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Studies on nonhomologous chromosome pairing in females and a maternally suppressed position-effect lethal… Harger, Hideh 1974

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• STUDIES ON NONHOMOLOGOUS CHROMOSOME PAIRING IN FEMALES AND A MATERNALLY SUPPRESSED POSITION-EFFECT LETHAL IN MALES OF DROSOPHILA MELANOGASTER by HIDEH HARGER B.Sc, Un i v e r s i t y of Teheran, Iran, 1964 M.Sc, Un i v e r s i t y of Teheran, Iran, 1968 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY In the Department of Zoology We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA August, 1974 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an a d v a n c e d d e g r e e at t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . A HARGER HIDE^ Department o f Zoology The U n i v e r s i t y o f B r i t i s h C o l u m b i a V a n c o u v e r 8, Canada i ABSTRACT In the absence of s t r u c t u r a l heterozygosity i n females of Drosophila melanogaster, the frequency of compound autosome nonsegregation i s generally i n the range of 0.5 to 5.0 percent. However, the frequency of nonsegregation for any given combination of compounds i s highly reproducible. This low frequency of nonsegregation i s commonly referred to as the spontaneous l e v e l of nonsegregation. By mating compound-3 females, with marked X-chromosomes, to d i f f e r e n t i a l l y marked compound-3 males, i t has been possible to demonstrate that nonsegregation of the compound autosomes i s d i r e c t l y correlated with and proportional to non-di s j u n c t i o n of the X-chromosomes. In addition, the majority of female progeny that are patroclinous f o r the p a i r of compound autosomes are matroclinous f o r both X-chromosomes and, i n most cases, the male progeny that are matroclinous f o r the p a i r of compound autosomes are patroclinous for the X-chromosome. From t h i s d i s t r i b u t i o n of X-chromosomes and compound autosomes i t i s evident that the simplest explanation for the recovery of exceptional progeny i s nonhomologous p a i r i n g between the compound autosomes and the X-chromosomes. The presence of crossover X-chromosomes i n a portion of nonsegregational exceptions indicates that not a l l nonsegregational progeny are derived following nonhomologous pa i r i n g of compound autosomes and X-chromosomes. Such independent non-segregational progeny might represent the true "spontaneous" l e v e l of nonsegregation. In contrast to nonsegregation of compound autosomes, with rare exceptions X-chromosome nondisjunction i s s t r i c t l y a function of t h e i r nonhomologous p a i r i n g with compound autosomes. i i The inverse c o r r e l a t i o n between X-chromosome recombination and X-chromosome nondisjunction not only provides added support to the concept of nonhomologous p a i r i n g but also agrees with the temporal sequence of G r e l l ' s (1962) d i s t r i b u t i v e p a i r i n g model. On the assumption that nondisjunction i s a function of nonhomologous pai r i n g , the observed d i s t r i b u t i o n of nondisjunctional X-chromosomes, r e l a t i v e to the assortment of compound autosomes, i s consistent with a t r i v a l e n t p a i r i n g model that views nondisjunction as p r i m a r i l y a consequence of both X-chromosomes p a i r i n g with, and segregating from, a sin g l e compound autosome. However, the formation of bivalents involving nonhomologues i s also a possible contributing f a c t o r . The evidence given here strongly favours the concept that nonhomologous d i s t r i b u t i v e p a i r i n g occurs at the centromeric region, rather than along the t o t a l length, of the chromosomes. In a number of experiments, where the male parents c a r r i e d the Muller-5 (M-5) X-c.hromosome, the number of patroclinous nondis j u n c t i o n a l (M-5/0) males recovered was much lower than the number of matroclinous, nondisjunctional (X/X/Y) females. This reduced v i a b i l i t y of M-5/0 males has been r e a l i z e d f o r several years and believed to r e f l e c t a p o s i t i o n - e f f e c t suppression of the ribosomal-RNA genes. However, the re s u l t s of these experiments reveal that M-5/0 l e t h a l i t y i s conditional and dependent on the parental o r i g i n of the M-5 chromosome. When the o r i g i n of t h i s chromosome i s paternal, M-5/0 males display very low v i a b i l i t y ; when the M-5 chromosome i s i n h e r i t e d maternally, the v i a b i l i t y expressed by M-5/0 males i s equal to that of M-5 males carrying a Y-chromosome. Moreover, M-5/0 l e t h a l i t y i s p a r t i a l l y suppressed by i i i certain combinations of compound autosomes, both when carried by the female parents and when carried by the M-5/0 progeny themselves. The direct and maternal suppressive effects of these compound autosomes are assumed to be caused either by a specific suppressor gene present on these compound autosomes or by a nonspecific effect exerted by their centromeric heterochromatin. To explain M-5/0 lethality and the maternal effect associated with i t , a speculative model, based on the inactivity of chromosomes in sperm and a possible cytoplasmic preconditioning in eggs of M-5 females, is proposed. iv TABLE OF CONTENTS PAGE ABSTRACT i TABLE OF CONTENTS iv LIST OF TABLES v i LIST OF FIGURES v i i i ACKNOWLEDGEMENT i x CHAPTER I. STUDIES ON NONHOMOLOGOUS CHROMOSOME PAIRING IN FEMALES OF DROSOPHILA MELANOGASTER Introduction 1 Materials and Methods 16 Description of chromosomes and mutations. ... 16 The construction of compound autosomes 16 The marker system used to study simultaneous meiotic events 18 General mating procedures 19 General outline for maintaining genetic uniformity between the experimental and control female lines 19 Results 22 Nondisjunction in compound-3 females 22 Nondisjunction in compound-2 females 43 Discussion 51 CHAPTER II. STUDIES ON A MATERNALLY SUPPRESSED POSITION-EFFECT LETHAL IN MALES OF DROSOPHILA MELANOGASTER 66 Introduction 67 Materials and Methods 75 Description of chromosomes and mutations. ... 75 The parental source of the Muller-5 (M-5) chromosome 75 V PAGE General mating procedures 76 Results 77 Discussion 96 LITERATURE CITED 102 APPENDIX I 109 1. The formation of the marked-X chromosome used i n recombination studies 109 2. The generation of compound-2 and compound-3 autosomes heterozygous for paracentric inversions 110 3. Transfer of a marked-X chromosome from a standard s t r a i n into a l i n e bearing compound autosomes I l l g 4. The introduction of the marked Y-chromosome, B Y, into a l l compound autosome strains 112 5. The representative of a cross used to minimize the genetic v a r i a b i l i t y between the females of the experimental and control series 113 6. The construction of experimental (heterozygous for autosomal inversions) and control females for crossover studies i n strains bearing standard autosomes 113 7. The use of the constructed stocks i n measuring recombination i n the proximal region of the X-chromosome i n the presence of inversions and the absence of compounds 114 8. Replacement, i n males, of the M-5 chromosome with a standard X-chromosome carrying the Bar (B) mutation. 114 9. A diagram indicating the regions of crossing-over and the products of exchange i n the X-chromosomes. .... 115 APPENDIX I I The generation of compound autosomal l i n e s with attached-X females and attached-XY males having no free Y-chromosome. 116 v i LIST OF TABLES TABLE PAGE CHAPTER I I. T o t a l number of progeny recovered as products of regular and exceptional meiotic events. 23—24 I I . D i s t r i b u t i o n of nonsegregating compound thirds i n nondisjunctional progeny. 28 I I I . Adjusted d i s t r i b u t i o n of the exceptional classes recorded as percent of the t o t a l progeny. 31 IV. Percent X-chromosome recombination i n d i f f e r e n t compound-third and standard l i n e s . 36 V. C o r r e l a t i o n between crossing-over i n the proximal region of the X-chromosome and t o t a l nondisjunctional events. 40 VI. Percent X-chromosome recombination i n d i f f e r e n t compound-second and standard l i n e s . 47 VII. Adjusted d i s t r i b u t i o n of the exceptional classes recorded as percent of the t o t a l progeny. 48 CHAPTER II I. A comparison of the r e l a t i v e recovery, i n various compound l i n e s , of X/0 and X/Y males with maternally derived M-5 X-chromosome., , 78 I l a . The e f f e c t of d i f f e r e n t compound-third autosomes and a Y-chromosome on the s u r v i v a l of maternally derived M-5 males. Sex r a t i o obtained when M-5/0 were derived from: M-5/M-5;C(3L)P2,ri;C(3R)VT43In(Payne)/eS female X XYS.TLy+/0jC(3L)VT23In(Payne)/se;C(3R)VT2J>cu male 79 l i b . The e f f e c t of d i f f e r e n t compound-third autosomes and a Y-chromosome on the s u r v i v a l of maternally derived M-5 males. Sex r a t i o obtained when M-5/Y males were derived from: M-5/M-5-C(3L)P2,vi;C(3R)VT4,In(Payne)/eS female X v i i TABLE PAGE X XIs' .ILy+/Y;C(3L)VT23In(Payne)/se;C(3R)VT23cu male 80 I I I . Relative s u r v i v a l , i n a standard autosome l i n e , of M-5/0 males whose X-chromosomes were maternal i n o r i g i n as compared to those with X-chromosomes that were paternally derived. 82 IVal. The e f f e c t of d i f f e r e n t combination of compound-thirds and t h e i r parental o r i g i n on the r e l a t i v e s u r v i v a l of M-5 males with paternally derived X-chromosomes. Sex r a t i o obtained when M-5/0 males were derived from: C(1)RM3 +/0;C(3L)P23ri;C(3R)VT43In(Payne)/eS female X M-5/Y;C(3L)VT2,In(Payne)/se;C(3R)VT23eu male 84 IVa2. A repeat of experiment I V a l . 85 IVb. Sex r a t i o obtained when M-5/Y males were derived from: C (1 )RM/+/Y>; C(3L)P23 ri; C (3R) VT43 In (Payne )/eS female X M-5/Y;C(3L)VT23In(Payne)/se;C(3R)VT23cu male 86 IVc. Sex r a t i o obtained when M-5/0 males were derived from: C (1 )RM3 +/0; C(3L) VT23 In (Payne)/se; C(3R) VT-2, cu female X M-S/Y;C(3L)P23ri;C(3R)VT43In(Payne)/eS male 87 IVd. Sex r a t i o obtained when M-5/Y males were derived from: C(l)RM3+/Y;C(3L)VT23In(Payne)/se;C(3R) VT2,ou female X M-S/I;C(3L)P23ri;C(3R)VT43In(Payne)/eS male 88 V. Cumulative sex r a t i o s obtained from t o t a l progeny recovered at three successive i n t e r v a l s following removalsof the parents. 92-93 v i i i LIST OF FIGURES FIGURE PAGE 1. The r e l a t i o n s h i p between nondisjunctional and non-segregational meiotic events i n compound-3 females. 34 2. The inverse c o r r e l a t i o n between percent crossing-over i n the proximal region (_f to the centromere) and t o t a l nondisjunction of the X-chromosome. 42 3. The t r i v a l e n t p a i r i n g model. 59 4. The double b i v a l e n t p a i r i n g model. 60 5. The si n g l e bivalent p a i r i n g model. 63 i x ACKNOWLEDGMENT I d e e p l y a p p r e c i a t e t h e a s s i s t a n c e o f Dr. D a v i d G. Holm d u r i n g t h e c o u r s e o f my s t u d i e s a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a . I n a d d i t i o n t o p r o v i d i n g r e s e a r c h f a c i l i t i e s and f i n a n c i a l s u p p o r t , Dr. Holm has g i v e n f r e e l y o f h i s t i m e i n a d v i s i n g and e n c o u r a g i n g me i n my r e s e a r c h and i n t h e p r e p a r a t i o n o f t h i s m a n u s c r i p t . I woul d a l s o l i k e t o thank Mr. A r t h u r J . H i l l i k e r f o r h i s c r i t i c a l r e a d i n g o f t h e m a n u s c r i p t and i n a d d i t i o n e x p r e s s my a p p r e c i a t i o n t o him and to Mr. W i l l i a m G. G i b s o n f o r many u s e f u l d i s c u s s i o n s . My th a n k s t o Dr. P e t e r A. L a r k i n f o r t h e b e n e f i t o f h i s s t a t i s t i c a l c o u n s e l . CHAPTER I STUDIES ON NONHOMOLOGOUS CHROMOSOME PAIRING IN FEMALES OF DROSOPHILA MELANOGASTER 1 INTRODUCTION In 1911, T. H. Morgan correlated sex-linked inheritance with the presence of heteromorphic sex-chromosomes in Drosophila. He demon-strated that genes for sex-linked characters are carried on the X-chromosome and that normally sons inherit their single X-chromosome from their mothers, while daughters inherit two X-chromosomes, one from each of their parents. Bridges (1913), while working in Morgan's laboratory, discovered an exception to regular sex-linked inheritance. He noticed, among the progeny recovered in a sex-linkage experiment, a few females that showed exactly the same sex-linked characteristics as their mothers (matroclinous), as well as a few males that were pheno-typically exactly l i k e their fathers (patroclinous). The recovery of matroclinous females and patroclinous males was viewed as a consequence of abnormal segregation of the X-chromosomes in females during f i r s t division of meiosis, hence they were referred to as nondisjunctional progeny. The patroclinous males, not having a Y-chromosome, were s t e r i l e , and therefore could not be investigated any further. The matroclinous females, however, when mated to normal males, gave rise to a higher frequency of nondisjunctional exceptions in the next generation. This increase was shown to result from the presence of a Y-chromosome in the genome of exceptional females and was interpret-ed as an effect of heterosynapsis of an X-chromosome with the Y. Bridges (1916) called nondisjunction of X-chromosomes i n X/X females primary nondisjunction and that of the X/X/Y females, secondary non-disjunction. Of the four types of zygotes arising from secondary nondisjunction 2 (XXX, XXY, XY and YY) only two (XXY females and XY males) w i l l result in viable progeny. The actual number of secondary nondisjunctional events, therefore, is approximately twice the observed number of exceptional progeny. Since Bridges (1916) postulated that pairing in X/X/Y females is exclusively bivalent in nature, he interpreted the frequency of 4.3 percent observed nondisjunction to reflect 16.5 percent pairing between one of the two X-chromosomes and the Y with the other X assorting at random. In support of his bivalent pairing theory, Bridges noted that nondisjunctional X-chromosomes were invariably noncrossovers. A second type of nondisjunction, referred to as equational non-disjunction, has also been observed (Bridges 1916, Merriam and Frost 1964). Equational nondisjunction implies nondisjunction of two sister chromatids at the second meiotic division, which results in the recovery, from heterozygous mothers, of daughters that are homozygous for markers carried by one of the maternal X-chromosomes. The X-chromosomes, following either primary or equational nondisjunction, can be of the crossover or non-crossover type (Bridges 1916, Merriam and Frost 1964), but products of secondary nondisjunction are invariably noncrossovers (Bridges 1916, Sturtevant and Beadle 1936, Grell 1962). Through cytological studies on oogonial c e l l s , Bridges (1921) demonstrated that the rarely occurring "diminished" mutants resulted from chromosome four primary nondisjunction producing haplo-four progeny. The frequency of this event was in-.the same range as primary nondisjunction of the X-chromosomes. In t r i p l o i d females of Drosophila melanogaster Bridges and Anderson (1925) found that synapsis generally involves a l l three X-chromosomes, 3 unlike X/X/Y females where only two sex chromosomes are inferred to be synapsed at any given time. Contrary to Bridges and Anderson's view, Beadle (1934) found that in attached-X triploids of Drbsbphila  melanogaster pairing i s mostly of the bivalent type. Inversion heterozygosity of the X-chromosomes has but a moderate influence on the frequency of primary nondisjunction (Sturtevant;. i n Morgan and Sturtevant 1944); however, secondary nondisjunction is greatly increased. Sturtevant and Beadle (1936) reported rates of secondary nondisjunction of greater than 50 percent in females heterozygous for multiple-break re-arrangements and Cooper (1948) reported that the increase in secondary non-disjunction was correlated with the complexity of the rearrangement involved. When the dissimilarity of the chromosomes is great enough to virtua l l y eliminate crossing-over, secondary nondisjunction approaches 80 percent (Cooper 1948). According to Bridges' bivalent-univalent pairing model;, the maximum possible nondisjunction in 2^ /X/Y females i s 50 percent, giving rise to the recovery of 33.3 percent exceptional progeny. To explain nondisjunctional rates of greater than 50 percent, Cooper (1948) proposed a model of trivalent formation in which he assumed that one X paired with the short arm of the Y-chromosome, while the second .X paired with the long arm. In support of his model, Cooper demonstrated that in the presence of a Y_-chromosome fragment, namely a Y-chromosome lacking either the short or the long arm, nondisjunction was never higher than 50 percent. In addition to the concept of trivalent pairing-complexes, Cooper (1948) also suggested that pairing between structurally hetero-^ zygous X-chromosomes i s a function of their proximal heterochromatin, allowing the heterochromatic Y-chromosome to become a very effective 4 pairing competitor. When females are heterozygous for both autosomal and X-chromosome inversions, a rather interesting picture emerges - secondary nondisjunction decreases (Morgan and Sturtevant 1944), but primary nondisjunction inc increases significantly (Morgan and Sturtevant 1944; Cooper, Zimmering and Kirvshenko 1955). Concomitant with the increased primary nondisjunction, Cooper ejt a l . (1955) observed an increased frequency of dominant lethality. They concluded that dominant lethality reflected autosomal nondisjunction and proposed that when only one pair of chromosomes in the genome is structurally heterozygous, segregation is normal; but when two or more pairs of chromosomes are structurally heterozygous, nonhomo-logous pairing.frequently occurs giving rise to aneuploidy and hence to dominant lethality. Similar findings were made by Terzaghi and Knapp (1960) in Drosophila pseudoobscura. Ramel (1961) confirmed autosomal nondisjunction as a cause of dominant lethality by mating X-irradiated males to females structurally heterozygous Both for the X and the second chromosomes. He recovered progeny that were matroclinous for the autosomes; clearly the products of nondisjunctional diplo-2 eggs f e r t i l i z e d by radiation-induced nullo-2 sperm. In the presence of structural heterozygosity for the second chromosomes alone, chromosome-2 nondisjunction is very low. There is a ten-fold increase in nondisjunction of the second chromosomes i f a Y_-chromosome is added to such females. In an additional test involving compound-X, nullo-Y females with structurally heterozygous second chromosomes, Ramel (1961) recovered only exceptional males with matro-clinous seconds that were the products of nonhomologous pairing, in the 5 female parent, between the compound-X chromosome and one of the two second chromosomes. Ramel further demonstrated that exchange between the two arms of compound-X chromosomes was not disturbed by t h e i r non-homologous p a i r i n g with the seconds. Ramel (1961) concluded that non-homologous p a i r i n g was r e s t r i c t e d to centromeres and the proximal heterochromatin. Sandler and N o v i t s k i (1956) demonstrated that triplo-^IV females, bearing standard X-chromosomes, produce a much higher frequency of matro-clinous progeny for the X-chromosomes than do diplo-IV females. The extra fourth chromosome, i n t h i s case, was assumed to nonhomologously pa i r with a noncrossover X-chromosome, thereby causing X-^chromosome nondisjunction. They suggested that regions of homology, which might reside i n the proximal heterochromatin, are shared between the X and the fourth chromosomes. They further stated that such regions might, i n f a c t , be shared between a l l chromosomes i n Drosophila melariogaster and that chromocenter formation might be a r e f l e c t i o n of t h i s homology, Lindsley