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P 1'" t‘ ‘. a"... 5;. u -. ~ "n E} 5; .'~ . 4‘ -an ,.'. '.Lu. ‘4‘ - .- 9.5-4 c" p. g 3:. n1" 7‘" 4". f U! .: V . I up . . ‘ ._ ‘ . ‘ ‘5 a? . ' T .. .3} J m I‘ :G-Ca‘u \J 9 r ‘I: g {is —- v—— 1‘77”; Ti This is to certify that the thesis entitled ISOLATION AND CHARACTERIZATION OF AN APHIDICOLIN-RESISTANT MUTATOR MUTANT OF CHINESE HAMSTER CELLS presented by Philip Kuocherng Liu has been accepted towards fulfillment of the requirements for _ELD.__degee in _G_Qn9_ti_cfi_ a“ J am r P Major professor Date Se tember 22 1981 0-7639 MSU LIBRARIES n RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. ——— ISOLATION AND CHARACTERIZATION OF AN APHIDICOLlN-REIST ANT MUTATOR MUTANT OF CHINESE HAMSI'ER CELLS By Philip Kuochemg Liu A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Genetics Interdepartmental Program 1981 © Capyright by Philip Kuocherng Liu 1981 ii ABSTRACT ISOLATION AND CHARACTERIZATION OF AN APHIDICOLIN-RESISTANT MUTATOR MUTANT OF CHINESE HAMSTER CELLS By Philip Kuocherng Liu Studies in T4 bacteriophage, prokaryotes and lower eukaryotes have demon- strated that DNA polymerase is involved in spontaneous and induced mutagenesis. However, due to the lack of suitable polymerase mutants, the role of the DNA replicative enzyme, polymerase a, in mutagenesis is unknown in mammalian somatic cells. A direct selection method using aphidicolin, a specific polymerase a inhibitor, has been used to isolate aphidicolin-resistant (aphr) mutants (Chang :31 £11., 1981, Somatic Cell Genet. 7:235-253) at a frequency of 5 per 107 clonable cells from a population of Chinese hamster V79 cells previously treated with bromodeoxy- uridine/black light—ultraviolet light (UV). One of these aphr mutants, i.e., aphr-ll, exhibits pleiotropic effects such as slow growth, thymidine (TdR) auxotrophy, a high sensitivity to UV, cytidine, TdR, deoxy-guanosine or -adenosine and a high frequency of site-specific bromodeoxyuridine-dependent chromosomal aberrations. Revertants which retain part of the mutant characteristics have been isolated. Further characterization of aphr-ll mutants is reported here. Philip Kuocherng Liu Subclones of aphr-ll mutant used in the present studies were aphr-ll-Z, aphr-ll- R2, aphr-ll-RPQ and ”hr-44295. Aphr-ll-Z cell line had similar pleiotrophic phenotypes as the aphr-ll cells. Of the revertants isolated, aphr-ll-RZ is a fast growing TdR auxotrOph and has similar deoxycytidine triphosphate (dCTP) levels as aphr-lr-Z cells; aphr-ll-RPAt and -RP5 cells are TdR prototrOphs. The mutagen- sensitivity was also determined in aphr-u-Z, aphr-li-RZ and V79 cells. As compared to the V79 cells, aphr-ll-Z was sensitive to UV, N-acetoxy-Z-acetylaminofluorene (NAcAAF) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), but not sensitive to X-ray and dimethyl sulfate (DMS), and aphr-ll-RZ was sensitive to NACAAF and MNNG, but not to UV, X-ray and DMS. The UV-induced mutability of aphr-ll-Z, aphr-ll-RZ and V79 cells was deter- mined in 3 genetic loci measuring forward mutations from sensitive to resistant to ouabain (oua), diphtheria toxin (DT) and 6-thioguanine (6T6). The results showed that the mutant was hypermutable for ouar and DTr mutations compared to V79 cells at the same UV dose or at the same survival level. The mutant exhibits a delayed expression of maximal frequencies of induced 6TGr mutants. .When maximal frequencies were compared at the same UV dose, the mutant also had higher 6TGr mutation frequencies. The revertant was similar to the V79 in UV sensitivity and mutability. The ability to repair UV damage, as measured by incorporation of 3H-TdR, in aphr-ll-Z, aphr-ll-RZ, aphr-Q-RPQ and the V79 cells was the same. Furthermore, the V79 cells showed liquid holding recoveries as measured by UV survival and induced mutation frequencies at ouaIr and DTr mutation loci, but aphr-ll-Z cells did not. Spontaneous mutation rates were also determined in aphr-ll-Z, aphr-lr-RZ, aphr-ll-RPll and -RP5 and V79 cells. The spontaneous mutation rates at 3 genetic markers determined by fluctuation analysis were l-M x 10'8 per cell per division in the V79 and aphr-ll-RZ cells, whereas those in the UV-sensitive aphr-ll variants were Philip Kuocherng Liu 5 to 30 times higher. Similar results were obtained in DTr and 6T6r loci using a modified Newcomb's multiple replating technique. It appears that the mutator activities of UV-sensitive aphr-ll variants are not locus specific and cannot be explained by TdR auxotrophic phenotype nor by dCTP pools. A defective enzymatic function in these mutator mutants that rendered DNA replication or repair "error-prone" is suggested. To my mother, S. Y. Chao and my dear friend, Dr. C. M. [(00 iii ACKNOWLEDGMENTS For their invaluable guidance, encouragement, support and friendship through- out the course of this study and my graduate training, I wish to express my appreciation to Dr. Chia-cheng Chang and Dr. James E. Trosko. l have benefited greatly from the perception and persistence shown by each of them. I thank Dr. Chang for providing me superior technical and intellectual training. I thank Dr. Trosko for his generous theoretical discussions and scientific freedom to pursue my ideas. I also thank the other members of my graduate committee, Dr. Veronica M. Maher and Dr. Loren Snyder for the interest and time that they contributed to this project and my education. I am deeply grateful for their advice and friendship. Special thanks to my colleagues and friends: Drs. Gen Tsushimoto, Peggy Wade, Ron Wilson, Thomas Glover, Larry Yotti, Roger Schultz, Stephen Warren and Andy Medcalf, as well as Ms. Judy Funston, Carolyn Hunt, Carol Lassila, Betty Dawson, Beth O'Malley, Gina Lu, Joyce Carter, Clara Rubinstein, Mr. Gary Ferenchick, and to Ms. Judy Copeman for her excellent typing assistance; last, but not least, to Ms. R. Severance who reviewed this manuscript and to Ms. Lily Chen for her assistance in computer programing. iv TA BLE OF CONTENTS LISTOFTABLES............. LISTOFFIGURES. . . . . . . . . . INTRODUCTION . LITERATURE REVIEW . . . . . . Biological Consequences of DNA Damage and Mutation. Analysis of Gene Mutation . . . . . . . . l. 2. CellsI_n_Vitro . . Biochemical Analysis of Gene Mutation Induced Mutagenesis in Mammalian Cells . . . l. 2. (by-cw Mechanism of Actions of Various Mutagens Repair Pathways . . . . . . . Mutations Due to an Unrepaired DNA Damage Mutations Due to a Misrepaired DNA Damage ADP-ribosylation and DNA Repair. Mutation Fixation . . Spontaneous Mutagenesis . . . 0 >11?er— 6. Isolation and Characterization of Mutagen-sensitive Mutants in Base-mispairing Hypothesis . . . . Base Slippage-realignment Hypothesis . Replicative Enzyme-dependent Hypothesis Post-replication Methylation . . Fidelity of Replication . . . . . A. Polymerase Base-selectivities. . Quantitative Mutation Assays in Mammalian Somatic B. Base Analyogues and Deoxyribonucleotide Pools. C. Uracil- DNA Glycosylase . . . . Mutator Activities Associated with Unknown Mechanisms MammalianCells. . . . . . . . . Aphidicolin-resistant Mutants . . l. 2. Use of Aphidicolin to Identify Eukaryotic Replication Aphidicolin: Mode of Action . . and/or Repair Enzyme(s) . . . . . ConcludingRemarks. . . . . . . . . Page viii 10 II 11 Ill 15 I7 19 20 20 21 23 24 26 26 28 29 30 32 33 33 35 37 MATERIALS AND METHODS. . . . . CellStrain . . . . . . Culture Medium . . . Culture Vessels and Incubation Conditions Cell Growth Rates . . . . . . Cell and Colony Counts . . . . Chemicals . Cytotoxicity and Cloning Efficiency Determinations Mutant Isolation. Deoxyribonucleoside Triphosphate Measurements Unsd'ieduled DNA Synthesis . . Mutation Induction . . . . I. Mutagenesis with Ultraviolet Light Radiation. 2. Mutagenesis with X-ray Radiation . Mutation Assays. . . . . . . Spontaneous Mutation Rate Determinations . l. Fluctuation Analysis . . 2. Multiple Replating Technique Mutation Frequency and Mutation Rate Calculations l. Mutation Frequencies . 2. Mutation Rates . . . . A. Fluctuation Analysis B. Multiple Replating Technique . RESULTS . . . . . Isolation of UV-sensitive Aphidicolin-resistant Mutant and Its Revertants . Growth Rates . . . Deoxyribonucleoside Triphosphate Pool Measurements . NutritionalRequirements . . Mutagen Sensitivities . . . . Effects of Benzamide and Caffeine on Survival of DNA Damage Cytotoxicities of Chemicals Used in Mutation Assays Ultraviolet Light Induced Mutation . l. Ouabain-resistant Mutations 2 Diphtheria Toxin-resistant Mutations . 3. 6-Thioguanine-resistant Mutations . . 4 Effect of Thymidine on Mutation Expression . X-ray Induced Mutation. . . . . Repair Capabilities Measured by Liquid Holding Recoveries and Unscheduled DNA Syntheses 1. Liquid Holding Recoveries by Conditioned Medium 2. Unsdieduled DNA Synthesis . vi 0 Spontaneous Mutation Rate Determinations . l. Fluctuation Analyses. . . 2. Multiple Replatings . DISCUSSION . . . . . . Induction of Aphidicolin-resistant Mutants . . Characterization of Aphidicolin-resistant Mutants . . Mutagen-sensitivity and Its Modification by Various Chemicals Mutability of the UV- sensitive Aphidicolin-resistant Mutants . SUM MARY APPENDICES APPENDIX A. DEOXYRIBONUCLEOSIDE TRIPHOSPHATE EXTRACTIONS AND MEASURMENTS Extractims O O O O O O O O O O O O O O O 0 Measurements 1. Reaction Reagents . . a. dATP and dTTP Assays. b. dGTP and dCTP Assays. 2. Escherichia coli DNA- polymerase I. 3. Assay Procedures. DNA Measurements . APPENDIX B. EXPRESSIONS OF OUA’, DTr AND 6mr MUTANTS . APPENDIX c. A HYPOTHESIS ON COMPARISONS OF SPONTANEOUS MUTATION RATES OBTAINED FROM Po ESTIMATIONS . . APPENDIX D. EXPERIMENTAL LIMITS OF THE FLUCTUATION ANALYSIS USING Po ESTIMATIONS . LIST OF REFE RENCES vii Page 98 98 102 108 108 108 113 117 125 127 127 128 128 128 129 129 129 130 131 138 1‘13 154 Table 10. 11. 12. 13. 14. 15. LIST OF TA BLES Characteristics of Mutation Assays at Ouar, DTr and 6TGr Loci . In Vitro Selection of Various Mammalian Somatic Cells and _ Forward Spontaneous Mutation Rates . . . . . Deoxyribonucleoside Triphosphate Pools in Aphr-mutants andV79Cells............. Relative Colony-forming Abilities of Aphr-mutants and V79 Cells in Medium Supplemented with 596 Dialyzed F CS and Various Deoxyribonucleosides at 37 C . . . . . . . . Relative Colony-forming Abilities of Aphr -mutants and V79 Cells in Mgdium Supplemented with Various Deoxyribonucleosides at 37and3l1C.............. Effects of 12-0- tetradecanoyl- 13- acetate (TPA), rBenzamide, or Caffeine on the Plating Efficiency (96) of Aphr -mutants and Wild Type V79 Cells . . . . . . . . Effects of Benzamide (2 mM) on the Relative Colony- forming Ability of Mutagen-treated Aphr -mutants and V79 Cells . p . . Effects of Caffeine on UV-irradiated Chinese Hamster Cells . Cytotoxicities of 6- Thioguanine, Ouabain and Diphtheria Toxin to Aph -mutants and V79 Cells In Media Supplemented with 5% FCS with or without 2 11M Thymidine . . . . . Stabilities of Spontaneous Oua', 6mr and DTr Mutants . . Recoveries of Ouabain-, 6-thioguanine- and Diphtheria Toxin- resistant Cells In Drug-sensitive Aph -mutants and V79 Cells . X-ray-induced Mutation Frequencies in Aphr -mutants and V79 Cells . Liquid Holding Recoveries (96 Survival) by Conditioned Medium in Aph -mutants and V79 Cells . . . . . . . . . Fluctuation Analysis for V79, Aphr-mutants at Ouar Locus. Fluctuation Analysis for V79, Aphr-mutants at DTr Locus . . . . viii Page 58 6O 61 69 70 72 73 75 76 91 92 99 100 Table Page 16. Fluctuation Analysis for V79, Aphr-mutants at 6TGr Locus . . . 101 I7. Spontaneous Mutation Rate Determinations (SMR-6TGr) Using Multiple Replating Technique . . . . . . . . . . . 104 18. Characterizations of Aphr-mutants and V79 Cells at 37°C . . . . 109 19. Spontaneous Mutation Rates (6TGr) in Chinese Hamster V79 Cells . 139 20. The Relative Mutation Rate in Aphr-mutants and V79 Cells . . . 1111 ix LIST OF FIGURES Figure 1. Colony-forming Abilities of Aphr-mutants and wild type V79 Cells in the Presence of Various Concentrations of Aphidicolin . . . . . . . . . . 2. Growth Rates of Aph r-4- 2 and the Wild Type V79 Cells In Growth Medium Supplemented with 5% FCS. . . . . . 3A. Survival Curves of UV- irradiated Aphr 41- 2, Aph r-li- R2 and Wild Type Chinese Hamster V79 Cells. . . . . 33. Survival Curves of UV-irradiated Aphr-4-2, mania-Rm and Wild Type V79 Cells in Medium with or without TdR . . ‘1. Survival Curves of UV-, X-ray-irradiated Aphr-mutants and Wild Type V79 Cells . 5. Survival Curves of DMS-, MNNG-, and NACAAF-treated Aphr- mutants and Wild Type V79 Cells . . . . . . . 6. Dose Responser of UV-induFed Ouar Mutation Frequencies in Apn 4+, Aph r-4- 2, Aph 41- R2 and V79 Cells . 7. Expressions of UV- induced Ouar Mutants in Aph r-4- 2 andV79Cells. . . . . . . . . 8. Expressions of UV- induced 011ar Mutants in Aph r-#- 2, Aph r-ll- R2 and V79 Cells. . . . . . . 9. UV-induced Ouar Mutant Frequencies of Aphr -t1, Aph r-#- 2, Aphr 41- R2 and V79 Cells as a Function of Cytotoxicities of UVDamage............... lO. Dose Response of rUV-induced DTr Mutation Frequencies in Aph 41-2, Aph -4-R2 and V79 Cells . . . . . . . . 11. Expression of UV-induced DTr Mutants in Aphr-ti-Z and v79 Cells. 12. UV-induced DTr Mutant Frequencies of Aphr-ll-Z, Aphr-‘l-RZ and V79 Cells as a Function of Cytotoxocities of UV Damage . . 13. Expressions of UV-induced 6TGr Mutants in Aphr-II-Z andV79Cells............... 55 57 64 65 66 67 77 78 79 81 82 83 85 Page Figure Page Ill. 15. 16. 17. 18. 19. 20. 21. 22. Expressions of UV-induced 6TGr Mutants in Aph r-4- 2 and V79Cells................86 Dose Response of UV-induced 6TGr Mutation Frequencies in Aph 4‘- 2 and V79 C8115 o o o o o o o o o o o o 88 UV- induced 6TGr Mutant Frequencies of Aph r-lI, Aphr 41- 2, Aphr 41- R2 and V79 Cells as a Function of Cytotoxicities ofUVDamage..............89 Survival Curves of UV- irradiated Aph r-4- 2, Aph r-l+- R2, Aphr -ll- RP5 and V79 Cells with or without Liquid Holding by Conditioned Medium for 12 Hours . . . . . 911 Mutatiorn Frequencies for Aph r-4- 2 and Wild Type V79 Cells at OuaorDTLocus... 96 UV-induced Unscheduled DNA Synthgsis as Meas red by 3H-TdR Incorporations for Aphr -‘l- 2, Aph 41- R2, Aph Ji- RP’4 and V79 Cells at 1.5 or 3.0 Hours after UV Irradiation . . . . 97 Spontaneous Mutation Rate Determinations for Aphr JI— 2, Aph r-‘l- R2 and V79 Cells at 6TGr Locus by Multiple Replating Technique................103 Spontaneous Mutation Rate Determinations for Aph r-ll- 2 and V79 CellsatDTrLocus.............106 PyrimidineMetabolisminMammalianCells . . . . . . . Ill xi INTRODUCTION The integrity of the genome requires a faithfulreplication and maintenance of deoxyribonucleic acids (DNA) in an organism. Mutations occur when the base sequences of DNA are altered or deleted. Mutations in somatic cells of the mammals have now been implicated in disease states, such as the carcinogenic (22,54,269,270), atherosclerotic (13) and aging processes (31,53,79,120,156,2#3,260). Recently, an increasing amount of attention has been focused on the toxicologic and mutagenic properties of chemicals present in our environment. Two observations have generated these interests. The first observation was the discovery that mutagenic compounds exist in our environment and most mutagens are carcinogenic in the mammals, and the second one was the awareness that environmental factors (epigenetic factors) are involved in the expression of genetic information during developmental processes (270,275). Thus, an understanding of mutagenesis and gene expression becomes essential for understanding the origin of chronic diseases. Advances in mutation research during the past two decades have revealed two major mechanisms of mutagenesis: DNA misrepair during repair synthesis and directly induced base mispairing during DNA replication (6(1-67). Mutants sensitive to mutagens or deficient in DNA repair are especially useful for the analysis of the mechanism of mutagenesis. Studies in bacterial mutants have provided information about the complexity of mutagenic pathways including evidence for the existence of both constitutive and inducible "error-free" and "error-prone" DNA repair enzymes (111,296), but evidence for these pathways remains to be ascertained in the mammalian systems. 2 Because of the lack of useful mutants in mammalian cells, most of the studies are limited to mutant cells derived from some human syndromes found or suspected to be defective in DNA repair (5,49,86,ll7,160,202,2l6,217,232,286,287). Several techniques have been reported recently whereby mutagen-sensitive or repair- defective mutants of mammalian cells in culture have been induced, isolated and partially characterized (34,233-235,264,265). Similar to prokaryotic systems, three types of repair mechanisms have been reported to exist in mammalian cells: i.e., excision, photoreactivation and post-replication (or bypass replication) repair path- ways (112). Studies with xeroderma pigmentosum and other normal or repair deficient mammalian cells have generally concluded that excision repair is an error- free process (167,186,234). Unlike bacterial cells, convincing evidence for the existence of an inducible error-prone repair mechanism in mammalian cells has not been demonstrated (37). Furthermore, the existence of a discrete post-replication repair system in mammalian cells has been questioned (50,51). However, in spite of the controversy on the mechanism of DNA repair pathways, some of those lesions seem to be substrates for mutation fixation. , It has been postulated that excision repair can be rendered error-prone if the deoxyribonucleoside triphosphates, i.e., DNA precursors, are not balanced (123). The effect of exogenous pyrimidines on induced mutagenesis could be attributed to enhanced base-mispairing (176) and/or inhibition of poly(ADP-ribose) polymerase which has been shown to be implicated in DNA repair process (70,71,237). The perturbation of nucleotide pools may also mediate spontaneous mutagenesis. Muta- tions induced by BrdU or MNNG have been shown to be modifiable by the presence of exogenous TdR or cytidine (61,207,208). Evidence suggests that bromodeoxy- uridine (BrdU) mutagenesis is determined by the concentration of BrdU to which the cells are exposed instead of by the amount of bromouracil substitutions (140,257). 3 Genetic control of spontaneous mutator activity has been reviewed in pro- karyotes (56,64-67,lll,303) and eukaryotes (115,282,283). The mutator activity of these genetic loci may be mediated through a diversified function such as altered DNA precursor pools (175,288), defective DNA polymerase (56,67,124,240,246,247), a defective activity in post-replication methylation' of DNA (90,91,171), or uracil- DNA glycosylase (69), so that errors of base mispairing are not recognized and effectively removed. Theoretically, misreplication might occur under three condi- tions: 3) the presence of a defective DNA polymerase; b) the presence of base analogues or base modification; and c) the imbalance of deoxyribonucleotide pools. The involvement of altered DNA polymerase and DNA precursor pools in the mutagenic process in mammalian cells was not demonstrated until recent years. In Chinese hamster cells, a high exogenous concentration of thymidine (TdR) is both toxic and mutagenic (23,257). On the other hand, Brennard and Fox (25) reported that an excess of TdR is toxic but not mutagenic to Chinese hamster cells. Evidence provided by Meuth e_t_ 31., (175) and Weinberg _e_t E" (288) suggests that an elevated dCTP level in the mammalian rodent cells may be associated with a spontaneous mutator activity. Although an elevated spontaneous mutation rate has been reported in cells derived from patients with Bloom's syndrome (97,282,283), the mechanism for this activity is not known. Aphidicolin, a tetracylic diterpenoid antibiotic obtained from a species of fungi, is a specific inhibitor of polymerase 01. The inhibition on purified a- polymerase is competitive with dCTP (106,125,193,l94). Studies in human cells indicate that aphidicolin inhibits repair replication (16,47,110) or removal of pyrimidine dimers in ultraviolet light irradiated cells (248), although conflicting evidence has also been reported (89,205,236). The drug appears to be useful for the selection of mutants that have altered DNA polymerase 01 (190,259) or nucleotide 1, pools (7,8,10,40,226). Both types of mutations are conceivably able to influence the fidelity and accuracy of DNA replication and repair. Recently, aphidicolin-resistant mutants have been isolated from several organisms (7,8,10,40,190,226,259). The mutants were found to have an elevated level of dATP (7) or altered DNA polymerase 01 activity (190,259). But none of them has been shown to be mutagen sensitive. In our laboratory, an UV sensitive aphidicolin-resistant mutant has been isolated (40). The mutant also possesses several other interesting phenotypes such as thymidine auxotrOphy, cytidine sensi- tivity and site specific BrdU-dependent chromosomal abberrations. The present study was undertaken to further characterize this mutant, especially to answer the following: a) is the induced mutability in this mutant different from that of V79 cells? D) does this mutant possess mutator activity? c) what are the phenotypes of its revertants? and d) what might be the basis for the phenotypic changes of this mutant? LITERATURE REVIEW Biological Consequences of DNA Damage and Mutation The observable biological consequences of DNA damage depend not only on the types of lesions encountered by the cell but also on the location of the lesions in relation to each other or to the replication fork. The lesions in eukaryotes can be produced in non-transcribable regions such as introns that affect transcription of DNA to RNA, RNA processing, or nothing at all (silent mutation). The lesions may produce disruption to replication if they occur near replicons and cause the eventual generation of a change in the complementary DNA sequence. They can be located in the DNA strand near another lesion, such as pyrimidine dimers in the opposite strand. If these two lesions are repaired simultaneously, the gaps generated may overlap each other, producing a lethal or mutagenic double-strand break. Clearly, simply identifying and quantifying lesions will not be sufficient to predict the biological consequences with certainty. Biologically, this alteration in DNA may result in cell death or gene mutation. The mutated gene may or may not show any biological effect. Some unexpressed mutations can be converted by some chemicals, such as tumor promoters, and become expressed (146,153,184,270,275). The reverse process can also be achieved by certain chemicals, such as antipromoters (141,270). The accumulated damage to DNA caused by mutagens is thought by some investigators to be a major contribu- tion to most of the chronic diseases found in technological societies, such as cancer (144,211,269-271), teratogenic events (178,230), atherosclerosis and heart disease (13), neurological disorders (3) and aging (31,79,120,211,260). 6 Analysis of Gene Mutation 1. Quantitative Mutation Assays in Mammalian Somatic Cells In Vitro The quantitative study of experimental mutagenesis in mammalian cells E! m was first reported independently in 1968 by three laboratories, all using Chinese hamster cells (42,138,238). These and subsequent developments in experi- mental mutagenesis, cell hybridization and intercellular gene transfer using in X1152 cell culture provide not only a simple method to study genetic control of muta- genesis and gene expression in human and mammalian cells, but also a new bioassay for environmental mutagens and carcinogens. The development of mutation assay systems requires that genetic markers for mutant selection be available. The selective systems in somatic cell genetics developed in the past have been thoroughly reviewed by Chu and Powell (46) and others (4). Most of the genetic markers used in mutation assays are drug resistant markers, such as resistance to ouabain (Na+/K+-ATPase), 6-thioguanine or 8- azaguanine (HGPRT), diphtheria toxin (EF-Z), a—amanitin (RNA polymerase), 2,6- diaminopurine or 8-azaadenine (APRT), BrdU (TK), toyocamyCin or tubercidin (adenosine kinase), aminopterin or methotrexate (dihydrofolate reductase), tricho- derm (60$ ribosome subunits), emetine (40$ ribosome subunits), thialysine (asn-tRNA synthetase), 5-f1uorotryptophan (tryptophan transport system), 3H-amino acid (affinity of aa—tRNA synthetase for amino acid), 2-deoxygalactose (galactokinase activity) and cycloleucine (methionine adenosyl transferase) [see Ref. 155 for review]. In Chinese hamster cells the most often used systems are 6TGr, ouar and DTr mutation loci (Tables I a 2). Mutagenesis at these loci is different in terms of the nature of mutation(s) (dominant or recessive), location of the gene(s) (autosomal or X-linked), mutant expression time, the cell-density effect, concentration and duration of the selective Ammry c_304 soc» nauamu< .mcommuse Amem.aem .oo_eoau .Nem.em~.mm~ aao_eo> oeo opu_mcou1__oo >3 pounce. 0A oc_cm:mo_zutm o>_mmooom uoxc_41x page: Aam~.ufi_ .R~_.me_.ae_.mm .Rm.~m.mm.mmv .Ntum mo co_um> t_uomc_ uuoCCm >mE _o>o_ omutx .>: Ahoy c_xou «Incuumu toc_coo< >9 nausea. o_1: m_umzuza_o acmc_EouoU .mEOm0u:< co_ummco_m Amm.nm.——v .mcommuae acaumuanOH um_;mosmum new ucm :o_um_nmc QE< u__o>u mc_~_co_ .chuOEOLQ >n nooan:_ uzosah no: "mean .E:_coE Imuas uc_oa oeau_au one .6222 .mzu c_ :0. +x .>n nausea. spin c_mnm:o acmc_Eou0u .mEOm0ua< ommaha >u_u_m_uoam Am>mov acom< mmoco>_mmouo¢ comDDOOS vauu0mm< commas: oe_h o>_uoo_om oucmc_eoo oEOmOEOLEQ mace; :o_mmOLOXm co_mm0caxm co_umu:z .me_uao _u04 coho ocm uho .cmzo um m>mmm< co_um»:z mo mu_um_c0uumcmzu .— o.nm# H..naac3 .>3tuzm_3 .