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Induced recombination in the proximal regions of chromosome 2 in females of Drosophila Melanogaster Tattersall, Philippa Jill 1981

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INDUCED RECOMBINATION IN THE PROXIMAL REGIONS OF CHROMOSOME 2. IN FEMALES OF DROSOPHILA MELANOGASTER by PHILIPPA J . TATTERSALL B . S c , U n i v e r s i t y of Concordia, 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE DEPARTMENT OF ZOOLOGY We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA 1981 © P h i l i p p a J . T a t t e r s a l l , 1981 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 requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the 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 study. I f u r t h e r agree 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 copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may be gran t e d by the head of my department o r by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying 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 allowed without my w r i t t e n p e r m i s s i o n . Department o f Zoology The U n i v e r s i t y o f B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date 2 5 F e b-> 1 9 8 1 DE-6 (2/79) i i ABSTRACT In order to study and compare spontaneous and r a d i a t i o n induced recombination frequencies of the euchromatic and heterochromatic regions of chromosome 2^ Drosophila melanogaster females were exposed to 0, 2, 3, 4, or 5 krads of gamma r a d i a t i o n . Exchange was measured i n the b - B l , B l - I t , l t - r l , r l - p k , and pk-cn i n t e r v a l s . The Tt-rl i n t e r v a l defines a wholly heterochromatic segment. A n a l y s i s o f the recombination data demonstrates that spontaneous recombination occurs i n the heterochromatic i n t e r v a l at a frequency of approximately 0.1% and the frequency of induced recombination i s p r i m a r i l y increased i n the heterochromatic i n t e r v a l . Moreover, the frequency o f recombination i n the heterochromatic i n t e r v a l i s c o r r e l a t e d w i t h the dose of r a d i a t i o n . There are s l i g h t o r no increases i n the recombination frequencies of the euchromatic segments and the regions c o n t a i n i n g the heterochromatic-euchromatic boundaries show responses intermediate to the heterochromatic and euchromatic regions. Te s t i n g of the recombinant chromosomes i n d i c a t e s that 22% of them are a s s o c i a t e d with r e c e s s i v e l e t h a l s . The a s s o c i a t i o n i s greater i n eggs l a i d the f i r s t f o u r days a f t e r r a d i a t i o n treatment than i n t h o s e l a i d f i v e -eleven days a f t e r the r a d i a t i o n treatment. I t i s p o s t u l a t e d t h a t induced recombination can occur v i a symmetrical as w e l l as asymmetrical interchange. The interchromosomal e f f e c t o f chromosome 3_, heterozygous f o r In(3LR)DcxF, on recombination i n chromosome 1_ has been s t u d i e d . The r e s u l t s show that i t s e f f e c t i s not s i g n i f i c a n t i n the heterochromatic regio n . Thus, the a l t e r a t i o n s i n the recombination frequencies owing to r a d i a t i o n treatment appear to be independent o f those owing to the interchromosomal e f f e c t . Recombination was al s o measured i n the presence of the hetero-chromatic d e f i c i e n c y - Df(2R)MS2^. The r e s u l t s i n d i c a t e t h a t the frequency of recombination i s decreased i n the chromosome arm c o n t a i n i n g the d e f i c i e n c y and i n the heterochromatic i n t e r v a l of the l e f t arm. The euchromatic regions of the opposite arm show a s l i g h t i n c r e a s e i n reconfr b i n a t i o n . A higher number of m u l t i p l e crossover progeny are recovered than would be expected according to map distances i n the presence o f the hetero-chromatic d e f i c i e n c y , Df(2R)MS2^, the heterozygous i n v e r s i o n , In(3LR)DcxF, and f o r double crossovers i n v o l v i n g the heterochromatic r e g i o n , but only a t high doses of r a d i a t i o n . i v ACKNOWLEDGEMENTS I wish to thank Dr. Holm f o r h i s guidance and sustained i n t e r e s t during the research, those working i n the U.B.C. Drosophila l a b o r a t o r i e s f o r t h e i r valuable suggestions and c r i t i c i s m s and L e i l a Harding f o r her help i n the preparation of the f i n a l d r a f t of t h i s t h e s i s . ABSTRACT ACKNOWLEDGEMENTS LIST OF TABLES LIST OF FIGURES INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION BIBLIOGRAPHY APPENDICES TABLE OF CONTENTS i i iv vi vi i 1 8 15 54 69 77 LIST OF.TABLES 1. D e s c r i p t i o n of Second Chromosome Genetic Markers Used i n the 9 Recombination Experiments 2. D e s c r i p t i o n of Chromosomal Rearrangements Used 10 3. Recombination Frequencies i n Proximal I n t e r v a l s of Chromosome 16 2_ f o r Experiment 1 4. Recombination Frequencies i n Proximal I n t e r v a l s of Chromosome 17 2_ f o r Experiment 2 5. Recombination Frequencies i n Proximal I n t e r v a l s of Chromosome 18 2 f o r Experiment 3 6. Recombination Frequencies i n Proximal I n t e r v a l s of Chromosome 19 2_ f o r Experiment 4 7. Recombination Frequencies i n Proximal I n t e r v a l s of Chromosome 20 2^  f o r Experiment 5 8. Recombination Frequencies i n Proximal I n t e r v a l s of Chromosome 30 2_ f o r Two Broods of Experiment 5 9. Recombination Frequencies of Experiment 3 R e l a t i v e to the Mean 37 of the Recombination Frequencies of Experiments 1, 2 and 4 10. Recombination Frequencies of DcxF/+ Females R e l a t i v e to +/+ 43 Females at Each Dose 11. C o e f f i c i e n t s of Coincidence and Numbers of Recombinant Chrom- 45 osomes Recovered 12. Tetrad Percentages f o r the Crossover Data 49 13. D i s t r i b u t i o n of Crossovers i n the Vt - r l _ Region of Chromosome .51 2_ and the Number of Crossover Events 14. Summary of Recessive Le t h a l s Present on l_t - r l _ Recombinant 53 Chromosomes Recovered from Experiments LIST OF FIGURES Schematic r e p r e s e n t a t i o n of the proximal regions of the second chromosome showing the genetic markers used and t h e i r report-ed map p o s i t i o n s Ratio of r a d i a t i o n induced recombination values i n each region of chromosome 2 to re s p e c t i v e c o n t r o l values f o r Experiment 1. Ratio of r a d i a t i o n induced recombination values i n each region of chromosome 2 to r e s p e c t i v e c o n t r o l values f o r Experiment 2 ~~ Ratio of r a d i a t i o n . inducedcreconibination;.va3ues. ini.each^region of chromosome _2_ to r e s p e c t i v e c o n t r o l values f o r Experiment 3 Ratio of r a d i a t i o n induced recombination values i n each region of chromosome 1_to r e s p e c t i v e c o n t r o l values f o r . b I t pk cn/BI rl;+/+ females of Experiment 4 Ratio of r a d i a t i o n induced recombination values i n .each region of chromosome _2_ to r e s p e c t i v e c o n t r o l values f o r b 1t pk cn/BI r l ; DcxF/+ females of Experiment 4 Ratio of r a d i a t i o n induced recombination values i n each region of chromosome ^ _ t o r e s p e c t i v e c o n t r o l values f o r b I t pk cn/BI rl;+/+ females of Experiment 5 Ratio of r a d i a t i o n induced recombination values i n each region of chromosome _2_ to r e s p e c t i v e c o n t r o l values f o r b 1t pk cn/BI r l ; DcxF/+ females of Experiment 5 Recombination frequencies of two broods i n Experiment 5 f o r b - B l , B1 -1t and I t - r l regions E f f e c t of Df(2R)MS2-*-Q on recombination i n chromosome 2 E f f e c t of In(3LR)DcxF on recombination i n chromosome 2_ i n Experiment 4 E f f e c t of In(3LR)DcxF on recombination i n chromosome 2 i n Experiment 5 E f f e c t of In(3LR)DcxF on recombination i n chromosome _2_ i n the f i r s t brood of Experiment 5 E f f e c t of In(3LR)DcxF on recombination i n chromosome_2_ i n the second brood of Experiment 5 1 INTRODUCTION Although the s t r u c t u r a l and f u n c t i o n a l p r o p e r t i e s o f heterochromatin have been i n v e s t i g a t e d e x t e n s i v e l y i n Drosophila melanogaster, many charac-t e r i s t i c s of t h i s genetic m a t e r i a l remain open to s p e c u l a t i o n . Heterochromatin was f i r s t described by He i t z (1933) as those regions of the chromosomes t h a t remain i n a p o s i t i v e heteropycnotic s t a t e during interphase... Euchromatic regions are s i m i l a r l y condensed only at l a t e prophase and metaphase. Brown (1966) c l a s s i f i e d heteropycnotic chromatin as e i t h e r f a c u l t a t i v e o r c o n s t i t u t i v e heterochromatin. C o n s t i t u t i v e heterochromatin i s c h a r a c t e r i s t i c a l l y found i n s i m i l a r regions o f d i f f e r e n t chromosomes i n eukaryotes. In Drosophila melanogaster i t i s p a r t i c u l a r l y prominent i n the regions surrounding the centromeres (Hannah, 1951). In metaphase chromo-somes of somatic t i s s u e s , heterochromatin comprises 1/5 - 1/4 of the length of the two metacentric autosomes, 1/3 - 1/2 of the X. chromosome and the whole of the Y_ chromosome (Cooper, 1965). The f i n d i n g that long r e i t e r a t e d regions of simple sequence DNA are present i n eu k a r y o t i c DNA ( B r i t t e n and Kohne, 1968) led others to l o c a l i z e r e p e t i t i v e o r s a t e l l i t e DNA sequences predominantly to the centromeric heterochromatin as wel l as to various s i t e s along the chromosome arms of Drosophila melanogaster (Snow and C a l l en, 1969; Rae, 1970; Jones and Robertson, 1970; G a l l e t a l . , 1971) . Peacock e t a]_. (1977) have detected seven species o f s a t e l l i t e DNA and determined that each chromosome has a s p e c i f i c arrangement of these r e p e t i t i v e sequences. Sequencing o f the s a t e l l i t e s (Endow e t aj_., 1975) has shown that the b a s i c repeating units are only 5-12 base p a i r s l o n g , thus c o n s i s t e n t with the concept that they do not code f o r a gene product. 2 E a r l y genetic studies r a r e l y l o c a l i z e d v i t a l genes w i t h i n centro-meric heterochromia t i n . Bridges (1916) described the heterochromatic Y_ chromosome as l a c k i n g genes e s s e n t i a l f o r v i a b i l i t y s i n c e i t s absence from the genome o f males only confers s t e r i l i t y . M u l l e r et aj_. (1937) concluded the heterochromatin of the _X chromosome was a l s o c o r r e l a t e d w i t h genetic i n e r t n e s s , although, more r e c e n t l y , S c h a l e t and Lefevre (1973) have suggested the presence o f suppressor of forked w i t h i n the proximal heterochromatin of the X. and the tandemly redundant rRNA gene complex (the bobbed locus) has been known f o r some time to occupy a p o s i t i o n i n the heterochromatin of the X_ and Y_ ( R i t o s s a and Spiegelman, 1965). This complex, however, may a c t u a l l y e x i s t as an i s l a n d of euchromatin w i t h i n the heterochromatic r e g i o n . Using the technique of compound second chromosome synthesis and detachment, Hi H i k e r and Holm (1975) generated proximal d e f i c i e n c i e s which uncovered r e c e s s i v e l e t h a l s i n both the l e f t and r i g h t heterochromatic blocks of chromosome 2_. A n a l y s i s of the r e c e s s i v e l e t h a l s by pseudodominance and complementation t e s t s demonstrated the ex i s t e n c e of at l e a s t two genetic l o c i i n 2R heterochromatin, one of which i s r o l l e d , and at l e a s t f o u r l o c i , i n c l u d i n g l i g h t , i n 2L_ hetero-chromatin. When the d e f i c i e n c i e s were test e d against EMS-induced mutations, Hi 1 1iker (1976) resolv e d a t o t a l o f 13 heterochromatic l o c i , i n c l u d i n g l i g h t and r o l l e d i n chromosome 2^ . He concluded that the l o c i i n 2L_ and 2R hetero-chromatin are not redundant. S i m i l a r l y , Baldwin and Suzuki (1971) have provided evidence f o r v i t a l l o c i i n the proximal heterochromatin of the t h i r d chromosome. Aneuploidy f o r heterochromatin can be t o l e r a t e d w i t h l i t t l e change i n the phenotype compared with aneuploidy f o r euchromatin, thereby supporting 3 the evidence f o r low genetic a c t i v i t y o f heterochromatin. M u l l e r et a l . (1937) found that d u p l i c a t i o n s and d e f i c i e n c i e s f o r -X heterochromatin i n c u r r e d no i n v i a b i l i t y and Yamamoto and Miklos (1977) found that d u p l i -c a t i n g the amount o f heterochromatin one to f o u r f o l d y i e l d e d f l i e s which were p h e n o t y p i c a l l y normal. While i t appears that f u n c t i o n a l genetic l o c i e x i s t i n heterochromatin, the m a j o r i t y of the genetic m a t e r i a l may be t r a n s c r i p t i o n a l l y i n a c t i v e . The observation that the recombination map length of the hetero-chromatic regions i s very short when compared with t h e i r m i t o t i c lengths l e d M u l l e r and P a i n t e r (1932) to