and Sandler (1956) put forward further evidence for p a i r i n g homology between the X and the fourth. They studied the behaviour of d i f f e r e n t heterochromatic free X-duplications i n attached-X females bearing no free Y-chromosome. Those duplications that did not p a i r with and segregate from the attached-X, segregated from one of the fourth chromosomes. A l l these duplications had the e n t i r e proximal heterochromatin of the X-chromosome. G r e l l (1957, 1959a) and G r e l l and G r e l l (1960) demonstrated that i n X/X/Y females heterozygous for a 3;4 t r a n s l o c a t i o n , designated T(.3;4) , and bearing a s i n g l e free fourth chromosome, the Y-chromosome segregated from the free fourth 83 percent of the time, and from the T4_ element 6 (that element of the translocation composed of the centromere and proximal heterochromatin of chromosome four attached to the right arm of chromosome three) 62 percent of the time. When the T4 element was eliminated from competition by generating females homozygous for T(3;4) the Y-4 segregation was increased to 92 percent. Grell (1959a) suggested that when more than two unpaired elements are present in the genome, such elements compete for association. This was clearly demonstrated by.the respective decrease and increase in Y-4 segregation in the presence and absence of T4_ as a pairing competitor. Equal recovery of the reciprocal classes demonstrated that they are products of meiotic segregation following association of the two elements (Grell and Grell 1960), That proximal heterochromatin i s not entirely responsible for nonhomologous pairing was indicated by the fact that in a female with, a Y-chromosome, a single free fourth and a T4, the T4_ chromosome segregates more frequently from the Y than from the fourth, although four and T4 have homologous centromeres and proximal heterochromatin (Grell and Grell 1960). Grell (1962 a, b) proposed a model to explain secondary nondisjunction. According to her model, the f i r s t meiotic division in female Drosophila melanogaster consists of two separate phases. The f i r s t phase, called "exchange pairing", i s based on homology recognition and i s concerned with genetic exchange between homologous chromosomes. Nonhomologous pairing is not involved at this time. The second phase, which is sub-sequent to exchange but prior to disjunction, is called "distributive pairing". During the distributive pairing phase, crossover homologues remain paired, but noncrossover elements go to the "distributive pairing 7 pool". When two homologous chromosomes, owing to structural hetero-zygosity, are noncrossover chromosomes, and are the only members of the distributive pairing pool, regular disjunction w i l l result. Furthermore, i f two heterologues are the only members of the distributive pairing pool, they too w i l l regularly segregate. However, when there are more than two chromosomes in the distributive pairing pool, pairing i s competitive. Since distributive pairing is apparently not a function of homology, nonhomologous distributive pairing can occur, one con-sequence of which is nondisjunction of homologous chromosomes. According to Grell's distributive pairing hypothesis, since non-homologous pairing only involves those chromosomes that failed to undergo exchange, i t neither alters the rate of exchange nor increases the number of nonexchange tetrads. With respect to secondary nondisjunction, specifically, the Y-chromosome distributively pairs with one or both nonexchange X-chromosomes subsequent to the exchange pairing phase. Inversion heterozygosity for the X-chromosomes suppresses the frequency of exchange thereby increasing, in X/X/X females, the proportion of non-crossover X-chromosomes available to the Y_ as nonhomologous pairing partners (Sturtevant and Beadle 1936, Cooper 1948, Grell 1962 a). In contrast to the Grell (1962) hypothesis, Merriam (1967) postulated that nonhomologous association, leading to nondisjunction of homologues, occurred prior to exchange. Merriam based this conclusion on his observation of a.decrease in crossing-over in the proximal region of the X-chromosome in X/X/Y females. He reasoned that the Y-chromosome interfered with exchange pairing of the _X's and that the X^proximal exchange value was restored in the presence of inversion heterozygosity 8 for the autosomes because the latter provided alternative nonhomologous pairing partners for the Y. Repeating Merriam's work, Grell (1970) disclosed that a proximal decrease in X-chromosome crossing-over i s accompanied by a distal increase. Therefore, the overall frequency of exchange i s not disturbed and the effect of the Y on crossing-over between X-chromosomes i s an interchromosomal effect rather than an interference of the Y with X--chromosome exchange pairing. Grell (1970) also found that the presence of an autosomal inversion in X/X/Y females failed to correct the proximal crossover depression occasioned by the Y_. Nevertheless, a high degree of nonhomologous pairing between the Y-chromosome and the rearranged autosome was evident. Based on these observations, Grell discounted Merriam's findings and concluded that nonhomologous pairing does not precede but follows the pairing of homologues for exchange. Another alternative to the Grell (1962 a) distributive pairing model was offered by Novitski (1964). His model involves the association of a l l chromosomes, prior to exchange, in a chromocentral configuration. Following pairing for exchange, homologous chromosomes separate from the chromocenter; however, those chromosomes which, for one or another reason, cannot successfully engage in pairing for exchange, maintain the chromocentral association and, by default, pair with either homologues of nonhomologues. Noting the temporal sequences of meiotic events described in Novitski's model, Grell (1967) concluded that his model was, in fact, consistent with the distributive pairing hypothesis. While distributive pairing forces appear to be independent of chromosome homology (Grell 1962, 1967, 1970), rules governing preferen-t i a l pairing under a competitive situation have been described (Grell 9 1964, 1967, 1970). According to the distributive pairing model, the small fourth chromosomes are regular members of the distributive pairing pool, although their presence has no noticeable effect on the distribution of larger chromosomes that enter the distributive pairing phase. However, upon introducing a free-X duplication of size similar to that of the fourths, chromosome four disjunction decreases sharply (Grell 1964). That distributive pairing is a function of size recognition, at least for the smaller chromosomes, was clearly revealed through a series of competitive studies involving X-duplications of varying sizes (Grell 1964 a). The highest degree of pairing recognition occurs between elements whose size ratio (based on mitotic metaphase measurements) equals one. Sturtevant (1934, 1936), when studying triplo-IV females in which the fourth chromosomes were either structurally or genetically different from one another, suggested that segregation of the three fourth chromosomes from a trivalent pairing complex resulted in preferential distribution such that one particular fourth usually migrated to the haplo-IV pole. He related this preference to variable pairing a f f i n i t i e s of different fourth chromosomes, and, accordingly, arranged such chromosomes in an increasing series, according to the tendency of each to go to the haplo-IV pole at meiosis. Although Sturtevant did report preferential segregation among the fourth chromosomes, he did not present any mechanism responsible for their behaviour. It was Grell (1964 b) who related this phenomenon to slight size differences among the fourth chromosomes employed. Recently, Moore and Grell CL972 a) discovered, in studies involving females bearing compound—4 chromosomes3 that size recognition was determined by overall length and not arm length. 10 As an addendum to the genetic studies, cytological observation of ob'gonial cells indicate a correlation between size and mitotic pairing, although homology recognition also appears to be an important factor (Grell 1967, Grell and Day 1970). When the distributive pairing pool contains three elements of dissimilar size, the assortment of chromosomes implies trivalent pairing complexes, with the chromosome of intermediate length assuming a directing role (Grell 1964 a, 1964 b). The maximum frequency of trivalent formation apparently occurs when the length of the middle element i s the geometric mean of the lengths of the other two. In addition to chromosome size, the position of the centromere also appears to serve as a governing factor in determining the distribution of chromosomes i n a nonhomologous competitive pairing situation (Moore and Grell, 1972 b). Evidently, when the largest of three chromosomes i s metacentric and the size ratio between the inter-mediate acrocentric chromosome and the largest chromosome i s greater than 5/9, the metacentric chromosome provides the directing role in segregating from a trivalent association. The present study has focused attention upon an exceptional class of meiotic events i n females bearing compound autosomes. Compound autosomes are generated through the attachment of homologous chromosome arms to a common centromere. For the large metacentric autosomes of Drosophila, the attachment of homologous arms essentially converts a pair of metacentric homologues into a pair of metacentric heterologues. While compound autosomes (as well as the reverse metacentric compound-X) can be considered analogous to the isochromosomes originally described by Darlington (1939), owing to the nature of their formation, pseudo-isochromosome was i n i t i a l l y adopted as a term of description (Lewis 1972). 11 The original compound autosomes were constructed under the guidance of E.B. Lewis by I.E. Rasmussen, E. Orias and P. Deal (Rasmussen 1960; see Holm 1974 for a discussion on the methods used by E.B. Lewis and collaborators). Since that time, numerous compound-2 and compound-3 chromosomes have been recovered from X-ray treated females and males; they also arise infrequently as spontaneous events (Chadov 1970). The nature of compound autosome formation was i n i t i a l l y viewed as a translocation-like event where an acentric fragment became joined to an homologous centromere bearing free arm (Rasmussen 1960). Although other mechanisms have been proposed (Bateman 1968), recent experimental evidence clearly demonstrated that compounds arise through a translocation-l i k e or interchange event (Holm, Gavin, Kowalishyn and Yeomans, in preparation). The earliest report on the use of compound autosomes (McCloskey 1966) suggested a regular meiotic segregation of C(3L)RM (compound-3 l e f t , reversed metacentric) from C(3R)RM (compound-3 right, reversed metacen-tric) as well as C(2L)RM from C(2R)RM. It was soon discovered, however, that while the frequency was low (normally less than 5 percent), exceptional progeny, either patroclinous or matroclinous for the complementary pair of compound-thirds, were recovered. Moreover, inversion heterozygosity for chromosome-2 or the presence of a compound-X in C(3L);C(3R) females resulted in a greatly increased production of progeny inheriting both compounds from either one or the other parent (Baldwin and Chovnick 1967, Holm et a l . 1967, Holm 1969). Similar observations were made in studies involving C(2L);C(2R) lines (Holm 1969,E.Grell 1970). These findings led to the discovery that, in males, 12 C(3L) and C(3R) assort randomly during meiosis (Holm 1969, 1974). While nullosomic and disomic sperm are also produced by compound-2 males, the frequency of egg hatch, which is used as a measure of compound autosome assortment in males, is generally greater than expected under the assumption of random assortment (Holm 1969, 1974; Clark and Sobels 1973; Evans 1971). The meiotic behaviour of compound chromosomes in females has been acknowledged as supporting the general terms of the distributive pairing model (Grell 1963, 1970; Holm 1969', 1974). However, unlike the other chromosomes that assort s t r i c t l y as a function of distributive pairing properties, the compound-X (Welshons 1955) and the compound-third (Baldwin and Chovnick 1967) with isosequential arms engage in exchange pairing with the same regularity as standard chromosomes. Nevertheless, compound chromosomes are regular members of the distributive pairing phase and exchange pairing within these chromosomes has no effect on the pairing properties regulating their distribution in the f i r s t meiotic division (Grell 1963, Holm 1969, 1973). The specific feature of compound autosome behaviour that initiated this study was the recovery of significant numbers of nonsegregational (that i s matroclinous and patroclinous) progeny from females whose genomes were free of any known heterologous rearrangements. Frequencies of nonsegregation from approximately 0.5 to 5.0 percent have been recorded. For any given complementary pair of compound autosomes, however, the frequency remains relatively constant. The occurrence of nonsegregation without any known cause has been referred to as the spontaneous level (Leigh and Sobels 1970) and the suggestion was made (Liitolf 1972) that 13 the apparent "stock specific" frequencies may reflect different size duplications and deficiencies of the centromeric region arising as a consequence of compound formation. While, on the one hand, "spontaneous" nonsegregation may reside in peculiar properties of the compound autosomes per se, on the other hand, since compounds are considered regular members of the distributive pairing phase, nonsegregation might result almost exclusively from non-homologous pairing interactions. These two concepts, in fact, may not be mutually exclusive. For instance, structural differences within the proximal heterochromatin might have some bearing on nonhomologous pairing competition. The question to ask, therefore, i s : in the absence of structural heterozygosity, what chromosomes provide pairing alternatives to the compound autosomes? In addition to the compounds, the fourth chromosomes, as noted previously, are recognized as regular members of the distributive pairing pool (Grell 1964). According to the size model, however, their small size should prevent them from competing with the compound autosomes. This view i s supported by the fact that nonsegregation of a compound-X and a free Y rarely occurs. Exchange pairing should almost totally exclude the isosequential standard second chromosomes (in compound-3 females) or the thirds (in compound-2 females), whereas, recalling the frequency of secondary nondisjunction for the X-chromosomes, one realizes that a considerable fraction of these chromosomes f a i l to engage in crossing-over. I f , instead of viewing i t as an event solely dependent on the presence of a free Y, secondary nondisjunction i s considered as resulting from nonspecific pairing of the X-chromosomes with non-14 homologues, compound autosomes should provide as competitive a pairing alternative to the noncrossover X-chromosomes as does a free Y. Such nonhomologous pairing interactions would be expected, therefore, not only to produce gametes nondisjunctional for the X-chromosomes, but also gametes nonsegregational for the compound autosomes. Furthermore, the frequency of nonsegregation, which appears to be characteristic for any given li n e , might, in fact, indicate an interchromosomal effect as a property of certain compound autosomes. The experiments described in this paper were designed to examine the above concepts, namely that nonsegregation of compound autosomes arises as a consequence of nonhomologous pairing with the X-chromosomes. While there remains a small but significant fraction of nonsegregational events yet to be attributed to some causal agent, the results of this study clearly show a positive correlation between nonsegregation and nondisjunction. Furthermore, the negative correlation between exchange and nondisjunction implies that, to a large extent, nonsegregation of the compounds i s dependent on the availability of noncrossover it-ch romo somes. Before proceeding to the next section, I wish to direct the readers' attention to the definition of the terms "nondisjunction" and "non-segregation" as they are used in this paper. Nondisjunction is conventionally used to describe a situation in which a pair of homologous chromosomes, because they failed to migrate to opposite poles during the meiotic division, are recovered in the same gamete. Compound autosomes, which essentially represent pairs of heterologues, when recovered in a single gamete are termed nonsegregational. This term has been adopted 15 as an operational definition, not to replace the more conventional terminology, but rather to provide a clear distinction between events involving compound autosomes, which segregate only through nonhomologous pairing, from those events involving true homologues. 16 MATERIALS AND METHODS Description of chromosomes and mutations: The genetic markers used 2 in t h i s study include: yellow (y_: 1-0.0); yellow-2 (y_ : 1-0.0); white apricot (w : 1-1.5); forked ( f : 1-56.7); Bar (B: 1-57.0); carnation (car: 1-62.5); held out (ho: 2-4.0); Curly (Cy_: 2-6.1); plexus (p_x: 2-100.5); Roughened (R: 3-1.4); Lyra (Ly: 3-40.5); radius incompletus ( r i : 3-47.0); curled (cu: 3-50.0) and Hairless (H: 3-69.5). Eight autosomal and two X-chromosome inversions were u t i l i z e d . The autosomal inversions employed were In(3L)Payne and In(3R)Payne, paracentric inversions of chromosome three; In(3LR)TM3,Sb Ser and In(3LR)DcxF, per i c e n t r i c multiple-break t h i r d chromosome inversions; In(2L)Cy and In(2R)Cy, paracentric inversions of the l e f t and right arms repectively of chromosome two; and In(2LR)SMI,Cy, a pericentric multiple break inversion of chromosome two. The two X-chromosome inversions were In(l)65 and Muller-5. The l a t t e r i s a multiple-break . . . T . / , v . S1L 8R.C SI 8 a_ inversion with the genetic constitution I n ( l ) s c sc +S,sc sc w B. A homozygous wild-type reversed metacentric attached X-chromosome, S C(1)RM,+; and the Y-chromosome, B Y, marked by a small duplication of S the X-chromosome bearing the B_ marker, were also employed. A general description of a l l markers, rearrangements, the attached-X and the marked Y-chromosome can be found i n Lindsley and G r e l l (1968). The construction of compound autosomes: Compound autosomes constructed s p e c i f i c a l l y for use i n the present study were recovered by treating v i r g i n females, carrying appropriately marked autosomes, with 3500 rads of gamma radiation from a ^ Co source, and mating them 17 to males carrying d i f f e r e n t i a l l y marked compounds f o r e i t h e r chromosomes-2 or -3. The only surviving progeny from these matings w i l l be e i t h e r those who, through nondisjunction of the r a d i a t i o n treated p a i r of autosomes, i n h e r i t both homologues from t h e i r mother (matroclinous exceptions) or both compounds from t h e i r father (patroclinous exceptions) or those i n h e r i t i n g a newly formed compound from t h e i r mother and the complementary compound from t h e i r father (Holm 1969, 1974). From females s t r u c t u r a l l y heterozygous f o r autosomal inversions [Iri(3L)Payne, In(3R)Payne and In(2L+2R)Cy], compound autosomes were generated for the purpose of examining compound associated interchromosomal e f f e c t s . Therefore, such new compounds are heterozygous f o r the inversions employed. S t r u c t u r a l l y homozygous compound autosomes were also construct-ed and used throughout these experiments as a series of r e l a t i v e controls. Three compound autosomes [C(2R)P1,C(3L)P2 and C(3L)P5] were constructed i n the laboratory of E.B. Lewis at Pasadena. The general terminology suggested by Lindsley and G r e l l (1968) has been adopted to designate the genotypic c o n s t i t u t i o n of compound autosomes. To demonstrate how the terminology f o r compound autosomes i s to be interpreted, the following pair of compound thirds have been selected: C(3L)RM,VT2,In(3L)Payne/se;C(3R)RM,VT2,cu. This reads as follows: Compound-3 L e f t , Reversed Metacentric, Vancouver Tabatabaie-2, Inversion-3 L e f t Payne/sepia; Compound-3 Right, Reversed Metacentric, Vancouver Tabatabaie-2, homozygous curled. The code VT2 represents the l o c a t i o n of construction, the name of the person who constructed the compound and the number serves to i d e n t i f y a p a r t i c u l a r compound autosome within a s e r i e s . 