San Fernando Valley State College EFFECTS OF THE CENTROMERE AND HETEROCHROMATIN ON RECOMBINATION IN DROSOPHILA MELANOGASTER A thesis submitted in partial satisfaction of the requirements for the degree of Master of Science in Biology by Joan Lily Bartholomew August, 1969 The thesis of Joan Lily Bartholomew is approved: San Fernando Valley State College August, 1969 ii. ACKN OWLEDGJ:.:lENT 8 For the quality of his advice and direction and for his unfailing forbearance, grateful to Dr. I am George Lefevre, iii Jr. TABLE OF CONTENTS Page 'LIST OF TABlES ' ABSTRACT • i • • • • • • • • • • • • • • • • v • • • • • vi • • • • • • 1 • • • • • • • 5 • • • • • • • • • • • • • • • • • • • • • • • • Chapter I. INTRODUCTION. II. REVIEW OF THE LITERATURE III. IV. v. MATERIALS AND METHODS • • • • • • • • • 9 RESULTS • • • • • • • • • 13 • • • • • • • • • 18 • • • 28 • • DISCUSSION. • • Recombination homozygotes Recombination homozygotes Recombination zygotes Recombination zygotes BIBLIOGRAPHY • • • • • • • • • • • in inversion sc8 in inversion rstJ in wVco heteroVco homoin 'tf.__ • • iv • • • • • • • • LIST OF TABLES Table 1 2 3 Page Crossing Over in Homozygous sc8 Inversion Crossing Over in Homozygous rstJ Inversion The Effect of wVco on Recombination in the Third Chromosome • • • • • • • • • • • v • • • • • • • • • • • • • 14 • • • • 15 • • • • 17 ('"··--·-----···· ' , I ABSTRACT. EFFECTS OF THE CENTROMERE AND HETEROCHROMATIN ON RECOMBINATION IN DROSOPHILA MELANOGASTER by Joan Lily Bartholomew Master of Science in Biology August, 1969 Effects of the centric heterochromatin and the centromere on recombination frequency were investigated with the use of two paracentric inversions of the X chromosome and a heterochromatic, pericentric inversion of the third chromosome. The results clearly support the hypothesis that crossing over is inhibited in regions immediately adjacent to the centromere; a decrease from standard values is obtained when regions are placed, by inversion, nearer the centromere than is normally the case, and an increase when regions are moved away from the centromere. The influence of centric heterochromatin is not so easily assessed. Relative to standard values in normal chromosomes, increased recombination was obtained between :markers spanning distally inserted centric heterochromatin with the two X chromosome inversions. Although these data suggest that crossing over occurs more readily once the heterochromatin is removed from centromeric .... ... . vi ... ...·-·--·-·-··-----·-··-·--·--..- . . .. inhihition,.that these values might. result from. stimula tion of euchromatic exchange can not be excluded, Interpretation of the data obtained with the third chromosome inversion presents similar problems, Increased recombination in the interval that includes the rearranged centric heterochromatin was observed, but it was not possible to determine whether the exchanges actually occurred in the heterochromatin or in the adjacent euchromatic regions. i Further work utilizing heterochro- matically located loci is required before the problem can be fully resolved. vii CHAPTER I INTRODUCTION Early prophase chromosomes characteristically 'possess extensive regions in the vicinity of the centromere having a dense compact configuration, evidenced b� ·their stainability (Heitz, 1928) . The term heterochromatin is commonly used to distinguish these regions from the remaining euchromatin. Cooper (1959) persuasively argued that this terminology is inappropriate since it implies a condition of permanent structural difference between the two regions, rather than an "out-of-phaseness" of temporal events. Although his argument has merit, the terms heterochromatin and euchromatin remain useful in referring to specific chromosomal regions; they will be so used below, implying, however, only that the regions are cytologically distinguishable. In addition to the �ifference in configuration between distal and proximal areas, comparison of genetic and cytological maps, necessarily constructed from independent data, suggests an inhibition of crossing over in the neighborhood of the centromere (Dobzhansky, 1930, 1930a, 1932). There is little doubt that genes in close proximity to the centromere exhibit remarkably little crossing over. Beadle (1932) w�s among the first to 2. report on this effect, basing his conclusions on crossover data for genes of the_right arm