Recombinogenic Effects of Suppressors of Position-Effect Variegation in Drosophila

Corresponding author: Gunter Reuter, Biologicum, Martin Luther University, D-06120 Halle, Weinbergweg 10, Germany., reuter{at}genetik.uni-halle.de (E-mail)

Communicating editor: T. C. KAUFMAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Compact chromatin structure, induction of gene silencing in position-effect variegation (PEV), and crossing-over suppression are typical features of heterochromatin. To identify genes affecting crossing-over suppression by heterochromatin we tested PEV suppressor mutations for their effects on crossing over in pericentromeric regions of Drosophila autosomes. From the 46 mutations (28 loci) studied, 16 Su(var) mutations of the nine genes Su(var)2-1, Su(var)2-2, Su(var)2-5, Su(var)2-10, Su(var)2-14, Su(var)2-15, Su(var)3-3, Su(var)3-7, and Su(var)3-9 significantly increase in heterozygotes or by additive effects in double and triple heterozygotes crossing over in the ri-p^p region of chromosome 3. Su(var)2-2⁰¹ and Su(var)2-14⁰¹ display the strongest recombinogenic effects and were also shown to enhance recombination within the light-rolled heterochromatic region of chromosome 2. The dominant recombinogenic effects of Su(var) mutations are most pronounced in proximal euchromatin and are accompanied with significant reduction of meiotic nondisjunction. Our data suggest that crossing-over suppression by heterochromatin is controlled at chromatin structure as well as illustrate the possible effects of heterochromatin on total crossing-over frequencies in the genome.

CROSSING over is almost excluded in pericentromeric heterochromatin and strongly suppressed in adjacent euchromatic regions. This negative effect on crossing over is one of the characteristic features of heterochromatin (BAKER 1958 ). Significant discrepancies in relative lengths between genetic and cytological maps were first described in Drosophila by MULLER and PAINTER 1929 and DOBZHANSKY 1930 . The strong decrease in occurrence of crossing over per unit of absolute distance within pericentromeric regions was first ascribed by MATHER 1939 to the influence of heterochromatin. The exact relationship between polytene and genetic map positions has been determined in Drosophila from loci that have been accurately mapped by both techniques (cf. LINDSLEY and SANDLER 1977 ; ISING and BLOCK 1981 ; ASHBURNER 1989 ). Genetic analysis of regional constraints on meiotic exchange in Drosophila proved that compared to the remainder of the euchromatin crossing over is virtually excluded in all heterochromatic regions and significantly suppressed in regions near heterochromatin and the telomeres (MATHER 1939 ; ROBERTS 1965 ; CARPENTER and BAKER 1982 ; SZAUTER 1984 ). These studies also indicated the existence of different control systems for crossing-over suppression in heterochromatin and for specifying distribution of exchange within the euchromatin. Independent of centromere effects on crossing over (BEADLE 1932 ; MATHER 1939 ; YAMAMOTO and MIKLOS 1978 ), constraints on exchange in proximal euchromatic regions flanking heterochromatin are still established in the lack of proximity to the centromere (BAKER 1958 ; SZAUTER 1984 ). This suggests that crossing-over suppression within proximal regions might be caused by heterochromatin.

This pattern is not unique to Drosophila and in many other organisms it has been demonstrated that crossing over is significantly suppressed at pericentromeric regions (cf. JOHN and LEWIS 1965 ; COMINGS 1972 ; YUNIS and YASMINEH 1972 ). Structural differences between synaptonemal complexes of euchromatic and heterochromatic regions have been reported for different organisms (CARPENTER 1975 ; STACK 1984 ). In general, synaptonemal complexes of heterochromatic regions are characterized by a several times higher DNA density per axis length (for review see ZICKLER and KLECKNER 1999 ).

Physical recombination maps were elaborated by analyzing distribution and frequency of recombination nodules or by immunological detection of individual proteins involved in reciprocal recombination along synaptonemal complexes in animals and plants (CARPENTER 1979 ; STACK 1984 ; SHERMAN and STACK 1995 ; ANDERSON et al. 1999 ). These studies provided further evidence for strong restriction of recombination to the euchromatic portions of bivalents, which might simply indicate that heterochromatic regions are not accessible to the recombination machinery.