conclude that m e i o t i c exchange i s confined to the euchromatic portions of the chromosomes. T h e i r observations have been supported by Baker (1958) and Roberts (1965) who a t t r i b u t e t h i s c h a r a c t e r i s t i c to the condensed s t a t e o f heterochromatin at the time that recombination occurs. A l s o , the reduction i n recombination w i t h i n euchromatic regions when they are moved next to centromeric heterochromatin (Beadle, 1932) has been termed by Mather (1936) as the "centromere e f f e c t " . Thompson (1964) proposed t h a t the basis of t h i s e f f e c t i s a f u n c t i o n of the r e p u l s i o n of the centromeric regions a f t e r p a i r i n g of the homologues. Oksala's (1958) bouquet model, where the centromeric regions of a l l chromosomes are i n c l o s e proximity to each o t h e r , proposes that non-homologous p a i r i n g occurs i n the heterochromatic regions so t h a t recom-b i n a t i o n i n these areas i s g r e a t l y reduced. As a consequence the proximal euchromatic segments are f r e q u e n t l y paired and t h i s accounts f o r the reduced frequency of recombination found i n those regions. There have been sp e c u l a t i o n s as to the f u n c t i o n of c o n s t i t u t i v e heterochromatin, although few are supported by experimental evidence. Under r e p l i c a t i o n of DNA during p o l y t e n i z a t i o n i n s a l i v a r y gland chromo-4 somes suggests i t i s of l i t t l e importance but the e v o l u t i o n a r y maintainance of i t suggests i t i s v i t a l . Heterochromatin may contain r e g u l a t o r y genes which play a r o l e i n p o s i t i o n - e f f e c t v a r i e g a t i o n (Hannah, 1951; Baker, 1968), may be i n v o l v e d i n chromosome p a i r i n g and segregation (reviewed i n Yunis and Yasmineh, 1971), and may c o n t r i b u t e to the c o n t r o l of recombination (John and M i k l o s , 1979). The response i n Drosophila melanogaster females to agents which modify the recombination process i s w e l l c h a r a c t e r i z e d . The e f f e c t s of temperature (Plough, 1917; G r e l l , 1978), age (Bergner, 1925), mineral content (Browning, 1949) and r a d i a t i o n ( M u l l e r , 1925; W h i t t i n g h i l l , 1951; Herskowitz and Abrahamson, 1957; Williamson e_t aj_., 1970) have been found to change the frequencies of recombination. As described by Kikkawa (1934), X-rays cause a "proximal increase and a compensatory d i s t a l decrease". Recombination i n males, i n which spontaneous recombination i s r a r e , i s also known to f o l l o w s i m i l a r regional responses ( F r i e s e n , 1937; W h i t t i n g h i l l , 1937; Puro, 1966; Hannah-Alava, 1968). Radiation has been shown to a l t e r the recombination process i n females so that exchange i s increased p r i m a r i l y i n the regions proximal to the centromere. This phenomenon was f i r s t observed by Mavor and Svenson (1924) f o r the centromeric region of the second chromosome. M u l l e r (1925) showed that the e f f e c t a l s o occurred i n the euchromatic regions adjacent to the centromeric regions of both the second and t h i r d chromosomes and that the magnitude of the e f f e c t was dependent on the dose - a higher dose g i v i n g a greater response. S i m i l a r f i n d i n g s have been reported by Herskowitz and Abrahamson (1957) and by Yost and Benneyan (1957). W h i t t i n g h i l l (1951) and 5 Yeomans (1972) also noted t h a t r a d i a t i o n caused a decrease i n recombination i n the more d i s t a l euchromatic segments. None of the published s t u d i e s , however, give c o n c l u s i v e evidence t h a t induced recombination i n the proximal i n t e r v a l s occurs p r i m a r i l y w i t h i n heterochromatin. The o r i g i n of induced recombination can be explained by two hypotheses. The f i r s t suggests that induced recombination takes place at i d e n t i c a l places on homologous chromosomes, g i v i n g r i s e to m e i o t i c - l i k e exchange events. Radiation may thus act i n d i r e c t l y on the normal recombination process found i n Drosophila females. A l t e r n a t i v e l y , induced recombinations are l i k e t r a n s l o c a t i o n s except the interchanges i n v o l v e breaks at d i f f e r e n t points on homologous chromosomes. Therefore, t h i s unequal exchange produces d e f i c i e n c i e s and d u p l i c a t i o n s i n the recombinant chromosomes. Evidence i n favor of induced recombination o c c u r r i n g v i a symmetrical interchange i s provided by s t u d i e s which i n d i c a t e t h a t recombinant chromo-somes are not a s s o c i a t e d with l e t h a l s o r a b e r r a t i o n s , p a r t i c u l a r l y i n the regions i n which exchange occurred. Mglinets (1973) recovered 149 recom-binant t h i r d chromosomes from i r r a d i a t e d females. Of these 149 chromosomes, only 11 of those contained rearrangements. However, s i n c e the breakpoints of the rearrangements occurred i n the same i n t e r v a l s , he concluded that induced exchange could i n v o l v e homologous or non-homologous points depending on the s t a t e o f p a i r i n g at the time of r a d i a t i o n treatment. He a l s o found that the rearranged recombinant chromosomes were recovered w i t h the highest frequency i n the e a r l i e s t broods. Williamson et al_. (1970) found that none of the 97 recombinant f o u r t h chromosomes they t e s t e d c a r r i e d r e c e s s i v e l e t h a l s , but the i n d u c t i o n of recombination was shown to be dependent on a 2 - h i t phenomenon. In Drosophila males, r a d i a t i o n induced recombination i n 6 chromosomes 2_ and 3^  i s u s u a l l y not associated with the i n d u c t i o n of l e t h a l mutations (Patterson and Suche, 1934; F r i e s e n , 1937). Evidence i n support of the second a l t e r n a t i v e i s more s u b s t a n t i v e . Herskowitz and Abrahamson (1957) demonstrated, by varying the i n t e n s i t y of r a d i a t i o n , that the i n d u c t i o n of recombination i n the chromosome of females i s a r e s u l t of a m u l t i - h i t event. They a l s o found that unequal exchange chromosomes were recovered more f r e q u e n t l y from eggs l a i d the f i r s t four days a f t e r the time of r a d i a t i o n - a f i n d i n g which p a r a l l e l s t hat of Mglinets (1973). O l i v i e r i and O l i v i e r i (1964) have shown t h a t , i n males, induced recombination i s a l s o dose dependent. Hannah-Alava (1968) concluded t h a t the i n d u c t i o n of recombination was s i m i l a r to the i n d u c t i o n of t r a n s l o c a t i o n s since she found that induced recombinant chromosomes i n males had a higher frequency of associated l e t h a l s than d i d non-recombinant chromosomes. The e f f e c t s of nonhomologous chromosomes on recombination, or interchromosomal e f f e c t s , were i n i t i a l l y observed by Sturtevant (1915). He discovered the coincidence of a heterozygous i n v e r s i o n i n the t h i r d chromosome and increased recombination i n chromosome 2^ Since then, an interchromosomal e f f e c t has been observed f o r t r a n s l o c a t i o n s (Hinton, 1965; Williamson, 1966), compound chromosomes (S u z u k i , 1962), t r i p l o i d y ( S c h u l t z and R e d f i e l d , 1951), d u p l i c a t i o n s ( K a l i s c h , 1973) and d e l e t i o n s (Yamomoto, 1979). The e f f e c t of i n v e r s i o n h e terozygosity has been w e l l documented (Morgan e t a l _ . , 1933; Morgan et_ a l _ . , 1943; Ramel, 1962; R e d f i e l d , 1964). The c h a r a c t e r i s t i c response observed i s one where marked increases are l i m i t e d to regions that normally e x h i b i t a low crossover frequency - regions that span the centromere and the d i s t a l ends of the chromosome arms. The medial regions of the arms are l e s s a f f e c t e d . This 7 a n a l y s i s has been l i m i t e d by the a v a i l a b i l i t y of appropriate genetic markers. An increase i n the frequency o f m u l t i p l e exchanges with a concomitant decrease i n chromosome i n t e r f e r e n c e accompanies the i n t e r -chromosomal e f f e c t ( S t e i n b e r g , 1936; S c h u l t z and R e d f i e l d , 1951; S u z u k i , 1963; Yamamoto, 1979). Mather (1936) suggested t h a t the change i n i n t e r f e r e n c e would a f f e c t l o c a l i z a t i o n of recombination events so t h a t they would occur more f r e q u e n t l y near the centromere and d i s t a l ends. Other attempts to e x p l a i n the mechanism causing an interchromosomal e f f e c t have centered on theories c o n s i d e r i n g the mechanical (Schultz and R e d f i e l d , 1951; Cooper et a l _ . , 1955; Oksala, 1958; Lucchesi and S u z u k i , 1968) or the p h y s i o l o g i c a l (Mather, 1936; Yost and Benneyan, 1957) e f f e c t s of the presence of a heterozygous i n v e r s i o n i n the genome. The present study was i n i t i a t e d to c h a r a c t e r i z e the centromeric heterochromatin of chromosome 2 of Drosophila melanogaster with respect to i t s recombinational p r o p e r t i e s . The l o c a l i z a t i o n of the l i g h t and rol1ed genes to the l e f t and r i g h t heterochromatin r e s p e c t i v e l y (Hi 11iker and Holm, 1975) defined a heterochromatic i n t e r v a l t h a t could be compared with euchromatic i n t e r v a l s f o r recombi n a t i o n a l responses to r a d i a t i o n , i n v e r s i o n h e t e r o z y g o s i t y and heterochromatic d e f i c i e n c e s . By analyzing any recombinant chromosomes derived from t h i s study, one can attempt to determine the nature of the induced exchange. 8 MATERIALS AND METHODS Tables 1 and 2 summarize a l l Drosophila melanogaster second chromosome mutations and s p e c i a l chromosomes used i n t h i s study. Recombination has been measured i n the proximal region of chromosome 2^  using the f o l l o w i n g markers: b_ - black (48.5), BJ[ - B r i s t l e (54.8), I t - l i g h t (55.0), r l - r o l l e d (55.1), pk - p r i c k l e (55.2), and cn -cinnabar (57.5) (map p o s i t i o n s are those reported i n Li n d s l e y and G r e l l , 1968). Figure 1 depicts the p o s i t i o n s o f these l o c i and shows that the regions of study are e i t h e r predominantly heterochromatic or euchromatic i n nature. Detachment st u d i e s on compound 2_ chromosomes place the l t -locus i n the d i s t a l segment o f the l e f t heterochromatic block and the r l locus i n the approximate middle of the 2R heterochromatin (Hi 11iker and Holm, 1975). The balancer chromosome, In(3LR)DcxF, used to i n v e s t i g a t e interchromosomal e f f e c t s , appears to suppress recombination along the e n t i r e length o f chromosome 3_. Mating procedure V i r g i n females heterozygous f o r markers on chromosome 2_ were c o l l e c t e d w i t h i n e i g h t hours of e c l o s i o n and i r r a d i a t e d immediately with 0- ( c o n t r o l ) , 2, 3, 4 or 5 krads o f gamma r a d i a t i o n . The source of r a d i a t i o n was a cobalt-60 Gammaeel 1 i n the U.B.C. Chemistry Department. The females were then aged f o r three days a f t e r which they were mated s i n g l y i n s h e l l v i a l s w i t h two or more males o f a homozygous genotype. Mating continued f o r seven days i n Ex p e r i -ments 1-4 and, i n Experiment 5, f o r f o u r days i n brood 1 and seven days i n brood 2. The numbers and phenotypes o f F-j f l i e s were determined u n t i l the 9 TABLE 1 D e s c r i p t i o n of Second Chromosome Genetic Markers Used i n the Recombination Experiments Symbol Name Map P o s i t i o n D e s c r i p t i o n b_ black 2 - 48.5= black body B]_ B r i s t l e 2 - 54.8 short b r i s t l e s , homozygous l e t h a l cn cinnabar 2 - 57.5 b r i g h t red eye, c o l o r -l e s s o c e l l i I t 1ight 2 - 55.0 y e l l o w i s h - p i n k eyes pk p r i c k l e 2 - 55.2 a n t e r i o r l y slanted c o s t a l h a i r s pr 1 purple 2 - 54.5 purple eyes r i r o l l e d 2 - 55.1 r o l l e d wing edges 10 TABLE 2 D e s c r i p t i o n of Chromosomal Rearrangements Used Symbol D e s c r i p t i o n In(3LR)DcxF M u l t i p l e - b r e a k i n v e r s i o n of chromosome 3 that c a r r i e s the dominant mutation, Dichaete Df(2R)MS2 1 0 D e f i c i e n c y of 2R heterochromatin i n c l u d i n g the locus f o r r o l l e d ; t h i n b r i s t l e d ; homozygous l e t h a l D f ( 2 L ) C D e f i c i e n c y of 2L_ heterochromatin i n c l u d i n g the locus f o r l i g h t ; g e n e r a t e d by the detachment of C(2L); C(2R) chromosomes (see H i l l i k e r and Holm, 1975) 11 FIGURE 1. Schematic r e p r e s e n t a t i o n of the proximal regions of the second chromosome showing the genetic markers used and t h e i r reported map p o s i t i o n s . 