18 Since newly constructed compounds are the products of independent events, and therefore have differnent breakpoints, each compound i s maintained i n a separate stock. Hence, a l l the members of a compound-bearing l i n e are i d e n t i c a l , at least i n regard to the proximal region. The procedures for transferring marked chromosomes from strains with standard autosomes to the compound l i n e s , and other crosses involved i n the formation of stocks used throughout the experiments, are demon-strated i n f u l l d e t a i l i n Appendix I. The marker system used to study simultaneous meiotic events: Crossing-over was measured i n the proximal region of the X-chromosome. The X-chromosome used for t h i s study carries a duplication [Dp(l;l) sc^ "*"] of the d i s t a l t i p of the X-chromosome marked by the wild type a l l e l e of yellow, y^\ This duplication, which i s attached to the short arm of the X, was o r i g i n a l l y derived as an aneuploid recombinant from a heterozygote for the pericentric inversion, I n ( l L R ) s c ^ (Lindsley and G r e l l 1968). + 2 Females with the X-chromosomal constitution, y f car.y /y , were mated to males whose X-chromosomes were marked with the dominant mutation, Bar (B). By using such combinations of markers i n the male and female parents, matroclinous and patroclinous progeny resulting from non-disjunction of X-chromosomes i n females, as we l l as regular progeny bearing crossover or noncrossover X-chromosomes, can be simultaneously examined. Males and females used i n the main experiments carried differentially-marked compound autosomes, therefore, regular and non-segregational progeny for the compound autosomes could be readily i d e n t i f i e d . 19 General mating procedures: In each of the experiments, single females were mated with two males of the appropriate genotype in 25 mm x 95 mm shell vials containing standard cornmeal, sugar, yeast and agar medium with propionic and phosphoric acid added as mold inhibitors. Parents were transferred through three successive broods of 4, 3 and 3 days, for a total of 10 days of egg laying. Vials were numbered so that the meiotic behaviour of chromosomes for any given female could be followed throughout the total time of egg laying. A l l the experiments were conducted at 24° + 1°C for the duration of egg laying and develop-ment. Following eclosion, a l l progeny were counted and classified according to their phenotypes. General outline for maintaining genetic uniformity between the  experimental and control female lines: While the design of these experiments was directed primarily at examining the hypothesis that spontaneous compound autosome nonsegregation was the effect of non-homologous pairing interactions involving the X-chromosomes, the use of compound autosomes heterozygous for inversions was viewed as a means of comparing the interchromosomal effect (Lucchessi and Suzuki 1968) to the incidence of nonsegregation. Assuming, a p r i o r i , that compound-associated inversions would influence the frequency of crossing-over on the X-chromosomes, each experimental cross involving females carrying autosomal inversions was accompanied by a cross in which the female parents carried isosequential compound (or standard) autosomes. One of the crosses, i n every experiment involving compound-3 autosomes, used females carrying the same pair of isosequential compounds, C(3L)P2,ri and C(3R)VT2,cu. However, as demonstrated below, these 20 females were derived from different parental lines for each test. The second cross, i n each study, involved females heterozygous for an inversion in either C(3L) or C(3R) alone, or in both. Since the genetic background, excluding the effects of rearrangements, can influence, to varying degrees, the frequency of exchange, the females for each pair of crosses were recovered from the same parental cross. By following this procedure, the differences in genetic background should reside primarily within the compounds themselves. The outline below provides an example of the general procedures followed: y f eav.y+/y f car.y+;C(3DVT23In(3DPayne/se;C(3R)VT23cu females X y2/BSI; C ( 3D P23 ri; C(3R) VT43 In(3R) Payne/eS males (a) Experiment: females bearing structurally heterozygous compound autosomes. + 2 <? y f car.y /y ;C(3L)VT2,In(3L)Payne/se;C(3R)VT43In(3R)Payne/e females X B/Y;C(3DP23ri;C(3R)VT23ou males (b) Control: females bearing isosequential compound autosomes. + 2 y f oav.y /y ;C( 3DP23ri;C( 3R)VT23cu females X B/Y; C(3L) VT23 In(3D Payne/se: C(3R) VT4, In (3R)Payne/eS males It w i l l be noted from the example shown above that the male parents in the experimental cross carry the same pair of compound 21 autosomes as the female parents in the control series, and vice versa. Identical procedures were followed for the study of compound seconds and, similarly, for the general controls using standard autosomes with and without inversion heterozygosity. 22 RESULTS Nondisjunction in compound-3 females: Throughout the present study, three meiotic events in females of Drosophila melanogaster were simultaneously examined: (1) crossing-over i n the proximal region of the X-chromosome; (2) the meiotic distribution of X-chromosomes; and (3) the segregation pattern of compound autosomes. As a general control, crossing-over and nondisjunction were studied i n standard autosomal lines structurally homozygous, or heterozygous for inversions that were carried by the corresponding compound thirds. By replacing the standard autosomes with structurally homozygous compounds i t was possible to investigate the interchromosomal effects of the compound autosomes per se on the frequency of proximal exchange in the X-chromosome, as well as their possible role in X-chromosome non-disjunction. Additionally, by substituting the isosequential compounds with compounds that are structurally heterozygous, i t was possible to examine the interchromosomal effects of inversions associated with compound autosomes. The parental genotypes and the total number of regular and exception-al progeny are recorded in Table I. Females in a l l standard and compound autosomal lines used in these experiments were coisogenic for the heterozygous pair of proximally-marked X-chromosomes. To f a c i l i t a t e the identification of nondisjunctional progeny, the male parents in each cross carried an X-chromosome marked by the dominant Bar (B) mutation. The B_ marker for the f i r s t six experiments was included in the multiple-break inversion, Muller-5 (M-5), whereas, in the last four experiments, i t was carried by a standard X. The reason for this change in paternal Table I. Total number of progeny recovered as products of regular and exceptional meiotic events. Exp. No. Sex Parents Genotype + 2 Female y / oav.y /y ;In (3D Payne/'In (3R)Payne Male M-5/Y;+/+ Number of progeny Regular Exceptional Independent N.D.-X N.S.-C(3) 10866 4 Associated N.D. & N.S. + 2 l a Female y f oav.y /y ;Ly R/H Male M-5/Y;+/+ 6899 0 2 Female y f cav.y+/y2;C(3L)VT23InP/se;C(3R)VT23cu 8992 8 Male M-5/Y;C(3L)P2,ri;C(3R)VT4,InP/eS 2a Female y f oar.y+/y2;C(3L)P2Jri;C(3R)VT23cu 7395 94 Male M- 5/Y; C(3L)VT23 InP/s e; C ( 3R) VT4, InP/e S 32 102 20 162 Female y f car.y+/y2;C(3L)P2svi;C(3R)VT4,InP/eS 6771 41 79 74 M a l e M-5/Y;C(3L)VT23InP/se;C(3R)VT23eu Table I continued 3a v Female y f oav.y~/y2;C(3DP23rijC(3R)VT2,cu 5933 44 59 78 Male M-S/Y;C(3DVT23InP/se;C(3R)VT43InP/eS 4 Female y f oav.y+/y2;C(3DP53InP3ve h th/+;C(3R)VT23cu7999 30 20 28 Male B/Y;C(3L)P23ri;C(3R)VT43InP/eS 4a Female y f cav.y+/y2;C(3L)P23ri;C(3R)VT23cu 6905 182 43 193 Male B/Y;C(3L)VT23InP/se;C(3R)VT43InP/eS I O 5 Female y f oav.y /y ;C(3L)VT23InP/se;C(3R)VT43InP/eS 8530 15 20 15 Male B/Y; C(3L)P23ri;C(3R) VT2,cu 5a Female y f oav.y+/y2;C(3L)P23rl;C(3R)VT23cu 5740 74 19 88 Male B/Y;C(3L)VT23InP/se;C(3R)VT43InP/eS N.D. = Nondisjunctional;N.S. = Nonsegregational A * InP =•= Inversions (3D or (3R)Payne 25 X-chromosome w i l l be discussed later. Since the parents in crosses 1 and l a carried standard autosomes, only crossing-over and nondisjunction of the X-chromosomes could be followed. The remaining crosses involved males and females bearing differentially-marked compound autosomes, thereby permitting, in addition to nondisjunctional X-chromosomes, the recovery of nonsegregational compound autosomes. For each experimental cross involving females heterozygous for inversions on their standard or compound autosomes (1, 2, 3, 4 and 5) there is a corresponding control in which the females carry standard or compound autosomes with isosequential arms (la, 2a, 3a, 4a and 5a). The females used in each pair of crosses, such as 1 and l a , were recovered as sibling progeny. Therefore, except for the rearranged autosomes, these females share a common genetic background. Two types of progeny are recorded in Table I: regular and exceptional. Regular progeny arise from the normal distribution of chromosomes during meiosis in females and consist either of males who receive their X and one autosome from their mothers and the other autosome from their fathers, or of females who receive one X and one autosome from each of their two parents. The exceptional progeny recovered in those experiments involving compound autosomes are recorded as either independent or associated events. The independent exceptionals include individuals who either receive both compounds from one of the parents, but show the normal inheritance of X-chromosomes, referred to as independent nonsegregationals, or receive one compound from each parent, but inherit both maternal X's, i f female, or the paternal X, i f male. The latter are called independent nondisjunctionals. The 26 associated class of exceptions consists either of females with both maternal X's and paternal compounds or of males with a paternal X and both maternal compounds. Therefore, they are nondisjunctional as well as nonsegregational. Included in the associated group (as shown in Table II) are rare progeny who inherit the products both of ^-non-disjunction and compound-nonsegregation from the same parent. An examination of Table I reveals that in the absence of either compound autosomes or heterozygous inversions no exceptional progeny were recovered (cross l a ) . When the standard third chromosomes were trans-heterozygotes for In(3L)Payne/In(3R)Payne, four nondisjunctional exceptions were encountered. While the above differences are not significant, and even though the Payne inversions are known to increase X-chromosome recombination (Schultz and Redfield 1951), i t is interesting note that nondisjunctional exceptions were recovered only in association with these rearrangements. Pooling the data from crosses 1 and l a , the frequency of X-chromosome exceptions is in the same range as that previously reported for primary nondisjunction (Bridges 1916, Merriam and Frost 1964) . From females carrying compound autosomes, the recovery of non-disjunctional progeny was greatly increased. This increase cannot be related to secondary nondisjunction caused by the presence of a Y-chromosome, because the possibility of X/X/Y females was excluded by the use of the dominantly marked Y-chromosome, B Y, in the males of the original stocks from which a l l the experimental and control females originated. As shown in Table I, a very close correlation exists between total nondisjunction and total compound autosome nonsegregation. 27 This c o r r e l a t i o n implies an interdependence between the two events. Moreover, the observed frequency of associated nondisjunction was equal to or greater than that obtained for independent nondisjunction i n every case. This further demonstrates that the majority of such exceptionals a r i s e from an i n t e r a c t i o n between the X-chromosomes and the compound autosomes during oogenesis. I f associated exceptions were the product of two independent meiotic events, one would expect the frequency of t h e i r occurrence to be equal to the product of the frequency of the two independent classes. This, however, i s not the case, as the r e s u l t s c l e a r l y demonstrate (Table I I I ) . Furthermore, since t h e i r rate of occurrence i s f a r greater than the frequency of primary nondisjunctional events, not a l l of the independent nondisjunction-a l s could have originated i n the absence of compound-autosomal interference. The r e l a t i o n s h i p between the two meiotic events, as a product of nonhomologous p a i r i n g , i s well demonstrated i n Table I I . Within t h i s table the nondisjunctional exceptions are divided into two classes: patroclinous males and matroclinous females. Each of these two classes i s further subdivided to include progeny that are e i t h e r matroclinous or patroclinous f o r the nonsegregational compound autosomes. The values l i s t e d i n the t h i r d ;. column of Table II indicate the t o t a l number of nondisjunction&Lprogeny recovered. Columns 4 and 5 r e f e r to the portion of the nondisjunctional progeny that were also nonsegregational. A close examination of the l a s t two columns, excluding those rare progeny marked by an a s t e r i s k , reveals that progeny matroclinous f o r the X-chromosomes were patroclinous f o r the compounds, and vic e versa. Although two p a i r s of chromosomes are involved, the rare exceptions recovered 28 Table I I . Distribution of nonsegregating compound thirds in nondisjunction-al progeny. Exp. Number Sex Nondisjunctional progeny Nonsegregational for C(3) Total Matroclinous Patroclinous 2a 3a 4a 5a Male Female •Male Female Male Female Male Female Male Female Male Female Male Female Male Female 1 27 33 223 43 72 15 107 26 32 209 166 18 12 96 66 0 1 13 1 31 * 1 9 1 9 •k 2 95 0 7 3 53 ft 0 19 0 148 1 41 0 68 0 17 1 97 * 2 3 * 1 33 Progeny arising as products of nullo-X; nullo-C(3) eggs or diplo-Xj diplo-C(3) eggs. 29 as either matroclinous or patroclinous both for the X and the compound autosomes (the 4-0 class in Table III) occur with a frequency close to that expected for primary nondisjunction of the X only. The mechanism giving rise to the 4-0 exceptions is not made evident by the present results. Throughout the rest of this paper, the associated class refers only to those progeny inheriting the products of irregular meiotic events in which the nondisjunctional X-chromosomes assorted from the nonsegregating compound autosomes. The distribution pattern revealed in Table II indicates non-homologous association between X-chromosomes and compound autosomes. This could give rise to eggs that are diplo-X and nullo compound-3 or nullo-X and diplo compound-3. Such eggs w i l l become viable zygotes only when f e r t i l i z e d by the respective complementary sperm. For the f i r s t four experiments recorded in Table II (exp. 2, 2a, 3 and 3a), where M-5 males were used, a disproportionate number of nondisjunctional males and females was recovered. Since the M-5 chromosome was employed as a balancer for introducing the marked X-chromosome into the compound stocks, i t was chosen as a convenient dominantly marked chromosome for the purpose of identifying the nondisjunctional progeny. However, owing to the low v i a b i l i t y of M-5/0 males (Hess 1962; Baker 1971) exceptional progeny arising as a function of nonhomologous pairing between the X and compound chromosomes was inadequately demonstrated. Therefore, to provide for a relatively equal recovery of reciprocal classes, the M-5 chromosome was substituted, in the last four experiments, by a dominantly marked standard-X. In experiment 3, i t w i l l be noted, the observed discrepancy was not as pronounced as i t was for the other 30 three experiments involving the M-5 X-chromosome. This departure from the observed pattern, assumed to be influenced by the structural nature of the combination of compounds involved in that particular experiment, was investigated further and has provided the subject for the second chapter of this thesis. The use of the standard X-chromosome in males of the last four crosses (Table II) clearly demonstrates that reciprocal classes are produced in approximately equal frequencies. This provides evidence in support of nonhomologous pairing between compounds and the _X-chromosomes as the cause of nondisjunction. To make relative comparison, the data from a l l experiments were adjusted by subtracting the total for nondisjunctional males and doubling the total for nondisjunctional females. This treatment of the data w i l l correct both for the low vi a b i l i t y of M-5/0 males and for the possibility of chromosome loss. Note that the associated, but not the independent, nonsegregational exceptions are also included in this adjustment. A correlation was made between different classes of exceptions. This correlation i s represented in Table III, where the experiments are arranged in a decreasing frequency with respect to total nondisjunction. The experiment numbers list e d in column one of Table III refer to the corresponding crosses l i s t e d in Table I. A comparison of columns 2 and 3 reveals that, while there is a relative correlation between the rates of total nondisjunction and total nonsegregation, inconsistencies do appear, notably in experiment number 3. However, by subtracting independent nonsegregation (column 5) from total nonsegregation (column 3), which equals the class of associated exceptions (column 6), a number of interesting features emerge. First, a comparison of total Table III. Adjusted distribution of the exceptional classes recorded as percent of the total progeny. Exp. m n • * - r , j Associated N.D.-X** No. Total exceptional progeny Independent ' ^ g _rj(3)~ N.D.-X N.S.-C(3) N.D.-X N.S.-C(3) XX €(3) h < 2a 5.61 5.03 1.86 1.28 3.75 0.01 4a 4.56 3.25 1.89 0.59 2.66 0.01 3a 3.44 3.17 1.22 0.95 2.22 0.02 5a 2.24 1.47 1.08 0.32 1.15 0.03 3 2.05 2.33 0.85 1.12 1.20 0.03 4 0.79 0.71 0.32 0.24 0.47 0.02 2 0.59 0.79 0.15 0.35 0.44 0.01 5 0.27 0.37 0.13 0.23 0.13 0.06 N.D. »= nondisjunctional; N.S. « nonsegregational ** XX €(3) * Nondisjunctional males or females who inherit their X-chromosome(s) from one parent and both their compound autosomes from the other parent. ** 4 0 =>= Progeny inheriting both their X-chromosome(s) and compound autosomes from one of their parents. 