Homolog pairing and segregation in Drosophila meiosis.

Pairing of homologous chromosomes is fundamental to their reliable segregation during meiosis I and thus underlies sexual reproduction. In most eukaryotes homolog pairing is confined to prophase of meiosis I and is accompanied by frequent exchanges, known as crossovers, between homologous chromatids. Crossovers give rise to chiasmata, stable interhomolog connectors that are required for bipolar orientation (orientation to opposite poles) of homologs during meiosis I. Drosophila is unique among model eukaryotes in exhibiting regular homolog pairing in mitotic as well as meiotic cells. I review the results of recent molecular studies of pairing in both mitosis and meiosis in Drosophila. These studies show that homolog pairing is continuous between pre-meiotic mitosis and meiosis but that pairing frequencies and patterns are altered during the mitotic-meiotic transition. They also show that, with the exception of X-Y pairing in male meiosis, which is mediated specifically by the 240-bp rDNA spacer repeats, chromosome pairing is not restricted to specific sites in either mitosis or meiosis. Instead, virtually all chromosome regions, both heterochromatic and euchromatic, exhibit autonomous pairing capacity. Mutations that reduce the frequencies of both mitotic and meiotic pairing have been recently described, but no mutations that abolish pairing completely have been discovered, and the genetic control of pairing in Drosophila remains to be elucidated.

A role of the Drosophila homeless gene in repression of Stellate in male meiosis.

The homeless gene of Drosophila melanogaster encodes a member of the DE-H family of ATPase and RNA helicase proteins. Loss-of-function homeless mutations were previously found to cause female sterility with numerous defects in oogenesis, including improper formation of both the anterior-posterior and dorsal-ventral axes and failure to transport and localize key RNAs required for axis formation. One homeless mutation was also found to affect male meiosis, causing elevated X-Y nondisjunction. Here we further analyze the role of homeless in male meiosis. We show that homeless mutations cause a variety of defects in male meiosis including nondisjunction of the X-Y and 2-2 pair, Y chromosome marker loss, meiotic drive, chromosome fragmentation, chromatin bridges at anaphase, and tripolar meiosis. In addition, homeless mutations interact with an X chromosomal factor to cause complete male sterility. These phenotypes are similar to those caused by deletion of the Suppressor of Stellate [Su(Ste)] locus. Like Su(Ste) deficiencies, homeless mutants also exhibit crystals in primary spermatocytes and derepression of the X-linked Stellate locus. To determine whether the regulatory role of hls is specific for Stellate or includes other repeated sequences as well, we compared testis RNA levels for nine transposable elements and found that all but one, copia, were expressed at the same levels in hls mutants and wild type. Copia, however, was strongly derepressed in hls mutant males. We conclude that hls functions along with Su(Ste) and other recently described genes to repress the Stellate locus in spermatocytes, and that it may also play a role in repressing certain other repeated sequences.

On the roles of heterochromatin and euchromatin in meiosis in drosophila: mapping chromosomal pairing sites and testing candidate mutations for effects on X-Y nondisjunction and meiotic drive in male meiosis.

Mapping of pairing sites involved in meiotic homolog disjunction in Drosophila has led to conflicting hypotheses about the nature of such sites and the role of heterochromatin in meiotic pairing. In the female-specific distributive system, pairing regions appear to be exclusively heterochromatic and map to broad regions encompassing many different sequences. In male meiosis, autosomal pairing sites appear to be distributed broadly within euchromatin but to be absent from heterochromatin, whereas the X-pairing site maps in the centric heterochromatin. The X site has been shown to coincide with the intergenic spacer (IGS) repeats within the rDNA arrays shared between the X and Y. It has not been clear whether the heterochromatic location of this pairing site has any significance. A novel assay for genic modifiers of X-Y chromosome pairing was developed based on the intermediate nondisjunction levels observed in males whose X chromosome lacks the native pairing site but contains two transgenic insertions of single rDNA genes. This assay was used to test several mutations in Su(var) (Suppressor of position effect variegation), PcG (Polycomb-Group) recombination defective, and repair-defective genes. No strong effects on disjunction were seen. However, the tests did uncover several mutations that suppress or enhance the meiotic drive (distorted X-Y recovery ratio) that accompanies X-Y pairing failure.

