GI L.-- 1 "l RECOMBINATION MECHANISMS AND TEE CONTROLS OF GENE-CONVERSION IN ASCOBOLUS IMMERSUS by AGLAIA GHIKAS B.Sc. (NATURAL SCIENCES, UNIVERSITY OF ATHENS, GREECE) M.Sc. (GENETICS, WEIZMANN INSTITUTE OF SCIENCE - TEL-AVIV UNIVERSITY, ISRAEL). A thesis submitted in part fulfilment of the requirements for the degree of Doctor of Philosophy of the University of London. Department of Botany and Plant Technology, Imperial College of Science and Technology, London S.W.7. JANUARY, 1978 Dedicated to My Parents CONSTANTINOS & IRENE AT1IANASIADIS for they cherished the life they passed to me 3 ABSTRACT In this work with Ascobolus immersus, gene conversion at the w-78 site and of various newly isolated mutations was studied. For the w-78 site two new narrower ratio classes were detected, namely 6:2(2), 2:6(2), with numbers in parentheses giving the number of pairs of non-identical sister-spores contained in the respective octal'. The 6:2(2), 2:6(2) as well as the 4:4(2) octads detected here were used in the estimation of corresponding-site interference coincidence coefficients which showed that in agreement,with previous findings, corresponding- site interference in Ascobolus is weak. The relative frequencies of symmetrical and asymmetrical hybrid DNA at the w-78 site were investigated. It was concluded that hybrid DNA in this site occurs mainly symmetrically, in two chromatids, without excluding the possibility of asymmetrical hybrid DNA occurring, but to a smaller extent. A new method was developed for carrying out this kind of project, which will allow the study of all possible arrangements of the w-78 site and its gene conversion controlling factors. The gene conversion spectra of 40 mutants were studied in crosses to different wild-type strains. It was found that the type of conversion spectrum shown by a mutant often differs drastically from cross to cross, to the degree that there are mutants showing the spectrum of a frameshift in one cross and that of a base substitution in another cross. Moreover it is found that there is an intergradation of the conversion spectra in connection with the relative frequencies of postmeiotic segregation, with no sharp distinction between frameshift and base substitution mutations. The data are discussed within the framework provided by the current literature, on the bearing of both the chemical nature of the mutant and of other genetic factors on the type of spectrum shown by the mutant in a cross. 4 CONTNTS Page No. ABSTRACT 3 TABLE OF CONTENTS 4 LIST OF PLATES 8 LIST OF TABLES 9 LIST OF FIGURES 11 GENERAL INTRODUCTION 12 LITERATURE SURVEY 17 1. Genetic recombination 17 2. Gene conversion 18 3. Fidelity of gene conversion 19 4. The models: their evolution and predictions 20 i)"Dual hybrid chromatid" models 25 ii)"Single hybrid chromatid" models 27 5. Recombination related phenomena 29 0. The genetic control and factors affecting, recombination 32 7. Corresponding-site events 40 GENERAL MATERIALS AND METHODS 45 I General materials 45 1. The organism 45 2. The stocks 49 3. The Chemicals 52 4. Incubators 52 II General methods 53 1. Media 53 2. Sterilization, inoculation, crossing techniques collection of spores, scoring, germination of ascospores and photography 54 3. Calculations 55 PART A Gene conversion at the w-78 locus of Ascobolus i mme rs us 56 SECTION I The detection of 4:4(2), 6:2(2) and 2:6(2) segregations and their use in corresponding-site interference studies 57 5 Page No. 1. INTRODUCTION 57 a) Statement of problems Thvestigated 57 b) The markers 57 i) Properties of pfr-1 and fpr mutants 58 ii) Properties of gr-3 and w-78 mutants 62 c) The detection system 64 i) The detection of 4:4(2) 69 ii) The detection of 6:2(2) 75 iii) The detection of 2:6(2) 78 iv) The detection of other unique narrower ratio classes 81 2. RESULTS 83 a) 4:4(2) 86 b) 6:2(2) and 2:6(2) 89 3. DISCUSSION 93 a) aberrant 4:4 octads 93 b) The detection of 6+:2m(2) and 2+:6m(2) 96 c) Wider ratio octads 97 d) Corresponding-site events 98 4. CONCLUSIONS 99 SECTION II Analysis of the 5:3 and 3:5 classes in IICF x LCF monohybrid crosses 101 1. INTRODUCTION 101 2. THEORETICAL CONSIDERATIONS 102 a) Cross: LCF wild-type x IICF w-78 102 b) Cross: IICF wild-type x LCF w-78 105 3. RESULTS 109 118 4. DISCUSSION 5. CONCLUSIONS 129 6 Page No. SECTION III Background Studies 130 1. The nature of w-78 phenotype 130 a)The development of the "collapsing" character 130 b) w-78 in crosses to other white mutants 134 2. The re-isolation