< Co uum_a .3_3 .ACVHNIo_xN Rammuv _am .53: xum_m\:ncm c_ v 3:6 .622: .mzm ooaouoauoue_ .E 36.5 x» .¢< Co O3mo_mcm mum mucumm m3» Ame—V mic—x: oxu .mzm on“ .czocxc: .u .>o» x< .co_um_mcmuu mc_u3u A~e_._e_ eo_uooeo_o .ee_.mmv m-o_xm-_ ozu .mzm e_oau na_a_ae_ .t ezu we: Aa_no5u o_5av m-e_xm-m AmR>v 3:6 .n_muaae>n c_0u0ca 5v .3xu Amm_.mm_v e-o_xm 3 oaaoz .u no ouo_a A_m~v qua—x: mmo .53: c_ u_o_w_uoam acomm any we c\o mucom< m3004 m m3oocMucoam m__ou commu3: :uOOmum .mLDOD m ~o>_uuo_om —u_uocuu maumz co_umu3z m3oocmucoam vumzu0u ucm n__ou u_um50m cm__65562 mao_um> mo co_uuo_om can.) 14. .N o_nm5 .Amm—v c_304 EOL$ UUHQQU< om .co>_m m_ “CV >oc03vocm OOO3oc_ .o_nm__m>m mum name 0: can: .co_umuocom Ema __oo can mm co>_m m_ mumc co_umu35 maoocmucoam .m .u_o_a3ocm _ H cw .o_o_a_v “cw .m__ou _m__ocu_ao cm53; u_qm .53: .m__oo >um>o Emum5m; "ozu .m__oo mc3_ caum5mc omoc_cu "4:9 .mumm_n0un_w :m53c ”.0ca_u .53: .m .ucm__ uo_o_>muu_3 ">2 .oc_e_cm3mOmOLu_ctzIOLu_51.21_>:uoetz "ozz: .0umc0m_3mocmcu05 _>zuo “mzm .m .oc_coum u< .o:_u_5>cu ”5 .oc_m0coum ux< .oc_cm3m "u .3 oucmc_EOuoO ”no .mmoco>_mmou0c “c .m .oc_coumm~mtm "<~xoou050un "seem mc_u_uuoa3u "map mc_u>56oo>ou ”>05 moc_u050 "hzm mc_x0u mmoc_m3uom .m uhnu "x5 .ommc_x o:_m0cooo ux< .u_c3n3m 050m0n_c we: "we: .Ntcouumm co_umwco_o "Ntuw .Ommmh<153_mmuoa153_o0m «ommah< .Ommcowmcmuu05am02a oc_cm3m o:_;u:mxon>z ”page: .— .~ 0.5m» o» menacuooa [\3 ran 10 agents, and medium composition of competitive analogues. A brief summary of the comparison of ouar, DTr, and 6TGr mutation assay systems is presented in Table l. 2. Biochemical Analysis of Gene Mutation In addition to the biological assay systems mentioned above, a viral probe with an amber mutation can be used to study, quantitatively, the reversion of this mutation to wild type by infection of viruses to host cells. An excessive reactivation of viruses, as indicated by viral survival or cell death, is a result of changes in the amber mutation code. Deoxyribonucleic acid sequencing of this amber code, biochemically, would reveal the nature of mutation, such as transition, transversion, frameshift, deletion or duplication of base-pairs in the amber triplet code. With the advances in the restriction endonuclease enzymology, one can use viral probes without the amber mutation, rather, the profile-changes in restriction endonuclease sensitive-sites of the surviving virus after viral infection of host cells are studied. Furthermore, with DNA sequencing techniques, mutations in the introns of human hemoglobin genes of thalassemic patients have been studied (12,145) in order to understand whether these mutations cause differential expres- sion during transcription or mRNA splicing. Biochemical studies Of DNA sequences can be compared with biological studies so that a correlation can be drawn to reveal the mechanisms of thalassemia disorders in the hum ans. Mutations can arise from two processes: spontaneous and induced muta- genesis. Spontaneous mutations usually occur during DNA replication when altera- tions in base-pairing properties are not recognized and effectively corrected. Induced mutations arise from treatments of mutagens and are relatively better understood compared to spontaneous mutations. These two types of mutations in mammalian cells will be discussed separately. 1'65 0"] ~.1 511 C31 123 ll Induced Mutagenesis in Mammalian Cells Mutagenesis must be regarded as a biological process in which a chemical change in DNA is the primary and initial step. Induced mutations are produced as a result of the treatment of organisms with mutagens and usually are recovered at much higher frequencies. The idea that mutations result from errors in repair is an old one, because a class of mutations (i.e., chromosomal aberrations) has been thought to be the result of abnormal arrangement of broken chromosomes during reunion (75,76,121,147,228,229). A more recent hypothesis, which suggests that induced gene mutations arise as a result of error in repair due to base-pair alterations, has been developed by Witkin (295). Recent observations of bacteria seem to indicate that some mutations also result from repair replication error or post-replication repair error. In this section I will concentrate on recent observa- tions on mutations due to repair in mammalian cells. For repair and mutagenesis in bacteria, the literature has been reviewed by Hanawalt _e_t_ g“ (111), Kimball (142), Schendel (23l),and Witkins (296). I. Mechanism of Actions of Various Mutagens. Ultraviolet light (UV), at a wavelength of about 254 nm generated by a germicidal lamp, primarily produces cyclobutane rings in adjacent pyrimidines, while ionizing radiation generates strand breaks. Unlike physical mutagens mentioned above, each chemical mutagen produces its own specific array of lesions. However, DNA damage can be grouped into two categories: noncoding and coding lesions. Strand breaks, intercalations and cross-links do not seem to be recognized as a coding base during DNA replication. Alkylating agents, such as aflatoxin Bl’ benzo(a)pyrene [B(a)P], 2-acetylaminofluorene (AAF), and UV, can cause bulky lesions in DNA. Among them, pyrimidine dimers produced by UV are the most studied lesion. Proflavin, acridine orange and ethidium bromide can intercalate into a DNA helix. Lastly, mitomycin C, nitrous acid and bifunctional nitrogen mustard 12 are known to react, chemically, with both strands of a helix causing a strand-to- strand cross-link. Psoralen requires photochemical reaction to cause cross-links. Ionizing radiation may cause cross-links if single-strand nicks on the opposite DNA strand are one base apart. Coding lesions generally are produced by base analogues, while miscoding is related to alkylated base damage. These lesions occur by: a) incorporations of base analogues; b) thermal or chemical deamination of adenine or cytosine residues already in DNA that produces a hypoxanthine or uracil base; and c) alkylation of a base to alter base pairing properties during DNA replication. Classification and mechanisms of chemical mutagens according to their reaction with DNA have been reviewed by J.J. Roberts (220). Chemical mutagens can react with DNA directly or indirectly. Direct mutagens are biological alkylating agents, such as B-propiolactone, sulfur and nitrogen mustards, alkyl sulfonate, alkyl nitrosamide, alkyl nitrosamidine; and arylalkylating agents, such as 7-bromomethyl benz(a)-anthracene. Indirect mutagens are synthetic chemicals, such as polycyclic hydrocarbons, aromatic amines, azo dyes, nitrosamines, 4- nitroquinoline-l-oxide, urethane, ethionine; and natural products Such as cycasin, the pyrrolizidine alkaloids aflatoxin, saf role and various antibiotics. Chemical mutagens undergo many chemical and/or enzymatic reactions in cells and react as electrophilic reagents with nucleophilic sites in DNA, RNA, and proteins in cells. Direct mutagens are themselves electTOphilic. Indirect mutagens undergo metabolic activation to become electrophilic derivatives. The activation is also dependent on a cytochrome P-450-dependent membrane-based monoxygenase system whidt may be subjected to genetic control. Nucleophilic sites (which if involved in the formation of normal base-pairings are underlined) are in the 96, N3, N7, and C8 positions of guanine, the l_\l_l, N3, and N7 positions of adenine, the NI, N_3 2 and 9 position of cytosine, the N2 and 94 positions of thymine, and the S atoms of l3 methionine and cysteine, the C3 position of tryosine, and the N1 and N3 positions of histidine. Chemical mutagens can react with the phosphate groups in polynucleo- tides to form phosphotriesters. The reactions of chemical mutagens that have been shown to occur following direct reaction of DNA i_n_ _vi_t13 are: single base substitutions, depurinations, single- and double-strand breaks, esterification of phosphate groups, inter- and intrastrand cross-links and cross-links of DNA and proteins. Nitrous acid removes the amino group from adenine and cytosine which become hypoxanthine and uracil respec- tively. Deamination of adenine to hypoxanthine or of cytosine to uracil causes transition, while deamination of guanine to xanthine causes inactivation of DNA. Hydroxylamine and other hydroxyl (OH) donors also cause transition by addition of OH to the amino group of cytosine, so that cytosine undergoes a tautomeric shift to pair with adenine. Alkylating agents, such as nitrogen mustard and ethyl ethanesulfonate, may produce mutations by a) addition of methyl or ethyl group to guanine which behaves like adenine analogues and pairs with thymine; b) depurination; and c) inter- or intrastrand cross-links. Special emphasis is given to N-methyl-N'-nitro-N-nitro- soguanidine (MNNG) and dimethyl sulfate (DMS), which have been used in the present studies. Both MNNG and DMS are direct acting alkylating compounds. It is believed MNNG has a low Swain-Scott substrate constant (_s_) of 0.42, and the ratio of 6 7 O MeG to N MeG in DNA by MNNG is 0.1 (154) as compared to the §_ constant of 6MeG/N7Mec; ratio of 0.011 by DMS (170, 188): MNNG is a more 0.86 and an O potent mutagen than DMS is (170). Mutagenicity of O6MeG in DNA has been thought to be due to its miscoding properties while the presence of N7MeG does not lead to mispairings (170). Compounds that require metabolic activation bind to specific sites of DNA. For example, activated AAF binds to C8 guanine (220). The modified bases are selec We 11:11: 5i D 14 shifted out of the double helix, while the fixed carcinogen is inserted in its place (83,148) and may cause inhibition of DNA methylation (209). Ionizing radiation induces changes in DNA mainly through free radical reactions that produce modified deoxyribose residues, modified bases and strand breaks. Another class of mutagen is that of metal carcinogens (266), e.g., beryllium, a metal carcinogenic to animals and mutagenic to animal cells in culture (215). Beryllium inhibits the editing of 3'-5' exonuclease activity of Iii 1113923 DNA polymerase, and decreases the fidelity of avian myelobastosis virus reverse trans- criptase; beryllium (2 mM) can cause misincorporation of dAMP by purified human HeLa a, B, and y DNA polymerases. Beryllium can increase the misincorporation rate of three human polymerases in UV-irradiated poly(dC) template. Since no editing exonuclease activity can be detected with purified mammalian a, B and 7 DNA polymerases, the effect of beryllium may be due to a diminished nucleotide selectivity of polymerase, to the binding of beryllium to the template or to nucleoside triphosphates. 2. Repair Pathways. Damaged DNA either prevents cellular proliferation (cell death) or it results in faulty protein production or regulation (mutations). Damaged DNA can be overcome by DNA repair (photoreactivation, base excision repair, nucleotide excision repair and post-replication repair). Repairable damages in DNA are missing, incorrect or altered bases, interstrand cross-links and strand breaks. The efficiency of DNA repair depends on several factor(s), such as repair enzymes, energy source (visible light in photoreactivation pathway), DNA precursors and post-replication methyla- tion as suggested from studies in prokaryotes. Failure in one causes misrepair or no repair at all; thus cell death or mutation occurs. The maintenance of the genetic integrity is the result of repair processes. There are two forms of repair, depending upon the requirement of light (light repair and Gar exist Ir lulCilo 1535.111 repair iatc‘n ( ”IUClEO‘ pricier. lie lei r) with h, 15 and dark repair). It has been demonstrated that a photorepair pathway seems to exist in some mammals (Dr. M. H. Wade, personal communication). However, the function of photorepair is not well understood in mammalian cells, which are not usually exposed to light. Maybe it is an analogue to the appendix in humans. Dark repair involves base excision repair, nucleotide excisionirepair and post-replication repair. The pathway of each repair is discussed in detail by several investigators (51,84,111, 112,114,142,220,231,296). Excision repair is characterized by short patch (X-rays) or long patch (UV and AAF) synthesis, depending upon the number of nucleotides excised. In either case, after the DNA lesions are excised (as long as the damage is confined to one strand), the other undamaged strand is used as a template for repair replication. Repair of DNA generally has two consequences; one is "error-free" and the other is "error-prone". "Error-free" repair mechanisms prevent lethality and mutation fixation, whereas "error-prone" repair removes only the lethal components of DNA damage. Photorepair in bacteria and dark repair (excision repair) in mammalian cells are believed to be "error-free" (51,111,169,220) if sufficient time is allowed for cells to perform repair before DNA replication. Thus, mutation frequency and/or lethality of irradiated cells can be reduced by "liquid holding" (48,107-109,137,160,167,168,186,241,286,287,299). 3. Mutations Due to An Unrepaired DNA Damag. Our understanding of excision repair (nucleotide excision) comes from studies with human diploid fibroblasts of normal and xeroderma pigmentosum (XP) individ- uals. The XP cells of A, C, and D groups have a reduced level of the incision activity in the excision repair pathway. These types of cells have higher induced mutation frequencies. If excision repair is error free and occurs mainly before fixation by DNA replication, then when DNA synthesis (replication) is inhibited, there will be more damaged DNA removed and repaired. Therefore, lethality and mutation frequency 16 will decrease. Indeed, liquid holding of UV-irradiated normal diploid fibroblasts reduces lethality and mutation frequency, whereas it did not change either bio- logical end points in UV-irradiated XP cells (group A) (167-169). A similar conclusion (that excision repair is error-free in Chinese hamster V79 cells) was reached by Nakano e_t- 31., (186). Furthermore, the role of thymine dimers in determining the biological effects of UV irradiation is less clear in mammalian cells than it is in bacteria. Excision of dimers has been reported for many cell lines, but ability to excise dimers does not seem to be associated with lower UV sensitivity, except in the case of certain repair deficient XP human cells and mutagen sensitive V79 mutants (167-169, 233-235). If induced mutations are the result of unrepaired damaged DNA in cells, one can then measure and correlate mutation frequency and DNA adducts. There will be a direct relationship between increasing mutation frequency and mutagenic effec- tiveness. Although not all DNA adducts are responsible for mutation, one can compare the mutation frequency and the nature of adducts. For example, from 51: udies by Newbold, gt a_l., (188) carcinogenicity (as measured by mutagenicity) of aliphatic alkylating agents, such as the alkylnitrosamides and the alkylmethane- Sulphonates, is correlated with their ability to alkylate the nucleophilic oxygen a toms in DNA, particularly the O6-atom of guanine. The O6-alkylation of guanine is 1il-<>se) polymerase, such as benzamide and nicotinamide, enhance UDS of UV- inad iated repair proficient cells (2,18,73,165,179,187,196,212); e) inhibitors of post- relDlication repair, such as caffeine and theOphylline, also are inhibitors of the polymerase (213); and f) benzamide causes enhanced cytotoxic effects of MNNG a "<1 DMS to the wild type v79 cells (Liu, unpublished results), while caffeine causes e “ha-need cytotoxicity and mutagenicity in UV-irradiated V79 cells (36,37). To date, 20 no studies in correlating mutation induction and the effect of benzamide have been reported. 6. Mutation Fixation. To understand the relationship between mutation to repair and to replication is to determine the time of fixation or stabilization of mutation relative to these cellular events. Photoreversibility of pyrimidine dimers in bacterial system has been used to determine or infer the time of fixation of UV-induced mutations. Studies in E: £911 indicated mutations are fixed prior to or at replication (214). Another method for drawing an inference about fixation is to apply the mutagen at various times relative to DNA replication. This method was used with synchronized cells to infer that mutations induced by several different agents (UV, X-rays, N AcAAF) were fixed either solely or at least most efficiently at replication. The yield of mutations is highest when the cells were treated in late G1, significantly less when the treatment was given earlier in G1, and is the least when in G2. A lower cytotoxicity and mutation yield will result when there is a longer interval between mutagen-treatment and DNA replication (107,137,159). Using this inference, a number of different agents and conditions, such as metabolic inhibitor 0!" non-nutrient conditions, can influence the mutation yield produced by a given mu tagen. Delayed DNA replication by the conditioned medium has been reported to enhance repair capacity of a normal excision repair (error-free) pathway by reducing mu tation frequencies and increasing survivals (48,160,186). However, this treatment lb"‘<>late base at the replication fork causes a deletion, while slippage in the ter minal base of the daughter strand causes an addition, and frameshifts therefore I . 33' be dependent on a replication enzyme complex. 24 3. Replicative Enzyme-dependent Hypothesis. Studies in bacteriophage T4 mutants have identified a number of genes whose functions in DNA replication and repair have been implicated in the genetic determination of both spontaneous and induced mutageneses. Mutant genes having mutator or antimutator effect are (65-67): genes p35 (generalized repair and recombination), genes 3 (pyrimidine dimer exonuclease), genes h_m (unknown func- tion), genes 30 (DNA ligase), genes 32 (Albert's protein which functions in DNA replication, recombination and repair), genes 42 and t_c_l (pyrimidine metabolism, 42 codes for dCMP hydroxymethylase, Ed codes for thymidine synthetase to synthesize hydroxymethyl dCTP and dTTP respectively), and gene 43 (DNA polymerases). Extensive review on mutator mutants of T4 phage has been given by Drake (65-67) and Rosamond (223). For mutator mutants in prokaryotes, Cox (56) and Hanawalt gt _a._l_-, (111) have also presented a thorough review. While there are reports indicating replication-independent mutations may occur in prokaryotes and lower eukaryotes (6), there is no such evidence suggesting the existence of a replication-independent mutation in mammalian somatic cells. Although recent reports indicate that transposable elements in corn and fungi (6,1 64) do control expressions of certain phenotypes that may be viewed as the re plication-independent mutation through recombination, the existence of transpo- sable elements in mammalian cells is unknown. Mutation-like events due to e x tranuclear DNA, e.g., virus, can be detected as genetic variation and regarded as r e pli cation-independent mutation. For example, integration and loop-out of viral D N A in cellular DNA can possibly introduce duplications or deletions by non-precise exCision of viral DNA. Recent demonstrations on three distinct genes in human DN A related to the transforming genes of mammalian sarcoma retroviruses may be of im portance for elucidation of this mechanism on cell growth, differentiation, and hero plastic transformation (150,297,300). 25 Evidence supporting enzyme(s)-dependent mutation mechanism is from studies in T4 bacteriophage, prokaryotes and lower eukaryotes (20,56,67,115). The replica- tion-dependent spontaneous mutation produces most frameshifts (20,67) and accounts for the majority of spontaneous mutation in these organisms. Works from mutator gene analysis of T4 phage (65-67), bacteria (56,111), yeast (115) and in m fidelity anlaysis of DNA replication enzyme(s), including that from mammalian cells (254), clearly suggest that DNA polymerase, DNA precursors, and post-replication methylation enzymes may play an active role in the selection of bases or correction of base mispairings during replication. The following review will concentrate on mutation mechanism assumed to be single base-pair alterations. 4. Post-replication Methylation In prokaryotes, there has been demonstrated that post-replication methylation of base residues (N6 of adenine, C5 of cytosine) prevents cleavage of DNA by nucleases. The methylation provides a mechanism with which foreign DNA or mismatched base pairs are recognized and effectively excised. The methylation also provides a protective function so that foreign DNA is eliminated from the host DNA. Mutants such as gafl'(_da_m_-3 or dam-4) or ESE-(901919171) with defective] deficient methylation function, have been reported to contain single-stranded breaks which are amplified in dim-3 chlAlZ and gait-3 1i -7 double mutants (171). Combinations of gig-3 with p_o_lA, [_e_c.A, [_e_gB and 522C are lethal. Mutant dam-3 has increased UV-sensitivity, spontaneous mutation and spontaneous induction of prephage. It appears that the function of a gain-specific enzyme also involves "mis- match" repair such that m’ strains are deficient in a post-replicative error- avoidance pathway which allows a specific elimination of mismatdied lesions. A new approach has been used (91) to isolate mutants with a second mutation defective in adenine-methylation-instructed mismatdi correction in a dam mutant. 26 Because 513m mutants are Z-aminopurine sensitive due to the excision of unmethyl- ated, mismatched base pairs, a 9‘32 strain with a second mutation lacking mismatch correction could be expected to restore resistance to Z-aminopurine in a _clafl mutant with the mutator properties. The methylation of eukaryotic DNA has been reviewed by Ehrlich and Wang (74). The methylation of eukaryotic DNA may control transcription, and function in maintenance of chromosome structure, repair of DNA, hotspot mutagenesis, and oncogenic transformation. However, no mammalian mutant with a defective "SEE" or "1cm" specific enzyme has been reported. Recent demonstration on reactivation of an inactive X chromosome by 5-azacytidine provides a new research direction (180). 