those of humans, are unaMe to nucleotides inserted in Ihe average repaired region and excise more than a small percentage of UV-induced the kvel of repair in tht xeroderma pigmentosum eel's. pyrtlpyr. Howr.rr. they are able to tolerate a certain number of pyrt Ipyr because they possess a postrepli- I. I. I) Rcon jnd R. II. Stlkm.Cim-rr Kef 34. "3IH J3».« cation repair mechanism similar to that already de scribed in human cells *"' Colony-forming ability in the Rl 11 rat cell line is three times more sensitive to DNA DAMAGE AND REPAIR KV 4NQ0 treatment man is the Fl 11 cell line. The sole 4-NITOOQUNOUNE-l -OXIDE IN NORMAL AND difference between the two cell lines is that Rill has a RAU9CHER LEUKEMUV1RUS4NFECTED Ramcher leukemia virus integrated into its genome. RAT CELL UNES Hence the presence of this virus has conferred a sensitivity to 4N00. Some differences in prereplicaiion Raymond Waters.* F. M. Faulcon. repair after 4NQ0 treatment are observed. The virus- + Giampero DiMayorca and James D. Regan infected cell appears f by bromodeoxvuridine photolysis 4-Nilroqumoline-'-oxide is a potent carcinogen1 and assay) to be somewhat defective in this repair mode, produces damage to DNA either directly or via a although the magnitude of these differences, while metabolite. Primarily purines, especially guanine, are consistent, is not great. We are now in the process of * *•»>•* nr.rWj"'* ~^ • '.'"Vliiw : * "— •••~*****'&EFBK&X&IQ* V* studying whether the postrephcation repair of 4NQ0 can be tested by adding various concentrations of damage in the RIM and Fill cell lines is influenced by methylchrysenes to the celb during the period ot repair the presence of this cancer virus and. if so. to what replication. extent. The 5-methylchrysene appeared to be a strong inhibi tor of repair of 254-nm radiation-induced damage at a 5 'ftotldoetofal nncstigalor supported by twhtootnct 3322 dose of I0' M. The 4-methyl isomer was less active fnm MV moiocy MCTMM. OftNl. to One Vtmtraty of but still inhibitory. Furiher investigation, however, has Tennessee. KnoxviBt. revealed an increase in molecular weight of the control i I'mvenity of lftno» School of McdKme. (V7p. 6061 2. DNA from moi wt * 230 X 10* to mot wt 3 439 X 10* 1. IL F. Strtcfc, R. M. C. Sa». and Y. Kawazoc. .toftnv 229. in the samples receiving both mcthylchrysene and 416(1971). 6X 10* ergs/mm2 of 313-nm radiation, indicating 2. H. Fndo. T. OBO. and T. Supmura (ed*>. Ofimiwr «JN/ Vofagicaf .4rt*j»c «if -tyuroqumolmr-l-oxidr. Spfinfct-Vcrtac. pholodynamic cross-linking of this DNA. This shift was New York. 1971.101 pp. seen with both isomers, but. since moiecuia weights 3. J. S. Paul. P. <)"B Moalfomcfy. Jr. and J. B. Low*. this large or larger are not resolved on they gradients, CmctrRn JI.4U (1971). any differences in cmss-linking between the methyl- 4. I. C. FdkiKr and I. Kaofoba*. /. Aurferinf. 9*. 144* chryscnes wre not measured. The apparent differences (19*8) 5. N. Yamamoto. S. Fakoda. and H. Takrb*. Camrr Rt*. 30. in repair replication inhibition shown by these two 2532(1971*). compounds could be artitactuaJ in an assay and actually 6. M. Ikenaa. II. Itfeikawa-Ryo. and S. Kendo. / Mai. Biol. due to differences *n cross-linking characteristics of the 92.341(1975). different rsumers. These pholodynamic cross-linking 7. II. I SdUb and R. II. C. San. Jfafci/ R. t 13.279 (1971). characteristics are now being investigated. S. J. I). Regan and R. B. Setkw. Cornr /c«. 34. 33IS (1974). 9. i. ¥.. Of-j»cr and ('.. M- Thomas. JBnrn>-m. Butfihyt. Res. 1. D. Hoffman. W. F. BondineM. and F. L. Wjrndct. Scirmc Common. 36.203)1969). 1*3. 215(19741. 10. A. R. Lchmann./ »M Kuf. 66. 319 (1972>. 2. J. D. kegan. R. B. SeMow. and R. P. Ley. /*»* \afl Atml. Sci. LSA 6*. 70* (19711 EFFECTS OF ISOMERIC METHYLCHRYSENES COUPLING OF TRANSPORT IN CULTURED ON DNA OF HUMAN CELLS HUMAN CELLS A. A. Francis and James D. Regan Emily Tate Brake and J. S.Cook Methykhrysenes are present in '.>bacco smoke and in Changes in thr Na*-K* transport system may be lower concentrations in other pollutants, including indirectly linked to uptake of other metabolites: specifi gasoline engine exhaust. Various isomeric forms of cally, amino acid uptake may be linked to the mem methylchrysenes have been tested for carcinogenicity in brane potential or to the Na* gradient. mice: among these the 5-methyl isomer was found to be With the nontransformed human strain HSWP wc a strong carcinogen, whereas the 4-mcthyl isomei was have been measuring the transport icsponse lo serum inactive.1 While investigating the effects of these two stimulation of quiescent cells. Our preliminary results methylchrysene isomers on the repair of DNA in human are that the K* turnover doubles in both the transport cells using the 5-bromodeoxyuridinc photolysis tech and leak components within minutes of stimulation, nique.3 wc found that these chemicals cross-link the and. as reported last year, these remain at the elevated DNA photodynamkally. level and proportional lo cell size throughout the Cells are labeled with |JH| thymidine and subsequent cell cycle. |'*C) thymidine and are exposed to 200 ergs/mm2 of The quiescent cells, as observed by scanning electron 254-nm radbtion. Thr 'H-labcled cells are then allowed microscopy, are quite flat and smooth. The early lo undergo repair replication in the presence of BrdUrd. transport changes are mtt accompanied by an increase the ,4('-labeled cells with thymidine. After a repair in microvilli that might indicate an increase in surface period of IK hr. the cells are mixed and irradiated with area: the microvilli arise later in the cell cycle. Since (his 313-nm light, which photolyzcs the BrdUrd-containing stimulated transport yields no net change in cell DNA. The cells arc lyscd on alkaline sucrose density electrolytes, it has no immediately obvious physiologi gradients and the difference in the molecular weights cal significance. However, the enhanced passive K flux used to follow the repair process. Inhibition of repair suggests an enhanced membrane potential, which in KM 1S5 I turn would inciease the electrochemical force on Na* such induction in net occurs. Their argument is based and on any Na*-gradient-coupled transport such as that (in part) on the fact that ceils incubated for long of OMuninoisobulyric acid (AIB). An enhanced uptake pciiods in low (<*£#) concentiaiions of pH) ouabain of AIB in stimulated cells is in fact observed. The for several days and then challenged with high I serum-siunulaiion effect can be mimicked by treating <2X |0~7 M) [3H)ouabain for a few minutes show the celb with valinomycin. a drug which enhances the more cell-associated radioactivity dun controls. We passive K flux; as expected, valinomycin also enhances agree with the data, but fine* the excess ouabain to be AIB uptake in HSWP cells. This argument gives meaning inside the cell (internalization: see below). We have to the repeatedly observed enhanced electrolyte fluxes examine** the induction question by treating cells w in other stimulated systems such as PHA-stimulatcd follows: ,«) short challenge with 2 X 10'7 M ouabain lymphocytes and SV40-transformeU 3T3 eels.1 ~ i (control for total number of binding sites); (*) growth of ceib 48 hr in 10** M [3H|ouabam, K, being 3 X 10 "• M; (c) growth of cells as in b followed by 1. P. Wright M. R. Oumd. aad J. G. KapkM. Exp. CttKrs. challenge as in m. In each case. *H per cell was 79, «7 (1973). 