Male sterility and meiotic drive associated with sex chromosome rearrangements in Drosophila. Role of X-Y pairing.

In Drosophila melanogaster, deletions of the pericentromeric X heterochromatin cause X-Y nondisjunction, reduced male fertility and distorted sperm recovery ratios (meiotic drive) in combination with a normal Y chromosome and interact with Y-autosome translocations (T(Y;A)) to cause complete male sterility. The pericentromeric heterochromatin has been shown to contain the male-specific X-Y meiotic pairing sites, which consist mostly of a 240-bp repeated sequence in the intergenic spacers (IGS) of the rDNA repeats. The experiments in this paper address the relationship between X-Y pairing failure and the meiotic drive and sterility effects of Xh deletions. X-linked insertions either of complete rDNA repeats or of rDNA fragments that contain the IGS were found to suppress X-Y nondisjunction and meiotic drive in Xh-/Y males, and to restore fertility to Xh-/T(Y;A) males for eight of nine tested Y-autosome translocations. rDNA fragments devoid of IGS repeats proved incapable of suppressing either meiotic drive or chromosomal sterility. These results indicate that the various spermatogenic disruptions associated with X heterochromatic deletions are all consequences of X-Y pairing failure. We interpret these findings in terms of a novel model in which misalignment of chromosomes triggers a checkpoint that acts by disabling the spermatids that derive from affected spermatocytes.

Pairing sites and the role of chromosome pairing in meiosis and spermatogenesis in male Drosophila.

Mechanistic and regulatory aspects of meiotic chromosome pairing and segregation have received increasing attention in recent years. This review is concerned with the role of chromosomal sites and chromosome organization in pairing and sperm development in Drosophila. Two major topics are reviewed. The first concerns the distribution and identification of meiotic pairing sites in male Drosophila. Cytogenetic data show that pairing sites are distributed widely in the euchromatin of autosomes but are absent from centromeric heterochromatin. The reverse distribution holds for the X, where the major pairing site is located in the central region of the centric heterochromatin, co-mapping with the rDNA locus. Recent transgenic studies have demonstrated that this pairing site consists mainly of a 240-bp repeated sequence in the intergenic spacers of the rDNA repeats. These spacer repeats contain RNA polymerase I promoters, which must be functional for the repeats to have pairing activity, suggesting a mechanistic connection between pairing and transcription. The general idea that pairing sites coincide with transcribed sequences is discussed. The second major topic involves the effects of sex chromosome rearrangements on spermiogenesis. A variety of rearrangements involving the sex chromosomes, including heterochromatic deletions and translocations with autosomes, have been shown to lead either to meiotic drive or to sterility. Recent evidence strongly implicates the X chromosome pairing site in the etiology of these effects. These findings are discussed in terms of a novel model that interprets the spermiogenic disruptions associated with sex chromosome rearrangements as resulting from disabling of spermatids due to triggering of a checkpoint concerned with monitoring chromosome alignment at meiotic metaphase.

Roles of rDNA spacer and transcription unit-sequences in X-Y meiotic chromosome pairing in Drosophila melanogaster males.

