DNA repeated sequences may be involved in synaptonemal complex formation

Synaptonemal complexes (SCs) are intranuclear structures that facilitate the reversible lateral synapsis of homologous chromosomes in the course of meiosis. It is still unclear which DNA nucleotide sequences are responsible for the attachment of chromatin to SC lateral elements. Considering the features of the dispersed repeated sequences (RSs), it is possible to assume that they participate in the structure and functional organization of the meiotic chromosomes. Using numerical analysis, we have investigated the relationship between the RS and the distribution of meiotic recombination events in mouse chromosome 1. Using in situ hybridization on spread mouse spermatocytes, we have examined the arrangement of different types of RSs relative to SCs. Hybridization signals of B1(Alu), B2, and minisatellite probes were localized predominantly in SCs regions. Based on the results, we proposed a model of meiotic chromosome organization. According to the model, RSs participate in the attachment of chromatin loops to SCs.     

Differential distribution and association of repeat DNA sequences in the lateral element of the synaptonemal complex in rat spermatocytes

The synaptonemal complex (SC) is an evolutionarily conserved structure that mediates synapsis of homologous chromosomes during meiotic prophase I. Previous studies have established that the chromatin of homologous chromosomes is organized in loops that are attached to the lateral elements (LEs) of the SC. The characterization of the genomic sequences associated with LEs of the SC represents an important step toward understanding meiotic chromosome organization and function. To isolate these genomic sequences, we performed chromatin immunoprecipitation assays in rat spermatocytes using an antibody against SYCP3, a major structural component of the LEs of the SC. Our results demonstrated the reproducible and exclusive isolation of repeat deoxyribonucleic acid (DNA) sequences, in particular long interspersed elements, short interspersed elements, long terminal direct repeats, satellite, and simple repeats. The association of these repeat sequences to the LEs of the SC was confirmed by in situ hybridization of meiotic nuclei shown by both light and electron microscopy. Signals were also detected over the chromatin surrounding SCs and in small loops protruding from the lateral elements into the SC central region. We propose that genomic repeat DNA sequences play a key role in anchoring the chromosome to the protein scaffold of the SC. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

DNA repeated sequences may be involved in the synaptonemal complexes formation

Synatonemal complexes (SCs) are the intranuclear structures which facilitate reversible lateral synapsis of the homologous chromosomes in the course of meiosis. It is still unclear which DNA nucleotide sequences are responsible for the chromatin attachment to the SC lateral elements. Considering the features of the dispersed repeated sequences (RS) it is worth to assume their participation in the structure functional organization of the meiotic chromosome. Using numerical analysis we have investigated the relationship between RS and the distribution of events of the meiotic recombination in mouse chromosome 1. Using in situ hybridization on spread mouse spermatocytes, we have demonstrated the arrangement of different types of RS relative to SCs. Hybridization signals of B1(Alu), B2, and minisatellite probes were localizating predominantly in the SCs regions. Our results allow us to suggest the model of the meiotic chromosome organization with the RS as the sequences, participating in the attachment of chromatin loops and SCs.     

Direct visualization of the genomic distribution and organization of two cervid centromeric satellite DNA families

Several repetitive DNA fragments were generated from PCR amplifications of caribou DNA using primer sequences derived from the white-tailed deer satellite II DNA clone OvDII. Two fragments, designated Rt-0.5 and Rt-0.7, were sequenced and found to have 96% sequence similarity. These caribou clones also had 85% sequence similarity with OvDII. Multiple-colored fluorescence in situ hybridization (FISH) studies with satellite I and satellite II DNA probes to caribou metaphase chromosomes and extended chromatin fibers provided direct visualization of the genomic organization of these two satellite DNA families, with the following findings: (1) Cervid satellite I DNA is confined to the centromeric regions of the acrocentric autosomes, whereas satellite II DNA is found at the centromeric regions of all chromosomes except for the Y. (2) For most acrocentric chromosomes, the satellite I signal appeared to be medially located at the primary constriction, in contrast to that of satellite II, which appeared to be oriented toward the lateral sides as two separate fluorescent dots. (3) The satellite II clone Rt-0.7 appeared to be enriched in the centromeric region of the caribou X chromosome, a pair of biarmed autosomes, and a number of other acrocentric autosomes. (4) Fiber-FISH demonstrated that the satellite I and satellite II arrays were juxtaposed. On highly extended chromatin fibers, the total length of the hybridization signals for the two satellite DNA arrays often reached 300-400 microm. The length of a given satellite II array usually reached 200 microm, corresponding to 2 x 10(3) kb of DNA in a given centromere.     

