Sex Heterochromatin Research Articles

Structural and Evolutionary Relationships in the Giant Sex Chromosomes of Three Microtus Species.

The genus Microtus has high karyotypic diversity. The existence of notable differences in the length of its sex chromosomes contributes to this variation. Variations in size are attributed to the enlargement of their heterochromatin content, which is of such magnitude in some species that they are referred to as “giant sex chromosomes”. Here, we perform an intra- and interspecific analysis of the molecular composition of the heterochromatic blocks in three species with giant sex chromosomes (Microtus chrotorrhinus, M. cabrerae and M. agrestis). Our results show that the heterochromatic content is very similar in both the X and Y chromosomes of M. chrotorrhinus, and that their molecular composition is more closely related to the heterochromatic blocks of M. agrestis than to the sex heterochromatin of M. cabrerae; however, species-specific differences do clearly exist. Interestingly, the euchromatic regions of the X chromosome of all three of these species share a homologous region composed of heterochromatic-related sequences. Our results therefore reinforce the idea that certain similarities in the original organization of these X chromosomes could have facilitated their later enlargement.

Epigenetic modifications in sex heterochromatin of vole rodents.

The genome of some vole rodents contains large blocks of heterochromatin coupled to the sex chromosomes. While the DNA content of these heterochromatic blocks has been extensively analyzed, little is known about the epigenetic modifications controlling their structure and dynamics. To better understand its organization and functions within the nucleus, we have compared the distribution pattern of several epigenetic marks in cells from two species, Microtus agrestis and Microtus cabrerae. We first could show that the heterochromatic blocks are identifiable within the nuclei due to their AT enrichment detectable by DAPI staining. By immunostaining analyses, we demonstrated that enrichment in H3K9me3 and HP1, depletion of DNA methylation as well as H4K8ac and H3K4me2, are major conserved epigenetic features of this heterochromatin in both sex chromosomes. Furthermore, we provide evidence of transcriptional activity for some repeated DNAs in cultivated cells. These transcripts are partially polyadenylated and their levels are not altered during mitotic arrest. In summary, we show here that enrichment in H3K9me3 and HP1, DNA demethylation, and transcriptional activity are major epigenetic features of sex heterochromatin in vole rodents.

New method for visualization of C-heterochromatin in synaptonemal complex spreads

DAPI staining of the metaphase chromosomes pretreated with barium hydroxide generates a C-like banding pattern. In this work a protocol for visualizing similar pattern at the synaptonemal complex (SC) spreads after immunostaining is suggested. This method was used to visualize centromeric and sex heterochromatin at the SC spreads of guppy fish (Poecilia reticulata Peters, 1859). The efficiency of this method was further confirmed at SC spreads of the northern red-backed vole (Myodes rutilus (Pallas, 1779)), the guinea pig (Cavia porcellus (Linnaeus, 1758)), and the pigmy shrew (Sorex minutus Linnaeus, 1766).

Identification of the female-determining region of the W chromosome in Bombyx mori

The W chromosome of the silkworm Bombyx mori is devoid of functional genes, except for the putative female-determining gene (Fem). To localize Fem, we investigated the presence of W-specific DNA markers on strains in which an autosomal fragment containing dominant marker genes was attached to the W chromosome. We produced new W-chromosomal fragments from the existing Zebra-W strain (T(W;3)Ze chromosome) by X-irradiation, and then carried out deletion mapping of these and sex-limited yellow cocoon strains (T(W;2)Y-Chu, -Abe and -Ban types) from different Japanese stock centers. Of 12 RAPD markers identified in the normal W chromosomes of most silkworm strains in Japan, the newly irradiated W(B-YL-YS)Ze chromosome contained three, the T(W;2)Y-Chu chromosome contained six, and the T(W;2)Y-Abe and -Ban chromosomes contained only one (W-Rikishi). To investigate the ability of the reduced W-chromosome translocation fragments to form heterochromatin bodies, which are found in nuclei of normal adult female sucking stomachs, we examined cells of the normal type p50 strain and the T(W;2)Y-Chu and -Abe strains. A single sex heterochromatin body was found in nuclei of p50 females, whereas we detected only small sex heterochromatin bodies in the T(W;2)Y-Chu strain and no sex heterochromatin body in the T(W;2)Y-Abe strain. Since adult females of all strains were normal and fertile, we conclude that only extremely limited region, containing the W-Rikishi RAPD sequence of the W chromosome, is required to determine femaleness. Based on a comparison of the normal W-chromosome and 7 translocation and W-deletion strains we present a map of Fem relative to the 12 W-specific RAPD markers.

