Features of the chromocenter structure in Drosophila melanogaster larva cells, mutant for genes C(3)G and NOD

Homolog pairing, chromosome morphology, and chromosome disjunction in the first meiotic division were studied in the oocytes of c(3)G/c(3)G female Drosophila melanogaster at developmental stages 3-4 and 14. It was found that homologs were completely or partly paired in some cells (about 20% in either case). The lengths of chromosomes in +/+, +/c(3)G, and c(3)G/c(3)G cells were at a ratio of 1.0:1.6:2.2. The chromocenters of homozygous cells had an abnormal structure. There was no meiotic block in metaphase 1, and chromosomes only segregated equally in about 80% of anaphases of the first meiotic division. The data obtained correspond to the abnormal variants of the formation of the chromocenter in c(3)G/c(3)G females that could be predicted based on the two-ring structure of the chromocenter. The mechanism of the effect of the homo- and heterozygosity for the hypomorphic mutation c(3)G on the formation of the synaptonemal complex (SC) and crossing over frequency was suggested. In nod/nod homozygous females, asynapsis of pericentromeric regions of homologs was observed in the chromocenter. It was assumed that NOD kinezin is necessary at the last stages of pairing of the pericentromeric regions of homologs and formation of the coordinating bonds between them.     

Role of the Chromocenter in Nonrandom Meiotic Segregation of Nonhomologous Chromosomes inDrosophila melanogasterFemales

The evidence supporting universal significance of physical links between pericentromeric regions of homologous chromosomes for their bipolar orientation during the first meiotic division is discussed. The pericentromeric chiasmata between homologs or (in the absence of the latter) chromocentric links between nonhomologs, which are preserved until prometaphase, compensate for the disturbed binding between homologous pericentromeric regions in both structural or locus mutants. When the links between nonhomologs are involved, interchromosomal effects on chromosome disjunction and nonhomologous pairing were revealed by the genetic methods. An explanation suggested for genetic events observed during Drosophilameiosis conforms with the original, cytogenetically proved model of the orderly two-ring chromocenter formation and reorganization.     

Puzzling Chromosome Behavior in Triploid Females of Drosophila melanogaster

Based on a particular formation of the chromocenter and trivalents in triploid Drosophila females, as well as on asynapsis in pericentromeric regions (which is a result of trivalent competition), an explanation for the increased frequency of crossing over and nonrandom segregation of the X chromosomes and autosomes in the first meiotic division is suggested. It is proposed that a delay in pairing of the pericentromeric heterochromatic chromosome regions combined into a single chromocenter leads to the following: (1) formation of the heteroduplex structures (X structures) takes more time and, consequently, their number and the frequency of crossing over in the paired chromosome regions increases; (2) in nonhomologous chromosomes, the chromocentral connections, which normally degrade in prometaphase, are retained to fulfill a function of coorientation during the first meiotic division.     

Bivalent individualization during chromosome territory formation in Drosophila spermatocytes by controlled condensin II protein activity and additional force generators

Reduction of genome ploidy from diploid to haploid necessitates stable pairing of homologous chromosomes into bivalents before the start of the first meiotic division. Importantly, this chromosome pairing must avoid interlocking of non-homologous chromosomes. In spermatocytes of Drosophila melanogaster, where homolog pairing does not involve synaptonemal complex formation and crossovers, associations between non-homologous chromosomes are broken up by chromosome territory formation in early spermatocytes. Extensive non-homologous associations arise from the coalescence of the large blocks of pericentromeric heterochromatin into a chromocenter and from centromere clustering. Nevertheless, during territory formation, bivalents are moved apart into spatially separate subnuclear regions. The condensin II subunits, Cap-D3 and Cap-H2, have been implicated, but the remarkable separation of bivalents during interphase might require more than just condensin II. For further characterization of this process, we have applied time-lapse imaging using fluorescent markers of centromeres, telomeres and DNA satellites in pericentromeric heterochromatin. We describe the dynamics of the disruption of centromere clusters and the chromocenter in normal spermatocytes. Mutations in Cap-D3 and Cap-H2 abolish chromocenter disruption, resulting in excessive chromosome missegregation during M I. Chromocenter persistence in the mutants is not mediated by the special system, which conjoins homologs in compensation for the absence of crossovers in Drosophila spermatocytes. However, overexpression of Cap-H2 precluded conjunction between autosomal homologs, resulting in random segregation of univalents. Interestingly, Cap-D3 and Cap-H2 mutant spermatocytes displayed conspicuous stretching of the chromocenter, as well as occasional chromocenter disruption, suggesting that territory formation might involve forces unrelated to condensin II. While the molecular basis of these forces remains to be clarified, they are not destroyed by inhibitors of F actin and microtubules. Our results indicate that condensin II activity promotes chromosome territory formation in co-operation with additional force generators and that careful co-ordination with alternative homolog conjunction is crucial.     