Telomere and centromere DNA are associated with the cores of meiotic prophase chromosomes

Mouse (Mus musculus) whole-mount, surface-spread, meiotic prophase chromosomes have an axial which extend chromatin loops. This arrangement permits a novel approach to the analysis of chromosome structure. Using in situ hybridization, the types of DNA sequences preferentially associated with the SC and the types located primarily in the chromatin loops can be determined. With biotinylated probes, detected by avidin conjugated to FITC, we present evidence for differential chromatin-SC interaction. The telomere sequence (TTAGGG)_n is associated exclusively with the two ends of each autosomal SC rather than with the chromatin loops. The minor satellite DNA sequences are predominantly localized to the centromeric region of the SC, as defined by CREST serum anti-centromere antibodies. In contrast, the major satellite DNA probe hybridizes to the chromatin loops of the centromeric heterochromatin, and a probe containing a LINE sequence hybridizes to chromatin loops in general with no obvious preference for the SC. These observations demonstrate that, depending on the type of DNA sequence, the chromatin has different properties in regard to its association with the SC.D.P. Bazett-Jones     

Anti-topoisomerase II recognizes meiotic chromosome cores

At meiotic prophase the chromatin becomes arranged in loops on newly formed chromosome cores. The cores of homologous chromosomes become aligned in parallel and thus form the synaptonemal complex (SC), a structure found in the meiocytes of nearly all recombinationally competent, sexually reproducing organisms. We report that two polyclonal antibodies against topoisomerase II (topo II), which recognize the mitotic metaphase chromosome scaffold give, at pachytene, a positive immunocytological reaction with the chromatin and, predominantly, with the cores and centromeric regions of the paired chromosomes. It therefore appears that during meiotic prophase, topo II — a DNA-binding enzyme implicated in transient double-strand breaks, chromosome condensation, and anaphase separation — is associated with the chromatin and SCs of the pachytene and diplotene chromosomes.     

Organization of highly repeated sequences in surface-spread pachytene chromosomes of rye.

A method of preparing two-dimensional surface spreads of plant synaptonemal complexes (SCs) associated with fluorescence in situ hybridization (FISH) has been applied to analyze the location and organization of five different highly repeated DNA sequences in rye. Our observations indicate that, depending on the type of sequence, the chromatin displays different types of organization. Telomeric sequences were seen tightly associated with the SC while other repetitive DNA sequences were found to form loops that are associated with SCs only at their bases. On the contrary, the FISH signal of a centromeric satellite had a granular appearance, reflecting that the hybridization occurs only with parts of the chromatin loops.     

Visualization of chromosome territories in interphase nuclei of ovarian nurse cells in Calliphora erythrocephala Mg. (Diptera: Calliphoridae)

Analysis of localization of chromosomes 2, 3, and 6 of Calliphora erythrocephala Mg. in ovarian nurse cell nuclei with different chromatin structure has shown that the regions of DNA probe hybridization reduced with increasing chromatin compaction. Hybridization of DNA probes of chromosomes 3 and 6 to secondary reticular nuclei demonstrated that chromosomes retain their territories in the nuclei when the chromatin acquires a reticular structure. These results suggest regular organization of the chromosomal apparatus at all stages of the endomitotic cycle, including the stage of highly polyploid reticular nuclei. FISH of DNA probe of the chromosome 2 telomeric region to secondary reticular nuclei revealed a peripheral distribution of the signal. Zones of more intensive DNA probe hybridization have been distinguished. These zones probably are the regions of accumulation of telomeric and (or) centromeric chromosome regions.     

