A Novel MS7 Repeat Specific for Heterochromatin of Sex Chromosomes in Voles of Genus Microtus, Group arvalis: Genomic Organization and Chromosomal Localization

The tandemly arranged MS4 repeat with monomeric units of 4.1 kb is species-specifically distributed in heterochromatin of sex chromosomes of four common vole species of genus Microtus, group arvalis. In this work, we studied the genomic organization of the MS4 homolog in euchromatin of the X chromosome of M. arvalis. It has been shown by analyzing the phage genomic clones that one MS4 copy makes a part of a monomeric unit exceeding 8.5 kb that also includes a new MS7 repeat and, possibly, LINE fragments. MS7 is located together with MS4 in heterochromatin of common vole sex chromosomes, but in a substantially lesser amount. Probably, as a result of an evolutionary transition of an original repeat from euchromatin of the X chromosome to heterochromatin of the Y chromosome, MS4 underwent multiple amplification, and MS7 spread throughout heterochromatin, being surrounded by the MS4 tandem arrays.     

Organization of extended repeats in heterochromatin in sex chromosomes in the common vole species Microtus group "arvalis"

Two long repeats, MS3 and MS4, are predominantly located in sex-chromosomal heterochromatin in common vole species. Their tandem arrangement was revealed by means of the PCR analysis of genomic DNAs of four Microtus species and by restriction mapping of clones selected from a M. rossiaemeridionalis genomic library. Several mobile elements proved incorporated in a monomeric unit of each repeat and amplified together with its other components. In addition, LINE inserts were found in MS4 tandem arrays. The copy number of both repeats per haploid genome was estimated at 100-300 for euchromatin and 20,000-40,000 for the M. rossiaemeridionalis genome. The repeats were assumed to be the major component of sex-chromosomal heterochromatin DNA.     

Organization of Long Repeats of Sex-Chromosomal Heterochromatin in Common Vole Species

Two long repeats, MS3 and MS4, are predominantly located in sex-chromosomal heterochromatin in common vole species [1]. Their tandem arrangement was revealed by means of the PCR analysis of genomic DNAs of four Microtus species and by restriction mapping of clones selected from a M. rossiaemeridionalis genomic library. Several mobile elements proved to be incorporated in a monomeric unit of each repeat and amplified together with its other components. In addition, LINE inserts were found in MS4 tandem arrays. The copy number of both repeats per haploid genome was estimated at 100–300 for euchromatin and 20,000–40,000 for the M. rossiaemeridionalis genome. The repeats were assumed to be the major component of sex-chromosomal heterochromatin DNA.     

Organization and chromosomal localization of a B1-like containing repeat of Microtus subarvalis

A repetitive DNA sequence, MS2, was isolated from EcoRI-digested genomic DNA of the vole, Microtus subarvalis. The fragment was cloned and sequenced. Sequence analysis of this 1194-bp fragment revealed a 156-bp region demonstrating a 55% homology with the mouse B1 repeat. The remaining MS2 sequence shows no significant homology with other known GenBank sequences. The results of in situ hybridization of MS2 on vole metaphase chromosomes indicate the fragment is confined to heterochromatin blocks of the sex chromosomes in all but one species (M. arvalis). Distribution of MS2 sequences provides evidence for heterogeneity of the giant heterochromatin blocks of the XY Chromosomes (Chrs) in voles, for the unique cluster-like localization of MS2 within these blocks. Received: 10 October 1995 / Accepted: 30 March 1996     

Sister chromatid exchange points in the heterochromatin and euchromatin regions of Chinese hedgehog chromosomes

Summary The Chinese hedgehog has a diploid chromosome number of 48 in which there are eleven pairs of telo- or subtelocentric autosomes, twelve pairs of meta- or submetacentric autosomes, a metacentric X chromosome and a telocentric Y chromosome. The heterochromatin is almost completely distributed in five large distal segments of chromosomes nos. 9 to 12 and no. 18. There is no positive C-band in the centromeres of the chromosomes except for the X chromosome which has a small, weakly stained C-band in the centromere. In Chinese hedgehog cells 52.1% of SCEs are found at the junction between the euchromatin and the heterochromatin, 39.5% in the heterochromatin and 8.4% in the auchromatin. The SCE number per unit C-band is double the SCE number per unit euchromatin. The SCE rate in the heterochromatin or euchromatin regions is not proportional to their chromosome length and can be quite different between different pairs of the chromosomes. Our results indicate that there is a non-uniform distribution of the SCEs in the Chinese hedgehog cells.     