3 2. t. K. Loaf, personal determined as well as H per S'-nudeotidase in the 3. H. Kinlbtif and E Mayhrw. J. Bid. Cktm. 2M. 100 isolated membranes (Table 35). We conclude from this 097S>. and similar experiments that the enhanced ouabain per cell docs not reflect increased surface binding sites: in TURNOVER OF OUABAIN-MNMNC long-term experiments most of the ('H) ouabain is not SITES IN HeLa CELLS found with the membranes. Similarly, growth of the cells m low K* (0 5 toM) slows growth but does not J. S. Cook. Emily Tate Brake. increase ouabain-binding sites per nucleotidase m the andP.C.Wifl* membranes. We thus find no evidence for specific Using I'Hj ouabain both as a ligand to find and count induction on the membranes of K'-depleted cefls. This Na*-K* iransporl enzyme and as a specific inactivator is an important conclusion for what follows. of thai enzyme, we have continued the study of the Cells pulse-labeled with |'H) ouabain at low enough turnover of ouahain-binding sites on the HeLa cell concentrations so that growth is not noticeably in surface. We make extensive use of the Atkinson- hibited, slowly (hours) lose a fraction of the label into Summers sucrose-gradient technique' for isolation of the medium by simple dissociation. Thb dissociation the membranes, which may be prepared in less than I rate as measured by ouabain bound to isolated mem hr. Since the isolation is carried out in the cold, branes iv the same, within experimental error, as the virtually no bound ouabain is lost during this time. The rate measured in whole celb whose protein turnover is preparation contains I -4% cell protein, no detectable slopped by treatment with NaNj and 2-deoxyglucose. glucose-6-phosphatase. less than 2% of the total acid- The latter treatment rapidly and effectively depletes ?hosphatasc. and a very small amount of DNA: 5'- cells of ATP. since 2-deoxyglucose is readily phosphory- nucleotidase is enhanced 30- to 80-fold in specific lated but yields no new ATP via metabolism. Allowing activity. We use the latter as our quantitative marker. for this dissociation, we find thai ouabain binding sites As an explanation of our earlier data, we had are taken into the cells at a rate of ~i5%/hr. The suggested2 that the inactivating effects of ouabain may ('H* ouabain so internalized is found in the non- specifically induce the enhanced synthesis of ouabain- membrane fractions and is bound to a site of molecular binding sites: Brardman el ai.SA have concluded that weight >25.000: i.e.. it is too large to be free ouabain TaMe 35. Short- and htng-lmn |,H| to HcU celts 'H per cell 'll per S'-niKkoiiduc Treatment on isolated membranes (percent of control) (percent of control) (#) 2 x I0"7 M |}H|ouabain. 90 rain (control) 100 100 (A) 2-oay crowih in I0~" M ['HIouabain 60 IS (r) h. then* 146 99 186 but with sedimentation properties of a tysosome. At a independent of whether transport is quiescent, as in rate about equal to the internalization rate, the ouabain glucose-fed confluent cells, or sicnubied by serum or is released within the cytoplasm, presumably by the starvation. Binding is not inhibited by phloridzin. degradation of the binding site. Eventually the drug hexoses. or cytochalasin B. although dsethylsiimeslerol escapes in its original form into the medium. is as effective as phioretin itself. The number uf From the above, a three-compartment kinetic model phloreiins per cell at saturation is 3 X 10*. which far has been constructed for flow of the drug after its exceeds the number of all membrane proteins. Our binding to the cell surface, the compartment* being working hypothesis is that phlorctin (and diethybtil- membrane, cytoplasm, and medium. Given data only besterol) do not interact with the transport molecules for the total cdl-associated drug as a function of lime, a at their active site but specifically mteract with the livid compute! program. CRICF.5 has computed the optimal matrix in which the transport proteins are embedded. Tit for all the kinetic and compartment parameters, Cytochatasin B has a Kt for glucose transport of 0.1 yielding rate constants for dissociation, internalization, u.V in HSWP cells. Its JH derivative binds rapidly at all and escape which agree well with those independently tcmperatuic*. At the A'/ there are or. 1.5 X If/ derived from fractionation and ATP-deptetion studies. mokcuies bound per cell. The diaccute derivative of We conclude that the Na*-K* transport enzyme on cytochalasin B binds very extensively but is inactive the surface of HeLa cells is turning over with a rate against glucose transport and does not affect cell constant of -0.07 hr~*. w about twice per cell cycle, morphology. even at high doses. and that its synthesis a not enhanced or derepressed by In glucose-starved cells, where the glucose transport depleting the cell of its alkali-cation substrate. system is stimulated (see last year's report), the morphological effects of cytochalasin B at I yM are readily demonstrated. Once the morphology is altered, *Foudoctoral itwsliplor supported by subcuntiati 3322 removal of the cylochalasin B does not lead to reversal from the Biology Dmsson. ORNL. to the University of Tennessee, Knoxvnfe. to normal morphology until glucose is restored to Ihc 1. t. H. Atkinson and P. W. Summers. / Mo/. Chem. 246. medium. Presumably the restoration of normal micro 3162(1971). filament structure requires glucose, whereas Ihc other 2. G. L. Vjojjun and J. S. Cook. Aw Salt. Acml. Sri. USA metabolites in Eagle's basal medium are not ulilizable 69,262? 11972). for this reversal: this observation is still under investi i. 1. J. Boardman. J. F. Lamb, and D. McCatt. / rtyjwrf. 229.619(1972). gation. 4. L. Boardman. M. Knelt, i. F. Lamb. J. T. Newton, and J. M. fbtson./ tkyiM. 241. 771 11974). 5. J. r. Chandler. D. K. HOI. and II. O. Spi»ey. C«mp»r. 'Student at Ihc IT Oak Rrfpr Graduate School of Bio Homed. Kej. 5.515 (1972). medical Sciences. GLUCOSE TRANSPORT IN CULTURED PROPERTIES OF AN INHIBITOR OF MOUSE HUMAN CELLS LEUKEMIA VIRUS INFECTION ASSOCIATED WITH THE rr-l GENE D.W.Salter* and J. S.Cook R. W. Tennant. Bonnie Schluter.* F. E. Myer. We have used ndioacirve compounds to investigate Wcn-Kuang Yang, and Arthur Brown* binding of known inhibitors of glucose transport in the diploid human fibroblasi HSWP. (This work was greatly The h'vl locus of mice is the major determinant of impeded by (he finding, since confirmed by the resistance to most naturally occurring leukemia viruses. manufacturer, that commercially available |3 H) phlorid- The two alleles of the gene, h'v-l" and AV-/*. recipro zin and |'H|phlore(in are highly impure products. The cally restrict N- and B-tropic leukemia virus infection in phloridzin was initially 4% pure: further purification by viro and in cell culture. We have reported evidence for a the manufacturer gave a 100% pure contaminant with cellular product, apparently specified by Fv-I gene no detectable phloridzin. The phloretin is 90% ex alleles, which specifically and reciprocally transfers changeable 'H but can be readily purified by lyophili- resistance to mouse leukemia viruses.1 Inhibitors have zaiinn.) Phlorctin hinds In HSWP cells with a lw«- also been obtained from soluble extracts of eml'ryo cell component curve, ihc saturable component having a cultures of several mouse strains, and the inhibition -vas dissuciaiion constant equal to ihc K/ for