Visualization of Chromosome Territories in Interphase Nuclei of Ovarian Nurse Cells in <Emphasis Type="Italic">Calliphora erythrocephala</Emphasis> Mg. (Diptera: Calliphoridae)

Analysis of localization of chromosomes 2, 3, and 6 of Calliphora erythrocephala Mg. in ovarian nurse cell nuclei with different chromatin structure has shown that the regions of DNA probe hybridization reduced with increasing chromatin compaction. Hybridization of DNA probes of chromosomes 3 and 6 to secondary reticular nuclei demonstrated that chromosomes retain their territories in the nuclei when the chromatin acquires a reticular structure. These results suggest regular organization of the chromosomal apparatus at all stages of the endomitotic cycle, including the stage of highly polyploid reticular nuclei. FISH of DNA probe of the chromosome 2 telomeric region to secondary reticular nuclei revealed a peripheral distribution of the signal. Zones of more intensive DNA probe hybridization have been distinguished. These zones probably are the regions of accumulation of telomeric and (or) centromeric chromosome regions.     

Chromosomal localisation of DNA sequences in condensed and dispersed human chromatin.

The DNAs purified from condensed and dispersed human chromatin were used as templates for the in vitro synthesis of ³H-labelled complementary RNAs (cRNAs). These cRNAs were hybridised in situ to preparations of fixed human metaphase chromosomes which had previously been stained with quinacrine and photographed with fluorescent (UV) light. Autoradiographs of the hybridised chromosomes were stained and photographed and the results analysed by comparison of the fluorescence photographs with the autoradiographs. This method allowed positive identification of every chromosomal site of hybridisation and quantitative analysis of grain distribution over a number of metaphase spreads. The cRNA transcribed from condensed chromatin DNA (cRNA_C) hybridised mainly to a limited number of sites close to or including centromeric heterochromatin (C-bands) and also to the brightly fluorescent regions of the Y chromosome. Many of these C-band regions are known to contain satellite DNAs, indicating that the repeated DNA in the condensed chromatin fraction consists largely, if not entirely, of satellite sequences. The cRNA transcribed from dispersed chromatin DNA (cRNA_D) does not contain satellite DNAs and hybridised more generally over the chromosome arms. However, the main sites of hybridisation with cRNA_D included the C-bands in the Y chromosome and autosomes, i.e. those regions which bound cRNA_C. This suggests that nonsatellite repeated DNA sequences may be associated with satellite DNAs in the chromosomes. No general correlation between the distribution of either kind of cRNA and the overall level of quinacrine fluorescence in chromosomes or chromosome arms was detectable, nor could the dispersed fraction be equated with cytological euchromatin, since it hybridised in many sites which appear heterochromatic. However, there was a suggestion that some non-fluorescing Q-bands bound cRNA_D preferentially. The differences which were found between the distribution of the cRNAs from the two chromatin fractions may be associated with differences in genetic activity.     

The species and chromosomal distribution of the centromeric alpha-satellite I sequence from sheep in the tribe Caprini and other Bovidae

The evolution of chromosomes in species in the family Bovidae includes fusion and fission of chromosome arms (giving different numbers of acrocentric and metacentric chromosomes with a relatively conserved total number of arms) and evolution in both DNA sequence and copy number of the pericentromeric alpha-satellite I repetitive DNA sequence. Here, a probe representing the sheep alpha-satellite I sequence was isolated and hybridized to genomic DNA digests and metaphase chromosomes from various Bovidae species. The probe was highly homologous to the centromeric sequence in all species in the tribe Caprini, including sheep (Ovis aries), goat (Capra hircus) and the aoudad or Barbary sheep (Amnotragus lervia), but showed no detectable hybridization to the alpha-satellite I sequence present in the tribe Bovini and at most very weak to species in the tribes Hippotragini, Alcelaphini or Aepycerotini. The sex chromosomes of sheep, goat and aoudad did not contain detectable alpha-satellite I sequence; in sheep, one of the three metacentric autosomal chromosomes does not carry the sequence, while in aoudad, it is essentially absent in three large autosomal pairs as well as the large metacentric chromosome pair. The satellite probes can be used as robust chromosome and karyotype markers of evolution among tribes and increase the resolution of the evolutionary tree at the base of the Artiodactyla.     