Simple GA C T A repeats characterize the X chromosomal heterochromatin of Microtus agrestis,European field vole (Rodentia,Cricetidae)

The sex chromosomes of Microtus agrestis are extremely large due to the accumulation of constitutive heterochromatin. We have identified two prominent satellite bands of 2.0 and 2.8 kb in length after HaeIII and HinfI restriction enzyme digestion of genomic DNA, respectively. These satellites are located on the heterochromatic long arm of the X chromosome as shown using Microtus x mouse somatic cell hybrids. By in-gel hybridization with oligonucleotide probes, the organization of the two satellites was studied: among the many copies of the simple tandem tetranucleotide repeat GATA are interspersed rare single GACA tetramers. One of the satellites also harbours related GGAT simple tandem repeats. In situ hybridizations with plasmid-carried or oligonucleotide GA _C ^T A probes show clustered silver grains on the long and short arm of the X chromosome. Interspersion of differently organized (GATA)_n elements is also demonstrable in the autosomal complement and on the Y chromosome. These results are discussed in the context of the evolution of vertebrate sex chromosomes in relation to heterochromatin and simple repetitive DNA sequences.     

A deletion at the mouse Xist gene exposes trans-effects that alter the heterochromatin of the inactive X chromosome and the replication time and DNA stability of both X chromosomes

The inactive X chromosome of female mammals displays several properties of heterochromatin including late replication, histone H4 hypoacetylation, histone H3 hypomethylation at lysine-4, and methylated CpG islands. We show that cre-Lox-mediated excision of 21 kb from both Xist alleles in female mouse fibroblasts led to the appearance of two histone modifications throughout the inactive X chromosome usually associated with euchromatin: histone H4 acetylation and histone H3 lysine-4 methylation. Despite these euchromatic properties, the inactive X chromosome was replicated even later in S phase than in wild-type female cells. Homozygosity for the deletion also caused regions of the active X chromosome that are associated with very high concentrations of LINE-1 elements to be replicated very late in S phase. Extreme late replication is a property of fragile sites and the 21-kb deletions destabilized the DNA of both X chromosomes, leading to deletions and translocations. This was accompanied by the phosphorylation of p53 at serine-15, an event that occurs in response to DNA damage, and the accumulation of gamma-H2AX, a histone involved in DNA repair, on the X chromosome. The Xist locus therefore maintains the DNA stability of both X chromosomes.     

Pseudoautosomal Region 1 Length Polymorphism in the Human Population

The human sex chromosomes differ in sequence, except for the pseudoautosomal regions (PAR) at the terminus of the short and the long arms, denoted as PAR1 and PAR2. The boundary between PAR1 and the unique X and Y sequences was established during the divergence of the great apes. During a copy number variation screen, we noted a paternally inherited chromosome X duplication in 15 independent families. Subsequent genomic analysis demonstrated that an insertional translocation of X chromosomal sequence into theMa Y chromosome generates an extended PAR. The insertion is generated by non-allelic homologous recombination between a 548 bp LTR6B repeat within the Y chromosome PAR1 and a second LTR6B repeat located 105 kb from the PAR boundary on the X chromosome. The identification of the reciprocal deletion on the X chromosome in one family and the occurrence of the variant in different chromosome Y haplogroups demonstrate this is a recurrent genomic rearrangement in the human population. This finding represents a novel mechanism shaping sex chromosomal evolution.     

Gene for structural proteins of the SMC family in the common vole Microtus arvalis]

Genes for four subfamilies of SMC (structural maintenance of chromosomes) proteins have been isolated from the genome of a common vole Microtus arvalis. The high degree of homology between representatives of each SMC protein subfamily of different classes of organisms has been demonstrated. The full-sized copy of a mammalian gene encoding SMC4 protein has been isolated and analyzed for the first time. The SMC proteins enter into the composition of complexes responsible for cohesion of sister chromatids, formation of mitotic chromosomes, recombination, DNA repair, and regulation of gene expression. We discuss the possible participation of the SMC proteins in inactivation of the X chromosome in mammalian females. Common voles of genus Microtus group "arvalis" serve a unique model for the study of the inactivation process.     

Structural organization and functional analysis of centromeric DNA in the fission yeast Schizosaccharomyces pombe.