5. Fidelity of Replication A. Polymerase Base-selectivities. The fidelity of replication concerns the accuracy with which incorrect base- pairs are excluded during or immediately after deoxyribonucleoside triphosphate (dNTP) incorporation. Mismatchings due to tautomeric forms of the bases have been estimated from the tautomeric equilibrium constants of the bases, and are higher than the observed 12 1112 mutation rate in T4-phage (2 x 10'10) per base pair (21,67). Hydrogen bonding alone cannot explain the observed fidelity of replication. It is, therefore, proposed that DNA polymerase may participate in the selection of a correct base during the base incorporation step and a 3'-5' exonuclease activity may be involved in removal of the misincorporated base. Evidence supporting these base selectivities of polymerase was gained by isolating T4 phage mutants defective in DNA polymerase that also have a temper- ature sensitive mutator or antimutator phenotype (67,82,118,250-253). In $52 studies on the DNA polymerase from this mutant suggest that the polymerase is 27 responsible for the elevated mutation rate, and that the purified polymerase incorporates incorrect bases into a template four times higher than does a wild-type polymerase. Base selectivity of the polymerase in a mutator T4 mutant shows mutator activities and temperature sensitivity for DNA replication, i.e., the misincorporation of nucleotides increases with increasing temperature by a factor of six as compared to the wild type polymerase. Studies on higher eukaryotes provide further support for the base selection activity of polymerase during DNA replication. Weymouth and Loeb (292) demon- strated that the misincorporation frequency of the 13 £119 system by polymerae is 1.25 x 10’“. This frequency may reflect mispairings at the first catalytic step of replication because the polymerase does not seem to have error-correction properties, such as the associated 3'-5' exonuclease in prokaryotes. Springgate and Loeb (254) further reported that a polymerase from acute lymphoblastic leukemic cells produces a tenfold higher polymerization error than the polymerase from normal lymphocytes does. This inaccurate incorporation of bases is indicative of possible alteration in the polymerase base selectivity. Studies on T4 polymerase mutants indicate there are at least two types of mutations on polymerase that can alter base selectivity. If base selectivity of polymerase is involved in replication-dependent spontaneous mutations, the poly- merase must contain binding sites for both the template base and the entering monomer dNTP. The first hypothetical copy-error mechanism as proposed by Drake (65) relates to the monomer base acceptor. A mutation of this type generally can recognize the template base but incorrectly accepts a different base residue, e.g., accepts guanine instead of adenine to couple with a thymine, and causes a transition mutation. Mutants of this type have a high transition rate but a low transversion rate, and would be sensitive to certain chemical mutagens because the probability of accepting a chemically altered base would be increased. A second hypothetical Z8 copy-error mechanism by a polymerase mutant relates to communications between two binding sites. A structurally altered polymerase of this type might produce transitions as well as transversions. This type of polymerase mutant might not be sensitive to chemical mutagens. Studies on T4 polymerase mutants generally support these two hypotheses (21,65). However, in mammalian somatic cells no such polymerase mutants have been reported, though a temperature sensitive mutant of mouse FM3A cells may be defective in one of the two subunits of polymerase (274). Other enzymes that have been implicated in producing mutator phenotype are the associated 3'-5' exonuclease activities in polymerase of bacteriophage, prokary- otes and polymerase II of yeast, and ligase of T4 phage (66). MD5 mutants possibly are defective in exonuclease activities (111,135,268). Purified eukaryotic replicative polymerase 01 does not have this associated 3'-5' exonuclease, and mutation of this activity on a separate protein has not been reported in the eukar yotes. B. Base Analogues and Deoxyribnucleotide Pools Alterations in DNA precursors have been demonstrated to change spontaneous mutation rates or induced mutation frequencies in prokaryotes and eukaryotes (61,67,175,l76,198,207,208,257). In prokaryotes and T4 phage, thymidine (TdR) deprivation causes an enhanced mutation rate either by mass action or by inhibition of pyrimidine metabolic pathways (26,66,140). Thymidineless mutagenesis is syner- gistic with Z-aminopurine (ZAP) mutagenesis in bacteriophage T4 (123,222). It has been suggested from accumulated evidence that perturbation of normal deoxyribo- nucleotide triphosphate pools (95,119,151) and mispairs due to base analogues in DNA, lead to mutagenic effects of 5'-bromodeoxyuridine (BrdU) and ZAP. Hopkins and Goodman (123) further suggested that the configuration of the neighboring nucleotides surrounding the base analogue mispair is responsible for the antipolarity of BrdU and ZAP mutageneses. In mammalian cells, evidence has been shown that BrdL been not 29 BrdU mutagenesis is mediated by the concentration of BrdU to which the cells have been exposed instead of the amount of bromouracil substitution (140). This hypothesis is consistent with the hypothesis that triphosphates of BrdU, TdR, 2'- deoxyadenosine (AdR) or Z'-deoxyguanosine (GdR) inhibit cytosine diphosphate (CDP) ribonucleotide reductase and decrease deoxycytidine triphosphate (dCTP) pools (174). It is this imbalance of pyrimidine pools whida mediates the base (or base analogue) mutagenesis. Recent evidence suggests that in the rodent cells, endogenously elevated dCTP pools cause an increase in spontaneous mutation rate in ouabain- and 6-thioguanine- resistant loci (175,288). This elevated mutation rate seems to be locus specific (does not occur in emetine resistant locus or reversion of proline auxotrophs to proto- trophs). Additions of TdR to the culture medium so that the dCTP/dTTP ratio is returned to normal, reverse the mutagenic effect. Furthermore, excess exogenous AdR, GdR or TdR in the culture medium of V79 Chinese hamster cells is both toxic and mutagenic (23,257). It appears that an imbalance of dNTP pools causes mass action so that mispairings are elevated, as proposed by Drake (66) and others. On the other hand, Brennard and Fox (25) reported exogenous TdR is toxic but not mutagenic. Hydroxyurea, inhibitor of CDP ribonucleotide reductase, or cytosine arabinoside (araC), inhibitor of the reductase or DNA polymerase, is not mutagenic (23). Although 3'-5' exonuclease activities do not reside on the replicative enzyme, it is generally believed that this editing enzyme exists in a replication complex with polymerase in the mammalian systems. The reason that exonuclease activities did not correct the mispairing due to increased dCTP by mass action is unknown. C. Uracil-DNA (flcosylase Duncan and Weiss (69) reported that spontaneous mutation to nalidixic acid or to rifampicin resistance is increased 5-fold in uracil-DNA glycosylase (395) mutants of E; ggl_i. In addition, G-C to A-T transition in MA reversion analysis is increased 30 by l5-fold and this type of transition mutation seems to be specific. Moreover, uracil-containing phage are viable in Egg- bacteria, thus suggesting that the uracil is not removed from matured DNA, contrary to what is generally believed (95,112). It is suggested that uracil-DNA glycosylase appears to edit (mutagenic) deaminated cytosine residues, but does not prevent uracil misincorpOration into DNA as AU base pair. The function of the 223 gene seems to preclude the use of uracil as a normal DNA constituent. 6. Mutator Activities Associated with Unknown Mechanisms Reports from studies in _E_._ coli K-12 mutator mutants, as summarized by Cox (56) and Hanawalt gt g” (111), indicate that elevated spontaneous mutations occur in mutations in genes such as MD: uer, dam, dcm, dnaQ, mutD, mutL, mutR, 115$, _m_u_t_T and MU, in which gv_rD, 915E and MU may be at the same gene. Mutants of the _rtltltD gene cause generalized hypermutability (transitions, trans- versions and frameshifts) which is stimulated by TdR (2 11M) in the TdR+ strain and suppressed by n_al_A, or gtlmD genes. Mutants of mgtT genes are specific mutator mutants (transversions only) while transitions and transversions occur in pits, r_n_utR, mu_tL and MU (u_er). Furthermore, po_lA mutants (polymermase I mutation) with mu_t-S, R, -L, or -D background increase frameshift mutation rate, and muta- tions in polymerase 111 (EC) also have an elevated frameshift mutation rate. In lower eukaryotes such as yeast, there are four interrelated repair pathways (24,116); and mutator activities in yeast are dependent on channelling DNA lesions to repair pathways which may be "error-prone" when there is a deficiency in "error- free" repair pathways (115). A mutable gene in maize, reported by McClintock (164), has now been demonstrated to be a transposable element which regulates expressions of genes. Such a transposable element has not been demonstrated in mammalian cells. 31 Regarding the mammalian somatic cell system, three reports to date indicate that an elevated spontaneous mutation rate has been observed in TdR- Chinese hamster ovary cells (175), in ribonucleotide reductase mutants of mouse lympho- sarcoma cells (288) and in diploid fibroblasts from patients with Bloom's syndrome (BS) (97,282). The hypermutabilities of the former two- mutants are associated with an endogenously elevated dCTP level while that of the latter is unknown. The mutator activities of the rodent mutants are locus specific (6TGr and ouar loci), while that of BS fibroblasts are found in 6TGr (282) and DTr (97) loci. Emerit and Cerutti (78) and Warren (283) reported that the concentrates of culture medium from BS fibroblasts contain a clastogenic factor which causes 6TGr mutation and chromosomal aberrations but not ouar mutation in the wild type V79 cells. Specula- tion has been that BS fibroblasts lack a detoxification factor and that this clastogen accumulates in the culture medium. In humans, cells having spontaneous mutations as defined by chromosomal instabilities, are found in individuals with Fanconi's anemia (FA), Bloom's syndrome, and Ataxia-Telangiectasia (AT). Fibroblasts from these patients are sensitive to mutagens such as X-ray (FA, AT), and/or uv (BS). Other human fibroblasts with i_n thtg mutagen-sensitivity are found in patients with xeroderma pigmentosum (UV and UV-like mutagens); hereditary retinoblastoma (X-ray); and Cockayne's [UV, EMS, (279)] and Gardner's [X-rays, mitomycin C, UV and no liquid holding recoveries (160)] syndromes. All of these patients have an increased incidence of spontaneous and/or induced cancer [see reviews by Arlett and Lehmann (5), Paterson and Smith (202)]. Recent studies, using restriction endonuclease mapping on human hemoglobin genes from thalassemic or sickle cell anemic patients (12,145), suggest that a thalassemias are due to a deletion of a gene loci, possibly caused by an unequal crossing-over between tandemly repeated 0 genes; and that a distinctive polymorph- ism ckle to base stbstitution in a HpaI cleavage site of the 85 gene occurs in 60 to 80 32 percent of sickle cell anemic patients with this gene. As to 8 thalassemia, it is more complicated. In 8+thalassemias (low production of normal 8 globin from a decreased amount of 8 mRNA), the defect is due to either defective regulation of transcription of 8 globin genes or abnormal processing of mRNA. In 8° thalassemias (with no normal 8 globin or mRNA), the defect is probably at the level of defective transcription, abnormal mRNA processing, deletions or base substitution in the 8 structural gene. For example, some patients with 8° thalassemias have either untranslatable 8 globin mRNA, or specific nonsense mutation in the 8 mRNA. In 68 thalassemia and hereditary persistence of fetal hemoglobin (HPFH), synthesis of 6 and 8 globin does not occur, while only fetal hemoglobin is synthesized. Using recombinant DNA cloning techniques, the deletion of 6 and 8 globin genes from 68 thalassemic and HPFH patients have been mapped and shown to regulate the relative expression of y globin genes. It appears that mutations on the introns of human genomes also affect the expressions of structural genes and give rise to hereditary disorders in the humans. Isolation and Characterization of Mutagen- . Sensitive Mutants in Mammalian Cells A number of methods have been used to induce, and select for mutagen- sensitive mutants in mammalian cells [see review by R. Schultz (233)]. The methods are: a) nonselective procedures with massive screening or replica plating tech- niques (l52,ZZ7,255,267) to avoid the harsh processes necessary for mutant enrich- ment; b) selection techniques using differential incorporations of BrdU and subse- quent exposure to visible light so that excision repair proficient cells become lethal (133,143,218). The second method provides some success in the isolation of repair deficient mutants. But a combination of BrdU incorporation and black light- exposure is also mutagenic as reported by Chu gt Q" (45). A more recent 33 development using irradiated viruses to select for UV-sensitive and repair deficient mutants which cannot undergo host cell reactivation of the virus and thus survives through selection. This viral reactivation may be a better technique, except that certain viruses have a host cell specificity and the kind of viruses that can be used is also limited. For example, Chinese hamster cells do not appear to incorporate the viruses commonly used for such studies (233). An alternative method of enrichment and selection of mutagen sensitive and excision repair deficient mutants has been described by Schultz gt gl_., (233,235). In this method, wild type V79 cells were induced to mutate by BrdU/black light and UV (20 J/mz). The two mutagenic inductions are necessary if one assumes that gene(s) responsible for the repair function is (are) autosomally recessive (Z64). Selections of repair deficient mutants were carried out by the use of differential incorporation of 3H-TdR immediately following exposures to low dose UV (10 J/mz) in a replication- inhibited population. After incubation withBH-TdR, the selection was performed at 4°C to accumulate 3 H damage. Following selection, the surviving cells were incubated at 34°C in a medium containing hypoxanthine, aminopterin and thymidine so that thymidine kinase deficient cells were eliminated. Using this method, Schultz gt gt" (233-235) have isolated several DNA repair deficient mutants which have been shown to be mutagen sensitive and have mutagen-induced hypermutability. However, none of the DNA repair mutants is reported to be deficient in polymerase or spontaneously hypermutable. Aphidicolin-resistant Mutants l. Aphidicolin: Mode of Action. The tetracyclic diterpenoid aphidicolin (‘3on31101): m.p. 227-233°C) is an antibiotic and antiviral metabolite from Cephalosporium aphidicola Petch (28,59). It is a potent inhibitor of nuclear and mitochondrial DNA synthesis and it strongly 34 inhibits the growth of herpes simplex, vaccinia, SV40 and adenoviruses (30,106,149, 162, 166,204). It inhibits DNA but not RNA or protein synthesis (110,131,294). Aphidicolin is active against iododeoxyuridine-resistant herpes virus and does not induce mutation in herpes virus or prokaryotes directly (30) or indirectly through metabolic activation by rat liver microsomes (203). When aphidicolin (0.5 uM) was added to the culture medium during mutant expression or selection of 6TGr cells, aphidicolin did not enhance recoveries of 6TGr mutants (Liu, unpublished results). It suggests that aphidicolin does not have mutagenic activities. Aphidicolin has been shown to be a specific inhibitor of polymerase 11 (131,193- 195,294) but not of polymerase 8 or y (129-131,294) at levels which inhibit mitosis (130,131,205). It does inhibit polymerase II of yeast [unlike most polymerases in eukaryotes, it has 3'-5' exonuclease activities (210)] but it has no effect on polymerases from E: gig or T4 phage (206). The fact that vaccinia virus DNA polymerase, which is a single polypeptide chain, is sensitive to aphidicolin suggests a direct interaction between polymerase and aphidicolin. On the other hand, that the sensitivity of yeast DNA polymerases I and II and of sea urchin DNA polymerase a to aphidicolin decreases upon purification (131,210) also indicates an interaction between aphidicolin and an accessary subunit. Recent demonstration of an aphidicolin-sensitive protein stimulatory factor for DNA polymerase (I from rat giant trophoblast cells or calf thymus (136,298) supports the indirect interaction hypothesis. The effect on polymerase a of this stimulatory factor is abolished by aphidicolin but is unaffected by 2',3'-dideoxythymidine triphosphate (inhibitor of 8-, y polymerase). Kinetic analyses of the mechanism of inhibition on purified polymerase 11 by aphidicolin, using activated DNA as template/primer, show that the inhibition is competitive with respect to dCTP (106,193,194,206), noncompetitive with respect to other dNTPs and uncompetitive with DNA. When synthetic homopolymers are used, 35 the degree of sensitivity of DNA polymerase 11 to aphidicolin is modified (193). DNA synthesis in isolated nuclei of sea urchins, perm eabilized mouse FM3A cells or intact cells is inhibited competitively with all four dNTP, but noncompetitively with the individual dNTP (9,194) or with three of the four dNTP (193). In adenovirus-infected KB cells, the inhibition by aphidicolin to the endogenous DNA polymerase activity is competitive with dTTP (106). The competitive interaction suggests that the inhibition of DNA replication by aphidicolin is brought about by inhibiting the binding of one or more of the dNTP to DNA polymerase. This is so because aphidicolin probably binds only to DNA polymerase molecules which have not yet bound dNTP molecules, and forms a catalytically inactive aphidicolin-polymerase complex. 2. Use of Aphidicolin to Identify Eukaryotic Replication and/or Repair Enzyme(s). Five lines of evidence suggest that a or a-like DNA polymerase is required for DNA replication. The first is that a-polymerase activity increases during the S phase when DNA undergoes replication, or in actively dividing cells (27,58,126,13l, 161, 205, 289-291). Secondly, aphidicolin inhibits DNA replication and cell growth i_n X122! and a-like, not 8-, or y-like polymerase i_n 11.119 (27,47,204,205). Thirdly, the fact that the concentration of aphidicolin required to inhibit DNA replication i_n 1319 is almost identical to the concentration required to inhibit DNA polymerase a i_n 3.13.521 further suggests that the enzyme is the target of aphidicolin (205). Moreover, aphidicolin does not have the inhibitory effects similar to inhibitors of 8-,y- polymerase, such as dideoxy-GTP or -TTP [ddTTP (57,136,166,298)]. This suggests that 8- and y-polymerases are probably not replicative enzymes. Lastly, animal cells resistant to aphidicolin either have an increased level of DNA polymerase 11 (190), or contain an aphidicolin-resistant DNA polymerase-a (259). Indirect evidence from studies on sister chromatid exchange (SCE) in a Chinese hamster V79 cell line suggests that aphidicolin acts on the replication fork 36 (139). Ishii and Bender (132) reported that DNA polymerization inhibitors, such as aphidicolin, hydroxyurea, and cytosine arabinoside (araC), increase spontaneous SCE frequency and have a synergistic effect on UV-induced SCE frequency, while ddTTP, neocarzinostatin, and novabiocin have no effect, and cycloheximide decrease both SCE frequencies. The most interesting findings come from the studies on the effects of aphidicolin during unscheduled DNA synthesis (UDS) in mutagen-treated (UV, MNNG, MNU) mammalian cells. Independent investigations from four laboratories (16,47,110,248) showed that aphidicolin inhibits UDS in human diploid fibroblasts (47,248), normal lymphocytes (16), and HeLa cells (110). While these studies were performed in replication-inhibited cells [replication is inhibited by hydroxyurea and/or araC (110,248), or by cell contact in confluent culture (47)], the results from mitotic cells show that aphidicolin does not inhibit UDS in UV-irradiated HeLa cells (89,205), bleomycin treated permeable SR-C3H/He or mouse ascites sarcoma cells nor in isolated rat liver nuclei (236). Rather, the later studies indicate that aphidicolin can be substituted for hydroxyurea to inhibit scheduled DNA synthesis and that it allows measurement of repair synthesis (205). Attempts have been made in many laboratories to isolate aphidicolin-resistant mutant cells. Most mutants were induced by MNNG (7,8,10,190), EMS (226,259), or by combined treatments with BrdU/black light and UV (40). Among these mutants, aphidicolin resistance is reported to be associated with a) an altered polymerase 11 (259); b) an inducible amplification of polymerase a (190); and c) an alteration in dNTP pools, such as i) by an elevation in dATP (7); ii) thermolabile thymidylate synthetase (8); iii) by alteration (10) or iv) by amplification (226) of ribonucleotide reductase; and v) by an elevation of dCTP or all four dNTP (40) in the rodent cells. An elevated UV-induced cytotoxicity and mutability are reported in an aphidicolin resistant mutant from the V79 Chinese hamster cell line (40). The mutant (aphr-4, 37 or aphr-4-Z) isolated by Chang gt in is reported to be: a) slow growth; b) auxotro- phic to TdR, deoxycytidine or deoxyuridine; c) sensitive to UV light; d) hyper- mutable by UV irradiation, e) sensitive to AdR, GdR and TdR (100 11M); 1) of having high BrdU-dependent chromosomal aberrations; g) a high SCE frequency; and h) a high "reversion" rate. One of the "revertants", aphr-4-RZ, is not sensitive to UV and has a fast growth rate. Also, it is resistant to aphidicolin and has the same dCTP pool as the original mutant. Thus, the UV sensitivity in the aphr-4-2 probably is not associated with the endogenous dCTP levels. Concluding Remarks With the exception of large deletions, insertions, and inversions, both spon- taneous and induced mutations arise from errors in DNA synthesis during repair or replication. On the basis of the mechanism of mutagen action, there are two groups of mutagens: a) base modifiers (BrdU, ZAP), and b) DNA attackers: these agents, directly (MNNG) or indirectly (AAF) through metabolic activation, modify DNA or attack DNA, cause bulky DNA lesions and produce bulky distortions in DNA. They cause miSpairings and inhibit DNA synthesis. The lesions, if not repaired or if misrepaired, cause lethality and mutation. The fidelity of DNA replication could be achieved by at least two enzymatic mechanisms: a) nucleotide selectivity of DNA polymerases, i.e., the slower the polymerization rate, the longer the checking time between nucleotide pairs, and the higher the fidelity; b) "proof-reading" or "editing" by the 3'-5' exonuclease activity. In mammalian cells, this latter activity is separated from the enzyme harboring polymerization activity. Unlike spontaneous mutagenesis, where DNA polymerases are mistaken by the tautomeric and isomeric forms of nucleotides, induced mutagenesis is provoked by mutagen-induced modifications of DNA template strands. Excision repair of potentially lethal and/or mutagenic DNA lesions can occur only in double-strand 38 DNA, and then only when the segment of the complementary strand is intact for excision repair to be "error-free". In the case of lesions in the single-strand DNA and in the case of nonrepaired DNA lesions encountered by the DNA replication machinery, there is no way for DNA polymerase to produce an "error-f ree" repair. Error-prone repair pathway is inferred from studies in bacterial systems and is thought to involve post-replication repair and/or polymerase fidelity. In mammalian cells, due to lack of suitable mutants, the speculative existence of an error-prone repair pathway has not yet been verified. Enzymatic functions to ensure the accuracy of normal DNA replication are base selectivity, editing, uracil-DNA glycosylase, post-replication methylation, and DNA precursor pools. Although mutants defective in polymerase 01 have been isolated, mutator activities are reported to be associated with endogenously elevated dCTP. Aphidicolin is a specific inhibitor of polymerase a and aphidicolin resistant mutants have been isolated. One of the mutants (aphr-4-2) is sensitive to UV and is hypermutable at ouar locus. The enhanced UV-sensitivity in this mutant is not associated with the endogenous dNTP. MATERIALS AND METHODS Cell Strain A partially transformed aneuploid lung fibroblast line (V79) derived from a male Chinese hamster, Cricetulus griseus (2n=22), (80) was used as the parental cells for isolation of potentially mutant cell lines. This cell line has an indefinite life span and can be grown in cultures attached to the bottom surface of plastic or glass culture flasks or plates. These V79 cells divide every 14-16 hours and can form colonies from single cells with a cloning efficiency of 7096 or more. The parental cell line will be regarded as the wild type cell line and used as the control group to study the nutritional requirements and the mutabilities of the mutants in this dissertation. Culture Medium Unless otherwise indicated, all cells in the experiments to be described below were grown in modified Eagle's Minimum Essential Medium (MEM) (72) with Earle's salts supplemented with a 5096 increase of all vitamins, all essential amino acids except glutamine, a 100% increase of all non-essential amino acids and 1 mM sodium pyruvate. The concentration of sodium bicarbonate in the medium was adjusted to 1.0 gm/L. This medium was referred to as growth medium. The selection medium for ouabain-resistant mutants or 6-thioguanine resistant mutants was the same as the growth medium except that it was supplemented with 1 mM ouabain or 10 ug/ml 6-thioguanine (Sigma Chemical Co.). The medium used in the experiment studying 39 40 liquid holding recoveries was prepared from Eagle's MEM (GIBCO, Grand Island, New York, Cat. No. F-ll) containing 1.5 gm/L sodium bicarbonate. The medium was sterilized by a positive pressure filtration (Nuclepore Corporation, Pleasanton, California) and stored in a dark, cold room at 4°C. Prior to use, each bottle of 500 ml medium was supplemented with fetal calf serum (FCS, 5% v/v; GIBCO, Grand Island, New York; Flow Laboratories, Inc., Rockville, Maryland; Pel Freeze, Rogers, Arkansas; or Sterile Systems, Inc., Logan, Utah) which had been stored at -200C, thawed and heat inactivated, if not heat inactivated by the manufacturers, at 56°C for 30 minutes prior to use. In addition, penicillin G (100 units per ml) and streptomycin (100 pg per ml) (Eli Lilly and Co., Indianapolis, Indiana) were added to the medium. For testing the nutritional requirements of the aphr-4 variants, dialyzed FCS prepared with an Amicon hollow fiber dialyzer (HIPIO, Amicon Corporation) was used. Conditioned medium was prepared (186) by growing confluent wild type cells [5-6x105 per cmz; 20 ml of medium per flask (75 cm2)] in this Eagle's MEM supplemented with 10% FCS. Twenty-four hours later, the conditioned medium was pooled, sterilized by negative filtration (0.22 11M, Falcon Plastics, Oxard, California) immediately and stored at -20°c until use. Culture Vessels and Incubation Conditions Stock cell cultures were grown in plastic flasks (Corning Glass Works, Corning, New York). The cells were grown in fresh medium, either by refeeding or subculturing to ensure a log phase growth, the day before an experiment. Cells were dissociated with 0.01% crystalline trypsin (Sigma Chemistry Co.) in phosphate buffered saline (PBS) without calcium and magnesium ions but with 0.196 (wt/v) methylcellulose. Unless otherwise indicated, cells were grown in plastic culture dishes (9 cm, Corning Glass Works) and incubated in water-jacketed incubators 41 which provide a stable temperature of 37°C with humidified air and 5% C02. The wild type cells have a generation time of 14-16 hours at log phase growth (233). Cell Growth Rates For each cell line, approximately 0.5-1 x 105 cells were plated in a sufficient number of culture dishes at time zero; two plates from each cell line were trypsinized at various times thereafter and counted. The values were averaged. Cell and Colony Counts After trypsinization, cells were suspended in medium and stored on ice and counted with a hem acytometer. Serial dilutions were carried out to obtain cells at different concentrations. Less than a four-hour-exposure to cold did not signif- icantly affect cloning efficiencies. For all experiments, cells were scored after 7 (the V79 cells) or 12 (slow growing mutants) days of growth. Colonies were rinsed with saline, fixed with 95% ethanol and stained with Giemsa (2.5% Giemsa in 3% methanol, Gurrs Improved R68, Sarle, Santa Monica, California). Only colonies with 30 or more cells were visually scored using a colony counter (American Optical Company). Chemicals Unless otherwise indicated, all chemicals were dissolved in PBS. The following chemicals were obtained from Sigma