The location of highly repetitious DNA in the somatic chromosomes of Drosophila melanogaster

In situ hybridization of Drosophila melanogaster somatic chromosomes has been used to demonstrate the near exact correspondence between the location of highly repetitious DNA and classically defined constitutive heterochromatin. The Y chromosome, in particular, is heavily labeled even by cRNA transcribed from female (XX) DNA templates (i.e., DNA from female Drosophila with 2 Xs and 2 sets of autosomes). This observation confirms earlier reports that the Y chromosome contains repeated DNA sequences that are shared by other chromosomes. In grain counting experiments the Y chromosome shows significantly heavier label than any other chromosome when hybridized with cRNA from XY DNA templates (i.e., DNA from male Drosophila with 1 X and 1 Y plus 2 sets of autosomes). However, the preferential labeling of the Y is abolished if the cRNA is derived from XX DNA. We interpret these results as indicating the presence of a class of Y chromosome specific repeated DNA in D. melanogaster. The relative inefficiency of the X chromosome in binding cRNA from XY and XYY DNA templates, coupled with its ability to bind XX derived cRNA, may also indicate the presence of an X chromosome specific repeated DNA.     

Semi-automatic laser beam microdissection of the Y chromosome and analysis of Y chromosome DNA in a dioecious plant, Silene latifolia

Silene latifolia has heteromorphic sex chromosomes, the X and Y chromosomes. The Y chromosome, which is thought to carry the male determining gene, was isolated by UV laser microdissection and amplified by degenerate oligonucleotide-primed PCR. In situ chromosome suppression of the amplified Y chromosome DNA in the presence of female genomic DNA as a competitor showed that the microdissected Y chromosome DNA did not specifically hybridize to the Y chromosome, but hybridized to all chromosomes. This result suggests that the Y chromosome does not contain Y chromosome-enriched repetitive sequences. A repetitive sequence in the microdissected Y chromosome, RMY1, was isolated while screening repetitive sequences in the amplified Y chromosome. Part of the nucleotide sequence shared a similarity to that of X-43.1, which was isolated from microdissected X chromosomes. Since fluorescence in situ hybridization analysis with RMY1 demonstrated that RMY1 was localized at the ends of the chromosome, RMY1 may be a subtelomeric repetitive sequence. Regarding the sex chromosomes, RMY1 was detected at both ends of the X chromosome and at one end near the pseudoautosomal region of the Y chromosome. The different localization of RMY1 on the sex chromosomes provides a clue to the problem of how the sex chromosomes arose from autosomes.     

Actin-like sequences are present on human X and Y chromosomes.

The human genome contains greater than 20 actin-related sequences, six of which at least are expressed as protein. We have shown by blot hybridization the presence of actin-like sequences on both the X and the Y chromosomes. These sequences can be detected in HindIII digests of genomic DNA, using as probe cDNA clones corresponding to human alpha skeletal actin or to a hamster (beta or gamma) cytoskeletal actin; they show more homology to the latter probe. The actin probes also detect a polymorphic DNA fragment showing autosomal inheritance with a frequency for the major allele of 0.55 in the population studied. The X-linked actin sequence has been assigned to a centromeric region between Xp11 and Xq11 by hybridization to DNAs from a panel of human-mouse hybrid cell lines, and thus lies outside the postulated region of homology between the X and Y chromosomes. The Y-linked actin sequence can serve as a marker to analyse anomalies of sex determination or of gametogenesis in man. It was found in all XY males studied but was absent from the genomic DNA of four unrelated 'XX male' subjects and two XX hermaphrodites. This shows that the region of chromosome Y which contains the actin sequence is not translocated onto the X chromosome (or onto autosomes) in these patients.     

Isolation and characterization of an alphoid centromeric repeat family from the human Y chromosome

A collection of human Y-derived cosmid clones was screened with a plasmid insert containing a member of the human X chromosome alphoid repeat family, DXZ1. Two positive cosmids were isolated and the repeats they contained were investigated by Southern blotting, in situ hybridization and sequence analysis. On hybridization to human genomic DNAs, the expected cross-hybridization characteristic of all alphoid sequences was seen and, in addition, a 5500 base EcoRI fragment was found to be characteristic of a Y-specific alphoid repeat. Dosage experiments demonstrated that there are about 100 copies of this 5500 base EcoRI alphoid fragment on the Y chromosome. Studies utilizing DNA from human-mouse hybrids containing only portions of the Y chromosome and in situ hybridizations to chromosome spreads demonstrated the Y centromeric localization of the 5500 base repeat. Cross-hybridization to autosomes 13, 14 and 15 was also seen; however, these chromosomes lacked detectable copies of the 5500 base EcoRI repeat sequence arrangement. Sequence analysis of portions of the Y repeat and portions of the DXZ1 repeat demonstrated about 70% homology to each other and of each to the human consensus alphoid sequence. The 5500 base EcoRI fragment was not seen in gorilla, orangutan or chimpanzee male DNA.     