Distribution of L1-retroposons on the giant sex chromosomes of Microtus cabrerae (Arvicolidae, Rodentia): functional and evolutionary implications

Long interspersed nuclear elements (L1 or LINE-1) are the most abundant and active retroposons in the mammalian genome. Traditionally, the bulk of L1 sequences have been explained by the 'selfish DNA' hypothesis; however, recently it has been also argued that L1s could play an important role in genome and gene organizations. The non-random chromosomal distribution of these retroelements is a striking feature considered to reflect this functionality. In the present study we have cloned and analyzed three different L1 fragments from the genome of the rodent Microtus cabrerae. In addition, we have examined the chromosomal distribution of this L1 in several species of Microtus, a very interesting group owing to the presence in some species of enlarged ('giant') sex chromosomes. Interestingly, in all species analyzed, L1-retroposons have preferentially accumulated on both the giant- and the normal-sized sex chromosomes compared with the autosomes. Also we have demonstrated that L1-retroposons are not similarly distributed among the heterochromatic blocks of the giant sex chromosomes in M. cabrerae and M. agrestis, which suggest that L1 retroposition and amplification over the sex heterochromatin have been different and independent processes in each species. Finally, we proposed that the main factors responsible for the L1 distribution on the mammalian sex chromosomes are the heterochromatic nature of the Y chromosome and the possible role of L1 sequences during the X-inactivation process.

Sex chromatin body as a marker of radiation-induced sex chromosome aberrations in the potato tuber moth, Phthorimaea operculella (Lepidoptera: Gelechiidae)

Genetic sexing technique based on the construction of a balanced lethal strain (BLS) has been proposed for Phthorimaea operculella (Zeller). The isolation of female with T(W;Z) translocation is a fundamental step to develop such a strain. Gamma irradiation was used to induce the requested translocations. The availability of sex-linked morphological marker is required to facilitate the detection of such mutations. Since a visible sex-linked marker has not been found in P. operculella, the main objective of our study was therefore to determine the possibility of using sex heterochromatin body as a marker to identify the required translocated females. The appearance of sex heterochromatin body and the analysis of sex chromosomes in F1 females of irradiated P. operculella females were investigated. The percentage of abnormality in sex heterochromatin body in highly polyploid Malpighian tubule nuclei was positively correlated with the applied dose. Based on the appearance of this body, three mutant lines were isolated: elongated, small and fragmented lines. W chromosome was easily distinguished from Z chromosome when the analysis of pachytene sex chromosome bivalents of P. operculella females was carried out. The aberrations involved in W chromosome directly influenced the appearance of sex heterochromatin body in highly polyploid somatic cells of the isolated mutant lines. The results showed that sex heterochromatin could be used as sex determination and cytogenetic marker in P. operculella.

X chromosome painting in Microtus: Origin and evolution of the giant sex chromosomes

Sex chromosomes in species of the genus Microtus present some characteristic features that make them a very interesting group to study sex chromosome composition and evolution. M. cabrerae and M. agrestis have enlarged sex chromosomes (known as 'giant sex chromosomes') due to the presence of large heterochromatic blocks. By chromosome microdissection, we have generated probes from the X chromosome of both species and hybridized on chromosomes from six Microtus and one Arvicola species. Our results demonstrated that euchromatic regions of X chromosomes in Microtus are highly conserved, as occurs in other mammalian groups. The sex chromosomes heterochromatic blocks are probably originated by fast amplification of different sequences, each with an independent origin and evolution in each species. For this reason, the sex heterochromatin in Microtus species is highly heterogeneous within species (with different composition for the Y and X heterochromatic regions in M. cabrerae) and between species (as the composition of M. agrestis and M. cabrerae sex heterochromatin is different). In addition, the X chromosome painting results on autosomes of several species suggest that, during karyotypic evolution of the genus Microtus, some rearrangements have probably occurred between sex chromosomes and autosomes.

Cytogenetic analysis shows that the unusually large chromosome in the sex-limited pB silkworm (Bombyx mori) strain consists of three chromosomes.

