Organization and evolution of alpha satellite DNA from human chromosome 11

The human alpha satellite repetitive DNA family is organized as distinct chromosomal subsets located at the centromeric regions of each human chromosome. Here, we describe a subset of the alpha satellite which is localized to human chromosome 11. The principal unit of repetition of this alpha satellite subset is an 850 bp XbaI fragment composed of five tandem diverged alphoid monomers, each 171 bp in length. The pentamer repeat units are themselves tandemly reiterated, present in 500 copies per chromosome 11. In filter hybridization experiments, the Alpha 11 probes are specific for the centromeric alpha satellite sequences of human chromosome 11. The complete nucleotide sequences of two independent copies of the XbaI pentamer reveal a pentameric configuration shared with the alphoid repeats of chromosomes 17 and X, consistent with the existence of an ancestral pentameric repeat common to the centromeric arrays of at least these three human chromosomes.     

Replication pattern of the X and Y chromosomes in partially synchronized human lymphocyte cultures

The replication pattern of the X and Y chromosomes at the beginning of the synthetic phase was studied in human lymphocyte cultures partially synchronized by the addition of 5-fluoro-2-deoxyuridine (FUdR). The data were evaluated statistically by an analysis of the distribution of silver grain counts over the X and Y chromosomes. —In cells from normal females, one of the X chromosomes began replication later than any other chromosomes of the complement. The short arm of the late replicating X chromosome started replication earlier than the long arm. The telomeric region of the short arm was a preferential site of DNA synthesis at the beginning of replication. —In partially synchronized lymphocyte cultures from a patient with the XXY syndrome, the Y chromosome started replication together with the late replicating X chromosome. The Y chromosome most frequently replicated synchronously with the short arm of the X. The centromeric region of the Y chromosome initiated synthesis before the telomeric region and appeared to replicate synchronously with the telomeric region of the short arm of the X. These findings are discussed with reference to the pairing of the X and Y chromosomes at meiosis.Supported in part by the National Institute of Health Research Grant HD-01979 and National Foundation Birth Defects Research Grant CRCS-40. Dr. Knight was a predoctoral fellow under National Institute of Health Training Program HD-00049-09.     

In situ hybridization to somatic metaphase chromosomes of potato

Summary An in situ hybridization procedure was developed for mitotic potato chromosomes by using a potato 24S rDNA probe. This repetitive sequence hybridized to the nucleolar organizer region (NOR) of chromosome 2 in 95%–100% of the metaphase plates. Another repetitive sequence (P5), isolated from the interdihaploid potato HH578, gave a ladderpattern in genomic Southern's of Solanum tuberosum and Solanum phureja, but not in those of Solanum brevidens and two Nicotiana species. This sequence hybridized predominantly on telomeric and centromeric regions of all chromosomes, although chromosomes 7, 8, 10 and 11 were not always labeled clearly.     

Longitudinal differentiation of metaphase chromosomes of Indian muntjac as studied by restriction enzyme digestion,in situ hybridization with cloned DNA probes and distamycin A plus DAPI fluorescence staining

The longitudinal differentiation of metaphase chromosomes of the Indian muntjac was studied by digestion with restriction enzymes, in situ hybridization with cloned DNA probes and distamycin A plus DAPI (4-6-diamidino-2-phenylindole) fluorescence staining. The centromeric regions of chromosomes 3 and 3 + X of a male Indian muntjac cell line were distinct from each other and different from those of other chromosomes. Digestion with a combination of EcoRI^* and Sau3A revealed a pattern corresponding to that of C-banding. Digestion with AluI, EcoRII or RsaI yielded a band specific to the centromeric region only in chromosomes 3 and 3 + X. Furthermore, HinfI digestion yielded only a band at the centromeric region of chromosome 3, whereas DA-DAPI staining revealed a single band limited to the extreme end of the C-band heterochromatin of the short arm of 3 + X. These results suggest that centromeres of Indian muntjac chromosomes contain at least four different types of repetitive DNA. Such diversity in heterochromatin was also confirmed by in situ hybridization using specific DNA probes isolated and cloned from highly repetitive DNA families. Heterozygosity between chromosome homologs was revealed by restriction enzyme banding. Evidence is presented for the presence of nucleolus organizer regions (NORs) on the long arm of chromosome 1 as well as on the secondary constrictions of 3 and 3 + X.Abbreviations DA distamycin A - DAPI 4-6-diamidino-2-phenylindole - NOR(s) nucleolus organizer region(s) - PBS phosphate-buffered saline - PI propidium iodide     