of chromosome 3 ;that had been translocated to the fourth chromosome. :Studies of the recombinational prop,rties of various ,other chromosomal rearrangements hav� corroborated Beadle's conclusions (Offerman and Mulller, 1932; Gruneberg, il935; Mather, 1939; Thompson, 1963), ��d the concept of an inhibitory "centromere effect" is now widely accepted. The question of an effect of heterochromatin itself 1on crossing over still arouses controversy: is the absence iof crossing over in heterochromatin a result of its i iproximity to the centromere, or does its configuration at ithe time of synapsis preclude exchange? Offerman and JMuller (1932), after study of the scute-8 (sc8) and !de1ta-49 (dl-49) inversions, as we11 as several X-4 trans :locations, concluded that proximity to the centromere I I .controls recombination frequencies in euchromatin but not I 1 ! 11n the heterochromatin, this material being refractory ) 1to crossing over wherever located. Later, both Grlineberg • ! (1935) and Mather (1939) presented data, obtained from ;studies of inversion roughest-3 (rst3 ) homozygotes, that ,they felt could best be accounted for by assuming an 'increased level of crossing over in the distally displaced heterochromatin. An objection to this interpretation, however, was raised by Baker (1958) on the basis of his study of a Drosophila virilis stock having a 3-5 reciprocal translocation. He suggested that increased crossing over 3 ,in euchromatin adjacent to displaced heterochromatin was .an alternative and more plausible explanation for the re sults of Gruneberg and Mather. Support for this hypothesis came from Braver (1957) , who observed a significant in crease in recombination in the uninverted yellow (y) to :white (�) interval in rstJ homozygotes. Schultz and 'Redfield (1951) had earlier speculated on the importance :of heterochromatic regions on chromosomal exchange. They !postulated that factors influencing "the polarized pattern 'of pairing at meiosis" could affect crossing over, ,heterochromatic association being one of these factors. The availability of a rather different kind of chromosomal rearrangement from those previously analyzed ! ' makes appropriate a further investigation of the problem. This rearrangement, duplication white-variegated-cobbled (Dp wVco) , consists of a pericentric inversion in the :third chromosome whose breakpoints are essentially confined to the heterochromatin. In addition, a short euchromatic section of the X-chromosome containing the �white locus is inte�polated between the breaks in chromosome .J. · Despite this complication, the inversion basically involves only the heterochromatic region of the third .chromosome. The euchromatic loci in the third chromosome, whose crossover intervals were tested, are not moved from itheir normal locations. Their linear sequence, physical distance from one another (except in the case of the two spanning the centric region) , and distance from chromosome 4 .ends remain unchanged. Only their relative proximities to the centromeres have been altered. This paper reports on the results of a study of this chromosome, and, in addition, on a reinvestigation of recombination in the i rst3 and sc8 inversions, all carried out under identical conditions to permit valid comparison. CHAPTER II REVIEW OF THE LITERATURE Investigations of the recombinational properties of centric regions of Drosophila melanogaster have continued since Dobzhansky, in a series of papers in 19.30 and 19.32, 'demonstrated a discrepancy between the genetic and cyto 'logical maps of this organism. The cytological maps were ,constructed using a variety of deficiencies and transloca i :tions, the genetic maps from recombination data. Although 'the two methods resulted in the same linear sequences for the loci studied, the relative distances separating them :as determined by cytological methods did not correlate well with crossover data. Intervals in the vicinity of the centromere were shorter genetically than intervals of comparable cytological length in distal regions. ,Earlier studies by Heitz (1928) had shown that prophase ! chromosomes exhibit extensive centric regions that stain more deeply than distal areas for which he proposed the term heterochromatic solely on the basis of its staining characteristics. Dobzhansky (19.32) pointed out that ,the discrepancies between genetic and cytological maps :could be accounted for by assuming "that crossing over is not equally frequent in the different regions of the chromosome." 