48.5 BI I t r l pk 54.8 55.0 55.1 55.3 cn 57.5 13 nineteenth day a f t e r the parents had been introduced. A l l matings were c a r r i e d out at 25°C+1°C on standard cornmeal, sugar and y e a s t Drosophila medium c o n t a i n i n g p r o p i o n i c a c i d as ;ia mold i n h i b i t o r . D e s c r i p t i o n s o f Experiments Five d i f f e r e n t s t u d i e s of recombination were performed using the mating procedure described: Experiment 1. To examine and compare recombination frequencies i n the euchromatic and heterochromatic regio n s , less than 8 hour o l d r l / b I t pk cn females were exposed to 0 ( c o n t r o l ) , 2, 3 or 4 krads of gamma r a d i a t i o n and, a f t e r three days, mated wi th b_ Vt £k_ cn/b Vt £k cn_ males. Experiment 2. To f u r t h e r define the b_ - Vt region and to observe the e f f e c t o f a change i n the maternal genotype, recombination wasr-. s i mi l a r l y measured by s c o r i n g the F-j progeny from BI r l / b 11 pk cn females which were t r e a t e d as i n Experiment 1. Experiment 3. To study the e f f e c t o f a heterochromatic d e f i c i e n c y on recombination, Df(2R)MS2^/b I t pk cn parental females were t r e a t e d as i n Experiment 1. Experiment 4. To i n v e s t i g a t e the e f f e c t o f a heterologous i n v e r s i o n on the frequency of spontaneous and r a d i a t i o n induced recombination i n the second chromosome, s i b l i n g females of the genotypes, BI r l / b I t pk cn; In(3LR)DCxF/+ and ; 14 Bl r l / b I t pk cn; +/+, were t r e a t e d with 0 ( c o n t r o l ) , 2, 3 or 4 krads of gamma r a d i a t i o n and mated with b I t pk cn/b I t pk cn; +/_+ males. A b_ Vt r l _ recombinant chromosome recovered from the co n t r o l s e r i e s i n t h i s experiment was used i n the subsequent crosses. Experiment 5. Females of the same genotype as used i n Experiment 4 were t r e a t e d with 0 ( c o n t r o l ) , 2, 4 or 5 krads of gamma r a d i a t i o n and mated with b I t r l / b I t r l males. To observe the v a r i a t i o n i n response of recombin-a t i o n at d i f f e r e n t stages of development of the oocyte at the time of r a d i a t i o n exposure, t r a n s f e r s were made once i n eleven days to give two broods of four and seven days. Recombinant chromosomes f o r the l t - r l region were t e s t e d over Df(2R)MS2J0 and over Df(2L)C* to confirm the presence or absence of d e f i c i e n c i e s i n that region a r i s i n g from recombin-a t i o n . Progeny Te s t i n g Progeny r e s u l t i n g from recombination between Vt and p_k were t e s t r crossed to r l / r l i n d i v i d u a l s i n Experiments 1, 2 and 4 and to b pr r l cn/ b_ p_r r l _ cn_ mates i n Experiment 3 to determine whether the event occurred between Vt and r l _ or between r l _ and p_k. In Experiment 5, t e s t c r o s s e s to i n d i v i d u a l s homozygous f o r the b_ Vt r l chromosome provided a d i r e c t measure of recombination i n the 1 t - r l region. A l l f a m i l i e s i n Experiments 1, 2, 3 and 4 i n which a 1t-pk recombinant chromosome was not t e s t a b l e were removed from the data. 15 RESULTS For each of the f i v e experiments and at each o f the r a d i a t i o n doses used, the frequencies of recombination ( i n p e r c e n t ) , f o r each ge n e t i c i n t e r v a l examined on chromosome 2^ , and the t o t a l number of progeny recovered are entered i n Tables 3 to 7. The 95% confidence i n t e r v a l s are a l s o given f o r each value. Since c a l c u l a t i n g the frequencies of recombination between Vt and r i and between r]_ and p_k required progeny t e s t i n g of recombinants between Vt and p_k, a l l c a l c u l a t i o n s are based on data which exclude f a m i l i e s i n which Vt - p_k_ recombinants could not be t e s t e d . This treatment of the data gives recombination frequencies that d i f f e r i n s i g n i f i c a n t l y from estimated frequencies obtained when those f a m i l i e s i n which progeny t e s t i n g had been unsuccessful are i n c l u d e d . The recombination data f o r a l l crosses are presented i n Appendix I . Contingency chi square values have been c a l c u l a t e d to t e s t f o r d i f f e r e n c e s among the c o n t r o l s e r i e s f o r each experiment. The c a l c u l a t i o n s are given i n Appendix 2. Comparisons among Experiments 1, 2, 3 and 4 reveal a homogeneous d i s t r i b u t i o n o f recombinant products. Thus, t h e i r experimental values can be compared. However, the c o n t r o l data of Experiment 5 are homogeneous only to t h a t of Experiment 4. Regional Responses to R a d i a t i o n . Contrary to previous f i n d i n g s (Baker, 1958; Robert, 1965), spontaneous exchange occurred i n the heterochromatic region f l a n k e d by the genetic markers Vt and r l _ . The distance between Vt and r l _ , as c a l c u l a t e d by the mean of the recombination frequencies obtained i n Experiments 1, 2 and 4 TABLE 3 Recombination Frequencies i n Proximal I n t e r v a l s of Chromosome 2 for : Experiment 1 Treatment o f Female Parent I n t e r v a l 0 2 krad 3 krad 4 krad b - I t 5.80+0.70* 6.5U0.82 7.OHO.68 7.90il.30 I t - r l * * 0.12±0.10 0.43±0.22 0.79±0.22 1.08±0.48 r l - pk** 1.47±0.36 1.49±0.40 1.7H0.34 3.01+0.80 pk - cn 0.84±0.26 0.80±0.28 0.73±0.22 0.84±0.42 ' t o t a l map length 8.23±0.84 9.28±0.98 10.24±0.82 12.83±1.64 no. of progeny 4294 3501 5447 1659 * 95% confidence l i m i t s c a l c u l a t e d by 2/pq/n where p = recombination frequency, q = frequency o f non recombinants and N = number of progeny k* Values f o r Tt - r l _ and r l _ - p_k_ (regions were obtained from progeny t e s t i n g 1 TABLE 4 Recombination Frequencies i n Proximal I n t e r v a l s o f Chromosome 2 f o r Experiment 2 Treatment o f Female Parent I n t e r v a l 0 2 krad 3 krad 4 krad b - BI 6.36±1.04 5.63+1.00 4.34±0.74 6.18±0.94 BI - I t 0.18±0.18 0.05+0.10 0.03±0.06 0.12+0.14 I t - r l * 0.23±0.20 0.48±0.30 0.63+0.28 1.52±0.48 r l - pk* 1.56±0.52 1.75+0.56 2.17±0.52 3.25±0.68 pk - cn 0.87±0.38 0.87±0.40 0.67±0.28 0.87±0.36 t o t a l map length 9-.20±l .24 8.78±1.24 7.84±0.98 11.94±1.32 no.of progeny 2186 2062 2993 2428 *Values obtained from progeny t e s t i n g TABLE 5 Recombination Frequencies i n Proximal I n t e r v a l s o f Chromosome 2 f o r Experiment 3 Treatment of Female Parent I n t e r v a l 0 2 krad 3 krad 4 krad b - I t 6.99±0.70 5.74±0.58 6.30±0.60 9.31±0.76 I t - M* 0.07±0.08 0.14±0.10 0.21±0.10 0.37±0.16 M - pk* •-. - 0.03±0.04 0.12±0.08 pk - cn 0.06±0.06 0.05+0.06 0.03±0.04 0.09±0.08 " ' t o t a l map length 7.12±0.70 5.93±0.58 6.57+0.60 9.89±0.78 no.of progeny 5392 6465 6634 5906 *Values obtained from progeny t e s t i n g 19 TABLE 6 Recombination Frequencies i n Proximal I n t e r v a l s of Chromosome 2 f o r Experiment 4 Treatment of b I t pk cn/BI r l ; +/+ Female Parent I n t e r v a l 0. , -; • 2 krad 3 krad 4 krad b - BI 5.75+0.96 4.81+0.78 3.62±0.72 5.22+1.40 BI - I t 0.26+0.20 0.14+0.14 - -I t - r l * 0.04+0.08 0.28+0.18 0.31+0.22 0.61+0.48 r l - pk* 1.2110.44 1.82+0.48 1.58±0.48 2.15+0.90 pk - cn 0.52+0.28 0.38+0.22 0.31±0.22 0.21+0.28 t o t a l map length 7.78+1.12 7.39+0.98 5.82±0.92 8.19±1.76 no. of progeny 2313 2909 2595 977 Treatment of b I t pk cn/BI r l ; DcxF/+ Female Parent I n t e r v a l 0 2 krad 3 krad 4 krad b - BI 13.8711.52 14.40+1.58 8.80+1.22 13.44+1.90 BI - I t 0.2010.18 0.16+0.16 - -I t - r l * 0.1010.12 0.21+0.20 0.2910.22 0:7310.44 r l - pk* 3.0410.70 3.5110.78 3.2710.74 3.9111.02 pk - cn 0.80+0.36 0.58+0.32 0.63+0.32 0.4910.36 t o t a l map length 18.01+1.72 18.86+1.80 12.9811.48 18.57+2.22 no. of progeny 2005 1882 2080 1228 *Values obtained from progeny t e s t i n g 20 TABLE 7 Recombination Frequencies i n Proximal I n t e r v a l s of Chromosome 2 f o r Experiment 5 Treatment of b_ Vt p_k cn/BI r l ; +/+ Female Parent I n t e r v a l 0 2 krad 4 krad 5 krad b - Bl 4.88±0.62 4 .26±0 .58 5 .29+0 .64 6 .52±0'.78 Bl - I t 0.26+0.14 0 .30±0 .16 0 .48±0 .20 0 .82±0.28 I t - r l 0.02±0.04 0 .16±0 .12 0 .42±0 .18 0 .82±0.28 t o t a l map.„ length 5.16±0'.62 4 .73+0 .60 6 .19±0 .70 8 .17±0.88 no. of progeny 4978 4933 4801 3895 Treatment o f b_ Vt p_k cn/BI r l ; DcxF/+ Female Parent b - Bl 12.33±1.20 8.81 ±0.92 12.02±1.54 13.06±1.64 Bl - I t 0.60+0.26 0.87±0.30 1.13+0.48 1 .54+0.56 I t - r l 0.07+0.08 0.14+0.12 0.56+0.34 1.11±0.48 total map length .12.99 ±1.22 9.81+0.98 13.7U1.64 15 .7U1.80 no. of progeny 3018 3691 1772 1623 21 i s 0.1 map u n i t s . The map distances f o r the b_ - B]_ and Bj_ - Vt regions are i n agreement w i t h those p r e v i o u s l y published - 6.3 and 0.2 r e s p e c t i v e l y ( L i n d s l e y and G r e l l , 1968). Although the t o t a l distance between Vt and cn i s a l s o i n accordance with the published map d i s t a n c e , the data from Experiments 1, 2 and 4 c l e a r l y i n d i c a t e that pj< i s f u r t h e r from r l _ (1.4 m.u.) and c l o s e r to cn (0.7 m.u.) than the reported values (0.2 m.u. and 2.2 m.u. re s p e c t i v e l y ) . I t i s evident that the responses to r a d i a t i o n d i f f e r among the regions and vary with the dose o f gamma r a d i a t i o n . These d i f f e r e n c e s are p a r t i c u l a r l y apparent i n Figures 2 to 8 which show the changes i n the recombination frequencies owing to r a d i a t i o n . The experimental values (P-j) f o r each region are d i v i d e d by the c o n t r o l values (Pg) f o r the same region and the q u o t i e n t i s p l o t t e d as a f u n c t i o n of the r a d i a t i o n dose a p p l i e d . I f the values compared are not d i f f e r e n t , a r a t i o o f 1 i s obtained. The 95% confidence l i m i t s o f the r a t i o s ( S u z u k i , 1962) have been c a l c u l a t e d by: 95% Conf. Lim. s = P^PQ • e- 2 j v a r ( I n P ] - I n P Q ) where Var ( I n P r I n P 0 ) = [(1-P ] )/P ]N ] ] - [(1-PQJ/PQNQ] and N-| = number of progeny o f the r a d i a t i o n t r e a t e d females N2 = number of progeny of the c o n t r o l females. The greatest change i n the recombination frequencies from the c o n t r o l s i s shown i n the heterochromatic Vt - r j _ r egion. A f t e r the lowest dose o f 2 krad i s a p p l i e d , recombination values are 2 - 6 f o l d more frequent than those observed i n the c o n t r o l s and as the dose o f r a d i a t i o n i s r a i s e d , f u r t h e r increases are noted. A p o i n t of maximal increase i s not apparent when doses of 2 to 5 krads are used u n l i k e the r e s u l t s of Bateman and Chandley (1965) 22 5-H 4H 3-\ 2-\ — 2 krad — — 3 krad — 4 krad T b - l t pk-cn l t - r l r l -pk. REGION FIGURE 2. Ratio of r a d i a t i o n induced recombination values in each region of chromosome _2 to respect i ve contro l values f o r Experiment 1 FIGURE 3. Ratio of r a d i a t i o n induced recombination values i n each r e -gion of chromosome _2_ to r e s p e c t i v e c o n t r o l values f o r Experiment 2. 24 b-lt l t - M M-pk pk-cn REGION FIGURE 4. Ratio of r a d i a t i o n induced recombiantion values i n each region of chromosome_2_ to r e s p e c t i v e c o n t r o l values f o r Experiment 3. Confidence l i m i t s cannot be c a l c u l a t e d f o r the M-pk region because no spontaneous recombination occurred. 25 15H 144 — 2 krad — 3 krad . 4 krad b-Bl b-lt l t - r l rl-pk pk-cn REGION FIGURE 5. Ratio of r ad i a t i on induced recombination values in each region of chromosome 2_to respect i ve contro l values f o r b It pk cn/ Bl_rl_ ; _+/+ females of Experiment 4. FIGURE 6. Ratio of rad iat ion induced recombination values in each region of chromosome 2_ to respective control values for b I t pk cn/ BJ_ rl_ ; DCxF/ + females in Experiment 4. 27 4H s 2H 20-^  8-^ 6-^ P l / P o 5H 3 H M _ 2 krad — 4 krad 5 krad b-Bl BI-It REGION l t - r l FIGURE 7. Ratio of r a d i a t i o n induced recombination values in each region of chromosome_2_ to respect i ve contro l values f o r b I t pk cn/ Bl_ rl_; +/+ females of Experiment 5. 28 17-1 16H 8H 7H 6H 5H P 7 / P J o 3-4 2H H 7 • i ./ •i 7 i i 7 .» / ./ — - 2 krad 1 •/ 4 krad 1 7 •• • • 5 krad i • • T b-Bl 1 r~ Bl -H I t - r l REGION FIGURE 8. Ratio of r a d i a t i o n induced recombination values i n each region of chromosome 2_to r e s p e c t i v e c o n t r o l values f o r b I t pk cn/BI r l ; DcxF/+ females of Experiment 5. 