Partner choice in heterologous chromosome segregation of the Y chromosome in competitive situations in the oocyte of Drosophila melanogaster

Heterologous segregation of the Y chromosome and secondary non-disjunction of the X chromosomes in female meiosis of Drosophila melanogaster was investigated in ten different crosses where different constellations of translocation/inversion or translocation/translocation systems of the large autosomes were present in the female parent. It appeared that the Y chromosome always segregates from the shortest of the possible heterologous pairing partners. This may be due to size-dependent mechanism of so-called distributive disjunction or to the possibility that the shorter the chromosome element is, the more easily it moves in the nucleus of the oocyte. Secondary non-disjunction of the X chromosomes appeared to be lower the more possible autosomal pairing partners the Y chromosome had, suggesting that the autosomes effectively compete with the X chromosomes for pairing with the Y chromosome. An alternative explanation is that, due to interchromosomal effect on recombination, crossing over in the X chromosomes was different in different experiments.     

Cytological dissection of sex chromosome heterochromatin of Drosophila hydei

Prophase chromosomes of Drosophila hydei were stained with 0.5 g/ml Hoechst 33258 and examined under a fluorescence microscope. While autosomal and X chromosome heterochromatin are homogeneously fluorescent, the entirely heterochromatic Y chromosome exhibits an extremely fine longitudinal differentiation, being subdivided into 18 different regions defined by the degree of fluorescence and the presence of constrictions. Thus high resolution Hoechst banding of prophase chromosomes provides a tool comparable to polytene chromosomes for the cytogenetic analysis of the Y chromosome of D. hydei. — D. hydei heterochromatin was further characterized by Hoechst staining of chromosomes exposed to 5-bromodeoxyuridine for one round of DNA replication. After this treatment the pericentromeric autosomal heterochromatin, the X heterochromatin and the Y chromosome exhibit numerous regions of lateral asymmetry. Moreover, while the heterochromatic short arms of the major autosomes show simple lateral asymmetry, the X and the Y heterochromatin exhibit complex patterns of contralateral asymmetry. These observations, coupled with the data on the molecular content of D. hydei heterochromatin, give some insight into the chromosomal organization of highly and moderately repetitive heterochromatic DNA.     

The relation of exchange to nondisjunction in heterologous chromosome pairs in the Drosophila female

An analysis of the relationships in Drosophila melanogaster between one set of homologues (third chromosome) undergoing crossing over and a second, independent set (X chromosome) undergoing nondisjunction shows that the nondisjunctional set almost invariably segregates from one of the members of the crossover set and not the other. The results seem contradictory to the expectations based on the "distributive pairing hypothesis" according to which nondisjunctional (i.e., noncrossover) elements form a "distributive pool" whose members behave independently of those which have been involved in exchange.     

There are two mechanisms of achiasmate segregation in Drosophila females, one of which requires heterochromatic homology.

There are numerous examples of the regular segregation of achiasmate chromosomes at meiosis I in Drosophila melanogaster females. Classically, the choice of achiasmate segregational partners has been thought to be independent of homology, but rather made on the basis of availability or similarities in size and shape. To the contrary, we show here that heterochromatic homology plays a primary role in ensuring the proper segregation of achiasmate homologs. We observe that the heterochromatin of chromosome 4 functions as, or contains, a meiotic pairing site. We show that free duplications carrying the 4th chromosome pericentric heterochromatin induce high frequencies of 4th chromosome nondisjunction regardless of their size. Moreover, a duplication from which some of the 4th chromosome heterochromatin has been removed is unable to induce 4th chromosome nondisjunction. Similarly, in the absence of either euchromatic homology or a size similarity, duplications bearing the X chromosome heterochromatin also disrupt the segregation of two achiasmate X chromosome centromeres. Although heterochromatic regions are sufficient to conjoin nonexchange homologues, we confirm that the segregation of heterologous chromosomes is determined by size, shape, and availability. The meiotic mutation Axs differentiates between these two processes of achiasmate centromere coorientation by disrupting only the homology-dependent mechanism. Thus there are two different mechanisms by which achiasmate segregational partners are chosen. We propose that the absence of diplotene-diakinesis during female meiosis allows heterochromatic pairings to persist until prometaphase and thus to co-orient homologous centromeres. We also propose that heterologous disjunctions result from a separate and homology-independent process that likely occurs during prometaphase. The latter process, which may not require the physical association of segregational partners, is similar to those observed in many insects, in Saccharomyces cerevisiae and in C. elegans males. We also suggest that the physical basis of this process may reflect known properties of the Drosophila meiotic spindle.     