To study whether suppression of crossing over in proximal euchromatin and its exclusion from heterochromatic regions can be correlated in Drosophila with higher order chromatin organization of heterochromatin we studied the effect of dominant suppressor mutations of position-effect variegation (PEV) on crossing over. These mutations strongly suppress heterochromatin-induced gene silencing in PEV (REUTER and WOLFF 1981 ; SINCLAIR et al. 1983 ; WUSTMANN et al. 1989 ) and may cause changes of chromatin structure within heterochromatin (GRIGLIATTI 1991 ; REUTER and SPIERER 1992 ; WEILER and WAKIMOTO 1995 ). Consequently, Su(var) mutations that change chromatin structure within heterochromatin might modify constraints on exchange within heterochromatin and the flanking proximal euchromatic regions.

Almost all of the recombination-defective mutations identified until now are recessive and affect the frequency as well as placement of crossing over within euchromatic regions (SANDLER et al. 1968 ; BAKER and CARPENTER 1972 ; BAKER and HALL 1976 ; BAKER et al. 1976 ; SEKELSKY et al. 1999 ). In most of these mutations reduction of crossing over is correlated with an increase of nondisjunction, which reflects the important function of crossing over in the disjunction of bivalents (HAWLEY 1988 ). Another class of meiotic mutations affects only chromosome disjunction (BAKER and HALL 1976 ; SEKELSKY et al. 1999 ). Finally, the meiotic mutations identified can be differentiated into three groups: mutations defective in the recombination-based pathway, segregation-defective mutations, and mutations that affect the achiasmate system. In spite of the relatively small number of chromosomes and mutations tested in the different screens, the high rate of meiotic mutations recovered indicates that the known meiotic genes in Drosophila represent only a fraction of the total number of genes causally connected with the control of meiotic processes (HAWLEY 1988 , HAWLEY 1993 ).

Here we describe the dominant effects of suppressor of PEV mutations on crossing over in pericentromeric regions. Out of the 46 mutations tested, 16 mutations representing nine different genes significantly increase crossing over in proximal regions, likely by reducing the suppressive effect of heterochromatin on crossing over. These modifier of PEV mutations represent a new class of dominant meiotic mutations. They show the strongest effects in proximal euchromatin and enhance recombination within heterochromatin but show only weak effects on distal euchromatic regions. The increase of crossing over within proximal regions is positively correlated with a decrease of nondisjunction or chromosome loss of chromosome 2 in meiosis. Su(var) mutations affect heterochromatin-induced gene silencing in PEV and their effect on recombination might be mediated through changes in chromatin structure. This new class of meiotic mutations implicates a link between heterochromatic chromatin organization and suppression of meiotic recombination.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila cultures and stocks:
Flies were reared on standard medium at 25°. Except where noted chromosomes and mutations are described in LINDSLEY and ZIMM 1992 and FLYBASE 2001 . All crosses were carried out at 25°. The suppressor mutations of PEV were isolated on the basis of their dominant suppressor effect on white variegation in In(1)w^m4 (REUTER and WOLFF 1981 ; REUTER et al. 1986 ; WUSTMANN et al. 1989 ; SINCLAIR et al. 1992 ). Su(var) mutations studied are listed in Table 1. None of the mutations displayed any dominant effects on viability. Transgenic lines with additional genomic copies of Su(var)2-5, Su(var)3-7, and Su(var)3-9 were used to study dosage-dependent effects of the heterochromatin-associated proteins HP1, SU(VAR)3-7, and SU(VAR)3-9 on crossing over. The P{GS[ry⁺] Su(var)2-5 1.9 kb} genomic DNA fragment is described in SCHOTTA and REUTER 2000 . Transgenic lines carrying two additional genomic copies of Su(var)3-7 were constructed by remobilization of P{[ry⁺] Su(var)3-7 6.5 kb} (REUTER et al. 1990 ) in genotypes carrying the TM3, ry^RK Sb e P[(ry⁺)2-3](99B) balancer chromosome, which contains a stable source of transposase (REUTER et al. 1993 ). Genomic copies of Su(var)3-9 were introduced by P{[ry⁺] Su(var)3-9 11 kb} (TSCHIERSCH et al. 1994 ). In the chromosome designated 2x P{[ry⁺] Su(var)3-9 11 kb} two additional genomic copies of Su(var)3-9 are inserted in 46F and 55DE on 2R.

The loss-of-function mutation Su(var)2-5⁰⁵ is described in WUSTMANN et al. 1989 and EISSENBERG et al. 1992 . Su(var)3-9⁰⁶ is characterized by an insertion of an 6-kb DNA fragment. In Su(var)3-9⁰⁶ homozygotes the Su(var)3-9-specific transcript cannot be detected after reverse PCR analysis (V. KRAUSS, G. SCHOTTA and G. REUTER, unpublished data). Df(3R)AceHD1 uncovers the Su(var)3-7 locus (REUTER et al. 1990 ) and allowed us to study haplo-dependent effects of the Su(var)3-7 gene on crossing over in the ri-p^p region.