6 Dobzhansky's findings raised the following question: was the reduced frequency of recombinations near the centromere due to the heterochromatic material .or to an inhibitory effect of the centromere? � � Beadle ·(1932) investigated recombination frequencies in a stock 'having a reciprocal translocation between the third and fourth chromosomes. Because of the extremely small size of the fourth chromosome, this rearrangement necessarily positioned the genes of the translocated right arm of chromosome 3 much closer to a centromere than had been 'the case in its normal configuration. A significant reduction in recombination frequency in the curlec to stripe and stripe to ebony-sooty intervals led Beadle to conclude that crossing over was inhibited as a result of proximity to the centromere. Offerman and Muller (1932), after investigating recombination in several translocations and two X-chromosome inversions, also concluded ;that euchromatic crossing over was inhibited by proximity to the centromere. In addition, they reported that low crossing over was characteristic of heterochromatic .regions regardless of their positions relative to the centromere. A more recent consideration of spindle fibre inhibition by Thompson (1963). using a 3-4 translocation heterozygote, led him to suggest that following synapsis, mutual repulsion of homologous centromeres just prior to ·the time of exchange results in low recombination . frequencies in adjacent regions. 7 These investigations, together with similar work by Grtmeberg (1935) and Mather (1939), served to establish :the concept of an inhibitory effect of the centromere. However, the possibility of an additional heterochromatic ,effect remained a source of controversy. Both Mather :and Gr8neberg had utilized the roughest-3 inversion of the X-chromosome in their recombination studies. In this inversion the right break occurs in the centric hetero :chromatic material. This results in the intercalation of icentric heterochromatin between euchromatic material at :a distal location just to the right of the white locus. !Increased crossing over between markers spanning th is now distally located heterochromatin was observed by oth workers, who attributed the result to exchange taking place in the heterochromatin at a much higher ,frequency than occurs in the uninverted chromosome. ! This interpretation, however, was challenged by :Baker (1958). A stock of Drosophila virilis having a 1reciprocal translocation between chromosomes 3 and 5 allowed him to investigate crossing over in heterochromatin :far removed from the centromere. · Observing little or no lexchange in this material, he suggested that the increased recombination noted by both Gruneberg and Mather could have been due to a stimulation of crossing over in the euchromatic regions adjacent to the inverted heterochromatin. Earlier work by Braver (1956, 1957) provided data which supported Baker's proposal. Using inversion roughest-3 8 homozygotes, Braver obtained recombination frequencies as high as 6.6% in the distal uninverted yellow-white interval adjacent to the inverted heterochromatin. standard value is 1. 5%. ) (The These findings, however, did not result in a final settlement of the problem since Schalet (1967) , using homozygotes for the scute-8 'inversion in which the heterochromatically located bobbed : locus is inverted, reported the occurrence of crossing �ver within the bobbed locus. CHAPTER III MATERIALS AND METHODS The stocks used in the course of this investigation are described below, together with the markers used in conjunction with each. More complete descriptions of the 'markers will be found in both Bridges and Brehme (1944) ;and Lindsley and Grell (1968). Inversion (1) roughest-) (rst3): an X-ray induced inversion of the X chromosome discovered by Gruneberg (1935). Cytological analysis by Emmens (1937) placed the left break after salivary chromosome band JCJ (Bridges� 1938 revised map) and the right break in the centric heterochromatin, probably in 20B. The bobbed (bb, 1-66.0) locus was orginally thought to be included in the inversion, but genetic analysis by Lindsley (1954) showed this not to be the case. One rstJ stock used carried the markers yellow (�, 1-0.0); white60i29(w60j.29, 1-1. 