A test of distributive pairing in Zea mays

Zea mays plants containing two extra chromosomes were analyzed to determine if distributive pairing takes place in maize. No interaction between two univalent chromosomes generated in such plants could be detected at diakinesis or metaphase I. The chromosome disjunction in such plants was random at anaphase I. Furthermore, it was found that two chromosomes which are found as univalents in essentially 100 percent of the meiotic cells assorted randomly to the progeny. These experiments duplicated as closely as possible certain of Grell's (1962) experiments on distributive pairing. The author could detect no evidence for distributive pairing in maize. It was concluded that distributive pairing either does not occur in maize or that it occurs with a far lower efficiency than it does in Drosophila melanogaster females. Speculations on the reasons for these differences are included.Research supported in part by a grant (GM82-08) from the National Institutes of Health, U. S. Public Health Service.     

Dicentric chromosomes and the inactivation of the centromere

Summary The origin and behavior of human dicentric chromosomes are reviewed. Most dicentrics between two non-homologous or two homologous chromosomes (isodicentrics), which are permanent members of a chromosome complement, probably originate from segregation of an adjacent quadriradial; such configurations are the result of a chromatid translocation between two nonhomologous chromosomes, or they represent an adjacent counterpart of a mitotic chiasma. The segregation of such a quadriradial may also give rise to a cell line monosomic for the chromosome concerned (e.g., a 45,X line). Contrary to the generally held opinion, isodicentrics rarely result from an isolocal break in two chromatids followed by rejoining of sister chromatids. In this case the daughter centromeres go to opposite poles in the next anaphase, and the resulting bridge breaks at a random point. This mechanism, therefore, leads to the formation of an isodicentric chromosome only if the two centromeres are close together, or if one centromere is immediately inactivated. Observations on the origin of dicentrics in Bloom syndrome support these conclusions. One centromere is permanently inactivated in most dicentric chromosomes, and even when the dicentric breaks into two chromosomes, the centromere is not reactivated. The appearance and behavior of the acentric X chromosomes show that their centromeres are similarly inactivated and not prematurely divided. Two Bloom syndrome lymphocytes, one with an extra chromosome 2 and the other with an extra chromosome 7, each having an inactivated centromere, show that this can also happen in monocentric autosomes.     

There are two mechanisms of achiasmate segregation in Drosophila females,one of which requires heterochromatic homology

There are numerous examples of the regular segregation of achiasmate chromosomes at meiosis I in Drosophila melanogaster females. Classically, the choice of achiasmate segregational partners has been thought to be independent of homology, but rather made on the basis of availability or similarities in size and shape. To the contrary, we show here that heterochromatic homology plays a primary role in ensuring the proper segregation of achiasmate homologs. We observe that the heterochromatin of chromosome 4 functions as, or contains, a meiotic pairing site. We show that free duplications carrying the 4th chromosome pericentric heterochromatin induce high frequencies of 4th chromosome nondisjunction regardless of their size. Moreover, a duplication from which some of the 4th chromosome heterochromatin has been removed is unable to induce 4th chromosome nondisjunction. Similarly, in the absence of either euchromatic homology or a size similarity, duplications bearing the X chromosome heterochromatin also disrupt the segregation of two achiasmate X chromosome centromeres. Although heterochromatic regions are sufficient to conjoin nonexchange homologues, we confirm that the segregation of heterologous chromosomes is determined by size, shape, and availability. The meiotic mutation Axs differentiates between these two processes of achiasmate centromere coorientation by disrupting only the homology-dependent mechanism. Thus there are two different mechanisms by which achiasmate segregational partners are chosen. We propose that the absence of diplotene-diakinesis during female meiosis allows heterochromatic pairings to persist until prometaphase and thus to co-orient homologous centromeres. We also propose that heterologous disjunctions result from a separate and homology-independent process that likely occurs during prometaphase. The latter process, which may not require the physical association of segregational partners, is similar to those observed in many insects, in Saccharomyces cerevisiae and in C. elegans males. We also suggest that the physical basis of this process may reflect known properties of the Drosophila meiotic spindle. © 1993 Wiley-Liss, Inc.     