Crossover analysis:
We performed a series of crosses to identify Su(var) mutations with a dominant recombinogenic effect.

Cross 1: For a systematic test of 43 Su(var) mutations on crossing over in the ri-p^p region females heterozygous for a Su(var) mutation on chromosome 2 or 3 and the marker mutations radius incompletus (ri) and pink peach (p^p) flanking the pericentromeric region of chromosome 3 [Su(var)/+; ri p^p/++ or Su(var) + +/+ ri p^p] were crossed to homozygous ri p^p males. In offspring crossing over between ++/ri p^p was scored. Freshly hatched females were mated on the same day. After 4 days of egg laying the parents were transferred four times to fresh culture vials every second day. Altogether five consecutive egg-laying periods were established until females were 14 days old. The progeny of each egg-laying period were analyzed separately. This procedure allowed us to determine age-dependent effects on crossing over.

Double heterozygotes were constructed by crosses of flies with the following genotypes: Su(var)2-X/In(2L)t+In(2R)Cy, Cy Roi cn² bw^45a or^45a sp²; ri p^p females to Su(var)2-Y/In(2L)t+In(2R)Cy, Cy Roi cn² bw^45a or^45a sp²; ++ males or Su(var)2-X Su(var)2-Y/In(2L)t+In(2R)Cy, Cy Roi cn² bw^45a or^45a sp² females to homozygous ri p^p males. For construction of triple heterozygotes Su(var)2-X Su(var)2-Y/In(2L)t+In(2R)Cy, Cy Roi cn² bw^45a or^45a sp² females were crossed to Su(var)2-Z/In(2L)t+In(2R)Cy, Cy Roi cn² bw^45a or^45a sp²; ri p^p males [X, Y, and Z denote different Su(var) genes]. For several of the combinations reciprocal crosses were also performed.

Cross 2: For Su(var) mutations with a dominant recombinogenic effect in the ri-p^p interval we measured crossing-over frequencies along chromosome 3 by using the marker mutations sepia (se) 3-26.5, radius incompletus (ri) 3-46.8, spineless (ss) 3-58.8, ebony (e) 3-70.7, and rough (ro) 3-91.1. Females Su(var)/+; se ri ss e ro/++++ were crossed to se ri ss e ro homozygous males and offspring were analyzed for crossing-over frequencies in the four genetic intervals defined by the recessive visible marker mutations. Offspring were collected for three egg-laying periods (days 0–4, 6–7, and 11–12).

Cross 3: Crossing over in the tip of the X chromosomes was analyzed between yellow (y) 1-0.0 and split (spl) 1-3.0 by crossing y spl/+ +; Su(var)/+ females to y spl/Y; +/+ males.

Cross 4: Recombination in heterochromatin was studied between the heterochromatic genes light (lt) and rolled (rl). As a flanking marker stw³ located in proximal euchromatin of 2R was used. Su(var)2-2⁰¹ and Su(var)2-14⁰¹/In(2L)t+In(2R)Cy, Cy Roi cn² bw^45a or^45a sp² females were crossed to lt Df(2R)rl^10a stw³/In(2LR)CyO males. Offspring Su(var)2-2⁰¹ or Su(var)2-14⁰¹/lt Df(2R)rl^10a stw³ females were crossed to lt rl stw³ homozygous males. Crossing over was measured in offspring produced during the first 5 days of egg laying. Df(2R)rl^10a was kindly provided by Ernst Hafen. Heterozygous Df(2R)rl^10a/rl flies show complete penetrance of the rolled mutant phenotype.