Labeling of the centromeric region on human chromosome 8 by in situ hybridization

Summary Probe DNA that binds preferentially to the centromeric region of human chromosomes 8 was synthesized. Alpha satellite probe DNA molecules were selectively amplified from sorter-purified human chromosomes 8 by in vitro DNA amplification using the polymerase chain reaction (PCR). Probe labeling was performed during PCR by incorporation of biotinylated deoxyuridine. In situ hybridization of unpurified probe DNA comprised of alpha satellite monomer and higher molecular weight DNA fragments with metaphase chromosome spreads showed binding to the centromeric regions of numerous chromosomes. However, blocking with unlabeled total human alphoid DNA dramatically reduced crosshybridization to chromosomes other than 8. Under these conditions, the degenerate probe DNA allowed unambiguous visualization of domains occupied by centromeric DNA of chromosome 8 in metaphase spreads and interphase cell nuclei, thus greatly facilitating the detection of numerical chromosome aberrations in tumor cells. In situ hybridization of size-fractionated alpha satellite DNA identified the monomeric fraction as the major cause of crosshybridization. Alpha satellite dimers and higher molecular weight DNA fragments showed relatively high specificity for human chromosomes 8.     

Analysis of alphoid DNA variation and kinetochore size in human chromosome 21: evidence against pathological significance of alphoid satellite DNA diminutions.

Centromeric alpha satellite DNA sequences are linked to the kinetochore CENP-B proteins and therefore may be involved in the centromeric function. The high heterogeneity of size of the alphoid blocks raises the question of whether small amount of alphoid DNA or "deletion" of this block may have a pathological significance in the human centromere. In the present study, we analysed the correlation between size variations of alphoid DNA and kinetochore sizes in human chromosome 21 by molecular cytogenetic and immunochemical techniques. FISH analyses of alpha satellite DNA sizes in chromosome 21 homologues correlated well with the variation of their physical size as determined by pulsed field gel electrophoresis (PFGE). By contrast, the immunostaining study of the same homologous chromosomes with antikinetochore antibodies suggested that there is no positive correlation between the alpha satellite DNA block and kinetochore sizes. FISH analysis of chromosome 21-specific alphoid DNA and immunostaining of kinetochore extended interphase chromatin fibers indicate that centromeric kinetochore-specific proteins bind to restricted areas of centromeric DNA arrays. Thus, probably, restricted regions of centromeric DNA play an important role in kinetochore formation, centromeric function and abnormal chromosome segregation leading to non-disjunction.     

Molecular cytogenetic characterization of 17 rob(13q14q) Robertsonian translocations by FISH, narrowing the region containing the breakpoints.

We have characterized 17 rob(13q14q) Robertsonian translocations, using six molecular probes that hybridize to the repetitive sequences of the centromeric and shortarm regions of the five acrocentric chromosomes by FISH. The rearrangements include six de novo rearrangements and the chromosomally normal parents, five maternally and three paternally inherited translocations, and three translocations of unknown origin. The D21Z1/D13Z1 and D14Z1/D22Z1 centromeric alpha-satellite DNA probes showed all rob(13q14q) chromosomes to be dicentric. The rDNA probes did not show hybridization on any of the 17 cases studied. The pTRS-47 satellite III DNA probe specific for chromosomes 14 and 22 was retained around the breakpoints in all cases. However, the pTRS-63 satellite III DNA probe specific for chromosome 14 did not show any signals on the translocation chromosomes examined. In 16 of 17 translocations studied, strong hybridization signals on the translocations were detected with the pTRI-6 satellite I DNA probe specific for chromosome 13. All parents of the six de novo rob(13q14q), including one whose pTRI-6 sequence was lost, showed strong positive hybridization signals on each pair of chromosomes 14 and 13, with pTRS-47, pTRS-63, and pTRI-6. Therefore, the translocation breakpoints in the majority of rob(13q14q) are between the pTRS-47 and pTRS-63 sequences in the p11 region of chromosome 14 and between the pTRI-6 and rDNA sequences within the p11 region of chromosome 13.     

Structure of DNA near long tandem arrays of alpha satellite DNA at the centromere of human chromosome 7.