transport specific for the mouse leukemia virus host range (">,< (10 IS ii\f). The amount bound at saturation is restricted by Ihc appropriate h'r-l allele. Inhibitory 1*7 activity could also be extracted from tissues of adult cDNA was synthesized by detergent-disnipteJ. purified take (liver, muscle, kidney I which reciprocally m- AKR virus using a 'H-rabeied thymidine triphosphate bibiied N- or B-trnptc but not NB-tropic virus, which is and the other three nucleoside triphosphates unlabeled. not natunly sensitive to this gene. Actinomycin D was used to decrease the transenption Direct association of the inhibitor with the Fw-I locus of a double-stranded product. was established by independently determining the The cDNA product is 95% single stranded as assayed segration of tlx alkies m the Fj generation embryos of by hydroxyapatiie chromatography, is approximately an Fr-I** X fr-/** mating and the inhibitory activity 3S in size, and has a specific activity of 4 J X I07 of extracts obtained from each embryo. The products cpm/pg. The cDNA has been hybridized to excess of five separate matings. compt*4ng a total of 47 purified 70S AKR virus RNA, fractionated by embryos, were retcslcd for direct resistance to N- or hydroxyapatiie. and alkaline hydrolyzed to remove the B-tropk virus and the ability of extracts of cultured viral RNA. This virus-specific cDNA is the probe for embryo cells to transfer resistance to either virus. detecting proviral DNA and will be used to assay virus Resistance to either virus designated the homozygous expression in Fr-I permissive and nonpermissivc cells. individuals, and resistance to both viruses designated the heterozygotes. The segregation of the alleles closely *NIH Prcdoctoial Trainee. Department of Microbiology, approximated the expected 1.2:1 ratio, and the mhibi- Unncnity of Ttiwcuee. KnoxviBe 3791*. •ory activity extracted from the cells segregated accord 1. R. W. Tenant. F. E. Mjrcr.and L. McCrath./nr. / Cancer ing to the Fr-I genotype. 14.304(1974). Studies directed toward the mechanism of the Fr-I 2. T. G. Krontim. R_ Soewo. ami B. N. Fn*b. /tor. \atl Acad. Set. VSA. 70. 2549 (1973). restriction and the nature of the inhibitor are in 3. A. S. Htnat. F. Beaner. I.. Cbo. and D. Baltimore. / Vinl. progress. 12,659(1973). 4. M. M. Sveda. B. N. B*Wfe. and R.Soeiro. OH 2. 271 (1974). 'Department of Microbwtop. University of Tcnaraer. Knoxvifle 37916. EARLY EVENTS IN MURINE LEUKEMIA VIRUS I. K. W. Temnni. B. Schhrtcr. W.-K. Yam, and A. Brown. #ror. JVtlt Acad. Sri. USA. 71,4241 -45 (1974). INFECTION OF CULTURED CELLS* J.M.Quartes* L.R. Boone.' D.P. Alison, and R. W. Tennani EFFECT OF Fr-/ GENE ON MURINE LEUKEMIA VIRUS PROVIRAL Murine leukemia viruses show host-range restrictions DNA SYNTHESIS which are determined at least in part by an active ceil function which occurs after attachment and penetration L. R Boone,* C. C. Laveilc. and R. W. Teraiani of the virus into the cell. These mouse viruses may be We are currently investigating the steps in the classified as "ecotrooic" if they will infect other mouse replication cycle of murine leukemia viruses which may celts or as "xenotropic" if they infect only cells of be sensitive to the Fv-l pent restriction. Earlier work species other than mice. Within the ecotropic class, the from this laboratory has indicated that an early event viruses are furtrrr subdivided into "N-lropic." those after infection is probably involved.1 Others have which infect crib of mice like the NIH/Swiss. and reported.2 4 and we also have found, that the ad "B-tropic." those which infect cells like the BALB/c sorption process is not involved in this host range line. restriction. Our aim now is to investigate the tran We are attempting to determine whether these re scription of the infecting viral RNA into proviral DNA strictions involve differences in the kinetics of attach in cells which are permissive or restrictive. The process ment and penetration, in uncrating the virus after of reverse transcription seems a likely step for regu penetration, or in differential virus degradation in lation of virus infection by the Fv-l gene. Our approach permissive and restrictive cells. Our approach has been is to isolate low-molecular-wetghi DNA at early time to determine the fate of labeled virus during the catty points after infection and assay by hybridization for periods of infection of various cell types. Partially unintegraied proviral DNA. Our recent efforts arc purified |,H|uridir*-lar«cied viruses of N type (AKR) concerned with synthesizing and characterizing a Ml- and B type (BALBV3T3) were used. Preliminary work labeled complementary DNA (cDN4) to use as a using labeled xenotropic virus from BALB cells has molecular probe for the detection of viral DNA. The been inconclusive due to lack of a satisfactory source of 188 virus. We have verified the work of other laboratories BALB'3T3 cell system. This test system was chosen which showed the restriction of N and B celb is not a because the celb are sensitive to potential carcinogens (unction of the eel membrane. Attachment rales were and also contain endogenous leukemia viruses which essentially identical on these and several other cell can be activated by chemicab such as halogenated types. There b some indication, however, that the virus pyrimidines and protein inhibitors. may be less tightly bound by one human cell line In work completed to date we have found thai (W138) and may elute more easily from these celb than nitrosation can cause profound changes in the biohfic from mouse celb. This b being further investigated. We acthrity of a parent pesticide. Nitrosocarbaryi. but run have prepared labeled virus preparations of several carbaryi. caused morphological transformation of the different known particle numbers, infertivitv titers, BALB fibroblasts. The transformed celb differed (torn radioactivity, and particfe-to-infeciive-virus ratios. parental control celb by growth to higher saturation These reagents are currently being used to obtain densities, loss of contact inhibition, change in morphol kinetic dat* on the rate of attachment and degradation ogy, and growth in soft agar I Fig. 20). The transformed of virus and the number and specificity of the attach celb formed tumors at the site of injection in normal ment sites of permissive and restrictive celb. newborn and irradiated weanling mice and alhymk nude mice, bu; uniranslormed control cells did not form tumors. Neither carbaryi. nilrosocarbaryl. nor the *Rotarch sfMM&uxcil by rite Vinn Cinccr rtocram •>•" ibr Natimol Oncer Institute *SC1 rVHtdoctoai Fdknr. *NIH ftcdocroral Tranwr. iVparimrni of Mkrubiokiry. Vtunrtity of Temrncr. Knmviitr 3791b. EFFECTS OF SELECTED PESTICIDES AND THEIR NITROSATED DERIVATIVES ON CELL TRANSFORMATION AND RNA TUMOR VIRUS EXHtESSION J. M. Quarks* and R. W. Termani Nitroso compounds are known to have carcinogenic activity in a wide variety of animal species, and several have been shown 10 have mutagenic and transforming activity in ceil-culiure systems. Certain nitroso com pounds, particularly niirosamines. may he important environmental carcinogens, due in part i» their ready formation from nitrite and secondary amines under conditions similar to those found in the mammalian stomach and to their common use throughout the environment. For example. Ekspuru «•/«/.' reported the formation of Y-niirosocarharyl from a common fond additive, sodium nitrite, and the widely used pesticide carbaryi. Many other commonly used pesti cides u.niain nitrogen atoms which can he nitrosaied to form V-miroso compounds which are similar in struc F|g. M. Ml Conwoi. mutrm 'ormrrf IUI JTJ crib. The orfh air cwmacr whiriirrtf.rpitiwJintd m mmtfttrt^ct. mi *>•« ture to known carcinogens. Therefore these compounds «r> CTHW.II im»>. I#> >t*wwBca*wjrMn«B*onne* BAI.l IT) form a class of chemkah which arc potentially hazard crib. Thr cdh have Iml rnnracl M-ifeinn*. air More Wen-Kuang Yang. R. W. Temtant. Bonnie Scfduier.