Chromosomal localization of the major satellite DNA family (FA-SAT) in the domestic cat

A major satellite DNA sequence was isolated from the cat genome and its sequencing data revealed homology to the FA-SAT family. In situ hybridization of the cat satellite DNA and telomeric sequences to cat chromosomes, together with staining of constitutive heterochromatin, allowed the physical mapping of the FA-SAT sequences, and also an overall constitutive heterochromatin study in cat chromosomes.     

Characterization and chromosomal distribution of satellite DNA sequences of the water buffalo (Bubalus bubalis).

Satellite DNA sequences were isolated from the water buffalo (Bubalus bubalis) after digestion with two restriction endonucleases, BamHI and StuI. These satellite DNAs of the water buffalo were classified into two types by sequence analysis: one had an approximately 1,400 bp tandem repeat unit with 79% similarity to the bovine satellite I DNA; the other had an approximately 700 bp tandem repeat unit with 81% similarity to the bovine satellite II DNA. The chromosomal distribution of the satellite DNAs were examined in the river-type and the swamp-type buffaloes with direct R-banding fluorescence in situ hybridization. Both the buffalo satellite DNAs were localized to the centromeric regions of all chromosomes in the two types of buffaloes. The hybridization signals with the buffalo satellite I DNA on the acrocentric autosomes and X chromosome were much stronger than that on the biarmed autosomes and Y chromosome, which corresponded to the distribution of C-band-positive centromeric heterochromatin. This centromere-specific satellite DNA also existed in the interstitial region of the long arm of chromosome 1 of the swamp-type buffalo, which was the junction of the telomere-centromere tandem fusion that divided the karyotype in the two types of buffaloes. The intensity of the hybridization signals with buffalo satellite II DNA was almost the same over all the chromosomes, including the Y chromosome, and no additional hybridization signal was found in noncentromeric sites.     

Homolog pairing and sister chromatid cohesion in heterochromatin in <Emphasis Type="Italic">Drosophila</Emphasis> male meiosis I

Drosophila males undergo meiosis without recombination or chiasmata but homologous chromosomes pair and disjoin regularly. The X–Y pair utilizes a specific repeated sequence within the heterochromatic ribosomal DNA blocks as a pairing site. No pairing sites have yet been identified for the autosomes. To search for such sites, we utilized probes targeting specific heterochromatic regions to assay heterochromatin pairing sequences and behavior in meiosis by fluorescence in situ hybridization (FISH). We found that the small fourth chromosome pairs at heterochromatic region 61 and associates with the X chromosome throughout prophase I. Homolog pairing of the fourth chromosome is disrupted when the homolog conjunction complex is perturbed by mutations in SNM or MNM. On the other hand, six tested heterochromatic regions of the major autosomes proved to be largely unpaired after early prophase I, suggesting that stable homolog pairing sites do not exist in heterochromatin of the major autosomes. Furthermore, FISH analysis revealed two distinct patterns of sister chromatid cohesion in heterochromatin: regions with stable cohesion and regions lacking cohesion. This suggests that meiotic sister chromatid cohesion is incomplete within heterochromatin and may occur at specific preferential sites.     

Flow cytometric quantification of human chromosome specific repetitive DNA sequences by single and bicolor fluorescent in situ hybridization to lymphocyte interphase nuclei

Fluorescent in situ hybridization allows for rapid and precise detection of specific nucleic acid sequences in interphase and metaphase cells. We applied fluorescent in situ hybridization to human lymphocyte interphase nuclei in suspension to determine differences in amounts of chromosome specific target sequences amongst individuals by dual beam flow cytometry. Biotinylated chromosome 1 and Y specific repetitive satellite DNA probes were used to measure chromosome 1 and Y polymorphism amongst eight healthy volunteers. The Y probe fluorescence was found to vary considerably in male volunteers (mean fluorescence 169, S.D. 35.6). It was also detectable in female volunteers (mean fluorescence 81, S.D. 10.7), because 5-10% of this repetitive sequence is located on autosomes. The Y probe fluorescence in males was correlated with the position of the Y chromosome cluster in bivariate flow karyotypes. When chromosome 1 polymorphism was studied, one person out of the group of eight appeared to be highly polymorphic, with a probe fluorescence 26% below the average. By means of fluorescent in situ hybridization on a glass slide and bivariate flow karyotyping, this 26% difference was found to be caused by a reduction of the centromere associated satellite DNA on one of the homologues of chromosome 1. The simultaneous hybridization to human lymphocyte interphase nuclei of biotinylated chromosome 1 specific repetitive DNA plus AAF-modified chromosome Y specific DNA was detected by triple beam flow cytometry. The bicolor double hybridized nuclei could be easily distinguished from the controls. When the sensitivity of this bicolor hybridization is improved, this approach could be useful for automatic detection of numerical chromosome aberrations, using one of the two probes as an internal control.     

