Interstitial deletions of repetitive DNA blocks in dicentric human Y chromosomes

Cytogenetic analysis of aberrant human Y chromosomes was done by fluorescence in situ hydbridization (FISH) with Y specific repetitive DNA probes. It revealed an interstitial deletion of different DNA blocks in two dicentric chromosome structures. One deletion includes the total alphoid DNA structure of one centromeric region. The second deletion includes the total repetitive DYZ5 DNA structure in the pericentromeric region of one short Y arm. Both dicentric Y chromosomes were iso(Yp) chromosomes with break and fusion point located in Yq11, the euchromatic part of the long Y arm. Their phenotypic appearance was abnormal, resembling small monocentric Yq-chromosomes in metaphase plates. Mosaic cell lines, usually included in karyotypes with dicentric Y chromosomes, were not observed. It is assumed that both deletion events suppress the kinetochore activity in one Y centromeric region and thus stabilize its dicentric structure. Local interstitial deletion events had not been described in dicentric human Y chromosomes, but are common in dicentric yeast chromosomes. This raises the question of whether deletion events in dicentric human chromosomes are rare or restricted to the Y chromosome or also represent a general possibility for stabilization of a dicentric chromosome structure in human.     

Highly repetitive component α and related alphoid DNAs in man and monkeys

The genomes of Old-World, New-World, and prosimian primates contain members of a large class of highly repetitive DNAs that are related to one another and to component DNA of the African green monkey by their sequence homologies and restriction site periodicities. The members, of this class of highly repetitive DNAs are termed the alphoid DNAs, after the prototypical member, component of the African green monkey which was the first such DNA to be identified (Maio, 1971) and sequenced (Rosenberg et al., 1978). The alphoid DNAs appear to be uniquely primate sequences. — From the restriction enzyme cleavage patterns and Southern blot hybridizations under different stringency conditions, the alphoid DNAs comprise multiple sequence families exhibiting varying degrees of homology to component DNA. They also share common elements in their restriction site periodicities (172 · n base-pairs), in the long-range organization of their repeating units, and in their banding behavior in CsCl and Cs₂SO₄ buoyant density gradients, in which they band within the bulk DNA as cryptic repetitive components. — In the three species from the Family Cercopithecidae examined, the alphoid DNAs represent the most abundant, tandemly repetitive sequence components, comprising about 24% of the African green monkey genome and 8 to 10% of the Rhesus monkey and baboon genomes. In restriction digests, the bulk of the alphoid DNAs among the Cercopithecidae appeared quantitatively reduced to a simple series of arithmetic segments based on a 172 base-pair (bp) repeat. In contrast with these simple restriction patterns, complex patterns were observed when human alphoid DNAs were cleaved with restriction enzymes. Detailed analysis revealed that the human genome contains multiple alphoid sequence families which differ from one another both in their repeat sequence organization and in their degree of homology to the African green monkey component DNA. — The finding of alphoid sequences in other Old-World primate families, in a New-World monkey, and in a prosimian primate attests to the antiquity of these sequences in primate evolution and to the sequence conservatism of a large class of mammalian highly repetitive DNA. In addition, the relative conservatism exhibited by these sequences may distinguish the alphoid DNAs from more recently evolved highly repetitive components and satellite DNAs which have a more restricted taxonomical distribution.     

Toward a molecular paleontology of primate genomes

Families of related, but nonidentical repetitive DNA sequences, termed the alphoid DNAs, have been identified and characterized in representative species from seven major primate Families. The sequences appear as old as the primate Order itself: they are found in a prosimian (lemur), in a New World monkey, and in all Old World primates examined, including man. The alphoid DNAs are uniquely primate sequences and they may represent the most abundant repetitive DNAs in the primate genome. — A classification scheme for two major families of alphoid DNAs is proposed that is based upon restriction enzyme analysis and Southern blotting with radioactive probes prepared from component DNA (Maio, 1971) and from the human EcoRI dimer sequences (Manuelidis, 1976). The family of alphoid DNAs that hybridizes readily with component is termed the HindIII family of alphoid DNAs. This family shows an almost universal distribution among present-day primates. The family of DNA sequences that hybridizes readily with the human EcoRI dimer probe is termed the EcoRI dimer family of alphoid DNAs. This family may be restricted to the great apes and man. The two probes permitted the discrimination of different, but related alphoid families in present-day primates. Multiple alphoid sequence families are found within the genomes of individual primates and the major primate taxa can be characterized by the representations of the various alphoid DNAs within their genomes. — An Appendix is presented (Brown et al., 1981) indicating that competition hybridization effects may influence the autoradiographic banding patterns, and hence, the interpretations of Southern filter-transfer hybridizations when dealing with related repetitive sequences such as the alphoid DNAs that are present in abundance in eukaryotic genomes.     