Rescue analysis of Su(var)2-1 mutations:
Dp(2;2)10 [Dp(2;2) 31A; 32A] covers the Su(var)2-1 locus (WUSTMANN et al. 1989 ) and rescues the dominant suppressor effect of Su(var)2-1⁰¹, indicating a loss-of-function or hypomorphic nature of this allele. All the 11 Su(var)2-1 alleles were tested for rescue of the dominant suppressor effect in w^m4h; Dp(2;2)10/Su(var)2-1^X heterozygotes after a cross of w^m4h; Dp(2;2)10/In(2LR)CyO females to w^m4h/Y; Su(var)2-1^X males. Variegation for white was quantified by red eye pigment measurements. The effect of Su(var)2-1⁰⁴ and Su(var)2-1²⁰⁷ on crossing over between ri and p^p was studied in Su/+; ri p^p/+ + and Su/Dp(2;2)10; ri p^p/+ + females. Su/Dp(2;2)10; ri p^p/+ + females were generated by a cross of w^m4h; Su(var)2-1⁰⁴ or Su(var)2-1²⁰⁷/In(2LR)CyO; ri p^p/ri p^p females to w^m4h/Y; Dp(2;2)10/In(2LR)CyO males. Offspring w^m4h; Su(var)2-1⁰⁴ or Su(var)2-1²⁰⁷/Dp(2;2)10; ri p^p/+ + females were crossed to ri p^p homozygous males. Crossing over between ri and p^p was measured in offspring produced during a 6-day egg-laying period.

Deficiency Df(2L)TE301X1 (31A2; 31C1) uncovers the Su(var)2-1 locus (WUSTMANN et al. 1989 ). This deficiency is dominant female sterile.

Nondisjunction and chromosome loss:
Nondisjunction values reported are from crosses of w^m4h; Su(var)2-X/+ or w^m4h; Su(var)2-X/Su(var)2-Y females to C(2)EN, bw sp males. Flies were mated for 4 days and eggs laid within the following 5 days were counted. The ratio of nondisjunction + chromosome loss is calculated as the number of viable exceptions resulting from nondisjunction or chromosome loss during oogenesis compared to the total number of eggs produced. After normal segregation all progeny produced by a cross to 0/C(2)EN, bw sp males are lethal due to trisomy or monosomy for chromosome 2 [+/C(2)EN or +/0]. Diplo-2 oocytes are recovered if fertilized by nullo-2 sperm (wild type in phenotype). Conversely, nullo-2 oocytes result in viable offspring if fertilized by C(2)EN sperms (brown and speck in phenotype).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Heterochromatin-associated proteins and suppression of crossing over in the ri-p^p pericentromeric region:
The three heterochromatin-associated proteins HP1 (JAMES and ELGIN 1986 ; EISSENBERG et al. 1990 , EISSENBERG et al. 1992 ), SU(VAR)3-7 (REUTER et al. 1990 ; CLEARD et al. 1997 ), and SU(VAR)3-9 (TSCHIERSCH et al. 1994 ; SCHOTTA and REUTER 2000 ), which control heterochromatin-induced gene silencing in PEV, were identified with the help of Su(var) mutations. Mutations of these genes strongly suppress whereas additional gene copies enhance gene silencing in PEV (REUTER and SPIERER 1992 ). We tested loss-of-function alleles and overexpression lines of the three genes for haplo- and triplo-dependent effects on crossing over between ri and p^p, genetic markers in the proximal regions of 3L and 3R, respectively (SINCLAIR 1975 ). Df(3R)AceHD1 uncovers Su(var)3-7 and causes a significant increase in crossing over between ri and p^p whereas the loss-of-function mutations Su(var)2-5⁰⁵ and Su(var)3-9⁰⁶ do not show a significant effect (Table 2). However, involvement of HP1 and SU(VAR)3-9 in crossover suppression within proximal regions is indicated by a significant effect of the double heterozygote Su(var)2-5⁰⁵/+; +/Su(var)3-9⁰⁶ (Table 2). The dominant effect of a Su(var)3-7 deletion on crossing over in the ri-p^p region is not enhanced in combinations with Su(var)2-5⁰⁵ or Su(var)3-9⁰⁶. Additional genomic copies of Su(var)3-7 and Su(var)2-5, which strongly enhance PEV, result in a significant decrease of crossing over between ri and p^p (Table 2). Therefore, the effects of the heterochromatin-associated proteins HP1 and SU(VAR)3-7 on crossing over between ri and p^p are dosage dependent. The finding that both Su(var)2-5⁰⁵ and Su(var)3-9⁰⁶ cause only a dominant recombinogenic effect if combined into a double heterozygote indicates a rather complex control of crossing-over suppression within pericentromeric regions. To identify additional genes that are involved in the control of exchange within proximal regions we tested other dominant suppressor of PEV mutations for their effect on crossing over between ri and p^p.