The Cohesion Protein SOLO Associates with SMC1 and Is Required for Synapsis,Recombination, Homolog Bias and Cohesion and Pairing of Centromeres in Drosophila Meiosis

Cohesion between sister chromatids is mediated by cohesin and is essential for proper meiotic segregation of both sister chromatids and homologs. solo encodes a Drosophila meiosis-specific cohesion protein with no apparent sequence homology to cohesins that is required in male meiosis for centromere cohesion, proper orientation of sister centromeres and centromere enrichment of the cohesin subunit SMC1. In this study, we show that solo is involved in multiple aspects of meiosis in female Drosophila. Null mutations in solo caused the following phenotypes: 1) high frequencies of homolog and sister chromatid nondisjunction (NDJ) and sharply reduced frequencies of homolog exchange; 2) reduced transmission of a ring-X chromosome, an indicator of elevated frequencies of sister chromatid exchange (SCE); 3) premature loss of centromere pairing and cohesion during prophase I, as indicated by elevated foci counts of the centromere protein CID; 4) instability of the lateral elements (LE)s and central regions of synaptonemal complexes (SCs), as indicated by fragmented and spotty staining of the chromosome core/LE component SMC1 and the transverse filament protein C(3)G, respectively, at all stages of pachytene. SOLO and SMC1 are both enriched on centromeres throughout prophase I, co-align along the lateral elements of SCs and reciprocally co-immunoprecipitate from ovarian protein extracts. Our studies demonstrate that SOLO is closely associated with meiotic cohesin and required both for enrichment of cohesin on centromeres and stable assembly of cohesin into chromosome cores. These events underlie and are required for stable cohesion of centromeres, synapsis of homologous chromosomes, and a recombination mechanism that suppresses SCE to preferentially generate homolog crossovers (homolog bias). We propose that SOLO is a subunit of a specialized meiotic cohesin complex that mediates both centromeric and axial arm cohesion and promotes homolog bias as a component of chromosome cores.     

Telomere and centromere association tendencies in the human male metaphase complement

Summary Generalized distances between centromeres and between telomeres were statistically analyzed (x ² tests) in 100 trypsin-banded metaphase figures derived from normal males.Analysis of association tendencies on the first column of obtained c-c, p-p, q-p, and p-q histograms showed significant heterochromatin attraction not only between nonacrocentrics and acrocentrics but also between two nonacrocentric chromosome pairs (1 and 16). However since, not all c-heterochromatin-rich chromosomes were involved in associations (pair 5), and conversely, since chromosomes without an important centromeric heterochromatin block were involved in associations (pairs 8 and 11), it is probable that centromeric heterochromatin is not the only factor responsible for chromosome association. Moreover associations occur not only at the centromeres; in our circle analysis of the binding capacity of the telomeres or centromere of one chromosome pair with the telomeres or the centromeres of all other chromosome pairs, we also found preferential associations for T(4,13), T(9,15), T(11,15), T(13,19) T(15,19), T(17,18), T(17,22), and T(19,20).We therefore suggest that heterochromatin is not the only reason for chromosome association and that telomeres may also play an important part in this process.     

Temporal Characterization of Homology-Independent Centromere Coupling in Meiotic Prophase

Background

Over the past thirty years several reports of the pairing or association of non-homologous centromeres during meiotic prophase have appeared in the literature. Recently, the homology-independent pairwise association of centromeres, termed centromere coupling, was also reported in budding yeast. It seems paradoxical that centromeres would pair with non-homologous partners during a process intended to align homologous chromosomes, yet the conservation of this phenomenon across a wide range of species suggests it may play an important role in meiosis.

Principal Findings

To better define the role of this phenomenon in budding yeast, experiments were preformed to place centromere coupling within the context of landmark meiotic events. Soon after the initiation of the meiotic program, centromeres were found to re-organize from a single cluster into non-homologous couples. Centromere coupling is detected as soon as chromosome replication is finished and persists while the recombination protein Dmc1 is loaded onto the chromosomes, suggesting that centromere coupling persists through the time of double strand break formation. In the absence of the synaptonemal complex component, Zip1, centromere coupling was undetectable, at all times examined, confirming the essential role of this protein on this process. Finally, the timely release of centromere coupling depends on the recombination-initiating enzyme, Spo11, suggesting a connection between events in homologous pairing/recombination and the regulation of centromere coupling.