of "K" derived wild-types 135 a) Method of isolation 135 b) Tests of the "K" derived wild-types 139 3. Germination' and Fertility tests 143 a) Germination 143 b) Fertility 145 4. Search for outside markers 148 5. Attempts at higher temperature crosses 150 PART B The Study of white-spored mutants of different mutagenic origin 154 I INTRODUCTION 155 1. Problems investigated 155 2. Isolation of mutants 155 3. Induction of mutations 156 a) UV induced mutations 156 i)Induction with UV 156 ii) Frequency of UV induced mutations 157 b) NG induced mutations 160 i)Induction with NC 160 ii) Frequency of NG induced mutations 161 c) ICR induced mutations, 161 i)Induction with ICR 161 ii) Frequency of ICR induced mutations 162 4. Spontaneous mutations 162 II RESULTS 164 1. The mutants 164 2. Linkage relations 165 a)Locus I 170 b) Locus II 170 c)Locus III 171 d) Locus IV 171 7 Page No. 3. Conversion spectra 172 4. Aberrant asci with wicHr ratios 193 III DISCUSSION 199 Locus I 199 Locus II 205 Locus III 206 Locus IV 208 UV induced mutations 209 NG induced mutations 214 ICR170 induced mutations 214 Spontaneous mutations 216 Mutagen specificity and interchangeability of conversion spectra 217 Intergradation of conversion spectra 219 Wider ratio octads 223 IV CONCLUSIONS 224 OPEN PROBLEMS AND SUGGESTIONS FOR FURTHER WORK 1. The detection of 5:3(3)s and 3:5(3)s 22G 2. A new method for assessing the relative frequencies of symmetrical and asymmetrical hybrid DNA at a mutant site 229 3. Studies with the mutant UVKw8 236 4. Studies with some UV induced mutants 237 S. Studies with mutants of locus II 238 6. Studies with mutant BBm 239 LITERATURE CITED 240 ACKNOWLEDGEMENTS 251 8 LIST OF PLATES Page No. Plate 1 46 Plate 2 46 Plate 3 61 Plate 4 61 Plate 5 67 Plate 6 67 Plate 7 68 Plate 8 68 Plate 9 72 Plate 10 72 Plate 11 73 Plate 12 73 Plate 13 74 Plate 14 74 Plate 15 131 Plate 16 131 Plate 17 132 Plate 18 132 Plate 19 133 Plate 20 133 Plate 21 152 Plate 22 152 Plate 23 153 9 LIST OF TABLES Page No. Table No.l. 50 Table No.2. 59 Table No.3. 60 Table No.4. 66 Table No.5. 85 Table No.6. 91 Table No.7. 109 Table No.8. 112 Table No.9. 113 Table No.10. 114 Table No.11. 115 Table No.12. 117 Table No.13. 137 Table No.14. 141 Table No.15. 142 Table No.16. 144 Table No.17. 145 Table No.18. 157 Table No.19. 161 Table No.20. 162 Table No.21. 164 Table No.22. a) 167 b) 168 c) 169 Table No.23. 175 Table No.24. 176 Table No.25. 178 Table No.26. 179 Table No.27. 180 Table No.28. 182 Table No.29. 184 Table No.30. 185 Table No.31. 186 Table No.32. 187 Table No.33. 188 Table No.34. 189 Table No.35. 190 Table No.36. 191 Table No.37. 194 10 Page No. Table No.38. 196 Table No.39. 198 Table No.40. 218 Pedigree Chart 51 11 LIST OF FIGURES Page No. Figure 1 102 Figure 2 104 Figure 3 106 Figure 4 107 Figure 5 120 Figure 6 122 Figure 7 138 Figure 8 140 Figure 10 158 Figure 11 225 Figure 12 226 Figure 13 227 Figure 14 228 12 GENERAL INTRODUCTION The present work was carried out with the Discomycete fungus Ascobolus immersus and attempts to investigate problems concerning the mechanism and control of gene conversion. Specifically it deals with gene conversion at. the w-78 locus as well as at sites of other newly isolated spore colour mutants. Conversion at w-78 is controlled by the closely linked factor (P), which, when homozygous, (in monoallelic crosses) confers high conversion frequency (8-18%) while, when hetero- zygous, low conversion frequency (2-6%). Two more factors (K) and (91) also closely linked to w-78, confer low conversion frequency in all cases for w-78. Work on this locus has been done in the past by Emerson and Yu-Sun (1967), who first established the high and low converting strains and partly the controlling factors involved, by Lamb (19724LambgWickramaratne (1973) who detected the occurrence of genuine wider ratio octads namely 7:1, 1:7, 8:0 and 0:8, and Wickramaratne & Lamb (1975), who studied the determinants of gene conversion properties in Ascobolus immersus and induced changes in the gene conversion spectra of w-78. These studies have established, among other things,that a) conversion events at corresponding sites in both pairs of non-sister chromatids of a single bivalent may occur in Ascobolus immersus and b) that environmental and genetical factors may alter the gene conversion frequency and spectrum of a single mutant, so that it may be of spectrum class C or D, as these classes were defined by Leblon (1972a), according to the conditions involved.