The complex genetic basis of crossing-over suppression in pericentromeric regions:
Altogether 36 mutations of 15 other Su(var) genes were tested for their dominant effect on crossing-over suppression in the ri and p^p region of chromosome 3. Ten of these mutations representing six different loci displayed a significant dominant recombinogenic effect (Table 2). In heterozygotes of these mutations crossing over between ri and p^p is increased two- to threefold. The Su(var)2-2, Su(var)2-10, Su(var)2-14, and Su(var)2-15 loci are represented by only one mutant allele. Altogether 11 Su(var)2-1 and 10 Su(var)3-3 alleles have been tested. Three Su(var)2-1 and three Su(var)3-3 alleles were found to increase dominantly crossing over between ri and p^p. Additive dominant effects on crossing over were evaluated in double and triple mutant heterozygotes. With the only exception of Su(var)2-15⁰¹/Su(var)2-10⁰¹ and Su(var)2-1⁰⁴/Su(var)2-10⁰¹ additive effects were found in all the other double heterozygous combinations tested (Table 2). Differences in crossing-over frequencies are found for several trans-heterozygotes depending on maternal or paternal origin of the mutations. In trans-heterozygotes with Su(var)2-10⁰¹ paternal in origin only weak additive effects are found whereas in combinations where this mutation is maternally inherited stronger effects are observed (Table 2).

Triple combinations were furthermore used to reevaluate the effects of Su(var)2-5 and Su(var)2-1 alleles. For these studies a Su(var)2-2⁰¹ Su(var)2-1⁰⁴ double-mutant chromosome was constructed. The Su(var)2-2⁰¹ Su(var) 2-1⁰⁴ chromosome was combined with the four Su(var)2-5 alleles 01, 02, 04, and 05. With the only exception of the 01 allele all the other Su(var)2-5 mutations cause a further increase of crossing over between ri and p^p (Table 2). The effect of two extra genomic copies of Su(var)3-7 on crossing over between ri and p^p was also analyzed in heterozygotes with the Su(var)2-2⁰¹ Su(var)2-1⁰⁴ double-mutant chromosome. Additive effects of Su(var)2-2⁰¹ and Su(var)2-1⁰⁴ result in an about fivefold enhancement of crossing over between ri and p^p. In combination with two extra genomic Su(var)3-7 copies this effect is significantly reduced. Therefore, overexpression of heterochromatin protein SU(VAR)3-7 results in partial compensation of recombinogenic effects displayed by the Su(var)2-2⁰¹ and Su(var)2-1⁰⁴ mutations.

In combinations with the double-mutant chromosome Su(var)2-2⁰¹ Su(var)2-10⁰¹ the Su(var)2-1 alleles 01, 05, 214, and 42/9 were tested for possible recombinogenic effects. Although Su(var)2-1^42/9 alone does not show a recombinogenic effect it causes a further increase of crossing over between ri and p^p in the presence of Su(var)2-2⁰¹ and Su(var)2-10⁰¹. From the 11 Su(var)2-1 mutations tested three alleles show dominant recombinogenic effects in the ri-p^p region. Mutations of the Su(var)2-1 gene are characterized by an interesting spectrum of pleiotropic effects. In addition to their strong suppressor effect on PEV the mutations show butyrate sensitivity and display lethal interaction with Y chromosome heterochromatin (REUTER et al. 1982 ). Su(var)2-1 mutations furthermore reduce histone H4 deacetylation (DORN et al. 1986 ). To determine the genetic nature of Su(var)2-1 mutations we used Dp(2;2)10, which duplicates the Su(var)2-1 gene (WUSTMANN et al. 1989 ).