32 nondisjunctionals with associated exceptions demonstrates a close d i r e c t c o r r e l a t i o n between the frequency changes within these two classes. Second, the independent nondisjunctional class (column 4) also shows a close c o r r e l a t i o n both with the t o t a l nondisjunctionals and with the associated events. Furthermore, i t i s i n t e r e s t i n g to f i n d that i n many of these crosses the r a t i o of t o t a l nondisjunction to e i t h e r independent nondisjunction or the associated nonsegregation approaches 2:1. In contrast, an inspection of column 5, the independent nonsegregationals, does not show a consistent pattern of decreasing frequencies. The r e l a t i o n s h i p between nondisjunctional and nonsegregational events can be seen i n Figure 1. Slope A i n Figure 1 represents the regression c o e f f i c i e n t (h=0.80) for t o t a l nonsegregation r e l a t e d to t o t a l nondisjunction, while slope B i s the regression c o e f f i c i e n t (b~0.64) for the associated nonsegregation r e l a t e d to t o t a l nondisjunction. The c o e f f i c i e n t s were obtained through regression analyses using the arcsine transformation values of the frequencies (Rohlf and Sokal 1969). The regression c o e f f i c i e n t s for both curves are highly s i g n i f i c a n t , but a covariance analysis demonstrates that slope A i s not s i g n i f i c a n t l y d i f f e r e n t from slope B (P»=0.1). Nonetheless, the Y-intercepts for the two curves do d i f f e r s i g n i f i c a n t l y (P<0.01). The Y-intercept of curve B i s c l e a r l y consistent with the observation that primary nondisjunction i s extremely rare and that the increased frequencies of nondisjunction obtained are a function of the presence of compound autosomes, whereas, the Y-intercept of slope A supports the notion that some degree of compound autosome nonsegregation i s t r u l y independent of nondisjunction. A comparison of the d i s t r i b u t i o n of points about the two curves i n Figure 1 reveals that the variance of slope A i s far greater than that 33 of slope B. Since the difference between these two curves is a measure of observed independent nonsegregation, i t might be argued that only those nonsegregationals included in the associated class are due to nonhomologous pairing. If this argument i s correct, keeping in mind the rules of the distributive pairing model (Grell 1967), that i s , only noncrossover X-chromosomes w i l l nonhomologously pair, one would expect to recover products of exchange pairing in the independent non-segregational male progeny. Indeed this was the case. Since the independent nonsegregational males in any given experiment are few in number, the data from a l l 8 experiments were pooled to give 188 such exceptional males. From this group, 9 carried crossover X-chromosomes. The frequency of crossing-over among these exceptionals was equal to 4.78% with the 95% confidence interval extending from 2.22 - 8.88% (Stevens 1942). The mean frequency of crossing-over for regular males, obtained by pooling the data from a l l eight experiments, i s equal to 11.8% with the frequency varying from a low of 9.35% (experiment 4a) to a high of 14.87% (experiment 5, see Table IV). Although the upper limit of the confidence interval for the exchange value from exception-a l males i s lower than the frequencies of exchange obtained from regular progeny, the results suggest that at least some fraction of these independent nonsegregational events are derived in the absence of nonhomologous pairing with the X-chromosomes. Possibly they represent the true level of "spontaneous" nonsegregation. In contrast to the argument that, for the most part, independent nonsegregation of compound autosomes arises as a meiotic event not involving pairing interactions with the X-chromosomes, i t i s evident 34 FIGURE 1 The relationship between nondisjunctional and nonsegregational meiotic events in compound-3 females. Slope A (o- - - -o) represents the regression coefficient (b « 0.80) of total nonsegregation related to total nondisjunction. The Y-intercept of this curve is c »= 0.18. Slope B (© ©) represents the regression coefficient (b * 0.64) of associated nonsegregation related to total nondisjunction with a Y-intercept of c - -0.07. Percent Nonsegregational Progeny O -> co ^ o i G) 35 from the marked rise in nondisjunction witnessed in the present study that everything above the primary level, whether recovered as independent or associated events, was caused by the presence of compound autosomes. It follows, therefore, that i f nondisjunction i s s t r i c t l y a function of nonhomologous pairing involving compound autosomes, and yet the correlation between the two events should consider total nondisjunction with respect to only the associated nonsegregation, the frequency of nondisjunction occasioned by nonhomologous pairing is approximately double that of compound nonsegregation. From this one can recognize the clear possibility of a trivalent pairing event involving both X-chromosomes and one of the compound-autosomes. The second autosome, in such meiotic events, is assumed to segregate randomly. Trivalent pairing and other possibilities w i l l be considered at some length in the discussion. The distribution of crossover classes among the regular male progeny i s reported for a l l experiments, and their respective controls, in Table IV. A comparison between crosses 1 and 5, where females in both experiments were heterozygous for inversions (3L) and (3R) Payne, reveals that the presence of compound autosomes in cross 5 as compared to their absence in cross 1, did not appreciably influence the rate of crossirig-over in the proximal region of the X-chromosomes. Similar-l y , in comparing the controls for experiments 1 and 5 ( i . e . , l a and 5a) the frequencies of crossing-over are found to be the same. Therefore, i t would appear that the increase in crossing-over between the experimental crosses and their respective controls i s solely a function of an interchromosomal effect owing to the presence of inversions. Table IV. Percent X-chromosome recombination in different compound third and standard lines. Exp. No. Male progeny Percent crossing--over Percent increase NCO SCO DCO Total Region f-oav 1 Region 2 car-y Region 1+2 f-y 1 4820 792 2 5614 7.76 6.41 14.17 15.0 l a 3072 426 3 3501 7.16 5.16 12.32 2 3683 478 1 4162 6.67 4.85 11.52 8.4 2a 3235 381 2 3618 5.44 5.19 10.63 3 3273 482 2 3757 6.86 6.06 12.92 30.9 3a 2813 306 1 3120 5.48 4.39 9.87 4 3822 513 5 4340 6.40 5.64 12.04 28.8 4a 3099 316 2 3417 5.20 4.15 9.35 5 3704 645 1 4350 8.32 6.55 14.87 21.0 5a 2417 339 - 2756 6.89 5.40 12.29 37 and that compound autosomes per se do not influence the frequency of crossing-over, at least not in the proximal region of the X-chromosome. A comparison between experiments 2, 3, 4 and 5 would lead one to suggest that heterozygosity for both inversions has a greater inter-chromosomal effect than either inversion separately. However, the variation in X-chromosome exchange in the control series (2a-5a inclusive) is as great as that obtained between any given control and i t s respective experiment. Furthermore, as noted in the last column of Table IV, the relative increase brought about by inversions (_3R) and (3L) Payne alone (experiment 3 and 4) was greater than that caused by the presence of both inversions together (experiment 5). It i s interesting to find that although the females involved in a l l control crosses are coisogenic for the X and compound autosomes, the frequency of exchange between forked and the centromere (marked by y +) shows a 30% var i a b i l i t y . A similar observation has been reported by Valentin (1972), who related this phenomenon to possible variation in laboratory environment and genetic background. In this study variation in genetic background, by virtue of the- use of compound thirds, i s restricted to chromosomes two and four. Furthermore, i t should be noted that no obvious clustering that would represent gonial events was observed. Considering, therefore, a l l possible sources of variation in the results obtained from the crosses reported in Table IV, the only comparisons of consequence are those between experiments and their respective controls, such as 2 and 2a, or 3 and 3a, where the female parents were recovered as sibling progeny. It w i l l be recognized, nevertheless, that from any two such crosses the highest value of crossing-over i s always obtained from females heterozygous for inversions. 3 8 The compound-3L chromosome, C(3L)P5, heterozygous for inversion (3L)Payne, had been used in an earlier study by Baldwin and Chovnick (1967) who were carrying out a half-tetrad analysis on compound-3R. They reported that in females carrying C(3L)P5 the frequency of crossing-over in C(3R) was moderately decreased. The kind of study carried out by Baldwin and Chovnick made i t exceedingly d i f f i c u l t to recover, from the same parents, females to be used for controls as well as for experimental crosses. The control group would arise only as patroclinous nonsegregational exceptions. Since the compound-3L heterozygous for In(3L)Payne constructed for use in the present study fC(3L)VT2, experiment 2] demonstrated, in comparison with i t s respective control, an inter-chromosomal effect on X-chromosome recombination, i t was considered of interest to examine the compound-3L used by Baldwin and Chovnick (experiment 4). In comparing the results of experiment 4 with i t s control 4a, we find that C(3L)P5 is in fact associated with an increased frequency of exchange on the X-chromosome. Therefore, i t would appear that increased crossing-over on the X» occasioned by either of the Payne inversions linked to compound autosomes, i s a general phenomenon. According to the model for distributive pairing, compound autosomes are constant members of the distributive pairing pool. Therefore, these chromosomes would regularly be expected to provide alternative distributive pairing partners for any noncrossover X-chromosome. Addition-a l l y , compound autosomes can be heterozygous for inversions that, through the interchromosomal effect, increase the frequency of X-chromosome exchange, but should have no influence on the distributive pairing properties of the compounds. Structural alteration to the compounds 39 should result, therefore, in an inverse correlation between the two meiotic events: crossing-over and nondisjunction. That is,inversions carried by the compounds w i l l increase crossing-over and, as a consequence, w i l l decrease the fraction of X-chromosomes that segregate solely as a function of distributive pairing. Comparisons between percent crossing-over in the proximal-X region, marked by _f and Dp(l)y +, and the calculated percent nondisjunction are l i s t e d in Table V in a descending order of crossover frequencies. The values recorded in column 3 represent not the adjusted frequencies of observed nondisjunctional progeny, as given in Table I I I , but the calculated percent nondisjunction. These values were derived by modifying the formula [4(except. females)100/Total+excep. progeny . - percent nondisjunction] reported by Grell, e_t a l . (1966) to correct for the lethality of M-5 males as well as of X/X/x and 0/Y zygotes. Hence, the precent nondisjunction i s equal to 4(excep. females)100/Total -observed excep. males and females+4(excep. females). The correctness of this calculation depends on the assumption that nullo-X and diplo-X gametes are produced in a 1:1 ratio. A comparison between the values in columns 2 and 3 of Table V w i l l reveal the existence of an inverse correlation. When the frequency of crossing-over in the proximal region of the X-chromosome is high, the rate of recovery of nondisjunctional exceptions is low; as the frequency of crossing-over decreases, nondisjunction increases, although not uniformly. The regression analysis of these data is presented in Figure 2, with percent total nondisjunction regressed on percent crossing-over. The slope of this curve i s b = -1.52 with a correlation coefficient 4 0 Table V. Correlation between crossing-over in the proximal region of the X-chromosome and total nondisjunctional events. Exp. Number Percent Percent crossing-over nondisjunction f-y 14.87 0.55 12.92 4.03 5a 12.29 4.38 12.04 1.57 11.52 1.18 2a 10.63 10.63 3a 9.87 6,66 4a 9.35 8,72 41 significant at P=.05, but not at P=.01. The 95% confidence intervals were calculated both for crossing-over (horizontal bars) and nondisjunction (vertical bars), as displayed in Figure 2. An examination of this Figure w i l l demonstrate that, with the exception of experiment 2 and i t s control, 2a, the 95% confidence interval for any given experiment does not overlap i t s respective control. It should be noted that not only do the confidence intervals of experiment 2 and 2a overlap, they also f a l l the farthest from the line of regression. Owing to the d i f f i c u l t i e s caused by phenotypic interactions between different mutations, only a small region of the X-chromosome was studied. The correlation observed in Table V might have been considerably improved by studying, i f not the entire, a larger segment of the X-chromosome. However, i t should be re-emphasized that Payne inversions, as reported by Schultz and Redfield (1951) do in fact have a greater effect on recombination within the proximal region of the X-chromosome. If, in fact, the rate of nondisjunction is primarily determined by the availability of noncrossover X-chromosomes to the distributive pairing pool, then any great variation about the line of regression would be attributable to changes in environmental factors. On the other hand, distributive pairing could also be influenced by the structural properties of compound autosomes arising as a consequence of their formation. It has been shown, for compound seconds, that formation arises from an interchange between either sister or nonsister chromatids of the standard chromosomes resulting in the attachment of an acentric fragment to a centromere-bearing homologous free arm. Hence, these interchanges, or translocation-like events, produce C(2L) and C(2R) 42 FIGURE 2 The inverse correlation between percent crossing-over in the proximal region (f_ to the centromere) and total nondisjunction of the X-chromosome is demonstrated by the regression coefficient (b = -1.52). The number given to each experiment is shown in parenthesis. The 95% confidence intervals are represented as horizontal bars for crossing-over and vertical bars for nondisjunction. 42a 4 3 chromosomes heterozygous for heterochromatic duplications and deficiencies that, in some cases, include the most proximal known genetic markers (Holm, Gavin, Kowalishyn and Yeomans, in preparation). Although the centric properties for any given compound autosome might be unique, exchange pairing between the isosequential attached arms appears to be regular throughout (Baldwin and Chovnick 1967, Holm 1969, 1973). Furthermore, exchange pairing within the compound chromosomes, appears not to influence their segregational behaviour. Therefore, distributive pairing as a function of the proximal heterochromatin of compound autosomes is offered as a distinct possibility; but since we have as yet not gained sufficient insight into the nature of distirbutive pairing in terms of forces, pairing recognition and duration, pairing efficiency as a cause of variable results must be considered with reservation. Despite the d i f f i c u l t i e s in providing a precise interpretation for the data given in Table V and Figure 2, the results of this study have clearly demonstrated that X-chromosome nondisjunction is a function of nonhomologous pairing and the inverse correlation between X-chromosome c r o s s i n g - o v e r and n o n d i s j u n c t i o n i s in keeping with the theory that non-homologous pairing is dependent on the presence of nonexchange X-chromosomes. It follows, therefore, in support of R. Grell (1970)} that distributive pairing forces must operate subsequent to the exchange pairing event. Nondisjunction in compound-2 females: In view of their size and morphology, one would suggest that the meiotic behaviour of compound second autosomes could be predicted on the basis of results obtained from studies using compound-thirds. While, in fact, the meiotic behaviour 44 of both pairs of autosomes is similar, differences have been noted. The most striking difference is the frequency of egg hatch encountered in compound-2 strains, which is greater and far more variable than that obtained with strains bearing compound-thirds. Although not rigorously demonstrated, these differences are suggested to reflect pairing properties operative during spermatogenesis (Holm 1969, 1974; Clark and Sobels 1973). Regardless of these problems, a study parallel to the one reported above for compound-thirds was conducted on two strains carrying compound-2 autosomes. The two strains of females used in this study were recovered simultaneously as sibling progeny derived from a cross between y f car.y +/y f car.y+;C(2L)VT5,In(2L)Cy/lt;C(2R)Pl,px females and y2/Y;C(2L)VT1,ho;C(2R)VT2,In(2R)Cy jbw 4 5 asp 2or 4 5 a/stw 3 males. In this way (consistent with the procedures followed for compound-3 strains) a genetic uniformity, excluding the compound autosomes, was shared by the two lines of females used for comparing compound-autosome associated interchromosomal effects. From the above cross, females heterozygous for In(2L)Cy and In(2R)Cy, carried by C(2L) and C(2R), respectively, represent the experimental group, while those carrying isosequential C(2L)VTl,ho and C(2R)Pl,px provide the controls, Male parents i n both crosses carry the dominant X-liiiked eye marker, 13, and the opposite pair of compounds to that carried by the females so that regular and exceptional progeny in both crosses not only could be identified but also would inherit the same combination of the four compound-2 autosomes. In addition to the crosses involving compound seconds, and as a general control, parallel studies were conducted involving standard 45 second chromosomes. In one of the latter two crosses the females were heterozygous for In(2L+2R)Cy; the females in the second (control) cross were homozygous for the standard linear sequence of chromosome two. As with the compound lines, the structurally homozygous and heterozygous females arose from the same parental lines. The genotype of the parents and the results from these studies are presented in Tables VI and VII. Two features of the interchromosomal effect related to the Cy inversions should f i r s t be considered. Although the Cy_ inversions have a pronounced interchromosomal effect on the distal segment of the X-chromosome, according to Schultz and Redfield (1951) these chromosome-2 associated inversions are considerably less effective than inversion Payne on increasing crossing-over in the proximal region of the X. Because of the peculiar interchromosomal effect of the Cv_ inversions, Ramel and Valentin (1966) excluded the proximal-X when they reported that inversions Cy_, 2-left and 2-right, showed greater effect when coupled (in cis configuration) than when in repulsion (i.e. in trans configuration). While previous findings were not being disregarded, i t was considered best to involve the same X-chromosomes in studies comparing the effects of compound-2 with compound-3. Furthermore, In(2L+2R)Cy, the cis rather than the trans configuration, was used in the standard series since this same inversion has been employed in generating C(2L) and C(2R) heterozygous for Cy_ inversions. The results recorded in Table VI under the heading "percent crossing-over" are consistent with the findings from the inversion Payne studies in revealing that the frequency of proximal-X exchange 46 i s increased relative to the respective control, both in standard and compound-2 females bearing the Cy_ inversions. Furthermore, as indicated by the previous results on compound thirds, interchromosomal effects by compound autosomes per se are not evident. It i s also of interest to find that the interchromosomal effect attributed to the compound-associated inversions is restricted to region 1 (i.e., f-car) while, in contrast, an increase in both regions was realized with the double paracentric inversion in the standard strain (see Table VI). In comparing the relative interchromosomal effect in the standard-bearing line with that of the compound line, i t is tempting to interpret the results as supportive evidence that coupling of the l e f t and right paracentric inversions enhances their influence on crossing-over in the X-However, as shown by the present and many other studies, the inter-chromosomal effect i s to a large degree dependent on the overall genetic background. Moreover, crosses involving standard chromosomes were not carried out simultaneously with the compound studies. Therefore, a model describing relative increases as a function of linkage relationships cannot be supported at present. An examination of the values recorded under the heading "total nondisjunction" (Table VII) immediately indicates that nondisjunction of the X-chromosomes increases markedly in the presence of compound-2 chromosomes. It w i l l be noted that out of approximately 23,000 standard progeny, one only arose from a nondisjunctional event; interestingly, that single exception came from an inversion bearing parent. While this increased nondisjunction is generally in keeping with the results obtained using compound-thirds, the isosequential compound-seconds appear to be Table VI. Percent X-chromosome recombination in different compound second and standard lines. Exp. No. Parents Sex .Genotype + 2 Female y-y /y ;In(2L+2R)Cy/+ Male M-S/Y;+/+ Tptal males 5813 Percent crossing-over region 1 7.79 region 2 5.62 regions 1+2 13.41 Percent increase 23.9 + 2 6a Female y-y /y ;+/+ Male M-5/Y;+/+ + 2 7 Female y-y /y :C(2L)Cy/lt;C(2R)Cy/stw Male B/Y:C(2L)ho:C(2R)px 7a Female y-y+/y2;C(2L)ho;C(2R)px Male B/Y;C(2L)Cy/lt;C(2R)Cy/stw 5801 4244 5971 6.36 6.19 4.87 4.46 4.47 4.48 10.82 10.66 9.35 14.0 y-y = X-chromosome marked with y f oav.y C(2L)Cy/lt = C(2L)VT5,In(2L)Cy/lt C(2R)Cy/stw = C(2R)VT23In(2R)Cy/sti/ Table VII. Adjusted distribution of the exceptional classes recorded as percent of the total progeny. Exp. No. * Total exceptional progeny Independent Associated N.D.