* 'NCI rVmdoctoral Kdttm. J. O. Kiggans* F. E. Myer. and Arthur Brown* I. R. Hnpara and W. Lipjmky. FortJ Cotmrl Tmieal II. «07tl973». We have previously demonstrated thai mouse embryo culture celb which belong to a given Fv-I genotype and HOST-MEDIATED M Y1VO-IS YTTKO art susceptible to an ecoiropic MuLV can be rendered ASSAY FOR CHEMICAL CARCINOGENS partially resistant to the infection of tins virus by treatment with ceB-free extracts made from ceRs of a J. M. Quarles.* Cynthia K. Schenley. different h'v-l genotype which is resbtani to the and R. W. Termani infection.1 we describe our recent results concerning The identification of potential chemicd carcinogens biochemical properties of the active components in the requires rapid and effective screening tests. In rimy ceil extracts. ceN-culiure systems, using morphologic changes in die Our initial studies of the inhibiiory activity were all celb as an indication of carcinogenicity, may not delect performed with embryo culture cefb homogemzed by potential carcinogens which require metabolic activa sunication. Dbruption of the eels by hypotonic treat tion. Dr. J. A. DiPaoio of the National Cancer Institute ment and Dounce homogemzation was also found *o be developed an assay system which provides the necessary effective for the extraction of the Mubiior. Subcellular metabolic activation and retains the advantages of m fractions were obtained by differential cenlrifngaiion of rim> culture systems. In (his assay, pregnant hamsters the cell homogenate. Subsequent tests snowed dot nV are injected with the test chemical: fetuses, which have inhibitor activity was found predominantly in the thus been exposed transplaceniafly 10 the chemical and microsomal fraction and abo m the postnikrosoinai its metabolic products, are removed and prepared as cell mpernaiani. The iniumiioR of the vims infection by cultures. During subsequent pnwage of the fetal ceHs. these swVchntar fractions abo foNowed the f-r-l gene transformed ceus are detected and quauirtalc4 by properties of die ccBs. The presence of inhnwior examining colonies and scoring colony morphology. activity in the posinpcn^omal sspernaiant provided the f/e have ccnduciei approximatety SO assays with 25 ponaMiiy of further separalinn. Gel fdl ration on different chemicals and solvents using this system and Sephadex G-25 indkated that the inhibitor b of high have cor related morphological changes with growth in molecular weight. Chromatography on DEAE-ceMose soft agar. A series of kn.iwn positive chemicals |dtefhyt- and nwmnwoterMoar showed thai the inhibitor activity mfrosanune. urethan. hm/«f#lpyrenr. MirosMneihyt- spread in broad diffused patterns of ehrtinn. The urethanel and negative controls (untreated and solvent activity of the two suUeHuiw fractions also showed treated) were used to standardize ard evaluate the rebtive instability on storage. 190 Tests of the ceil extracts with various hydrotyiic gene at the molecular level, we have miiuHy focused on enzymes demonstrated that the inhibitor activity was |«) reverse transcription and 1*1 integration of provirus sensitive to inactivaiiun by ribomdeasts (but nut by DNA. These two biochemical functions occur at the deoxyribouudease or by proteases)- Together with the early stage of the virus infection, in which FT-I gene subcellular distribution, this observation suggested dial products appear to exert their effect- We describe stune the inhibitor molecules were presumably rutauHcleic experimental results rehied to these studies. acir? and might be isubied by bMchermcai procedures. AKr* murine leukemia virus, an N-tropic virus, was RNAs of Fr-f and Fr-f** embryo culture ceMs were rsobvr-1 from cuhure medium of a high-passage hue of isolated by phenol deprotemizatiun at pH 7.6 and pti AKR mouse embryo culture actively producing the 9.0 and subsequent ethanol precipitation. These RNA virus. RNA materials sard for ihe study were prepared preparations were able to transfer the specific virus by phenol extraction procedures from embryo primary resistance according to the Fr-I gene determinants of culture eds of N1H Swiss mice (carrying Fr-f ancle i their original cell materials, namely. RNA preparations afcd BALB/c mke (carrying Fr-f 'aHclcV. ihe RNA from Fr-/** ceils (or Fr-f eds) can induce increased preparations were previously shown to cuntain Fr-I- resistance to N-tropk lor B-tropk) MuLV infection in gene-specific nuubitory activity in the cefl-cHlure in susceptible mouse-cell cultures. The inhibitory activity fection system. Endogenous reverse transcription activ of the RNA preparation becomes mure stable after ity of the AKR viruses was not inhibited when the phenol extraction, presumably due to removal of total ceNutar RNA preparations were included in the endogenous ribonucteases from the extract. It is re reaction mixture up to OJ mg/ml. Utilization of sistant to heat inactivaiMMi at 37*C for 30 mm. 56'C synthetic template-primer. IrAt^-ldTl*. by the viral for I5 mm. and *0*C for 3 mm. and cm be stored for reverse transcriptase was markedly mhmitt-d by the some lime in a liquid-nitrogen freezer without change in ccuutar RNA: honevcr. both Fr-f and Fr-/' cell RNA activity. RNA preparations with high activity can also showed the same marked irduhitory activity on AKR be isolated from livers of young adult truce, mus viruses. The cehular RNA preparations were fraction providing sources for obtaining a large pool of the ated by sucrose-gradienl sedimentation, and ail RNA mhmitorf s). The mhrbiior acimry can be demonstrated fractions were found to have similar mhmitory effect both in the pH 7.6 and pH 9.0 fractions of RNA on the reverse transcription of irA^-ldTl*. The frac prepared by differential pH extraction with phenol: tions containrtg 20 28S RNA were poided for further quantitative titration suggested that more activity was study. Enzyme kinefic analyses demonsirated that the present in the pH **.0 fraction. Preliminary results Munition is competitive with regard to IrAfe'ldT),. showed that the active component sedimenied ai the Utilization of (rAV,'(rU\, and frA»»-(d*T»w as lem- 20 25S region of the sucrose gradient cenirifugaiinn. pble-prirner by the AKR-virus reverse transcriptase was Abo. preliminary results suggest that the participation also affected by the 20 2*S cellular RNA. but again of a certain vehicle is important for efficient uptake and the inhibition was nonspecific as far as ihe Fr-I gene is •aNzalion of the RNA in ihe medium by ihe culture concerned. cdts- A dnect examination of the possible effect of Fr-I gene on insertion and integration of pro'-irus DNA mio Rcsfafcii unVMM rif mr Vwu* faucet rViwram *4 ifcr host cell gtnunn. depends on ihe feasibdily of isolating Nafiomi Cancer HMiruir. biologically mfcuious DNA from cehs infected with *DrpwrmnH ot jUcfwuvnvnyEV. luwvTvry «r TemiCMCc. N-tropic and B-tropk murine leukemia viruses. Al Kumviur 37911. though mfectinus DNA of Rous sarcoma virus for chick I. It. W. TciMtMif. 