Chemical Company: IZ-O-tetradecanoyl phorbol-l3-acetate (TPA), hypoxanthine, thymidine (TdR), 5-bromodeoxyuridine (BrdU), caffeine, hydroxyurea, cytidine (CR), benzamide, cytosine-l-B-D-arabinofur- anoside (araC), 2'-deoxyadenosine (AdR), Z'-deoxyguanosine (GdR) and their 5'- triphosphates (dTTP, dCTP, dATP and dGTP). Aminopterin was from Nutritional 42 Biochemical Corporation and 6-thioguanine (2-amino-6-mercaptopurine, 6TG) was from ICN Pharmaceutical Inc. (Cleveland, Ohio). Purified diphtheria toxin (Lot No. D343, 2000 floculating unit per mililiter, 1 If = 2.5 ug of protein or 40-60 minimum lethal doses) was from Connaunght Laboratories Limited (Willowdale, Ontario, Canada, M2N5T8). Diphtheria toxin (DT) was divided into aliquots of 0.15 ml per glass vial and stored in liquid nitrogen until use. All other chemicals were stored according to recommendations by the manufacturers. Aphidicolin (C20H3404’ M.W.:338) was a gift from the Development Therapeutics Program, National Cancer Institute and was dissolved in 100% dimethylsulfoxide (DMSO, Mallinckrodt Inc., Paris, Kentucky). Solutions of 6TG, AdR, or GdR were prepared by dissolving the chemicals in NaOH (1N) and then in double distilled water (final pH=11.35). Benzamide and TPA were dissolved in 100% ethanol. N-Methy1-N'-nitro-N-nitro- soguanidine (MNNG) and dimethyl sulfate (DMS) were purchased from Aldrich Chemical Company, Inc., Milwaukee, Wisconsin. And N-acetoxy-Z-acetylamino- fluorene (NACAAF) was a gift from Dr. James Miller, University of Wisconsin. All chemical mutagens (MNNG, DMS and NACAAF) were dissolved in 100% DMSO and added onto cell culture immediately. All Chemical solutions were filter sterilized (except when 100% ethanol or DMSO was used as a solvent) with a disposable, negative pressure filter (0.22 11M, Falcon Plastic) or a positive pressure filter unit (Swinnex-GS, Millipore Corp., Bedford, Massachusetts) and stored in -20°C until use. The final concentration of 6TG used in mutant selection was 10 ug/ml (6 uM). For deoxyribonucleoside triphosphate measurements, primer template double strand poly(dI-dC)-poly(dI-dC) and poly(dA-dT)-poly(dA-dT) were from P-L Biochem- icals, Inc. (Milwaukee, Wisconsin) and _E_. go_li_ DNA-polymerase I (in 50% glycerol) was from Boehringer Mannheim. Tritiated-dATP (28 ci/mmol), -dGTP (11.7 ci/ mmol) and -dCTP (I9 ci/mmol) were from the Radiochemical Center (Amersham, 43 England) and 3H-dTTP (82.4 ci/mmol) was from New England Nuclear (Boston, Massachusetts). All templates, polymerase, and 3H-dNTP were stored at -20°C. CLtotoxicity and Cloning Efficiency Determinations Sensitivities of the cells to various forms of radiation and chemicals were determined by plating 400 or more cells per plate to allow an estimated 100-400 surviving colonies. Survival rates were determined in triplicate plates for each treatment. Four hours after cell plating, Chemicals in microliter (pl) quantities were added to the medium on the attached cells. For ultraviolet light (UV) irradiation, with the medium removed, the attached cells were irradiated with a germicidal lamp (General Electric, 25T8-Z5 w) which was positioned to (deliver 1.4 Joule per meter squared per second (J/m2/sec). The UV-irradiated cells were grown in 5% dialyzed FCS for 24 hours so that replication and repair of DNA were dependent entirely on endogenous nucleotides. The effects of caffeine (0.25-0.5 mM) or benzamide (2 mM) during DNA repair period were tested. Benzamide was added at the time of cell plating. Four hours later, chemical mutagen (MMNG, NACAAF or DMS) in ul volume was added or cells were irradiated with UV. Caffeine was added immediately after UV. Caffeine was changed every 4 days until cell colonies were fixed and scored. Benzamide was present for 4 days. Cells were grown in medium supplemented with 5% FCS when chemical mutagens were used. For X-ray irradiation, cells were diluted to various concentrations so that one ml contained the number of cells to be plated onto one plate. Cells were then suspended in ice-cold medium during X-ray irradiation (184 R/min, Z50 Kvolts, 20 mA with 3 mm Al filtration) and were plated and grown in medium with 5% FCS after irradiation. The doses of chemicals are expressed in micromoles (uM), or ug/ml whereas UV and X-ray doses are in Joule per square meter (J/mz) and rads (r) 44 respectively. To test the effect of benzamide (2 mM) on the survival of X-ray irradiated cells, the Chemical was added to the medium after irradiation and was removed 4 days later. Unless otherwise indicated, chemicals and medium were changed every four days. Cloning efficiencies of controls were determined concurrently by plating 1,200 cells in three plates without chemicals or radiations. Colony-forming abilities of control and treated cells were calculated by dividing the total number of colonies recovered by the total number of cells plated, for each treatment in each cell line. All experiments were repeated at least once. Mutant Isolation Portions of this work have been reported elsewhere (40,226). Mutants resistant to aphidicolin were induced in V79 cells (30x106) by the combined treatment of bromodeoxyuridine (1x104 M) and black light (40,45,235). After sufficient expression time (one week) the mutagenized cells were pooled, replated, and irradiated with UV (l6 J/mz). Seven days later, these cells (30 x 106) were selected with aphidicolin (1 M) for 23 days at a cell-density of I x 106 per plate (40). This concentration of aphidicolin (1.0 11M) was used because the wild type V79 cells are very sensitive to this drug, e.g., they have a relative plating efficiency (by single-cell platings) of 4 x104 at 0.5 11M of aphidicolin (40,226). A lengthy treatment (longer than 10 days) of V79 cells in mass-culture with 0.4 11M of aphidicolin resulted in no clonable cells (personal observation). The combined mutation induction was performed because it is assumed that gene(s) responsible for aphidicolin resistance (aphr) is (are) autosomal recessive. Four aphr colonies at a frequency of 4.5 x 10-7 survived the selection. The surviving colonies were isolated by removing the medium and attaching a glass cylinder with autoclaved adhesive (Cellseal, Fisher Scientific) around the colony; the encircled cells were dissociated from the plate surface with trypsin. One of the 45 aphr mutants, aphr-4, is a slow-growing thymidine auxotroph. It continuously gave rise to fast growing "revertants" when cells were grown in medium supplemented with 5% FCS. Some slow-growing thymidine prototrophic colonies were also recovered when cells were grown in medium supplemented with 5% dialyzed FCS. These revertants were isolated and characterized. The aphr-4 mutant has been recloned. One of the recloned population, aphr-4-2, has been thoroughly charac- terized in this study together with a fast-growing thymidine auxotrophic revertant aphr-4-RZ, and the slow-growing thymidine prototrophic revertants, aphr-4-RP4 and RP5. To reduce the frequency of fast-growing revertants, in the aphr-4-Z pOpula- tion, aphr-4-2 cells were grown in 1.0 11M aphidicolin for one week. A large population of these cells was frozen and stored in a liquid nitrogen tank. No cell culture was used for an extensive period of time: a new culture from storage stocks was initiated for each experiment. Deoxyribonucleoside Triphosphate Measurements The endogenous deoxyribonucleoside triphosphates (dNTP) were measured according to L. Skoog and his colleagues (157,245). To obtain partially synchronized (276,281) cell extracts, cells from confluent cultures were plated at a density of 1.5 x 106 per plate (9 cm, 2.3 x 10“ per cmz) in growth medium with 5% FCS one day before extraction. Collection of cells was made with a rubber policeman and dNTP was extracted by 60% methanol at -20°C overnight. The extracts were separated from cell debris with centrifugation (29X103 XG, Survall) and were lyOphilized with a lyOphilizer (Model 10-MR-TR, the Virtis Co., Gardiner, New York). The dried samples were further treated with perchloric acid (0.5 N) to reduce interference from enzymes (e.g. nucleases, nucleoside diphosphokinases and deoxyribonucleoside monophosphokinases which phOSphorylate dAMP, dGMP and 46 dCMP), and were then treated with potassium hydroxide (1.5 N) to neutralize perchloric acid. The extracts were further lyophilized and dissolved in double distilled water (1-2 ml), then stored in aliquots at -2o°c until dNTP determinations. The cell debris in the precipitation was used for determinations of DNA contents. The dNTP pools were measured, using the defined copolymers poly(dI-dC)- poly(dI-dC) and poly(dA-dT)-poly(dA‘-dT) as primer templates and _E_. 992 DNA polymerase I. This method works on the ability of DNA-polymerase in the presence of primer template to incorporate dNTP into an acid-insoluble product. When dCTP 3 was measured, poly(dI-dC)-poly(dI-dC) and excess H-dGTP were used. That is, when dCTP in the cell extracts was a limiting factor in the reaction, the extent of polymerization was measured by the incorporation of 3 H-dGTP into the primer template as an acid-insoluble product. The incorporation of radioactivity was directly prOportional to the amount of the limiting deoxyribonucleoside triphos- phate. When dGTP was measured, excess 3H-dCTP was used. For measurement of dATP or dTTP, primer template poly(dA-dT)-poly(dA-dT) and excess 3l~l.dTTP or 3H-dATP, respectively, were employed. The dNTP pools are expressed as picomole (pmol) per ug DNA for each nucleotide. For detailed dNTP measurment, see Appendix A. Unscheduled DNA Synthesis The quantitative assay for the excision repair capacities, that is, the unsched- uled DNA synthesis assay, was originally developed by Trosko and Yager (273), and it measures incision, excision and polymerization of DNA following damages by radiations or chemical mutagens. Cells were plated at a density of 3-6 x 106 per plate (6 cm, Corning Glass Works) in growth medium with 5% FCS and incubated overnight. The next day, cells were maintained in tyrosine deficient medium (GIBCO) and 5% dialyzed FCS for 24 hours; then they were maintained in a medium 47 lacking arginine and isoleuccine (GIBCO), but also supplemented with 5% dialyzed FCS (GIBCO), for another 48 hours. One hour prior to UV irradiation, hydroxyurea (5 mM, Sigma) was added to the medium to further arrest scheduled DNA synthesis. Before UV irradiation, the medium was collected and reserved. Cells were then exposed to 3H-TdR (5 “Ci/ml, 45 ci/mmol) in the reserved conditioned medium following UV irradiation. Cells were harvested (in PBS with a rubber policeman) after 1.5 or 3.0 hours of 3H-TdR incorporation, centrifuged and stored in the freezer for further analysis. The control cells received the same treatment except that they had no UV irradiation. All treatments were measured in duplicate or triplicate plates. Radioactivities were measured in DNA extracted by trichloracetic acid. The specific aCtiVitieS (dpm/ug DNA) of DNA repair capacities are expressed as the amount of radioactivities per unit of DNA. The radioactivities were measured in a counting fluid containing toluene and dioxene by a liquid scintillation spectrophotometer (LS 9000, Beckman). Mutation Induction 1. Mutagenesis with Ultraviolet Light Radiation. Cells were plated for attachment in medium supplemented with 5% FCS. Four hours later, culture medium was removed and cells were irradiated with various doses of UV from a germicidal lamp (General Electric, 25T8-Z5w) which delivers 1.4 J/mzlsec. Enough cells were plated so that each treatment resulted in survivors of 6 cells. Irradiated cells were then incubated in fresh medium at least 1 x 10 supplemented with 5% dialyzed FCS for 24 hours so that replication and repair of DNA were dependent entirely on endogenous nucleotides. After the DNA repair period, cells were grown in 5% FCS and refed with fresh medium every day to ensure optimal expression of mutants. In some experiments, thymidine (TdR) at 2-4 11M or aphidicolin at 0.5 11M was added to the growth medium 3 days after UV 48 irradiation to study the effect of TdR or aphidicolin on mutation expression. Survival rates of cells receiving the same UV doses were also determined concur- rently (see Cytotoxicity and Cloning Efficiency Determinations). In experiments to test the effects of error-free excision repair to the mutation induction by liquid holdings with conditioned medium, both the control and UV irradiated cells were incubated in the conditioned medium for various times during the DNA repair period, and then in the regular growth medium thereafter. The survival of UV irradiated cells was corrected by the plating efficiency of control cells which were grown in the same conditioned medium for the same duration but without the UV irradiation. When mutagenized cells reached 90% confluence, the cells were subcultured (one to 8 dilutions for the wild type and aphr-4-RZ cells; one to 4 dilutions for aphr- 4-2 cells) into flasks (75 cm2, Corning Glass Works) and similarly subcultured every two days thereafter. 2. Mutagenesis with X-ray Radiation. Cells were suspended in an ice-cold medium during X-ray irradiation (184 R/min), and then were grown in flasks (75 cm2, Corning Glass Works) with medium and 5% FCS. Cultures were never allowed to exceed 90% confluence without subculture and the growth medium was changed every day. Survival rates were determined concurrently. Mutation Assys Quantitative forward mutations were measured by 3 assay systems: mutations from sensitive to resistance to 6-thioguanine, ouabain and diphtheria toxin. The genetic loci involved are genes for hypoxanthine guanine phosphoribosyl transferase 49 (HGPRT), sodium-potassium adenosine triphosphotase (Na+/K+ ATPase), and elonga- tion factor-2 of protein synthesis respectively. Replating techniques were used throughout the experiments. After sufficient expression time, cells were trypsinized and replated at appropriate densities. Enough plates were used so that induced mutants were selected from a population of at least 106 cells. For ouabain resistant (ouar) cell selection, 4 x 105 cells were plated into each plate containing 1 mM ouabain (Sigma) with or without 4 11M TdR. For selection of 6TG'- mutants, cells were plated either at a density of I x 105 per plate (low cell-density selection) without TPA, or at a density of 1.0-1.5 x 106 per plate (high cell-density selection, for X-ray induced mutation) with TPA (0.01 ug/ ml, 4 days); and 6TGr mutants were selected by 6TG (6 uM) with or without 2 uM TdR. At these cell densities and drug concentrations, mutant-recoveries were more than 90% (37,38,92,l53,284,302). Selective medium was renewed every 3-4 days. For diphtheria toxin resistant (DTr) mutant selection, 1 x 105 cells were plated into each plate, and these cells were allowed to grow for 2 days so that each attached cell developed into 4-16 cell-stage. The medium was Changed and DT (0.1, 0.2 or 0.6 lf/ml) was added to each plate. The medium contained no nucleosides except those from 5% FCS. Four days later, DT was removed and colonies were allowed to grow for another 6-7 days. Under this selection procedure, the recovery of DTr colonies was identical to the recovery of those with DT present for the entire period of development (unpublished results). All resistant colonies were fixed with ethanol, stained with Giemsa and scored after 7,8 or 12 days of growth. Two or three replatings were performed in each experiment, and each experiment was also repeated at least twice. Some resistant colonies were isolated with a glass cylinder and trypsin, and retested for their resistance to individual drug or toxin. 50 Spontaneous Mutation Rate Determinations 1. Fluctuation Analysis. Spontaneous mutation rates in ouabain (ouar), diphtheria toxin (DTr) and 6- thioguanine (6TGr) resistant loci were determined by using fluctuation analysis (163,175) or modified Newcomb's replating technique (189,277,282). Clones derived from single cells of each cell line were isolated from culture dishes and were grown in 24-well plates (16 mm diameter per well, Costar, Massachusetts) or in plates (9 cm, Corning Glass Works, Corning, N.Y.) when more than 1x 106 were desired. Generally, 72 to 150 clones (replicate or sib cultures) were isolated per experiment and no more than 10 clones were from the same culture dish. When cells reached 90% confuence, these cultures were individually trypsinized and replated equally in three tissue culture plates. Mutants resistant to ouabain were selected by ouabain (1 mM) containing medium with 4 11M TdR that was changed every three days. For determinations of mutation rate at the 6TGr locus, cells were plated in triplicate plates (l-l.5 x 106 cells per plate) containing fresh medium and 2 11M TdR. Four hours later, TPA (Sigma Chemical Co., 10 ng/ml) and 6TG were added. The TPA was removed by medium change 4 days later. This concentration of TPA and duration of exposure are not toxic to V79 cells, but are able to eliminate metabolic cooperation which decreases the recovery of timr mutants (153,284,302). The 6- thioguanine or ouabain and TdR were present in the medium for the entire period of colony development (7 to 12 days). The DTr mutants were selected by a modified replating technique reported previously (234). Cells from each sib culture were plated at a density of less than 2x105 cells per plate; two days later, the medium was changed to a fresh one, and DT (0.6 lf/ml) was added. The toxin was removed four days later. The commonly used replating technique (39,92) was utilized in one experiment in which DT (0.2 lf/ml) was added four hours after replating and was present for three days. All DTr 51 clones thus selected were allowed an additional 6 days for colony development. No purine or pyrimidine except that in 5% FCS, was present in the medium. The cell number and plating efficiency were estimated simultaneously from a sample of randomly selected clones. The mutation rate was calculated according to Luria and Delbruck (163) using the Po estimation. 2. Multiple Replating Techrmue. Because expression of 6TGr or DTr mutants has a relatively long phenotypic lag, it is possible that the mutation rates in these loci may be underestimated. A modified Newcomb's technique (189,233,277,282) was utilized to determine the spon- taneous mutation rates at these loci. This technique consists of multiple mutation frequency determinations and concurrent cell-division monitorings at different times, and obtains the mutation rate from the increase in the mutation frequency of a cell population over a known number of cell divisions. A cell population of 2-5 x 107 cells derived from two single cells of each cell line was grown in medium supplemented with 5% FCS. For mutation frequency 6 determinations, a total of 2.4-3 x 10 cells from each cell line was replated for 6 cells per plate for 6TGr mutants; 4 x 105 per mutant selection (cell density: 1 x 10 plate for DTr mutants). 6-Thioguanine and TPA, or diphtheria toxin (0.6 lf/ml) were added four hours after cell plating. In addition to the cells that were plated for an 6 cells (aphr-4-2, 20 x 106; estimation of mutation frequency, a total of 10-20 x 10 V79, 10 x 106) was distributed to two culture flasks (150 cm2, Corning Glass Works). Cells in the flasks were subcultured when necessary and the total number of cells at each subculturing was determined as described above. The cell counts allow determinations of cell divisions occurring between two time points when mutation frequencies are estimated. The spontaneous mutation rate (a) was calculated from the equation: a=1n2[2(MFx-MFX_1)/ln(Nx/Nx_1)] where MF and N are mutation frequency and cell number respectively. The calculated mutation rate using this 52 equation allows a paired t-test comparison (249) between cell lines. All determina- tions were repeated. Mutation Frequency and Mutation Rate Calculations l. Mutation Frequencies. The frequency was determined by dividing the number of mutants selected from mutation assays by the total cells plated in eadi treatment, corrected by the plating efficiency. The frequency is expressed as numbers of mutants per 105 or 106 survivors (i.e., clonable cells). 2. Mutation Rates. A. Fluctuation Analysis. The average number of cell divisions (d) was calculated from the formulae: (Ni-No)/ln2, where Nf and No are final and initial cell numbers per replicate culture. The mutation rate was calculated on the basis of the mean number of mutants per replicate culture. There are three ways to obtain this mean: a) the Po estimation [mean = ln(l/Eo)] (163), where Po is the fraction 'of cultures with no mutants; b) the likely mean, CaNf/C, which is the average mutants per replicate culture assumed to arise from certain generations back when no mutation has yet occurred, and which can be obtained indirectly from the observed mean; and c) the arithmetic mean, r, which is the observed mutants per replicate culture. The means calculated from these methods are presented in RESULTS. Because r = aNfln(CaNf) (163) and Cr = CaNfln(CaNf) (32) where a is mutation rate and C is the number of replicate cultures, the mutation rate, a, is Cr/CNfln(CaNf) or CaNf/CNf. The value CaNf can be obtained from the tables provided by Capizzi and Jameson (32) if r and C are known. 53 The mutation rate was calculated from the equation: mean/cell divisions, and is expressed as mutants per cell per division. Only the mutation rate calculated by the _Po estimation has been presented. B. Multiple Replating Techniggg. The mutation rate is calculated from the equation derived from the original equation (the difference in mutation frequencies divided by the difference in cell numbers) by Newcomb (189,233,277,282). Because the number of mutants (m) observed in one particular replating time point (x) arise in one or more prior generation(s), mutation frequency (MFX) can be expressed as mx/l’sz and the difference in frequency would be mx/Vsz - mx-l/ysz-l’ or 2(MFx - MF ). The x-l change in cell generation is ln(Nx/Nx_l)/ln2 (282). The mutation rate can be obtained from [2(MFx - MFX_ 1)/ln(Nx/Nx-l)]ln2 (282). The mutation rate can also be calculated from the slope of the mutation frequency curve as a function of cell divisions (256). The mutation rate calculated from this equation is expressed as per cell per generation. RESULTS Isolation of UV-sensitive Aphidicolin-resistant Mutant and Its Revertants Cells (3 x 107), which were previously mutagenized with BrdU-black light and UV, were plated for selection with 1.0 uM aphidicolin. Four colonies survived the selection. One of these four aphr mutants is found to be sensitive to UV (40). This mutant (aphr-4) has been recloned. One of the recloned lines, aphr-4-2, was extensively studied in this dissertation. This UV sensitive mutant is slow-growing compared to V79 cells. Upon single cell plating, larger size colonies were observed. These were found to be TdR auxotrophic and UV-normal revertants. One of these revertants with a normal growth rate (designated aphr-4-R2) was chosen for study. In addition, when the mutant cells were plated in medium with 5% dialyzed FCS, few slow-growing survivors were found. These appear to be thymidine prototrophic revertants. Two revertants of this category designated aphr-4-RP4 and aphr-4-RP5 were also studied. The colony-forming abilities of these mutants and V79 cells in the presence of various concentrations of aphidicolin are shown in Figure l. The results indicate that aphr-4-Z and its revertants were resistant to aphidicolin compared to V79 cells. The V79 cells cannot grow in medium with more than 0.4 1.1M aphidicolin, whereas the revertants have more than 80% plating efficiency in the same medium. The figure also indicates that the colony-forming abilities of aphr-4-R2 cells decreased at 0.8 11M and those of aphr-4-2 and aphr-4-RP cells decreased at 1.0 11M aphidicolin. The results found here are consistent with the results reported by others in CHO (226) or V79 cells (40). 54 55 100 _§. \:\ r aph'-4-RP4 .| aph'-4-2 3i aph'-4-RP5 g 10 - Z 3 m 1 wild type 11 ”NJ-81 < 1 l l l T e 0 0.2 0.4 0.6 0.8 1.0 Aphidicolin Concentrations (1.1M) Figure l. Colony-forming abilities of aphr-mutants and wild type V79 cells in the presence of various concentrations of aphidicolin. Aphidicolin was present for the entire period of colony development. 56 Growth Rates Cells were plated at a density of 400 per plate (about 6 cells per cmz). Four hours after plating, 6 single cells per plate were Chosen for observation of cell division through the microsCOpe every 24 hours. The following results were found. The V79 cells divided every 14 hours, aphr-4-2 divided every 211 hours and aphr-4-R2 divided every 18 hours. The aphr-4-RP cells divided every 24 hours in the presence of 5% dialyzed FCS. In another experiment, the V79 and aphr-4-Z cells were plated in a density of 0.75 and 1.4 x 103 per cmz, respectively (0.5 and l x 105 cells per 9 cm plate) in medium with 5% FCS. Cells in duplicate plates were trypsinized and counted every 24 hours after plating. Figure 2 shows that aphr-4-2 cells double every 24 hours and the V79 cells every 14 hours. Cell counts at 72 hours showed non-exponential growth which may be due to nutrient depletion. Deoxyribonucleoside Triphosphate Pool Measurements The inhibition of aphidicolin on purified (it-polymerase has been shown to be competitive with deoxycytidine triphosphate (dCTP). To test the possibility that the resistance of aphr-mutants to aphidicolin might be due to elevated endogenous dCTP pools, the deoxyribonucleoside triphosphates (dNTP) were measured in aphr-4-2, aphr-4-R2 and the V79 cells. When the dNTP were extracted with methanol (60%) and assayed for dNTP levels, it was found that dCTP pools in aphr-4 and aphr-4-R2 are 3 to 8 times higher than those in V79 cells (40). However, when the methanol extracts were treated with 0.5 N perchloric acid which has been reported to eliminate enzymatic activities, such as nuclease, nucleoside diphosphokinase and deoxyribonucleoside monOphosphokinase involved in the conversion of dAMP, dGMP, and dCMP to its dNTP (192), the results (Table 3) were different from those reported previously. There appears to be no significant difference in dCTP in the V79, aphr-4-2, and aphr-4-R2 cells. In these experiments the exogenous dNTP was 57 10'5 - __ 0 wild type 2" = 14.4 hrs 0 — A aph '-4—2 2" = 24 hrs A I.