-X with N.S.-C(2)** Total progeny N.D.-X N.S.-C(2) N.D.-X N.S .-C(2) XX C(2) 4 — 0 6 0.009 - - - 11222 6a - - - - 11873 7 0.32 0.80 0.11 0 .59 0.21 0.017 9270 7a 0.69 0.47 0.42 0 .20 0.27 0.015 13268 * N.D. = nondisjunctional; N.S. = nonsegregational. XX €(2) = Those nondisjunctional males or females who inherit their X-chromosome(s) from one parent and both their compounds from the other parent. if 0 = Progeny inheriting both their X-chromosome(s) and compound autosomes from one of their parents. 00 49 less effective in competitive distributive pairing. However, considerably more information on the distribution of compound-2 chromosomes during spermatogenesis as well as on possible interchromosomal effects in the distal region of the X must be obtained before concepts of effective pairing demand attention. In comparing the relative rates of recovery of crossover and non-disjunctional progeny in the two compound lines, as in the experiments dealing with compound thirds, an inverse correlation was realized (compare the last two entries under percent crossing-over, experiment 7 and 7a in Table VI, with percent total nondisjunction recorded for experiments 7 and 7a in Table VII). While an examination of the whole X would be desirable, these results provide added support to the hypothesis that the interchromosomal effect reduces the portion of X-chromosomes available as nonhomologous pairing alternatives to the compound autosomes. An examination of the entries in Table VII reveals that total non-disjunction is divided into two groups: independent nondisjunction and (nonsegregation) associated nondisjunction. The number of independent and associated nondisjunctional progeny do not differ significantly from an expected ratio of 1:1, based on the trivalent pairing model suggested in the previous section. Again, this is assuming a l l non-disjunction i s a function of nonhomologous pairing events involving a compound autosome. Nonsegregation, however, is not viewed as totally dependent on pairing interactions with the X-chromosomes. On the contrary, considering the contrasting pattern of this event, independent non-segregation is interpreted as arising without influence by the X-chromosomes. Indeed, among the 29 independent nonsegregational male 50 progeny recovered, two inherited crossover-X chromosomes. This gives a crossover value of 6.9% with a 95% confidence interval (0.9 to 23.4) that clearly includes the values obtained from the regular class in either experiment. 51 DISCUSSION The concept of nonhomologous pairing as a mechanism responsible for the occurrence of exceptional meiotic events was studied and the following summarizes the results. X-chromosome nondisjunctional progeny were not recovered from Drosophila melanogaster females carrying isosequential pairs of standard autosomes. However, in the presence of structural heterozygosity for chromosome three, primary nondisjunction was increased to the level reported in the literature (Bridges 1916, Merriam and Frost 1964). The frequency of crossing-over in the proximal region of the X-chromosome was, at the same time, increased. This increase was attributed to inter-chromosomal effects exerted by the autosomal inversions employed. When standard third chromosomes were replaced by compound autosomes having isosequential arms, the frequency of exceptional progeny recovered from females carrying such compounds was greatly increased. The close correlation between the frequencies of the two types of exceptionals, namely, nondisjunctional for X-chromosomes and nonsegregational for compound autosomes, indicates an interdependence of the two events. In any given experiment, when the observed frequency of occurrence of the associated class, which was derived from the simultaneous non-disjunction of the X's and nonsegregation of the compound autosomes, is compared to the product of the frequencies for each of the exceptional events separately, the interdependence of the two variables is clearly established. This observation further supports the notion that the majority of exceptional progeny arises through nonhomologous pairing between the X-chromosomes and compound autosomes. Examination of 52 associated exceptionals, on the basis of their X-chromosome(s) and compound autosomes, demonstrated that with rare exceptions, (i.e., progeny who inherited both their X's and compounds from one parent), females matroclinous for X-chromosomes were patroclinous for compounds and males that inherited a paternal X, derived their compound autosomes from their mothers. From this distribution of X-chromosomes and compound autosomes i t becomes clearly evident that nonhomologous pairing provides the simplest and most probable explanation for these events. A comparison between different classes of exceptions and the regression analysis performed on their rate of occurrence demonstrated that a certain proportion of nonsegregationals, referred to as independents, were derived in the absence of nonhomologous pairing. This class, which, displayed a highly variable frequency of recovery in different experiments, may represent the true "spontaneous" level of nonsegregation. The presence of crossover X-chromosomes in this class further implies that they arose independently of pairing interactions with the X-chromosomes. Therefore, while a portion of nonsegregational exceptions cannot be related to known nonhomologous pairing events, the results clearly show a direct correlat-ion between nondisjunction and nonsegregation. It must be concluded from this study that with rare exceptions, and in contrast to non-segregation of compound autosomes, X-chromosome nondisjunction i s s t r i c t l y a function of nonhomologous pairing. This dependence of nondisjunction on nonhomologous pairing i s supported by the fact that the rate of recovery of nondisjunctionals in the absence of compounds did not exceed the primary level. When structurally heterozygous compounds were usedj X-chromosome 53 proximal exchange was increased, and this was accompanied by a decrease in the frequency of nondisjunctional progeny recovered. The inverse correlation between X-chromosome recombination and X-chromosome"non-disjunction not only provides added support for the concept of non-homologous pairing but also agrees with the temporal sequence of the distributive pairing model (Grell 1962, 1967). Although the availability of noncrossover X-chromosomes i s apparent-ly the most important factor in determining the extent of nonhomologous pairing, there is the possibility that compound autosomes display different distributive pairing efficiencies. At present there i s no direct evidence that such pairing efficiencies do exist. However, stock specific nonsegregational frequencies have been reported with no indication of interchromosomal effects. This stock specificity, i f i t is a property of the compounds per se, could be related to unique properties of the centromeric heterochromatin associated with the independent formation of each compound. The nondisjunctional values, obtained in the experiments reported here, are in the same range as the frequency of secondary nondisjunction observed for X/X/Y_ females (Bridges 1916). The use of a marked Y-chromosome (B Y) excluded the possibility that X/X/Y_ females contributed to the observed nondisjunctional class. Furthermore, the absence of non-*--segregational clusters indicates that the female parents selected for these experiments did not carry free Y-chromosomes. The term "secondary nondisjunction" is applied to the process by which nondisjunctional exceptions arise from X/X/Y females following heterosynapsis between the X-chromos omes and tlie Y, This process. 54 therefore, involves chromosomes that share at least a short segment of homology. The nonhomologous pairing observed here, on the other hand, is between chromosomes that are completely heterologous to one another: the term secondary nondisjunction is therefore not applicable in this case. However, the similarity between the nondisjunctional frequencies obtained here and those reported for secondary nondisjunction offers unquestionable support for the argument that secondary nondisjunction is not a product of competitive pairing, owing to segmental homology, but i s rather a product of a nonspecific pairing event that i n no way differs from the meiotic interactions that engage compound autosomes and non-crossover X-chromosomes. Before viewing these results specifically in terms of the distributive pairing model let us consider the possible involvement of the other chromosomes, namely, the seconds and the fourths, as alternative non-homologous pairing partners for the compound thirds. Although the fourth chromosomes are regular members of the distributive pairing pool and their meiotic assortment is solely a function of such pairing, because of their small size they do not serve as competitive pairing partners for larger chromosomes (Grell 1964a, b, 1967). Regarding the standard second chromosomesj when isosequential, they exchange and segregate regularly, therefore, their nondisjunctional rate is extremely low (Gavin and Holm 1972). Grell (1963) found that in the absence of autosomal structural heterozygosity, attached X-chromosomes regularly segregate from a free Y. From his work i t can be concluded that isosequential standard autosomes are rarely available for nonhomologous pairing. His studies further demonstrate that chromosome four has l i t t l e effect, i f any, on the non-.55 homologous pairing of compound chromosomes. The X-chromosomes, therefore, appear to be the only possible candidates for nonhomologous pairing with compound thirds; this, of course, in keeping with the distributive pairing model, should involve only the noncrossover X-chromosomes. Regarding Grell's (1962, 1967, 1969) distributive pairing model, exchange pairing precedes distributive pairing and only those chromosomes which did not exchange with their homologae w i l l enter the distributive pairing pool. Therefore, nondisjunctional progeny are expected to carry noncrossover X-chromosomes. While nondisjunctional female progeny in the present study were not tested for the possibility of being exchange products, no homozygosity for sex-linked markers was observed within this group. Therefore, nondisjunctional X-chromosomes were assumed to be non-crossovers, as is generally accepted. As mentioned previously in the Introduction, Merriam (1967) proposed an alternative to the distributive pairing model to explain the sequences of events associated with secondary nondisjunction. In his model, Merriam assumed that, in contrast with Grell's model, nonhomologous association between the X-chromosome and the Y^, which subsequently resulted in secondary nondisjunction, occurred prior to exchange between the X-chromosomes. Therefore, the frequency of crossing-over between the X-chromosomes would be decreased by the interference brought about through competitive pairing with the Y. To prove his assumption, he compared the frequency of crossing--over, in the proximal region of the X-chromosome, in X/X/X females with that of X/X females. His results revealed that in the presence of a" chromosome, proximal X-chromosome recombination was decreased. Furthermore,, Merriam demonstrated that inversion heterozygosity for the second 56 chromosome not only increased the rate of exchange in the proximal region of the X but also decreased the frequency of secondary nondisjunction. From these results he concluded that the inverted second provided an alternative nonhomologous chromosome that could successfully compete with the X-chromosomes as a pairing partner for the Y. As a consequence of removing the Y-chromosome, through pairing with the second, homologous pairing and hence exchange, between the X-chromosomes was facilitated. Grell (1970) repeated Merriam's study and confirmed the proximal decrease in X-chromosome recombination as shown by Merriam; but, at the same time, Grell discovered an accompanying distal increase with the net result an overall slight increase in total X-chromosome exchange. The frequency of nonexchange X-tetrads measured was almost the same in the presence or absence of the Y-chromosome. Moreover, Grell failed to find any increase in proximal-X exchange in the presence of an inverted second, although the nonrandom distribution of the inverted second chromosome and the Y suggested a very high frequency of pairing between these two heterologues. Grell (1970) suggested that the effect of the Y~.-chromosome on X-chromosome recombination i s interchromosomal rather than due to exchange interference brought about by competitive pairing. Therefore, in support of her earlier model, she concluded that exchange pairing and exchange precedes distributive pairing, and that only noncrossover X-chromosomes can pair nonhomologously with the Y-chromosome. Since, in the present study, compound autosomes rather than a free Y provide the source of pairing competition for the X-chromosomes, inversion heterozygosity between the attached-arms of the compounds w i l l not generate additional nonhomologous pairing alternatives, as i s the case 57 when autosomal inversions are introduced into X/X/Y females. Moreover, a structurally-rearranged compound autosome should be equally as effective at competitive pairing with the X-chromosomes as i s a compound autosome with isosequential arms. In keeping with Merriam*s argument, i f one considers that nonhomologous pairing interactions are competitive and precede exchage, then inversions residing on the compounds would not be expected to influence the degree of nonhomologous interactions and, hence, the frequency of nondisjunction. Furthermore, i t might also be suggested that compound-associated inversions would not increase the ratio of X-^  chromosomes having undergone exchange in the proximal region. Therefore, one would expect to obtain a similar nondisjunctional value from a l l crosses involving compounds, regardless of the structural nature of these autosomes. This, however, was not the case, as demonstrated by the data recorded in Table V. The presence of structural heterozygosity, without exception, increased the recombination value in the proximal region of the X-chromosome and, as an apparent direct consequence, decreased the frequency of nondisjunctional progeny recovered. On the basis of these findings, I conclude that, in clear agreement with Grell's distributive pairing model, exchange pairing precedes distributive pairing and only those chromosomes that, for one or another reason, f a i l to exchange pair with an independent homologue} engage in nonhomologous-pairing interactions. A reexamination of the data of Table I demonstrates that in the absence of compound autosomes nondisjunctional exceptions are rarely, i f ever, recovered. Therefore, the presence of compound autosomes in females is unquestionably responsible for the occurrence of such exception-58 al progeny. Compound autosomes, owing to their lack of independent exchange pairing partners, are believed to regularly assort as a function of the distributive pairing forces. It would appear, therefore, that like a Y-chromosome, compound autosomes are consistently available as alternative distributive pairing partners for nonexchange X-chromosomes, Although the nondisjunctional progeny are viewed as arising from nonhomologous pairing events involving X-chromosomes and compound thirds, two different classes are recovered. These two classes, as demonstrated in Table I I I , include: (1) those individuals arising from an event in which nondisjunctional X-chromosomes segregate from both compound autosomes (the associated class); and (2) those that appear to arise from a nondisjunctional event that i s independent of the distribution of compound autosomes (the independent nondisjunctional class), In comparing the frequency of the two nondisjunctional classes, we find that the percent recovery of the independent class i s always less than or equal to that of the associated class and, as interpreted from Figure 1, there is a very close linear relationship between the two. If we assume that nondisjunction i s s t r i c t l y a function of non-homologous pairing between the X-chromosomes and compound autosomes, the simplest model to explain the segregational pattern observed would be one of trivalent formation involving two X-chromosomes and one compound autosome, similar to Cooper's (1948) model for secondary nondisjunction (Figure 3). As can be seen from this Figure, i f the trivalent pairing model were considered as the only possible configuration to account for the recovery of nondisjunctional exceptions, then one would expect to obtain independent nondisjunctionals and those nondisjunctional events 59 FIGURE 3 The trivalent pairing model describing nondisjunction as the product of meiotic events in which both X-chromosomes associate, through nonhomologous pairing forces in the region of the centromere, with a single compound autosome, while the other compound assorts at random. According to this model, four types of gametes are expected: (1) - diplo-X and nullo-C(3) (2) - nullo-X and diplo-C(3) (3) - diplo-X and haplo-C(3) (4) - nullo-X and haplo-C(3). Type 1 and 2 eggs, i f f e r t i l i z e d by complementary sperm, w i l l give rise to associated nondisjunctional and nonsegregational progeny^ while type 3 and 4 w i l l only give rise to independent nondisjunctional individuals. As a consequence of trivalent pairing these two groups, i.e., associated exceptionals and independent nondisjunctionals, must be recovered in a 1:1 ratio. 6 0 FIGURE 4 The double b i v a l e n t p a i r i n g model where cent romer ic p a i r i n g of one X _chromosome and one compound autosome i s cons idered to commit the o ther X and compound to p a i r w i t h one another . In the event of such p a i r i n g four types of gametes are expected to be produced: (1) - d i p l o - X and nu l lo-C(3 ) (2) - n u l l o - X and d ip lo-C(3 ) (3) - haplo-X and haplo-C(3) (4) - haplo-X and hap lo-C(3 ) . F e r t i l i z a t i o n of type 1 and type 2 eggs, by the complementary sperm, w i l l r e s u l t i n fo rmat ion of a s s o c i a t e d n o n d i s j u n c t i o n a l and non«. s e g r e g a t i o n a l e x c e p t i o n s , w h i l e types 3 and 4 w i l l on ly g i ve r i s e to r egu l a r progeny. Th is model i s proposed to account f o r the h ighe r recovery of a s s o c i a t e d e x c e p t i o n a l as compared to independent non-d i s j u n c t i o n a l progeny. 