5) and carnation (�, 1-62.5); the other was unmarked. Inversion (1) scute-8 (sc8): an X-ray induced inversion of the X chromosome found by Noujdin (Bridges and Brehme, 1944). Genetic analysis showed the left break to be between achaete (�, 1-0.0+) 9 10 and sc, and the right break between bb and the spindle attachment. Muller and Prokofyeva (Bridges and Brehme, 1944) placed the left salivary· chromosome break between 1B2 and 1B3 and the right break to the right of 20B. One sc8 stock used carried yellow3ld (y3ld) and white-apricot (wa); the other �' vermilion (�, 1-33.0), and cross- veinless (QYJ 1-13.7). White-Variegated cobbled (w Vco): a pericentric inversion in the third chromosome with breaks at 77D4 and 81A, found by Clausen and analyzed by Schultz (Bridges and Brehme, 1944). In addition, a short piece of the X chromosome (2Cl-3C4) is inserted at 81A. Two marked wVco stocks were prepared. One carried the left arm markers hairy (h, 3-26. 5), thread (th, 3-43.2), scarlet (st, 3-44.0); the other the right arm markers pink peach (�, 3-48.0), curled (cu, 3-50. 0), stripe (g, 3-62. 0). . . . 258-45 (w258-45): Whlte Deflclency an X-ray induced deficiency of the X-chromosome originally described as deficient solely for 3Cl (Sutton, cited by Bridges and Brehme, 1944), but more recently described by Lefevre (1968) as deficient for 3B3 through 3C2. In addition, the stock maintained in this laboratory contains a second - �· 7 11 deficiency of unknown origin which extends approximately from 3A7 up to and including 3Bl (Lefevre, unpublished) . This doubly deficient chromosome was illustrated by Lefevre and Wilkins (1966) . This stock made it possible to obtain fertile females homozygous for wVco. Initial studies revealed that females (and males) having nondeficient X chromosomes Yeo ln homozygous cond·t· b ut carrylng � l lOn, though · · · viable, are not fertile. The following procedure was used in all experiments from which quantitative data on recombination were obtained. Virgin females were collected shortly after emergence and aged for 3 days. They were then mass-cultured with an excess of appropriate males for 48 hours, after which the males were discarded, The females were then placed individually in vials· and subcultured three times at two day intervals, being discarded on the 6th day. Counting and scoring of the progeny in each vial was discontinued after 17 days in order to avoid including F2 progeny in the counts. Except for short periods during which flies were subcultured or progeny examined, the culture vials, including those from which parental females were obtained, were maintained in incubators at 25°C. The breakpoints of the three inversions utilized in the course of this work are diagrammatically represented :below at Figure 1. sc8 rst3 X '<' l_ -� SC H --�----4i==------�---- -. _______} j �-----bb car v cv III R -- ----·------r--sr III ---- Figure l. sc8, I n(l) rst3 and In(J) vJVco Diagrammatic representation of In(l) __, - �� CHAPTER. IV RESULTS Recombination in homozygous X chromosome inversions. Recombination between markers spanning distally inverted centric heterochromatin, and in adjacent euchromatic regions, was measured in two X chromosome inversion homozygotes, sc8 and rstJ. As shown in Table 1, In (l)sc8 exhibited reduced crossing over in the cv-wa interval, which occupies a much more proximal position than it does in the normal chromosome. Conversely, recombination between y and � was higher than estimates based on the behavior of the uninverted chromosome would predict; the y-�interval, however, includes the distally inverted heterochromatin. The other two intervals tested (�-y; y-£Y) did not differ significantly from standard values. None of the values differed noticeably from those reported by Mather (1939). As seen in Table 2, In (l)rstJ exhibited higher recombination frequencies in both the y-� and �-£££ (which includes the inverted heterochromatin) intervals :than occur in the normal chromosomes. Although the present results are comparable with those obtained by Mather, crossing over between y and car was significantly lJ Table 1 Crossing Over in Homozygous sc8 Inversion y-ear 3.5+* 2 X =19,45 P<<.Ol 5. 8 (188) �· 2 =0,14 .7<P<. 8 v-cv 19. 3 2 X ::::2,41 .l<P<.2 cv-wa 12. 2 2 X ::::26.20 P<<.Ol 28.9 (937) 20, 2 (653) 6.9 (107) n=l559'H'" car-v 2 8.5 ----- --------------------·----------------------------------------------------�---------- Standard values X X 2=4. 