Meiotic pairing of the X and Y chromosomes in Drosophila melanogaster males is mediated by the rDNA repeats, which are present in two tandem clusters, one in the centric X heterochromatin and the other near the base of the short arm of the Y chromosome. Deletion of the X chromosomal rDNA cluster disrupts X-Y pairing and causes high frequences of X-Y nondisjunction. Pairing can be partly restored by insertions of cloned complete rRNA genes or by rDNA fragments that include the intergenic spacer (IGS) region. A 240 bp repeated sequence in the IGS was shown to be effective in promoting pairing when present at copy numbers above five. This study further defines the rDNA sequences involved in mediating pairing. Germline insertions of a P element construct containing most of the rDNA transcription unit but no promoter or IGS region were obtained. Two single-copy insertions and four two-copy insertions proved unable to stimulate X-Y disjunction when located on an rDNA-deficient X chromosome. In addition, three insertions of a P element construct consisting of the IGS and promoter regions of the rDNA were characterized molecularly. These three insertions had previously been shown to range in pairing ability from very weak to quite strong. Molecular analysis revealed that the three insertions also vary in copy number of the 240 bp IGS repeat and that these structural differences correlate with the differences in pairing ability. These data indicate that 240 bp repeats are considerably more effective than other regions of the rDNA in stimulating chromosome pairing.

The license to pair: identification of meiotic pairing sites in Drosophila.

Sites for pairing and segregation of achiasmatic bivalents have been characterized in both male and female meiosis in Drosophila melanogaster. The major sex chromosome pairing site in male meiosis corresponds to the intergenic spacer repeats of the rDNA arrays, which are located in the heterochromatin of the X and Y. The sex chromosome pairing sites in females are also heterochromatic, but involve different repeated sequences. In males, weak pairing sites are widely distributed along euchromatin but not heterochromatin of chromosome 2, an autosome. One strong site for male meiotic pairing has been identified on chromosome 2; it overlaps with the his locus, which contains the repetitive structural genes for the histones. In females the sites for pairing of chromosome 4, another autosome, are restricted to the heterochromatin. Thus for both sex chromosomes and autosomes, sites for achiasmatic pairing are heterochromatic in females but euchromatic (except for the rDNA) in males. The possible roles of sequence repetition and of transcription in chromosome pairing are discussed.

Meiotic recombination: a mechanism for tracking and eliminating mutations?

The function of meiotic recombination has remained controversial, despite recent inroads into mechanisms. Ideas concerning a possible role of recombination in the elimination or efficient incorporation of mutations have been backed by theoretical studies but have lacked empirical support. Recent investigations into the basis for local variations in recombination frequency in yeast have uncovered a strong association between recombination initiation sites and transcriptional regulatory sequences. Other recent studies indicate a strong correlation between transcription and mutation rates in yeast genes. Taken together, these data imply that distributions of recombination and mutation frequencies may be strongly correlated. This suggests that recombination may be targeted to genomic sites of high mutation frequency; such a 'mutation-tracking' function would clearly aid in the shuffling of mutations to break up unfavorable and create favorable allelic combinations. Moreover, recent insights into the mechanism of gene conversion in yeast reveal a very strong inherent bias in favor of alleles on the non-initiating homolog. Combined with mutation tracking, these findings suggest a novel and general mechanism by which allelic gene conversion may act to eliminate mutations.

A recA-like gene in Drosophila melanogaster that is expressed at high levels in female but not male meiotic tissues.

The RecA protein is the central enzyme in prokaryotic recombination. It catalyzes pairing and strand exchange between homologous DNA molecules, and functions in both DNA repair and genetic recombination. The RecA-like proteins Rad51 and Dmc1 of yeast are both required for meiotic recombination and the former is also necessary for repair of double-strand breaks in vegetative cells. Genes encoding Rad51 homologs have been isolated recently from several higher eukaryotes. This paper describes the isolation and molecular characterization of a genomic DNA fragment from Drosophila melanogaster containing the coding sequence for a RecA-like protein. This protein exhibits strong sequence homology with the Rad51 proteins of budding yeast, fission yeast, chickens, mouse and humans, and slightly less (but still strong) homology with yeast Dmc1. Both in situ hybridization and Southern analysis indicate that the Rad51 gene is present only once per genome in Drosophila (at 99D on chromosome arm 3R). However, there are at least three other fragments that cross-hybridize strongly at low stringency. RNA blotting analysis detects a single transcript of about 1.35 kb that is present throughout development at low levels. Transcript levels are induced at least tenfold in ovaries, as measured by RNase protection analysis, suggestive of a role in female meiosis. Transcript levels are significantly lower in testes than in bulk RNA of adult males, however, indicating that Rad51 may be repressed in meiosis of Drosophila males.