The role of the chromocenter in nonrandom meiotic segregation of nonhomologous chromosomes in Drosophila melanogaster females

The evidence supporting universal significance of physical links between pericentromeric regions of homologous chromosomes for their bipolar orientation during the first meiotic division is discussed. The pericentromeric chiasmata between homologs or (in the absence of the latter) chromocentric links between nonhomologs, which are preserved until prometaphase, compensate for the disturbed binding between homologous pericentromeric regions in both structural or locus mutants. When the links between nonhomologs are involved, interchromosomal effects on chromosome disjunction and nonhomologous pairing were revealed by the genetic methods. An explanation suggested for genetic events observed during Drosophila meiosis conforms with the original, cytogenetically proved model of the orderly two-ring chromocenter formation and reorganization.     

Puzzling chromosome behavior in triploid females of Drosophila melanogaster

Based on a particular formation of the chromocenter and trivalents in triploid Drosophila females, as well as on asynapsis in pericentromeric regions (which is a result of trivalent competition), an explanation for the increased frequency of crossing over and nonrandom segregation of the X chromosomes and autosomes in the first meiotic division is suggested. It is proposed that a delay in pairing of the pericentromeric heterochromatic chromosome regions combined into a single chromocenter leads to the following: (1) formation of the heteroduplex structures (X structures) takes more time and, consequently, their number and the frequency of crossing over in the paired chromosome regions increases; (2) in nonhomologous chromosomes, the chromocentral connections, which normally degrade in prometaphase, are retained to fulfill a function of coorientation during the first meiotic division.     

Chromocentral nature of interchromosome connections coorienting nonhomologous chromosomes in meiosis of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> females

Data are presented in favor of universal significance of physical connections between pericentromeric regions of homologs in their orientation to the opposite poles of the first meiotic division in Drosophila melanogaster. Disturbances in the formation of such connections caused by structural or locus mutations are compensated for by the presence of pericentromeric chiasmata between homologs or (in the case of their absence) by chromocentral connections between nonhomologs being preserved up to the prometaphase. In the latter case, an interchromosome effect on chromosome disjunction and nonhomologous pairing is registered by genetic methods. Inhibition of the formation of the division spindle fibers during prometaphase of meiosis 1 by the long-term action of colcemide promotes the retention of connections between paired nonexchanged homologs and between nonhomologous chromosomes with abnormal homologous pairing because of heterozygosity for numerous inversions and transpositions (X and autosome 2). These connections are registered cytologically. Cytologically registered are also connections between normal X chromosomes and metacentric compounds by the arms of autosome 2 (C(2L)RM, C(2R)RM), which is the known case of the interchromosome effect on chromosome nondisjunction. It is supposed that cytologically detected associations between compounds are realized through a normal mechanism, as a result of interaction and formation of orienting connections between the homologous pericentromeric regions of these compounds. Cytological evidence is presented for colocation of compounds in the chromocentrally organized nucleus of somatic and germline cells.     