Centromeric DNA in the fission yeast Schizosaccharomyces pombe was isolated by chromosome walking and by field inversion gel electrophoretic fractionation of large genomic DNA restriction fragments. The centromere regions of the three chromosomes were contained on three SalI fragments (120 kilobases [kb], chromosome III; 90 kb, chromosome II; and 50 kb, chromosome I). Each fragment contained several repetitive DNA sequences, including repeat K (6.4 kb), repeat L (6.0 kb), and repeat B, that occurred only in the three centromere regions. On chromosome II, these repeats were organized into a 35-kb inverted repeat that included one copy of K and L in each arm of the repeat. Site-directed integration of a plasmid containing the yeast LEU2 gene into K repeats at each of the centromeres or integration of an intact K repeat into a chromosome arm had no effect on mitotic or meiotic centromere function. The centromeric repeat sequences were not transcribed and possessed many of the properties of constitutive heterochromatin. Thus, S. pombe is an excellent model system for studies on the role of repetitive sequence elements in centromere function.     

Construction of male and female PAC genomic libraries suitable for identification of Y-chromosome-specific clones from the liverwort, Marchantia polymorpha

Unlike higher plants, the dioecious liverwort, Marchantia polymorpha, has uniquely small sex chromosomes, with X chromosomes present only in female gametophytes and Y chromosomes only in male gametophytes. We have constructed respective genomic libraries for male and female plantlets using a P1-derived artificial chromosome (pCYPAC2). With an average insert size of approximately 90 kb, each PAC library is estimated to cover the entire genome with a probability of more than 99.9%. Male-specific PAC clones were screened for by differential hybridization using male and female genomic DNAs as separate probes. Seventy male-specific PAC clones were identified. The male specificity of one of the clones, pMM4G7, was verified by Southern hybridization and PCR analysis. This clone was indeed located on the Y chromosome as verified by fluorescence in situ hybridization (FISH). This result shows that the Y chromosome contains unique sequences that are not present either on the X chromosome or any of the autosomes. Thus, the respective male and female libraries for M. polymorpha offer an opportunity to identify key genes involved in the process of sex differentiation and this unique system of sex determination.     

Integrated cytogenetic map of chromosome arm 4S of A. thaliana: structural organization of heterochromatic knob and centromere region

We have constructed an integrated cytogenetic map of chromosome arm 4S of Arabidopsis thaliana. The map shows the detailed positions of various multicopy and unique sequences relative to euchromatin and heterochromatin segments. A quantitative analysis of the map positions at subsequent meiotic stages revealed a striking pattern of spatial and temporal variation in chromatin condensation for euchromatin and heterochromatin. For example, the centromere region consists of three domains with distinguishable structural, molecular, and functional properties. We also characterized a conspicuous heterochromatic knob of approximately 700 kb that accommodates a tandem repeat and several dispersed pericentromere-specific repeats. Moreover, our data provide evidence for an inversion event that relocated pericentromeric sequences to an interstitial position, resulting in the heterochromatic knob.     

C-bands on chromosomes of four beetle species (Coleoptera: Carabidae, Silphidae, Elateridae, Scarabaeidae)

The C-banding pattern of Bembidion geniculatum, Silpha atrata, Prosternon tesselatum, and Epicometis hirta are presented. All analysed species have pracentromeric C-bands on autosomes and chromosome X but the widest ones are visible in the karyotype of B. geniculatum. In S. atrata, P. tesselatum, and E. hirta sex chromosome y is heterochromatic, only B. geniculatum having the Y chromosome wholly euchromatin. The results indicate that on the chromosomes of the investigated species do not have a terminal and an intercalar segments of heterochromatin.     

A male-specific DNA probe detects heterochromatin sequences in a familial Yq- chromosome.

Using a recombinant DNA probe, we have demonstrated the presence of residual 3.4-kilobase (kb) repeat sequences in a family with a Yq- chromosome. The heterochromatin of this Y variant was not readily detectable with conventional chromosome-banding techniques. These data suggest that the breakpoint of the deletion occurs at the heterochromatin region proximal to the euchromatin/heterochromatin junction.     

Localization of repetitive DNA in the chromosomes of <Emphasis Type="Italic">Microtus agrestis</Emphasis> by means of <Emphasis Type="Italic">in situ</Emphasis> hybridization

Heterochromatin in the European field vole, Microtus agrestis, was studied using a special staining technique and DNA/RNA in situ hybridization. The heterochromatin composed the proximal 1/4 of the short arm and the entire long arm of the X chromosome, practically the entire Y chromosome and the centromeric areas of the autosomes. By using the DNA/RNA in situ hybridization technique, repeated nucleotide sequences are shown to be in the heterochromatin of the sex chromosomes.     