The antimorphic nature of recombinogenic Su(var)2-1 alleles:
The results received for Su(var)2-5, Su(var)3-7, and Su(var)3-9 indicate a dosage-dependent effect of the heterochromatin-associated proteins on crossover suppression in the pericentromeric ri-p^p region. With the only exception of Su(var)2-10 (HARI et al. 2001 ), none of the other five Su(var) genes for which we identified alleles with dominant recombinogenic effects have been molecularly characterized until now. For Su(var)2-1 cytogenetic fine-structure mapping was performed (WUSTMANN et al. 1989 ; SINCLAIR et al. 1992 ; T. WESTPHAL, unpublished results) and the locus resides in 31B1. This region is duplicated by Dp(2;2)10. In Su(var)2-1⁰¹/Dp(2;2)10 heterozygotes the dominant suppressor effect of Su(var)2-1⁰¹ is completely rescued and variegation typical for w^m4h; +/+ control flies becomes visible (Table 3). This phenotypic reaction classifies Su(var)2-1⁰¹ as an amorphic or hypomorphic mutation. Dp(2;2)10 also rescues the dominant suppressor effect of Df(2L) TE301X1, which uncovers the Su(var)2-1 locus (data not shown). The deficiency Df(2L)TE301X1 causes dominant female sterility and cannot be analyzed for recombinogenic effects. From the 11 Su(var)2-1 mutations tested only the three alleles 04, 42/9, and 207 enhance crossing over in the ri-p^p region (Table 2). Results of rescue crosses with Dp(2;2)10 and Su(var)2-1 mutant alleles are summarized in Table 3. Complete rescue of the dominant suppressor effect is found in all Su(var)2-1 alleles without recombinogenic effects (01, 02, 03, 05, 06, 210, 214, and 215). The dominant suppressor effect is only partially rescued in Dp(2;2)10/Su(var)2-1^42/9 whereas Dp(2;2)10/Su(var)2-1⁰⁴ and Dp(2;2) 10/Su(var)2-1²⁰⁷ heterozygotes still express a suppressor phenotype (Table 3). These data suggest that all three recombinogenic Su(var)2-1 alleles are antimorphic in nature. This is also supported by the finding that Dp(2;2)10 does not rescue the recombinogenic effect of Su(var)2-1⁰⁴ and Su(var)2-1²⁰⁷ (data not shown).

Su(var) mutations enhance crossover in heterochromatin of the second chromosome:
Su(var)2-2⁰¹ and Su(var)2-14⁰¹ show the strongest effects on crossing over in the ri-p^p interval and were tested for their effects in the heterochromatic light-rolled region. The light gene is located within the 2L heterochromatin block h35 whereas rolled maps to 2R heterochromatin region h41 (DIMITRI 1991 ; BERGHELLA and DIMITRI 1996 ). In the control 0.006 cM were measured between lt and rl and 0.032 cM were determined for the rl and stw interval, which also includes the most proximal euchromatin of 2R (Table 4). Both Su(var)2-2⁰¹ and Su(var)2-14⁰¹ increase crossing over within heterochromatin as well as in the flanking proximal euchromatic region of 2R about four and three times, respectively (Table 4). All lt⁺ rl recombinant chromosomes carried stw as flanking marker whereas all lt rl⁺ recombinants were stw⁺, indicating reciprocal events. Recombination between lt and rl likely represents crossing over and not a single cluster of recombinants has been found. However, mitotic recombination after the stem cell division cannot be excluded.

Influence of Su(var) mutations on crossing over in the third chromosome and age-dependent effects:
To investigate the distribution of exchanges along the third chromosome the multiply marked se ri ss e ro chromosome subdividing the chromosome into four regions of 15–25 cM was used. The mutations do not change the frequency of crossing over in distal 3R between e and ro and with the exception of Su(var)2-1²⁰⁷ and Su(var)2-2⁰¹ show only weak effects in the ss-e interval (Table 5). All mutations significantly increase crossing over within the ri-ss interval. In Su(var)2-1⁰⁴, Su(var)2-1²⁰⁷, Su(var)2-2⁰¹, and Su(var)2-10⁰¹ the frequency of crossing over between se and ri is also increased. Furthermore, frequency of double crossing over in the se-ri and ri-ss intervals is significantly higher whereas the number of noncrossover random strands is significantly reduced (Table 5).

The data from Table 5 and Table 6 and their graphical summary in Fig 1 visualize the age-dependent changes in crossing-over frequencies along the third chromosome. Age-dependent effects on crossing-over frequencies were first described by BRIDGES 1927 . Most severe age-dependent reduction of crossing over is found within the ri-p^p region. The control frequency of crossing over in this region is 1.6 cM in 0- to 4-day-old females and becomes reduced to only 0.3 cM in 13- to 14-day-old females. With exception of Su(var)2-15⁰¹ and Su(var)2-10⁰¹ all mutations increase crossing over in the ri-p^p interval independent of females' age (Table 6). The total calculated map distance between se and ro varies between 79.4 and 61.1 if broods from 0- to 4- and 11- to 12-day-old females are compared (Table 5).

View larger version (18K): In this window In a new window Download PPT slide   Figure 1. Influence of female age on crossing-over frequencies in the se-ri, ri-pp, ss-e, and e-ro genetic intervals of chromosome 3. Data for +/+ control and Su(var)/+females are shown.