Isolation and identification of a novel tandemly repeated DNA sequence in the centromeric region of human chromosome 8

EcoRI subclones, designated as 50E1 and 50E4, were independently obtained from a cosmid clone previously mapped to the centromeric region of human chromosome 8. Southern blot hybridization analyses suggested that both subclones contain repetitive DNA sequences different from the chromosome 8 specific alphoid DNA. DNA sequence analysis of the 704 bp insert of 50E1 and the 1, 962 bp insert of 50E4 revealed that both inserts contained tandemly repeated units of 220 bp. Fluorescence in situ hybridization studies confirmed these two subclones to be specifically located on the centromeric region of chromosome 8. A 220 bp consensus sequence, derived from nine monomeric repeats, showed no significant homology to alphoid consensus sequences or to other currently known human centromeric DNA sequence. Furthermore, no significant homology was found with any other DNA sequence deposited in the EMBL or GenBank databases, indicating that this chromosome 8 specific repetitive DNA sequence is novel. From slot blot experiments it was estimated that 0.013% of the human genome comprises 1,750 of these monomeric repeats, residing on the centromeric region of chromosome 8 in tandem array(s).     

Chromosome banding in Amphibia. XV. Two types of Y chromosomes and heterochromatin hypervariabilty inGastrotheca pseustes (Anura,Hylidae)

The chromosomes of the newly discovered South American marsupial frogGastrotheca pseustes were analyzed by conventional methods and by various banding techniques. This species is characterized by XY/XX sex chromosomes and the existence of two different morphs of Y chromosomes. Whereas in type A males the XY^A chromosomes are still homomorphic, in type B males the Y^B chromosome displays a large heterochromatic region at the long arm telomere which is absent in the X. In male meiosis, the homomorphic XY^A chromosomes exhibit the same pairing configuration as the autosomal bivalents. On the other hand, the heteromorphic XY^B chromosomes form a sex bivalent by pairing their short arm telomeres in a characteristic end-to-end arrangement. Analysis of the karyotypes by C-banding and DNA base pair-specific fluorochromes reveals enormous interindividual size variability of the autosomal heterochromatin.     

Deletion of specific sequences or modification of centromeric chromatin are responsible for Y chromosome centromere inactivation

Summary Stable dicentric chromosomes behave as monocentrics because one of the centromeres is inactive. The cause of centromere inactivation is unknown; changes in centromere chromatin conformation and loss of centromeric DNA elements have been proposed as possible mechanisms. We studied the phenomenon of inactivation in two Y centromeres, having as a control genetically identical active Y centromeres. The two cases have the following karyotypes: 45,X/46,X,i(Y)(q12) and 46,XY/ 47,XY,+t(X;Y)(p22.3;p11.3). The analysis of the behaviour of the active and inactive Y chromosome centromeres after Da-Dapi staining, CREST immunofluorescence, and in situ hybridization with centromeric probes leads us to conclude that, in the case of the isochromosome, a true deletion of centromeric chromatin is responsible for its stability, whereas in the second case, stability of the dicentric (X;Y) is the result of centromere chromatin modification.     

DNA analyses of XX and XX-hypospadiac males

Fourteen 46,XX males were analyzed by Southern blot hybridization with seventeen different Y chromosome-derived DNA probes and by the polymerase chain reaction for an additional two sites on the short arm of Y. Eight 46,XX males possessed various segments of the short arm of the Y chromosome, including the sex determining region. The detected segments ranged from the two most distal loci to nearly the entire length of the short arm, viz., 10 out of 11 loci. None of the eight patients had hypospadia. Five out of the six remaining cases had hypospadia and no Y sequence was detected, suggesting the presence of a causative difference between hypospadiac and non-hypospadiac groups.     

Chromosome engineering: generation of mono- and dicentric isochromosomes in a somatic cell hybrid system