Structure of the Y chromosomal Su(Ste) locus in Drosophila melanogaster and evidence for localized recombination among repeats.

The structure of the Suppressor of Stellate [Su(Ste)] locus on the Drosophila melanogaster Y chromosome was examined by restriction analysis of both native and cloned genomic DNA. The locus consists of short subarrays of tandem repeats separated by members of other moderately repeated families. Both size variants and restriction variants proved to be common. Most repeats fell into two size classes-2.8 and 2.5 kb-but other size variants were also observed. Restriction variants showed a strong tendency to cluster, both at the gross level where some variants were present in only one of three subintervals of the locus, and at the fine level, where repeats from the same phage clone were significantly more similar than repeats from different clones. Restriction variants were shared freely among repeats of different size classes; however, size variants appeared to be randomly distributed among phage clones. These data indicate that recombination among tandem Su(Ste) repeats occurs at much higher frequencies between close neighbors than distant ones. In addition, they suggest that gene conversion rather than sister chromatid exchange may be the primary recombinational mechanism for spreading variation among repeats at the Su(Ste) locus.

Visualization of banded polytene chromosomes under epifluorescent illumination by counterstaining with ethidium bromide.

Current methods of localization of chromosomal antigens on polytene chromosomes of Drosophila melanogaster salivary glands by indirect immunofluorescence require comparison of two microscopic images of the same nucleus--a phase contrast or bright field image to visualize the chromosomes and a fluorescent image to locate the antibody. We have found that inclusion of the DNA-intercalating agent ethidium bromide in the mounting medium makes banded polytene chromosomes visible under epifluorescent illumination, eliminating the need for two images. The banding of the polytene chromosomes is clear enough to locate specific bands.

The distribution of male meiotic pairing sites on chromosome 2 of Drosophila melanogaster: meiotic pairing and segregation of 2-Y transpositions.

The distribution of meiotic pairing sites on a Drosophila melanogaster autosome was studied by characterizing patterns of prophase pairing and anaphase segregation in males heterozygous for a number of 2-Y transpositions, collectively covering all of chromosome arm 2R and one-fourth of chromosome arm 2L. It was found that all transpositions involving euchromatin from chromosome 2, even short stretches, increased the frequency of prophase I quadrivalents involving the sex and second chromosome bivalents above background levels. Quadrivalent frequencies were the same whether the males carried both elements of the transposition or just the Dp(2:Y) element along with two normal chromosome 2s, indicating that pairing is non-competitive. The frequency of quadrivalents was proportional to the size of the transposed region, suggesting that pairing sites are widely distributed on chromosome 2. Moreover, all but the smallest transpositions caused a detectable bias in the segregation ratio, in favor of alternate segregations, indicating that the prophase associations were effective in orienting centromeres to opposite poles. One transposition involving only heterochromatin of chromosome 2 had no effect on quadrivalent frequency, consistent with previous evidence that autosomal heterochromatin lacks meiotic pairing ability in males. One region at the base of chromosome arm 2L proved to be especially effective in stimulating quadrivalent formation and anaphase segregation, indicating the presence of a strong pairing site in this region. It is concluded that autosomal pairing in D. melanogaster males is based on general homology, despite the lack of homologous recombination.

Sex chromosomes, recombination, and chromatin conformation.

We review what is known about the transcriptional inactivation and condensation of heteromorphic sex chromosomes in contrast to the activation of homomorphic sex chromosomes during meiotic prephase in animals. We relate these cytological and transcriptional features to the recombination status of the sex chromosomes. We propose that sex chromosome condensation is a meiotic adaptation to prevent the initiation of potentially damaging recombination events in nonhomologous regions of the X and Y chromosome.