The effect of Su(var) mutations on crossing over in the distal y-spl region of the X chromosome:
In the more distal e-ro region of chromosome arm 3R the Su(var) mutations did not affect significantly the frequency of crossing over (Table 5). We furthermore studied their effect in the y-spl region at the tip of the X chromosome (Table 7). In this region only slight age-dependent effects on crossing over have been found and the data for consecutive egg-laying periods (0–12 days) were pooled. The data show that with the only exception of Su(var)2-10⁰¹ all the other mutations tested cause a weak but significant increase in crossing over. According to our data the recombinogenic effect of Su(var) mutations is most pronounced in proximal regions and the mutations show only weak effects in distal euchromatic regions. However, this increase in crossover frequencies might indicate that interchromosomal effects (reviewed in LUCCHESI and SUZUKI 1968 ) are observed and the Su(var) mutations might also affect other aspects of recombination related to heterochromatin.

The Su(var) mutations affect chromosome segregation in female meiosis:
Crossing over ensures segregation of homologous chromosomes in meiosis I. The frequency of nondisjunction or chromosome loss for the second chromosome in female meiosis was determined after crosses of w^m4h; Su(var)2-X/+ or w^m4h; Su(var)2-X/Su(var)2-Y females to C(2)EN, bw sp males (cf. MATERIALS AND METHODS). In the control 0.18% of all eggs laid developed to adults. These flies were either +/+ or C(2)EN, bw sp/0 in genotype. The +/+ flies can be caused only by nondisjunction whereas C(2)EN, bw sp/0 offspring could be due to nondisjunction or chromosome loss. All the Su(var) mutations that display recombinogenic effects significantly reduced nondisjunction and/or chromosome loss in female meiosis (Table 8). The observed reduction is positively correlated with the strength of their recombinogenic effects in the ri-p^p interval. In double-mutant heterozygotes additive effects are found. Our data indicate a possible correlation between the frequencies of crossing over in proximal regions and nondisjunction or chromosome loss.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Heterochromatin constitutes 30% of the Drosophila genome. It is mostly transcriptionally inert and silences euchromatic genes relocated next to it. In meiosis, the heterochromatin of all chromosomes is physically associated throughout prophase until meiosis I and this association is important for proper disjunction of achiasmate homologs (HAWLEY et al. 1992 ; DERNBURG et al. 1996 ; KARPEN et al. 1996 ). This function of heterochromatin is affected by mutations of the wings apart-like (wapl) gene, which dominantly affect achiasmate segregation and modify position-effect variegation (VERNI et al. 2000 ). Several other genes implicated in PEV modification like DmORC-2, Su(var)3-6, and Trithorax-like (Trl) have also been shown to play a role in chromosome condensation and segregation (GATTI and BAKER 1989 ; BAKSA et al. 1993 ; BHAT et al. 1996 , SEKELSKY et al. 1999 ). The cytological visible effects of Su(var)2-5 mutations indicate a function of the HP1 protein not only in heterochromatin but also in telomere stability (FANTI et al. 1998 ). All these genes are suggested to play a role in heterochromatin structure and function. Their mutant effects on mitotic and meiotic chromosome behavior underline the importance of heterochromatin in control of mitotic and meiotic processes. To identify new genes controlling heterochromatin and meiosis processes we studied the effect of Su(var) mutations on crossing over in pericentromeric regions. If crossing-over suppression in these regions is influenced by the compact chromatin structure of heterochromatin several of the products encoded by Su(var) genes should significantly affect crossing over in proximal euchromatin and recombination within heterochromatin. Because most of the Su(var) loci are essential genes, only dominant effects on crossing over can be recognized. In a genetic test of 46 Su(var) mutations we measured crossing over in the ri-p^p interval of chromosome 3, a region that was already well characterized in its recombination features (SINCLAIR 1975 ). Between the ri and p^p genes, which are located in proximal euchromatin of the 3L and 3R chromosome arms, respectively, 13–16 Mb DNA of heterochromatin is located (YAMAMOTO et al. 1990 ). The distance between the two genes in the genetic map is only 1.8 cM but comprises 26 Mb of DNA. In physical size this is almost identical to the euchromatic part of a complete chromosome arm spanning 50 cM. This discrepancy in map distances is caused by crossover suppression within heterochromatin and proximal euchromatic regions.