Conclusions

Based on our results we propose a role for centromere coupling in blocking interactions between homologous centromeres as recombination initiation is taking place.     

A dicentric chromosome of Drosophila melanogaster showing alternate centromere inactivation

Dicentric chromosomes are rarely found, because they interfere with normal cell division causing chromosome instability. By in situ hybridization of region-specific heterochromatic yeast artificial chromosomes we have found that the artificially generated C(1)A chromosome of Drosophila melanogaster has two potential centromeres: one carries all the sequences of the centromere of the Y chromosome and the other carries only a part of the Y centromeric region that is rich in telomere-related sequences. Immunostaining with anti-Bub1 (a kinetochore-specific marker) shows that, in spite of the differences in sequence, both centromeres can be active although as a rule only one at a time. In a small fraction of the chromosomes centromere inactivation is incomplete, giving rise to true dicentric chromosomes. The centromere inactivation is clonally inherited, providing a new example of epigenetic chromosome imprinting and the possibility of genetically dissecting this process. The involvement of telomere-related sequences in centromere function is discussed. Received: 15 September 1999; in revised form: 21 November 1999 / Accepted: 24 December 1999     

Electron microscopic band-interband pattern of the X chromosome in Drosophila hydei

The band-interband pattern of the salivary gland X chromosome in Drosophila hydei was studied by electron microscopy (EM) using the technique of surface-spread polytene (SSP) chromosome preparation. We observed 526 chromosome bands, i.e. 135 additional bands as compared with the original light microscopic chromosome map (Berendes 1963). Individual interband lengths and band thicknesses were measured for the entire X chromosome in electron micrographs of ten SSP chromosome preparations. Average values were used to plot an EM chromosome map. The average interband had an axial length of 0.38 m. Depending upon the extension of the DNA packing ratio in interbands, this indicates 1.1 kb of totally extended DNA or 3.8 kb, if a DNA packing ratio of 0.10 m/kb is assumed for SSP chromosomes (Kress et al. 1985).     

Chromatin organization in the male germ line of Drosophila hydei

The chromatin organization in developing germ cells of Drosophila hydei males was studied with the highly sensitive DNA stain DAPI (4, 6-diamidino-2-phenylindole dichloride). The prophase of meiosis I is characterized by decondensed chromosomes and only late during this stage do they condense rapidly. The sex chromosomes show allocycly. During postmeiotic development the final condensation of chromatin is preceded by a cycle of condensation and subsequent decondensation. Meiotic chromosomes were studied in more detail after orcein staining. Pairing sites of the sex chromosomes could be localized in the distal end of the heterochromatic arm of the X chromosome and distally in both arms of the Y chromosome. The various heterochromatic parts of the genome condense differentially in meiosis. Chromatin reorganization was studied cytochemically with antibodies raised against histones H1 and H2A of D. melanogaster. The core histone H2A is present in spermatid nuclei until the late elongation stage. However, histone H1 is not found in the chromatin later than the early primary spermatocyte stage. Thus, chromatin reorganization during spermatogenesis in D. hydei is complex. The process is discussed with regard to possible functions.     

Sequence of centromere separation: Analysis of mitotic chromosomes in man

Summary Mitotic chromosomes from human peripheral lymphocytes studied at the junction of metaphase and anaphase show that the centromeres of various chromosomes separate in a nonrandom, apparently genetically controlled sequence. It does not depend upon the position of the centromere in the chromosome, the length of the chromosome or total amount of detectable C-chromatin. In man, several chromosomes e.g. 18, 17, 2, separate very early. Such early cells do not include nos. 1, 13, 14, 15, and Y and very rarely nos. 21 and 22. The last separating chromosomes are those from group D, G, no. 1, 16, and Y. The possible implication of these findings in evolution, non-disjunction and the control of centromere separation sequences is discussed.This study was supported by a grant from National Institure of Health (GM 24591)     