29 who found a t h r e s h o l d i n response at 4 krad f o r a heterochromatic region on the X_ chromosome. Euchromatic recombination, i n d i c a t e d by the f r e -quencies of recovering pjk - cn_ recombinants, i s a l t e r e d s l i g h t l y by r a d i a t i o n treatment but i n no instances are the d i f f e r e n c e s s i g n i f i c a n t The heterochromatin-containing r e g i o n , r l _ - p_k on 2R, shows a response that i s intermediate to the wholly heterochromatic and euchromatic regions. L i t t l e or no increases i n the recombination frequencies occur at lower doses (2 and 3.krad) and increases are r e a l i z e d at a higher dose (4 krad). The response of the heterochromatin-containing r e g i o n , BJ_ - JJt on 2L_, i s s i m i l a r i n that there i s a moderate increase i n the recombination frequency at higher doses but the r e s u l t s are more v a r i a b l e . Recombination i n Experiment 5 i s evaluated by examining the progeny from two broods. The f i r s t brood c o n s i s t e d of progeny from eggs l a i d i n the f i r s t f o u r days of mating and correspond to eggs which were oocytes at the time of r a d i a t i o n treatment and thus were i n the process of or had completed spontaneous m e i o t i c c r o s s i n g over. The second brood contained the progeny from eggs l a i d 5 - 12 days a f t e r mating. Most of these eggs were oogonia or e a r l y oocytes when r a d i a t i o n was administered - stages at which spontaneous c r o s s i n g over had p o s s i b l y not y e t occurred. In Dro- sophi1 a melanogaster females, oocyte formation begins approximately 5 days before o v i p o s i t i o n of a mature oocyte and recombination supposedly precedes o v i p o s i t i o n by 3.3 to 4.5 days ( G r e l l , 1973). Table 8 presents the recombination frequencies i n Experiment 5 f o r the three t e s t e d regions i n two broods at each dose. Figure 9 d i a g r a m a t i c a l l y represents those frequencies f o r b I t pk cn/BI r1;+/+ female parents. A decrease i n the recombination frequency w i t h r a d i a t i o n treatment i s apparent i n the f i r s t brood i n the b_ - Bl_ r e g i o n . This f i n d -TABLE 8 30 Recombination Frequencies i n Proximal I n t e r v a l s o f Chromosome 2 f o r Two Broods of Experiment 5 Treatment of b_ Vt p_k cn/BI r l ; +/+_ Female Parent I n t e r v a l 2 krad 4 krad 5 krad 1st Brood b - Bl Bl - I t I t - r l t o t a l map length no. of progeny 2nd Brood b - Bl Bl - I t I t - r l t o t a l map length no. o f progeny 6.51+1.00 0.37±0.24 6.88±1.02 2427 3.33±0.72 0.16±0.16 0.04±0.08 3.53±0.74 2551 5.10±0.88 0.27±0.20 0.08+0.10 5i'45±0.90 2551 3.36±0.74 0.34±0.24 0.25±0.20 3.95±0.80 2382 4.56±0.98 0.38±0.28 0.54+0.34 5.48±1.06 1841 5.74±0.86 0.54±0.26G 0.34±0.20 6.62±0.92 2960 5.39±1.04 0.27±0.24 0.69±0.38 ,35±V 1873 12 7.57±1.16 1.34±0.48 0.94±0.42 .85±1. 2022 34 Treatment o f b_ Vt p_k cn/BI r l ; DcxF/+ Female Parent I n t e r v a l 2 krad 4 krad 5 krad 1st Brood b - Bl Bl - I t I t - r l t o t a l map length no. of progeny 2nd Brood b - Bl Bl - I t I t - r l t o t a l map length no. of progeny 14.17±1.90 1.04±0.52 0.07±0.14 15.28+0.90 1348 10.84±1.52 0.24±0.22 0.06±0.12 11.14+1.54 1670 9.25±1.30 1 .06±0.44 0.15+0.16 10.46+1.38 1990 8.29±1.34 0.65±0.38 0.12±0.16 9.06±1.40 1701 10.95±2.38 1.33±0.84 0.74±0.62 13.02±2.60 676 12.68±2.00 1.00±0.56 0.46±0.38 14.14±2.10 1096 11 .69±2.36 0.70±0.58 0.99+0.68 13.38+2.52 710 14.13+2 .26 2.r9±0.90 1.21±0.66 17.53+2.52 913 31 FIGURE 9. Recombination frequencies from two broods of b I t pk cn/BI r l ;»/+• female parents i n Experiment 5 f o r the b - B l , Bl - I t and I t - r± regions f i r s t brood — second brood OJ 33 ing i s i n accord with those of Puro (1969) and Yeomans (1972) who re-ported s i m i l a r decreases i n euchromatic regions when r a d i a t i o n treatment was a p p l i e d to oocytes which had presumably undergone spontaneous recom-b i n a t i o n . In the Bl_ - Vt region the frequencies do not change i n the f i r s t brood but they are r a i s e d with a r a d i a t i o n dose of 5 krad i n the second brood. U n l i k e the r e s u l t s f o r the b_ - Bl_ and Bl_ - Vt r e g i o n s , recombination frequencies f o r the J_t - r l _ region increase i n the f i r s t brood. E f f e c t o f BI mutation. Comparison of the recombination frequencies f o r Experiment 1 without the Bl_ mutation (Table 3) and Experiment 2 with Bl_ (Table 4) shows that i n no instances are the spontaneous values d i f f e r e n t f o r any r e g i o n . However, the P]/Pg values i n Appendix 3 i n d i c a t e t h a t treatment of 3 and 4 krads i n the presence of Bl_ lessens the enhancing e f f e c t of r a d i a t i o n on exchanges between lb and Vt - the region i n which the Bl_ mutation i s placed. E f f e c t of Df(2R)MS2 1 0 The MS2 1 0 d e f i c i e n c y reduces the r i g h t arm of chromosome 2_ to about 3/4 i t s normal length at m i t o t i c metaphase ( L i n d s l e y and G r e l l , 1968). I t has been defined as a 2R heterochromatic d e f i c i e n c y by c y t o l o g i c a l a n a l y s i s ( H i l l i k e r and Holm, 1975) and because i t enhances p o s i t i o n e f f e c t v a r i e g a -t i o n (Morgan, S c h u l t z and Curry, 1940; Hannah, 1951). However, i t s d i s t a l breakpoint r e l a t i v e to the heterochromatic-euchromatic j u n c t i o n i s not known ( H i l l i k e r and Holm, 1975). 34 The experiments u t i l i z i n g the Df(2R)MS2 chromosome y i e l d changes i n the regional recombination frequencies. Figure 10 depicts the recom-b i n a t i o n frequencies obtained f o r the b_ - Vt, Vt - p_k_ and pjk - c^ n regions i n Experiment 3 and the mean of the corresponding values found i n Experiments 1, 2 and 4. The presence o f the d e f i c i e n c y r e s u l t s i n a s i g n i f i c a n t decrease i n the frequency o f spontaneous exchange i n two regions. The predominantly heterochromatic region i n which the d e f i c i e n c y i s con-tained and which i n c l u d e s chromatin of both the r i g h t and l e f t arms shows a 95% decrease i n the frequency of recombination. A reduction representing a 92% decrease i s shown i n the euchromatic, p_k_ - cn_ region which i s located on the same r i g h t arm as the Minute d e f i c i e n c y . Yamamoto and Miklos (1978), using d i f f e r e n t s i z e s of heterochromatic d e l e t i o n s of the X chromosome, reported a s i m i l a r reduction i n recombination i n the X. The euchromatic region between b_ and Vt on the opposite arm of chromosome 2 responds w i t h a 14% inc r e a s e . Also a f f e c t e d are the frequencies o f induced recombination. Examination of the d i f f e r e n c e s i n the recombination frequencies at each dose i n Table 9 shows, i n the b_ - Vt regions, the d i f f e r e n c e s increase as the dose i s r a i s e d i n d i c a t i n g the e f f e c t o f r a d i a t i o n may be enhanced with i n c r e a s i n g dose when MS2^ i s present. In the p_k_ - cn_ i n t e r v a l the d i f f e r e n c e s remain constant. The Vt - p_k_ region seems to respond i n a diminished way to r a d i a t i o n when the d e f i c i e n c y i s present because the negative d i f f e r e n c e s i n c r e a s e . The frequencies obtained f o r the M - p k region (Table 5) at doses of 3 and 4 krad i n d i c a t e increases c h a r a c t e r i s t i c f o r regions c o n t a i n i n g heterochromatin. Therefore, the d i s t a l breakpoint of the MS2^ d e f i c i e n c y may be w i t h i n the heterochromatic block o f the r i g h t 35 FIGURE 10. E f f e c t of Df(2R)MS2 1 0 on recombination chromosome 2. Broken l i n e s are values obtained from the mean of the f r e q u e n c i e s i n Experiments 1, 2 and 4 • • b_ - l_t region A A Vt - p_k_ region ~° p_k - cn region DOSE (KRAD) 37 TABLE 9 Recombination Frequencies o f Experiment 3 R e l a t i v e to the Mean of the Recombination Frequencies of Experiments 1, 2 and 4 Treatment o f Female Parent I n t e r v a l 0 , 2 krad 3 krad 4 krad b - I t +0.87 +0.03 +1.30 +2.85 I t - pk -1.45 -1.94 -2.15 -3.39 pk - cn -0.68 -0.63 &0.54 -0.55 38 arm. Thus, only heterochromatin i s removed by t h i s d e l e t i o n . E f f e c t of In(3LR)DcxF. The interchromosomal e f f e c t of heterozygosity f o r a chromosome 3_ i n v e r s i o n , In(3LR)DcxF, on recombination i n the various proximal regions of chromosome 2_ was stu d i e d i n Experiments 4 and 5. Table 6 shows t h a t , i n Experiment 4, the presence of the DcxF chromo-some s i g n i f i c a n t l y increases the t o t a l b_ - cn map length i n the c o n t r o l females from 7.78 to 18.01. Increases i n the t o t a l map length are a l s o apparent i n the t r e a t e d females at each dose. Within the b_ - cn i n t e r v a l , the g r e a t e s t increases appear i n the euchromatic b_ - B]_ and the heterochromatin-c o n t a i n i n g r l _ - p_k regions. The increases noted i n the p_k - on region are not s i g n i f i c a n t at any dose. Increases i n the recombination frequency are al s o observed i n the l_t - r l _ region but, when they are present, they are not s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l values at any dose. The response of the l_t_ - r l _ region suggests that the presence o f a heterozygous i n v e r s i o n does not exert an interchromosomal e f f e c t i n t h i s heterochromatic segment of chromosome 2_. I f that were t r u e , the increases noted i n the r l - pk region could be a t t r i b u t e d to the e f f e c t o f In(3LR)DcxF on the euchromatic po r t i o n o f t h a t region. The data are i n accordance w i t h those o f Cooper (1946), Schultz and R e d f i e l d (1951), Ramel (1962) and Roberts (1965) which show that recombination i n centromeric regions i s not a f f e c t e d by a heterozygous i n v e r s i o n . The regional responses described are d i a g r a m a t i c a l l y represented i n Figures 11 to 14 i n which the r e l a t i v e d i f f e r e n c e s owing to DcxF/+-are considered. The r a t i o s are obtained by d i v i d i n g the recombination value (P^) obtained using the DcxF i n v e r s i o n by the value (P R) obtained without the — — c o n t r o l 2 krad - - 3 krad — — 4 krad i 1 1 1 1— b-Bl B l - l t l t - r l r l - p k pk-cn REGION FIGURE 11. E f f e c t of In(3LR)DcxF on recombination i n chromosome2 Experiment 4. Ordinate shows r a t i o of values obtained w i t h DcxF to without a t each dose. 4-, b-Bl B l - l t l t - r l REGION FIGURE 12. E f f e c t o f In (3LR)DcxF on recombination i n chromosome 2 Experiment 5. Ordinate shows r a t i o o f values obtained w i t h DcxF to without a t each dose. 41 b-Bl B l - l t l t - r l REGION FIGURE 13. E f f e c t of In(3LR)DcxF on recombination i n the f i r s t brood i n chromosome 2_ i n Experiment 5. 4j -BI B l - l t l t - r l REGION FIGURE 14. E f f e c t of In(3LR)DcxF on recombination i n the second brood i n chromosome 2_ i n Experiment 5. i n v e r s i o n at each dose. I f the values compared are not d i f f e r e n t , a r a t i o of 1 i s obtained. In Experiment 4, the changes observed f o r the b - B l and r j _ - pj< regions are higher than u n i t y i n both the c o n t r o l and the t r e a t e d s e r i e s (Figure 11) i n d i c a t i n g that the presence of the DcxF i n v e r s i o n increases the recombination frequencies i n those regions. The r e s u l t s obtained i n Experiment 5 a l s o show that In(3LR)DcxF increases recombination i n the b_ - Bl_ region and has no s i g n i f i c a n t e f f e c t i n the Vt - r l _ region (Figure 12). However, they d i f f e r from : those of Experiment 4 i n that the recombination frequency f o r the BJ_ -Vt region is: increased when the i n v e r s i o n i s present,at a dose of 2 krad. The brood a n a l y s i s i n Experiment 5 i n d i c a t e s that the PA/PB r a t i o s f o r the b_ - Bl_ region i s the same i n both of the two broods (Figures 13 and 14). In other words, the magnitude of the interchromosomal e f f e c t i s s i m i l a r i n both broods - a r e s u l t t h a t i s expected when the f a c t o r modifying the recombination frequencies i s an i n t r i n s i c one. To determine whether gamma r a d i a t i o n a f f e c t s DcxF/+ females and non-inversion females i n the same way or whether r a d i a t i o n (or DcxF) diminishes or enhances the e f f e c t of DcxF (or r a d i a t i o n ) , the d i f f e r e n c e s i n recombination frequencies were c a l c u l a t e d and are shown i n Table 10. In the i n t e r v a l s where an interchromosomal e f f e c t i s apparent (b - BI and r l _ - p_k_), as the dose i n c r e a s e s , the d i f f e r e n c e s i n the recombination frequencies remain v i r t u a l l y constant. The d i f f e r e n c e s i n the BI - I t region i n Experiment 5 increase as the dose i s r a i s e d i n d i c a t i n g a p o s s i b l e enhancement of induced recombination when the DcxF i n v e r s i o n i s present. The d i f f e r e n c e s i n the Vt - r l _ region a l s o seem to remain constant which c o n t r a d i c t s Lucchesi's (1966) f i n d i n g that heterochromatin i s rendered more s u s c e p t i b l e to r a d i a t i o n by i n v e r s i o n s . In Experiment 4 , the d i f f e r e n c e TABLE 10 Recombination Frequencies of DcxF/+ Females R e l a t i v e to +/+ Females at Each Dose Experiment 4 Treatment of Female Parent I n t e r v a l 0 2 krad 3 krad 4 krad b - BI +8.12 +9.62 +5.18 +8.22 BI- I t -0.06 -0.02 - _ I t - r l +0.06 -0.06 -0.02 +0.12 r l - pk +1.83 +1.69 +1.69 +1.76 pk- cn +0.28 +0.21 +0.32 +0.28 Experiment 5 Treatment of Female Parent I n t e r v a l 0 2 krad 4 krad 5 krad b - B l +7.45 +4.55 +6.73 +6.54 BI- I t +0.34 +0.56 +0.65 +0.72 I t - r l +0.05 -0.03 +0.15 +0.29 i s g r e a t e s t i n the b_ - Bl_ r e g i o n , l e s s i n the H_ - p_k region and the l e a s t i n the p_k - c_n re g i o n . This order of magnitude of the d i f f e r e n c e s i s the same as tha t found f o r the regions' recombination