A comparison of intra-and inter-gene recombination in the meiotic mutant c(3)G of drosophila melanogaster

In females homozygous for the meiotic mutant c(3)G, a reasonable amount of intergenic recombination was found to occur, while no intragenic recombination could be detected at the white locus. On the other hand, a significant increase in both inter-and intragenic recombination was observed in females heterozygous for the same mutation. These results are discussed in the context of the synaptonemal complex formation during chromosome pairing in the mutant.     

Genetic Control of the Formation and Reorganization of Chromocenter in Drosophila

The chromocenter integrates the entire Drosophilagenome into a unit. The formation and reorganization of chromocenter are genetically determined. Currently, several mutations affecting the structure of chromocenter have been described. In this work, I present evidence on the time of the formation and reorganization of chromocenter in mitotic and meiotic cells of females of the wild type and the ff16mutant line obtained by selection of mosaic clones produced from mitotic recombination of chromosomes in the dividing embryo cells. In females homozygous for this mutation, the second stage of the formation of chromocenter (joining two groups of nonhomologous chromosomes X-4 and 2-3 into a united ring structure =X=2=3=4=) is disturbed. The differences between the mitotic and meiotic reorganization of chromocenter and the role of chromocenter in the control of chromosome segregation are discussed.     

Survival ofDrosophila melanogaster Progeny after Prolonged Suppression of Pairing and Recombination in Autosome 3

Two laboratory strains of Drosophila melanogaster carrying autosome 3 with a meiotic mutation c(3)G, that is maintained since 1985 in various balancer chromosomes, were used to study progeny survival. The conditions of maintenance of these strains and the effect of c(3)G mutation completely suppress pairing and crossing over in autosome 3. In addition, selection pressure was reduced because of permanent heterozygosity, mediating mutation accumulation in the studied chromosome. In both strains, all homozygotes for autosome 3 (c(3)G/c(3)G) perished. The hybrid homozygotes carrying chromosomes with c(3)G mutation from different strains survived in 0.4 of the progeny. Higher viability was observed after normal pairing and meiotic recombination of the studied chromosome with the chromosome from the wild-type line. The possible nature of mutations accumulated after prolonged suppression of chromosome pairing and recombination is discussed.     

Chromosome pairing in meiosis of partially fertile wheat/rye hybrids

The normal course of meiosis depends on regular pairing of homologous chromosomes. In intergeneric hybrids, including those of wheat, there is no chromosome pairing because there are no homologs. In F₁ wheat/rye hybrids, pairing is largely prevented by the pairing homoeologous1 (Ph1) gene. In its presence, there are only rare instances of pairing; most chromosomes are univalent, and their orientation at metaphase I initiates different pathways of the meiotic cycle. The meiotic-like pathway includes a combination of the reductional and the equational + reductional steps at AI followed by the second division. The resulting gametes are mostly non-functional. The mitotic-like pathway involves equational division of univalents at AI and the absence of the second division. Any fertility of wheat/rye hybrids depends on the production of unreduced gametes arising from meiotic restitution (mitotic-like division). We examined the meiotic pairing in wheat/rye hybrids created from wheat lines with single rye chromosome substitutions and Ph1 present. This guaranteed F₁ meiosis with one pair of rye homologs. All hybrids formed bivalents, but proportions of meiocytes with bivalents varied. In the meiocytes where bivalents were present, there was a higher tendency for the meiotic-like pathway, while in meiocytes where bivalent pairing failed, the tendency was stronger for the mitotic-like pathway. Among the equationally dividing cells, we observed more than 90 % of meiocytes without bivalents, where rye homologs did not form bivalents, too. The data indicate a potential application of wheat/rye lines in producing genetic stocks of amphidiploids with designated genomic constitutions.     