Sex Chromosome Meiotic Drive in DROSOPHILA MELANOGASTER Males

In Drosophila melanogaster males, deficiency for X heterochromatin causes high X-Y nondisjunction and skewed sex chromosome segregation ratios (meiotic drive). Y and XY classes are recovered poorly because of sperm dysfunction. In this study it was found that X heterochromatic deficiencies disrupt recovery not only of the Y chromosome but also of the X and autosomes, that both heterochromatic and euchromatic regions of chromosomes are affected and that the "sensitivity" of a chromosome to meiotic drive is a function of its length. Two models to explain these results are considered. One is a competitive model that proposes that all chromosomes must compete for a scarce chromosome-binding material in Xh(-) males. The failure to observe competitive interactions among chromosome recovery probabilities rules out this model. The second is a pairing model which holds that normal spermiogenesis requires X-Y pairing at special heterochromatic pairing sites. Unsaturated pairing sites become gametic lethals. This model fails to account for autosomal sensitivity to meiotic drive. It is also contradicted by evidence that saturation of Y-pairing sites fails to suppress meiotic drive in Xh(- ) males and that extra X-pairing sites in an otherwise normal male do not induce drive. It is argued that meiotic drive results from separation of X euchromatin from X heterochromatin.     

Restriction enzyme digestion of heterochromatin inDrosophila nasuta

In situ digestion of metaphase and polytene chromosomes and of interphase nuclei in different cell types ofDrosophila nasuta with restriction enzymes revealed that enzymes like AluI, EcoRI, HaeIII, Sau3a and SinI did not affect Giemsa-stainability of heterochromatin while that of euchromatin was significantly reduced; TaqI and SalI digested both heterochromatin and euchromatin in mitotic chromosomes. Digestion of genomic DNA with AluI, EcoRI, HaeIII, Sau3a and KpnI left a 23 kb DNA band undigested in agarose gels while withTaqI, no such undigested band was seen. TheAluI resistant 23 kb DNA hybridized insitu specifically with the heterochromatic chromocentre. It appears that the digestibility of heterochromatin region in genome ofDrosophila nasuta with the tested restriction enzymes is dependent on the availability of their recognition sites.     

A case of spontaneous trisomy in the spermatocytes of Microtus arvalis

A triple synaptomal complex was observed between 3 small-sized chromosomes in 4 spermatocytes closely connected by intercellular bridges, of the common vole (Microtus arvalis L.). Other spermatocytes from the same and from 2 other males had a normal chromosome complement and pairing patterns. This finding was interpreted as the result of a single act of non-disjunction taking place in a spermatogonium. These data suggest that chromosome non-disjunction in premeiotic germ cells can be considered one of the causes of aneuploidy in mammals.     

Localization of repetitive DNA in the chromosomes of Microtus agrestis by means of in situ hybridization

Heterochromatin in the European field vole, Microtus agrestis, was studied using a special staining technique and DNA/RNA in situ hybridization. The heterochromatin composed the proximal 1/4 of the short arm and the entire long arm of the X chromosome, practically the entire Y chromosome and the centromeric areas of the autosomes. By using the DNA/RNA in situ hybridization technique, repeated nucleotide sequences are shown to be in the heterochromatin of the sex chromosomes.Supported in part by Research Grants DRG-1061 and 269 from the Damon Runyon Memorial Fund for Cancer Research, G-373 and G-267 from the Robert A. Welch Foundation.     

Karyology of the Savi pine vole, Microtus savii (De Sélys-Longchamps, 1838) (Rodentia, Arvicolidae): G-, C-, DA/DAPI-, and AluI-bands.

Specimens of the Savi pine vole (Microtus savii) were collected from three localities in central (Pisa and Viterbo) and southern Italy (Rosarno, Calabria) and were karyotyped using G-, C-, DA/DAPI-, and AluI-banding. All karyotypes had 2n = 54 chromosomes and seemingly identical autosomal banding. The sex chromosomes of the southern Italian specimens, M. savii brachycercus, showed additional large blocks of heterochromatin. In the northern specimens, M. savii savii, the X chromosome is metacentric, whereas in the southern specimens of M. savii brachycercus the X chromosome is a much larger submetacentric chromosome, and the Y chromosome is more than twice the size of the Y in the northern specimens. DA/DAPI staining reveals three levels of fluorescent intensity in the sex chromosomes of the Calabrian specimens. The sex chromosomes of M. savii brachycercus also have the only AluI bands seen in either chromosome set. These data suggest a heterogeneous origin and composition of the C-band regions of these chromosomes. Preliminary data suggest that fertility is reduced in crosses between the two karyomorphs.