frequencies. R e d f i e l d (1955) has noted t h a t the r e l a t i v e c o e f f i c i e n t s of c r o s s i n g over f o r regions remain the same when heterozygous i n v e r s i o n s are present. When the changes i n frequencies owing to r a d i a t i o n are considered (Figures 5 and 6 ) , the r a t i o s for.DcxF_/+ females do not d i f f e r from the r e s p e c t i v e values f o r the +_/+ females i n any region at any dose. The s i m i l a r i t y of the changes i n the regional responses to r a d i a t i o n i n d i c a t e they are independent of the presence of a heterozygous i n v e r s i o n . A n a l y s i s of Int e r f e r e n c e Two c a l c u l a t i o n s , c o e f f i c i e n t s of coincidence and p a r t i a l t e t r a d a n a l y s i s , are used to t e s t whether the changes i n the patterns of recombina-: t i o n frequency caused by r a d i a t i o n , the DcxF i n v e r s i o n and the HS2^ def-i c i e n c y are ass o c i a t e d with changes i n chromosome i n t e r f e r e n c e . The c o e f f i c i e n t s of coincidence f o r double exchanges i n v o l v i n g adjacent and non-adjacent i n t e r v a l s and the numbers of double exchange progeny recovered are given i n Table 11. As the coincidence value r i s e s from 0 to 1, i n t e r f e r e n c e approaches zero. When the value i s gr e a t e r than. 1, i n t e r f e r e n c e i s negative. In t e r f e r e n c e i s u s u a l l y p o s i t i v e i n the c o n t r o l s when double exchanges i n v o l v e the heterochromatic Vt - r j _ or Vt - M regions. When i n t e r f e r e n c e i s negative i n the Vt - r l _ r e g i o n , i t u s u a l l y occurs at the higher doses. S i n c l a i r (1977) demonstrated t h a t m u l t i p l e , spontaneous TABLE 11 45 C o e f f i c i e n t s of Coincidence and Numbers o f Recombinant Chromosomes Recovered C. of C. (Nos. of Recombinant Chromosomes) I n t e r v a l s 0 Treatment o f Female Parent 2 krad 3 krad 4 krad Expt. 1 b - l t ,1 t - r l b - l t , r l - p k b-1t,pk-cn 3.45(1) 1.02(1) 1.37(5) 1.18(4) 0.96(2) 0.99(3) 0.61(4) 0.36(1) 0.70(1) 1.01(4) Expt. 2 b - B l , 1 t - r l b-Bl , r l - p k b-Bl ,pk-cn B l - l t , l t - r l B l - l t 5 r l - p k B l - l t , p k - c h 1 t - r l , p k - c n 1.48(3) 3.31(4) 0.99(1) 16.07(1) 28.76(1) 1.42(4) 2.30(2) 8.31(1) 1.80(4) 1 .02(5) 22.48(1) 38.54(1) 3.21(1) Expt. 3 b - l t 5 l t - M b-lt,M-pk b - l t , p k - c n 4.79(1) 5.80(1) 7.93(1) 0.49(1) 6.14(4) 6.44(3) Expt. 4 +/+ b-Bl , r l - p k b-Bl ,pk-cn 0.62(1) 0.39(1) 1.46(1) 3.46(2) 0.67(1) 3.45(1) DcxF/+ b-Bl , B l - l t b-Bl , l t - r l . b-Bl ,r l - p k b-Bl ,pk-cn B l - l t , r l - p k rl-pk,pk-cn 1.44(1) 0.69(6) 1.27(3) 6.37(1) 1.87(1) 0.63(6) 0.63(1) 1.14(7) 1.65(2) 0.47(3) 2.48(2) TABLE 11 (cont'd) 46 C. of C. (Nos. of Recombinant Chromosomes) I n t e r v a l s Treatment of Female Parent 2 krad 3 krad 4 krad Expt. 5 1st Brood +/+ b - B l , B l - l t b - B l , l t - r l 2.19(1) 3.70(1) 1.43(1) DcxF/+ b - B l , B l - l t 0.50(1) b - B l , l t - r l 7.06(1) B l - l t , l t - r l 31.59(1) 1.22(1) 2nd Brood +/+ b - B l , B l - l t b - B l , l t - r l DcxF/+ b - B l , B l - l t b - B l , l t - r l 7.50(1) 30.01(1) 1.74(1) 0.72(1) 0.49(1) 0.71(2) 0.64(1) \ 47 exchanges i n v o l v i n g a predominantly heterochromatic region and immediately adjacent euchromatic i n t e r v a l s of chromosome 3_ were c h a r a c t e r i z e d by high negative i n t e r f e r e n c e . Double exchanges i n the present study i n v o l v i n g the heterochromatic region are u s u a l l y accompanied by an exchange i n the long b_ - Vt region of the l e f t arm. Except i n Experiment 5, there are only two cases of double exchanges i n v o l v i n g adjacent regions on the same si d e o f the centromere ( i .e. Regions b_ - BJ_, BJ_ - Vt o r r l _ - p_k, p_k_ - cn). G e n e r a l l y , they occur i n the absence of r a d i a t i o n treatment and are c h a r a c t e r i z e d by negative i n t e r f e r e n c e . I f i n t e r f e r e n c e i s governed by the p h y s i c a l p r o ximity o f two r e g i o n s , i t i s expected t h a t the c o e f f i c i e n t of coincidence be lower f o r b_ - BJ_, r j _ - jpj< and BJ_ - Vt, r l _ - p_k^  recombinants than f o r b_ - BJ_, p_k - cn and Bl_ - Vt, p_k - cn recombinants. Indeed, the data show t h i s to be t r u e . Double exchanges i n v o l v i n g the two most d i s t a l segments (b_ - Vt and p_k - on) show negative i n t e r f e r e n c e i n the c o n t r o l s . Other authors have found recombination occurs independently i n regions on opposite sides o f the centromere (Graubard, 1934; Bole-Gowda e t a l , 1962). Results w i t h the MS2^ d e f i c i e n c y i n d i c a t e i n t e r f e r e n c e i s decreased f o r double exchanges i n the d i s t a l euchromatic regions despite the lowered recombination frequency i n the pj< - on region noted p r e v i o u s l y . The c o e f f i c i e n t s o f coincidence f o r the i n v e r s i o n s e r i e s i n Experiment 4 exceed those of the non-inversion s e r i e s i n the c o n t r o l s with the exception of b - BJ_; p_k_ - cn double exchanges. A l s o , when the l t - r l region i s i n v o l v e d , the coincidence values are 0 i n both s e r i e s . Coincidence i n chromosome 2 i s a l s o r a i s e d when a heterozygous X. chromosome i n v e r s i o n i s used (Ramel, 1962). The higher values are not maintained w i t h r a d i a t i o n treatment except f o r double exchanges i n v o l v i n g the b_ - BJ_ and r l _ - p_k_ regions at 4 krads. No consistent brood effect is noticed for the coincidence values in Experiment 5. Partial tetrad analysis of the data was carried out using the formula for distribution of crossovers in tetrads derived by Weinstein (Steinberg, 1936) which assumes no chromatid interference: TET = 8(TC0 - 4QC0) DET = 4(DC0.- 3TC0 + 6QC0) SET = 2(SCO - 2DC0 + 3TC0 - 4QC0) NET = NCO - SC0.+ DCO * TCO + QCO where NET = frequency of no exchange tetrads, SET = frequency of single exchange tetrads, etc. NCO = frequency of noncrossover progeny, SCO = frequency of single crossover progeny, etc. The analysis is listed in Table 12. The negative DET values which are obtained in Experiment 5 are owing to the exceptional recovery of triple crossover progeny. The computations of the data from the crosses using In(3LR)DcxF indicate that the increases in recombination are accompan ied by a reduction of those tetrads showing no crossovers and an increased frequency of those having single and double crossovers at each dose. In other cases, involving interchromosomal effects on recombination, similar decreases of NETs and increases of SETs and DETs have been found (Redfield, 1957; Suzuki, 1962, 1963: Yamamoto, 1979). The results indicate that the heterozygous inversion does facilitate exchange in the b_ - cn segment of chromosome 2. The effect of radiation on tetrad distribution is seen to occur only at high doses (4 and 5 krads) where higher rank tetrads are slightly increased as NETs are decreased. Results from Experiment 5 indicate that the increases in the SET frquency occur predominantly in the second brood. TABLE 12 Tetrad Percentages f o r the Crossover Data Treatment of Female Parent Tetrad 0 2 krad 3 krad 4 krad Expt. 1 NET 84.31 82.12 89.28 75.52 SET 14.94 17.20 19.30 23.28 DET 0.76 0.68 0.60 1.20 Expt.2 NET 82.88 82.88 85.37 78.08 SET 16.24 16.02 13.82 19.96 DET 0.48 0.76 0.56 1.96 TET 0.40 - 0.24 -Expt.3 NET 85.84 88.22 86.92 80.77 SET 14.08 11.70 13.00 18.68 DET 0.08 0.08 0.08 0.56 Expt.4 NET 84.78 85.49 88.68 83.62 +/+ SET 14.86 14.10 11.00 16.38 DET 0.36 0.40 0.32 -DcxF/+ NET 65.98 63.76 75.30 65.14 SET 31.62 34.76 23.44 32.58 DET 2.40 1.48 1.36 2.28 0 2 krad 4 krad 5 krad Expt.5 1 s t Brood NET 86.24 89.10 89.68 87.67 +/+ SET 13.76 10.90 10.76 11.84 DET - - -1.12 0.44 TET - - 0.88 -DcxF/+ NET 70.03 89.65 74.26 71.27 SET 29.38 20.50 25.94 25.64 DET 0.60 0.20 -1.80 0.56 TET - - 1.20 -2 n d Brood NET 93.26 92.28 93.44 80.52 +/+ SET 6.42 7.40 12.98 19.28 DET 0.32 -0.48 0.12 0.20 TET - 0.32 - -DcxF/+ NET 77.72 81.90 72.08 66.26 SET 22.28 18.10 27.56 32.42 DET - - 0.36 1.32 50 The g r e a t e s t change i n DET frequency w i t h r a d i a t i o n occurs i n Experiment 3 where a seven f o l d increase i s seen w i t h a dose o f 4 krad. However, the frequency of DETs, when the heterochromatic d e f i c i e n c y i s present, i s much lower than those of the other experiments. M i t o t i c vs M e i o t i c Recombination. Gonial exchanges have been demonstrated to occur a f t e r r a d i a t i o n both i n females and i n males ( W h i t t i n g h i l l , 1955). He based his conclusion on the f i n d i n g s o f crossover c l u s t e r s and the i n e q u a l i t y i n recovery of r e c i p r o c a l c l a s s e s . I f a s i n g l e induced exchange i n the l_t - r l _ i n t e r v a l occurred i n a premeiotic d i v i s i o n , subsequent d i v i s i o n s would r e s u l t i n c l u s t e r s of the same or r e c i p r o c a l exchanges i n the gametes. Table 13 gives the numbers o f crossover progeny and the numbers of crossover events i n the l_t - r l _ region f o r the f i v e experiments. For these c a l c u l a t i o n s , a c l u s t e r of progeny a r i s i n g i n a s i n g l e c u l t u r e would be compared as a s i n g l e recombination event. In no instances are the c o n t r o l s e r i e s assoc-i a t e d w i t h c l u s t e r i n g of crossovers. There i s some c l u s t e r i n g w i t h the r a d i a t i o n t r e a t e d s e r i e s but, because the data f i t a Poisson d i s t r i b u t i o n , i t can not be concluded that the number of crossovers exceed the number of crossover events. The r e s u l t s from the i n v e r s i o n s e r i e s do not give an i n d i c a t i o n that In(3LR)DcxF causes crossovers to be d i s t r i b u t e d as c l u s t e r s . Thus, the data support conclusions by Ramel (1962) and Cooper (1946) t h a t the e f f e c t of a heterozygous i n v e r s i o n occurs at the time of medosis and the e f f e c t o f r a d i a t i o n may take place at a premeiotic stage or l a t e r . TABLE 13 D i s t r i b u t i o n of Crossovers i n 1 t - r l Region of Chromosome 2 and Number of Crossover Events Crossovers (Crossover Events)* Treatment of Female Parent Experiment 0 2 krad 3 krad 4( krad 1 4(4) 14(12) 40(31) 17(15) 2 4(4) 10(9) 17(15) 31(26) 3 4(4) 9(8) 14(14) •21(18) 4 +/+ 1(1) 8(8) 8(6) 6(5) DcxF/+ 2(2) 4(4) 6(6) 7(6) 5 1st Brood 4 krad 5 krad +/+ - 2(2) 7(6) 12(11) DcxF/+ - 2(2) 4(3) 6(6) 2 n d Brood +/+ _ 5(5) 9(9) 19(16) DcxF/+ K l ) 2(2) 5(5) 10(8) * Crossover Event= each c l u s t e r counted as one event 52 A n a l y s i s of Recombinant Chromosomes. Table 14 summarizes-the d i s t r i b u t i o n of r e c e s s i v e l e t h a l s a s s o ciated with the Vt - r l _ recombinant chromosomes recovered i n Experiment 5. Of 37 chromosomes t e s t e d , 8 had a p u t a t i v e d e f i c i e n c y f o r a v i t a l locus i n the heterochromatin uncovered e i t h e r by. D f ( 2 L ) C or by Df(2R)MS2 1 0. I f the p u t a t i v e d e f i c i e n c i e s arose from unequal exchange then there should be an equal number of chromosomes with d u p l i c a t i o n s . This means that approximately 43% of the exchanges i n the heterochromatic region i n v o l v e d unequal breaks encompassing at l e a s t one l o c u s . Although the numbers are s m a l l , more d e f i c i e n t chromosomes were recovered.in the f i r s t broods o f both the b Vt p_k cn/BJ_ r l _ ; +/+ and b_ Vt p_k cn/BI l l ; PcxF/+ females. TABLE 14 Summary of Recessive Lethals Present on l t - r l Recombinant Chromosomes Recovered from Experiment 5 Genotype of Parent Brood Dose Numbers of Recombinant Numbers Chromosomes Tested Nos. with L t . Hetero. D e f i c i e n c y Nos. with Rt. Hetero. Defi ciency b Vt p_k cn/BJ_ rj_;+/+ i s t >nd b Vt p_k cn/Bl_ r]_; DcxF/+ i s t 2nd 0 2 4 5 0 2 4 5 0 2 4 5 0 2 4 5 2 10 13 1 6 10 19 1 3 5 7 1 2 5 11 1 4 4 1 1 5 8 3 3 Totals 96 37 54 DISCUSSION The absence of m e i o t i c recombination has been described as one of the d i s t i n g u i s h i n g c h a r a c t e r i s t i c s of the heterochromatic regions i n Drosophila melanogaster (M u l l e r and P a i n t e r , 1932; Herskowitz, Schalet and Del Van Reuter, 1962; Roberts, 1965). Nonetheless, there have been i n d i c a t i o n s t h a t i t may occur i n heterochromatin at very low f r e q u e n c i e s . G r e l l (1978) reported a 0.06% frequency of spontaneous recombination between Vt and stw on chromosome 2 i n her 7 day c o n t r o l samples of exper-iments i n which she compared heat and' an interchromosomal e f f e c t on recom-b i n a t i o n . A spontaneous frequency of 0.3% for. the same region was found by Yeomans (1972). W h i t t i n g h i l l and Hinton (1950) a l s o performed exper-iments which detected r a r e , spontaneous recombinants i n the centromeric region of chromosome 2. In each case, however, i t could not be r u l e d out t h a t the exchange was o c c u r r i n g i n the small euchromatic segment included between the f l a n k i n g markers. Unexpectedly, the present work has revealed evidence f o r the occurrence of spontaneous m e i o t i c recombination i n the heterochromatin of the second chromosome. In each of the four experiments recombination was observed between Vt and r l _ . The'-.