0 SCMMCT. Vr.*K. vJNy. jnrf .•%. vfcwww. *nc W«f And Sr VZA 71.4141 «I974>. cets has been isnbied from rat cets transft>rmed Hv ihb *irus. similar results have ycl lo be reported for Ihe EFFECTS Of CELLULAR RNA ON REVERSE MuLV system. Recently, this infection system TRANSCRIPTASE ACTIVITY: ("iransfection**) has been demonstrated in human cells PRELKnJNARVgTUDtES' with DNA isolated from RD-114-virus-mlecled cells, and we have confirmed ihe results. Attempts t<» develop In-Chang Hsu.* wen-Kuang Yang. procedures of iransfeciion in the MuLV system arc ir» R. W. Termant. and Arthur Brown? progress. Previous studies have unhealed that the active ingredi ent in Fr-l-aMtk ccM extract responsible for specific resislance to murine leukemia virus infection is RNA in ftewjnn mnnsnrvd by ibe Vire* Cancer rVn^nNii of fur nature. To study the mechanism of action of the Fr-I NCI. 19! *tta«Wt«nl mm^ttpjUH. CamnnrwiB Jti^mftiam No. same husi-cdi inipbm (N-trupic). and gave improved VA «521*. Inna ihr Ml. efficiency oi endogenous reverse transcription. tDcaamatM «f >hvT.*»*iy>. t'aavcrntv of Tcaamer. KmnvMc 37*14. 'Unearth nuuMfitu' bv thr Viras Cancer fnfjjta at Ifcr OOMMNED USE OF HYMtOCORTISONE NCI. AND INSULIN FOR ntODUCIKN Or »-« *t*»f:«nctonl iamqBiH*. Canrinnututt» Tnmag Gnat No. MURINE LEUKEMIA VIRUS* CA nS29t froa the MCI. trV>u4octnnl iwmajntor vutauma h* subcoatract 3322 Wen-Kuan* Yaig. lb-Chang Hsu.* L. C. Waim. fnm the Bnlnf? Dmvm. OHM. to me Vawcrorr of Beth C. Mumn.t D. W. Fountain* L.G. Hardm.and Den-Mei Yang* Fur bnichrmical studies of RKA iwnor viruses, a large TRYrTOTHAN «NA PRIMES REVERSE quantity of virus maicrial is generally required. Abu. TRANSCRRmON OF AVIAN MYELORLAST05IS vims preparation should be of considerable purity and VIRUS f AMV) 3SS RNA IS VtTKO* unwind degradation for analyses since intact nnclcic- L C. Waters. Reth C. Mutm.* Ti Ho. acid structure b critical. Tins is often difficult in the Wen-Kuang Yang, and L C. Hardin asc of RNA lumnr viruses produced by ibe culture cew*. whirfi are gencuwy of low yield and known to be Several hues of evidence mdWate that 4S RNA degraded rapidly in the culture medium. In our labora molecules initiate (or primer reverse transcription of tory, we have devised a procedure to obtain from tumor virus RNA to DNA m rim: These primer RNAs cultured AKR mouse embryo high-passage eel lines can be dissociated from the viral 70S or 35S RNA only wfHcienl quantity of the virus for the purpose of our at relatively high temperatures. Tins Association coin h» chemical studies. The procedure is briefly described cides with die loss of template-prrrier activity as a- foRows: AKR ecus are transferred from culture measured m riir>> The 70S-associaied 4S RNA has dishes mlo n*Her tubes and grown to ctmfluency m the several properties of iRNA. uMer drum. Hydrocortisone, at the concentration .if The properties of the primer molecules indicate a 10 * -V. is then included in the culture medium. great degree of civrnpieraentanty between them and die Harvest of viruses b abn started using 3- to 4-hr viral 35S RNA. We used lbs property to show that daytime medb and 15- to Ift-hr overnight medium by a specific ceRnbr 4S RNA molecules wil hybridize with dbcwimuous and a continuous sucrose densily gradi AMV 35S RNA m rim:' A major proportion of die 4S ent teninfugaiion. At 5 "* days bier. 100 nrT ml of RNA which hybridizes b iRNA. and of die iRNA. more nmlm f llelm. Eli Lnry) b also included in the medium than ten rs Njeniified as tryptophan |RNA.: The in addition to hydrocortbone. and the virus harvest hybrid formed m rim> between AMV 35S RNA and continues for the next 2 3 weeks. Addition of hydro tryptophan iRNA. but not lysine, methionine, and cortisone m the medium has two effect: f#» increasing other umdeniifkd tRNAs. was as efficient a lemplate- the virus yield two- to threefold and IM preventing de primer tor DN.V synthesb m rim< as -as the native tachment of the confluent cell sheet from the tube wall AMV 70S RNA.2 toed on these results and those Addition of insulin I week later evidently stimulate* obtained by Dr. J. E. Pahlberg and civwotkers at the growth of the edb to ovcrconflueucy. which apparently University of Wisconsa* with the Rous sarcoma trims. enhances the virus-producing function, and a further mukaiinns are that tryptophan iRNA b important in increase of two- t«> fivefold yield iff virtue* b often the nrpbeatjon of avian RNA tumor viruses. obtained. With the use of hydrocortisone, medium We arc currently using the purified components, i.c. ndimit per roller tone can he decreased to 15 ml at 3- viral 35S RNA. reverse transcriptase, and tryptophan to 4-hr intervals and to 30 ml overnight, thus saving iRNA. to study the mechanism of repKcatinn of these medium and also the process of ccninfugarion. In a viruses. typical operation -M 11 ruder tubes and 3-week harvest. we obtained a hit of AKR viruses containing about 10 nut protein. Effect of msuhn b wry much dependent on 'Research jappimcd ti* far Vim Cancer Pforraw) of ibr lime of addition and state of culture ceNs. Our results MCI showed that AKR viruses produced by this procedure 'IWalncioMl wwwuai.T wnsinted by Mtxonrravt 3322 were of cofcsiderahk specific infectious tiler, of the from the hintory Dwwirm. ORNI. in the VtmrnHf of ttimrmtt. KnnxvuV. 192 1. L C. UMOV ». C. Matin. T. Hit. and *.-k. Ya««. synthess m rino. we haw shown that tryptophan mucktm Hop*?* Hex Ctmumm. M. 4X9 97 tl»74>. tRNA isolated from chick cells wiU hybridi/c 2. L. C. Wains. tt. C. MMUM. T. H». aad ».-K. Vaf. IVo, with AMV 35S RNA m rim.. The hybrid, but Wrf. 4ont Sn. VSA. 72.2155 S« l W7S>. neither tryptophan tRNA m>i 35S RNA alone, is a very e ficirni template-primer fur UNA syn rUMHCATION OF CMCK-CELL thesis in rriru.' To show a unique association TRYPTOPHAN ANA BY RffC-5 between tryptophan tRNA and A.MV 35S RNA m the COLUMN CltROMATOGRAPHY* isolated virus panicles would lend strung support to the L C. Waters. Beth C. Mulhn.* Wen-huar^ v r-r notion that tins tRNA is bioloexaUy important as - J. L. Nichol.* and L G. Hardin primer of DNA synthesis at AMV. In the present study we have made two assumptions: Nonacylated tryptophan iRNA ehites early from *f> the potential prime' is a tnologicalh active tRNA. RPC-5. However, tryptopnyl iRNA is >•* of the last and (nl it is thermally dissociated from viral 706 or 35S tRNAs eiuted from ike column. We exploited this RNA at a temperature which B consistent with the heat unique behavior to identify tryptophan tRNA as a inactivation pattern for the native temptate-prnner | 70S major tRNA component which hybridizes to AMV 35S RNA f The -IS RNA within the vniua was isolated as RNA m rim:' By first chromatographing nuttacylated three fractions: |«) Tree 4S RNA" which is not clucKen-livef tRNA followed by rechrumatography >ti associated win the viral 70S RNA: (ftI "60"C 70S the tryptophan tRNA region of the Inst culur.m after associated 45 RNA" that which is dissociated from try ptophylation. we haw purified chick-hver trypto the 70S RNA by brain* to t>0 ( <<-) "t>0 HOC phan tRNA >o homogtneity .* Oligonucleotide finger- 7QS-associated 4S RNA* that which b obtained by prmts of "P-labeled tryptophan tRNA purified by the healmg the 70S ,H 35S RNA obtained at «0 to HOC. method described are identical to those repotted for The RNA sa.