Evolution of a human Y chromosome-specific repeated sequence

The structure and evolution of a repetitive sequence on the human Y chromosome has been studied by restriction enzyme analysis of both total DNA and the isolated sequence. The sequence is shown to cross-hybridize to sequences in female DNA forming unstable duplexes. Mouse/human cell hybrids have been used to investigate the pattern of sequence homology on the X chromosome and some autosomes. We conclude that this sequence is related to human satellite III, but shows considerable differences in structure.     

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     

RAPD markers encoding retrotransposable elements are linked to the male sex in Cannabis sativa L.

Male-associated DNA sequences were analyzed in Cannabis sativa L. (hemp), a dioecious plant with heteromorphic sex chromosomes. DNA was isolated from male and female plants and subjected to random amplified polymorphic DNA analysis. Of 120 primers, 17 yielded 400 to 1500-bp fragments detectable in male, but not female, plants. These fragments were cloned and used as probes in gel-blot analysis of genomic DNA. When male and female DNA was hybridized with 2 of these male-specific fragments, MADC(male-associated DNA sequences in C. sativa)3 and MADC4, particularly intense bands specific to male plants were detected in addition to bands common to both sexes. The MADC3 and MADC4 sequences were shown to encode gag/pol polyproteins of copia-like retrotransposons. Fluorescence in situ hybridization with MADC3 and MADC4 as probes revealed a number of intense signals on the Y chromosome as well as dispersed signals on all chromosomes. The gel-blot analysis and fluorescence in situ hybridization results presented here support the hypothesis that accumulation of retrotransposable elements on the Y chromosome might be 1 cause of heteromorphism of sex chromosomes.     

Studies of He-T DNA sequences in the pericentric regions of Drosophila chromosomes

He-T DNA is a complex set of repeated DNA sequences with sharply defined locations in the polytene chromosomes of Drosophila melanogaster. He-T sequences are found only in the chromocenter and in the terminal (telomere) band on each chromosome arm. Both of these regions appear to be heterochromatic and He-T sequences are never detected in the euchromatic arms of the chromosomes (Young et al. 1983). In the study reported here, in situ hybridization to metaphase chromosomes was used to study the association of He-T DNA with heterochromatic regions that are under-replicated in polytene chromosomes. Although the metaphase Y chromosome appears to be uniformly heterochromatic, He-T DNA hybridization is concentrated in the pericentric region of both normal and deleted Y chromosomes. He-T DNA hybridization is also concentrated in the pericentric regions of the autosomes. Much lower levels of He-T sequences were found in pericentric regions of normal X chromosomes; however compound X chromosomes, constructed by exchanges involving Y chromosomes, had large amounts of He-T DNA, presumably residual Y sequences. The apparent co-localization of He-T sequences with satellite DNAs in pericentric heterochromatin of metaphase chromosomes contrasts with the segregation of satellite DNA to alpha heterochromatin while He-T sequences hybridize to beta heterochromatin in polytene nuclei. This comparison suggests that satellite sequences do not exist as a single block within each chromosome but have interspersed regions of other sequences, including He-T DNA. If this is so, we assume that the satellite DNA blocks must associate during polytenization, leaving the interspersed sequences looped out to form beta heterochromatin. DNA from D. melanogaster has many restriction fragments with homology to He-T sequences. Some of these fragments are found only on the Y. Two of the repeated He-T family restriction fragments are found entirely on the short arm of the Y, predominantly in the pericentric region. Under conditions of moderate stringency, a subset of He-T DNA sequences cross-hybridizes with DNA from D. simulans and D. miranda. In each species, a large fraction of the cross-hybridizing sequences is on the Y chromosome.     