“ j— < .J O. I I.“ Q U) _l _l 111 0 4 l—— 3 x 104 l l 1 o 24 as 72 TIME AFTER CELL PLATING (hrs) Figure 2. Growth rates for aphr -4- 2 and the wild type V79 cells in the growth medium supplemented with 5% F CS 2n =doubling time. a. C. 58 Table 3. Deoxyribonucleoside Triphosphate Pools in Aphr-mutants and V79 Cellsa'b' Cell lines dCTP dTTP dATP dGTP (Experiment #1) V79 15.6 .2.8 2.24 0.02 aphr—4-R2 7.9 2.9 0.94 0.04 (Experiment #21 V79 4.9 2.4 - - aphr—4-2 8.6 1.25 - — aphr-4-R2 4.0 10.0 - - V79 with Markersc' 17 7.5 - - Cells in confluency were plated at a density of 1.0—1.5 11 106 per plate to obtain log phase growth. One day (24 h) after replating in 5% FCS, cells were harvested; and dNTP were extrac- ted and assayed according to methods described in the text. pmole/ug DNA. -: not assayed. Deoxyribonucleotides (pmole) were added to V79 cells during cell harvesting: dCTP, 10; dTTP, 5. 59 also added to the wild type V79 cells during harvesting. The results indicate that exogenous dCTP and dTTP were not degraded by treatment with 0.5 N perchloric acid. Nutritional Requirements In a series of studies, Chang e_t _a_l., (40) have reported that aphr-4 requires deoxyuridine, deoxycytidine or thymidine for growth in medium with dialyzed FCS. It appears that the mutant is a thymidine auxotroph. Results presented in Tables 4 and 5 confirm that aphr-4-2 and aphr-4-R2 cannot grow in medium with 5% dialyzed FCS. In one experiment, aphr-4-2 had 38% colony-forming ability compared to 0.3% for aphr-4-R2. This may be due to the presence of prototrophic revertants in aphr- 4-2 cell population. When the medium was supplemented with 2 or 4 uM TdR, these two mutants grew as well as the wild type V79 cells. Addition of 5 11M of adenine deoxyribonucleoside (AdR) or guanine deoxyribonucleoside (GdR) did not support the growth of aphr-4-2 or aphr-4-R2 cell in 5% dFCS. Additions of TdR, AdR or GdR at a concentration of 100 (TM resulted in drastic cell killing for aphr-4-2 and aphr-4-R2. The cytotoxic effects of TdR, AdR or GdR at 100 11M were significantly less in V79 than in aphr cells. Additions of TdR (2 uM) to the medium, supplemented with AdR (5 or 100 uM) or GdR (5 1.1M), reversed the cytotoxic effects of AdR or GdR. Thymidine at 2 11M, however, did not reverse the cytotoxicity of 100 11M GdR. It is clear that aphr-4-2 and aphr-4- R2 are auxotrophic to TdR but are also sensitive to 100 11M TdR or GdR, and to 100 11M AdR if TdR (2 uM) is not added. Since fast-growing revertants of aphr-4 are all TdR auxotrophs and aphr-4 may harbor more than two mutations, it is possible that a second type of revertant exists. In order to search for this second type of prototrophic revertant, aphr-4-2 cells (400 cells/plate) were grown in medium supplemented with 5% dialyzed fetal 60 “mo .oe_moo_u3c0n_c>xooe .eONmON ace .1 N_m>_>u3m ucuuuoa mum econ caucumOLQ mu_3mo¢ oc_cm3m "xeu .ue_moo_u3c0n_c>xooe oc_coem ¢e< .oc_e_u>u .oc_e_eaca “gee .uc05ao_u>oe >co_ou mo eo_uoa au_ucu ecu Lo» moe_moo_u3con_u>xooe m3o_um> ecm mum eo~>_m_e um nu_3 euuc050_na3m 53_eo5 c_ oc__ __ou some $0 m__ou oo~._ mc_um_q >9 eoc_5u0uoe mum: mu_u___nm mc_5comt>co_oo o>_um_0c 0:5 - - mN - ea. c o - e . - m.e ee_ mm a NT a- can - - we - eN a o - e - oc— cc_ co. oc— mN> :* uc05_COQXm e mN as aN - - - a - a o - ee— _ NT a- can a a: m mm - - - e.o - a.o m._ - ee— m._ N a- can oN mm mm cap - r - _m - am co. - cc. co. mN> me acu5_COQXu m mm _w no. mm c o Nm m.o me e m__ co. NN maT a- can em m__ mm mm. m e 5 mm N NN e mm— ea. em aaT a- can a me as me ea. c e a e a o NN ee— m.e NT a- can _N Ne. mm N__ N e ..e NN e mN N Na ee— mm N a- can om mm mm No. as. c NN MN mm _N om Nm co. am mN> we uc05_uoax~ c No— mu a_p 1 t t t t t t 1 oo_ maxi :1 5am N c__ mN N_. - - - - - - - - co. aaT a- can a em me aN - - - - - - - - co. NT 4. can N Nm _c mm - - - - - - - - ee— N a- can mm Nm aN ma. - - - - r - - - cc. mN> 111. —* uc05_COQXm zncc_ 23m taco. 23m Nzaavxee 25a znoo_ 23m taco. 23m zucc— za: :3N «cu ¢o< Nz5eemu go «no ¢c< me» an» :3 N eu_3 ocoz noc__ __uu uo um um moe_moo_u3con_c>xooo m3o.um> ecm mum eou>_m.o Mm :u.3 emuc050_aa3m 53_eoz c. m__ou m~> ecm mucmu35t za< mo mo.u_._n< mc.5cowt>co_oo o>.um_o¢ .: a_nmh 61 Table 5. Relative Colony-forming Abilities of Aphr-mutants and V79 Cells in Meglum Suppgemented with Various Deoxyribonucleosides at 37 C and 34 C Medium supplemented with 5% dialyzed Cell lines (0C) 5% dialyzed FCS FCS + 2 uM TdR None AdR GdR Brdu None AdR GdR 100 uh 100 uM 100 uM 100 uM 100 uh V79 37 92 0 0 0 100 68 54 34 100 2 0 0 100 40 12 aphr-4-2 37 21 o 0 0 100 54 3 34 18 0 0 o 100 16 3 aph'-4-R2 37 0.1 o 0 o 100 29 o 34 o 0 0 0 100 23 0 aph'-4-RP4 37 76 0 0 0 100 58 o 34 53 o 0 0 100 23 o 5% dialyzed FCS 52 FCS thymidine (PMIV Cytidine 4mM AraC (pH) TdR 2 4 100 - (4uM) 0.2 0.5 V79 37 100 84 90 0 74 17 0 34 100 74 7o 0 100 - 0 aphr-4-2 37 100 97 7 0 10 66 o 34 100 69 0 0 3 - 0 aphr-4-R2 37 100 100 0.4 o 100 0 34 100 98 0 0 20 - 0 aphr-4-RP4 37 100 100 13 0 2 48 0 34 100 70 3 0 l7 - 0 The colony-forming abilities were determined by plating 1200 cells of each cell line in medium with various deoxyribonucleo- sides at either 340 or 37°C for the entire period of colony development. BrdU = bromodeoxyuridine. AraC - cytosine-l-B-D- arabinofuranoside. Results are percent survival; -, not tested. 62 calf serum. Ten surviving colonies were isolated two weeks later. All of them are sensitive to UV-irradiation as aphr-4-2 cells are (data not shown). Two of the ten prototrophic revertants (aphr-4-RP) were tested for their nutritional requirements. The results presented in Tables 4 and 5 indicate that aphr-4-RP4, and -RP5 grew well in medium with 5% dialyzed FCS, and that like the parental mutant they were sensitive to 100 11M of TdR, AdR or GdR in the absence of 2 11M TdR. Thymidine at 2 11M reversed the cytotoxicity of AdR for both aphr-4-RP revertants. Aphr-4- RP5 is sensitive to GdR (100 11M) whether or not TdR is present. In one of three experiments, aphr-4-RP4 was not sensitive to GdR (100 11M) when TdR was present. The reason for the discrepancy is not clear. To test whether the mutation expression is temperature sensitive, the nutri- tional requirements of aphr-4-Z, and aphr-4-R2 were observed at both 34°C and 37°C. Table 5 shows that at 34°C, the nutritional requirements, and TdR-, AdR-, and GdR-sensitivities of aphr mutant and revertants, were not at variance with the phenotype expressed at 37°C. When bromodeoxyuridine (BrdU) at 100 uM was added to growth medium supplemented with 5% dialyzed FCS, all the aphr mutants and revertants were as sensitive as the V79 cells to BrdU at 34°C or 37°C (Table 5). It appears that aphr-4- 2, its revertants and the wild type cells are not deficient in thymidine kinase. Table 5 also shows that aphr-4-2 and its revertants were as sensitive to 0.5 11M cytosine arabinoside (araC) as the wild type. Because aphr—4-Z and aphr-4-R2 cells are TdR auxotrophs and sensitive to TdR and GdR, they may be defective in ribonucleoside diphosphate reductase. Robert de Saint Vincent gt a_l., (278) reported that in a Chinese hamster CCL39 mutant deficient in deoxycytidine monOphosphate (dCMP) deaminase, a TdR auxotrophic phenotype can be created if uridine diphosphate reductase is inhibited by a high concentration of cytidine. Chang e_t gl_., (40) reported that the wild type V79 cells 63 used in our laboratory become thymidine auxotrophic in the presence of a high concentration of cytidine. The survivals of aphr-4-Z, aphr-4-RZ, aphr-4-RP4, -RP5 and the wild type V79 cells in 4 mM cytidine, with or without 4 uM TdR, were compared. The results, presented in Table 4, show that all cell lines could not grow in 4 mM cytidine. Additions of 4 uM TdR reversed the Cytotoxicities of cytidine in the wild type, aphr-4-R2 and aphr-4-RP5, but not in those of aphr-4-2 and aphr- 4-RP4. When cells were grown at 34°C, TdR partially reversed the cytotoxicity of cytidine in aphr-4-R2 (20% at 34°C compared to 100% survival at 37°C). Mutagen Sensitivities Since the aphr mutant could be due to a mutation at the gene encoding DNA polymerase a and since a-polymerase could be involved in the repair process, I was interested in the mutagen sensitivity of the aphr mutant. The results may provide some preliminary indication concerning the molecular basis of mutation. For this purpose the repair capabilities of aphr-4-2 and aphr-4-R2 were measured by determining the survival of these mutants after exposure to direct acting mutagens such as UV, X-rays, dimethyl sulfate (DMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-acetoxy-Z-acetylaminofluorene (NACAAF). Figures 3A and 3B show the results from 6 determinations using UV as a mutagen. As shown in Figures 3A and 3B aphr-4-R2 was as sensitive to UV as the wild type V79 cells, whereas aphr-4- Z and aphr-4-RP4 cells were more sensitive to UV irradiation. In one experiment, V79 cells had a higher UV survival (Fig. 4) than they did in the 6 previous experiments (Figs. 3A,3B); the reason is not known. The survival of the aphr-4 mutants and the wild type cells after X-ray irradiations or DMS-treatment is shown in Figures 4 and 5A respectively. The results clearly show that aphr-4-2 and aphr-4- R2 were not more sensitive to X-rays or DMS than were V79 cells. At doses higher than 500 rads, aphr-4-R2 had a slightly lower survival than V79 cells. When MNNG 64 100 F *3 30 1— 10 \ 1 wild type \ aph' -4-R2 IjjTj 1 I Survival PM (0 l / \ II aph' -4-2 IIIFI /~ I _ .1 .3 '- _1 1 l 1 A 0 4.2 8.4 12.6 16.8 uv dose (J/m2) Figure 3A. Survival curves of UV-irradiated aphr-4-2 (A), aphr-4-R2 (D) and wild type Chinese hamster V79 cells (0). Cells were allowed to repair in the growth medium supplemented with 5% dFCS for one day and then were grown in the same medium supplemented with 5% FCS. Mean and standard errors of 4 experiments are shown. 65 100 30 10 Survival (°/o) ,3 v 1 1 l l J o 4.2 8.4 12.6 16.8 UV dose (J/m2) Figure 3B. Survival curves of UV-irradiated aphr-4-2 (A,A), aphr-4-RP4 (v) and wild type V79 cells (0,.) in medium with (closed symbols) or without (open symbols) TdR (4 11M). 66 100 H‘l non' 4M 30 >- 3 W ’— \ 20 )- r ,_ a 3 __ \ '- l" \ i— \ 3, g A “I ‘ 2 ‘0 ’- . P— F I 09.0 uv Control . 0.1-unmet I i— O I ‘ no.“ uv - Contain-o. p 0 mid typo o l 1 b— I -. ’ 6 "WI-2 ‘ l L r) 09""‘l2 I J' p— } i- J 1 L L 2 i ll cl 4 1 1 _1 4 J O ‘ '3 4 '9 ' 0 100 200 300 000 500 600 700 , 1 UV now 3.01 14.1608. lads Figure 4. Survival curves of UV-(left), X-ray-(right) irradiated aphr-mutants and wild type V79 cells. Benzamide (2 mM) was added during the repair period for 3 days in medium supplemented with 5% FCS. 67 .mUm can 53 3958233 E3608 532w 2: 5 998 m .3 voted :32 wctzu 823m 83 :25 my mEEmNcmm .mzvu mu> ~93 33 28 macmqudcam Bump: AUvum<OcmueumL “tum ac.um~l Uta CO umbioim. k0 waveguw .w 0~Obh 69 .A~e\s e.mv >3 eu_z ee.m_eeee_ see; n__ou .e .moumu__a_eu mo >uco_u_mmu mEum—a cameo; 05 mm vmucmmoea new: .3 33¢ ....cman—gmu >co_ou mo voted 33cm 05 .8”— acomoea mm: o:_ommmu .>_o>_uumamme m>mu ¢ ucm m Lo; ucommea mew: (ah new on_Em~com .mc_um_aoe eoumm mesa; : nouum cum: mc_ommmu new u_mcon m um ovum—a new: m_.ou .m emm see ems see cum amo gum emu mm um om mm _m om on um «e .uaxm so am _m am _* .uexm ~-:-emmm m~> N-s-eeem m~> A_e\mn _e.ev ~-s-eeam ~-:-eeem m~> ine-eeam Nis-eeam m~> ze m.e Axe m~.ev «e_eeeeu _oeueou .m me we Nm om mm on em.o _s me am a: mm me m~.e . i . om mm mu a me mm mm Nm _N am me e Ne mN mo RN _m _m um c _* .uexu mmumnummm ~-e-eeee m~> ~¢-s-eemm ~-e-eeem m~> oe__ __eu Axe Ne ee_Em~eem eu_z _oeueou A” omznv .< om__ou mm> ma>h 6..) new mucmuasi ca< mo ANV >ucu_u_mmu mc_um_m on» :0 oc_ommmum Lo .mu.Em~com .A0cmumumeumh :o ~_ mo muuommm .o e_nmh 70 Table 7. Effects of Benzamide (2 mM) on the Relative Coalony-forming Ability of Mutagen-treated Aph -mutants and V79 Cells Mutagen (dose) Ratiob UV (J/mz) V79 aphr-0-2 aphr-0-R2 0 1.00 1.00 0.96 0.2 .95 0.98 0.83 8.0 .98 0.79 0.89 12.6 1.00 0.56 0.62 16.8 1.00 0.77 0.59 X-ray (rads) 0 1.00 1.00 1.00 100 0.99 0.82 0.77 300 0.96 0.60 0.70 500 0.76 0.56 0.05 700 0.88 0.38 0.50 MNNG (ug/mi) 0 1.00 1.00 1.00 0.2 1.00 .75 0.60 0.0 .58 .32 0.10 0.8 <.02 <.01 <.01 1.6 <.01 <.01 <.01 NACAAF (pg/ml) 0 1.00 1.00 1.00 0.1 0.99 0.91 0.93 0.2 1.00 0.72 0.90 0.5 1.11 0.90 1.07 1.0 0.87 - C ' 1.50 DMS (um) 0 1.00 1.00 1.00 80 .09 .59 .50 100 .51 .00 .25 120 .38 .38 .20 a. Benzamide (2 mM) was added to medium with 52 FCS in triplicate plates at the time of cell plating. Four hours later, cells (> 1200 cells/3 plates) were exposed to mutagens except X-rays. Cells were exposed to benzamide one hour after X-ray irradiation. Benzamide was present for 2-0 days after mutagen treatments. b. Ratio - Survival (2) with benzamide/survival (2) without benzamide. c. Not determined. 71 UV, X-ray or MNNG. Benzamide essentially gave similar enhancements of cell- killings to V79 and aphr-0-2 cells when DMS was used. it did not enhance the lethal effect of NACAAF in any cell line. Table 8 shows the effect of caffeine (0.25 or 0.5 mM) on the survival of UV- irradiated V79 and aphr cells. The concentrations of caffeine used were not toxic to V79 and aphr-tl-RZ cells, but they appear to be slightly toxic to the aphr-ll-Z cells (Table 6). Although caffeine at either 0.25 or 0.5 mM enhances the lethal effect of UV in both cell lines, it appears that the effect was slightly greater in aphr-0-2 cells than in V79 cells. This is shown (Table 8) by the ratio of survival with caffeine to survival without caffeine at low UV doses (0.2-12.6 J/mz), and the difference may not be statistically significant. Cytotoxicities of Chemicals Used in Mutation Assays ln mutation assay studies, mutants were selected with different selecting agents in the presence or absence of exogenous thymidine or TPA. Experiments were carried out to determine whether there would be a differential effect of these chemicals on the plating efficiency of mutant and V79 cells. Table 6 shows that TPA at a concentration of 0.01 ug/ml did not affect the plating efficiency in V79 and aphr-ll-Z cells, whether they were irradiated or not irradiated by UV (8.0 J/mz). Table 9 shows that the three selection agents, i.e., ouabain (oua), 6-thioguanine (6TG) or diphtheria toxin (DT) are equally toxic to aphr- 0-2, aphr-ll-RZ and V79 cells. Additions of TdR to oua, 6TG and DT media did not alter the cell-killing effect of these drugs. Aphr-0-2 and aphr-0-RPO cells apparently had a high frequency of spontaneous mutants resistant to oua, 6TG or DT. Resistant cells selected by these drugs were also isolated and grown in 20-well plates in the absence of the drugs for several generations, before being retested 72 Table 8. Effects of Caffeine on UV-irradiated Chinese Hamster Cells1 Expt. No. Cell lines UV dose Survival (2) (J/mz) Control + Caffeine2‘3' Ratio“ 1 v79 0 100 100 1.00 0.2 58 05 .76 8.0 01 . 6.0 .16 12.6 13 0.6 .05 16.8 3 0.2 .07 aphr-0-2 0 100 100 1.00 0.2 55 32 .58 8.1 22 5 .23 12.6 7 1 .11 16.8 2 0.1 .05 2 V79 0 100 100 1.00 1.2 98 77 .79 8.0 17 22 .17 12.6 11 5 .15 16.8 3 1 .33 aphr-0-2 0 100 100 1.00 0.2 67 00 .60 8.0 22 7 .32 12.6 3 1 .33 16.8 0.7 0.3 .13 aphr-0-R2 0 100 100 1.00 0.2 95 63 .66 8.1 51 27 .53 12.6 19 8 .12 16.8 7 2 .29 Cells were grown in 5% dFCS i caffeine for 20 hours after UV-irradia- tion, then were grown in medium with 5% FCS and caffeine. Expt. #1 caffeine = 0. 5 mM, plating efficiency (2) without UV: V79 60 (control), 07 (with caffeine), aphr -0- 2: 50 (control), 31 (with caffeine). Expt. #2 caffeine = 0.25 mM, plating efficiency (2) without UV: V79 88 (control) and 70 (with caffeine), aphr -0- 2: 59 (control) and 05 (with caffeine), aphr --:0R2 88 (control) and 81 (with caffeine). Ratio - Z survival with caffeine/2 survival without caffeine. If; - 1 left! C. m.~mu mk> DEM .1 1:1 C_¢£GJO .QC.CGJQO.£F10 &O n ocuauaxOuOuxu .m 0:1: 73 .mc_um_a __00 00000 m>00 N 00 meno: : 00000 m0: he :05} uncmcu go: 0.0 he no >u_u_xo~00>u 05h .00um0u yo: 0 0.: .m__0u 0_nm_> o. 500 m0_co_ou m0 00mm0eQX0 0003 me0nE:z : .0mc0cu 53.002 >n 00>OE0L mm? c050 m>0u : e00 uc0m0ea mm: e_xOu e_c0cusq_c .000: 0003 m__0u mo. x m0 .m__00 0_nm_> m .m__0u 0_nm_> m op 00a m0mco_00 m0 00mm0LQx0 0003 mc0nE:c 020 new 00um_a 0L0: m__0u m o_ 00a m0_co_ou mo mc0n53c m0 00mm0cax0 0L0: mc0n53c 0:0 0:0 n0um_a 0003 m__0u mop x mg a. x N._u .uc0an_0>0u >co_ou mo uo_c0a 0e_uc0 0:0 .00 mango _m:0_>_0:_ mc_c_0uc00 0_00E c_ A50 my m0um_q 0um_a_eu c_ m__0u mc_um_a >9 00c_ee0u00 0003 m0_u___am mc_50001>co_ou 0chm 0.: NN NN — a a + u.e e, _m s e N - seeie-eeem 0.: Nv N m _ N + 0.: mv mv _v _v _ i Nmiaiezam m m Nv Nv N m mN o. m. N. + N s m m N m_ N. e. m m - Nieieeem 00um0u mco_umeuc0ucou qv : Nv N N + __m e_ me.o eeeu was. ev e m m Nv - mN> e._ m.e N.e ..e me.e e._ m.e N. e m z: N .efi_e\esv e_xoe e_eeeuee_e .u.mAxev e_memso .e.mflz:v «e_eesmo_ee-e ee_e_2>ee ee_4 __eu sy_z u0uc0S0_aa:m e_u0: «e_e_e>ee :1 N usoeu_: eo eu_z mum ”m e0_z eeueeee_aesm h_eez e_ m__eu mN> eee mucmuasieca< cu e_xOF a_e0nuca_o 0:0 e_mnmao .0c_cm:mo_ch1c mo m0_u_u_xouo»>u .m 0.20h 70 with the same selective agent. Table 10 shows that all mutants tested are stable and heritable for their mutant phenotype. Two 6TGr mutants from aphr-0-RPO were resistant to HAT medium (06). To determine whether cell densities used for mutation assays in the present studies would affect the recoveries of ouar, 6TGr or DTr mutants, reconstruction experiments were carried out, using spontaneous or UV induced mutant clones. Table 11 shows that when ouar cells were cocultivated with ouas cells and selected by ouabain medium, the recovery rate of ouar was more than 8096, compared to the experiment in which only ouar cells were plated. The recovery of 6TGr or DTr mutants from the cocultivating of mutants and 6TGs or DTs cells was 100%. The experiment using DTr mutants, derived from aphr-0-2, gave the same results. Ultraviolet Light Induced Mutation A preliminary mutation study at the ouabain-resistant locus has been reported by Chang it. 31., (00). These studies, using an i_q §_i_t_Ll_ technique, have shown that aphr-0-2 is hypermutable at the Na+/K+-ATPase locus. in the studies presented in this thesis, a replating technique was used to determine the mutation frequencies in three loci, i.e., ouabain-, diphtheria toxin- and 6—thioguanine- resistant loci. l. Ouabain-resistant Mutations. The mutation frequencies at the optimal expression time for different UV doses from 0 experiments are presented in Figure 6. Results within each individual experiment or from the combined data clearly showed that the UV-sensitive aphidicolin-resistant mutant, aphr-ll-Z, is hypermutable at the Na+/K+-ATPase locus. The range of mutability of aphr-ll-RZ was found to be overlapping with that of V79 cells. Figures 7 and 8 show the mutation frequencies at different expression times from two representative experiments. in general, the mutation expression time at this locus is not significantly different between mutant cell and V79 lines. 75 Table 10. Stabilities of Spontaneous Ouar, 6TGr and DTr Mutants A. r Oua Clones Medium Supplemented with 5% F05 Ouabain (1 mM), 52 FCS Cell line None 0 uM thymidine None 0 uM thymidine V79 13 13 13 13 aphr-0-2 39 39 39 39 18 18 18 18 17 17 17 17 aphr-0-R2 6 6 6 6 B. 6TGr Clones Medium Supplemented with 5% FCS None 6TG (6 uM) HAT 5% dFCS V79 9 9 0 7 aphr-0-2 8 8 0 8 aphr-0-R2 7 7 0 0 aphr-0-RPO 9 9 2 8 C. DTr Clones Medium Supplemented with 5% FCS 52 FCS + 0T (2 lf/ml) aphr-0-2 22 22 aphr-0-RPO 9 9 Clones were isolated individually with a glass cylinder and re- plated onto muti-well plates in medium without selection agents. Three days later, individual selection agents were added to the The numbers presented here are the well with fresh medium. number of viable clones. HAT: medium contains hypoxanthine, aminopterin and thymidine (06). 76 .A0um_a Eu m Lou c—x_ . . V m who .mc—x_ .mohm “mo—x: .mmao “0000.0 m__0u 0>_0_mc0m 00 L0nE:z N .mc_um_a __00 L0 . . .m0c. . 000 mL30n 0 0 ._ __00 0>_u_mc0m __0 c_ >uc0_u_mw0 mc_um_a >0 uNHW0mwou0mwmzmquwu wwwuuo_0m m__0u .— Aee_xv N: A N1 1 am :N .umuawcwm>:v “m ANieiLzamV mc.x_ Leo_xv mm c .e N1 1 can . mp .03M0cwunoamv wm Amlaleamv aopxm a .0 ea AmeeLS __eu .eam. L . .Lov ho 0c_4 . Ace—V :m ANlci cam Lam A __0uv who N.m2004 e_xOF 0_L0;u;a_o .... _m .u0uauc%1>:v om ANiqchamv :o_x:.m Lee—v _m LN-e- e66 am e .e N L . _m amusee_->=v em AmN>V se_xm N .6 who Am0c_4 __0u .mc_m. L . .Lov who 0:. Aamv :m ANx a L A .4 __00V woke Numsuou 0c_cm:mo_chim ... 0m .UQUDWCWJ>MV om AN¢1:IL£QMV mo—xm.N Aoa.v we AN-4- ea. c .6 m4 .Umuancfll>=v om A~1J1Lcamv mo—XN.N Loe_v NM LN-s- e66 m e .6 RN .m300cmuaoamv ”m AN1:1Lcamv mc—XN.N Ace—V —0 AmN> .UQUDUC_I>DV cm a .0 mm Amm> .u0uauc_1>=v om Amm>v mo_x:—.m Aowv MN Am~> .m300cmucoamv cm a n mN AmN> .maoocmucoamv om AmN>V mo_xqp.m o .0 mac AhmeLS __0u .me_m1 L . _Lov 0:0 0 ANV 00L0>000¢ m__0u L A e_g __0uv mmao Num3004 e_mnmao .. 0um_m L0m m__0u _ m__0u mm) 0cm mucmuaei ca< 0>_u_mc0m1m:La c. m ”0 ucmum mlec xOP m Lona I . . _I I . 2030 m0 m0.L0>000¢ .—— 0 _ I —nm Induced ouar mutants per 106 survivors Figure 6. 77 350 ~ A 300 - 250 - 2. /’ 200 1- I A x 150 - ’ M I ‘ a 100 - ° 2/ , I A ‘ uv dose (J/m2) Dose response of UV-induced ouar mutation frequencies in aphr-0 (X), aphr-0-2 (A’A), aphr-1-R2 (13,-) and V79 (0,.) cells. Mutagenized cells were grown in medium with (closed symbols) or without (open symbols) 0 mM TdR during expression and selection per' . The background frequencies in all cell lines were less than 20 x 10’ and were subtracted from the induced frequencies (0 experiments). 78 500— ‘L’ 400 - A 3 \ E ‘ A 1llJ/m2 6: \ . '9 .. o 300 ‘ \A/ "' x g \\ IIA 7J/m2 \ I 2 ‘4’ g 200 - a E w o—--- 14J 2 g )— o”’ 0 /m 100 2 ’,o ----- o 7J/m o”’ ...L ' 0.1/m2 o l g” T 8 J 0 2 4 6 8 10 Expression time (days) Figure 7. Expressions of UV-induced ouar mutants in aphr-0-2 (A,A) and V79 (0,.) cells. Results from control (closed symbols) and UV-irradiated cells (open symbols, 7 ll/m2 and 10 J/mz) are presented. 79 300 '- 2 g /A\ “3" 2001- ’/ \ a A \\ \ «a \\ 3 \\ 3 e A110/1112 a. 2. 5 2 _ cl— —- — — — — — —- a / a 11 J/m f 100- / 0 3 / 0 //°\\ 1- / \ ~ 0 \ \ \ ~611J/m2 0.1/1112 0H,: L—L L 1 I 4 0 8 10 12 14 16 18 Expression time (days) Figure 8. Expressions of UV-induced ouar mutants in aphr-0-2 (A ,A), aphr-0-R2 (0,!) and V79 (0,.) cells. Results from control (closed symbol) and UV irradiated cells (open symbols, ll J/mz) are presented. 80 When mutation frequencies are presented as a function of cytotoxicity of UV damage (Fig. 9), aphr-0-2 showed a higher mutation frequency than did V79 and aphr-0-R2 at the same level of survival. The results are similar to those plotted according to UV doses, except that a biphasic curve was obtained for aphr-0-2 (Fig. 9) instead of a linear curve (Fig. 6). 2. Diphtheria Toxin-resistant Mutations. The UV-induced mutability of aphr-ll-Z and its revertant, aphr-0-R2, was also studied in the assay system involving mutations from diphtheria toxin sensitivity to resistance. The results (N=0) presented in Fig. 10 indicate that aphr-0-2 is also hypermutable at the diphtheria toxin-resistant locus. Similar to a previous report (230), the optimal mutation expression time in this system was about 5 to 7 days for both wild type V79 and the mutant cell lines (Fig. 11). When mutation frequencies were plotted according to survivals in logarithmic scales, the aphr-0-2 was found to have a higher mutation frequency than both the wild type and its revertant at the same survival level (Fig. 12). Unlike the ouabain-resistant mutations, the mutation induction curve at the diphtheria toxin-resistant locus is linear instead of biphasic. 3. 6-Thioguanine-resistant Mutations. The hypoxanthine guanine phosphoribosyl transferase (HGPRT) mutation assay system in the Chinese hamster cell is perhaps the most commonly used and the best described 1_n_ Vitro mammalian assay system. The prominent features of the system are the cell density effect due to metabolic cooperation, the long expression time (about 7 days) and the stable expression of mutation frequencies after the Optimal expression time. This mutation assay system was also used (low cell-density selection) to study the mutability of aphr mutants. The results showed (Figs. 13 and 10) that the wild type V79 cells indeed had stable mutation frequencies after the six-day expression time. Unexpectedly, the mutation frequencies of the mutant, UV-induced ouar mutants per 105 survivors. Figure 9. 25 81 A 20 v. A 15 A A A 10 ° 0 O a O a 5 I O O 8 ' 0 O k 1 1 100 30 10 3 Survival (°/o) UV-induced ouar mutant frequencies of aphr-0 (X), aphr-ll-Z (A ), aphr- 0-R2 (D) and V79 (0) cells as a function of cytotoxicities of UV damage. Mutagenized cells were grown in medium with (closed symbols) or without (open symbols) 0 llM TdR during expression and selection period. DTr mutants par105 survivors Figure 10. 82 800 r- 700 '- 600 '- 500 - / 400 - / 300 — 7’ 200 - / 100 / l l I l L 0 2 4 6 8 10 12 14 UV dose (J/mz) Dose res onse of UV-induced DTr mutation frequencies in aphr-0-2 (A), aph ~0-R2 (0) and V79 (O) cells. The background frequencies were not subtracted from induced mutation frequencies. 83 400— 2! .