The most common isochromosome found in humans involves the long arm of the X, i(Xq), and is associated with a subset of Turner syndrome cases. To study the formation and behavior of isochromosomes in a more tractable experimental system, we have developed a somatic cell hybrid model system that allows for the selection of mono- or dicentric isochromosomes involving the short arm of the X, i(Xp). Simultaneous positive and negative counterselection of a mouse/human somatic cell hybrid containing a human X chromosome, selecting for retention of the UBE1 locus in Xp but against the HPRT locus in Xq, results in a variety of abnormalities of the X chromosome involving deletions of Xq. We have generated 70 such ”Pushmi-Pullyu” hybrids derived from seven independent X chromosomes. Cytogenetic analysis of these hybrids using fluorescence in situ hybridization showed i(Xp) chromosomes in ∼19% of the hybrids. Southern blot and polymerase chain reaction analyses of the Pushmi-Pullyu hybrids revealed a distribution of breakpoints along Xq. The distance between the centromeres of the dicentric i(Xp)s generated ranged from ∼2 Mb to ∼20 Mb. To examine centromeric activity in these dicentric i(Xp)s, we used indirect immunofluorescence with antibodies to centromere protein E (CENP-E). CENP-E was detected at only one of the centromeres of a dicentric i(Xp) with ∼2–3 Mb of Xq DNA. In contrast, CENP-E was detected at both centromeres of a dicentric i(Xp) with ∼14 Mb of Xq DNA. Two other dicentric i(Xp) chromosomes were heterogeneous with respect to centromeric activity, suggesting that centromeric activity and chromosome stability of dicentric chromosomes may be more complicated than previously thought. The Pushmi-Pullyu model system presented in this study may provide a tool for examining the structure and function of mammalian centromeres. Received: 15 December 1998; in revised form: 2 March 1999 / Accepted: 5 April 1999     

Complete characterization of a large marker chromosome by reverse and forward chromosome painting

Marker chromosome are small supernumerary chromosomes that are sometimes associated with developmental abnormalities. Hence, the genes involved in such cases provide an interesting approach to understanding developmental abnormalities in man. As a first step towards isolating such sequences, marker chromosomes need complete characterization. By combining chromosome isolation by flow sorting and the degenerate oligonucleotide primed — polymerase chain reaction, we have constructed a DNA library specific for a marker chromosome found in a child with severe developmental abnormalities. We used fluorescent in situ hybridization of the library onto normal metaphase spreads (reverse chromosome painting) and were thus able to determine that the marker consists of the centromeric part of chromosome 7, the telomeric region of the long arm of chromosome 5 and the telomeric region of the short arm of the X-chromosome. Subsequently, we hybridized normal chromosome-specific libraries of the relevant chromosomes onto metaphases containing the marker chromosome (forward chromosome painting) and could in this manner establish the precise location of the different chromosome regions on the marker chromosome itself. This is a general approach suitable for outlining marker chromosomes in detail, and will aid the identification of the genes involved.     

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.     

Resolution of Dicentric Chromosomes by Ty-Mediated Recombination in Yeast

We have integrated a plasmid containing a yeast centromere, CEN5, into the HIS4 region of chromosome III by transformation. Of the three transformant colonies examined, none contained a dicentric chromosome, but all contained a rearranged chromosome III. In one transformant, rearrangement occurred by homologous recombination between two Ty elements; one on the left arm and the other on the right arm of chromosome III. This event produced a ring chromosome (ring chromosome III) of about 60 kb consisting of CEN3 and all other sequences between the two Ty elements. In addition, a linear chromosome (chromosome IIIA) consisting of sequences distal to the two Ty elements including CEN5, but lacking 60 kb of sequences from the centromeric region, was produced. Two other transformants also contain a similarly altered linear chromosome III as well as an apparently normal copy of chromosome III. These results suggest that dicentric chromosomes cannot be maintained in yeast and that dicentric structures must be resolved for the cell to survive.--The meiotic segregation properties of ring chromosome III and linear chromosome IIIA were examined in diploid cells which also contained a normal chromosome III. Chromosome IIIA and normal chromosome III disjoined normally, indicating that homology or parallel location of the centromeric regions of these chromosomes are not essential for proper meiotic segregation. In contrast, the 60-kb ring chromosome III, which is homologous to the centromeric region of the normal chromosome III, did not appear to pair with fidelity with chromosome III.     

Cytogenetics of the chinese giant salamander,Andrias davidianus (Blanchard): the evolutionary significance of cryptobranchoid karyotypes