Evidence that intergenic spacer repeats of Drosophila melanogaster rRNA genes function as X-Y pairing sites in male meiosis, and a general model for achiasmatic pairing.

In Drosophila melanogaster males, X-Y meiotic chromosome pairing is mediated by the nucleolus organizers (NOs) which are located in the X heterochromatin (Xh) and near the Y centromere. Deficiencies for Xh disrupt X-Y meiotic pairing and cause high frequencies of X-Y nondisjunction. Insertion of cloned rRNA genes on an Xh- chromosome partially restores normal X-Y pairing and disjunction. To map the sequences within an inserted, X-linked rRNA gene responsible for stimulating X-Y pairing, partial deletions were generated by P element-mediated destabilization of the insert. Complete deletions of the rRNA transcription unit did not interfere with the ability to stimulate X-Y pairing as long as most of the intergenic spacer (IGS) remained. Within groups of deletions that lacked the entire transcription unit and differed only in length of residual IGS material, pairing ability was proportional to the dose of 240-bp intergenic spacer repeats. Deletions of the complete rRNA transcription unit or the 28S sequences alone blocked nucleolus formation, as determined by binding of an antinucleolar antibody, yet did not interfere with pairing ability, suggesting that X-Y pairing may not be mechanistically related to nucleolus formation. A model for achiasmatic pairing in Drosophila males based upon the combined action of topoisomerase I and a strand transferase is proposed.

Promoter-containing ribosomal DNA fragments function as X-Y meiotic pairing sites in D. melanogaster males.

The Drosophila melanogaster ribosomal DNA (rDNA) functions as an X-Y meiotic pairing site. Deletions encompassing the X chromosomal rDNA block (located in the heterochromatin) disrupt X-Y pairing and disjunction. Insertions of single, complete rRNA genes at ectopic locations on the heterochromatically deficient X partially restore X-Y pairing capacity. This study was undertaken to test fragments of an rDNA repeat for the ability to stimulate X-Y pairing and disjunction and to test for relationships between pairing capacity and two other phenotypes associated with rDNA insertions: transcription and the ability to organize a nucleolus. Insertions of three different fragments, all of which retained the rDNA promoter and upstream spacer sequences and which differed among each other in the length of downstream sequences, were obtained by P-element mediated transformation. One of the fragments is truncated only 140bp downstream from the promoter. Insertions of all three fragments proved capable of stimulating X-Y disjunction. Double insertions were substantially more effective than single insertions. RNA/PCR analysis was used to show that transcripts initiated at the inserted rDNA promoters are present in testis RNA from all insertions. Treatment with an antinucleolar antibody revealed that none of the insertions was associated with a mininucleolus. Thus promoter-containing rDNA fragments are autonomously capable of being transcribed and of functioning as X-Y pairing sites, but not of forming a mini-nucleolus.

Drosophila ribosomal RNA genes function as an X-Y pairing site during male meiosis.

In Drosophila melanogaster males, the sex chromosomes pair during meiosis in the centric X heterochromatin and at the base of the short arm of the Y (YS), in the vicinity of the nucleolus organizers. X chromosomes deficient for the pairing region segregate randomly from the Y. In this report we show that a single ribosomal RNA (rRNA) gene stimulates X-Y pairing and disjunction when inserted onto a heterochromatically deficient X chromosome by P element-mediated transformation. We also show that insert-containing X chromosomes pair at the site of insertion, that autosomal rDNA inserts do not affect X-Y pairing or disjunction, and that the strength of an X pairing site is proportional to the dose of ectopic rRNA genes. These results demonstrate that rRNA genes can promote X-Y pairing and disjunction and imply that the nucleolus organizers function as X-Y pairing sites in wild-type Drosophila males.