We have discovered an inordinately large chromosome pair at the pachytene stage in the oocyte of the sex-limited pB (black larval marking) silkworm (Bombyx mori) strain (TWPB). We have analyzed the composition and arrangement of this large chromosome. A genetic linkage analysis shows that the large chromosome is made up of the W chromosome, the second chromosome fragment (pB fragment), and the fifth chromosome (linkage group) containing at least the region from map position 0.0 to 40.8. We also observed a sex heterochromatin body (SB) that we deduced to be made up of condensed W chromosomes. The number of SBs in each female nucleus among the sucking stomach cells of the TWPB strain was variable. Evidently, the W chromosome of the TWPB strain is attached to another chromosome. The composition of the W chromosome, the second chromosome fragment, and the fifth chromosome was studied through linkage analysis for these three chromosomes. We used two strains derived from the TWPB strain, the sex-limited pM (moricaud larval marking)-like (TWPML) and the autosomal pM-like (T5PML). The results show that the TWPML strain originates through a detachment of the fifth chromosome from the large chromosome of the TWPB strain, and the T5PML strain originates through a detachment of the W chromosome from that. Accordingly, the large chromosome of the TWPB strain is arranged in the order W chromosome--second chromosome fragment--fifth chromosome.

Molecular characterization of the complex sex-chromosome heterochromatin in the rodent Microtus chrotorrhinus.

The sex chromosomes of Microtus chrotorrhinus are unusually large compared to those of other mammals, comprising about 20% of the karyotype and containing substantial amounts of constitutive heterochromatin. Previous studies have described two highly repeated DNA families (MSAT-160 and MSAT-2570) that localize to this heterochromatin (Modi, 1992, 1993c). The present report describes a third satellite DNA family (termed MSAT-21) in M. chrotorrhinus that is also located in the sex heterochromatin. This repeat consists of diverged copies (average similarity = 75%) of a tandemly repeated 21-mer. Southern blotting of MSAT-21 revealed that although some higher order (5-20 kb) repeats do exist, none has spread throughout an appreciable portion of the genome. Pulsed field gel experiments indicated that most of the larger arrays (50-700 kb) of all three satellite families are distributed across numerous size classes, suggesting that the three repeats are interspersed with one another in this heterochromatin. Analysis of a boundary between MSAT-21 and MSAT-160 showed that the junction monomers of each satellite are intact and that a pentanucleotide has apparently been transferred from MSAT-21 to MSAT-160 via recombination. Sequence comparisons of MSAT-160 with another rodent satellite and with the U3 region of the Rous sarcoma virus (RSV) long terminal repeat identified inverted repeats and similarities with viral enhancer domains in the rodent sequences. Additionally, the MSAT-21 consensus was found to be similar to the R region of RSV, suggesting a retroviral ancestor for these rodent repeated DNA families.

Unequal crossing over and heterochromatin exchange in the X-Y bivalents of the deer mouse,Peromyscus beatae

Differences in length of the heterochromatic short arms of the X and Y chromosomes in individuals of Peromyscus beatae are hypothesized to result from unequal crossing over. To test this hypothesis, we examined patterns of synapsis, chiasma formation, and segregation for male P. beatae which were either heterozygous or homozygous for the amount of short-arm sex heterochromatin. Synaptonemal complex analysis demonstrated that mitotic differences in heterochromatic short-arm lengths between the X and Y chromosomes were reflected in early pachynema as corresponding differences in axial element lengths within the pairing region of the sex bivalent. These length differences were subsequently eliminated by synaptic adjustment such that by late pachynema, the synaptonemal complex configurations of the XY bivalent of heterozygotes were not differentiable from those of homozygotes. Crossing over between the heterochromatic short arms of the XY bivalent was documented by the routine appearance of a single chiasma in this region during diakinesis/metaphase I. Sex heterochromatin heterozygotes were characterized by the presence of asymmetrical chiasma between the X and Y short arms at diakinesis/metaphase I and sex chromosomes with unequal chromatid lengths at metaphase II. These data corroborate our hypothesis on the role of unequal crossing over in the production and propagation of X and Y heterochromatin variation and suggest that, in some cases, crossing over can occur during the process of synaptic adjustment.

The organ-specific rRNA gene number in Drosophila hydei is controlled by sex heterochromatin

Genotypes of Drosophila hydei having deficiencies of specific sections of X or Y chromosomal heterochromatin are characterized by heterogeneous rRNA gene numbers in their nonpolyploid organs. Depending on which sex heterochromatin portion is deleted, the rDNA amount in either brain or thoracic muscle cells or in both is increased threefold. This “rDNA overreplication” cannot be a “compensation” for a deficiency in ribosomal gene number, since the phenomenon also occurs in genotypes with a high initial rRNA gene numbers.