60a .61 associated with nonsegregating compound autosomes with equal frequencies. To explain the observed differences, however, we might consider a situation in which segregation was s t r i c t l y a function of bivalent pairing (Figure 4). As diagrammed in Figure 4, when one X-chromosome pairs with one compound autosome, the second X w i l l be committed to pair with the second compound autosome. The only exceptional progeny recovered from this type of nonhomologous pairing, therefore, would be those of the associated class. Since in six of the eight experiments reported in Table I I I , associated exceptions were recovered with a greater frequency than independent nondisjunctionals, i t i s possible that double bivalent pairing has contributed to the overall results. However, i t is quite evident that the model described in Figure 4 cannot be of major consideration. If an attempt is made to demonstrate the same kind of relationship between independent nonsegregationals and associated exceptions, as previously shown for nondisjunctional progeny, one f a i l s to find such a clear linear correlation (refer to Table I I I ) . Furthermore, i f one assumes, in agreement with the distributive pairing model, that non^ homologous pairing only involves nonexchange X-chromosomes, and that nonsegregation i s s t r i c t l y a function of nonhomologous pairing events involving the X's, no crossover X-chromosome w i l l be expected among independent nonsegregationals; but in fact, they were encountered in this class. Since the frequency of exchange in this class i s not as high as that obtained from regular males, a certain proportion of the independent nonsegregational progeny may not be independent of non-homologous pairing. Therefore, a third model i s considered which may 62 possibly contribute to these exceptional events (Figure 5). According to this third model, one X-chromosome w i l l pair with one compound autosome., while the remaining X and compound autosome segregate at random. Such pairing, as demonstrated in Figure 5, w i l l give rise to independent nonsegregational progeny as well as to independent non^ disjunctional and associated progeny. As with the double bivalent model described above, the events described in Figure 5 must be of limited occurrence. Although other pairing configurations involving the four available elements in the distributive pool are conceivable, the distribution of chromosomes resulting from such pairing configurations would be inconsistent with the data reported above. Considering the trivalent model presented in Figure 3, the compound autosome, which is a metacentric chromosome, i s assumed to segregate from the two acrocentric X-chromosomes. Similar patterns of chromosome assortment have been reported by Moore and Grell (1972b) , According to these authors, in a competitive situation, the directing role among the three members of a distributive pairing pool i s determined on the basis of chromosomal length; that i s , in a trivalent complex, when the ratio of the intermediate acrocentric to the large metacentric i s not less than 5/9, the metacentric chromosome w i l l assume the directing role. Although the exact length of the chromosomes utilized in the present study was not determined, the data obtained indicate a high degree of trivalent formation with the metacentric compound directing the acro-centric X-chromosomes to the opposite pole. In view of the fact that isosequential compound autosomes enter the distributive pairing pool with synapsed arms, in experiments involving such chromosomes, the 63 FIGURE 5 The single bivalent pairing model where centromeric pairing of only one X-chromosome with one compound autosome occurs, while the other !X and compound are randomly distributed. Pairing of this nature should result in the production of the following eggs: (1) - diplo-X and haplo--C(3) (2) - nullo-X and haplo--C(3) (3) - haplo-X and nullo--C(3) (4) - haplo-X and diplo-- c o x (5) - nullo-X and diplo--C(3) (6) - diplo-X and nullo~C(3) (7) - haplo-X and haplo--C(3) (8) - haplo-X and haplo--C(3), Fertilization of these eggs by complementary sperm w i l l result in the recovery of independent nondisjunctional progeny from types 1 and 2, independent nonsegregational progeny from types 3 and 4 S associated exceptions from types 5 and 6 and regular individuals from types 7 and 8. This model is offered as an explanation for the recovery of independent nonsegregationals. Although the occurrence of crossover X-chromosomes among this class supports the notion that the majority of such individuals are derived independent of interactions with: X-chromosomes, a minor proportion of them might be produced following single divalent pairing. 64 difference, i f any, between the physical lengths of the four elements as they enter the pool should be negligible. The question that arises, in connection with this, i s whether the important factor in distributive pairing of such elements is the total length or the total mass of each chromosome. If we apply the size rule of Moore and Grell CL972a) to my results, then total length, rather than arm length, should determine distributive pairing a f f i n i t y . If this rule, based on studies of compound four chromosomes, can be applied to the larger autosomes and the" X-chromosomes, one would not expect pairing competition between metacentric compounds and the acrocentric X-chromosomes. In contrast, i f arm length is- the determining factor, trivalent pairing between two X-chromosomes and a single compound autosome might occur. However, when compounds enter the distributive pairing phase their isosequential arms are synapsed. Therefore, i f distributive pairing is a function of pairing along the total length of the chromosome, there should be preferential assortment of the X-chromosomes from structurally heterozygous compounds. This clearly i s not the case. Segregation with equal frequency occurs from either isosequential or structurally heterozygous compounds when both are present in the same genome. Although valid for the behaviour of small chromosomes, the size rule i s of dubious value when dealing with larger chromosomes. However, i f total mass rather than total length regulates distributive pairing forces, the size model may have some application to my observations. Having considered these different p o s s i b i l i t i e s , one also could assume that distributive pairing of the X—ch'romosomes and compound 65 autosomes is a function of their proximal heterochromatin; that i s , one X-chromosome pairing with the proximal heterochromatic block to one side of the centromere of the compound autosome, while the other X pairs with the heterochromatic segment on the opposite side. If this assumption is correct, the euchromatic length of a synapsed isosequential compound w i l l not make i t any less efficient in nonhomologous pairing with the X-chromosomes than a structurally heterozygous compound which- w i l l only partially synapse. Furthermore, i f various compound autosomes possess different degrees of distributive pairing a f f i n i t y , this could be explained by pairing of the proximal heterochromatic region in these compounds. This argument is based on the notion that structural organization within the proximal heterochromatin, as a function of formation, i s unique for any given compound autosome. A similar pairing modelP operating by centromeric forces between compound thirds in females of Drosophila melanogaster, was proposed by Holm (1969, 1974), On the basis of my evidence, I suggest that centromeric non-*>-homologous pairing of the X-chromosomes and compound autosomes, following exchange pairing of a l l chromosomes, i s responsible for the production of nondisjunctional and nonsegregational exceptions. Certainly ? the observed distribution of nondisjunctional X-chromosomes, relative to the assortment of compound autosomes, i s consistent with the model that views nondisjunction as a consequence of both X-chrbmosomes pairing with t and segregating from, a single compound autosome. However, the formation of bivalents involving nonhomologues i s also a possible contributing factor. CHAPTER I I STUDIES ON A MATERNALLY SUPPRESSED POSITION-EFFECT LETHAL IN MALES OF DROSOPHILA MELANOGASTER 67 INTRODUCTION The greatly reduced v i a b i l i t y of the Muller-5 (M-5) inversion g [ a product of crossing-over between inversions scute-8 (sc ) and scute-Si SI (sc ) ] , in X/0 males noted in the f i r s t chapter was f i r s t reported by Hess (1962), who also noted similar effects for In(l)sc /0 males. Although these inverted X-chromosomes were f u l l y viable in X/Y males, Hess also discovered that v i a b i l i t y was partially restored in the presence of Y-chromosome fragments. He suggested, therefore, that lethality associated with the scute inversion is caused by a position-effect variegation owing to the euchromatic-heterochromatic breakpoints involved in the rearrangement of the X-chromosome. The influence of the Y_-chromosome fragments in restoring v i a b i l i t y was assumed to be due to their modifying effect, as previously observed in association with position-effect variegation. Position-effect variegation has been the subject of extensive investigations since Sturtevant (1925) f i r s t reported this phenomenon. However, i t was Bridges and Morgan (1923) who originally observed this effect, without realizing i t s significance. They observed, while studying the properties of duplication PHI (a duplication of the second chromosome including the region from the arc locus to the tip of the right arm translocated to the third-chromosome) , that when PHI was present in f l i e s with second chromosomes homozygous for the recessive alleles corresponding to the wild-type genes carried by the PHI duplication, the phenotype of such f l i e s appeared wild-type except for the penetration of a very weak plexus phenotype. They reported that in the region of partial trisomy, as explained above, a l l homozygous recessive genes were covered 68 by their respective dominant alleles carried on the PHI duplication except for the plexus locus where, they suggested, partial dominance was displayed by two recessive mutant alleles. In relation to the breakpoint of the duplication, plexus was the most proximal locus among a l l recessive mutations studied by Morgan and Bridges. As w i l l be discussed later, this i s an important requirement for the occurrence of the position-effect variegation. The term "position-effect", as f i r s t used by Sturtevant (1925), was in reference to diverse stable phenotypes expressed by two different alleles of the Bar (B) locus owing to their cis or trans position with respect to one another. He reported that when the two Bar alleles , _B and _Bj\ are on the same chromosome (cis), their phenotypic expression is greater than when they are on opposite homologues (trans). Sturtevant also interpreted the observation of Bridges and Morgan (1923) , on plexus expression, to be caused by the position of the gene on the chromosome. Since the time of Sturtevant's discovery, numerous studies done on position-effect have revealed several novel properties of this phenomenon. A rearrangement that moves a euchromatic gene to a heterochromatic position, or vice versa, usually impairs the expression of the gene. Since the suppression of the normal activity of the gene involved i s generally variable in different c e l l s , the mutation i s expressed i n an individual as somatic mosaicism (Schultz 1936). Position-effect of this type, which causes somatic i n s t a b i l i t y , i s referred to as variegated or V-type position-effect which i s distinct from the stable position-effect (S-type) observed at the Bar locus by Sturtevant (1925). In most cases, the rearrangement causing position-effect variegation must be located 69 in cis position with respect to the wild-type allele of the gene influenced. The variegated phenotype is observed only when the influenced gene is heterozygous for i t s recessive, mutant a l l e l e . This, however, is not the case-if the rearranged chromosome is in a trans position with respect to the wild-type a l l e l e . For example, where R stands for rearrangement, R(w+)/w w i l l display variegated white eyes but R(w)/w+ w i l l be wild-type for eye colour (Muller 1930). In Drosophila, a number of genes are known that display somatic mosaicism. These of course must be autonomous in their action. Although, as noted above, most cases of variegation are recessive and w i l l be expressed only when the recessive al l e l e i s present, dominant position-effect variegations are also known (Lewis 1950). The most notable is brown variegated associated with euchromatic-heterochromatic rearrange-ments that move the bw+ locus, on chromosome-2, to a heterochromatic position. In an individual with the genotype R(bw+)/bw+, the expression of the brown gene is variegated even though two doses of bw+ are present. Moreover, R(bw+)/bw i s almost phenotypically identical with homozygous brown (Glass, 1933). The cis-trans position does not appear to operate in the case of the brown locus; while bw/bw+ is wild-type in phenotype, R(bw)/bw+ is variegated (Slatis 1955 b). The peach (pe) gene in Drosophila v i r i l i s (Baker 1953) and the light (It) gene in Drosophila melanogaster (Hessler 1958) are examples of genes normally located in heterochromatin whose phenotypic expressions are variegated when, owing to rearrangements, they are moved to a euchromatic environment. The suppressive effect of heterochromatin on the expression of 70 euchromatic genes that are placed next to i t , through rearrangements, spreads from the point of the break. While the suppressive action is greatest on those genes most proximal to the breakpoint, suppression can spread distally along the chromosome for a considerable distance (Demerec and Slizynska 1937; Schultz 1941). The degree of suppression appears to decrease as the distance from the breakpoint increases. Genetic and environmental factors are known to modify the degree of variegation. These include: addition or deletion of heterochromatin, change in temperature, and structural alterations to the genome. Addition of a Y-chromosome to the genome of an individual whose variegated gene i s normally in euchromatin suppresses variegation (i.e., variegation is suppressed i n X/Y/Y males and X/X/Y females). Removal of the Y-chromosome, on the other hand, enhances variegation (i.e., variegation is enhanced in X/O males) (Gowen and Gay 1933 a). Changes in the quantity of heterochromatin of the X and the fourth chromosome (Noujdin, as quoted by Lewis 1950) and chromosome-2 (Morgan et _al. 1940) similarly appear to affect the expression of the V-type position-effect (reviewed in Lewis 1950 and Hannah 1951). The effect of extra heterochromatin on variegation of the light (It)locus normally located in or near heterochromatin appears to be opposite to that observed with euchromatic genes (Schultz 1936, Baker and Rein 1962). Hence, addition of extra heterochromatic material to light variegated f l i e s enhances the variegation and the removal of i t suppresses the variegated phenotype. Since the peach locus of Drosophila v i r i l i s (Schneider 1962) and the cubitus interruptus (ci) locus on the fourth chromosome in Drosophila melanogaster (Grell 1959) behave in a similar manner to that of euchromatic genes with regards to their inter-71 action with extra heterochromatin, the light locus appears to be peculiar in i t s response to additional heterochromatin. Temperature also affects the extent of variegation (Gowen and Gay, 1933 b): high temperature suppresses and low temperature enhances variegation. Finally, the presence of a deficiency appears to modify the expression of variegated genes. Gersh and Ephrussi (1946) showed that deficiencies near the white locus influenced the phenotypic expression of white variegated f l i e s . Parental genotype is also known to have a modifying effect on the frequency of the variegated phenotype among the progeny. This was f i r s t discovered by Noujdin (1944) who studied the variegated phenotype 8 3P associated with In(l)sc and In(l)y He reported that the proportion of the progeny that showed variegation for br i s t l e mutations was considerably lower when recovered from homozygous sc female parents than when g recovered from heterozygous sc /+ female parents. When homozygous 8 sc mothers carried the extra heterochromatic element, XR[T(1;4)A1, the proximal portion of the X inserted into the fourth chromosome,] the fraction of variegated individuals was reduced even further. This reduction in variegation occurred even though the heterochromatic element was not inherited by the progeny examined. A second case of a maternal effect on variegation was reported by Liining (1954) who examined variegation for yellow body colour in M-5 heterozygous females. Spofford (1959) examined the extent of the white variegated phenotype in progeny of f l i e s where either the male or the female parent carried a 20-band duplication of the X-chromosome, including the w^  locus, inserted into the proximal heterochromatin of 3L. This duplication i s designated Dp(w™). Spofford 72 reported that the extent of eye pigmentation (i.e., suppression of variegation) in the progeny was greatest when Dp(wm) was paternal rather than maternal in origin. This, however, was "residually" influenced by the Y-chromosome constitution of the mother. Further examination of Dp(wm) by Hessler (1961) resulted in the recovery of two different "states" for this duplication. The f i r s t state, referred to as Dp_£, in general produced a greater degree of pigmentation in progeny than did the second 3. a. state, Dp . When inherited paternally, the Dp bearing progeny have more pigmentation than those receiving the Dp from their mothers. This parental effect i s reversed for Dp^, namely, the maternally inherited Dp^ is more effective i n pigmentation than the paternally inherited one. Hessler also reported that homozygosity, as compared to heterozygosity for the duplication in female parents, was more effective in pigmentation of heterozygous duplication bearing progeny. The parental effect and maternal homozygosity are, confined to one generation, however, and a "grand parental" effect does not exist. The difference in parental f a effect between Dp and Dp 'was later found to be caused by a suppressor gene (Su-V) located on the same chromosome as Dp^ but recombinationally separable from i t (Spofford 1962, 1967). Since the extent of variegation in the progeny of a Su-V bearing male is not affected by this suppressor gene, the effect of the Su-V gene appears to be s t r i c t l y maternal. Furthermore, the maternal effect of Su-V gene on the amount of eye pigment in the progeny is the same whether the progeny do or do not inherit the Su-V gene. The variegation is suppressed more when an individual i s homozygous for Su-V than heterozygous. Thus, the suppressor gene lacks complete dominance. When the two "states" of the 73 duplication were tested again under controlled conditions for Su-V, the f a "parental source" differences between Dp and Dp was not observed. Instead, both duplications behaved similarly, i.e., more pigmentation was produced in the progeny when the duplication was inherited paternally. The wild type a l l e l e of the Su-V gene appeared to enhance variegation. Cohen (1962) demonstrated that other l o c i within the Dp(wm) showed a similar response to the different "states" of the duplication. On the basis of Cohen's results, Spofford (1967) concluded that the effect of the Su-V locus was a general effect on a l l the l o c i contained in the re-arrangement studied. A similar modifier gene, also located on the third chromosome, but with a dominant maternal effect on suppression of variegation, was discovered by Schultz (Morgan, Bridges and Schultz 1937). Schneider (1962) examined the maternal effect associated with the position-effect variegation of the peach (pe) locus in Drosophila v i r i l i s . She reported that heterozygous progeny of homozygous R(Pe+) females are always more pigmented than those of either heterozygous mothers or homozygous fathers and heterozygous fathers. Schneider also found for one particular Y-autosomal translocation that the presence of a Y-chromosome in the mother suppressed the variegation in the male progeny, even though this Y-chromosome was not inherited by the suppressed individuals. During studies on the nature of spontaneous compound autosome nonsegregation (reported in the f i r s t chapter of this thesis), I observed that the v i a b i l i t y of M-5/0 males was considerably enhanced in association with certain combinations of compound-thirds. In considering the observations of Hess (1962) and Baker (1971) that lethality of scute 74 inversions, including the Muller-5 (M-5) chromosome, is only partially suppressed by fragments of the Y_-chromosome and moreover, in view of Baker's (1968, 1971) suggestion that the lethality of scute inversions i s a V-type position effect which possibly suppresses the expression of ribosomal-RNA genes,it i s of interest to examine the extent to which compound-autosomes can restore the v i a b i l i t y of M-5/0 males. The results of this study demonstrate not only that the v i a b i l i t y of M-5/0 males i s enhanced by a suppressive effect associated with certain compound-autosomes but also, and of greater interest, that the lethality of M-5/0 males is conditional and dependent on the parental origin of the inverted X-chromosome. 