Effects of several meiotic mutations on female meiosis in maize

A modified enzyme digestion technique of ovary isolation followed by staining and squash preparation has allowed us to observe female meiosis in normal maize meiotically dividing megaspore mother cells (MMCs). The first meiotic division in megasporogenesis of maize is not distinguishable from that in mi-crosporogenesis. The second female meiotic division is characterized as follows: (1) the two products of the first meiotic division do not simultaneously enter into the second meiotic division; as a rule, the chalazal-most cell enters division earlier than the micropylar one, (2) often the second of the two products does not proceed with meiosis, but degenerates, and (3) only a single haploid meiotic product of the tetrad remains alive, and this cell proceeds with three rounds of mitoses without any intervening cell wall formation to produce the eight-nucleate embryo sac. This technique has allowed us to study the effects of five meiotic mutations (aml, aml-pral, afdl, dsy *-9101, and dvl) on female meiosis in maize. The effects of the two alleles of the aml gene (aml and aml-pral) and of the afdl and dsy *-9101mutations are the same in both male and female meiosis. The aml allele prevents the entrance of MMCs into meiosis and meiosis is replaced by mitosis; the aml-pral permits MMCs to enter into meiosis, but their progress is stopped at early prophase I stages. The afdl gene is responsible for substitution of the first meiotic (reductional) division by an equational division including the segregation of sister chromatid centromeres at anaphase I. The dsy * -9101 gene exhibits abnormal chromosome pairing; paired homologous chromosomes are visible at pachytene, but only univalents are observed at diakinesis and metaphase I stages. These mutation specific patterns of abnormal meiosis are responsible for the bisexual sterility of these meiotic mutants. The abnormal divergent shape of the spindle apparatus and the resulting abnormal segregation of homologous chromosomes observed in micro-sporogenesis in plants homozygous for the dv1 mutation have not been found in meiosis of megasporogenesis. Only male sterility is induced by the dv1 gene in the homozygous condition. © 1993 Wiley-Liss, Inc.     

Genetic control of formation and reorganization of the chromocenter in drosophila

The chromocenter integrates the entire Drosophila genome into a unit. The formation and reorganization of chromocenter are genetically determined. Currently, several mutations affecting the structure of chromocenter have been described. In this work, I present evidence on the time of the formation and reorganization of chromocenter in mitotic and meiotic cells of females of the wild type and the ff16 mutant line obtained by selection of mosaic clones produced from mitotic recombination of chromosomes in the dividing embryo cells. In females homozygous for this mutation, the second stage of the formation of chromocenter (joining two groups of nonhomologous chromosomes X-4 and 2-4 into a united ring structure -X-2-3-4-) is disturbed. The differences between the mitotic and meiotic reorganization of chromocenter and the role of chromocenter in the control of chromosome segregation are discussed.     

A cytological approach to studying meiotic recombination and chromosome dynamics in Arabidopsis thaliana male meiocytes in three dimensions

During meiotic prophase I chromosomes undergo dramatic conformational changes that accompany chromosome condensation, pairing and recombination between homologs. These changes include the anchoring of telomeres to the nuclear envelope and their clustering to form a bouquet. In plants, these events have been studied and illustrated in intact meiocytes of species with large genomes. Arabidopsis thaliana is an excellent genetic model in which major molecular pathways that control synapsis and recombination between homologs have been uncovered. Yet the study of chromosome dynamics is hampered by current cytological methods that disrupt the three‐dimensional (3D) architecture of the nucleus. Here we set up a protocol to preserve the 3D configuration of A. thaliana meiocytes. We showed that this technique is compatible with the use of a variety of antibodies that label structural and recombination proteins and were able to highlight the presence of clustered synapsis initiation centers at the nuclear periphery. By using fluorescence in situ hybridization we also studied the behavior of chromosomes during pre‐meiotic G₂ and prophase I, revealing the existence of a telomere bouquet during A. thaliana male meiosis. In addition we showed that the number of telomeres in a bouquet and its volume vary greatly, thus revealing the complexity of telomere behavior during meiotic prophase I. Finally, by using probes that label subtelomeric regions of individual chromosomes, we revealed differential localization behaviors of chromosome ends. Our protocol opens new areas of research for investigating chromosome dynamics in A. thaliana meiocytes.     

mei-2, a mutagen-sensitive mutant of Neurospora defective in chromosome pairing and meiotic recombination

Summary A Neurospora crassa mutation, mei-2, affecting meiosis and mutagen sensitivity, was characterized for its effect on meiotic recombination and chromosome pairing. Results from homozygous mei-2 crosses involving distant markers on the same chromosome demonstrated a drastic reduction in meiotic recombination. However, mitotic recombination continued to occur. Cytological observations indicated that pairing of homologous chromosomes in zygotene was greatly reduced or absent, resulting in aberrant segregation at anaphase I and often at subsequent divisions as well. The few mature ascospores produced were frequently disomic for one or more chromosomes.     