2 3 ,02<P<.05 X 4.99 n=l424 18.24 n=4046 7.09 n::.4046 2:::1,30 .2<P<o3 Inversion homozygotes - Mather (1939) * ** X / "'-- ;/ 2=0,09 o7<P<. 8 This value is used as a standard since it represents the ----car-bb interval in the normal chromosome. Since an appropr Aately marked backcross chromosome was not available, recombination between cv and w could be detected only in male progeny. - - x2 and P values lie between entries the significance of whose difference they test. f-1 ·� 15 Table 2 Crossing Over in Homozygous rst3 Inversion y-w Standard values 2=38.3 p <<. 01 w-ear 3.7+* . 2 X =288. 3 P<<. Ol y-ear 5.2 (by addition) 2 X =28. 56 P<<O. Ol 2. 8 (270) n=9699 12.3 (707) n=5759** 14.6 (by addition) 2 X =1. 60 0. 3>P> 0.2 1.5 X y w6o:r"" 29rst3car + + rst) + Inversion homozygotes - Mather (1939) Inversion homozygotes '- Braver (1957) . 15.6 (n=l999) x2=12.6 P<<O.Ol 5.1 (averaged) 13.4 (averaged) Inversion homozygotes - Gruneberg (1935) * This estimated value includes the 0.2% between � and rst plus 3. 5% between � and the right break is to the left of bb. 18.5 (by addition) 18.6 (n=7296) recombination bb, although ** Since the presence of -w masks the expression of car, recombination between w and car could be detected only-in w+ progeny. x2 and P values lie between entries the significance of whose difference they test. 16 .lower in �his �xperiment thari in those reported by Braver (1957) and Gruneberg (1935) . It is perhaps appropriate ;to note that cytological examination of the salivary 'gland chromosomes of both rst3 stocks used in this cross revealed no autosomal inversions which might have produced ; an interchromosomal stimulati6n of crossing over. Recombination in wVco. The effects of the presence of the wvco inversion on recombination in the third chromosome are recorded in Table 3. Control values are reported for two separate crosses, one having all the markers in coupling, the second with the left arm markers in repulsion to those of the right arm. In all cases the values obtained were lower than standard values. Nevertheless, the presence of wVco appears to stimulate recombination in both the st-� and �-£Q intervals; whereas, the intervals tested in the left arm are unaffected or, possibly, slightly depressed. The stimulatory effect is noticeable in the inversion heterozygo.tes, but is quite pronounced in the homozygotes. Table 3 The Effect of wVco on Recombination in the Third Chromosome th-st h-th Standard values Controls !Lth �t J.2P C!:-! + + + + + n=4877 h th st + + �u n=3782 Average rrr;vers-ion ·heterozygotes i y_ w258 -45 h th_.§J 12p__£Q + + +WVc o+ + + + n=2 883 i .: Inversion homoz.ygotes + . w258-4·5 -h th st wVco v- + p co �758:::-r r:s- + + + w p cu y w n=l627 - � � st-pP ------ -pP-cu 16.7 0. 8 4.0 2, 0 14. 2 (694) 0.3 (17) 0.8 (37) l. 1 (54) 15. 8 (598) 0.5 (18) 1. 6 (59) 1.0 (37) 14. 9 0. 4 1.1 1.1 0. 03 (1) l. 6 (46) 2,4 (69) 0 9. 0 (146) 7.8 (127) ·-- · ·· 9. 8 (281) - - � � 10,0 (163) }-J "-'1 CHAPTER V DISCUSSION Recombination in Inversion sc8 Homozygotes Support for the hypothesis that recombination is 'inhibited by the centromere, first proposed by Beadle in 19.32, is provided by the results obtained for the £:;L-Wa ,. terva1 1n the ,1n . . 8 1nvers lOne .§.2_ . The reduction in exchange 1frequency between the two markers, as compared with standard values, is highly significant and is most reasoni ably explained by the assumption of a centromere effect. 1As can be seen in Table 1, the present results are in good agreement with those of Mather (19.39) , who also [ascribed thi� diminution to a centromere effect. The results obtained for the �-�and y-£:;L intervals also compare well with those reported by Mather. The region between� and�' in the inverted chromosome, includes virtually all of the proximal heterochromatin, :and shows significantly more recombination than do the comparable chromosomal regions in the normal configuration. In view of the inhibitory influence of the centromere on crossing over between £::!.. and wa in their new proximal ,Positions, it seems reasonable to attribute increased recombination in the�-� interval to a release from centromere inhibition and to conclude that the euchromatic 18 19 portion of this interval, at least, crosses over more freely than it do.es in the normal chromosome. Recombination in Inversion rstJ Homozygotes Since only distal intervals