Differences in the meiotic pairing behavior of gonosomal heterochromatin between female and male Microtus agrestis: implications for the mechanism of heterochromatin amplification on the X and Y

It is generally thought that pairing and recombination between the X and Y chromosome in eutherian mammals is important for the occurrence of normal meiotic division and the production of functional gametes. Microtus agrestis is one of the examples whose giant and heterochromatin-rich sex chromosomes fail to establish a durable association at any stage of the first meiotic division in males. In contrast, in females, synapsis starts in the euchromatic short arm and pairing progresses unidirectionally and continues until both X chromosomes have paired completely, as can be demonstrated by the use of fluorescence in situ hybridization with a sequence confined to the non-centromeric, gonosomal heterochromatin. However, compared with euchromatin, this association is apparently ephemeral and breaks off precociously in the pachytene and metaphase I stages. We demonstrate that a middle repetitive element is localized interspersed in the noncentromeric heterochromatin of both X and Y, except the telomeric region of the Y. No differences could be detected at the molecular level between male and female DNA, indicating that at least the bulk of these elements are organized in the same manner on the X and Y. Our data imply that the loss of synapsis and recombination between the X and Y might have preceded the process of heterochromatin amplification in the course of Microtinae evolution. Since asynapsed elements are particularly susceptible to DNA strand breaks during prophase I, DNA repair of double-strand breaks involving heterochromatic segments of the X and Y could have resulted in translocations of larger segments from the X to the Y or vice versa during the course of chromosome evolution of the gonosomes, explaining the homology at the molecular level between the heterochromatin of the asynaptic X and Y in M. agrestis.     

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.     

New evidence for tandem chromosome fusions in the karyotypic evolution of Asian muntjacs

A clone of highly repetitive DNA, designated C5, was isolated from DNA of female Chinese muntjac cells. The nucleotide sequence of this clone is 80%–85% homologous to that of the satellite IA clone and other highly repetitive DNA clones previously obtained from the Indian muntjac. Using C5 as a probe for in situ hybridizations to chromosome preparations of cells of both the Chinese and Indian muntjacs, we were able to show that these repeated sequences occur in centromeric heterochromatin of the chromosomes of both Chinese and indian muntjac species. More significantly, non-random clusters of hybridization signals were detected on the arms of chromosomes of the Indian muntjac. These latter hybridization sites are postulated to be regions of interstitial heterochromatin and could be the remnants of centromeric heterochromatin from ancestral Chinese muntjac chromosomes. Our observations provide new supportive evidence for the tandem chromosome fusion theory that has been proposed for the evolution of the Indian muntjac karyotype.by P.B. Moens     

Heterochromatin differentiation shows the pathways of karyotypic evolution in Israeli mole rats (Spalax, Spalacidae, Rodentia)

C-banding, base-specific fluorochrome staining (CMA3/DA/DAPI), and comparative genomic hybridization (CGH) were used to analyze the constitutive heterochromatin in two Israeli Spalax species, S. galili (2n = 52) and S. judai (2n = 60). It was shown that C-positive centromeric heterochromatin and some telomeric sites comprise GC-rich DNA sequences in both species. Comparative genomic in situ hybridization revealed slight qualitative differences in highly repetitive sequences in the two Spalax species. Eight acrocentric pairs in S. judai that are involved in Robertsonian rearrangements, possessed composite heterochromatin with a preference of S. judai highly repetitive sequences in the proximal region. Heterochromatin of the sex chromosomes, two biarmed homologous pairs (4 and 5) in both species, and acrocentric chromosomes from the group with a variable centromere position in S. judai was entirely species-specific. The high level of homology in the composition of heterochromatin may relate to the recent divergence of Israeli Spalax. Interspecies heterochromatin differences are discussed in the context of possible mechanisms in the Spalax chromosome evolution.     

Application of cloned satellite DNA sequences to molecular-cytogenetic analysis of constitutive heterochromatin heteromorphisms in man

Summary The cloned alpha-satellite DNA sequences were used to evaluate the specificity and possible variability of repetitive DNA in constitutive heterochromatin of human chromosomes. Five probes with high specificity to individual chromosomes (chromosomes 3, 11, 17, 18, and X) were in situ hybridized to metaphase chromosomes of different individuals. The stable position of alpha-satellite DNA sequences in heterochromatic regions of particular chromosomes was found. Therefore, the chromosome-specific alpha-satellite DNA sequences may be used as molecular markers for heterochromatic regions of certain human chromosomes. The homologous chromosomes of many individuals were characterized by cytologically visible heteromorphisms of hybridization intensity with chromosome-specific alpha-satellite DNA sequences. A special analysis of hybridization between homologues with morphological differences provided the evidence for a high resolution power of the in situ hybridization technique for evaluation of chromosome heteromorphisms. The approaches for detection of heteromorphisms in cases without morphological differences between homologues are discussed. The results obtained indicate that constitutive heterochromatin of human chromosomes has a variable amount of alphasatellite DNA sequences. In situ hybridization of cloned satellite DNA sequences may be used as a new general approach to analysis of chromosome heteromorphisms in man.     