Evidence That Masking of Synapsis Imperfections Counterbalances Quality Control to Promote Efficient Meiosis

Reduction in ploidy to generate haploid gametes during sexual reproduction is accomplished by the specialized cell division program of meiosis. Pairing between homologous chromosomes and assembly of the synaptonemal complex at their interface (synapsis) represent intermediate steps in the meiotic program that are essential to form crossover recombination-based linkages between homologs, which in turn enable segregation of the homologs to opposite poles at the meiosis I division. Here, we challenge the mechanisms of pairing and synapsis during C. elegans meiosis by disrupting the normal 1∶1 correspondence between homologs through karyotype manipulation. Using a combination of cytological tools, including S-phase labeling to specifically identify X chromosome territories in highly synchronous cohorts of nuclei and 3D rendering to visualize meiotic chromosome structures and organization, our analysis of trisomic (triplo-X) and polyploid meiosis provides insight into the principles governing pairing and synapsis and how the meiotic program is “wired” to maximize successful sexual reproduction. We show that chromosomes sort into homologous groups regardless of chromosome number, then preferentially achieve pairwise synapsis during a period of active chromosome mobilization. Further, comparisons of synapsis configurations in triplo-X germ cells that are proficient or defective for initiating recombination suggest a role for recombination in restricting chromosomal interactions to a pairwise state. Increased numbers of homologs prolong markers of the chromosome mobilization phase and/or boost germline apoptosis, consistent with triggering quality control mechanisms that promote resolution of synapsis problems and/or cull meiocytes containing synapsis defects. However, we also uncover evidence for the existence of mechanisms that “mask” defects, thus allowing resumption of prophase progression and survival of germ cells despite some asynapsis. We propose that coupling of saturable masking mechanisms with stringent quality controls maximizes meiotic success by making progression and survival dependent on achieving a level of synapsis sufficient for crossover formation without requiring perfect synapsis.     

Cytological studies of heterochromatin function in the Drosophila melanogaster male: autosomal meiotic pairing

In Drosophila melanogaster it is now documented that the different satellite DNA sequences make up the majority of the centromeric heterochromatin of all chromosomes. The most popular hypothesis on this class of DNA is that satellite DNA itself is important to the pairing processes of chromosomes. Evidence in support of such a hypothesis is, however, circumstantial. This hypothesis has been evaluated by direct cytological examination of the meiotic behaviour of heterochromatically and/or euchromatically rearranged autosomes in the male. It was found that neither substantial deletions nor rearrangements of the autosomal heterochromatin cause any disruption of meiotic pairing. Autosomal pairing depends on homologs retaining sufficient euchromatic homology. This is the first clear demonstration that the highly repeated satellite DNA sequences in the heterochromatin of the second, third and fourth chromosomes are not important in meiotic pairing, but rather that some euchromatic homology in the autosomes is essential to ensure a regular meiotic process. These results on the autosomes, when taken in conjunction with our previous studies on sex chromosome pairing, clearly indicate that satellite DNA is not crucial for male meiotic chromosome pairing of any member of the D. melanogaster genome.     

Molecular Mechanisms of Homologous Chromosome Pairing and Segregation in Plants

In most eukaryotic species, three basic steps of pairing, recombination and synapsis occur during prophase of meiosis I. Homologous chromosomal pairing and recombination are essential for accurate segregation of chromosomes. In contrast to the well-studied processes such as recombination and synapsis, many aspects of chromosome pairing are still obscure. Recent progress in several species indicates that the telomere bouquet formation can facilitate homologous chromosome pairing by bringing chromosome ends into close proximity, but the sole presence of telomere clustering is not sufficient for recognizing homologous pairs. On the other hand, accurate segregation of the genetic material from parent to offspring during meiosis is dependent on the segregation of homologs in the reductional meiotic division （MI） with sister kinetochores exhibiting mono-orientation from the same pole, and the segregation of sister chromatids during the equational meiotic division （MII） with kinetochores showing bi-orientation from the two poles. The underlying mechanism of orientation and segregation is still unclear. Here we focus on recent studies in plants and other species that provide insight into how chromosomes find their partners and mechanisms mediating chromosomal segregation.     