>n4es are anunoacylaied with a mixture of ~spot l~ RNA isolated from, and shown to prime ONA trn win-labeled amino acids, the ammoacyl-iRNAs inf synthesis m. Rous sarcoma vans.' By this method, pure lated, and die ammo acids discharged and analyzed with tryptophan iRNA can easily be prepared for use in an amino acid anaty/er. In the manner a qualitative and studying the buxbnmcal mechanisms by which RNA semiquantitative estimate of the tRNAs within a sample tumor viruses replicate. can be made. Relative to that m myeloblasts. AMV "free" 4S RNA was characterized by very low quan 'Research imffutltd by the Vim Cancer ftoEiam of ihr tities of ghitamaie. vahne. and tyrosine tRNAs. Transfer NCI. RNAs accepting all I" amino acids with the exception *fk>sMjoctoraJ inn inaafa* MMUrfca) by wbtimlfKi 3322 ot tyrosine *'K shown to be present in Ihe 70S- tram ihr Bioiocy Dnnva. OR XI. IO the I wwitmy of assuciated 4S RNA which dissociates at oQTC". The Temrucc. KaowiRc. bulk of the TOS-assocuied -IS RNA was dissociated at ^nrmnaKM of Mkrobjobcr a"* hmmmmolatf. Dab Vmt- venMv Unheal Center. Dnfew. North Camhna 2270b. hO C at low ionic strength with a ctmcunHtani conver- 1. I.C. Waters. R. C. MoOta. T. Ho. and * k. Vane. I»w snin ot 70S RNA to 35S RNA. However, under these *»»/. AcmL Si. I S.4 72.2155 Stt1*751. cunditions. more than 75- of the original template- 2. I. C. »al 'tVntaVtctunl awntaploi laaamica1 by sribraMnct 3322 AN / V rTTRO LYIVHQCYTE-PKOIATED from ihc Wtntngr Dvriao*. OU.NL. to thr Vmmamy at CYTOTOXICITY ASSAY IMNC Tcaarncc. fcaoKnur. LABELED ADENOSINE* I. L C. tolas. ». C. Mama. T. Ha. aa* «.-K. Yjaj. AIM. VMI 4 Our study on m riro bioassay for transfer factor actmty has been initiated as a collaborative effort with SELECTIVE ASSOCIATION OF PltOUNE C. David NoveHi's group, who have been concentrating tRNA WITH THE 1S5 RNA OF TIC on separation and purification of transfer factonU) foe AUt MURINE LEUKcMU VIRUS/* VIVO* cci-mednted immunity agamst various human cancers, L. C. Waters. Beth C. Muum.* E. C. BartiiT. hfciudmg osteogenic sarcomas ciaied 4S RNA. which dis pentonealy at 3-week intervals for 3 4 times. Ten days sociates between nO and 80*T. tryptophan, lysme. after the last injection, lymphocytes were obtained argnwne. threonme and or serme. ghitamaie. proline, from peritoneal washings, ptnphtial blood, or the fh/cme. alanme. isnleucme. and leucine tRNAs were spleen of the rats. The lymphocytes, with appropriate repeatedh demonstrated However, with the exception controls, weir used to examine the apptkabibty of «>f proline iRNA. none are present at a level scaler various m rUrv cytotoxicity methods, such as nacro- than \?>. Profane iRNA comprises 50 (NTV of the piate test (to count the reduction of target ceH 1 identified tRNAs m this fraction. By analogy with the number i. lododeoxyundme labeling and release snethod avian tumor viruses and since our prehrnmary result* Imeasurmg release of radsotodine tadioactivity from with Rauscber murine kukenua vims ab» indicate target tumor ceVsl. Unmanme Ldnhng assays (DNA probne iRNA. it is possible thai m murine RNA tumor synthesis and breakdown), and "Cr labeling and reuses, profane iRNA ought be me primer molecule release assay tdetecuRg release of cytoplasmic mole Currently we are attempting to demonstrate a primer cules with * 'Cr from target ceBsl. Three criteria used to fuuctnm for profane tRNA nr nir» assess thr applicabnif> were sunpttcny I for handhng large numbers of sample), sensitivity (for muting with smart eel anaaiiiy of hnmhucytes). and itpiJuWibmiy 'InaKk \mffanrt uv rut Vm (JKO T*-*p*« «f ibr • lot dependable quantitative meamrement). Most of the wri. methods luifuVd one « two but not aX three criteria, tFml*Kiu»l M*«nnMnr «aaa>«ief tf wkoimrji;< till rmm rhr tMngr Hnw*. CIRKI l«» Ikr Immtrmy «4 in the case of the human breast canonfane ALAB. thr Tcunmar. Kim\*in». "Cr nmfaod was found to be smtplest -id gmrraHy 1. L ('. WaMt. U. C . Nanm. T. HO. ml ».-k. \*H- **»» dependable, but tins method has the ihiadvantagrs of Wrt M-ml.*«. ISA 72. »155 54 tl«73i. mti hrmg physnni>pcal and of grvmg a ligh background 2. it. WjKTt. ». (. Mriwa. \. »;. fa*rt. n. t. r. ani (spimianemis release of radwactivrry) upon piohmgrd L. (1. Hardm. INK rcpnn. i. L. C. WJICT*. t*Khrm a¥naftr< •>» ('.»*ff»* . ai arrt» mcvniion of target-cell cultures. Thus, starching for an 194 dtemate method other itun the conventional cytotox Cteditvit mntunrhus. Yigfta smt-srs. I'Sex etmtpmaa. icity methods seemed desirable. We have found that Ijtpmms amnknnu. /brnrrnrrta Ijicama). Mcantr improvement or the iabebng and release method van he nA'ikv. Atttizzig Jiumhym. Ctrrh ttlmfuounmi. ken- achieved by using adenosine, which can be uxor ponied neJm nrtismnw. and hvtrpis gUnJulti*. Seeds from MIIO a large nucleotide pool and which also serves as a these plants were puKeri/ed and extracted with a precursor tor RNA and IX tftataWturji •ncsqptor iffurted by mbcmiltact 3322 no hmding to the lectin gel. showed no binding upon lima Ihc u*>luc> Ditnn. (MM., lo the lunrac; ul rechromaiogfaphy. It) The balding, however, was Tcaarocr. kaw.vrw*. almost totally VTCveraMe. even with the ehition of solution containing o-meihyl-D-manmnafc (specific to PRELIMMARY STUDIES OF MURINE Con Al and A'-acetyl-u-gjiactosainmel specific for SBHi. LEUKEMIA VIRUS SURFACE PROTEINS Thus, the kctin gets prepared by using plant lectins of BY LECTIN AFFINJTY CHROMATOCRAFMY* strong binding affinity were shown to be not suitable for the purpose of uubtmg the glycoproteins of RNA D. W. Fountain* ami Wen-Kuang Yang tumor viruses. Research is in progress to use plant Vital envelope proteins. particularly glycoproteins, kcuns winch show weak and reversible binding prop pby an important role in the iniualun of mfectiun of erties. These include lectins from Lent admmm susceptible crib by RNA tumor viruses. In addition to seeds and /honWhs nnjavn varieties. pfuvnhng ales for the miiai adsorption of vmts particles lo the or! membrane, these envelope compo 'Uneven jpunwn tv Ike OU.NL f'pbmo^ Stank* nents tmwphoiuevauy utenufnMe as spikes or knobs ••n the virion meinbranel are also associated with the 'fbnaWiani in ii%iii»r mppuncd b» lavmairact 3322 type species anil group specificny of virus aniuvnicny. IIMB ihr nwhef Innawn. ORNL. 10 ihc Catrermy ot Tr—uiri. fca»\«aV- Although mnminological approaches have provided I. H. icsht. R. Kan. aad H. D. Kkatt. /. tin. VmaL 14. I much informaiioii on vnal protenis their relative II*72IL abundance, ktcatioa ut the vwnm. and retain* aninxmc 1. I. tofaU jaa f. tbftmam.J. t**. Vint. 14. 103 H972>. properties and cross reaewntses they semam difficult to obtam at any large ouanirty of purified materials by conventional biocbenwcal separation techniques and are LECTIN CONTENT OF FOURTEEN ihws poorly defined Inuchenncaly. VARIETIES OF SOYBEAN* The ahriiry of plant lectms to bmd K> and anjhMinate D.