The centromeric regions of human chromosomes contain long tracts of tandemly repeated DNA, of which the most extensively characterized is alpha satellite. In a screen for additional centromeric DNA sequences, four phage clones were obtained which contain alpha satellite as well as other sequences not usually found associated with tandemly repeated alpha satellite DNA, including L1 repetitive elements, an Alu element, and a novel AT-rich repeated sequence. The alpha satellite DNA contained within these clones does not demonstrate the higher-order repeat structure typical of tandemly repeated alpha satellite. Two of the clones contain inversions; instead of the usual head-to-tail arrangement of alpha satellite monomers, the direction of the monomers changes partway through each clone. The presence of both inversions was confirmed in human genomic DNA by polymerase chain reaction amplification of the inverted regions. One phage clone contains a junction between alpha satellite DNA and a novel low-copy repeated sequence. The junction between the two types of DNA is abrupt and the junction sequence is characterized by the presence of runs of A's and T's, yielding an overall base composition of 65% AT with local areas > 80% AT. The AT-rich sequence is found in multiple copies on chromosome 7 and homologous sequences are found in (peri)centromeric locations on other human chromosomes, including chromosomes 1, 2, and 16. As such, the AT-rich sequence adjacent to alpha satellite DNA provides a tool for the further study of the DNA from this region of the chromosome. The phage clones examined are located within the same 3.3-Mb SstII restriction fragment on chromosome 7 as the two previously described alpha satellite arrays, D7Z1 and D7Z2. These new clones demonstrate that centromeric repetitive DNA, at least on chromosome 7, may be more heterogeneous in composition and organization than had previously been thought.     

Selective digestion of mouse metaphase chromosomes

Metaphase chromosomes prepared from colcemid-treated mouse L929 cells by non-ionic detergent lysis exhibit distinct heterochromatic centromere regions and associated kinetochores when viewed by whole mount electron microscopy. Deoxyribonuclease I treatment of these chromosomes results in the preferential digestion of the chromosomal arms leaving the centromeric heterochromatin and kinetochores apparently intact. Enrichment in centromere material after DNase I digestion was quantitated by examining the increase in 10,000xg pellets of the 1.691 g/cc satellite DNA relative to main band DNA. This satellite species has been localized at the centromeres of mouse chromosomes by in situ hybridization. From our analysis it was determined that DNase I digestion results in a five to six-fold increase in centromeric material. In contrast to the effect of DNase I, micrococcal nuclease was found to be less selective in its action. Digestion with this enzyme solubilized both chromosome arms and centromeres leaving only a small amount of chromatin and intact kinetochores.     

Oligonucleotide-priming methods for the chromosome-specific labelling of alpha satellite DNA in situ

It is demonstrated that either general staining of the centromeric regions of all primate chromosomes, or selective staining of the centromeric region of specific chromosomes, may be obtained in preparations of metaphase chromosomes by probing specifically for different regions within the alpha satellite DNA monomer. In order to exploit observed patterns of sequence variation within the monomer for this purpose, we have developed two new DNA analysis methods. In PRimed IN Situ labelling (PRINS), synthetic oligonucleotides derived from subsections of the monomer are hybridized to the chromosomes. The oligonucleotides then serve as primers for the in situ incorporation of biotin-labelled nucleotides catalysed by Klenow polymerase. Incorporated biotin is visualized with fluorescein isothiocyanate-labelled avidin (FITC-avidin). In Primed Amplification Labelling (PAL), biotin-labelled hybridization probes are produced in a polymerase chain reaction (PCR, Saiki et al. 1985), in which two synthetic oligonucleotide primers anneal within the same monomer. With the right choice of primers libraries of labelled probes derived from most monomers present as templates are produced. If DNA from a specific chromosome is used as template, then the resulting probe mixture gives stronger and more chromosome-specific signals in in situ hybridization experiments than does a cloned alpha satellite DNA probe derived from the same chromosome. The results obtained indicate that the alpha-repeat monomer is composed of regions with different degrees of chromosome specificity.     

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     

Non-isotopical labeling of murine heterochromatin in situ by hybridization with in vitro-synthesized biotinylated gamma (major) satellite DNA

Degenerate probe DNA, homologous to part of the 234-bp repeated mouse gamma (major) satellite DNA, was generated by primer-directed in vitro DNA amplification using the polymerase chain reaction with oligonucleotide primers that anneal in the most conserved parts of the repeat. Probe labeling with biotin was performed during DNA polymerization. In situ hybridization of probe DNA with metaphase chromosome preparations showed exclusive binding of probe molecules to the centromeric region of mouse chromosomes. We applied the probe DNA for labeling of mouse heterochromatin in metaphase chromosomes, as well as interphase cell nuclei, and compared results of probe visualization using avidin tagged with either fluorescein or alkaline phosphatase in combination with a chromogenic substrate.