75 MATERIALS AND METHODS Description of chromosomes and mutations: The genetic markers, X-chromosome and autosomal inversions and the homozygous wild-type attached X-chromosome used in this study are described in the previous 2 S L + chapter of this thesis. An attached-X.Y chromosome (y v mal Y .Y y ) and a reversed metacentric compound X-chromosome [C(l)RM,y v bb] were also employed (for a complete description of these chromosomes see Lindsley and Grell 1968). The methods followed in the construction of compound autosomes, as well as the terminology used to describe these chromosomes, are also explained in the f i r s t part of this thesis. The procedure for generating compound autosomal lines with attached-X females and attached-X.Y males not bearing free Y-chromosomes (i.e., XX/O females and X.Y/O males) i s demonstrated in Appendix II. The parental source of the Muller-5 (M-5) chromosome: In the f i r s t set of experiments (Tables I - l i b inclusive), homozygous M-5 females, carrying different combinations of compound thirds, were crossed to X.Y males that either carried the same compounds as females or carried compounds that were differentially marked. Each set of experiments involved two crosses: one involving X.Y males with a free Y_, the other involving X.Y males without a free Y. Therefore, the maternally derived M-5 male progeny of one cross is nullo-Y_ (M-5/0) as compared to the M-5/Y males derived from the second cross. The M-5/Y and M-5/0 sons w i l l inherit different combinations of compound thirds from their respective parents. Therefore, in different experiments their v i a b i l i t y , 76 in the presence or absence of the Y-chromosome in combination with various compounds, can be studied. V i a b i l i t y i s expressed as a sex ratio; i.e., the ratio of male/ female progeny recovered in any given experiment. In the second set of experiments (Tables IVal-IVd inclusive) attached-X females, with and without a free Y_, were crossed to M-5 males. Here the M-5/Y and M-5/0 sons have derived their X-chromosome paternally, and since the parents carry different compound-third chromosomes in each cross, M-5 male progeny w i l l inherit different combinations of compound autosomes. As a control, a l l of the above experiments involving either M-5 females and X.Y males or attached-X females and M-5 males were conducted using standard third chromosomes (Table I I I ) , therefore, the v i a b i l i t y of M-5/0 as compared to M-5/Y sons could be measured in the absence of any influence by the compound autosomes. In the last experiment (Table I I I ) , Oregon-R wild-type females were crossed to X.Y/O males to establish the sex ratio of X/0 males carrying a standard wild-type X-chromosome. General mating procedures: In each of the experiments, single females were mated with two males, of the appropriate genotype, in 25 mm x 95 mm shell vials containing standard cornmeal, sugar, yeast and agar medium with propionic and phosphoric acid added as mold inhibitors. Parents were removed after 6 days of egg laying. A l l the experiments were conducted at 24° + 1°C. The progeny were counted and recorded at days 11, 14 and 17 following the removal of parents. From each experimental cross, a sample of M-5 male progeny were tested for f e r t i l i t y . 77 RESULTS The M-5/0 male progeny obtained in the experimental crosses reported in the f i r s t chapter of this thesis were products of exception-al meiotic events. It was necessary, therefore, to design experiments in which M-5/0 males were regular products of meiosis so that a more accurate measure of suppressed X/0 lethality could be obtained. Two schemes were available to produce X/0 males: (1) crossing C(l)RM/0 to M-5/Y males and (2) crossing M-5/M-5 females to X.Y/O males. The f i r s t method had the disadvantage of reducing the recovery of progeny by one half (owing to the production of XX/X and Y/0 zygotes), which in compound-autosome strains results in an overall reduction of progeny recovered to one eighth. Hence, as an i n i t i a l approach I used the second scheme, namely mating homozygous M-5; compound-3 females with attached-X.Y/O; compound-3 males to produce, as regular products, females that were heterozygous M-5/attached-X.Y and males that were of the M-5/0 genotype. The v i a b i l i t y of M-5/0 males, therefore, could be expressed as the ratio of male to female progeny. Along with each experiment, a control was conducted in which the attached-X.Y fathers carried a free Y-chromosome. A l l male progeny from the control series, therefore, inherited a Y-chromosome, i.e., they were M-5/Y in genotype. Since the latter males are expected to be f u l l y viable, their rate of v i a b i l i t y was used as a standard level for measuring the relative v i a b i l i t y of M—5/0 males. As reported in the previous chapter, the recovery of M-5/0 males relative to the matroclinous females was apparently influenced by the TABLE I. A comparison of the relative recovery, in various compound lines, of X/0 and X/Y males with maternally derived M-5 X-chromosome. Parental Genotype Number of Progeny Sex ratio Male Female Male Female Total XY S.Y Ly+/0;C(3DPayne;C(3R)ou M-5/M-5;C(3DPayne;C(3R)cu 431 908 1339 0.475 XY S.Y Ly+/Y;C(3DPayne;C(3R)cu M-5/M^5;C(3D Payne;C(3R)ou 1372 1219 2591 1.126 XY S.Y Ly +/0;C(3Dri;C(3R)Payne M-5/M-5;C(3Dri;C(3R) Payne 563 482 1045 1.168 XY S.Y Ljy+/Y;C(3Dvi;C(3R)Payne M-5/M-5;C (3Dvi;C(3R)Payne 554 530 1084 1.045 79 TABLE I l a . The effect of different compound third autosomes and a Y-chromosome on the survival of maternally derived M-5 males. Sex ratio obtained when M-5/0 males were derived from: M-5/M-S;C(3L)P2,ri;C(3R)VT4,In(Payne)/eS female X XIs.YLy+/0;C(3DVT2,In(Payne)/se;C(3R)VT2,cu male Progeny Sex ratio Compound autosomes Male Female C(3L)ri/C(3R)cu 143 266 0.538 C(3D Payne;C(3R)Payne 356 376 0.947 Total 499 642 0.777 80 TABLE l i b . The effect of different compound third autosomes and a Y-chromosome on the survival of maternally derived M-5 males. Sex ratio obtained when M-5/Y males were derived from: M-5/M-5;C(3L)P2,H;C(3R)VT43In(Payne)/eS female XY8. ILy+/Y;C(3D VT2,In(PayneJ/se;C(3R)VT2icu male Progeny Sex ratio Compound autosomes Male Female C(3L)ri\C(SR)au 484 349 1.387 C(3D Payne;C(3R)Payne 928 451 2.058 Total 1412 800 1.765 81 combination of the various compound autosomes used. To test the possible role of compound-third autosomes i n restoring M-5/0 v i a b i l i t y , a series of experiments were conducted i n which M-5/0 males were recovered with a l l possible combinations of compound autosomes used i n the previous study. Surprisingly, the recovery of M-5/0 males from these experiments was much higher than expected. In some of the crosses the sex r a t i o approached and even exceeded that obtained i n the respective control (Tables I-IIb i n c l u s i v e ) . There are two features of these crosses that d i f f e r from experiments previously reported: (1) the M-5/0 progeny inherited t h e i r X-chromosome from thei r mothers and (2) the parents carried compound autosomes. Therefore, to separate any possible maternal effect from suppression that may be caused by compound autosomes, comparable experiments were carried out using standard autosomal l i n e s . The f i r s t two crosses reported i n Table I I I demonstrate, consistent with the results reported by Hess (1962) and Baker (1971), that the recovery of M-5/0 male progeny, from crosses between M-5/Y males and C(1)RM/0 females, i s s i g n i f i c a n t l y lower than the recovery of the M-5 male progeny carrying a Y-chromosome (compare cross 1 with cross 2 i n Table I I I ) . However, when the M-5 chromosome i s maternally derived, the v i a b i l i t y of M-5/0 male progeny i s equal to that of M-5/Y male progeny. Moreover, the sex r a t i o i n both cases i s approximately one. That the sex rat i o obtained i n the l a t t e r two crosses probably does not r e f l e c t reduced v i a b i l i t y of X.Y/X females i s revealed by the l a s t cross reported i n Table I I I , i n which the male progeny were wild-type X/0. Furthermore, a sample of M-5 82 TABLE I I I . Relative survival, in a standard autosome lin e , of M-5/0 males whose X-chromosomes were maternal in origin as compared to those with X-chromosomes that were paternally derived. Parental Genotype Male Female M-5/Y;+/+ M-5/Y;+/+ C(l)RM,yvbb/0;+/+ C(l)RM/yvbb/Y;+/+ Xl8.YLy+/0;+/+ M-5/M~5;+/+. XY5. YLy+/Y;+/+ M-5/M-5:+/+ XYS.YLy+/0;+/+ +/+;+/+ Number of progeny . Sex ratio Male Female Total 506 1856 1603 1384 2362 0.273 2987 1.158 1924 1892 862 784 3816 1.017 1646 1.099 2691 2466 5157 1.091 83 male progeny from a l l experimental crosses were tested for f e r t i l i t y and i n every case were found to be s t e r i l e . The r e s u l t s i n Table III c l e a r l y demonstrate that l e t h a l i t y associated with M-5/0 males i s c o n d i t i o n a l and determined by the parental o r i g i n of the M-5 chromosome. Having established that the v i a b i l i t y of M-5/0 males i s a maternal e f f e c t , we can now return to the suppression of l e t h a l i t y occasioned by the presence of compound autosomes. Since the high recovery of M-5/0 males reported i n Tables I-IIb i n c l u s i v e , i s p r i m a r i l y due to a maternal e f f e c t , i t i s impossible to e s t a b l i s h the e f f e c t of compound autosomes on the v i a b i l i t y of these males. Therefore, i t was necessary to conduct experiments i n which M-5 compound-third males were crossed to attached— X; compound-third bearing females. As i n the f i r s t cross recorded i n Table I I I , t h i s w i l l r e s u l t i n the recovery of M-5 males who have i n h e r i t e d t h e i r X-chromosome pater n a l l y . These r e s u l t s are recorded i n Tables IVal-IVd i n c l u s i v e . Owing to the high nonsegregation of compound autosomes i n the presence of an attached-X chromosome i n females (Holm, personal communication), nonsegregational progeny, those who i n h e r i t e i t h e r the paternal or the maternal pair of compound autosomes, w i l l represent a s i g n i f i c a n t proportion of the population. Therefore, among the progeny of any one cross, M-5/0 or M-5/Y males w i l l be recovered, t h e o r e t i c a l l y , with four d i f f e r e n t combinations of compound autosomes. Tables IVal and IVa2 represent repeats of the same cross. Here attached-X; nullo-Y females were mated with M-5'/Y males. The compound-t h i r d chromosomes are d i f f e r e n t i a l l y marked i n the two sexes, therefore, 84 TABLE IVal. The effect of different combinations of compound-thirds and their parental origin on the relative survival of M-5 males with paternally derived X-chromosomes. Sex ratio obtained when M-5/0 males were derived from: C(I)RM3+/0;C(3L)P23 vi;C(3R)VT43In(Pay ne)/e female X M-5/Y:C(3L)VT23In(Payne)/se;C(3R)VT23 cu male Progeny Sex ratio Inherited compound Male autosomes Female C(3L)ri;C(3R)cu 131 348 0, .376 Segregational C(3L)Payne;C(3R)Payne 134 397 0 .338 C(3L)ri;C(3R)Payne Non-segregational .c(ZL)Payne;C(3R)ou 329 2 362 0 .904 Total 594 1109 0 .536 Percent non-segregational 55.39 32.82 35 TABLE IVa2. (A repeat of experiment IVal) Sex ratio obtained when M-5/0 males were derived from: C(1)RM3 +/0;C(3L)P23vi;C(SR)VT4,In(Payne)/eS female X M-5/Y;C(3DVT2,In(Payne)/se;C(3R)VT2 ^cu male Progeny Sex ratio Inherited compound autosomes Male Female C(3Dvi;C(3R)ou Segregational C(3L)Payne;C(3R)Payne 126 114 -391 471 0. 0. 322 242 Non_ C ( 3D vi;C(3R) Payne segregational b b C (3D Payne; C(3R)cu 412 495 0. 832 Total 652 1357 0. 480 Percent non-segregational 63.19 36.48 86 TABLE IVb. Sex ratio obtained when M-5/Y males were derived from: C(l)RM3+/Y;C(3L)P23ri;C(3R)VT43In(Payne)/se female X M-5/Y;C(3L)VT23In(Payne)/se;C(3R)VT23cu male Progeny Sex ratio Inherited compound autosomes Male Female C(3L)vi;C(3R)cu 418 434 0 .963 Segregational C ( 3D Payne; C ( 3R) Payne 533 357 1 .493 Non-segregational C ( 3D ri;C(3R) Payne C(3L)Payne;C(3R)au 296 32 77 163 1 .367 Total 1279 1031 1 .241 Percent non-segregational 25.65 23.28 87 TABLE IVc. Sex r a t i o obtained when M-5/0 males were derived from: C(1)RM3 +/0;C(3L)VT2,In(Payne)/se;C(3R)VT2,cu female X M-5/Y;C(3DP2,ri;C(3R)VT4,In(Payne)/eS male Progeny Sex r a t i o Inherited compound autosomes Male Female C(3L)ri;C(3R)cu 39 529 0. .074 Segregational G ( 3D Payne; C(3R) Payne 109 506 0, .215 Non-segregational C ( 3D vi;C(3R) Payne C(3D Payne; C(3R)cu 48 1055 3 0 .045 Total 196 2093 0 .094 Percent non-segregational 24.49 50.55 88 TABLE IVd. Sex ratio obtained when M-5/Y males were derived from: C(1)RM3 +/I;C(3L) VT2,In(Payne)/se;C(3R) VT2,cu female X M-5/Y;C(3L)P2,vi;C(3R)VT43In(Payne)/eS male Progeny Sex ratio Inherited compound autosomes •Male • Female Segregational C(3L)vi;C(3R)cu .C(3L)Payne;C(3R)Payne 431 424 389 459 1. 0. 108 924 Non-segregational C( 3D vi;C(3R) Payne C(3DPayneiC(3R)eu 12 108 268 34 0. 397 Total 975 1150 0. 848 Percent non-segregational 12.31 26.26 89 two types of regular and two types of exceptional progeny are recovered. Considering the regular (segregational) progeny for any combination of compounds, the number of M-5/0 males recovered was much lower than the number of attached-X females. Therefore, i t seems that even in compound autosomal lines, the maternal effect associated with the v i a b i l i t y of M-5/0 i s present. However, when nonsegregational progeny are examined, the lethality of M-5/0 males appears to be suppressed. Owing to the non-homologous pairing interactions between the attached-X chromosome and compound autosomes, with rare exceptions, a l l nonsegregational male progeny are matroclinous for the compound-thirds, while a l l non-segregational female progeny are patroclinous. Although they do not carry the same pair of compounds, the sex ratio for the nonsegregational class can be obtained only by dividing the number of males with maternally derived compounds by the number of females bearing paternally derived compound autosomes. The restored v i a b i l i t y of M-5/0 males in the nonsegregational class may be attributed to the specific compounds present in their genome. This i s documented by comparing the nonsegregational classes in Tables IVal and IVa2 to that recorded in Table IVc, M-5/0 males in combination with C(3L)Payne;C(3R)cu are very low in v i a b i l i t y (0.045 - Table IVc) while those with C(3L)ri;C(3R)Payne are highly viable (0.904 and 0,832 respectively - Tables IVal and IVa2). In Table IVc the parental combination of compounds i s reversed as compared to that of Table IVal and IVa2. The low v i a b i l i t y of M-5/0 males recorded in this Table can be observed in the regular progeny as well as in the nonsegregational progeny. This further supports the fact 90 that C(3L)ri;C(3R)Payne is the only combination of compound-thirds, among those studied, that suppresses lethality of M-5/0 males. It can be seen that this compound-associated suppression is also maternally affected. In a cross where C(3L)ri;C(3R)Payne are carried by the mother (Table IVal and IVa2), survival of the M-5/0 male progeny with any combination of compounds i s , in comparison, much higher than the respective class recovered from a cross in which C(3L)ri;C(3R)Payne is carried by the father (Table IVc). This property could be the result of a suppressor gene carried by C(3L)ri or C(3R)Payne, or a nonspecific effect exerted by extra heterochromatic material gained by such chromosomes during their construction. The latter possibility i s supported by the fact that compound autosomes are generated through translocation—type events that result i n duplications and deficiencies of their proximal region (Holm, Gavin, Kowalishyn and Yeomans, in preparation). Tables IVb and IVd demonstrate the control crosses conducted for experiments reported in Tables IVal (and IVa2) and IVc, respectively. Control crosses involve attached-X females who carry a free Y-chromosome. When such females are crossed to M-5/Y males, male progeny w i l l be M-5/Y with the M-5 chromosome of paternal and the Y-chromosome of maternal, origin. An examination of Tables IVb and IVd reveals the effect of the Y-chromosome in restoring the v i a b i l i t y of M-5 males. Note that the percent nonsegregation is reduced in these crosses. This is due to competition of the Y-chromosome for nonhomologous pairing with, the attached-X and the compound-thirds (Grell 1970). M-5/0 males that live to the adult stage have an extended period 91 of development as compared to their sisters. This was demonstrated by calculating the sex ratio in three successive counts at eleven, fourteen and seventeen days following the removal of parents from a number of experimental and control crosses (see Table V). When the M-5 chromosome was inherited paternally, using standard lines (cross 1, Table V), the recovery of M-5/0 males was greatly increased in the third count, i.e., 17 days after the removal of parents. The sex ratio for this cross was increased from .017 at day eleven to .273 at day seventeen. This represents an increase, in the sex ratio, of approximately 16-fold. Similar extended development times of M-5/0 males were observed when the M-5 chromosome was inherited either maternally or paternally in experimental crosses involving compound-third autosomal lines. However, the rate of increase in sex ratio in compound lines was much lower than that observed for the standard strains. The sex ratio for the M-5/Y males, on the other hand, appears to remain relatively constant throughout the 3 counts in most of the controls, with the majority of the progeny eclosing by day 14. Slow development is one of the phenotypic traits associated with bobbed mutants. According to Ritossa et a l . (1966), bobbed mutants manifest deficiencies for ribosomal-RNA genes located in the nucleolus organizer region. Therefore, position-effect suppression of r-RNA genes that results in reduced synthesis of ribosomal-RNA should also produce phenotypic effects similar to the bobbed deficiencies (Baker 1971, Nix 19 73). While the bobbed b r i s t l e phenotype commonly associated with deficiencies in the nucleolus organizer region was not evident in any TABLE V. Cumulative sex ratios obtained from total progeny recovered at three successive intervals following removal of the parents. Parental genotype Progeny recovered Cumulative sex