An important function in the control of chromatin structure in heterochromatin could be attributed to the heterochromatin-associated proteins HP1, SU(VAR)3-7, and SU(VAR)3-9. A deletion of Su(var)3-7 dominantly increases whereas overexpression results in a further decrease of crossing over in the ri-p^p interval. Also, overexpression of HP1 reduces crossing over. An affect of Su(var)3-9 on crossing over is found in a double heterozygote with Su(var)2-5⁰⁵. Possible DNA binding of SU(VAR)3-7 and anchoring of heterochromatin protein complexes containing HP1 and SU(VAR)3-9 could explain the dosage-dependent effects of the Su(var)3-7 gene on crossing over in the pericentromeric ri-p^p region. The results indicate involvement of heterochromatin protein complexes in crossing over suppression within pericentromeric regions; their moderate effects, however, implicate at the same time a rather complex genetic control of these processes. This is supported by our findings that at least six more Su(var) genes are involved in crossing-over suppression within proximal regions. Mutations of Su(var)2-1, Su(var)2-2, Su(var)2-10, Su(var)2-14, Su(var)2-15, and Su(var)3-3 genes increase crossing over in the ri-p^p region. Additive effects were found in double and triple heterozygotes of these mutations. The highest frequency of crossing over between ri and p^p was 14.1 cM in the Su(var)2-2⁰¹ Su(var)2-1⁰⁴/Su(var)2-5⁰² triple mutant combination. An effect on recombination within heterochromatin could be shown for the Su(var)2-2⁰¹ and Su(var)2-14⁰¹ mutations.

These recombinogenic effects of Su(var) mutations might be attributed to changes in the compact chromatin structure in heterochromatin. Such a model explains crossing-over suppression in proximal euchromatin by assuming cis-acting effects of heterochromatin. In heterozygotes of Su(var)2-2⁰¹ the total map distances between the genes se and ro on the third chromosome enlarge by >20 cM.

In Su(var)2-1 all the alleles with recombinogenic effects were shown to be antimorphic in nature. No homozygotes could be tested because of recessive female sterility. The antimorphic alleles could reduce the actual amount of wild-type function in mutant heterozygotes below a significant threshold by interference with the function of the wild-type product. Antimorphic mutations therefore might represent important tools for genetic dissection of regulatory components of crossing-over suppression by heterochromatin, which are otherwise essential for viability and/or fertility.

Crossing-over reduction results in an increase of nondisjunction because crossing over ensures regular disjunction of bivalents (HAWLEY 1988 ). Our results indicate that inversely an increase of crossing over within proximal regions might result in a decrease of nondisjunction. KOEHLER et al. 1996 studied the recombinational history of spontaneous X chromosomal nondisjunction in Drosophila females. They found that spontaneous meiosis I nondisjunction occurred primarily in oocytes with nonexchange chromosomes. From the few nondisjoining exchange bivalents most had distal crossovers. These results indicate that proximal crossovers are important for regular homolog segregation. However, the much less common meiosis II nondisjunction occurred only in oocytes with proximal exchange. Also, in humans most trisomy events for chromosomes 16 and 21 arise from meiosis I nondisjunction and show reduced proximal recombination, supporting that proximal exchanges are more effective at ensuring regular segregation. As in Drosophila, meiosis II nondisjunction in humans is associated with increased exchange in the proximal region (LAMB et al. 1996 ). The increase of crossing over in proximal regions caused by Su(var) mutations might significantly reduce the frequency of achiasmate bivalents and meiosis I nondisjunction. Although we cannot differentiate between meiosis I and meiosis II nondisjunction, our data indicate that an increase in proximal exchange must not necessarily result in an increase of meiosis II nondisjunction.

The simultaneous effect of dominant Su(var) mutations on both crossing over and nondisjunction might indicate a role of heterochromatin in optimizing meiotic processes like crossing over and disjunction of homologous chromosomes. This is also supported by interchromosomal effects of X heterochromatin deletions on recombination frequencies in chromosome 3 (YAMAMOTO 1979 ). The observed recombinogenic effects of Su(var) mutations might reflect dosage-dependent effects of their gene products in establishment of a compact higher order chromatin structure within heterochromatin, which might be responsible for crossing-over suppression in proximal regions. The mutations will serve as useful tools for molecular analysis of these processes.

	ACKNOWLEDGMENTS

We are grateful to our colleagues Drs. A. Hilliker and J. Szabad for discussions and their critical comments on the manuscript. We are grateful to G. Schotta for his help in statistical analysis. We thank Drs. E. Hafen, T. Grigliatti, J. Szabad, and the Bloomington Drosophila Stock Center for mutant strains. We also thank M. Kube for excellent technical assistance. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Re911/1-4), Land Sachsen-Anhalt, and European Union (meiosis network) to G.R.

Manuscript received January 25, 2001; Accepted for publication November 20, 2001.

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