^frequencies ranged from 0.02 to 0.23%. While the frequency of recombination i n heterochromatin i s low, i t s consistency s t r o n g l y suggests t h a t i t occurs r e g u l a r l y . I t i s not p o s s i b l e to a s c e r t a i n from the r e s u l t s whether recombination between Vt and r l _ occurs w i t h i n the c o n s t i t u t i v e heterochromatin or whether i t occurs i n regions of n o n - r e p e t i t i v e sequences. Although Peacock et a l . (1977) have c a l c u l a t e d that n o n - s a t e l l i t e sequences i n heterochromatin occur 55 r a r e l y , i f at a l l , H i l l i k e r (1976) demonstrated, by using EMS induced p o i n t mutations, that at l e a s t 13 n o n - r e p e t i t i v e genetic l o c i , of which I t and r l _ are two that have v i s i b l e phenotypes, r e s i d e i n the hetero-chromatin of chromosome 2. Since at l e a s t 5, and p o s s i b l y 7, complemen-t a t i o n groups have been i d e n t i f i e d between Vt and r l _ , recombination i n ' these regions may be l i m i t e d to the genes themselves. Recombination be-tween homologous, n o n - r e p e t i t i v e l o c i should give r i s e to exchange chrom-osomes that are homozygous v i a b l e . The homozygous v i a b l e b_ Vt rj_ chrom-osome recovered i n Experiment 4 may be a r e s u l t of t h i s type of exchange. I t i s a l s o p o s s i b l e t h a t recombination occurs w i t h i n p a i r e d s a t e l l i t e sequences that l e a d s " t o l i t t l e or no a l t e r a t i o n i n the amount of s a t e l l i t e DNA. A l t e r n a t i v e l y , the presence of r e i t e r a t e d sequences i n heterochrom-a t i n may lead to m i s p a i r i n g and recombination between non-homologous genetic l o c i . This unequal exchange event would r e s u l t i n chromosomes c a r r y i n g d u p l i c a t i o n s or d e f i c i e n c i e s o f s a t e l l i t e DNA. G e l b a r t and Chovnick (1979) estimate that spontaneous unequal exchange occurs once f o r every 500 m e i o t i c d i v i s i o n s g i v i n g credence to the p o s s i b i l i t y of the event o c c u r r i n g between v i t a l l o c i . The recombinant chromosome f o r the I t - r l region recovered spontaneously i n Experiment 5 contained a p u t a t i v e d e f i c i e n c y i n the r i g h t heterochromatic block and, thus,may have a r i s e n from unequal exchange. Although the numbers of chromosomes which had undergone spontaneous recombination i n the Vt - r l _ i n t e r v a l were recovered i n very low numbers, the r e s u l t s show that unequal exchange can occur spontaneously i n hetero-chromatin of chromosome 2. 56 Radiation has been found by other workers to have a d i f f e r e n t i a l e f f e c t on recombination. Increases i n recombination have been induced p r i m a r i l y i n the proximal regions. In the present work, the e f f e c t s o f r a d i a t i o n are more e x p l i c i t l y c h a r a c t e r i z e d s i n c e the proximal b - cn region of chromosome 2 i s subdivided i n t o regions which d i f f e r i n t h e i r p roximity to the centromere and i n t h e i r euchromatin and heterochromatin content. The r e s u l t s s u b s t a n t i a t e the previous f i n d i n g s t h a t there i s a p o s i t i v e c o r r e l a t i o n between the proximity of the region to the centromeric heterochromatin and an induced increase i n recombination. However, the degree o f response can be c o r r e l a t e d w i t h the amount of heterochromatin r e l a t i v e to the amount of euchromatin i n the region s t u d i e d . The region around the centromere and co n t a i n i n g only heterochromatin showed the greate s t response. Increases of l e s s e r magnitude are noted i n adjacent regions. A decrease i n recombination i n the euchromatic regions has been described by others ( W h i t t i n g h i 1 1 , 1951). This i s not observed i n the present experiments and i s probably owing to the proximity of the o u t s i d e markers to the heterochromatic boundaries. An intermediate response i n regions c l o s e to the c e n t r i c region has been explained by Yeomans (1972) as being the r e s u l t of a summation of a d i f f e r e n t i a l e f f e c t across a reg i o n , i . e . a decrease i n one p o r t i o n and an increase i n another p o r t i o n more s u s c e p t i b l e to r a d i a t i o n induced recombination. The brood a n a l y s i s i n Experiment 5 reveals t h a t , indeed, w i t h i n a given i n t e r v a l , increases and decreases i n recombination occur. Greater increases are seen i n regions comprised of more heterochromatin. The rate and magnitude of the 57 changes appear to be dependent on the region tes t e d and the dose of r a d i a t i o n . The reduction of recombination i n euchromatic segments of the chromosome f o l l o w i n g r a d i a t i o n treatment has been reported by various authors with the response being dependent on the dose ( W h i t t i n g h i l l , 1951; Bateman.and Chandley, 1965) and on the region tes t e d ( M u l l e r , 1925; W h i t t i n g h i l l , 1951; Bateman and Chandley, 1965; Roberts, 1969). Most previous evidence, through brood a n a l y s i s , i n d i c a t e s t h a t the re-ductions occur when r a d i a t i o n i s a p p l i e d to oogonia or oocytes t h a t have not y e t undergone m e i o t i c exchange. The decreases may thus r e s u l t from r a d i a t i o n e f f e c t s p e r s i s t i n g through c e l l d i v i s i o n s and a l t e r i n g the sub-sequent exchange process. The r e s u l t s presented i n t h i s study f o r the p_ - Bl region show that r a d i a t i o n causes a decrease i n recombination i n the f i r s t brood o n l y , probably a f t e r spontaneous c r o s s i n g over has occurred (or has been determined). S i m i l a r r e s u l t s have been documented by Puro (1969) i n the st_ - i_n region of chromosome _3 and by Yeomans (1972) i n the b_ - pjr region of chromosome 2. I t has been suggested that r a d i a t i o n may act to a l t e r the p a i r i n g r e l a t i o n s h i p by d i s r u p t i o n of p a i r i n g i n the d i s t a l (Bateman and Chandley, 1965) or medial (Haendle, 1979) regions. Although i n d u c t i o n of separation of p a i r e d euchromatic regions would ex-p l a i n r e duction of recombination i n those regions p r i o r to the recombina-t i o n events t a k i n g . p l a c e , i t does not e x p l a i n the r e d u c t i o n seen when the treatment i s a p p l i e d a f t e r completion of spontaneous c r o s s i n g over. Yeomans (1972) proposed t h a t a s e l e c t i v e l o s s of exchange homologues i n 58 e a r l y broods may be o c c u r r i n g . I t i s p o s s i b l e that induced exchanges may be more l i k e l y to occur i n regions which have already undergone the spontaneous process (e.g. because c e r t a i n pre-conditions have been met) and thus n u l l i f y the o r i g i n a l event. The use of a d d i t i o n a l markers which f u r t h e r define the regions s t u d i e d would t e s t t h i s e x p l a n a t i o n . A d d i t i o n a l l y , a n a l y s i s with broods of a s h o r t e r d u r a t i o n would f u r t h e r resolve the time of occurrence r e l a t i v e to the stage of oogenesis o f the t r e a t e d egg. The brood a n a l y s i s presented i n the present work also i n d i c a t e s t h a t r a d i a t i o n induces increases i n the wholly heterochromatic region of approximately equal magnitude i n both broods. The reduction of the recombination frequency found f o r the euchromatic i n t e r v a l i n the f i r s t brood i s not apparent i n the heterochromatic i n t e r v a l . Increases i n broods o c c u r r i n g too e a r l y to be a gonial event have also been noted f o r pre-dominantly heterochromatic regions on the ^(chromosome (Herskowitz and Abrahamson, 1957; Robert!,1969). The non s i g n i f i c a n t increases between I t and stw found by Yeomans (1972) during the f i r s t f o u r days of egglaying may be a r e s u l t o f f i r s t brood decreases i n the euchromatic p o r t i o n of t h a t regio n . Although increases owing to an e f f e c t o f r a d i a t i o n on the centromere i t s e l f cannot be r u l e d out, the f i n d i n g t h a t increases are not confined to the c e n t r i c region suggests that the magnitude of the response i s c o r r e l a t e d with the heterochromatic content of the r e g i o n . Roberts (1969) showed that r a d i a t i o n induces exchange p r i m a r i l y i n an a c e n t r i c , heterochromatic region Q o f the X chromosome moved d i s t a l l y by the i n v e r s i o n , I n ( l ) s c • The higher s e n s i t i v i t y i n the present study of the b_ - Vt region compared w i t h the 59 pjk - cn_ region may be r e l a t e d to the presence of i n t e r c a l a r y heterochromatin. Hannah (1951) gives evidence that the i n t e r v a l between b_ and Vt has more repeated sequences than does the p_k - cn_ i n t e r v a l . Regional d i f f e r e n c e s i n the r e l a t i v e increases of recombination are apparent i n the Experimental/Control values of the present data. That the re g i o n a l d i f f e r e n c e s r e f l e c t a higher s e n s i t i v i t y to r a d i a t i o n o f the proximal regions i s supported by other experiments. Induced recombination occurs p r i m a r i l y i n the proximal regions i n Drosophila males where i t can be measured without background spontaneous recombination ( M g l i n e t s , 1972). When spontaneous recombination i s suppressed i n females by the presence of the c(3)G gene, induced recombination i n the chromosome mostly occurs i n the heterochromatic region (Roberts, 1969). C y t o l o g i c a l a n a l y s i s using banding techniques- has i n d i c a t e d that aberrations induced by r a d i a t i o n occur more f r e q u e n t l y i n and near the c e n t r i c regions of the chromosomes (Holmberg and Jonassen, 1973; San.Roman and Bobrow, 1973; Buckton, 1976) although proof t h a t recombination i s r e l a t e d to those a b e r r a t i o n s i s y e t to be provided,. . I t has a l s o been argued t h a t r a d i a t i o n a f f e c t s a l l p o r t i o n s of the chromosome randomly with the increases being apparent only i n the i n t e r v a l s c h a r a c t e r i z e d by l i t t l e or no spontaneous c r o s s i n g over. Unequal exchanges may be more f r e q u e n t l y recovered when they occur i n heterochromatin i f the r e s u l t i n g d u p l i c a t i o n s and d e f i c i e n c i e s are l e s s detrimental than when they are present i n euchromatin. The f i n d i n g s that aneuploidy f o r heterochromatin i s t o l e r a t e d with l e s s detrimental e f f e c t s than aneuploidy f o r euchromatin ( L i n d s l e y et a l _ . , 1972; Yamamoto and M i k l o s , 1977) lends support to t h a t premise. 