ratio Sex F C(l)RM3y v bb/0;+/+ M M-5/Y;-h/+ F C(l)RM3y v bb/Y;+/+ M M-5/Y;+/+ F C(1)RM3 +/0;C(3L)P23 ri; C(3R) VT43 InP/e M M- 5/Y;C(3L)VT2,InP/se;C(3R)VT23cu F C(1)RM3+/0;C(3L)P23ri;C(3R)VT43InP/e M M-5/Y;C(3L)VT23InP/se; C(3R)VT23cu F C(1)RM3 +/Y;C(3L)P23ri;C(3R)VT43InP/e M M-5/Y;C(3L)VT23InP/se;C(3R)VT23cu M F M F M F M F Days following removal Total of parents 11 14 17 29 302 175 506 0.017 0.180 0.273 1710 131 15 1856 M 1513 90 0 1603 1.123 1.158 1.158 F 1347 37 0 1384 393 195 6 594 0.366 0.532 0.536 1074 32 3 1109 318 301 33 652 0.302 0.450 0.477 1054 299 4 1357 1132 133 14 1279 1.184 1.237 1.241 956 67 9 1032 TABLE V. Continued F C(DRM3+/0;C( 3D VT23 InP/se; C(3R)VT23eu M 134 M M-5/Y;C(3DP23ri;C(3R)VT43InP/e S F 2079 F C(DRM3+/Y;C(3DVT23InP/se;C(3R)VT23ou M 805 M M-5/Y;C(3DP23ri;C(3R)VT43InP/e S F 1018 F M-5/M-5/;C(3D VT23 InP/se; C(3R)VT23ou M 313 M X. Y/0;C(3D VT23InP/se; C(3RJVT23eu F 894 F M-5/M-5;C(3DVT23InP/se;C(3R)VT23cu M 946 M X.Y/Y;C(3DVT23 InP/se; C(3R)VT23ou F 1019 F M-5/M-5;C(3DP23ri;C(3R)VT43InP/e S M 353 M X.Y/0;C(3D VT23InP/se; C(3R)VT23cu F 627 F M-5/M-5;C(3DP23vi;C(3R)VT43InP/e S M 786 M X.Y/Y;C(3DVT23InP/se;C(3R)VT23ou F 601 F +/+;+/+ M 1518 M X.Y/0;+/+ F 1340 F = female parent and female progeny M = male parent and male progeny • 12 0 -196 0.089 0.094 0.094 12 0 2091 159 11 975 0.791 0.843 0.848 125 7 1150 115 3 431 0.350 0.472 0.475 12 2 908 422 4 1372 0.928 1.124 1.126 198 2 1219 137 4 499 0.571 0.772 0.777 14 2 643 606 20 1412 1.308 1.749 1.765 195 4 800 1157 16 2691 1.133 1.094 1.091 1103 23 2466 InP = Inversions (3D or (3R)Payne 94 of the M-5/0 males, these males did display a strong variegation for some of the genetic markers that flank the distal euchromatic breakpoint of the scute inversions. These markers include: achaete (ac), scute (sc), brachymacrochaetae (brc) and sawtooth (saw) (see Lindsley and Grell 1968 for complete descriptions of these mutations). Mutations at these four l o c i modify the br i s t l e and hair pattern of eyes, head, thorax, wings and abdomen. While a wide variation in the mosaic pattern of b r i s t l e mutation was observed on M-5/0 males, the following trend in the pattern was common to a l l crosses: (1) the absence of one, or more, macrochaeta on the scutellum with the br i s t l e pattern on the remainder of the thorax, the head and the wings, normal; (2) the absence of three or a l l four macrochaetae on the scutellum, but extra macrochaetae (dorsocentral and occasionally post alars) on the posterior region of the thorax; this pattern is sometimes accompanied by a sawtooth effect of the marginal hairs on the wings; (3) reduction in the numbers of macrochaetae on the scutellum and on the posterior thorax (a combined expression of the scute and achaete mutations) always associated with a mosaic pattern for the absence of microchaetae on the thorax, and frequently accompanied by the sawtooth-wing effect. It i s also possible that the mosaic pattern for the absence of microchaetae on the dorsal thorax is an expression of the gene outheld (at) which maps distally but very close to bobbed (the nucleolus organizer region). It i s important to note that these variegated patterns for b r i s t l e mutations were observed for M-5/0 males deriving their X-chromosomes paternally as well as those deriving their X-chromosomes maternally. 95 As far as the expression of these bristle genes i s concerned, no maternal effect was noted. However, M-5/0 males with the paternally derived X-chromosome, especially those recovered from the cross involving standard autosomes, were extremely feeble, while those with a maternal M-5 chromosome appeared fu l l y viable and as active as wild-type males. In experiments where M-5/0 males had inherited their X-chromosome paternally, i t was important to avoid any delay in examining the offspring. Delay would have resulted in an apparent reduction in the recovery of M-5/0 males. 96 DISCUSSION An examination of the lethality associated with the M-5 chromosome reveals a very interesting phenomenon: lethality of M-5/0 males i s conditional and determined by the parental origin of the M-5 chromosome. When the origin of this chromosome is paternal, M-5/0 males display very low v i a b i l i t y . However, when the M-5 chromosome i s inherited maternally, the v i a b i l i t y expressed by M-5/0 males i s equal to that of M-5 males carrying a Y-chromosome. The M-5 chromosome, a crossover product of two different scute inversions, contains the proximal (or right) breakpoint of inversion 8R scute-8 (sc ) and the distal (or left) break of inversion scute-Si S1L 8 (sc ). Lethality of X/0 males carrying the sc inversion, as well as S1L 8R males carrying the M-5 (sc , sc ) chromosome, was previously reported by Hess (1962) who related the lethality associated with these inverted chromosomes to a position-effect variegation. Baker (1968, 1971) examined a number of different scute inversions with break-points in the proximal heterochromatin. He found scute inversions to be lethal in X/0 males only i f the inversion included the nucleolus organizer; i.e., the nucleolus organizer was moved from the region of the centromere to the tip of the long arm of the X. While the lethality associated with some of these inversions was suppressed partially by a duplication for the tip of the X-chromosome, total suppression of lethality was obtained for a l l inversions only when males carried a complete Y-chromosome. Since a l l scute lethals involved the displacement of the nucleolus organizer (a tandemly repeated region of the X-chromosome coding for 97 18S and 28S ribosomal-RNA, Ritossa and Spiegelman 1965) and their lethality was totally suppressed by Y-chromosomes, that also carry a nucleolus organizer, Baker (1971) proposed that the lethality was at least p a r t i a l -ly due to suppression of transcription of the ribosomal genes. This concept received support from the work of Nix (1973) who discovered a SI significant reduction in the total RNA of sc /0 male larvae. The problem, however, is confounded by the fact that, on one hand, a Y_ fragment (Y ) carrying the nucleolus organizer only partially suppresses sc-associated male lethality and, on the other hand, a complete Y-chromosome deficient for the nucleolus organizer also partially suppresses lethality (Baker 1971). Since the Y-chromosome serves to suppress position-effect variegation, Baker suggested that scute inversions suppress the action of v i a b i l i t y genes at the base of the X as well as r-DNA. In the present study, although I found lethality of M-5/0 males to be completely suppressed when the source of the X-chromosome was maternal, variegation for the distal l y located b r i s t l e markers (achaete, scute, brachymacrochaetae and sawtooth), and possibly for the proximal marker, outheld (ot), was independent of the parental source of the X-chromosome. Evidently the maternal effect was restricted to the genes causing lethality. One additional feature of the M-5/0 males,in comparison to their sisters and M-5/Y males, was their prolonged development time. This is a characteristic of bobbed (bb) mutants, which are partial deficiencies for genes of the nucleolus organizer (Ritossa e_t a l . 1966). Although the rate of development was extended for both maternally and paternally derived M-5/0 males, i t was significantly reduced in-, the former. It i s possible, therefore, that the maternal effect i s operating at the level 98 of ribosomal-RNA synthesis. If , in accordance with Baker (1971) and Nix (1973), variegation of r-RNA genes i s assumed to be the major factor responsible for lethality of sc/O males, then one has to speculate on a model to explain the maternal effect involved in suppressing such lethality. In a sc/O zygote, with a maternal X-chromosome, both cytoplasm and X-chromosome are inherit-ed from the mother. This might, in fact, have an important bearing on the activity of sc-chromosomes in males, as lethality i s encountered only when cytoplasm and sc-chromosome are of different origin. The cytoplasm of the oocyte supplies the zygote with sufficient ribosomes, which were synthesized during oogenesis, to support the early stages of embryonic development. The ribosomal-RNA in the egg i s transcribed not only by the oocyte nucleus but also by the polyploid nuclei of the nurse cells that form the egg chamber. The RNA i s transferred from nurse c e l l to egg cytoplasm by interconnecting, cytoplasmic bridges (see King 1970 for review). It has been demonstrated by VonBorstel and Rekemeyer (1958) that f e r t i l i z e d eggs, even in the absence of sex-chromosomes, are able to undergo at least 10 to 12 nuclear divisions. It has also been demonstrated that RNA synthesis probably does not begin prior to the blastoderm stage of development (see Fristrom 1970 for review). Since homozygous scute females appear to be completely viable (Baker 1971), i t can be assumed that the eggs from such females carry approximately the same amount of ribosomal material as the eggs produced by wild-type females. This assumption would lead one to believe that the difference in v i a b i l i t y of paternally and maternally derived M-5/0 males i s of nuclear origin. 99 Inactivation of the X-chromosome in heterogametic male organisms during spermatogenesis has been discussed by Lifschytz (1971). Such inactivation apparently occurs by heterochromatization, in the f i r s t meiotic prophase of spermatogenesis, and results i n reduced RNA synthesis. Gould-Somero (1974) has also demonstrated that developing spermatids do not synthesize RNA. Furthermore, Lindsley and Grell (1969) reported that a developing spermatid does not require chromosomes to mature normally, although the presence of the complete genome is v i t a l to the primary spermatocyte. On the basis of the evidence given above, a number of suggestions may be offered to explain the maternal effect. F i r s t , there may be a differential rate of activation of maternally and paternally derived X-chromosomes. The sc/O embryos, with a paternally derived X-chromosome, require considerable time before the X i s activated. Even then the activity i s suppressed owing to position-effect variegation. Although the eggs carry sufficient ribosomes to support synthesis through the f i r s t 10-12 nuclear divisions, delayed synthesis of r-RNA by the suppressed r-DNA of the male X-chromosome w i l l not permit the organism to advance through the c r i t i c a l stages of development. This, however, could be altered by the degree of suppression. When the activity of fewer genes is affected by the rearrangement, more r-RNA would be synthesized which would delay the time of death in such males. In some cases where suppression of r-RNA genes i s slight, adults would emerge but would be weak, malformed and possibly l i v e for only a short while. sc/O males with maternally derived ^ -chromosomes, on the other hand, receive an X-chromosome that can probably transcribe sufficient r-RNA, even under the partially suppressed conditions, so that by the time the 100 stored ooplasmic ribosomes are exhausted, the embryo w i l l synthesize sufficient ribosomes of i t s own to help i t through the c r i t i c a l stages of development. A second possibility i s a preconditioning of the cytoplasm in homozygous scute females. This might be accomplished through an increase in the amount of RNA poured into the oocytes by the nurse ce l l s . Therefore, cytoplasm of these eggs might carry more ribosomes than the wild-type cytoplasm of the sc/O zygotes bearing a paternally derived X-chromosome. Finally, such preconditioned cytoplasm might interact with the suppressed X-chromosome in the sc/O males, resulting in acceleration of r-RNA transcription by these chromosomes. Although the suggestions offered above would f i t my experimental results, confirmation would require extensive cytological and biochemical investigation. The fact that certain combinations of compound-third chromosomes appear to suppress variegation more than others might reflect a non-specific effect exerted by extra heterochromatin carried by such compounds. This suppressive effect also appears to involve a maternal influence. The amount of proximal heterochromatin carried by compound autosomes is known to be variable owing to duplications and deficiencies arising from the translocation type event involved in their construction. The existence of extra heterochromatic material in those compound-third chromosomes which suppressed lethality the most might be cytologically demonstrable. 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X y f cav.y+/Yd> y f oav.y+/M-Ss X M-5/Y& y f oav.y /M-5s X y f oav.y /Jo* T y f oav.y /y f oav.y o and y f oav.y+/Ycf y = yellow body colour. 1-0.0 / = forked b r i s t l e . 1-56.7 cav= carnation eye colour. 1-62.5 y+ = duplication of the distal tip of the X-chromosome translocated to the short arm. This chromosome was originally derived from In(lLE)soV1 (Lindsley and Grell 1968). 110 2. The generation of compound-2 and compound-3 autosomes heterozygous for paracentric inversions. (a) Generation of C(3D heterozygous for Jn('3DPayne. In (3D Payne/Ly RQ X se/se & 3500 rads. In(3DPayne/se9 X C(3L)P2tri;C(3R)VKl3e a<? C(3DVT23In(3L)Payne/se;C(3R)VKlte ? $<? (b) Generation of C(3R) heterozygous for In(3R)Payne. In(3R)Payne/H9  x e S/e S<? 3500 rads. . In(3R)Payne/e 9 x C(3DP23ri;C(3R)SH43ca K-pn <? C(3D P23 ri; C ( 3R) VT4t In ( 3R) Payne/e" (c) Generation of C(2D heterozygous for In(2DCy and C(2R) hetero-zygous for In(2R)Cy, Bl/In(2L+2R)Cy3Cy bw 45asp 2or 45aS X It stu 3/lt stwV 3500 rads. 45a 2 *45a In(2L+2R)Cy3Cy bw^spor^/lt stw$x C(2DP2,b}C(2R)Pl3px cf C(2D VT53 In (2D Cy/lt; C(2R)Pl3 px and C(2DPl3 b; C(2R) VT23 In(2R)Cyt bw 45asp 2or 45a/sti/ I l l 3. Transfer of a marked X-chromosome from a standard strain into a line bearing compound autosomes. y f oar.y+/M-S;TM33Sb Ser/+$ x C(3L)P23ri;C(3R)VT4}In(3R)Payne/e Scf I) y f car.y+/Y;C(3L)P23ri;C(3R)VT43In(3R)Payne/eS <? II) y f oar.y+/M-5;C(3L)P23ri;C(3R)VT43In(3R)payne/eSJ III) M- 5/+; C(3L)P23ri;C( 3R) VT43 In ( 3R) Payne /eS ° Female number II was recovered as a simultaneous nondisjunction of the X-and third-chromosomes. The following two crosses were made using the progeny recovered from the above cross: (a) y f car.y+/Y;C(3L)P23ri;C(3R)VT43In(3R)Payne/eS & X y f oar. y+/M-5;C(3L)P23ri;C(3R) VT43In(3R)Payne/eS 9 y f oar.y+/y f car.y+;C(3L)P23ri;C(3R)VT43In(3R)Payne/eS $ y f oar.y+/Y;C(3L)P23vi;C(3R)VT43In(3R)Payne/eS & (b) M-5/+; C(3L)P23 ri; C(3R) VT43 In (3R)Payne/eS $ X y f cca>.y+/Y;C(3L)P23ri;C(3R)VT43In(3R)Payne/e Sd' y f oar.y+/M-S;C(3L)P23ri;C(3R)VT43In(3R)Payne/eS? y f oar.y+/y f oar.y+o X SMl3Cy/+;TM33Sb Ser/+ d> y f oar.y+/Y;SMl3Cy/+;TM3Sb Ser/+* X M-5/M^5 ? and X 112 X M-5/Y; C(3L)P23ri; C(3R) VT43In(3R)Payne/eS d* M-5/M-5;C(3L)P23 ri;C(3R)VT4,In(3R)Payne/eS ? and M-5/Y;C(3L)P23ri;C(3R) VT4,In(3R)Payne/eS d* Crossing y f oar.y+/Y;SMl3CY/+ males with compound-2 females resulted iff-the construction of the following C(2) stocks: (a) y f aar.y+/y f car.y+;C(2L)VT53In(2L)Cy/lt;C(2R)PlJpx y (b) M-5/M-5;C(2L)VTS,In(2L)Cy/lt;C(2R)Pl3px ? 2 In a similar way a y -marked X—chromosome was introduced into C(3) and C(2) lines: y2/y2;C(3L)P23ri;C(3R)VT23ou o y2/y2;C(2L)VTl3ho;C(2R)Pl3px y S 4. The introduction of the marked J-chromosome, B Y, into a l l compound autosome strains was accomplished as shown in the following example. The use of this marked Y-chromosome permitted the exclusion of X/X/Y females from a l l experimental crosses. y f oar.y+/Y;C(3L)P23ri;C(3R)VT43In(3R)Payne/eS (single male) X C(l)RM3+/BSY;C(3L)P23ri;C(3R)SH4(b)3ca K-pn (three females) y f oar.y+/BSY;C(3L)P23ri;C(3R)VT43In(3R)Payne/eS (two males) X y f oar.y+/y f oar.y+;C(3L)P23ri;C(3R)VT43In(3R)Payne/eS (single female) y f car.y+/BSY;C(3L)P23ri;C(3R)VT43In(3R)Payne/eSd' and 113 and y f oav.y+/y f oar.y+;C(3DP23ri;C(3R)VT43In(3R)Payne/eS 5 . The following is a representative cross used to minimize the genetic va r i a b i l i t y between the females of the experimental and control series. y f cav.y+/y f oar.y+;C(3DVT23In(3DPayne/se;C(3R)VT23cu 9: X y 2/BSY3- C(3L) P23 ri; C(3R) VT4, In ( 3R) Payne/eB<? (a) Experiment: y f oar.y+/y2;C(3L)VT23In(3DPayne/se;C(3R)VT43In(3R)Payne/e° $ X B/Y; C(3L)P23ri;C(3R) VT23 cu*i (b) Control: y f car.y /y ;C(3L)P23ri;C(3R)VT23ou9 X B/Y; C ( 3D VT23 In ( 3D Payne/se; C ( 3R) VT43 In ( 3R) Payne/eSd 6. The construction of experimental (heterozygous for autosomal inversions) and control females for crossover studies in strains bearing standard autosomes. M-5/Y;+/+d> X Inf3R)Payne/H2 . y2/y% X DexF/l(3)S12-<? MS/MS* X DGXF/K3)Sl2 M-5/+;H/+$ X y /Y;DcxF/+& M-5/+;DexF/+$ X In(3R)Payne/H<f M-5/y ;H/DaxF$ X M-5/Y;DcxF/In('3R)Payned1 To p i M-S/y ;In(3R)Payne/H$ X y /Y;In(3R)Payne/R d* y2/y2;In(3R)Payne/H2 and y2/Y;In(3R)Payne/Hdt 1 1 4 Using a similar procedure the following stock was constructed: + + y f oar.y /y f oar.y ;In(3DPayne/Ly R $ and y f oar.y+/Y:In(3DPayne/Ly„_R The two stocks constructed above were used to measure recombination in the proximal region of the X-chromosome in the presence of inversions and the absence of compounds: + + 2 y f oar.y /y f oar.y ;In(3R)Payne/Hi X y /YjIn( :3DPayne/Ey (a) Experiment: + 2 y f oar.y /y ; In (3R) Payne/In (3D Payne $ X M-5/Y;+/+ d» (b) Control: y f oar.y+/y2jH/Ly R9 X M-5/Yj+/+* Replacement, in males, of the Af-5-chromosome with a standard Z-chromosome carrying the Bar(B) mutation: B/B% X +/Y;SMlsCy/+;TM3,Sb Ser/+* B/Y; SMI ,Cy/+* and B/Y; TM3, Sb Ser/+ <* B/Y;TM33Sb Ser/+* X M- 5/M-5g B/M-5;TM33Sb Ser/+S x +/Y;C(3DP23ri;C(3R)VT4,In(3R)Payne/e S& " B/Y:C(3DP2sri;C(3R) VT43 In (3R) Payne/e Stf X M-5/M-5; C(3D P23 ri;C(3R) VT4, In(3R)Payne/e Sg B/M-5; C(3DP23ri; C(3R) VT4, In (3R)Payne/e S$ X 7. 8 . 115 X M-5/Y; C(3L)P2, vi; C(3R) VT43 In( 3R) Payne/e<? B/M-5; C(3L)P23 vi; C(3R) VT4, In( 3R) Payne/e j X B/Y; C(3L)P2,vi;C(3R) VT4,In(3R)Payne/eS * B/B;C(3L)P2,vi;C(3R) VT4, In(3R)Payne/e $ and B/Y; C(3L) P2, vi; C(3R) VT4, In (3R) Payne/eS & The following diagram indicates the regions of crossing-over and the products of exchange. + y f cav y O — + (1) + (2) Genotypes Phenotypes N C O S C O (region one) S C O (region two) D C O (region one and two) y f + oav.y (f oav) 2 y + + ( y2 ) y f + (y f) 2 y + + cav. y (car) y f oav (y f cav) y + + / ( + ) y f * / ( f ) 2 y + cav ( y2 cav) 116 APPENDIX II The generation of compound autosomal lines with attached-X females and attached-XY males having no free y-chromosome. (a) M-S/M-5;+/+^X +/Y;DcxF/TM3,Sb Sev* M-5/Y;TM33Sb Sev/+&X X.Y/X.Y;+/+ ? M-5/X. Y;TM33Sb Sev/+ ? X +/Y;C(3L)P23 vi;C(3R) VT23aud1 M-5/+;C(3L)P23vi;C(3R)VT23cu?X X.Y/Y;C(3L)P23vi;C(3R)VT23cu & M-5/X. Y; C(3L)P23 vi; C(3R) VT23cu$ X M-5/Y; C(3L)P23 vi; C(3R) VT23 cu<? M-5/X. Y;C(3L)P23 vi;C(3R) VT23 cu% X X. Y/Y;C(3L)P23 vi;C(3R) VT23cutf X. Y/X. Y;C(3L)P23vi;C(3R)VT23cus and X. Y/Y;C(3L)P2,vi;C(3R)VT23cu<? C(l)RM/Y; +/+ J X +/Y; TM33 Sb Sev/+ d» C(l)RM/Y;TM33Sb Sev/+$X +/Y;C(3L)P23vi;C(3R)VT23cu& C(1JRM/Y;C(3L)P23vi;C(3R) VT23ou$ X +/B Y;C(3L)P23vi;C(3R)VT23cu& C(1)RM/B Y;C(3L)P23 vi; C(3R) VT23cu? X X. Y/Y;C(3L)P23vi;C(3R) VT23cu <* X. Y/B°Y;C(3L)P23vi;C(3R) VT23cud1 117 CU)RM/B SY;C(3L)P23ri;C(3R)VT23cu X X. Y/B SY;C(3L)P23ri;C(3R)VT23cu Owing to nonhomologous pairing interactions involving compound-X, B Y and compound ( 3 ) during oogenesis, eggs w i l l be produced that are nullo for both the compound-X and the B Y-chromosome. Fertilization of these eggs by X.Y bearing sperm w i l l generate males of the following genotype: X. Y/0; C ( 3D P23 ri; C ( 3R) VT2; cu c ? The production of compound-X, nullo-Y females arise from the following crosses: X. Y/0; C(3D P23^ri^ X. Y/X. Y; C(3D P23 ri; C ( 3R) VT23 cu J X. Y/0; CC3DP2,ri;C(3R) VT23cu*X C(1)RM/B SY;C(3DP23ri;C(3R) VT23cu ? C(l)RM/0; C(3DP23ri; C(3R) VT23cu$X X. Y/0;C(3DP23 ri;C(3R) VT23 cu & C(DRM/0; C(3DP23 ri; C(3R) VT23 cu J arid X. Y/0; C(3D P23 ri;C(3R) VT23 cu & 


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