The Genetic Analysis of Distributive Segregation in Drosophila Melanogaster. I. Isolation and Characterization of Aberrant X Segregation (Axs), a Mutation Defective in Chromosome Partner Choice

We describe the isolation and characterization of Aberrant X segregation (Axs), a dominant female-specific meiotic mutation. Although Axs has little or no effect on the frequency or distribution of exchange, or on the disjunction of exchange bivalents, nonexchange X chromosomes undergo nondisjunction at high frequencies in Axs/+ and Axs/Axs females. This increased X chromosome nondisjunction is shown to be a consequence of an Axs-induced defect in distributive segregation. In Axs-bearing females, fourth chromosome nondisjunction is observed only in the presence of nonexchange X chromosomes and is argued to be the result of improper X and fourth chromosome associations within the distributive system. In XX females bearing a compound fourth chromosome, the frequency of nonhomologous disjunction of the X chromosomes from the compound fourth chromosome is shown to account for at least 80% of the total X nondisjunction observed. In addition, Axs diminishes or ablates the capacity of nonexchange X chromosomes to form trivalents in females bearing either a Y chromosome or a small free duplication for the X. Axs also impairs compound X from Y segregation. The effect of Axs on these segregations parallels the defects observed for homologous nonexchange X chromosome disjunction in Axs females. In addition to its dramatic effects on the X chromosome, Axs exerts a similar effect on the segregation of a major autosome. We conclude that Axs defines a locus required for proper homolog disjunction within the distributive system.     

Sequence of centromere separation: generation of unstable multicentric chromosmes in a rat cell line

A transformed cell line, B₁, of cerebral endothelial origin from the Wistar-Kyoto male rat has chromatid and chromosome type bridges in virtually every cell. It exhibits various dicentric and polycentric chromosomes. Most dicentrics are symmetric isochromosomes. Certain isodicentrics are present in a fair segment of the cell population; however, almost all cells have some newly arising isodicentrics. The live cells show a lengthened prometaphase. Anaphase is also retarded possibly due to the occurrence of bridges. At anaphase some multicentrics split at only one centromere. When pulled to the two poles the unsplit centromeres and the distal chromosome segment form a side arm bridge. Another mechanism appears to be a total lack of separation of daughter centromeres at meta-anaphase (meiotic-like behavior of mitotic chromosomes). This is realized by the pulling of each of the two unsplit centromeres to opposite poles and results in bridges with both sister chromatids running parallel to each other. A break at corresponding weak points in the two sister chromatids followed by rejoining can form a dicentric isochromosome. A third mechanism, the breakage-fusion-bridge cycle, is also operative but would not produce isodicentrics. In the case of the first two mechanisms some or all centromeres apparently split between telophase and onset of the following DNA synthesis rather than at the usual time at late metaphase. These observations may suggest some previously unknown behavior of multicentric chromosomes during mitosis.     

The spatial localization of homologous chromosomes in human fibroblasts at mitosis

Chromosomes from ten human male fibroblast metaphases were completely reconstructed from electron micrographs of serially sectioned material. Chromosome centromere positions were determined by finding the three-dimensional coordinates of the centromere midpoint. The data set showed the identity of nine chromosome types (chromosomes 1, 2, 3, 6, 9, 16, 17, 18 and the Y chromosome) preserved as they are positioned in vivo. The results indicate that there is (1) no significant association of the homologous chromosomes examined, (2) a significant tendency for a central location of the Y chromosome and of chromosome 18, (3) a significant tendency for a peripheral location of chromosome 6, (4) no significant tendency for homologous chromosomes to reorganize as metaphase advances and (5) no significant differential condensation across the metaphase plate. Therefore, the only organization pattern observed for the centromeres of the homologous chromosomes studied is some sorting by size across the metaphase plate. These results may be typical of dividing cell types. Different chromosome arrangements are found in some non-dividing cell types (e.g. mammalian brain cells). The different distributions of chromosomes in different cell types can be considered as forms of nuclear differentiation. It is postulated that nuclear differentiation may be related to cell differentiation.     