A Cis-Acting Locus That Promotes Crossing over between X Chromosomes in Caenorhabditis Elegans

This study reports the characterization of a cis-acting locus on the Caenorhabditis elegans X chromosome that is crucial for promoting normal levels of crossing over specifically between the X homologs and for ensuring their proper disjunction at meiosis I. The function of this locus is disrupted by the mutation me8, which maps to the extreme left end of the X chromosome within the region previously implicated by studies of X;A translocations and X duplications to contain a meiotic pairing site. Hermaphrodites homozygous for a deletion of the locus (Df/Df) or heterozygous for a deletion and the me8 mutation (me8/Df) exhibit extremely high levels of X chromosome nondisjunction at the reductional division; this is correlated with a sharp decrease in crossing over between the X homologs as evidenced both by reductions in genetic map distances and by the presence of achiasmate chromosomes in cytological preparations of oocyte nuclei. Duplications of the wild-type region that are unlinked to the X chromosome cannot complement the recombination and disjunction defects in trans, indicating that this region must be present in cis to the X chromosome to ensure normal levels of crossing over and proper homolog disjunction. me8 homozygotes exhibit an altered distribution of crossovers along the X chromosome that suggests a defect in processivity along the X chromosome of an event that initiates at the chromosome end. Models are discussed in which the cis-acting locus deleted by the Dfs functions as a meiotic pairing center that recruits trans-acting factors onto the chromosomes to nucleate assembly of a crossover-competent complex between the X homologs. This pairing center might function in the process of homolog recognition, or in the initiation of homologous synapsis.     

Genetic studies on heterochromatin in Drosophila melanogaster and their implications for the functions of satellite DNA

In Drosophila melanogaster the centromeric heterochromatin of all chromosomes consists almost entirely of several different satellite DNA sequences. In view of this we have examined by genetic means the meiotic consequences of X chromosomes with partial deletions of their heterochromatin, and have found that the amount and position of recombination on each heterochromatically deleted X is substantially different from that of a normal X. It appears that the amount of heterochromatin is important in modifying the centromere effect on recombination. — In all the deleted Xs tested, chromosome segregation is not appreciably altered from that of a nondeleted control chromosome. Thus satellite DNA does not appear to be an important factor in determining the regular segregation of sex chromosomes in Drosophila. Additionally, since X chromosomes with massive satellite DNA deficiencies are able to participate in a chromocenter within salivary gland nuclei, a major role of satellite DNA in chromocenter formation in this tissue is also quite unlikely. — In order to examine the mechanisms by which the amount of satellite DNA is increased or decreased in vivo, we have measured cytologically the frequency of spontaneous sister chromatid exchanges in a ring Y chromosome which is entirely heterochromatic and consists almost exclusively of satellite DNA. In larval neuroblast cells the frequency of spontaneous SCE in this Y is approximately 0.3% per cell division. Since there is no meiotic recombination in D. melanogaster males and since meiotic recombination in the female does not occur in heterochromatin, our results provide a minimum estimate of the in vivo frequency of SCE in C-banded heterochromatin (which is predominantly simple sequence DNA), without the usual complications of substituted base analogs, incorporated radioactive label or substantial genetic content. — We emphasise that: (a) satellite DNA is not implicated in any major way in recognition processes such as meiotic homologue recognition or chromocenter formation in salivaries, (b) there is likely to be continuous variation in the amount of satellite DNA between individuals of a species; and (c) the amount of satellite DNA can have a crucial functional role in the meiotic recombination system.