were considered in the ;investigation of crossing over in rstJ, inhibition in the 1vicinity of the centromere was not detectable. However, .an increase over standard values was observed in both of .the distal intervals (�-� and �-�) investigated. Although no real difference exists between the present results and those of Mather, they are significantly lower than those obtained by Braver (1957) and Gruneberg (1935) . The reason for this difference can not be ascertained beyond doubt, but several possible explanations can be suggested. A difference in the environmental conditions is one of these; temperature, for example, has been shown by several investigators, including Mather, to be a factor governing recombination frequency, particularly in heterochromatic regions. Also, Schultz and Redfield 1 (1951) have shown that the age of the mother is a factor influencing exchange frequency. Finally, there is the possibility of undetected autosomal rearrangements stimulating crossing over in the X chromosome. With this latter possibility in mind, the salivary gland chromosomes of the stocks used in this experiment were examined, but no abnormalities were found. The high frequency of exchange observed between � 20 and w in the uninverted euchromatic tip provides support for the proposal of Baker (1958) that stimulation of crossing over in euchromatin adjacent to inverted heterochromatin was a plausible interpretation of the results .obtained by Mather and Gruneberg. Nevertheless, the surprisingly high crossover values observed for the �-� �nterval, which includes the inverted proximal hetero �hromatin, is difficult to explain on the basis of euchromatic exchange alone. Bridges (19J7) introduced a method for quantifying regional differences in recombination along a chromosome by finding the ratio of crossover frequency to salivary hromosome length. Lefevre and Moore (1968) utilized a modification of this scheme, calculating coefficients of �r.ossing over on the basis of crossover units per salivary gland chromosome band. This latter method is useful in �etermining the extent of effects resulting from. chromo s' omal rearrangements such as the inversions used in this work. When the normal X chromosome is considered as a �hole, a value of 0.07 crossover units per band is obtained, The euchromatic region present between £§£ and the centric heterochromatin gives a slightly lower value of 0. 06, possibly as a result of centromeric influence. 12. J% recombination between � and � If the in the rst 3 inver sion is attributed solely to euchromatic crossing over, a coefficient of 0.23 crossover units per band would be 21 'require?; this value would be four times higher than normal for the interval. Thus, it seems probable that .some crossing over is occurring within the inverted heterochromatin itself, even though virtually none occurs .in the proximal heterochromatin of normal chromosomes. Evidence that heterochromatin is actually capable of recombination was provided by Schalet (1967), who reported on the occurrence of crossing over within the bb locus . sc8 homozygotes. ln A second problem arises, however, when one compares the rst3 results with those with sc8• Essentially the .same ammount of euchromatin is present in the y-� interval of sc8 as in the �-£§£ interval of rst3. In the latter case, appreciably less proximal heterochromatin is intercalated qetween the two markers, yet exchange occurs much more frequently, 12. 3% versus 5. 8%. Various explanations for this apparent inconsistency might be suggested. One feature differentiating the two .inversions is the proximity of the left break to the :chromosome tip. If the telomere acts as a suppressor :of crossing over, in a manner analogous to that of the centromere, there should be less recombination in the �-£§£region of sc8 than in the �-� interval of rst3. The ';L-� results in rst3 support this argument since, even with the increase obtained, the crossover coefficient for this region remains at a low value of 0.02 units per :band. Nevertheless, much of this is mere speculation and a model_might equally well be proposed in which all of the increased recombination is due solely to exchange in the euchromatic region, with the amount of stimulation being inversely proportional to the amount of inter .calated heterochromatin. Some support for this latter ,idea may be obtained from a consideration of recombination :in the standard chromosome in regions such as JC, where ithere is evidence for a relatively small amount of intercalary heterochromatin (Kaufmann, 1946) and the 'coefficient of crossing over is exceptionally high. Recombination in wVco Heterozygotes Analysis of the results obtained with the wVco inversion is complicated by the fact that the control values are low as compared with standard map distances. Probably, this is a consequence of the experimental conditions, since cytological examination revealed no chromosomal abnormalities. However, all crosses were carried out under the same conditions and, thus, comparison of control and experimental data should be valid. In the wvco heterozygotes, recombination in the left 1arm appears to be inhibited. There are two possible 1explanations for this effect. Reduced recombination within, and adjacent to, heterozygous euchromatic inver ·Sions is an expected and frequently observed effect. Thus the decrease for the h-th and th-st intervals may :be due to the wVco inversion extending somewhat into the 23 euchromatin of the left arm at ??D. However, this in', version also results in bringing the centromere into .closer proximity to the left arm markers than is true in the normal configuration, so that the reduced crossing :over might be attributed to an inhibitory centromere effect. The inversion also affected recombination between ·� and £.Q in the right arm, where the heterozygote showed a doubling in exchange frequency as compared with the ,controls. An increase was also observed for the st-� 'interval, which spans the centromere and centric heterochromatin. Interpretation of these latter two results is difficult. Thompson (1963) has proposed that pairing rof the centromeres at synapsis, and repulsion prior ! :to the time of exchange, could be responsible for the inhibitory effect of these structures. His model suggests a possible explanation for the increased recom1bination in the centric region and right arm of wvco !heterozygotes, since synapsis of homologous centromeres could conceivably be interrupted as a consequence of the inversion. In this case, however, one could not 'attribute the decrease in the left arm to centromeric inhibition. The data for all four of the tested intervals rcan be correlated by assuming an intrachromosomal effect: inhibition of crossing over in the left arm resulting in a stimulation of exchange in the right arm. 24 Vc o Rec ombination in Y:!_ Homozygotes The dec rease in rec ombination in the left arm 'intervals of the homozygote was no greater than that ;observed in the heterozygote. However� the dec rease in the homozygote c an not be a c onsequenc e of struc tural dissimilarity between the two c hromosomes. The c entro- mere effec t is now a muc h more probable explanation. The results for the right arm intervals are extremely inter:esting. A c onsiderable inc rease over both c ontrol and standard values was observed in both the �-� and st-� :intervals. Sinc e the c hromatic material between not been altered in any way, � and £Q has the inc rease must result from the influenc e of other c hromosomal regions; but is it due to a reduc tion in c entromeric inhibition or to a stimulation of euc hromatic exc hange by inverted hetero 'c hromatin? Although it would be c onsistent to interpret this as heteroc hromatic stimulation c omparable to that of the �-� interval of inversion rst3, a few simple c alc ulations suggest that in this c ase a large part of !the inc rease is probably due to a reduc tion in the inhi'bitory influenc e of the c entromere. c hromosome 3 c ontains :is about 58.4 1178 salivary c hromosome bands and map units long, ation c oeffic ient of 0.05 The right arm of whic h gives a mean rec ombin map units per salivary band ,for the right arm as a whole. While �(48.0) and £..1!(50.0) 25 have no� been precisely located cytologically, separated by approximately 150 Thus, 0.01 a low coefficient of they are salivary chromosome bands. map units per band is characteristic for this region in the standard chromosome. The recombination obtained for the interval in ?.8% Vco homozygous w brings the coefficient back up to the normal average of 0.05, which suggests that a release 'from inhibition by the increased distance from the cen :tromere was responsible for the change. 