A COMPARATIVE STUDY OF INTER AND INTRALOCUS RECOMBINATION IN DROSOPHILA. by DOROTHY JANE STUART SCHOLEFIELD .Sc. (Hons.), University of British Columbia, 19 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in the Department of ZOOLOGY We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA December, 19 65 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that per• mission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives„ It is understood that copying or publi• cation of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver 8, Canada Date Qt*- - i - ABSTRACT The effect of different treatments on crossing over between and within genes at the tip of the X chromosome of Drosophila melanogaster was studied to determine whether exchange in the two regions occurs by different mechanisms. In response to autosomal inversions, y -radiation, and heat shock, crossing over of both types was altered in the same direction and to a comparable extent. This would be expected if there were only one crossover mechanism involved. There was some difference in response of inter- locus and intralocus crossing over after mitomycin C injection but, since the effect on interlocus crossing over in two separate regions was not consistent, the significance of this result is questionable. Although double crossing over involving the two interlocus regions was very rare doubles involving an inter and an intralocus region were recovered. The association of exchanges within a gene with a crossover between genes might indicate that there are two noninterfering mechanisms, or that multiple exchanges occur in short effectively paired regions. A further experiment designed to detect switch regions is outlined. - ii - TABLE OP CONTENTS PAGE INTRODUCTION 1 METHODS and MATERIALS 3 Series 1. Autosomal Inversions 6 Series 2. Cis-Trans Effect 7 Series 3. y -Radiation 7 Series 4. Heat Shock 10 Series 5. Mitomycin C Injection 11 RESULTS 12 DISCUSSION 28 SUMMARY 31 LITERATURE CITED 32 - iii - LIST OF TABLES TABLE PAGE 1 Phenotype scored in each crossover class. 5 2 Crossover values in each region in females heterozygous for different autosomal inversions. 21 3 Crossover values in each region in females with different marker combin• ations. 22 4 Crossover values in each region in radiated females. 23 5 Crossover values in each region in heat shocked females. 24 6 Crossover values in each region in mitomycin C and saline injected females. 25 7 Cumulative sterility values of mitomycin C and saline injected females. 26 8 Summary of expected and observed double crossover types in each test series. 27 - iv - LIST OF FIGURES FIGURE PAGE 1 Mating procedure for obtaining sibling females with different combinations of inversions. 9 2 Ratios of crossover values in each region for each treatment (P^) to their respective control value (P0). 16 3 Number of females with values in each crossover percentage class for Region 1, Series 1. 18 4 Number of females with values in each crossover percentage class for Region 3, Series 1. 20 - v - ACKNOWLEDGEMENT S I wish to thank particularly Dr. David T. Suzuki for his encouragement, inspiration and guidance while this study was being carried out. I am grateful to Dr. Harold Batho and Mr. Ken Yuen of the British Columbia Cancer Institute, for their assistance in radiating the flies. I wish to thank all the members of the laboratory for the help they have given at various stages of the work, particularly Miss Susie Hayashi and Mr. Geoffrey Foster for their extensive help in scoring. - 1 - INTRODUCTION Classically, crossing over was considered to be an event occurring between genes and exhibiting high positive interference within short map distances. By increasing the resolution in crossover analysis, Lewis (1945) and Green and Green (1949) established that intragenic recombination did occur but at a frequency too low to have been detected previously. This finding has been corroborated many times in Drosophila and in microorganisms. High negative interference has in many cases (Pritchard, 1955 and 1960; Streisinger and Franklin, 1956; and Chase and Doermann, 1958) been associated with the phenomenon of intralocus recombination. These observations contradict basic assumptions of the classical crossover model. In order to resolve this contradiction, Lewis (19 63) distinguishes between two classes of events which would occur by different mechanisms: classical interlocus crossing over which is characterized by positive inter• ference in short regions, and intralocus crossing over which is associated with negative interference in short regions. On the other hand, Pritchard (1955 and 1960) invokes an hypothesis