The Synaptonemal Complex Protein Zip1 Promotes Bi-Orientation of Centromeres at Meiosis I

In meiosis I, homologous chromosomes become paired and then separate from one another to opposite poles of the spindle. In humans, errors in this process are a leading cause of birth defects, mental retardation, and infertility. In most organisms, crossing-over, or exchange, between the homologous partners provides a link that promotes their proper, bipolar, attachment to the spindle. Attachment of both partners to the same pole can sometimes be corrected during a delay that is triggered by the spindle checkpoint. Studies of non-exchange chromosomes have shown that centromere pairing serves as an alternative to exchange by orienting the centromeres for proper microtubule attachment. Here, we demonstrate a new role for the synaptonemal complex protein Zip1. Zip1 localizes to the centromeres of non-exchange chromosomes in pachytene and mediates centromere pairing and segregation of the partners at meiosis I. Exchange chromosomes were also found to experience Zip1-dependent pairing at their centromeres. Zip1 was found to persist at centromeres, after synaptonemal complex disassembly, remaining there until microtubule attachment. Disruption of this centromere pairing, in spindle checkpoint mutants, randomized the segregation of exchange chromosomes. These results demonstrate that Zip1-mediated pairing of exchange chromosome centromeres promotes an initial, bipolar attachment of microtubules. This activity of Zip1 lessens the load on the spindle checkpoint, greatly reducing the chance that the cell will exit the checkpoint delay with an improperly oriented chromosome pair. Thus exchange, the spindle checkpoint, and centromere pairing are complementary mechanisms that ensure the proper segregation of homologous partners at meiosis I.     

Terminal regions of wheat chromosomes select their pairing partners in meiosis

Many plant species, including important crops like wheat, are polyploids that carry more than two sets of genetically related chromosomes capable of meiotic pairing. To safeguard a diploid-like behavior at meiosis, many polyploids evolved genetic loci that suppress incorrect pairing and recombination of homeologues. The Ph1 locus in wheat was proposed to ensure homologous pairing by controlling the specificity of centromere associations that precede chromosome pairing. Using wheat chromosomes that carry rye centromeres, we show that the centromere associations in early meiosis are not based on homology and that the Ph1 locus has no effect on such associations. Although centromeres indeed undergo a switch from nonhomologous to homologous associations in meiosis, this process is driven by the terminally initiated synapsis. The centromere has no effect on metaphase I chiasmate chromosome associations: homologs with identical or different centromeres, in the presence and absence of Ph1, pair the same. A FISH analysis of the behavior of centromeres and distal chromomeres in telocentric and bi-armed chromosomes demonstrates that it is not the centromeric, but rather the subtelomeric, regions that are involved in the correct partner recognition and selection.     

Specific recognition and differential affinity of meiotic X-Y pairing sites in Lucilia cuprina males (Diptera: Calliphoridae)

Meiotic pairing of X and Y chromosomes in male Lucilia cuprina was studied by cytological observation of normal, rearranged and deficient sex chromosome karyotypes in spermatogenesis. Two X-Y pairing regions located distally in each arm of the X and Y chromosomes were defined. Contrasting with findings in Drosophila melanogaster, these pairing regions show specific recognition of their partners. By studying rearranged sex chromosomes short arm pairing was localised to their distal ends, closely associated with secondary constrictions containing nucleolar organisers in both sex chromosomes. Short arm pairing is very tight and not greatly disrupted by chromosome rearrangement, deficiency for the Y chromosome long arm or the presence of supernumerary X chromosomes. The pairing region of the long arms could not be precisely localised but probably also occurs at their distal ends. Pairing between the long arm sites is much weaker and is easily disrupted by chromosome rearrangement, failing completely in flies deficient for the Y chromosome short arm. No cytologically visible pairing was seen between X chromosomes and the remainder of the Y. In males with an extra X chromosome, the ends of both X chromosomes pair to form multivalents with normal and rearranged Y chromosomes provided the Y short arm is present, otherwise an independent X chromosome bivalent is formed. The mechanism of pairing in male Lucilia sex chromosomes thus seems to depend on specific loci of distinctive structure within the X and Y heterochromatin. Comparison of cytological and genetic data shows that increasing cytological pairing failure is matched by higher genetic X-Y nondisjunction but that the former occurs at much higher levels. In some karyotypes cytologically observed X-Y pairing failure is not matched by high frequencies of nondisjunction presumably because weak pairing associations are disrupted during slide preparation.