60 The r e s u l t s of Experiment 5 demonstrate that induced recombination between Vt and r]_ i s associated with a heterochromatic d e f i c i e n c y f o r a gene e s s e n t i a l f o r v i a b i l i t y i n 22% of the tested chromosomes. Hetero-chromatic d e f i c i e n c i e s are found less f r e q u e n t l y i n recombinant chromosomes from oocytes which were t r e a t e d at an e a r l y stage o f development than i n chromosomes from ooytes t r e a t e d at a l a t e r stage. King (1952) and Herskowitz and Abrahamson (1957) demonstrated t h a t ; a f t e r r a d i a t i o n treatment o f females, the frequency of r e c e s s i v e l e t h a l s decreases propor-t i o n a l l y i n successive broods. They suggest induced recombination i n eggs l a i d i n l a t e r broods i s more l i k e l y to occur between homologous l o c i . In a c y t o l o g i c a l and genetic a n a l y s i s o f induced recombination i n i r r a d i a t e d Drosophila males, Mglinets (1972) showed t h a t chromosomes recom-binant f o r centromeric and adjacent regions o f chromosome 3_ are a s s o c i a t e d l e s s f r e q u e n t l y with rearrangements than are chromosomes recombinant f o r euchromatic regions and t h a t crossovers induced at gonial stages are not associated w i t h rearrangements. He proposed t h a t the occurrence of recom-b i n a t i o n at homologous or non-homologous s i t e s depends- on the s t a t e of chromosome p a i r i n g at the time of r a d i a t i o n . The present study reveals t h a t induced recombination i n heterochromatin, l i k e spontaneous recombination i n heterochromatin, r e s u l t s i n chromosomes which may o r may not c a r r y d e f i c i e n c i e s f o r a v i t a l locus and suggests t h a t both equal and unequal induced exchange occurs w i t h i n the centromeric heterochromatin. Recombination s t u d i e s u t i l i z i n g a screening method which s e l e c t e d f o r induced exchanges w i t h i n the heterochromatic region of chromosome 2_ would increase the r e s o l u t i o n of t h i s method of i n v e s t i g a t i o n . For example, females c o n t a i n i n g two EMS p o i n t mutations l i n k e d i n trans i n the l e f t or 61 r i g h t heterochromatin could be i r r a d i a t e d and crossed to males heterozygous f o r a d e f i c i e n c y o f the chosen heterochromatic block and f o r a dominant temperature s e n s i t i v e l e t h a l mutation. At the r e s t r i c t i v e temperature, the only s u r v i v i n g progeny would be those with the w i l d type a l l e l e s l i n k e d because of an exchange event. Since unequal exchange can occur, t h i s method would probably measure the exchange frequency between the l e t h a l a l l e l e and the euchromatin i n one chromosome and the centromere and the l e t h a l a l l e l e i n i t s homologue. I t i s p o s s i b l e that an unequal exchange occurs between two nonhomologous points - one being l o c a t e d i n heterochromatin, the other i n euchromatin. The recombinant chromosome would thus be d e f i c i e n t f o r both a p o r t i o n of heterochromatin and euchromatin. However, the low v i a b i l i t y of heterozygous euchromatic d e f i c i e n c i e s suggests that recombination i n heterochromatin i s e x c l u s i v e o f recombination i n euchromatin. Evidence e x i s t s which i n d i c a t e s t h a t r a d i a t i o n can induce recombination i n the gonial stage o f oogenesis ( W h i t t i n g h i l l , 1951). Since heterochromatin, p r i m a r i l y , appears to be a f f e c t e d by r a d i a t i o n , i t may be c h a r a c t e r i z e d by the a b i l i t y to undergo oogonial recombination. W h i t t i n g h i l l (1951) suggested that gonial exchange would be manifested by a c l u s t e r i n g of crossover progeny and a nonrandomness of the frequency of recovery of r e c i p r o c a l crossover c l a s s e s . Also, gonial exchange and subsequent m e i o t i c exchange would r e s u l t i n the recovery of rare m u l t i p l e crossovers at unexpected frequencies ( W h i & t i n g h i l l , 1955; Suzuki et a l , 1966). Although minimal c l u s t e r i n g , evident i n the data, does not provide c o n c l u s i v e evidence i n support of gonial exchange, the f i n d i n g that increased recombination i n the hetero-chromatic region may be accompanied by reduced i n t e r f e r e n c e at high doses lends support to the occurrence of g o n i a l recombination. Radiation treatment using G r e l l ' s "pupal system" (1973) may help to i d e n t i f y the time periods at which recombination i s induced, p a r t i c u l a r l y i n r e l a t i o n to the time of occurrence of m e i o t i c c r o s s i n g over. The interchromosomal e f f e c t , namely t h a t i n v e r s i o n h e t e r o z y g o s i t y i n one pa r t of the genome increases the frequency of recombination i n the remainder o f the genome, has been s t u d i e d i n an attempt to determine some of the i n t e r r e l a t i o n s h i p s of nonhomologous.chromosomes on recombination. I t i s known th a t the increase i n recombination caused by heterozygous i n v e r s i o n s i s u s u a l l y not uniform along the chromosomes but the p a u c i t y of heterochromatic markers has led most previous workers to describe r e -combination as being maximally enhanced i n the regions of centromeric heterochromatin (Morgan, Bridges and S c h u l t z , 1933; Morgan, R e d f i e l d and Morgan, 1943; V a l e n t i n , 1972). However, Ramel (1962) reported that the heterozygous M u l l e r - 5 i n v e r s i o n increased recombination i n chromosome 2 w i t h the most marked e f f e c t being i n the centromeric b_.- p_r region . The centromere, however, i s lo c a t e d i n the adjacent p_r - £ region where he found much l e s s of an incr e a s e . G r e l l (1978) has al s o f a i l e d to f i n d an interchromosomal e f f e c t between Vt and s„tw using a UBx i n v e r s i o n of chromo-some 3_. Further c h a r a c t e r i z a t i o n of the patter n of recombination obtained with a heterozygous i n v e r s i o n i s p o s s i b l e i n the present study s i n c e the a v a i l a b i l i t y of genetic markers on chromosome 2 make i t p o s s i b l e to define s h o r t e r proximal i n t e r v a l s . An interchromosomal e f f e c t i s apparent i n the b_ - cn region and there i s a d e f i n i t e s p e c i f i c i t y of the behaviour of the d i f f e r e n t segments. The increases i n recombination i n the b_ - c_n i n t e r v a l are confined to the regions adjacent to the centromeric Vt - r]_ re g i o n . An 63 experiment by S c h u l t z and R e d f i e l d (1951) which compared two corresponding d i s t a l regions of the same c y t o l o g i c a l length on the X_ chromosome suggests that the absence of an interchromosomal e f f e c t w i t h i n the l_t - r j _ region i s probably due to i t s heterochromatic nature and not to the presence of the centromere. One region i n t h e i r experiment contained an a c e n t r i c por-t i o n of X_ heterochromatin moved d i s t a l l y by I n ( l ) r s t 3 , the other d i d not. The region with heterochromatin showed a comparatively small interchromosom-al e f f e c t . Although i t appears t h a t heterochromatin does not respond to a heterozygous i n v e r s i o n , the response of euchromatic regions may depend upon t h e i r p r o ximity to heterochromatin. The importance of the d i s t r i b u t i o n and the amount of heterochromatin i n the genome on the interchromosomal e f f e c t has been acknowledged (Suzuki, 1963; R e d f i e l d , 1964; Yamamoto, 1979). The d e s c r i p t i o n of the c h a r a c t e r i s t i c response to s t r u c t u r a l hetero-z y g o s i t y o f f e r e d p r e v i o u s l y can thus be modified with the information pro-vided by the present work. There i s an increase i n the recombination f r e -quency i n the euchromatic regions adjacent to the centromeric heterochromatin, l e s s of an e f f e c t i n the medial regions of the chromosome arms and increased recombination i n the d i s t a l regions. The e f f e c t of In(3LR)DcxF and gamma r a d i a t i o n were a l s o used con-c u r r e n t l y i n the present work to study t h e i r combined a l t e r a t i o n s i n the recombination f r e q u e n c i e s . The two e f f e c t s have been p r e v i o u s l y i n v e s t i g a t e d by Lucchesi (1966). He found that the presence of a heterozygous autosomal i n v e r s i o n increases induced recombination between an attached X_ and Y_ chromosomes and suggested that i n v e r s i o n s may increase the s u s c e p t i b i l i t y of heterochromatin to breakage by r a d i a t i o n . However, h i s study lacks independent c o n t r o l r e s u l t s . The lack of i n t e r a c t i o n between the i n t e r -chromosomal and r a d i a t i o n e f f e c t s noted i n the present r e s u l t s and the d i f f e r e n c e s between them i n the regions a f f e c t e d and the degree of response suggest that they a l t e r recombination by d i f f e r e n t mechanisms. Mather (1936) suggested that p a i r s of homologous chromosomes compete f o r a f i x e d number of exchanges c h a r a c t e r i s t i c f o r the genome. Roberts (1969) supported a proposal by Howard-Flanders and T h e r i o t (1966) that an enzyme(s) e s s e n t i a l f o r c r o s s i n g over and r e p a i r of r a d i a t i o n damage i s present i n the nucleus i n a l i m i t e d q u a n t i t y . The d i f f i c u l t y of p a i r i n g i n the homologues c o n t a i n i n g the heterozygous i n v e r s i o n may reduce recom-b i n a t i o n and increase the a v a i l a b i l i t y of the enzyme(s) to other regions of the genome. But, i f increased recombination owing to an interchromosomal e f f e c t and r e p a i r o f r a d i a t i o n damage are common enzyme(s)-mediated s t e p s , there would be a negative c o r r e l a t i o n between induced exchange i n hetero-chromatin and an interchromosomal e f f e c t i n euchromatin. The present f i n d -ings t h a t increases owing to the DcxF i n v e r s i o n and r a d i a t i o n do not l i m i t each o t h e r , do not support t h a t premise. As explained f o r induced exchanges, the concomitant decreases i n chromosome i n t e r f e r e n c e and increases i n m u l t i p l e exchange frequency w i t h the interchromosomal e f f e c t could be explained by the occurrence of suc-cessiv e s i n g l e recombinations. A gonial exchange followed by a m e i o t i c exchange would produce an apparent double exchange chromosome ( W h i t t i n g h i l l , 1955; Suzuki e t a l . , 1966). The higher than expected recovery of those chromosomes would r e s u l t i n an apparent reduction i n i n t e r f e r e n c e . However, the lack of a c l u s t e r i n g phenomenon, although not c o n c l u s i v e , suggests t h a t the DcxF i n v e r s i o n does not have an e f f e c t on g o n i a l recombination. Ramel (1962) used c o e f f i c i e n t s . o f v a r i a t i o n to show that heterozygous i n v e r s i o n s do not produce a high crossover v a r i a b i l i t y i n females - expected i f recombination occurs g o n i a l l y . He a l s o i n t e r p r e t e d h i s r e s u l t s as i n d i c a t i o n s that the interchromosomal e f f e c t s are l i m i t e d to meiosis. I t i s apparent from Experiment 3 which u t i l i z e s the MS2^Q def-i c i e n c y , that the amount of heterochromatin i n chromosome 2_ a f f e c t s intrachromosomal recombination. The experiments by Yamamoto and Miklos (1978) which i n v e s t i g a t e d an intrachromosomal e f f e c t i n d i c a t e that d e l e t i o n s of the _X heterochromatin cause a reduction i n spontaneous recombination of the t e l o c e n t r i c X_ chromosome. The proximal euchromatic region i n t h e i r study showed more of a decrease than the d i s t a l one. The present work with the metacentric chromosome 2 gives evidence that the reduction i n recombination, which i s marked, i s l i m i t e d to the chrom-osome arm c o n t a i n i n g the heterochromatic d e l e t i o n . Removal of the r i g h t c e n t r i c heterochromatin may mean th a t the proximity of the remaining 2R euchromatin to the centromere subjects i t to a stronger "centromere e f f e c t " . The centromere e f f e c t i s based on observations by Beadle (1932) and Offerman and M u l l e r (1932) who found that recombination i n euchromatin i s reduced when i t i s placed c l o s e r to the centromere. Mather (1939) determined t h a t the reduction was dependent on the distance of the eu-chromatic region from the centromere. Heterochromatin could thus be a c t -ing as a passive spacer so t h a t euchromatin i s subjected to the i n h i b i t o r y e f f e c t s of the centromere on recombination. Other t h e r i e s suggest t h a t heterochromatin may act more d i r e c t l y . For example, S c h u l t z and R e d f i e l d (1951) and Suzuki (1962) demonstrated t h a t both the d i s t r i b u t i o n and the amount of heterochromatin can a l t e r 66 recombination. Since chromosome i n t e r f e r e n c e i s reduced i n the presence of the MS21Q d e f i c i e n c y , i t seems t h a t the degree of i n t e r f e r e n c e could be d i r e c t l y c o r r e l a t e d to the amount of c e n t r i c heterochromatin. Another explanation f o r the reduction of recombination i n the chromosome arm co n t a i n i n g the d e f i c i e n c y could be that there may, be a s e l e c t i v e l o s s of exchange homologues f o r the M - cn_ i n t e r v a l thereby g i v i n g spurious decreases i n the recombination frequency f o r that r e g i o n . The loss of those exchange chromosomes should lead to a lower frequency of SETs i n Experiment 3 than those o f the other experiments. The lack o f an apparent d i f f e r e n c e i n SET values argues against the " s e l e c t i v e l o s s " a l t e r n a t i v e . Recently, Yamamoto (1979) demonstrated that d e l e t i o n s of X_ chro-mosome heterochromatin increases the recombination frequency i n chromosome 3_. He suggests that an a l t e r a t i o n i n the amount of heterochromatin i n a chromosome would a f f e c t recombination i n the other chromosomes. In the present r e s u l t s , the 14% increase i n the recombination frequency of the b_ - Vt region i n the l e f t arm of chromosome 2 when the MS2^ d e f i c i e n c y i s present, suggests t h a t a heterochromatic d e f i c i e n c y can not only i n -crease recombination i n other chromosomes of the genome, but, when i t i s present on an arm of a metacentric chromosome, i t can increase recom-b i n a t i o n i n the other arm of the same chromosome. . P o s i t i v e chromosome i n t e r f e r e n c e i s a c h a r a c t e r i s t i c of r e c i p r o c a l exchange i n adjacent euchromatic i n t e r v a l s i n Drosophila. In i n t e r v a l s wherethe recombination frequency i