- O .2 g 300— /4\\ 1” / \ “3’ ‘ // ‘\‘ 11J/ 2 t ,/ '“ 3 200_ ‘ .9 C 2 P 3 E E 100- r; _ 2’47__°~ § fl; 0.1/"‘12 _ “‘ ~6 11J/m2 .L_/; 1 ~L LOJ/mz 0 5 7 . 9 Expression time (days) Figure 11. Expression of UV-induced DTr mutants in aphr-0-2 (A) and V79 (0) cells. Results from control (closed symbols) and UV-irradiated cells (open symbols, ll J/mz) are shown. UV-lnduced DTr mutants per 105 survivors Figure 12. 80 d Survival (°/o) UV-induced DTr mutant frequencies of aphr-0-2 (A), aphr-0-R2 (CI) and V79 (0) cells as a function of cytotoxicities of UV damage. Results from 0 experiments are shown. Background frequencies have been subtracted. 85 500 F- A _ f 0 J/m2 I 400 — / /' 2 " I A/ / 4 J/m2 300 — I z / A _ A // A/ 2 200 1— 5 O 3 .. z 3 10 ca 100 — 3 g .. a 0 J/m2 E I 1 Ad 2 3 E 9 a g, 300 1— e ’ g—-—-_ 8 J/m2 o 200 — O ---~~ - ‘8 4 J/m2 100 - .. 8\3 0 J/m2 l 1 l 1 o 2 4 e 8 Expression time (days) Figure 13. Expressions of UV-induced 6TGr mutants in aphr-ll-Z (A,A) and V79 (0,.) cells. Mutagenized cells were grown in medium with (closed symbols) or without (Open symbols) 2 11M TdR during expression and selection period. 86 300 " Aph'-4-2 ,A 14.1/m2 600 - I/ / ’ - 7J/m2 / ,I“ A’ ,”’ 400 '- A” 200 - .... 0.1 2 ‘L/L l 1 k 1 l L l L/m] 6-TG' mutants per 106 survivors 600 - Wild-type O-"“""'-'o 14.1/m2 400 i- o ------- O 7J/m2‘ 200 i- 0.1/1m2 1 IL I a k 1 I ‘1 l L J 0 6 8 1o 12 14 Expression time (days) Figure 10. Expressions of UV-induced 6TGr mutants in aphr-0-2 (A) and V79 (0) cells. ' 87 aphr-0-2, continued to increase 8 days after UV-irradiation. This is exemplified by the result from two experiments shown in Figs. 13 and 10. Because the frequency of fast growing revertants will increase in the population of aphr-0-2 after an extended period of growth, no assay was done beyond 10 days after UV-irradiation. There- fore, the optimal expression time for aphr-0-2 cannot be determined from these results. However, when the maximum mutation frequencies of aphr-0-2 were compared with those of the wild type, aphr-0-2 clearly had a higher mutation frequency than did V79 for a given dose of UV (Fig. 15, reconstructed from Fig. 13). The revertant, aphr-0-R2, was similar to the wild type in UV mutability (data not shown). When all the mutation frequencies of the mutant, the revertant and the V79 cells were plotted according to their survival (Fig.16, N=0), a smooth curvilinear response curve, similar to that of a previous report using CHO (305) or V79 cells (300), was obtained. There appears to be no discernible difference between the wild type (V79) and the mutant (aphr-0-2), or its revertant (aphr-li-RZ). 0. Effect of Thymidine on Mutation Expression. Both aphr-0-2 and aphr-0-R2 are thymidine auxotrOphs. . They grow in a medium with 5% non-dialyzed FCS, but not with 5% dialyzed FCS. in the present experiments, the cells were grown in medium containing no exogenous nucleosides, but supplemented with 5% non-dialyzed FCS one day after UV-irradiation. In some experiments, exogenous TdR (2-0 uM) with or without AdR (2 11M), was added during expression and selection periods to determine whether exogenous deoxyribo- nucleosides affect mutation expression. The results shown in Figures 6, 9, l3, and 16 indicate that exogenous TdR did not affect mutation expression for ouar and 6TG,r mutants. The same results were obtained when aphidicolin (0.5 uM) or exogenous AdR was tested (data not shown). The data, concerning the expression of ouar, DTr and 6TGr mutants induced by UV, are shown in Appendix B. 88 .0020: 8300.0... 0:0 83.0050 wctsu m3. 2: N 238% :83 3053 Lo 23E? .0083 5:: E3006 E 9.6% 0.03 0:00 00~_:0m3:_2 6:00 Aflov a; 0:0 7 .qv NiaiLzam E 88:030.: 830sz L95 000:0:_i>3 No 00:080.. 080 .2 0.53m ANS: 68.. >3 0 e N o _ _ q \ l 8. o \ 1 SN 0 \4 1 Sn s l oov \ L can "'10 sionyuns 901 led stuetnul 191-9 peonpul 89 UV - induced G-TG' mutants per 105 survivors J 1 1 1 1 i 100 80 60 40 30 20 10 Survival (%) Figure 16. UV-induced 6TGr mutant frequencies of aphr-li (X), aphr-li-Z (A), aphr- li-RZ (0 ) and V79 (0) cells as a function of cytotoxicities of UV damage. Mutagenized cells were grown in medium with (closed symbols) or without (open symbols) 2 uM TdR. The background mutation frequen- cies have been subtracted from the induced mutation frequencies. Results from 14 experiments are presented. 9O X-ray Induced Mutation Table 12 shows the results from two experiments using X-ray as a mutagen. The results show that X-rays can induce mutation at two loci, namely 6TGr and DTr loci (39). Because the X-ray sensitivity of aphr-ll-Z is the same as that of the V79 cells, similar X-ray induced 6TG.r or DTr mutation frequency in both cell lines was expected. Table 12 shows that at an equal X-ray dose, 6TGr mutation frequencies in aphr-ll-Z cells were slightly lower than those in the V79 cells. On the other hand, DTr mutation frequencies were either the same for both cell lines, or lower in aphr- #-2 than in the wild type V79 cells. Repair Capabilities Measured by Liquid Holdilg Recoveries and Unscheduled DNA Syntheses 1. Liquid Holding Recoveries by Conditioned Medium. The mutants and the wild type V79 cells were tested for their ability to repair the UV-induced DNA damage. This was done by studying the reduction in cytotoxicity and mutation frequencies after DNA replication arrest by growing cells in the conditioned medium for various hours (186). It appears (Table 13) that the conditioned medium exposure reduced the plating efficiency of each cell line receiving no UV-irradiation (these cells being the control group). If plating efficiencies of the control group be taken as 100% survival, then, of the treated group receiving UV-irradiation, the wild type V79 cells showed enhancement of survivals at doses between l4.2-12.6 and at 12-20 hours of liquid holding (Table 13; Fig. 17). oh the other hand, aphr-ll-Z and aphr-ll-RP5 cells did not show improved survival at all: rather, the rate of survival decreased whether or not the conditioned medium was toxic. The TdR auxotrophic revertants, aphr-4- R2, improved survivals as it is in the wild type V79 cells. Figure 17 shows that the UV survivals of V79 and aphr-ll-RZ cells at two doses (8.4, 12.6 J/mz) were enhanced 91 Table 12. X-ray-induced Mutation Frequencies in Aphr-mutants and V79 Cells Expt. No. Cell X-ray Survival Expression Mutation Frequency lines dose time per 106 Survivors (Loci) (rads) (2) (days) (No. of Mutants) 1. (675’) v79 o 100 8 ' 26 (27) 735 7 265 (265) o 10 30 (110) 735 220 (207) aphr-h o 100 8 5 (7) 735 9 100 (198) o 10 3 (6) 735 137 (287) (01') V79 0 s 15 (23) 735 39 (138) 0 8 8 (8) 735 67 (67) aphr-h o 5 1 (1) 735 35 (32) 0 8 l (l) 735 37 (#0) 2. (676') V79 0 100 7 57 (103) 700 8 135 (353) o 9 , 71 (137) 700 262 (599) aphr-h-Z O 100 7 2 (5) 700 8 llh (I98) 0 9 3 (10) 700 116 (290) (DT') v79 0 7 1.4 (1) 700 74 (76) 0 9 <2 (0) 700 I83 (168) aphr-h-z o 7 6 (15) 700 137 (98) o 9 3 (3) 700 172 (163) Expt. No. low cell-density selection for 6TGr locus; DT-0.2 lf/ml. Expt. No. 2: high cell-density selection for 6TG locus; DT=0.6 lf/ml. Table 13. Experiment #l V79 aph'-h-2 Experiment #2 V79 aphr-h-Z aphr-h-Rz Experiment #3 V79 aphr-h-Z aphr-h-RZ aphr-h-Rps Liquid Holdin 92 Recoveries (2 Survival) by Conditioned Medium in Aph -mutants and V79 Cells UV2 (J/m ) 0 3 5 9 (hrS) o (110) (92) (110) o 100 100 100 7 hi 30 80‘ 0 (63) (#8) (18) o 100 100 100 7 1o 7 15 o 10 17 (hrS) 0 (88) - - o 100 13 11 29 29 o (59) o 100 13 3 h h 0 (88) o 100 l3 I9 29 2h 0 h 8 12 (hrs) 0 (#7) (A6) (55) (29) o 100 100 100 100 8.h 50 8k 7k 80 12.6 28 21 10 57 0 (48) (59) (50) (55) o 100 100 100 100 8.5 22 14 13 15 12.6 6 3 2 2 0 (61) (57) (Sh) (#7) o 100 lOO 100 100 8.h 60 75 98 90 12.6 in 52 till 55 0 (103) (99) (99) (110) o 100 100 100 100 8.5 46 55 59 56 12.6 19 18 20 21 93 Table 13. continued UV Experiment #9 (J/mz) 0 3 6 9 12 16 20 (hrs) V79 0 (90) (92) (76) (71) (73) (77) (75) 0 100 100 lOO 100 100 100 100 8.h 87 79 92 93 99 116 99 16.8 32 2) 31 35 35 33 32 aphr-‘l-Z 0 (85) (7‘1) (66) (66) (70) (52) (52) 0 100 100 100 100 100 100 100 h.2 8h 82 63 75 81 82 66 8.4 23 23 20 26 26 28 19 Numbers in parentheses are plating efficiencies (Z) of non UV- irradiated cells with the same treatment of conditioned medium; and these cells served as control. Colony-forming abilities were expressed as survival (2) calculated by dividing the plating efficiency of cells with UV-irradiation by that of the control cells. 94 100 30 10 Survival ( %) L.H. \ Control (12 h) o wildtypo 0 D aph'—4-R2 I \\ f A aph -4-2 A ‘ i> aph'-4-RP5 . 1 I l L 0 4.2 8.4 12.6 16.8 UV dose (J/rnz) Figure 17. Survival curves of UV-irradiated aphr-ll-Z, aphr-ll-RZ, aphr-ll-RPS and V79 cells with (closed) or without (open) liquid holding by conditioned medium for 12 hours. 95 upon liquid holding for 12 hours, whereas those of aphr-ll-Z or aphr-ll-RPS were reduced or had no change respectively. Since the UV survival of the wild type increased to 8096 after liquid holding for 12 hours from 5096 without liquid holding, the mutation frequency of ouar or DTr mutants in the wild type V79 and aphr-ll-Z cells was assayed. At a dose of 8.8 J/m2 (Fig. 18) UV induced 5O (ouar) or 80 (0'1") mutants per 106 clonable V79 cells (survivors), as compared to 130 (ouar) or 225 (DT') mutants per lo6 clonable aph'-a-2 cells. Upon liquid holding of l2 hours the mutation frequency was reduced to 10 (ouar) or O(DTr) per 106 clonable V79 cells, whereas the frequency was increased to 225 from 130 (ouar) or to 375 from 225 (DTr) per 106 clonable aphr-(l-Z cells (Fig. 18). The mutation enhancement after liquid holding in aphr-(l-Z was repeatable (data not shown) in a second experiment. At a dose of 16.8 J/mz, the 12-hour-liquid holding did not change uv survival (Table 13, Expt. {#1) or mutation frequency in the wild type V79 cells (data not shown). It appears that liquid holding recovery in the V79 cells was dose dependent. 2. Unscheduled DNA Syntheses. To study the capacity of excision repair in these cell lines, the unscheduled DNA synthesis (UDS) induced by UV was measured in confluent cultures treated with tyrosine-arginine-deficient medium and hydroxyurea to reduce the normal DNA synthesis. The method developed by Trosko and Yagger (273) detects the incision, excision and polymerization steps of excision repair by measuring the incorporation of 3H-thymidine into DNA. Figure 19 indicates that the UDS as measured by 3H- TdR incorporations at two UV doses of aphr-li-Z, aphr-li-RZ, aphr-ll-RPQ and V79 cells was the same whether measured for 1.5 or 3.0 hours of 3l-l--TdR incorporation after UV irradiation. 96 ;I a 400 - g, — g, 32 _OM' t; ._ or' 3 5." 2 ‘a E it :i 350.? e' - 7:— e g . 2251 g. g. -- E 3 “n g zoo — —— a a u. A '(r 5% .. i. .. 3a - "— .- §; 100 )— _ii- 2 L rm 5 5 7 a _J_ cl)- . '_E i S -- _ § 2 ,, _ __ \ é. \ 3 l- 4)- § 8 100 L _rL Figure 18. Mutation frequencies for aphr-ll-Z and V79 cells at ouar (left) or D‘l'r (right) locus. Mutation frequencies not induced by UV with or without liquid holding (L.H.) by conditioned medium were indicated as the background frequency and were located below the zero line. Mutation frequencies induced by UV without L.H. (Open box) served as control. UV-induced mutation frequencies with liquid holding of 12 hours (shaded box) were located above the zero line on the right side of each corgrol. Survivals are presented in experiment No. 3, Table 13, (UV = 8.4 J/m ) Specific Activity (0PM lug DNA) 97 60 - A 40 - v on o 20 - a o ./D - v o L l J l O 4 8 12 16 100 r- B '8 80)- 60)- a 40 - A — o 20 - g 0 l L IL J 0 4 8 12 16 uv dose(.l/m2) Figure 19. UV-induced unsdieduled DNA synthesis as measured bv 3H-TdR in- cOrporatlons for aphr-tl-Z (A). aph'-4-R2 (0). aphr-4-Rpa (v) and V79 (0) cells at 1.5 (A) or 3.0 (B) hours after UV irradiation. mi 98 Spontaneous Mutation Rate Determinations l. Fluctuation Analyses. Tables Ill-16 show the results of fluctuation analyses at ouar, DTr, and 6TGr loci. In experiments involving ouar mutant selection, the medium was supplemented with 4 uM TdR, although TdR has been shown not to affect the mutation frequency for ouar locus of V79, aphr-tl-Z and aphr-(l-RZ cells. The results presented in Table 14 show that aphr-ll-Z and aphr-(i-RPS cells had a higher spontaneous mutation rate than V79 and aphr-ll-RZ cells. Because the growth rates of aphr-(i-Z clones were not homogenous [three groups of clones were separated and their doubling times were approximately 24(F), 36(1), and more than (18 hours (5)], the assay of ouar mutation rates was performed separately in these three groups of cells. In spite of their growth characteristics, the spontaneous mutation rates in this locus were found to be higher in aph'-a-2 cells than in v79 or aphr-li-RZ cells. Spontaneous mutation rates, determined by fluctuation analysis, at DTr locus in v79, aphr-ll-Z, aphr-ll-RZ and aphr-Q-RPQ cells are shown in Table 15. The mutation rate obtained for the V79 was 2.!) and 8.5 x 15"8 per cell per division from two experiments. The UV sensitive aphr-li-Z and aphr—ll-RPll cells had a mutation rate 6 to 10 times higher than the wild type cells. As was found in the ouar locus, the revertant, aphr-ll-RZ, had a mutation rate of 8 x 10-9, slightly lower than that of V79 cells. Table 16 shows the results of mutation rate determination at the HGPRT locus. To eliminate metabolic cooperation between 6TGr and 6TGS cells, TPA was added to the medium during mutant selection so that more cells per culture could be assayed. This method not only reduces the experimental costs, but also allows for mutation rate determination in more cell divisions. This table shows that aphr-(l—Z and aphr-(l-RPQ had four- to nine-fold higher mutation rates than the wild type V79 cells (see Appendix C for hypothesis on comparisons between mutation rates calculated from Bo estimations). 99 Table 15. Fluctuation Analysis for V79, Aphr-mutants at Ouar Locus"2 Experiment #1 Cell line: v79 aphr-h—Z aphr-h-Rz aphr-h-RPS Replicate cultures (C): 32 #8 (I)3 41 #0 Cell numbers per culture: initial (No) 6 1 1 1 1 final (Nf) (x10 ) 6 2.7 0.9 1.5 6.9 divisions (d) (x10 ) 3.9 1.3 2.2 9.9 Po 0.88 0.58 0.98 0.075 ouar (total (5) (129) (1) (573) per culture: range 0-3 0-60 0-1 0-125 variance 0.35 77.2 0.02 782 mean 0.19 2.58 0.02 16.8 ln(l/Po) 0.13 0.50 0.02 2.6 CaNf/C 0.13 0.75 0.04 3.88 Mutation rate per cell '8 '8 '8 '8 per division: 3.3x10 hl.9x10 1.1x10 26.2x10 Experiment #2 Cell line: V79 aphr-h-z aphr-h-Rz Replicate cultures (C): 36 18(F)3 22(5)3 21(F)h 21(1)“ Cell division per culture (x10 ) 3.8 2.1 5.4 2.7 0.2 Po r 0.56 0.22 0.15 0.76 0.81 ua (total) (35) (103) (391) (10) (8) mean 0.972 5.72 17.8 0.08 0.19 ln(l/Po) 0.58 1.5 1.97 0.27 0.21 CaNf/C 0.37 1.85 3.97 0.27 0.16 Mutation rate per cell per division: 16x10“8 73x10'8 115x10'8 mm"8 5xlo'8 1. Enough numbers of replicate cultures originating from single cells were isolated from each cell line after 6- 10 days of growth. Each culture was then plated to 2 wells (16 mm diameter) in 24-well plates (Costar, Cambridge, Mass. ). When each culture reached nearly confluency, suffi- cient numbers of cultures were trypsinized individually with 1 ml trypsin (0.05%) and distributed equally into 3 plates (9 cm, Corning Glass, Corning, NY) for mutant selection. Another 6-10 cultures were individually counted, plated to 3 plates as above, and were recounted 5 hours later, or at the time of drug additions. Plating efficiency was 1002 for the wild type cells and 80% for aph r-h- 2 and its revertants. Nf/C reflects correction by plating efficiency. Mutation rate determi- nations were calculated from equations formulated by Luria and Delbruck (163) i.e., a a ln(l/Po)/divisions. 2. Selection lmM ouabain and 5 uM TdR. 3. Doubling time (hrs) - F, 25; l, 36, h. Doubling time (hrs) - F, 16; I, 18. s, 118. Tabl Expel Ce Rel Ce P0 of Fe: Cal Mu Expe| Ce Re; Ce 100 Table 15. Fluctuation Analysis for v79, Aphr-mutants at DT' Locu51°2°3° Experiment #1 Cell line: v79 aphr-0-2 aphr-0-R2 aphr-0-RP5 Replicate cultures (C): 26 22 37 28 Cell numbers per culture: initial (No) 6 1 1 1 1 final (Nf) (x10 )6 2.3 3.8 5 2.5 division (d) (x10 ) 3.3 5.0 7.2 3.6 Po 0.923 0.318 0.906 0.07 DTr (total) (2) (016) (6) (1797) Per culture: range 0-1 0-163 O-S 0-575 variance 0.07 1061.5 0.68 15520 mean 0.08 18.91 0.16 60.2 ln(l/Po) 0.08 1.15 0.06 2.60 CaNf/C 0.07 0.18 0.11 11.16 Mutation rate per cell -8 -8 _9 -8 per division: 2.0x10 21.2x10 7.7x10 73.9x10 Experiment #2 Cell line: V79 aphr-0-2 Replicate cultures (C): 30 30 Cell division per culture (x10 ) 3.6 3.6 Po 0.70 0.12 DTr (total) (19) (“29) mean 0.559 12.6 ln(l/Po) 0.307 2.10 CaNf/C 0.258 2.77 Mutation rate per cell -8 -8 per division: 8.5x10 59x10 See Table 10. Mutants (Experiment #1) were selected by DT [0.2 lf/ml (3 days)] for the V79 and aph -0- R2 cell lines. These mutants were kept in growth medium for additional 6 days without 0T until colonies were developed, whereas DTr from aph -0- 2 and aph -0- RPO were grown in fresh selective medium (DT - 0. 6 lf/ml) for the entire period of colony development. Experiment #2. DTr mutants were selected by _i__rl presence of DT (0.6 lf/ml) for 0 days. situ method in the Ta 101 Table 16. Fluctuation Analysis for V79, Aphr-mutants at 6TGr Locus1'2° Cell lines: V79 aphr-0-2 aphr-0-RPO Replicate cultures (C): 50 18* 26* 21 Cell numbers per culture: initiaI (No) 6 1 2 , 1 2 final (Nf) (x10 ) 6 3 1.7 3.1 2 divisions (d) (x10 ) 0.3 2.0 0.5 2.9 Po 0.91 0.778 0.385 0.762 6TGr (total) (11) (86) (1867) (111) Per culture: range 0-0 0-67 0-802 0-05 variance 0.53 230.3 37625.0 165 mean 0.200 0.78 71.8 5.03 1n(1/Po) 0.097 0.251 0.956 0.272 CaNf/C 0.113 1.06 12.03 1.55 Mutation rate per cell -8 -8 -8 -8 per division: 2.3x10 10.5x10 21x10 9.0x10 See Table 10. 2. Each replicate culture was originated from one or tevo single cells. Mutants were selected at a cell density of 1-1.5 x 10 cells per plate (9 cm). TPA (10 ng/ml) was present for four days, 6TG (6 pm) and TdR (2 uM) were present for the entire period of colony development. 2.11: than 1 rates. type ( single locus< than 1 dhdsio nmnbe aphr-h Mgher revert; 109 C6 When r ume-p rate (u Table J locUs ir indicgtG this 10c than 11K 81 In this . 102 2. Multiple Replating. Because the expression time of 6TGr and DTr mutations is longer (5-8 days) than that of ouar mutation, multiple replatings were also used to obtain mutation rates in 6TGr and DTr loci. To avoid a high frequency of fast growing revertants, such as the aphr-0-R2 type (cell doubling time 16-18 hours), a population derived from two slow growing single cells of aphr or V79 cell line was used. The mutation frequency in the 6TGr locus of V79 and aphr-li-RZ (Fig. 20) rose linearly when the cell divisions were less than 1014. 10 An equilibrium in mutation frequencies was reached beyond 10 cell- divisions in these two cell lines. This phenomenon is commonly observed when the number of initial cells is small (256). The mutation frequency in single cell clones of aphr-(i-Z reached this equilibrium earlier (approximately at 109 divisions), but was higher than the wild type V79 and aphr-li-RZ cells. Because the fast growing revertants in the aphr-ll-Z population rose from 196 at 107 cell-divisions to 10% at 109 cell-divisions, no mutation frequency determinations was made thereafter. When mutation rates, calculated from two mutation frequencies determined at two time-points in early cell-divisions, were compared, aphr-ll-Z had a higher mutation 6 per cell per generation) than the V79 or aphr-ll-RZ (1.0 x 10-6) cells. rate (10 x 10' Table 17 summarizes the spontaneous mutation rate determinations at the HGPRT locus in aphr-ll-RZ, aph'-tl-2 and the wild type v79 cells. The paired t—test (249) indicates that there was no significant difference in spontaneous mutation rates at this locus for aphr-ll-RZ and V79 cells, and that the rates are significantly lower than those of the UV-sensitive mutants aphr-ll-Z. Similar results were also obtained in DTr locus for aphr-ll-Z cells (Figure 21). In this experiment, only one DTr mutant from the wild type V79 cells (at 5 x 1010 cell-divisions) appeared in 5 mutation frequency determinations, whereas aphr-0-2 6 5 7 9 cells gave mutation frequencies ranging between 10' to 10' at 10 -10 cell- m¢o>.>¢3m Oo- KWQ m52(h3: .Uhc @30w2(> 201% Figure 103 CELL DIVISIONS m 8 107 10° 109 101° to“ to12 10‘3 to“ 1015 > g 1 1 1 1 1 1 1 3 30 __ o wildtypoll 47:10“) _ "5: D “IN-443219100") c 25 .... a, A emu-211x195) " g 20 _ (Initial Cell Number = 21 E “101111 cell-density selection ‘ .— g 15 1- -—1 ----- O E ‘0 _ .......... o 0 fl 0 ------- .—o in ————— a . ...... in '— ‘ z ( '5 <1 0 I 0) Figure 20. Spontaneous mutation rate determinations for aphr-li-Z (A), aphr-(l-RZ (a) and V79 (0) cells at the 6TGr locus by multiple replating ted'iniqu . Mutation frequencies were determined at low cell-density (*, 5 x10 , 60-72 plates) 'thout T PA (Experiment No. 1, Table 17) or at high cell- density (1 x 10 , 3 plates) with TPA (Experiment No. 2, Table 17). USU—c300? mc_um.amm a_a_u_32 mc_m: ALUholmImv n:0.0mc.ELmumo mumm £05000): maomtmutouw .N~ 0~omh 100 wtc— x c.. n c005 «to. x :.m mic. x o_.__ o.c_ x mo— mlc— x m . muo— x N— N— mc— x mm— mio— x N m 91:. x no m mc— x mo— : mic. x —m — me. x mm: Ntc— x mo.m n c008 oto— x o._ one. x m—.m wc— x mm: 515— x _ m can. x mm N me. x mo— - x 0 etc. mo — me. x _a— l slap x N mv mop x NN coherxxm Nu: Nz o-e_ x me.m ea. x x O 019— me m me. x x O 0-e_ _m _ me. x - N e-o_ x me.~ me. x X a X ere. we _ we. are. x N mv me. x r N _uz _z m.— A000_m 00a m__0u a o— x m .mco_uu0_0m xu_mc0or__0o 30_V _ .oz 0:05.00mxu ~e-e-ceao mn> m0c__ __0u 0:u_czu0h mc_um_q0¢ 0_a_u_:z mc_m= Auuhotmzmv meo_umc_eu0uoo 0umm co_umu:z maoocmuconm .N— 0_nmh 105 . 2—z\~zvc_2.0:1~053~E "0.00 0.00 5 0300300 050 O0 0503000 0000—30—00 0003 00000 03000:... 0c< 0:300:20. 100000 >0c0za000 co_000:E 000000 000 0m0_0 0:0 00 m0_0:0:0000 co_000:E 000 N0: 0 0: .0000530 __00 000 N2 a .2m .co_00c_s00000 0: £000 000 0000.0 m .000_a 00a oo_ x p 00 >0_mc00 ..00 0 00 m>00 0:00 000 a_E\m: _o.cv (ah c0_3 00>o_aE0 0003 000.000.00 >0_mc001__00 cm_:~ .00001m.000_0a >0 m0c__ __00 0000 c_ Amo.o v av 0:00000_0 >__00_0m_000m 0o: 000 0:0 co_000000m 00a __00 00a mco_000:s mm 00mm00Qx0 0003 00000 co_000ae 05h .0c0au000 00.00035 0000 000 0000—0 Nu 0o om .000_a 000 c— x my m0_0_mc00 __00 3o. 00 00000.00 0003 ohm o0 0:00m_m00 m__0u c _ mic. x _.c— u :005 010— x mo.m mic, x m.~ mo_ x m.m 019. x :m._ no. x mm.— mto— x mm.m mto_ x m.~ mo— x m.m miop x a.— mo. x m.m mic. x _o _ m1o_ x 0 _ mop x m m oto_ x am F mo_ x mm — . 0-e_ x em._ 0e. x mm._ - N Nierceao 510— x :m.m a 0005 sic— x mo.o 010. x mm.m 0.0. x m.m 010— x m—.m mop x m.m 010. x m~._ crop x mm.m a_o— x m.m 010— x m—.m mo. x m.m 1 wto— x m— 0 ma. x m m 1 N ~1o_ x mm.m mlo— x 54.. a_o— x 4._ mic. x m.._ mo_ x a.— 1 m1o_ x mF.— me. x 0.0 1 ~ mm) cohorxzm N0: NZ .0: _z m.~A0_mc001__00 cm_£v N .oz 0c0E_00QXm 000.. __0u 003000000 .5. 0.00h 106 CELL DIVISIONS m 107 10a 109 10‘0 10“ § 10 I I [rrTITr ] r [TUTTI] I I III!!!" I l IIIIT S 1- C 3 “1,0 .- 3 0 wild type($ 2x10'7) x - 11‘ to 0 A A aph'-4-211.7-4.1xlo'51 )- \ z \ 1‘. s— \ \ D \ a __ \ '5 _ ‘4 in 3 o .- A/A m 2 ( h - 3 ‘% 0.0 o --------- o ------------------- c-O ----- ¢--$ Figure 21. Spontaneous mutation rate determinations for aphr-li-Z (A) and V79 cells (0) a3 the DTr locus. The cell-density for each determination was 0 x 10 , 6 plates. 107 divisions. The spontaneous mutation rate at this locus, calculated for aphr-0-2, was 5 per cell per generation compared to < 2 x 10'7 for V79 cells. 2-0 x 10- The results from both loci suggest that mutations in aphr-(i-Z cells occurred at a higher rate in earlier cell-divisions than those in V79 or aphr-ll-RZ cells. This is reasonable in light of the appearance of fast-growing revertants in the mutant population. The spontaneous hypermutability of the UV-sensitive aphr-ll-Z cells detected by this method was consistent with that observed using fluctuation analyses. Together they showed the hypermutability of aphr-li-Z is not loci specific as reported in a different mutant (175) of CHO cells. DISCUSSION Induction of Aphidicolin-resistant Mutants Aphidicolin-resistant mutants were isolated from a BrdU/black-light and UV mutagenized population with a frequency of 0.5 x 10'7 (00). One of these mutants is UV-sensitive (uvs). The frequency for this uvs mutant is then 1.1 x 10”. The latter frequency is similar to that of MNNG-induced diaminOpurine resistant (dapr, APRT') mutants of CHO cells (01). In a cell line derived from a transitional cell cancer epithelium (2533, 2n=60-80 with two active X chromosomes and chromosome 16, Dr. C—c. Chang, unpublished results and see Refs. 77,96), 6TGr or dapr mutants were isolated at a frequency of 2 x 10'7 from a BrdU/black-light and UV muta- genized population (Liu, unpublished results). Because 6TGr or dapr is recessive, it takes two mutations for the expression of the mutant. The similarity in mutation induction frequency of the mutants mentioned above indicates that the aphidicolin resistance of aphr-ll might be recessive and that it takes at least two mutations to create such a mutant. Mutagen sensitive (UVS) repair deficient mutants reported by Thompson e_t a_l., (260,265) are also recessive, though the phenotypes of these mutants are clearly different. This UV-sensitive aphr mutant appears to be the first mutagen-sensitive mutant isolated from mammalian cells by a selective agent. Characterization of Aphidicolin-resistant Mutants Characterization 0f aph'Lq and its thymidine auxotrophic revertant has been reported (00). This dissertation carried out further characterization and confirma- tion. The results are presented in Table 18. 