Cytogenetic aspects of the cryptobranchid salamander Andrias davidianus of western China have been studied, including chromosome number and morphology, C-band patterns, meiosis, and the chromosomal localization of ribosomal 5S RNA genes. Our data regarding chromosome number (2n=60) and general chromosome morphology largely confirm the results of Morescalchi et al. (1977). The karyotype consists of 16 pairs of macrochromosomes that decrease gradually in relative length to 14 pairs of microchromosomes. Telocentric chromosomes are a conspicuous feature of the karyotype, representing more than half the genome. Differential staining reveals that all of the chromosomes, except four pairs of microchromosomes, have C-band heterochromatin in their centromeric regions, the amount varying irrespective of chromosome size. Faint bands of interstitial and telomeric C-band heterochromatin are found in mitotic chromosomes but are not seen in meiotic preparations. In C-banded mitotic preparations from a female, one of the smallest macrochromosome pairs is heteromorphic in respect to C-band heterochromatin and centromere position. In situ hybridization of an iodinated 5S RNA probe to meiotic chromosome preparations reveals that this repeated gene is clustered near the telomeric region of chromosome 7, a medium size telocentric, a location corresponding to a band of heterochromatin. Studies of spermatocytes indicate that the process of meiosis in A. davidianus closely resembles that of more advanced salamanders, and that the microchromosomes are meiotically stable. The significance of microchromosomes and chromosome morphology in the reorganization of salamander genomes during evolution is discussed on the basis of cytogenetic data available for A. davidianus and various other primitive and advanced salamanders.     

Construction of midget chromosomes in wheat.

To test the usefulness of breakage-fusion-bridge (BFB) cycles in generating new chromosome aberrations in bread wheat (Triticum aestivum L.) and to extend the range of aberrations available, a series of midget chromosomes was produced from the long arm of chromosome 1B. Using a reverse tandem duplication initiated chromatid type BFB cycle, the 1BL arm was broken and fused with centromeres of either chromosome 5BL or 1RS to form dicentric chromosomes. The 1R and 5B centromeres were broken by centric misdivision. Among the progenies of plants with dicentric chromosomes, two classes of monocentric chromosomes were selected: deficient chromosomes 1B and chromosomes that had 1RS or 5BL for one arm and various fragments of 1BL for the other arm. Following centric misdivision of these monocentrics, midget chromosomes 1BL were isolated: deficient and deletion telocentrics and telocentrics derived from interstitial regions of 1BL. By chance, one deficient chromosome 1BS and one deletion chromosome 1BS were identified in unrelated lines of the same wheat. Following centric misdivision of these chromosomes, two midget chromosomes covering the whole of 1BS were added to the set.     

Identification and molecular cytogenetic characterization of a novel complex Y chromosome rearrangement in a boy with disorder of sexual development

Ambiguous genitalia or disorder of the sexual development is a birth defect where the external genitals do not have the typical appearance of either a male or female. Here we report a boy with ambiguous genitalia and short stature. The cytogenetic analysis by G-banding revealed a small Y chromosome and an additional material on the 15p arm. Further, molecular cytogenetic analysis by Fluorescence in situ hybridization (FISH) using whole chromosome paint probes showed the presence of Y sequences on the 15p arm, confirming that it is a Y;15 translocation. Subsequent, FISH with centromere probe Y showed two signals depicting the presence of two centromeres and differing with a balanced translocation. The dicentric nature of the derivative 15 chromosome was confirmed by FISH with both 15 and Y centromeric probes. Further, the delineation of the Y chromosomal DNA was also done by quantitative real time PCR. Additional Y-short tandem repeat typing was performed to find out the extent of deletion on small Y chromosome. Fine mapping was carried out with 8 Y specific BAC clones which helped in defining the breakpoint regions. MLPA was performed to check the presence or absence of subtelomeric regions and SHOX regions on Y. Finally array CGH helped us in confirming the breakpoint regions. In our study we identified and characterized a novel complex Y chromosomal rearrangement with a complete deletion of the Yq region and duplication of the Yp region with one copy being translocated onto the15p arm. This is the first report of novel and unique Y complex rearrangement showing a deletion, duplication and a translocation in the same patient. The possible mechanism of the rearrangement and the phenotype–genotype correlation are discussed.     

Antibodies to defined histone epitopes reveal variations in chromatin conformation and underacetylation of centric heterochromatin in human metaphase chromosomes

Unfixed metaphase chromosome preparations from human lymphocyte cultures were immunofluorescently labelled using antibodies to defined histone epitopes. Both mouse monoclonal antibody HBC-7, raised against the N-terminal region of H2B, and rabbit serum R5/12, which recognizes H4 acetylated at Lys-12, gave non-uniform labelling patterns, whereas control antibodies against total histone fractions H4 and H1 produced homogeneous fluorescence. HBC-7 bound approximately uniformly to the bulk of the chromosomes, but the major heterochromatic domains of chromosomes 1, 9, 15, 16 and the Y showed significantly brighter fluorescence. Serum R5/12 indicated an overall reduction in acetylation of H4 in metaphase chromosomes compared with interphase nuclei, although some specific chromosomal locations had considerably elevated acetylation levels. Acetylation levels in the major heterochromatic domains appeared extremely low. To investigate further the differences noted in heterochromatin labelling, metaphases from cultures grown in the presence of various agents known to induce undercondensation of the major heterochromatic domains were similarly immunolabelled. Decondensed heterochromatin no longer exhibited higher than normal immunofluorescence levels with HBC-7. The higher resolution afforded by stretching the centromeric heterochromatin of chromosomes 1, 9 and 16 confirmed the low level of H4 acetylation in these domains. We consider the implications of these observations in relation to chromatin conformation and activity.by W.C. Earnshaw     