s 10% or l e s s , m u l t i p l e exchanges are u s u a l l y not recovered (Dennell and Keppy, 1979). Exceptions to t h i s have i n v o l v e d some of the heterochromatic regions i n the genome. For ex-ample, negative chromosome i n t e r f e r e n c e (expressed as c o e f f i c i e n t s of coincidence) has been reported f o r m u l t i p l e exchanges i n v o l v i n g the centromeric region of chromosome 2 (Morgan ejt a\_., 1925; Green, 1975; S i n c l a i r , 1975). The present f i n d i n g s that negative i n t e r f e r e n c e i s not c o n s i s t e n t l y c h a r a c t e r i s t i c of the centromeric region of chro-mosome 2 i n the c o n t r o l s e r i e s i n d i c a t e s that v a r i a t i o n s i n i n t e r f e r -ence s i m i l a r to those known to occur f o r the euchromatic regions ( G r e l l , 1978) may e x i s t among the heterochromatic regions of the genome. The p r o p o s i t i o n by S i n c l a i r (1975) t h a t the regions of the genome where spontaneous recombination frequency i s low may be char-a c t e r i z e d by negative i n t e r f e r e n c e i s not v a l i d f o r the centromeric region of chromosome 2. Negative i n t e r f e r e n c e does occur f o r regions on opposite arms of chromosome 2. This observation supports those by Graubard (1934) which i n d i c a t e t h a t recombination i n c l o s e l y l i n k e d regions i n d i f f e r -ent chromosome arms i s not s u b j e c t to i n t e r f e r e n c e . Although there i s a low recovery of m u l t i p l e recombinant chrom-osomes, i t i s apparent that at doses of 2 and.3 krad i n t e r f e r e n c e does not decrease as the recombination frequencies i n c r e a s e . When recombin-a t i o n i s a l t e r e d with age (Rendel, 1957), heat ( G r e l l and Day, 1974), X-rays (Parker, 1948) and X^  chromosome aberrations. (Ramel, 1962), the changes i n recombination frequency and c o e f f i c i e n t s of coincidence are a l s o not p a r a l l e l and suggest t h a t they are independently c o n t r o l l e d ( G r e l l , 1978). Although the heterochromatic region i t s e l f i s not s u b j e c t to negative i n t e r f e r e n c e , the amount of heterochromatin present seems to play a r o l e i n the degree of i n t e r f e r e n c e e x h i b i t e d . I n t e r f e r e n c e i s lowered when the c e n t r i c heterochromatin of 2R i s absent owing to the MS2^ d e f i c i e n c y . The 14% i n c rease i n recombination frequency i n the opposite arm may be a consequence of the reduced i n t e r f e r e n c e . Despite the lowered i n t e r f e r e n c e , the recombination frequency i n the r i g h t arm i s reduced and the t e t r a d a n a l y s i s i n d i c a t e s there i s no f a c i l i t a t i o n of exchange. The recent i d e n t i f i c a t i o n of the l i g h t and r o l l e d genes i n the l e f t and r i g h t heterochromatin r e s p e c t i v e l y has permitted a study which measures and compares recombination frequencies i n heterochromatic and euchromatic i n t e r v a l s . 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APPENDIX 1 Crossover Data from Control and Treated Female Parents of Experiment 1 Treatment of Female Parent Parental Genotypes  b I t pk cn l i S i n g l e Crossover Progeny Region Genotype b - l t b r i I t pk cn l t - r l b Vt r l _ pk cn r l - p k b i t r l pk cn pk-cn b I t pk r l cn Double Crossover Progeny Regions Genotype b - l t , l t - r l b pk cn Vt _rj_ b-11, r l -pk b_ r j _ p_k cn_ Vt b-l t , p k - c n b r l cn i l £k 0 1879 2070 112 129 2 2 29 29 18 16 1 4 1 2 2 krad 1404 1779 109 114 6 8 23 25 17 11 3 krad 2320 2577 177 197 13 27 42 47 17 22 4 krad 620 831 71 55 3 14 20 26 5 9 Totals 4294 3501 5447 1659 78 APPENDIX 1 cont'd i Crossover Data from Control and Treated Female Parents of Experiment 2 Treatment of Female Parent 0 2 krad 3 krad 4 krad Parental Genotypes  b Ttp_k cn ,968 853 1367 1042 B U I 1 0 2 5 1 0 2 5 1 4 0 0 1 1 0 8  S i n g l e Crossover Progeny Region Genotype b - B l b B J _ r l 67 59 66 67 U p J < c n 68 53 58 74 B l - l t b r l 1 1 BJ_ l t£j<cn - - - 1 l t - r l b I t H 3 7 10 18 Blp_k cn 1 3 7 13 r l - p k b Vt 21 21 32 41 B_lrlp_k cn 11 12 28 33 pk-cn b Vt p_k 7 7 7 7 Bj_rl£D_ 7 1 0 1 0 1 2  Double Crossover Progeny Regions Genotype b - B l , l t - r l J i l l - - - 4 b - B l , r l - p k b BJ_ r j _ P_k cn 1 - 2 Vt 2 4 3 b-Bl,pk-cn b BJ_ r l cn 3 1 - -VtpJ<_ 1 - 2 -B l - l t , l t - r l b_p_k cn 1 B l - l t , r l - p k b_ r l p_k cn_ 1 - - -B l - l t , p k - c n B l V t p _ k 1 - - 1 l t - r l , p k - c n k l l l l H L " " 1 1 T r i p l e Crossover Progeny B l - l t , l t - r l , r l - p k b 1 - 1 T o t a l s 2186 2062 2993 2428 APPENDIX 1 cont'd Crossover Data from Control and Treated Female Parents of Experiment 3 Treatment of Female Parent 0 2 krad 3 krad 4 krad Parental Genotypes  b Vtp_k cn 2371 3029 2954 2570 M 2638 3054 3245 2760 S i n g l e Crossover Progeny Region Genotype b - l t b_M 154 213 206 292 V t j j k cn 222 157 211 250 lt-M b U M 3 3 6 11 p_k cn 1 6 8 10 M-pk b I t - - - 1 M_ p_k_ cn_ - - 1 2 pk-cn b Vt p_k 2 2 1 1 M cn - - 1 1 Double Crossover Progeny Regions Genotype b - l t , l t - M Vt M 1 b-lt,M-pk b M D j c ' c n - - 1 3 Vt - - - 1 b - l t , p k - c n U £ k 1 1 - 3 :Totals 5392 6465 6634 5906 80 APPENDIX 1 cont'd Crossover Data from Control and Treated Female Parents of Experiment 4 Parental Genotypes Treatment of b I t pk cn/BI rl;+/+ Female-Parent 0 2 krad 3 krad 4 krad b U j A c n 741 1191 785 359 B J _ r l _ 1394 1504 1661 538 Sin g l e Crossover Progeny Region Genotype b-Bl b B l r l 60 .76 54 31 . -": ,, Vt £k £n 71 61 38 20 B l - l t b _rj_ 2 4 - -Bj_V££kcn_ 4 l t - r l b U H 1 6 3 3 BJ_ pk cn - 2 5 3 r l - p k b Vt 16 27 25 15 B_lr l£j<cn 11 25 15 6 pk-cn b V t j j k 3 2 5 1 BJ_ r l _ cn 8 8 2 1 Double Crossover Progeny Regions Genotype b - B l , r l - p k ; b BJ_ H. £k_ cn - 1 1 II 1 b-Bl,pk-cn; b BJ_ H e n 1 , - 1 -lt£k - 2 Tot a l s 2313 2909 2595 977 81 APPENDIX 1 cont'd Crossover Data from Control and Treated Female Parents of Experiment 4 Parental Genotypes Treatment of b I t pk cn/BI rl;DcxF/+ Females 0 2 krad 3 krad 4 krad b U r j k c n 669 493 722 314 B J _ r l _ 983 1041 1095 693 Single Crossover Progeny Region Genotype b-Bl b B l _ r l 138 167 101 97 U j j k cn 130 97 75 61 B l - l t b r l 1 2 - -BJ_ Vt pJicn. 2 1 - -1 t - r l b l t r l 2 1 2 2 BJ_ £k cn - 3 4 5 r l - p k b Vt 33 38 43 29 B l l l p i c n 22 22 20 16 pk-cn b Vtj j k . 5 3 4 1 BJ_ r l cn 8 7 9 3 Double Crossover Progeny Regions Genotype b - B l , B l - l t ; b B l J l p_k cn 1 - - -b-Bl ,1 t - r l ; b . B l p_k_ cn 1 J i l l 1 b - B l , r l - p k ; b BJ_ H_ pj< cn 1 2 1 2 Vt 5 4 6 1 b-Bl,pk-cn;b BJ_ r]_ on 1 1 - 2 U'ojc 2 Bl-1 t,rl-pk;b_ _r]_ jjk c_n 1 - - -rl-pk,pk-cn b_ Vt cn_ 1 - -Totals 2005" 1882" 2080 - H28" 82 APPENDIX 1 cont'd Crossover Data from Control and Treated Female Parents of Experiment 5 1st Brood Treatment of b I t pk cn/BI r l ; +/+ Females 0 2 krad 4 krad 5 krad Parental Genotypes  b_Vt 1198 1402. 1018 972 BI r l 1062 1010 727 784 Region Genotype b-Bl . - i b BJ_ r l _ 66 47 39 43 l_t 92 83 42 56 B l - l t b r i 3 3 1 2 1 1 i i 6 4 4 2 l t - r l b Vt r l - - 5 8 BJ_ - 2 2 4 Double Crossover Progeny Regions Genotype b-B l , B l - I t r i - - - 1 b - B l , l t - r l b B_i - - - 1 I t r j _ - - 1 -T r i p l e Crossover Progeny b - B K B l - I t , l t - r l + - - 2 -T o t a l s 2427 2551 1841 1873 APPENDIX 1 cont'd (Crossover Data from Control and Treated Female Parents of Experiment 5 2 n d Brood Treatment of b I t pk cn/BI r l ; +/+ Females  0 2 krad 4 krad 5 krad p a r e n t a l Genotypes  b It. 1324 1251 1581 1020 B i l l 1 1 3 9 1 0 3 9 1 1 8 4 8 0 4  S i n g l e Crossover Progeny Region Genotype b-Bl b _ B _ l r l 37 35 73 51 Vt 46 44 96 101 B l - l t b r l 2 1 9 11 Bj_ V t 1 6 7 15 l t - r l b Vt r l - 4 3 12 B_l - 1 6 7 Double Crossover Progeny Regions Genotype b - B l , B l - l t b BJ_Vt 1 - - -r l - - " 1 b - B l , l t - r l b_BJ_ - - 1 H H . 1 T r i p l e Crossover Progeny b - B l , B l - l t , l t - r l + - 1 Tot a l s 2551 2382 2960 2022 APPENDIX! cont'd Crossover Data from Control and Treated Female Parents of Experiment 5 1 s t . Brood Treatment of b I t pk cn/BI rl;DcxF/+ Females  Parental Genotypes  0^  2 krad 4 krad 5 krad b Vt 587 942 336 316 B l _ r l 557 841 254 282 Si n g l e Crossover Progeny Region Genotype b-Bl b _ B J _ r l 84 75 33 37 Vt 105 109 40 45 B l - l t b r l 6 7 4 2 B_lVt 7 13 4 3 l t - r l b Vt r l - 1 3 2 BJ_ - 1 1 4 Double Crossover Progeny Regions Genotype b - B l , B l - l t b B _ l V t 1 b - B l , l t - r l Vt r l 1 - - 1 Bl>-4t,VUrV:. ... b . - 1 T r i p l e Crossover Progeny b - B l , B l - l t , l t - r l + - - 1 Tot a l s 1348" 199TT ~676~ "TUT APPENDIX 1 cont'd Crossover Data from Control and Treated Female Parents of Experiment 5 2nd Brood Treatment of b I t pk cn/BI rl;DcxF/+ Females 0 . 2 krad 4 krad 5 krad Parental Genotypes b Vt 747 779 532 414 B_l r l _ 737 768 410 342 S i n g l e Crossover Progeny Region Genotype b-Bl b BJ_ r j _ 86 70 63 52 Vt 95 71 75 74 B l - l t b rl_ 3 3 5 5 BJ_ Vt 1 8 5 13 l t - r l b_ Vt r l _ 1 2 2 4 B_I - - 3 6 Double Crossover Progeny Regions Genotype b - B l , B l - l t b B_l U - - 1 1 r i - - - 1 b - B l , l t - r l Vt r l - - - 1 T o t a l s 1670 1701 1096 913 APPENDIX 2 Chi Square Test of Control Data example of method used No. . of Progeny Expt.2 Expt.4 Expt.2 Expt.4 Total E (O-E)^E E IO-E) £ ; Parentals 1993 2135 4128 2005 .7 0 .08 2122 .3 0 .08 S i n g l e Crossovers I n t e r v a l s b-Bl 135 131 266 129 .2 0, .26 136 .8 0, .26 B l - l t 1 6 7 3 .4 1, .69 3 .6 0, .67 1 t - r l 4 1 5 2 .4 1, .07 2 .6 0, .98 r l -pk 32 27 59 28, .7 0, .38 30 .3 0, .37 pk-cn 14 11 25 12, .1 0. .30 12 .9 0. .28 Double Crossovers I n t e r v a l s b - B l , r l - p k - 1 1 0. .5 0. .49 0, .5 0. .46 b-Bl,pk-cn 4 1 5 2, .4 1. .01 2, .6 0. .96 B l - l t , r l - p k 1 - 1 0. .5 0. .53 0. .5 0. .51 B l - l t , p k - c n 1 - 1 0. .5 0. ,53 0. .5 0. ,51 T r i p l e Crossovers B l - l t , l t - r l , r l - p k 1 - 1 0. ,5 0. 53 0. .5 0. 51 Total 2186 2313 4499 6. 87 5. 59 x 2 = 12.46 df - (2-1)(11-1) = 10 87 APPENDIX 2 cont'd Chi Square Test of Control Data Experiments Compared df X 2 1 and 2 8 10:. 56 1 and 4(+/+) 7 4.93 1 and 5J+/+) 3 6.79 2 and 4(+/+) 10 12.46 2 and 5J+/+) 5 19.82* I,2_,3, and 4(+/+) 6 10.96 4(+/+) and 5(+/+) 5 5.96 4(DcxF/+) and 5(DcxF/+) 5 9.52 * s i g n i f i c a n t a t o c = 0.05 APPENDIX 3 Ratio of Radiation Induced Crossover Values (P^) to the Control Values (PQ) Treatment of Female Parent Conf. Conf. Conf. Experiment I n t e r v a l 2 krad Lim. 3 krad Lim. 4 krad Lim. i b - l t i . i l ' l 1.2 \ A Q I A {•{ l t - r l 3.6 6 > 6 16.7 9 Q 24.4 r l - p k 1.0 J;* 1.2 2.1 ™ pk-cn i.o J;66 0.9 J;2 i.o l A 7 b-Bl 0.9 J;) 0.7 g-j . 1.0 J;| B l - l t 0.3 2-6 o.2 1.8 Q J 3.1 l t - r l 2.1 g.2 2 > 7 7.5 6 > g 17,1 r l - p k 1.1 J ; 8 1.4 2-1 2.1 \-\ 1 9 1 5 1 9 pk-cn 1.0 lQl 0.8 1.0 lQ-l 3 b - l t 0.8 o.9 J;° 1.3 1t-M 2.0 *•* 3.0 5.3 16.1 M-pk - ^ 0 0 pk-cn 0.8 0 - 5 3.0 1 5 6.2 4 + / + b-Bl 0.8 J;) 0.6 0.9 B l - l t 0.5 2.0 0.2 l t - r l 6 5 5 8 ' 3 < 7 2 6 4 - 8 14 2 1 3 2 " 4 1 1 1 °- 0.7 • 0.8 1.6 r l - p k 1.5 \ A 1.3 2.1 1.8 3.2 pk-cn 0.7 1'73 0.6 0.4 J ; 8 89 APPENDIX 3 cont'd Ratio of Radiation Induced Crossover Values ( P j ) to the Control Values(Pg.) Treatment of Female Parent Experiment I n t e r v a l Conf. 2 krad Lim. Conf. 3 krad Lim. Conf. 4 krad Lim. 4 DcxF/+ b-Bl 1.0 B l - l t 0.8 l t - r l 2.1 r l - p k 1.2 pk-cn 0.7 1.2 0.9 3.7 0.2 12.1 0.4 1.6 0.8 1.6 0.3 0.6 2.9 1.1 0.8 0.8 0.5 14.7 0.6 1.5 0.8 1.6 0.4 1.0 7.3 1.3 0.6 1.2 0.8 34.9 1.5 1.9 0.9 1.6 0.2 4 krad 5 krad 5 +/+ b-Bl B l - l t l t - r l 5 DcxF/+ b-Bl B l - l t l t - r l 0.9 1.2 8.1 0.7 1.5 2.1 1.0 0.7 2.5 0.6 67.9 1.0 0.8 0.6 2.6 0.8 10.5 0.4 1.1 20.9 1, 0, 3, 0. 162, 1.0 1.9 8.6 1.2 0.8 3.6 1.0 38.6 1.9 1.3 3.2 41.1 1.1 2.6 16.8 1.6 1.1 6.1 1.6 314.5 5.4 1.2 0.9 4.8 1.4 71.9. 3.8 90 APPENDIX -4 Values presented in Figure 10. Recombination Frequencies in Proximal Intervals of Chromosome 2 Treatment of Female Parent Interval 0 2 krad 3 krad 4 krad b - l t mean of Expts. 1,2,4. 6.12 5.71 5.00 6.46 Expt. 3. 6.99 5.74 6.30 9.31 1t-pk mean of Expts. 1,2,4. 1.52 2.08 2.39 3.88 Expt. 3. 0.07 0.14 0.24 0.49 pk-cn mean of Expts. 1,2,4 0.74 0.68 0.57 0.64 Expt. 3 0.06 0.05 0.03 0.09 91 APPENDIX 5 Values presented i n Figures 11-14. Ratio of Radiation Induced Crossover Values with In(3LR)DcxF ( P A ) to the Values without the Inversion (Pg) Figure 11 Treatment of Female Parent I n t e r v a l 0 2 krad 3 krad 4 krad b-Bl 2.41 3.01 2.43 2.57 B l - l t 0.78 1.15 _ _ l t - r l 0.40 0.77 0.94 1.19 r l -pk 2.51 1.92 2.07 1.82 pk-cn 1.54 1.54 2.03 2.39 Figure 12 0 2 krad 4 krad 5 krad b-Bl 2.53 2.07 2.27 2.00 B l - l t 2.31 2.85 2.36 1.87 l t - r l 3.30 0.83 1.35 1.35 Figure 13 b-Bl 2.18 1.82 2.40 2.17 B l - l t 2.80 3.85 3.50 2.64 l t - r l oo 1.94 1.36 1.42 Figure 14 b-Bl 3.26 2.47 2.21 1.87 B l - l t 1.52 1.93 1.86 1.64 l t - r l 1.54 0.47 1.35 1.28 

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