Mapping Simple Repeated DNA Sequences in Heterochromatin of Drosophila Melanogaster

Heterochromatin in Drosophila has unusual genetic, cytological and molecular properties. Highly repeated DNA sequences (satellites) are the principal component of heterochromatin. Using probes from cloned satellites, we have constructed a chromosome map of 10 highly repeated, simple DNA sequences in heterochromatin of mitotic chromosomes of Drosophila melanogaster. Despite extensive sequence homology among some satellites, chromosomal locations could be distinguished by stringent in situ hybridizations for each satellite. Only two of the localizations previously determined using gradient-purified bulk satellite probes are correct. Eight new satellite localizations are presented, providing a megabase-level chromosome map of one-quarter of the genome. Five major satellites each exhibit a multichromosome distribution, and five minor satellites hybridize to single sites on the Y chromosome. Satellites closely related in sequence are often located near one another on the same chromosome. About 80% of Y chromosome DNA is composed of nine simple repeated sequences, in particular (AAGAC)(n) (8 Mb), (AAGAG)(n) (7 Mb) and (AATAT)(n) (6 Mb). Similarly, more than 70% of the DNA in chromosome 2 heterochromatin is composed of five simple repeated sequences. We have also generated a high resolution map of satellites in chromosome 2 heterochromatin, using a series of translocation chromosomes whose breakpoints in heterochromatin were ordered by N-banding. Finally, staining and banding patterns of heterochromatic regions are correlated with the locations of specific repeated DNA sequences. The basis for the cytochemical heterogeneity in banding appears to depend exclusively on the different satellite DNAs present in heterochromatin.     

Molecular analysis of holocentric centromeres of Luzula species

Luzula spp, like the rest of the members of the Juncaceae family, have holocentric chromosomes. Using the rice 155-bp centromeric tandem repeat sequence (RCS2) as a probe, we have isolated and characterized a 178-bp tandem sequence repeat (LCS1) from Luzula nivea. The LCS1 sequence is present in all Luzula species tested so far (except L. pilosa) and like other satellite repeats found in heterochromatin, the cytosine residues are methylated within the LCS1 repeats. Using fluorescent in situ hybridization (FISH) experiments we have shown that there are at least 5 large clusters of LCS1 sequences distributed at heterochromatin regions along each of the 12 chromosomes of L. nivea. We have shown that a centromeric antibody Skp1 co-localizes with these heterochromatin regions and with the LCS1 sequences. This suggests that the LCS1 sequences are part of regions which function as centromeres on these holocentric chromosomes. Furthermore, using the BrdU assay to identify replication sites, we have shown that these heterochromatin sites containing LCS1 associate when being replicated in root interphase nuclei. Our results also show premeiotic chromosome association during anther development as indicated by single-copy BAC in situ and the presence of fewer LCS1 containing heterochromatin sites in these cells.     

Molecular cytogenetic research on the polymorphism of segments of the constitutive heterochromatin in human chromosomes

Cloned alpha-satellite DNA sequences were used to evaluate the specificity and possible variability of repetitive DNA in constitutive heterochromatin of human chromosomes. Five probes of high specificity to individual chromosomes (chromosomes 3, 11, 17, 18 and X) were hybridized in situ to metaphase chromosomes of different individuals. The stable position of alpha-satellite DNA sequences in definite heterochromatic regions of particular chromosomes was found. Therefore, the chromosome-specific alpha-satellite DNA sequences may be used as molecular markers for heterochromatic regions of certain human chromosomes. The significant interindividual differences in relative copy number of alpha-satellite DNA have been detected. The homologous chromosomes of many individuals were characterized by cytologically visible heteromorphisms, as shown by intensity of hybridization with chromosome-specific alpha-satellite DNA sequences. A special analysis of hybridization between homologues with morphological differences gives evidence for a high resolution power of in situ hybridization technique for evaluation of chromosome heteromorphisms. The approaches for detection of heteromorphisms in cases without morphological differences between homologues are discussed. The results obtained indicate that constitutive heterochromatin of human chromosomes is variable for amount of alpha-satellite DNA sequences. In situ hybridization of cloned satellite DNA sequences may be used as novel general approach to analysis of chromosome heteromorphisms in man.