«. Fountain:* M. Margaret Wimams.1 2 RXA tumor viruses' - suggests specific mteraclion* of Ven-Kuang Yang, and D. fc. Foard eiiematy located glycoproteins with IcclMs of drt- lereni Dr. J. V Hie The seeds were n'racted at km pH. and the 47 »yr Affinity huvdrng experiments usmg ike lectin gel jnummnim ndfate precnniable pnttem was Recovered prrparaiious and the '" l-tabeled nm potent prepara for onantHative and taaahtafive analysts of lectin con tions obtained the lonowtug pnhnnnary remits to I tent and acimry A radial nwmnnomffusion assay -»3ig Rejclmn of tnus proiem* and levim gel usually resulied an annsenim produced acjntst the lectm punfied from m hmdws. oi certain percentages ot the ladwacimiy to the l)MVi:7 culnrar of soybean unhealed that ike lectin gel |25 IT. for SMI and abont 5t»; lor although aR iif the extracts tested contained some levtm fun Al. 1*1 I be radsoatirve nm> protein* uflnvfc showed avinity. the seeds of cerlam vanelies nere depleted m 196 lectin content compared with oiheis I relative t« radial daltons reported earlier. SUS-evI dilution and SOS- data produced in this assay m response to standard poly aery lanude gel electrophoresis data suggest that the Cautions of puntied DoS-127 soybean lectin). The na:ivc Wctut is a tetramcr wi:h a subunit molecular lectin contents ranged front 41 j.-g per milligram <>f weight «>l approximately 2.».0U0. This lectin is the protein Itw/rnw "Chippewa") up to 550 ug per major isotectin component of DbH-127 soybean seeds milligram of protein iru/rfrar "Select Brag:"). Lectin and ts readily distinguishable from two minor isokctms activity of the etude extracts assayed by a standardized by m unretarded behavior on DEAL-cellulose at near hemagglutination test gave a ranked list oi soybean neutral pM. The two minor isolectins. although anti- varieties consistent with the ranking obtained Iron, the gemcally and eleclrophorctically idemicai to the Df.At immunof>pcal assay. breakthrough lectin, are released from DtAt-cciluluse Immunoelectrophoresis ot crude lectin extracts using by an elevated salt concenirati<>n and in this respect are DW-I27 soybtanJeciin .mtiserum indicated that an similai to the "major" isotectin previously reported by ajiiigenicallY similar lectin was present m seeds ot' each Lis t-r j/.' It thus seems likely that teeds of of the 14 varieties, and the relative intensity of different soybean varieties nay contain quite different precipitin fomution suggested that this lectin (or isolectins as their major lectin species. isoiectin complex | was responsible for the differences in lectin quantity bsened in the quantitative assays. 'I 197 hybrids with each traction. Similar results weie ob The development of :hese techiuqjes is particularly tained when hybridisation was examined b> single- significant !•> further analysis oi the autogenous im strand specific nuclease IS-11 assays. mune response in that purification of these proteins by !•' establish tuither that each fraction of mbuRit c synthesis ol virion protein appears to occur during this *Rrwjrch xippnricil t* UV Virus 1'iiwct Ft»gt;Mn <.bnj :i7.t| •nay be the first and critical pn>ducl of perturbation of the cellular genome by incorporation of Idl'rd. VIRUS ACTIVATION* J. Y lhleT and P. T. Kenney * RetaMfth wipffctrictl In ibe Virus However, antibodies prepared against total CNBr- 'Research supported by thr Virus Cancer Program of ihr cleaved fragments of the enzyme react only with NCI. subunits of the transferase. These results demonstrate 'Student at the UT-Oak Ridjce Graduate School of Bio that antibodies elicited by peptide fragments or sub medical Sciences. unils of tyrosine aminotransferase can recognize some sequential antigen determinants of the en/yme. Prelimi EFFECT OF VITAMIN B» DEFICIENCY nary results indicate that specific TAT polyribosomes ON THE SYNTHESIS AND DEGRADATION OF can be identified by such antibodies. HEPATIC TYROSINE AMINOTRANSFERASE* Kai-Lin Lee. P. L. Darke, and F. T. Kenney 'Research wpporlcd by the Vrus Cancer Program nf the ll has been suggested that the rate of dissociation of NCI. cefaclor of vitamin B»-dependent enzymes is the 200 rate-limiting step to control the degradation rate ot actinomycin D. It had been previously demonstrated in these enzymes in rnv. In agreement with this hypothe this laboratory that in ceils grown in the presence of sis, the half-life of hepatic serine dehydrase is prolonged low levels of actiiUHtiycin D 10.2 ug/nil). TAT by administration of pyrtduxine in rin> to B»-deficient synthesis is prevented but general protein synthesis is rats. In view of these observations and to substantiate not affected- We have found that the cessation of the role of cot actor in regulation of the enzyme level, degradation was not observed in cells grown in the we have studied the turnover rates of hepatic tyrosine presence of hydrocortisone and low levels of actino aminotransferase in B»-deficient rats. Weanling tats mycin D. thus indicating that the synthesis of TAT and were placed on B, -deficient diets, and control rats v*cre the transient prevention of degradation during its placed on regular Purina chow diets. During depletion induction ate coupled processes of coenzyme (vitamin B» deficiency) the total hepatic Experiments were also performed to find out if there TAT was less than that of normal rat liver: particularly. was any relationship between the amount of tin* the amount of holoertzyme was decreased in B»- needed to attain tuft induction of the enzyme -ind the detkient animals. These findings are in accordance with time at which degradation of the enzyme was pre the idea that the coenzyme is involved in the regulation vented. In order lo do this, cells were fully induced of the transaminase level in riro. Direct analysis of the with hydrocortisone, the hormone was then removed transferase synthesis and degradation by isutopic im for 3 hr. and finally the hormone was added back to the munochemical technique has revealed that the changes cells and the rate of enzyme degradation was measured. in TAT level in B* deficiency are primarily due to the If there was 3 fixed relationship between the lime at decreased rate of enzyme synthesis. The rates of which the hormone was added and the time at which enzyme degradation are the same in B»-deficient rats degradation was stopped, the degradation should have and control rats. These results clearly demonstrate that been shut off from 5 to 8 hr after the last addition of the cofactor. pyridoxal phosphate, does not protect hormone. If there is a relationship between the time at TAT apinst degradation in rmr. It can be envisioned which full enzyme induction is observed and the time at that, in riro. multiple steps are involved in the process which degradation is prevented, then the cessation of of protein degradation. The limiting step for such a degradation should recur within 3 hr after final process may be different from one protein to another. addition of the horrr.one. since the steady state is reached in this time. Normally, steady-state conditions are reached at about c 10 hr after addition of the 'Re-Kirch supported by the Virus Canter Prueram of (be hormone. Since no change in the rate of degradation NO. was observed at any t.me af:er the final addition of STUDIES ON THE DEGRADATION OF hydrocortisone, we have concluded that there is no HEPATIC TYROSINE AMINOTRANSFERASE fixed relationship between the time >f hormone addi DURING INDUCTION BY HYDROCORTISONE* tion and the cessation « f degradation ami. also, there is no relationship between the amount of lime necessary Nicholas Pomat-j.