108 109 Table 18. Characterizations of Aphr-mutants and V79 Cells at 37°C3 Aphr-Mutantsb Cell Lines V79 0-2 0-R2 0-RPO 0-RP5 1) AraC (0.5 uM) 2) Aphidicolin s .R R R R L050 (pH) (0.25) (0.6) (0.54) (0.8) (0.5) 3) Thymidine P A A p p 0) Thymidine (100 uM) R S S S S 5) Cytidine (0 mM) (with 8 uM TdR) N s N s N 6) BrdU (100 pm) 5 s s s - 7) CdR (100 pH) (with 2 uM TdR) N S S S S 8) Mutagen Sensitivities (+ Benzamide)c UV N(N) S(N) N(N) S(-) S(-) X-ray N(N) N(S) N(5) ‘ ' DMS N(S) N(S) N(S) - - MNNG N(S) S(S) S(SS) - - NACAAF N(N) S(NC) S(NC) - - 9) Mutability UV N H N - - X-rays N N - - - 10) UDS (UV) N N N N - 11) Liquid holding (12 hrs., UV) Recoveries: (Survival) NC (Mutation) D E - - - m U rn I Z I Z I I 12) Spontaneous Mutation Rate aA, Auxotroph; araC, Cytosine Arabinoside; BrdU, Bromodeoxy- uridine; D, Decreased; E, Enhanced; GdR, Deoxyguanosine; H, High; N, Normal; R, Resistant; S, Sensitive; 55, Very Sensitive; NC, No Change in Sensitivity; -, Not Tested. bSubclones of aphr-0; results were compared to those:of V79 cells. cCompared to those without benzamide. 110 Results of cytidine toxicity with or without thymidine (TdR), as suggested by Chang gt g. (00), demonstrated that a) the wild type V79 cells used in this study are probably deficient in deoxycytidine monophosphate (dCMP) deaminase, because in the presence of cytidine, wild type V79 cells become TdR auxotrophs as they do in a dCMP deaminase deficient mutant of CCL39 cell lines (278); b) the wild type V79 cells, due to deficiency in dCMP deaminase, possibly synthesize dTTP through the uridine diphosphate (UDP) reductase pathway. The fact that the wild type becomes a TdR auxotroph in the presence of cytidine, which elevates cytidine triphosphate and inhibits UDP reductase, strongly suggests that this UDP reductase pathway is the only one used by V79 cells to synthesize dTTP (Fig. 22). Aphr-0-2 and aphr-li- RPO cells were not able to grow in medium containing a high concentration of cytidine and a low concentration of thymidine. The mechanism for this cytidine toxicity is still unknown. Since thymidine was present in the medium, it is not likely due to dTTP deficiency. On the other hand, a high concentration of cytidine could increase dTTP or dCTP to a toxic concentration in these mutant cells, as postulated previously. This hypothesis can be tested by direct measurement of dNTP pools after'the addition of cytidine. The difference in responses between these mutants and V79 cells cannot be due to thymidine sensitivity, since aphr-ll-Z and aphr-(l-RZ are similar in thymidine sensitivity but different in cytidine sensitivity. Revertants aphr-(i-RZ and aphr-0-RP5 are similar to V79 cells in cytidine sensitivity but different in aphidicolin sensitivity. Therefore, thymidine auxotrophy and aphidicolin sensitivity are not necessarily associated with cytidine sensitivity. In fact, two subgroups of aphr-ll can be classified according to different nucleoside sensitities: namely, TdR auxotrophs (aphr-(l-Z and aphr-ll-RZ) and TdR prototrophs (aphr-li-RP); and, in the presence of thymidine, cytidine sensitive (aphr-ll-Z and aphr-0-RPO) and cytidine resistant (aphr-ll-RZ and aphr-0-RP5). Because in the presence of TdR (2 11M) the aphr-0-2 cells have small numbers of surviving clones in the presence of 111 0:00 00:05:00.: 5 80:30.08 0.0.28.0: .2. 05000 0.00553 8 8" 3 09:0 08.020.00.53 .3: l 02... 26: 00 000000053 d “0.00.1 0200 p — p 05 atoillvulvaseo Al 02:0 N 050 as 05 000:. . 03.000.80.032 0:” 00004 “02.208000 0.000050% " 00.00.02.000 . . . 0:0 .000 . 0:0 0200 ll 0000 000 2.0 08:2 8.0000 ‘ 0.50 ‘ d 0002.3 05 0000 1.1111 .300 Alli-:30 1111 020 9.0002200 .: 0 0 112 TdR, AdR or GdR at 100 11M, it is possible that revertants resistant to GdR, AdR or TdR can also be isolated. In the present studies, TdR auxotrophic phenotype and cytidine toxicity are not reversible at a lower temperature in aphr-ll-Z and in its thymidine auxotrophic revertants, aphr-ll-RZ. It appears that the mutated enzyme for TdR auxotrophs is not temperature sensitive, as are other aphr mutants of mouse FM3A cells (8). In fact, cytotoxicity of cytidine for aphr-ll-RZ is even more drastic at 30°C in the presence of TdR than at 37°C. It is possible that UDP reductase of aphr-ll-RZ becomes leaky at 30°C, or that the sensitivity resulting from the feedback inhibition of dCTP is higher at 311°C than at 37°C. Guanine- or adenine-deoxynucleoside (GdR or AdR respectively) and thymi- dine sensitivities of the aphr mutant and its revertants are possibly a result of defects in the subunits of deoxyribonucleotide reductase. Mutants of mouse cells with sensitivities toward exogenous GdR, AdR or TdR have been reported to have a defective deoxyribonucleotide reductase (288). It has been suggested that this reductase is an enzyme complex of four subunits. If the original mutant, aphr-(l-Z, contains 2 mutations in the gene(s) coding for these subunits, these mutations may be unstable and easily give rise to revertants. Alternatively, if aphr-ll-Z resulted from one mutation but is a non-locus-specific mutator mutant as shown in this study, it can introduce additional mutations which suppress the mutant phenotype such as growth rate, cytidine- or mutagen-sensitivities. From results of the present studies, it is not possible to distinguish between these two alternatives, although the suppressor mutation may be a favorable explanation. Deoxycytidine triphosphate (dCTP) pools of aphr -11—2 and aphr-ll-RZ found in this research are different from those reported previously (00). The dCTP pools of both aphr-(l-Z and aphr-ll-RZ were reported to be 6 to 8 times higher than those of wild type V79 cells (00). In the present studies, the procedure of biochemical assay 113 is the same as in previous experiments except that during the collection of cell extracts: a) three cell lines were partially synchronized by confluency, twenty-four hours before harvesting; b) the methanol extracts were treated with 0.5 N perchlo- ric acid to eliminate enzymatic activities which have been reported to interfere with dNTP measurements (192); and c) the dNTP markers externally added to wild type cell extracts survived treatments of 0.5 N perchloric acid. Because intra- cellular dNTP levels are finely tuned according to cell cycles, the possibility exists that cell extracts were collected at different cell cycles and thus led to the discrepancy. Nevertheless, there seems to be no difference in dCTP pools between the mutant and revertant. The discrepancy of dCTP levels between the values reported ((10) and the values found here in both aphr-ll-Z and aphr-ll-RZ cells, does not affect the hypothesis on the mechanism of mutabilities in aphr-li-Z cells, that will be presented later. Although aphr-ll-Z cells have twofold higher dCTP pools than the wild type V79 cells, this result could be interpreted as due to a difference in growth rate. The result could explain: a) why there is the same araC sensitivity of aphr and V79 cells; and b) why aphr-0-2 and aphr-ll-RZ are not more resistant to MNNG or DMS than V79 cells. An elevated endogenous dCTP level would reduce the toxicity of MNNG, DMS or araC, and enhance the survival (00,170,176,278) in the presence of these chemicals. Mutagen-sensitivity and Its Modification by Various Chemicals Preliminary studies on mutagens other than UV irradiation suggest that aphr- 4-2 and aphr-(l-RZ are sensitive toward specific mutagens. As compared to the wild type V79 cells, these two aphr mutants are not more sensitive to DMS. Aphr-ll-Z and aphr-ll-RZ are sensitive to MNNG and NACAAF. The difference in X-ray 110 sensitivity of the v79, aphr-0-2 and aphr-0-R2 may not be significant. It is premature to draw any conclusion from these studies, but several speculative possibilities will be discussed. Dimethyl sulfate produces methylated adenine or guanine as does MNNG, and the repair mode is base excision repair [short patch repair of X-ray-like mutagens (ll1,112,1l0,200,220,220,262)]. The DNA lesions caused by NACAAF are generally bulky and are repaired by long patch nucleotide excision repair (UV-like mutagens) (111,112,200,220), though AAF adducts on DNA may cause inhibition to methylation (209). It appears that aphr-0-2 and aphr-0-R2 are not nucleotide repair deficient, because UV-induced unscheduled DNA synthesis (UDS) in these two mutants is the same as in the V79 cells. Aphr-0-2 is possibly not a repair mutant but rather it may possess a defective nucleotide excision repair pathway which is rendered error-prone by a certain mechanism. In the case when chemical mutagens are used, the DNA lesions are different with different mutagens. Other than DNA adducts and single-strand breaks, ionizing radiation cuases free radicals which interact with cellular protein; MNNG reacts with the thio group of proteins and DMS reacts with the oxygen atom of the macromolecules (150). These adducts can possibly produce lethal effects. On the other hand, when DNA methylation adducts are compared, MNNG causes more O6MeG (796 of total methylated bases) than DMS does of O6MeG (0.396 of total methylated bases) at the same level of survival (150,173,188). It is believed that O6MeG is involved in mispairings which are responsible for lethal and mutagenic effects. Extracellular thymidine has been shown to enhance cytotoxicity of MNNG 6MeG (170). Because both aphr-0-2 and aphr-0-R2 by increasing mispairings with O are TdR auxotrophs, and dTTP pools of these cells in medium supplemented with 5% FCS are similar to the wild type V79 cells (00, and this report), the hypersensitivity of aphr-0-2 and aphr-ll-RZ to MNNG cannot be due to endogenous dTTP pools. 115 The higher sensitivity of aph'-11-2 and aphr-0-R2 to MNNG could be due to the mispairing pr0perties of O6MeG, or to corrections of this mispairing. Because 6 O MeG is considered to be responsible for mutagenicity (188), and if the observed 6MeG, aphr-mutants would have a hypersensivity in the present study is due to O MNNG-induced hypermutability. Indeed, they are hypermutable at ouar and DTr loci (data not shown). While aphr-0-2 is sensitive to both UV and NACAAF, aphr-0-R2 has different sensitivities toward UV and NACAAF. No clear conclusion can be made from this observation. Repair of UV and NACAAF cells is not additive in V79 Chinese hamster cells (1) indicating different repair enzymes are necessary to repair these two types of damage. The DNA repair mutants of V79 cells reported by Schultz 9131., (233- 235) show different sensitivities to UV, X-ray, NACAAF or MNNG. Compared to wild type V79 cells, the mutant strain UVS7 (deficient in UV-induced UDS) is UV sensitive and hypermutable for UV-induced mutations, but not sensitive to NACAAF, or to X-ray; strain UVr23 is UV resistant, but normal in NACAAF or X-ray sensitivity. Other strains, UVS0O and UVS00, are sensitive to UV, X-ray, MNNG and NACAAF (generalized mutagen-sensitivity). Data from the mutagen sensitivity in aphr-0-2 and aphr-0-R2 further suggest that they are different from those nucleo- tide excision repair mutants (e.g., UVS7) reported by Schultz _t 21: In an attempt to further investigate possible mechanisms of genetic defects in aphr mutants, the role of benzamide, a potent inhibitor of poly(ADP-ribose) polymerase, was tested during DNA repair period on cell survivals. The enhance- ment of cytotoxicity of N-methyl-N-nitrosourea (187), DMS (70,237) and ionizing radiation (187) by inhibitors of poly(ADP-ribose) polymerase (212) has been shown to result from the inhibition of poly(ADP-ribose) synthesis. In the present studies, benzamide significantly enhances the cytotoxicity of MNNG and DMS in wild type V79 cells; and this agrees with the results of Jacobson 116 gt a: (130), who reported that the rejoining of MNNG-induced strand breaks is defective in nicotinamide adenine dinucleotide (NAD) depleted mouse 3T3 cells. It also agrees with the results of Durkacz st 211., (70,71) and Shall gt 3., (237), who reported that 3-aminobenzamide enhances cytotoxicity of DMS and prevents strand rejoining in mouse L1210 leukaemia lymphoblast cells. Benzamide slightly enhances lethality of X-ray (less than 1 krad)—irradiated V79 wild type cells and this is in agreement with Nduka gt a_l., (187) who observed that 3-aminobenzamide also slightly reduces survival of 7 radiation (less than 1 krad)-treated L1210 cells. Benzamide inhibits poly(ADP-ribose) polymerase which can be stimulated by single-strand breaks in a dose-responsive manner (10,15). Excision repair of UV- and NACAAF-induced damage, and ionizing radiation at low doses might not sufficiently activate poly(ADP-ribose) synthesis; therefore, any inhibition may not be detectable by measuring cell survival. The difference in cytotoxicity enhancements by benzamide in these two alkylating agents and low dose ionizing radiation treated V79 cells, may also be due to specific DNA lesions caused by an alkylating agent that activate poly(ADP-ribose) polymerase. The reason for benzamide's greater enhancement of MNNG- or X-ray-induced cytotoxicity in aphr-0-2 and aphr-0-R2 may be twofold: a) poly(ADP-ribose) poly- merase inhibitors have a synergistic effect on cytotoxicity in partially NAD depleted cells (130,237); and b) alternatively, benzamide has been shown to inhibit break rejoining in DMS- (70,71), or MNU- (237) induced gaps. If enzyme(s) involved in repair of single-strand breaks for aphr-0-2 or aphr-0-R2 is (are) defective, gap closure (break rejoining) could proceed more slowly in the presence of benzamide and thus reduce cell survival. Perhaps ADP-ribosylation of nuclear protein by poly(ADP-ribose) polymerase may have a functional role in the cellular repair mechanism, either by modification of the chromatin structure or by specific modulation of the DNA repair step (17,18,70,71,73,90,165,213,270). 117 Caffeine, at the two doses tested, seems to potentiate the cytotoxic effect of UV in these three cell lines. Caffeine is proposed to be inhibitory to "post- replication" repair; it reduces cell survival, and it enhances or has no effect on mutations in V79 cells (36,37). It seems that the caffeine-sensitive repair step(s) may not be different in these three cell lines. Al-ternativey, caffeine is a weak inhibitor of poly(ADP-ribose) polymerase, but has a greater UV-cytotoxicity enhancement than benzamide does. This suggests that the caffeine sensitive DNA repair step is possibly different from the benzamide sensitive step. Furthermore, caffeine has been shown to reduce DNA-chain growth (239,263) and to enhance the number of leplicons, or to alter purine pools (287). Because aphr-0-2 cells exhibit slightly greater caffeine sensitivity than V79 or aphr-0-R2 cells do, the increased number of replicons or altered purine pools in aphr-(l-Z cells may increase their cytotoxicity (or mutation) by inhibiting the repair or by increasing mispairing during DNA repair. Further investigations are required to elucidate the roles of benzamide and caffeine in repair pathways. Mutability of the UV-sensitive Agilidicolin-resistant Mutant The results in UV-induced mutagenesis showed that: a) aphr-0-2 is hyper- mutable for ouar, DTr and 6TGr mutations when its maximum mutation frequencies were compared with those of V79 cells at the same UV dose; b) the thymidine auxotrophic revertant, aphr-ll-RZ is similar to V79 cells in UV-sensitivity and mutability; and c) when UV-induced mutation frequencies were compared at the same survival rate, the aphr-0-2 mutant showed a higher frequency than both V79 and its revertant at the ouar and DTr loci, but not at the 6TGr locus. Of the three mutation assay systems used in our experiments, the 6TGr mutants are considered to be the results of nonsense, deletion, frameshift or point mutations at the HGPRT locus; whereas ouar mutants reflect missense (point) 118 mutations at the Na+lK+-ATPase locus. The DTr mutants of V79 cells are not very well characterized. In Chinese hamster ovary (CHO-K1) cells two types of DTr mutants have been described: permeability-mutants with impaired uptakes of DT and translation-mutants with altered elongation factor 2 (EF-Z) of protein synthesis (68). The latter type of mutants can be selected with a high concentration of DT, such as the one used in the present studies. Similar to 6TGr mutants, but unlike ouar mutants, DTr mutants, which are resistant to high concentrations of DT, can be induced by X-rays. This is contrary to the prediction that the DTr mutant (EF-Z mutants) may be similar to ouar mutants, since the gene products of both loci are essential for the survival of cells. The fact that they are different may indicate that small deletions as well as missense mutations can cause DTr mutations. The observation that aphr-0-2 had a higher mutation frequency per unit of UV dose and per unit of survival at the Na+/K+-ATPase and EF-Z loci, may be considered as evidence that aphr-0-2 cells have a higher frequency of UV—induced missense mutations. Because the expression times for 6TG mutations are different for aphr-0-2 and wild type V79 cells, it is difficult to draw a clear conclusion from the results. The delayed expression of UV-induced 6TGr mutation in aphr-0-2 may be a real phenomenon, or it may be due to an indirect effect of some other mechanisms not related to mutation expression, such as selection advantage for 6TGr mutants. If the phenomenon is real, the aphr-0—2 can be considered as hypermutable per UV dose, also, at the HGPRT locus. If it is an indirect effect, then aphr-0-2 apparently exhibits differential mutability at different loci. In aphr-0-2 cells, X-ray induced 6TGr mutation frequency does not show delay expression. Therefore, the delayed UV-induced 6TGr mutation appears to be mutagen specific. Because X-ray radiation generates deletion type mutations, it appears that aphr-0-2 cells are not sensitive to deletion type mutagens (X-rays). As 119 shown in the DTr mutation locus, aphr-0-2 is not hypermutable after X-ray irradiation. The hypermutability of aphr-0—2 after UV irradiation may be specific in point mutation. Elkind (75,76) reported that radiation survivors repair most (if not all) of the strand breaks. It can be assumed that X-ray-induced mutations are fixed at the subsequent DNA replication in the survivors. On the other hand, UV-induced pyrimidine dimers are not totally removed and may be transferred to the daughter strand (60). These dimers served as mutation "substrates" for the next generations and may show a delayed-fixation in aphr-0-2, whose excision repair may be rendered error-prone. In prokaryotes, a mutation (mfd) in B/r strains of Escherichia coli may cause deficiency in UV-induced mutation frequency decline (85). This phenomenon may be similar to that observed in aphr-0-2, but the genetic basis may be different. Pyrimidine auxotrophs have been reported in mutants of fungi, N. 513553, _S_._ cerevisiae and i M (181), and of CHO cells (175). In the case of [_J; m, the UV, X-ray or nitrosoguanidine sensitivity is a direct result of pyrimidine auxotrophic mutation (181,182). A mutant of g m, pal-l, has a reduced level of thymidine nucleotides, but has no significant change in any other deoxyribonucleo tides. Photoreactivation did not alter this sensitivity. The pyll-l mutant does have UV-induced hypermutability (as measured by caesium-resistance), and is deficient in UV-induced mitotic recombination (due to inviability of recombinants). A diploid heteroallelic at the ml locus was UV sensitive but not deficient in UV-induced mitotic recombination. This UV-sensitivity of 1331-1 appears to be a result of failure in a repair DNA polymerase to fill post-excision single-strand gaps in the DNA. Mammalian repair deficient mutants with hypermutability of V79 Chinese hamster cells reported by Schultz (233-235) are not TdR auxotrophs and are not resistant to aphidicolin. They also show a reduced UDS after UV-irradiation. The 120 aphr-ll-Z hypermutability appears not to be associatd with a reduced UDS capacity as shown in Fig. 19. In the present studies, the thymidine auxotrophic revertant of aphr-ll regained not only its normal UV sensitivity, but also its normal mutability. From these results it is concluded that UV-sensitivity and mutability in aphr-0-2 are correlated and possibly controlled by a single gene. The fact that both aphr-ll-Z and aphr-0-R2 are thymidine auxotrophic and have similar levels of dCTP, but that only the former is UV-sensitive and hypermutable, may also be considered as evidence that the nucleotide pool in aphr-0-2 does not contribute to its UV-sensitivity and hyper- mutability. The exogenous TdR or AdR (2-0 11M) was also found not to affect UV- induced mutation expression. The basis for the UV-sensitivity and hypermutability of aphr-0-2 is not known. Our results indicate that it is not defective in excision repair, because UV-induced unscheduled DNA synthesis is normal in all three cell lines. In addition, aphr-0-2 is not hypermutable in X-ray-induced mutagenesis. Base excision repair may be as efficient as in the V79 cells. Unlike V79, the UV induced cytotoxic and mutagenic effects on aphr-0-2 appear not to be reduced by conditioned medium, whidl has been shown (186) to arrest V79 cell replication and enhance DNA excision repair following UV irradiation. Induced mutations in mammalian cells are considered results from unrepaired (by-pass repair) and/or misrepaired (excision repair) DNA damage. In normal diploid human fibroblasts and V79 cells, unrepaired damage can be overcome by liquid holding with conditioned medium so that the error-free excision repair is enhanced (168,186). Personal observations indicate that single cells (V79 and aphr-ll-Z) grown in the conditioned medium do not divide (data not shown). Together with the result reported by Nakano et al. (186), it is assumed that the excision repair pathway is enhanced in both cell lines daring liquid holding. The fact that the UV-induced 121 mutation frequency and cytotoxicity of V79 cells are reduced upon liquid holding (positive recoveries) indicates the excision repair process in V79 cells is error free. Furthermore, the fact that aphr-0-2 cells grow slower than V79 cells in the growth medium and show the negative liquid holding recoveries, i.e., both UV-induced mutation and cytotoxicity are enhanced, indicates the excision repair of aphr-0-2 cells is not error free, or this repair pathway has been rendered error-prone or mutagenic by certain mechanism(s). The possibility that aphr-0-2 could be an a—polymerase mutant has not been ruled out (00). If the mutant is, in fact, defective in a-polymerase, our data could be considered as evidence that a-polymerase is involved in the DNA repair process as indicated by previous reports (16,07,110,208). The DNA repair in aphr-0-2 must have been rendered error-prone (e.g., defective base selectivity) by the mutation, despite its slow growth, which normally favors error-f ree excision repair. The mutator activities of UV-sensitive aphr-0-2, aphr-0-RPO or aphr-0-RP5 in three genetic loci (ouar, DTr and 6TGr) are not locus specific and do not associate with the TdR auxotrophic phenotype. These observations are similar to those of UV- induced hypermutability of aphr-0-2. Therefore, the UV-sensitivity and mutability are possibly controlled by a single gene, as discussed previously. Mammalian variants that have an elevated mutation rate reported thus far, are from CHO TdR- mutants (175), mouse cells (288) and fibroblasts from patients with Bloom's syndrome (97,282). In human cells, the mechanism for the elevated mutation rate may be due to the presence of clastogenic material, which can be detected in the culture medium of Bloom's fibroblasts (78,283). In the rodents, the mutator activities are reported to be associated with either TdR auxotr0phic phenotype (175) and/or an elevated dCTP pool (288). These mutator activities are locus specific. Both mutator activities can be abolished by reduction of the ratio of dCTP to dTTP through additions of exogenous TdR. It appears that the mechanism 122 for this mutator activity is through a mass action of DNA precursor substrates and/or a deficiency in proofreading enzyme activity in CHO or mouse cells. Furthermore, the UV-induced cytotoxicity and mutagenicity have not been reported in these mutator mutants from the rodents. In the present studies, TPA was used to eliminate metabolic cOOperation commonly observed in quantitative mutation assay at 6TGr locus. The results from 6TGr mutation rate determination in the wild type V79 cells are similar to results reported by other investigators (see Table 2). The results clearly indicate that the mutator activities cannot be explained by the dNTP pools alone. Firstly, the aphr-ll-RZ cells have dCTP pools similar to those of aphr-0-2 cells, but the former cells are not hypermutable. Secondly, the mutator activities are not associated with TdR auxotrophic phenotype, because the UV sensitive TdR prototrophic revertants, aphr-0-RPO and -RP5, are also hypermutable. It appears that the mutator activities are associated with UV-sensitivities and UV- induced hypermutability. These aphr-(l variants were selected by a specific polymerase (1 inhibitor- aphidicolin. This inhibition is competitive with dCTP in the case of purified polymerase 01 (106,193,190), or with each of the dNTPs in isolated nuclei (190). A stimulatory factor of polymerase 01 has been isolated from mammalian cells (136). This stimulatory factor cannot reverse the inhibiting effect of aphidicolin on polymerase a. Aphidicolin has been used to isolate dNTP pool mutants in mammalian cells (7,8,10,226) or polymerase mutants in the fruit fly (259) and in mouse FM3A cells (190). However, none of them is mutagen sensitive or has an induced hypermutability. Because mutator phenotypes of UV-sensitive aphr mutants are not locus specific at three loci and continuously give rise to "revertants" of different phenotypes (cytidine sensitive vs. resistant in the presence of TdR; TdR auxotrophs vs. prototrophs), it appears that the mutation "gene(s)" is (are) a generalized (non-locus-specific) mutator. In this regard, the gene(s) is (are) similar 123 to the mutD gene of E: c_oli (55,56,62,206,207). The mutator phenotype of mutD gene is stimulated by T dR (2 uM). Whether or not aphr UVS mutator gene(s) is (are) also stimulated by TdR is unknown, because aphr-ll-Z is TdR' and the growth medium must contain TdR. Nevertheless, in one experiment when TdR (0 11M) was not added to ouabain medium, the spontaneous mutation rate was lower than for that of the wild type (data not shown). On the other hand, due to possible interference of TdR with toxin cytotoxicity, TdR was not added to selective medium during DTr mutation rate estimation, and aphr-0-2 is hypermutable at DTr locus. Further analysis using UVS TdR+ revertants is warranted. Even though aphr-0-2 mutants and the wild type V79 cells (00) have a polymerization activity which is as sensitive to the inhibition of aphidicolin, one could not eliminate a possible existence of defective enzymatic activities which are responsible for the observed mutator activities, i.e., site specific chromosomal aberration on the long arm of chromosome one (00) and UV-induced cytotoxicity and hypermutability. Eukaryotic mutator mutants that are sensitive to raditions (UV light) and chemical mutagens (methyl methanesulfonate) have been reported in yeast (115), and a mutagenic repair pathway seems to be responsible for'the observed hyper- mutability in these mutants (mt-3,0,5). Mammalian polymerase a differs from prokaryotic polymerase or yeast polymerase II in that the purified eukaryotic polymerase does not have an associated 3'-5' exonuclease activity which is involved in correction of mispaired bases. Nevertheless, polymerization error in a DNA polymerase from acute lymphoblastic leukemic cells is tenfold higher than that in a polymerase from normal cells (250). It has been suggested that this defect is in the base-selectivity of the polymerase. The UV-sensitive mutator aphr-0-2 cells could be defective in this selectivity, whereas a "reversion" or second mutation could have occurred in aphr-ll-RZ so that the selectivity is normal. The fact that aphr-0-2 cells gave rise to different kinds of reverents, and have an elevated non-locus specific 120 mutation rate favors the notion that revertants may be results of suppressor mutation in aphr-0-2 cells during normal semi-conservative DNA replication. This altered base-selectivity could also be responsible for a repair process which is "error-prone" or mutagenic in aphr-0-2 cells (301). The resulting mutation from this altered base selectivity would be a missense mutation such as that observed in ouar locus. The mechanism for the elevated spontaneous mutation rate in the UV-sensitive aphdicolin-resistant mutants remains unknown. The data indicate: a) aphr-0-2 is generalized mutator mutant; b) the mutator activities are associated with UV sensitivities but not with dNTP pools; c) UV-induced hypermutability of aph'-11-2 does not seem to be due to a defect in DNA repair; rather, it seems that the excision repair has been rendered error-prone; d) mutagen sensitivities of aphr-0-2 seem to be specific to mutagens whose adducts require long patch repair pathway; e) it appears that polymerase base-selectivity is involved in the hypermutability of aphr-0-2; and f) aphr-0-2 cells give rise to revertants which may allow in-depth characterization of the mutator gene(s). If the base-selectivity of polymerase in aphr-0-2 cells is indeed defective, these mutants provide new biological and biochemical means to study processes of DNA replication and mutagenesis in mammalian somatic cells. SUMMARY Isolation of mutagen-sensitive mutants in mammalian cells has been reported by several laboratories. Most of these mutants were selected from mutagenized cells by methods similar to replica plating technique. Recently a UV-sensitive mutant has been isolated by selection with aphidicolin, a specific inhibitor of DNA polymerase a. This mutant also exhibits other pleiotrophic effects such as thymidine auxotrophy, sensitivity to cytidine, slow growth and BrdU-dependent site- specific chromosomal aberrations. The present dissertation reports characteriztions and a detailed study of spontaneous and induced mutability of this mutant and its revertants. Compared to V79 cells, this mutant (aphr-0-2) was more sensitive to UV and NACAAF whose DNA adducts are believed to be repaired by the "long-patch" nucleotide excision pathway, but was not more sensitive to X-rays or MNNG whose lesions are repaired by the "short-patch" nucleotide excision pathway. The thymidine auxotrophic revertant and the mutant were sensitive to MNNG. In the previous report (00), the mutant was shown to contain no a-polymerase that is resistant, in crude extract measurement, to aphidicolin. In this study, the mutant was found to be normal in unscheduled DNA synthesis. However, unlike V79 cells, the mutant appears to increase mutation and cytotoxicity following UV damage by conditioned medium which arrests cell replication (186). The mutant was hypermutable at 3 loci (6TGr, ouar and DT') when UV-induced mutability are compared with V79 cells. Spontaneous mutation rates measured by two different methods also indicate that the mutant had an elevated spontaneous mutation rate at 3 loci. The thymidine auxotrophic revertant was not only normal in 125 126 UV-sensitivity but also normal in spontaneous and induced mutability. Therefore, it appears that a single gene controls both increased mutability and mutagen sensitiv- ity. The results also indicate that nucleotide pools or thymidine auxotrOphy are not correlated with UV-sensitivity and spontaneous or induced mutability. Furthermore, preliminary data showed that UV-induced mutability in the mutant was not reduced after conditioned medium treatment. This study thus identified an aphidicolin-resistant mutant that is mutagen sensitive and has elevated spontaneous and induced mutability, a phenomenon never reported before for any mammalian cell mutant. The molecular basis for the defect of this mutant is not known. We speculate that it is defective in certain enzyme related to both DNA replication and repair, the defect of the enzyme apparently rendered both replication and repair error prone. Further biochemical analysis of the properties of aphr-0-2 polymerase to define their relationship to the observed mutator activities is warranted. The significance of the results presented in this dissertation seems to be: a) mutator mutants can be isolated by aphidicolin; b) aphr-0-2 cells exhibit a generalized mutator mutation; c) the existence of mutator activity in this instance is not related to pyrimidine metabolism; d) ultraviolet light sensitivity and mutator activity in aphr mutants are possibly controlled by a single gene; e) evidence supporting the existence of an error-prone repair pathway; and f) new biochemical and biological means to elucidate the role of DNA polymerase(s) in somatic cell mutagenesis. APPENDICES APPENDIX A DEOXYRIBONUCLEOSIDE TRIPHOSPHATE EXTRACTIONS AND MEASUREMENTS APPENDIX A DEOXYRIBONUCLEOSIDE TRIPHOSPHATE EXTRACTIONS AND MEASUREMENTS Extractions Deoxyribonucleoside triphosphates (dNTP) were extracted from partially synchronized Chinese hamster V79 fibroblasts according to North St _a_l_., (192) and Warren (283). Fibroblasts from aphr-0—2, aph'-11—R2 and wild type v79 cell lines were grown in flasks (75 cm2, Corning Glass Work) until confluency. Twenty-four hours before cell harvesting, cells were released from confluency with 0.01% trypsin, counted and 3-0 x 106 cells were plated on to 3 plates (9 cm, Corning Glass Works) for each cell strain in duplicate sets. After 20 hours of incubation at 37°C in growth medium supplemented with 5% FCS, the cells were rinsed twice with ice- cold phosphate-buffered saline (PBS) and were harvested with a rubber policeman in ice-cold 60% methanol. The cells in 3 plates of each strain were pooled into a test tube and the volume was adjusted to approximately 10 ml with 6096 methanol, then were placed in -20°C overnight. Another set of wild type cells served as a control by additions of dCTP/dTTP or dGTP/dATP (concentrations were adjusted to 25-50 pmole per tube) to each pooled methanol-cell suspension immediately following cell harvesting. This group served as the control to test whether endogenous dNTP survived the extraction procedures. The methanol extracts were centrifuged (29,000 x G, Survall) and the supernatant was separated from the precipitate. The precipitate was saved for determination of DNA contents and this supernatant was 127 128 lyophilized to dryness in a lyophilizer (Virtis Co.). Each dry material was treated with 3 ml of 0.5 N perchloric acid to allow for enzyme inactivation at 0°C for half hour. The content in each tube was neutralized to pH:7 with 1.5N potassium hydroxide (0.705 ml) at 0°C for another half hour. The potassium chloride in the solution was discarded after centrifugation and the supernatant was again lyophil- ized to dryness. The dry powder was dissolved in 1 ml distilled water, centrifuged and the resulting supernatant was stored in 0 aliquots at -20°C for future dNTP m easurements. Measurements The amounts of dNTP in each extract of 1 ml sample were measured by incorporations of the complementary 3H-dNTP into the defined c0polymer of poly(dA-dT)-poly(dA-dT) or poly(dI-dC)-poly(dI-dC) and f: 928 DNA polymerase I. 1. Reaction Reagents a. dATP and dTTP Assays. The poly(dA-dTl-poly(dA-dT) copolymer of 1515 11M (10 optical density unit per ml) was diluted to 200 11M in 10 mM Tris-HCl (pH 8.0) and 20 mM KCI solution. For each sample of dATP assay, a total of 0.09 ml of the reaction reagents was prepared by mixing 0.00 ml of 50 mM glycine-KOH (pH 9.2), 0.02 ml of 75 mM MgCl 3 2, 0.02 ml of 200 uM poly(dA-dT)-poly(dA-dT) copolymer and 0.01 ml of 10 uM H-dTTP (2.5 uci/ml). For dTTP assay, 0.01 ml of 10 11M 3 3 H-dATP (7.5 u ci/ml) was used instead of H-dTTP. 129 b. dGTP and dCTP Assay. The poly(dI-dC)-poly(dI-dC) copolymer of 10 O.D./ml was diluted to 71.6 uM in the same solution as previously described. For each sample of dGTP assay, a total of 0.09 ml of the reaction reagents was prepared with 0.00 ml of 50 mM Tris- HCI (pH 8.0), 0.01 ml of 100 mM MgCl 0.02 ml of 71.6 uM of poly(dI-dC)-poly(dl- 2’ dC) and 0.01 ml of 10 11M 3H-dCTP (25 uci/ml). For dCTP assay, 0.01 ml of 10 uM 3H-dGTP (7.5 uci/ml) was used. 2. Escherichia coli DNA-polymerase l One part of the DNA-polymerase I (0505 units/ml) was diluted to 9.09 units/ml in a solution containing 25 parts of 1 M Tris-HCI (pH 8.0), 5 parts of 100 mM dithiothreitol, 5 parts of 10 mg/ml bovia serum albumin and 019 parts of distilled water. Generally, 0.5 ml of the above solution was made at 0°C immediately before dNTP assay. 3. Assay Procedures For each set of assays, a standard curve was obtained from known amounts of the dNTP in question. Each sample (0.01-0.08 m1) and reaction components (0.09 ml of reaction reagents and 0.01 ml of DNA polymerase solution) were mixed at 0°C and the final volume was adjusted to 0.2 ml with distilled water. The reaction tubes were then transfered to a 37°C waterbath. After incubation for 35 (for dATP, dTTP assays) or 00 (for dGTP, dCTP assays) minutes, the reaction was stopped by transferring the tube to an ice bath and additions of 3 ml of ice-cold 10% trichloroacetic acid with 1% sodium perphosphate (TCA-NaPPi) solution to each 130 tube. Thirty minutes later, each mixture was filtered through TCA-NaPPi- presoaked Whatman GF/C filters (2.0 cm, Whatman Ltd., England) under sampling- manifold vacuum (Millipore, Model 3025). Each tube and filter were rinsed 0 times with TCA-NaPPi solution, and twice with ice-cold 95% ethanol. The filters were then dried under a heat lamp. The dried filters were placed in scintillation vials containing 8 ml of a toluene-based counting fluid [12 gm of FPO-2,5diphenyloxozole, 0.3 gm of POPOP-l,0-bis-[2-(5-phenyloxazolyl)]-benzene and 3.0 L of toluene]. The radioactivity was measured in a Beckman LS 9000 liquid scintillation spectrophoto- meter. The amount of dNTP was calculated from the slope of the standard curve and the results were expressed as pmoles dNTP per ug DNA. DNA Measure ments The DNA content of methanol precipitates was determined according to the procedure described by Giles and Myers (87). The precipitates were suspended in 1 ml of distilled water and sonicated with a Branson sonifier (Sonifier Cell Disruptor, Heat Systems Co., New York) at a power level of 25 for 3—5 seconds. Equal volumes (0.5 ml) of the suspended sample and 20% perchloric acid were mixed and to this 1 ml mixture, 1 ml of 0% diphenylamine in glacial acetic acid (freshly prepared) and 0.05 ml of acetaldehyde (1.6 mg/ml) were added. The reaction mixture was further mixed with a vortex-genie (Model K-500-G Scientific Industries, Inc., Massachusetts) and incubated overnight at room temperature. The optical density of each sample was measured at 595 nm against a reagent blank on a spectrOphotometer (Stasar II, Gilford Instrument Laboratory, Inc., Ohio). The results were calculated from a standard curve determined simultaneously by using calf thymus DNA (between 5-00 1.1g). The results were expressed as ug DNA. APPENDIX B EXPRESSIONS OF OUAr, DT’, AND 6TGr MUTANTS 131 mmw .0NMV emu ea: .eoev 0mm ANN .eAN. NMN 0e~ .mmm. mmN .m. ..m.. cm. 0.. .mmNV meN e .00 m.m o .om.v «m mm: .mms. :me omm Aomev mum .cm ANNNV men .:e .0.00 cam omN Aucmv mm~ Nam .emav .ee 00. .aewv :m. .m. .e-v eMN o .00 m.0 o Am..v mm c.m .Nsmv m.m A...V mm me. .mmmv we. .emv mm o .e.v m .mv . e .0.. m ... e .m.m0 msm .mmmv .m. .amNV .m. .emv on .0.. m ... e.e ...0 m .e. e .mm.0 mo. ..m0 0. .av . .e0 e 0000 ouuaoe. 0.0 the mac A000000z 0o .Ozv m0o>_>0:m L w mez00v 0E_0 ANV ~E\0 0000000QXm _0>_>0:m >0 m x.ozumd< m0c__ __0u .oz .0axm 132 mmm .33 N0... ... 010.000 0N. ..Nm. mm. a. m.> mmm A.mmv mmm N 010000 mmm .quv mum u mm> o A... : 0 010500 a .m.. N. a. o ms> as: .m.mv an: 0. #10000 m—: AmNNV cm: :. mu> :oN Acm0v moN n 010500 NNN .mm.v mMN m m~> c As. m c #10500 e .0. .. N. e mN> :.m .mmqv NNm m a. {10:00 Nma .mum. :.m m. a. mu) N0. 1 .0... em. 0. N arceao Nam Ac.m. mom .0 m mm) o .m.. m co. 0 010500 c Amp. NN o. oo. o mh> : Aco_m00>00v 010000 c. m0_0_>_0_mc0m >2 o: 00 030 0000.00 0003 0000 0030 00.0 14. M Na: .mmmv mom «.0. #10500 .ee .mmm. eNm 0.0. m0> ... .N.m. 0N. 0.N. .-.eao mmm .0mnv mm: o.N. mu> mum Ammav .mm :.m 010000 emN .m... .0. 0.0 mN> ch ..oNV m.N N.: 010000 0m. Am::v mmN N.4 mn> o .m.v m. a 410500 c A..Nv m.. :. c mm> .N: Amoqv 0N: m.o. «10:00 mom Amuwv Nmm m.w. mn> N 00.0 ouuaoe. 00.0 00¢ case .aaoo. oe_o .0. Ne\e noe__ .oz co_mm00qu .0>.>0:m >0 ..0u .00Xm .000003z 00 .Ozv m0o>_>0:m o.\m._0u 0:00m.m0¢ o 133 .000 00 AN. 0 .000 00w .000 m.~ A0~0 .0 ..00 mm .00 m. .m0 ..000 000 mm: .m.00 .5: mom .mwsv 0.0 MNM .0mm. 0mm 0 .0N0 m. 0 A¢~0 m. 000 .0000 ..0 .0000 .am 0.: .0N00 000 ANJMV :N. m0m .0000 00m ..0.0 m0~ mNM .0000 0mm Amo~0 .0 o .0_0 0 .00. NN a .0N0 0. ANNV _. .mcqv 00m .~0~0 :N. ..000 NNN .00.. m. ..m0 0N .0.0 m. ..-0 Nmm ..m0 0.. ..mmv .mm .0.. mm AMNO __ ...0 m. L0.0 00u=0c_ L.30 0.0 0:0 Amuc0uaz .0 .020 mLo>_>L:m 0 o_\m__00 0:00m_mo¢ o m 0 .~ :.m NN 0.x .: 0.x oo. o m oo. o a. a. m n a .. c :. J. n n c m c 0. :. N a 0 c m a. m. :. mm m 3 N co. o 0 ea. c Am>muv .e_. .00 ~e\. co_mm0gqxu .0>_>L:m >3 «~-0-0;am 00c.— ..0u m .02 .uqu 13¢ 0 .0.. 0. .00. 00 .0.. 0. 0 0.0.0000 0 .0.. 0. .0. 0 .0. .v 0 00-0-0000 0 ..0. 00 .0.. 0 .0. 0 0. 0 00> 0.0 .000. 000 .000. 000 .00.. .0. - 0.0. «0-0-0000 0.0 .000. 000 .000. 000 .000. 00. 0 0.0. 0-0-0000 000 .000. 0.. .00. 00 .00. 00 0. 0.0. 00-0-0000 000 .000. 0.0 .00. 00. .00. .0 .. 0.0. 00> 0 .00. 0. .00. 00 .0. 0 00. 0 0.0.0000 0 .00. 00 .0. 0 ... . 00. 0 00-0-0000 0 .00. 00 .... 0. .0. 0 0 00. 0 00> 0 .00000_0000_ >0 00.00 0000 0 00.00.00000 2: 0 00.30 0.0 . .00. 000 0.0 «0-0-0000 000 .000. 000 0.0 0-0-0000 000 .000. 000 0.0 00> 0 .00. 00 0 0.0.0000 0 .00.. 00. 0. 0 00> 000 .000. 000 0.0 00-0-0000 000 .000. 000 0.0 0-0-0000 000 .000. 000 0.0 00> 0 .00. 00 0 0.0.0000 0 .00.. 00. 0 0 00> 000 .00.. 000 .00.. 000 0.0 00-0-0000 000 ..00. 000 .00.. 000 0.0 0-0-0000 000 .000. 000 .0.. 00 0.0 00> 0 .00. 00 .00. 00 0 0.0.0000 0 .00.. 00. .0. 0 0 0 00> .00.. 000 0.0 «0-0-0000 .00.. 000 0.0 0.0.0000 .00. 00 0.0 00> 0 0000 00030:. 0000 000 0000 .m>00. 05.0 A». ~E\0 00:.— .oz 00.0000axu .0>.>0:m >2 ..0u .ngm 0000003: 00 .Oz. m0o>_>0:m c.\m_.0u 00000.000 0 135 0. 0.0 .00..00. .00..0. .00..0 .00.00 00 0.0 - .00.00 .00.00 ..0.00 00 0.0 .000.000 .000.000 .00.00 .00.00 00 0.0 .00.00 .00.00 .00.00 .00.00 00 0.0 .00.00 .00.00 .0..0. ..0.0. 00 0.0 ..0...0. .00.00. .0.0 .0.0 00. 0 .0.0 .0.0 .0.. .0.. 00. 0 .0.0 .0..00 .0.0 .0.0 0 00. 0 .z: 0.00.+ 000- .z: 0.000+ 000- 000 0000 0000 >: .m.> 0000005 00 00.00000. 0000.. o: - .00.00_0000_ >0 00.00 0.00 0 0__ou_0_00< :0 0 000 .00. 000 .00. 000 .0. 00 0.0. 000 .000. 0.0 .000. 000 .000. 00. 0.0. 000 .000. 000 .00. 00 .00.. 0.. 0.0. ..0 .000. 000 .00. 00 .00. 00 0.0. 0 .00. 0. .00. 00 .... 0 0 0 .00. 00 .0. 0 ... .v 0 0 .00. 00 .0.. 0. .0. 0 0. 0 000 .000. 000 ..0.. 00. .00.. .0. 0.0. 0.0 .000. 000 .0.0. 000 .000. 000 0.0. 000 .000. 000 .00. 00 .00.. 0.. 0.0. 000 .000. 000 .00. 00 .00. 00 0.0. 0000 000000. 00.0 000 0000 .0000. 00.0 .0. 0500 .muc0uaz 00 .oz. m00>.>0:m 0 00.0000QXw .0>.>0:m >2 0.\0_.00 00000.000 0- 0-0 000 00-0000 00> 0- 0-0 000 00- 0-0 0000 000> 0- 0-0 000 00 0-0 0:00 000> 000.00 cu.3« 0T 0-0000 0- 0-00000 00- 0-00000 000> T 0-0000 00- 0-00000 000> «T 0-0 000 T 0-00000 00- 0-0 0000 000> 000.. ..00 0 0°: .uQXu 136 000 ..00. 000 0.0 .00.. 000 mmm .000. .0m ... .00.. 00. o .m.. 0. 0 .00. 00 0:0 ...0. mm: 00. .00. M00 .00 .00.. 000 me. .00. 00. 0 .0. m 0 .0.. .0 000 .00.. 000 0.. .00.. 00. 00. .0... 0M0 00 .00. .m 0.0 .00.. 0.0 m0 .00.. .0. 0.. .mm. mm. 0. .m0. 00 0 .00. 0 0 .0.. 0 0 .00. 00 0 .0. m .00 ..0.. M00 00. .00.. 00. .0. .00.. 000 00 .00. 0m 0m. .0... mm. mm .0m.. 0.. 0.. .00.. 00. 0 .0.. 0. 0 .0.. 0 0 .0.. m. 0 .00. cm 0 .0.. m 0000 000000. 000 000 000000_ 0000003: mo .02. 000>.>0:m L o o_\0_.0u 00000.00m + + + + + + I CD @— :0 um :m cc. co— cm as u no I Nm s cc— 0 oo— u + + + + + + 0030 znu A0>00V 05.0 A”. «00 00.00000Xm .0>.>0:m NNQ’J NNJ’4’ NNJ’J‘ O o o o coara-aococo-a-a-cocooo-Taooooccc:ooco NNJ’J E\0 >3 N 000.. ..0u ooz oHme 137 000 000 :0. m0 0 0 0 0 000 0.. 00. .0 0 0 0 0 000 .. 0 0 00m m0 0 0. 0 0 0 0 mm— mm 0 0 000 00 50 mm 0 0 0 0 000 0000 UUUDU—z “00000:: 00 .oz. 000>.>0:m .000.000 .m0.0.. .00.00 .0.0. .000.000 .00.00. .00.0.. .... .....000 .0.00 ..0..0 .0.00 0 Ame—VomN 00 :.m «:0-0000 .00.00 00 0.0. 00> .0..00 00 0 0.0.0000 .0.0 0 00 0 00> .0000: 0.. 00.0.00 0.00.0 .00..00.. 00 0.0 0-0-0000 .00.00 0m 0.0. 00> .00..0 00. 0 0.0.0000 .0.. 0 00. 0 00> 0. .mm—VNmN :.m 0-0-0000 .0.0m 0.0 00> .0..00 0 0.0.0000 .0.0. 0. 0 00> .00.00 0. 0.0 0-0-0000 .0..00 .0 0.0 00> .0.0 00. 0 0.0.0000 .0.0m 0 .0 0 00> .00000 0..00.0.oz 0.00.0 00.: .00.00. 0.0 0-0-0000 .0..00 0.0 00> .0.00 o 0-0-0000 .0... 0. 0 00> Am:.cm mm :.m N-cn0000 .0000 00 0.0 00> .00.0.00 .0.0 00. 0 0.0.0000 0.00.0. .0.0 0 00. 0 00> .. .000 z: a. 00:0 .0>00V 05.0 A”. 5\0 000.. .oz 00.00000xu .0>.>0:m ~>= ..0u .0axm o—\0..0u 00000.001 APPENDIX C A HYPOTHESIS ON COMPARISONS OF SPONTANEOUS MUTATION RATES OBTAINED FROM _Po ESTIMATIONS APPENDIX C A HYPOTHESIS ON COMPARISONS OF SPONTANEOUS MUTATION RATES OBTAINED FROM Bo ESTIMATIONS Mutations in mammalian somatic cells are rare events and are cell-division dependent. Mutation rates estimated from the fluctuation analysis vary from laboratories to laboratories and from experiments to experiments in the same cell line (Tables 2 and 19). Table 19 shows results from two experiments in whidt the 6TGr mutation rate was estimated from E0 calculation in V79 cells. In experiment No. 1, the mutation rate was calculated when 6TGr mutants were selected at low cell-divisions per replicate culture. It had a 30 of 0.9. In experiment No. 2, the 6TGr mutants were selected at high cell-divisions. It also had the same value of Bo as the previous experiment. However, the mutations rates estimated from the B0 are different. Because the cell line was the same in both experiments, it can be assumed that the expression of 6TG.r mutants was the same in both experiments, and the expression time contributed a similar effect in _Eo calculation. Furthermore, because the values of variance/mean in both experiments were not the same and were greater than one, the mutations were from random events. The differences in both experiments were the numbers of replicate cultures (C) and of cell divisions. To account for the difference in the observed mutation rates, a hypothesis is formulated and is described here. The limitations of mutation rate estimations using the go calculation in the fluctuation analysis are the go value and the numbers of cell divisions. The limits of 138 139 Table 19. Spontaneous Mutation Rates (6TGr) in Chinese Hamster V79 Cells.I Experiment No. 1 Experiment No. 2 Replicate cultures (C): 20 5h Cell numbers per culture: initial (No) 2 1 final (NF) (x106) 0.63 3 division (d) (x106) 0.91 A.3 go 0.9 0.91 6TGr (total) (7)2 (1113 Per culture: range 0-5 O-h variance 1.33 .53 mean 0.35 .20h var./mean h 2.6 ln(l/Eo) 0.1 0.097 CaNf/C 0.23 0.113 Mutation rate per cell _7 -8 per division: 1.1 x 10 2.3 x 10 a” 0.016 0.019 l. 2. 3. A. See Table lh. Low cell-density (l x 105 per plate) selections without TPA. High cell-density (l-l.5 x 106 per plate) selections with TPA. See footnote 2 on Table 16. R - [(Mut. rate - AL)/(AH - AL)], where AL and AH are low and high limits of mutation rate estimation respectively. See APPENDIX C. MO Bo are l/C and (C-l)/C, where C is the number of replicate cultures. No 30 calculation can be made when all the replicate cultures contain either at least one mutant (Bo = 0) or no mutants (go :1). For a given 30 value, the calculated mutation rate will be high if cell-division is low. Therefore, the mutation rate calculation is then limited by the experimental design, i.e., the numbers of replicate cultures and of cell divisions. For example, an experiment with replicate cultures of 20 and a cell division of 9 x 105 /culture (Table 19) allows the _Eo mutation rate 6 to 5.6x 10'8 [see Appendix D, in which N = number of estimation of 3.3 x 10' replicate cultures; D = cell divisions; MH and ML are high and low mean: ln(l/Eo), respectively, when Bo : UN and (N-l)/N; and AH and AL are high and low limits of mutation rates calculated from MH and ML respectively using A = ln(l/EOVD]. Thus, these two numbers are the limits for a mutation rate estimation using N = 20 and D = 9 x 105. In experiments using N = 54 and D = (1.3 x 106, limits of the mutation rate estimation would be 9.5 x 10-7 and 5 x 10-9. If we further assume the mutation (non-mutator mutation) is a rare event and arises from a similar mechanism in V79 cells, the mutation rate calculated should be in the lower range of the experimental limits. If we then define the g is the relative range and is the ratio of the difference between the observed mutation rate and the low limit to the experiment mutation rate limits, the 5 value should have a low and constant value (exceptions are when D is a very large number). For 7 6 example, the 3 value in Expt. No. l is 0.016 [(1.1 x lo' -5.6x 10‘3)/(3.3 x10- - 5.6 x 10‘3)] and it is similar to that of 0.019 in Expt. No. 2 [(2.3 x 10'3-5 x 10' 7-5 x 10-9) 9)/9.5 x 10' ]. Although the mutation rate obtained in Expt. No. l was 1.2 x 10"7 and was higher than that of Expt. No. 2 (2.3 x 10's), the 5 value suggests that these two mutation rates represent a similar mutation. If the mutation is not a rare event, such as a mutator mutation, the 5 value should be higher than that of "wild-type" V79 cells (Table 20). Conversely, an [#1 .N m_ menu—so oumu__ooc coo __ou _a_u_c_ .<¢h uaozu_3 co_uuo_om >u_mcoul__uo 304 .a_meee-__eu em_: .4< ocm 1< coozuon oucocomm_o one new mu_E__ _mucoe_coaxo och .>_o>_uuoamoc muumc co_umuae _mu_uoeoozu 30. new ;m_c ocm 4< ocm r< m>_o>_uooamoc U\A_luv 0 cm ocm U\_ u on up _Aoa\pvc_ u z_ memos zo— ocm :m_; can 4:.ocm :z “Auv menu—nu coo mco_m_>_n __ou u o “Auv macaw—nu uumo__aoc u z econ: .o x.ozmm¢< own .32.: 3305.698 93 ..o... 629(55 0 3m; c0333: .mm. .mom 50..» men on: Zoo ozu mo macs» uaooxo .w—am—.:_ mo_nflh 50cm oem moumc co_umuas oo>comno on» .Amu:=__ _mucoe_coaxu ozuv\Au_E__ _mucoetooxo so. on“ i 3a.. c0332: £03 83033 n 3: 03m... 3m 3an £5 E ooucomoeq 309.52 - - - _o~.e - a_e.o ~.oz eeee_eeaxm - emae.o - emo.e uso.o .e.eeae.e . .oz aeee_eeaxm eeoee - - - mmm.o - mue.e N .o: aeee_eeaxm _em.e - mee.o Nem.o - Nae.o _ .oz ueee_eeaxu eke - - a_vmo.e .Aaveao.o Amvem~.e .Aav~_m.o - ee_.e ~ .02 eees_eeaxm m_~.e - o A_Vem~.e __.o “Ne.e _ .oz beee_eeaxm ease mafia... images... 23-2% 3-2% are m9 .484 mmxmlmaqm em__eo m~> new eyesoaz-eea< e_ some eo_aeaaz e>_em_e¢ sec .eN e_ee» 142 antimutator mutation should have a lower value than that of the wild-type V79 cells. The R values calculated from mutation rate estimations in aphr-li-Z and aphr-ll-RZ generally agree with this hypothesis (Tables iii-16,20). A series of experiments has been carried out to verify this hypothesis. The hypothesis postulated that simple comparisons between the values of mutation rates obtained through the £0 estimation may be misled. The significance of this hypothesis would be that a) it includes experimental limits in comparisons of mutation rates; b) the 3 values allow a statistical analysis (e.g., paired _t_ test) for the significance of the observed mutation rate. 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