Jumping translocations in spontaneous abortions

Chromosome translocations involving one donor chromosome and multiple recipient chromosomes have been referred to as jumping translocations (JTs). Acquired JTs are commonly observed in cancer patients, mainly involving chromosome 1. Constitutional forms of JTs mostly involve the acrocentric chromosomes and their satellites and have been reported in patients with clinical abnormalities. Recognizable phenotypes resulting from these events have included Down, Prader-Willi, and DiGeorge syndromes. The presence of JTs in spontaneous abortions has not been previously described. The breakpoints of all JTs occur in areas rich in repetitive DNA (telomeric, centromeric, and nucleolus organizing regions). We report two different unstable chromosome rearrangements in samples derived from spontaneous abortions. The first case involved a chromosome 15 donor. The recipient chromosomes were 1, 9, 15, and 21, and the respective breakpoints were in either the heterochromatic regions or the centromeres. FISH studies confirmed that the breakpoints of the jumping 15 rearrangement did not involve the Prader-Willi region but originated at the centromere or in the proximal short arm. A second case of instability was observed with a rearrangement resulting from a presumed de novo 8;21 translocation. Three JT cell lines were observed. They consisted of a deleted 8p chromosome, a dicentric 8;21 translocation, and an 8q isochromosome. The instability regions appeared to be at the pericentromeric region of chromosome 8 and the satellite region of chromosome 21. Both cases proved to be de novo events. The unstable nature of the JT resulting in chromosomal imbalance most likely contributed to the fetal loss. It appears that JT events may predispose to chromosomal imbalance via nondisjunction and chromosomal rearrangement and, therefore, may be an unrecognized cause of fetal loss.     

Ultrastructure and division behaviour of dinoflagellate chromosomes

Chromosomes of Prorocentrum triestinum and P. micans have similar substructural and morphometrical values as revealed by electron microscopy of thin sections. However, differences were found between the species in mean length, volume and numerical density of chromosomes, and the volume of the chromosome complement, the nuclear volume and the chromosome number. When examined by a whole-mount procedure both Prorocentrum species have left-handed screw-like chromosomes which end in differentiated telomeres. The chromosomes divide sequentially from one telomere towards the other, presenting a Y and finally a V configuration. At the region where each chromosome divides nascent sister chromatids are connected by two bridges. Sister chromatids have similar quantitative values when compared with each other and with the still undivided chromosome, which suggests that both replication and division take place as coupled events.Supported by CAICYT, grant 2409/83     

Banded chromosomes of the owl monkey, Aotus trivirgatus.

Chromosomes of the owl monkey, Aotus trivirgatus, with 2n=54, 53, or 52, have been stained to show quinacrine (Q-) and Giemsa (G-) bands, and a karyotypic arrangement has been proposed based on lengths, centrometric index, and banding pattern. C-bands were present at the centromeric region of every chromosome and over the entire short arm of certain acrocentric chromosomes; 5-methylcytosine was concentrated in the same regions. Bright Q-bands at the telomeric ends of the short arms of some chromosomes probably represent a second type of repetitive DNA. Ag-staining showed that only the chromosomes bearing a secondary constriction are nucleolus organizer chromosomes.     

The characteristics of chromosomal distribution and of the variability of the multiply repeating DNA fraction of the genome in Bos taurus L.]

The uniform distribution of satellite DNA II and IV has been revealed using in situ hybridization and differential staining in centromeric regions of autosomes. The sex chromosomes have not found such nucleotide blocks. There is only minor satellite IV block inside Y chromosome short arm. The Y chromosome has got some (TG)n enriched blocks distributed also among other parts of genome and one copy of sequences like human ZFY gene. The high repetitive fraction of bovine genomic DNA have not revealed RFLP. However, the difference has been found by blot hybridization between genomic organization of satellite IV in cattle and yak chromosomal DNA. Non-Mendelian distribution of some such nucleotide blocks has been obtained for interspecies crosses of cattle and yak.