* Kai-Lin Lee. and F. T. Kenney to reach steady-state conditions and the time v. which We have extended our study on the degradation of degradation of the enzyme is prevented. TAT in the 11-35 hepatoma cell line during its induction Finally, an experiment was designed to determine by hydrocortisone. A detailed study of the rate of how long (he hormone he4 lo be present after the degradation of the enzyme by immunochemical tech addition of hydrocortisone to the ceils in order to see niques has revealed that no TAT degradation is ob the prevention of degradation. It has bren shown in this served between S and 8 hr after the addition of laboratory that the addition of progesterone to cells hydrocortisone. After 8 hr. degradation of the enzyme being grown on medium containing hydrocortisone will proceeds at a normal rate. prevent further induction of TAT by the hydrocorti We suggest that the phenomenon observed is caused sone. In this experiment, progesterone was added to by the inability of some component of the cellular cells at various times after the addition of hydrocorti degradative system to respond quickly enough to (he sone, and enzyme degradation was measured between 5 tremendous increase in synthesis of TAT in the pres and 7 hr after the initial addition of the hydrocortisone. ence of hydrocorliionc. To determine whether or not The results demonstrated that hydrocortisone must be this cessation of degradation is coupled lo the synthesis present for at least 3 hr in the ceil medium in order to of the cn/yme. we examined the enzyme degradation observe a cessation of degradation between 5 and 7 hr during hydrocortisone induction in the presence of after addition of the hormone. 201 Thus it is evident that the synthesis of TAT and the ml hormonal induction and die time at which the prevention of its degradation ate coupled processes. The effect is observed. hormone mist be present for at least 3 hr in order to observe this effect, and there is no fixed relationship 'Rocarcfc j»pyni»< hy the Vim Cancer Pragma «f ikc NCI. between the time at which the horauac is added and *r>nl*tctufal •wu^ejior lwyptitui by H*C—wart 3322 the lane at which the effect is observed- Finally, there iwm ike nialip Drraw*. ORNL. u» ntr Uaboraty of is no relationship between the time necessary to reach Teaatiacr. KaoxvuV AUTHOR INDEX CanaVr.fc.L.95 rarfnw.KOMnjBm.35 .D.P..22.I87 CjmcT.li.l-. 41.79 Mary Loo. 106 Cat**. J. *..*.. 176 Afdrey.W.M..24 .CA-.J6 Arnold.«. A.. 52 •an a. 14 Ani.J.R~52 tlvMit.Netwyn !..<•• atfantalL.4R.49.50 . V K-. 154.155.156.157.15*. 15*. 16* .E.G..9I.I92.19J Cote. •!»„ 105 • A.lr •!.!». 19 Cotyw Shane? I*. 154 i V.. 131 Cant. htVfcpm. 177 H. WE. 24.25 Cook. J. S.. 39.49.184.185.186 teford.NancyL.147 Conch. D.B.. 84 k. 163 Cn«jnW.r.<;..3; .J. at. 16 Ciean.D. %..7>.I7S Ci—n» . R. B.. 126.133.134.135.13*. 138.141.142 t. E. C„ 133 t V. 10.11 BKry.Darryi. 171 Dlwri.109.110. Ill .R.D..80 Dntje>.».E..|7* M t .63 Daniel. J. C.. Jr. 33 t. L It. 187 Dardm.KB..Jr..l53 •man. Deborah A.. 162.163 Uarke. r. L. 199 LMdaSu.85.ft7.S9 DM.S.K.,23.49 B.S..95 Brake. EMiy Tate. 39.40.184.185 DMU. A. K.. 20 Brake. I J. 40 Danjherty. J P.. 154.156.159 I. J. C. 66.67.69.71.73 Oaridtua. Konvfha A.. 154.15*. 157 .rbthdaA-.65.84.85 Day. J. ¥/.. 101 •row*. Arthur. 186.189.190 DtMa>-urca.(Ha«nprm. 183 Brown. J. r.. 48 Doherty.D.G..3.4.8l Brown. Peter. 7 Dwnom.J.V. 105.106 IMT. BeNjanm. 56 DMM.W.C..Jr.SO Ban. Frances M.. 56 BarttkC. A.. 156 . J. W.. 25.26.27 Edwards. M. E.. 59 Edter. R. E.. 108 Eastern. J. R.. 45 Cacheiro. Lucia H.. 151 Ekspwv. Ruiidii K.. 63 Cacfcuro. N L A.. 127.130.131.132 FJmhorsi. Barbara J.. 135 Can.KatnerineT.. 114.115.116.117.119 Cater. J. L. 82.103 CawwcR. Kcnd a L». 99 tpm-S.i.. 106 202 :o? fciKkm.lll.3J rto.t A..27.7* fcW«,llMM».»5 Nn.Ti.a2.191 Ifcipx Lata L-. 1*3 F Hawi.C A.. 3* FafcakW.IL.15 lllilil. J. *. »3.9|. 14*. 149.1*4.1*5.14*. 197 Ftocty. !.<;.. 24.19* Htatoje*. f C. 107 Facto*. F.IL. 77. IS3 HA. K-G~ 2B FMtoct.aV.153 H ihci. at. 54.59 rillnin.a-rr.3l JtoCTt.iTwiirj»i..35 r—••!». F. J. at M*.A-*..*5.«3.»4.»5.f*.*7.1».t9 FMKT.W D.44 M**4fca.M*plk-*.35 F«*f*. D F... 5*. 57,194.195 tto.pM1tot.l90.f9l r cmto. O *. 191.194.195.19k IMT. faMto «^ 114.115.117.119. |2S FWK*. A. A- HI »1. ItllM tto*Cltoj-Y«aB,l4a FtMkcif.lUtor.153 ltoto«tt.rtoM> IL. 123.125 r«p ILK..:I:JM I c R*.J.*..I9*.|97.|9» CtoNMt, F. K. 102. M3. 104. 105 Cnm.HMi4iF.l7fc.l77 MMLHIL.I2.I3 Grariaca. FjMbfc~ I W GCMMM. V. *.. 114.115.11*. 117.119.132 I GcwfcltoM L.*4.135 Jatto«nki.SwanKrr..33 rtoit.L.r.n«5. it* Jiinntow. K. Inwt. 14.15. Ifc. 17.90. 12* Condi. T. Caw*/*. **. 73 J*W.D». 109 run*—•• Urn W.. 147 liwtoc.>l.C,l*0.1*2 Confer*. C. F-. 151154.15» Jcao.JK.154 GccB. C H.99 GtA Rhoii F . I08L 101.102 JoMib 19- Mflttt., •>$ CMncwct. R. A.. 1*. 179. ISt Criflto.G.D.S.9.10 AafcU.G-.43 vtjVnRL H. K., pw GtoM.Gcoftoll.123.132 K Kiajaato. Koaiaro. 01. *9 H K«%. FJtotcta M.. 119.120.123 ifa^m.r T.no.in fancy. F. T. 196 197.198.199.200 Kkyw.J.X..27 HcMfRMH. Jawn. 49 T'iipii rr ir tfci.J WSJ KtoM.R.F.62.63.65 HCMWWF D.5.2S N«W.ILf. 170 KirUamW 1.153 KtoH.W.C..I54.l55.l5S H*MCM».G.D.. 153 Hccto.LC. 191.192.193 aajajaaj^ 9> 94™C^W^^^9PPP# ^f^97^ ^w& KrofjnuMLL. 150 HKMHI.F.C..47.4S K«MJ.Hk«*i.S: Mr/o. R. L. 172 L Hccfcrr. L I.. 24.25 HeHd.J W. 171 LaManfJJa. C. M.. 47 HrfcnMN».G.IL.fQ9.110 U*dk-.G C. 169. ;g7 HMMMJCT. K. P.. 54.55 Ler.Kai-Lw.l9S. 199.200 Wnckv Orraiof F.. 62 Lrr.W H.4I.79 Htodi.C.P..S5.86.9l.92 Urf.J.L. 102 204 MC. Ill (TNt* J. P.. 84.87.88 One*, j. A„ 168 Owns. J. &. 138.140 PJL0.CS. 134 i T-. 143 •a. 5.6.7 Parker. C.L. 6 Pjrket.DoruaKtV.28 njrMC. HUM SL. O7. GW ~ N K.I6I It M„ 53.54 S*db«L65 w-U.tS.S6 Eft. 148.149.150.151 .AM CI 76.177 .CM..IS2.ISS ,D.H,m .W.J„IS2 .W.E..42 .Peter. 29 .Peter. 32.33.34.3S .P. R~. 5.6.7 iM.. 92.93.94.9$ r.p..i62 rVfv.IL A~ 90.91.92.9S. 12*. 127.192.193 LJ-I *»2 .R. J. 67.69.71 ,T.W„Jr„|46 .C.C-.27 RD.28 .R.H..34 QmrksJ. M.. Jr.. 168.170.171.187. ISS. IS9 .T J.55.164.165 . 21.22 .€!?*&. 128.129.132 . fcrtC. I J. 191.192. 193 Rafter. J. J.. 172 .S..28 Ratw.RO..SI.S2.l33 bL.S2.64 RJI.W F.34 Mrvptky.J. R.I4 ML. 48.51.51 Myer. F E . 186.189 i D.. 4. S. 26.41.77.78.79.80. SI. 182.183.184 ReynoMi. R. J.. 41 Joyce C. 12.176.177 NcucslKiin.r*l. 176.177.179.181 J C.85.86.87 Nenm. Sitter Mary Pad. 9 .Nrckobs.32.3S Skhol.J. L.I92 RoosSartnraC. 22 Nidwi. Joanne M.. 199 Rwin.l.R..82 Niyop.SK .20.21 RHWI. Ume 8.90.126.127.128.129.130.131.132, Norton. I. Utile. 48 133 Nweii.G ftmd.8.9.10 RMwi.1l'. L..90.119.120.121.123.12S. 126.127. 133.134 Oakberg. E. F.. 143.144 OvcH.T.T. 162.163 SnHer.D W. 186 Owns. Ada L. 35 SauerfreM. LonC. 154.155.158 Onus. D. E.. 35 SdMriey.CyMlMK..I89 205 TywddL R. L 154.156.157 TyndLPttftkaD.. 144 U Iflrir*, SL L140.161.162.172 Vmluwomd. ftca* H. 30 Uad.Mkro.lt. 19.20.24 53 Cswlya M-. 90.123.126.127 25.36.27 . ARSIS E.. 173 r. BL A_ 33.106.107.100.109 . •%- VOW A-. »W MbraF..i34.l35.139 , A. H.. 29 R-E_ 139.141.142 •.l_CI72 .35 LC- 13.191.192.193 .R.S.52 .70.103 DR.. 21.10? Wa.CH-.44.45.46 R.A^I09 «M*«fer.A.J, IS •dicyL.12 Wdrit.&R_l04 .S-SL.4S.49 .MwySL.l65.l66.l67 .136 A.W..7 .J.R..I60. R«6yD.t2 T.C D.4i.56.»2 PC. 40. IS5 .M. P.. 12.13 194.195 r.K. E. 117 . Maty l_ 177 SsHtJov MMS|H A.. 15 T.CI5.16 MlM. NROOCCIN. 13 I.WUW.S2 M«f*«tS..I30.l3l.l32 Wright. EfCffine 0.. 35 P. A.. 42.43.44 .GaiP..9S TJK. Franco M.. 57 Y«moka.LH..37 Tajrlav.HW.. ISI.IS2 YaMj.Dm-ftfa,S.I9l.l93 Tayk*. S. A.. 20 Yan* Wen-Kuan*. 10.56.57.148. ISO. 109.190. TcMtey.ArkcP..24.25 191.192.193.194.195.196 TCMMH. R. W.. 154.16S. 170.171. ISO. IS7. ISS. Ycifcr. MM (MM L-. 59 109.190 Yrfm. J. M. 161.162.171.172.173 TaMtr.J.E.. 17 Toller. J. R.. 4 Triplet i. L.L.56 Twf.T-C.45 uWcs. Henry, 3