'course, All of this, of depends on the assumption that the calculated oefficients represent an index of centromeric inhibition. The st-� interval also showed a marked increase crossing over. Included in this interval, the centromere and centric heterochromatin, !euchromatic portion of the X chromosome, bute somewhat to the increase. ·however, only about It is worth noting, far from the this region represents a genetic length of 1.0 but in its new, less. is a short which may contri that even in its n6rmal location, centromere, in addition Thus, crossover units (Lefevre and Moore, more proximal po�ition, 196a), probably much we must explain at least a doubling of ,recombination frequency over standard values in this ;interval, ; perhaps more if the control figures presented here are representative of the true exchange frequency .under the applied experimental conditions. In the left :arm of the inverted chromosome there is a short ' heterochromatic region followed by a euchromatic region 26 of approximately :the st locus. 280 While this euchromatic region, .standard conditions, ,between st and bands between the centromere and � contributes 3.7 of the (Lindsley and Grell, under 4. 0 1968), map units it appears Vco most probable that in the w inversion the closer proximity of the centromere would act to reduce exchange • .The observed reduction in the more distal left arm inter ,vals supports this contention. This leaves the chromatin :present between the centromere and the right arm, � locus, in the as the most likely area showing increased crossing over. Unfortunately it i's not possible to i idifferentiate between euchromatic and heterochromatic . ; exchange in the system under discussion, can not say, with certainty, and so we which of the two chromosomal regions is responsible for the increase. doubt that There is no euchromatic bands between the hetero 170 chromatic junction and the � locus could accommodate 1this amount of recombination without displaying any unusual characteristics. Even a full 8% in this interval alone, would give just units per band, which as we have seen, recombination, 0.05 crossover is typical of JR ··as a whole. In conclusion, the data presented here do not fully resolve the problem of whether heterochromatin is in its�lf refractory to crossing over. by inversion, In regions where, it has been removed from the vicinity of the centromere, there is an increase in recombination 27 frequency. At least part of this increase is due to increased exchange in the adjacent euchromatin: heterochromatic exchange, possible. : in as well, increased remains distinctly The problem can be finally resolved only when, systems such as those described abov�recombination ' 'is measured between markers actually located in the heterochromatin. 28 B IBLI OG·RAPHY Bridges, c. B., 1937 correspondences between li��age maps and salivary chromosome structure as illustrated in the tip of chromosome 2R of Drosophila melanogaster. Cytologia Fujii Jubilei Volwnen, 74'5-755. Pt. 2: ---- , 1938 A revised map of the salivary gland X chromosome of Drosophila melanogaster. J. Hered. 29; 1 l-13. , and K. Brehme, 1944 The mutants of Droso£hila melanogaster. Carnegie Inst. \'lash. Publ. 552• _______ cooper, K. w., 1959 Cytogenetic analysis of major heterochromatic elements (Especially Xh and Y) in. Drosophila melanogaster, and the theory of T1heterochromatin11• Ohromosoma (Berl.) .!.Q.: 53 5 - 588. 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Serv. 43: 141. ---- , and L. l.J:oore, 1968 Recombination in regions adjacent to deletions in the X c hromos ome of Genetics .2§: 557 -571. Drosophila melanogaster. ____ Lindsley, D. L., 1954 On the position of the right break point of In(l) wm4 and In(l) rst3. Dros. Inform. Serv. 28: 130. , and E. H . Grell, 1968 Genetic variations of Drosophila melanogaster. Carnegie Inst. \'lash. Publ.: 6 27. ---- Mather, K., 1939 Crossing over and heterochromatin in the X chromosome of Droso£hila melanozaster. Genetics 24: 413-435· Offerman, C. A. and H. J. £duller, 1932 Regional differencies in crossing over as a function of the chromosome structure. Proc. 6th Int. Gong. Genet. g: 143-145. ; Schalet, A., 1967 Crossing over within the bobbed locus in sc8 homozygotes (Abstr.} Genetics 56: 587. 1 30 Schultz J., and H. Redfield, 1951 Interchromosomal effects on crossing over in Drosophilaw cold Spring Harbor Sym;po Quant. Biol. 16: 175-197. Thompson, P. 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