of short effectively paired regions within which multiple exchanges readily occur. If markers are included within such a region, double and triple crossovers - 2 - could be detected but if markers surround a switch region only odd number exchanges would be detectable and these would appear as single crossovers. Thus he accounts for the correlation of intralocus recombination with high negative interference, as well as classical interlocus crossing over by a single mechanism. The present study, involving two recessive pseudoalleles at the Notch locus and closely linked outside markers, was carried out in an attempt to determine whether a One or a Two mechanism hypothesis applies to crossing over in Drosophila. Certain treatments are known which alter inter- genic crossover frequencies. If there is only one mechanism by which both intralocus and interlocus crossing over occur, then intralocus crossover frequencies should be altered in the same way and to a similar extent as interlocus crossover frequencies by each treatment. If there are two different mechanisms then one treatment might not necessarily affect recombination of each type in the same way. - 3 - METHODS and MATERIALS Crossing over at the tip of the X chromosome of Drosophila melanogaster was studied using the following markers (followed by map distances as listed by Bridges and Brehme, 1944): apricot - wf; (1.5), facet-notchoid - fano (3.0±), split - s£l (3.0±) and ruby - rb (7.5). Welshons (1958) showed that fano and spl were separable, fano being localized 0.03 units to the left of spl. Separate Series were run testing four treatments known to affect crossing over: presence of autosomal inversions, y-radiation, heat shock and mitomycin C injection. In each Series, recombination in the intralocus (fano - spl) and interlocus(wa - fano and spl - rb) regions was measured concomitantly. Individual wa + + rb/+ fa110 spl + or wa + spl +/ + fano + rb females 12-18 hours old were testcrossed to three wa fano spl rb/Y males in shell vials. After six days (Brood I) the parents were discarded or transferred to a second vial. The flies were again transferred after three days (Brood II) and discarded after six days (Brood III). All offspring of each female were recorded separately for each brood. The phenotypes of each class of crossover chromosome are shown in Table 1. Three of the double Key to symbols: w - white s - smooth + - wild r - rough o - orange d - delta i rb - ruby e - easy I c - by chance TABLE 1 PHENOTYPE SCORED IN EACH CROSSOVER CLASS, w + + rb fa no spl + CROSSOVER TYPE GENOTYPE PHENOTYPE RECOGNITION eye eye colour texture wing a Non w + + rb w s + no + fa spl + + r d single: 1 wa fano Spi + o r d e (inter) + + + rb rb s + e wa + spl + o r + e (intra) + fano + rb rb s d e wa + + + o s + e (inter) + fano spl rb rb r d e double: 1,2 wa fano + rb w s d c (inter:intra) + + spl + + r + c 1,3 wa fano spl rb w r d e + + + + + s e (inter:inter) wa + spl rb — w r + c 2,3 + fa no + + + s d e (intra:inter) - 6 - crossover (DCO) classes (wa fa spl rb, + + + +, and + fano + +) could be recognized at all times. The other three DCO classes can be distinguished from the parental classes only by a detailed examination of all wild or white-eyed flies. Since the significance of DCO events was not anticipated at the beginning of the study, time was not taken to look for these three DCO classes and as a result they were recognized only by chance. All three of these classes are of the intra:interlocus type. The lines in Table 1 indicate the parental class with which these DCOs would be confused. Crosses were made at 24° ± 2° C on a Standard Cal Tech Drosophila medium. Series 1. Autosomal Inversions Steinberg (193 6) found that autosomal inversions increase crossing over in the X chromosome. The regions affected to the greatest extent are at the tip and adjacent to the centromere (Schultz and Redfield, 1951). In this study the interchromosomal effect of the inversions SM-1 and Ubx"*"^^ on crossing over at the tip of the X was determined. SM-1 is a series of inversions of the second chromsome, homozygous lethal and associated with a 130 dominant curly wing phenotype. Ubx is a series of - 7 - inversions of the third chromosome, also homozygous lethal and associated with a dominant enlargement of the halteres. Sibling females heterozygous for the X chromosome markers and carrying different combinations of the inversions were recovered as shown in Figure 1. Crossing over was studied only in Brood I. Series 2. Cis-Trans Effect Altenburg and Browning (19 65) found that exchange values within a cistron are different when pseudoalleles are linked in cis or trans.