Repeated sequences in the DNA of Drosophila and their localization in giant chromosomes

It is shown by isopycnic density gradient centrifugation that the DNAs of the sibling species Drosophila hydei, Drosophila neohydei and Drosophila pseudoneohydei differ regarding the numbers and proportions of satellite DNA bands. An overwhelming proportion of all repetitive nucleotide sequences of the DNA is contained in these satellite fractions. The majority of the satellites are species specific despite the close phylogenetic and cytological relationship between the three species studied. — By in situ hybridization experiments it is demonstrated that the various satellite sequences occupy different positions within the chromosomes. All types of localization patterns, from a wide spread occurrence in all chromosomes to an apparent restriction to kinetochore regions of single chromosomes, have been observed. Main band DNA, on the other hand, in its hybridization behavior reflects the DNA distribution according to the banding pattern in giant chromosomes. Generally satellite sequences seem to be included in -heterochromatic chromosome regions but no relation to the heterochromatin of the Y-chromosome was found. — Renaturation studies support various evidence that satellite sequences occur in tandemly repetitious units. At least some of this repetitious material seems to be linked to non-satellite DNA sequences or to DNA of other satellites.     

植物功能着丝粒DNA研究进展

着丝粒（centromere）是真核生物染色体的重要功能结构。在细胞有丝分裂和减数分裂过程中,着丝粒通过招募动粒蛋白行使功能,保障染色体正确分离和传递。真核生物中,含有着丝粒特异组蛋白的CenH3区域被定义为功能着丝粒区,即真正意义上的着丝粒。近年来,借助染色质免疫沉淀技术,人们对功能着丝粒DNA开展了深入研究,揭示其组成、结构及演化特征,并发现功能着丝粒区存在具有转录活性的基因,且部分基因具有重要生物学功能。由于存在大量重复DNA,着丝粒演化之谜一直未能完全揭示。对植物功能着丝粒DNA序列研究进展进行了概述,并重点阐述了着丝粒重复DNA研究的新方法和新进展,以期为深入开展相关研究提供借鉴。     

Mapping of rRNA genes and telomeric sequences in Danube salmon (<Emphasis Type="Italic">Hucho hucho</Emphasis>) chromosomes using primed in situ labeling technique (PRINS)

In the current paper we described the application of primed in situ (PRINS) labeling approach for the chromosomal mapping of repetitive DNA sequences in Danube salmon (Hucho hucho) (2n = 82, NF = 112). PRINS was successfully performed with primers enabling amplification of 5S rRNA genes (minor rDNAs), NOR building DNA sequences (major rDNAs), and telomeric sequences. Two loci of 5S rRNA were observed on distinct chromosome pairs; the minor arrays were located interstitially on the long (q) arms of two large metacentrics (chromosomes No. 3) and the large clusters of 5S rDNAs were assigned to the short (p) arms of two subtelocentric chromosomes No. 18. Major rDNA clusters were observed on the p-arms of two submeta-subtelocentric chromosomes No. 10. These chromosomal areas were built with GC-rich chromatin what was proved in the course of chromomycin A(3) (CMA(3)) staining performed sequentially. Major and minor rDNA families were not co-localized in the Danube salmon chromosomes.The distinct hybridization signals at the ends of all the chromosomes were provided in the course of PRINS with (CCCTAA)( n ) primer. The chromosomal localization of rRNA genes and telomeric DNA sequences was discussed in the context of Salmonidae karyotype evolution.     

Chromosomal Mapping of Repetitive DNAs in the Grasshopper Abracris flavolineata Reveal Possible Ancestry of the B Chromosome and H3 Histone Spreading

Supernumerary chromosomes (B chromosomes) occur in approximately 15% of eukaryote species. Although these chromosomes have been extensively studied, knowledge concerning their specific molecular composition is lacking in most cases. The accumulation of repetitive DNAs is one remarkable characteristic of B chromosomes, and the occurrence of distinct types of multigene families, satellite DNAs and some transposable elements have been reported. Here, we describe the organization of repetitive DNAs in the A complement and B chromosome system in the grasshopper species Abracris flavolineata using classical cytogenetic techniques and FISH analysis using probes for five multigene families, telomeric repeats and repetitive C₀t-1 DNA fractions. The 18S rRNA and H3 histone multigene families are highly variable and well distributed in A. flavolineata chromosomes, which contrasts with the conservation of U snRNA genes and less variable distribution of 5S rDNA sequences. The H3 histone gene was an extensively distributed with clusters occurring in all chromosomes. Repetitive DNAs were concentrated in C-positive regions, including the pericentromeric region and small chromosomal arms, with some occurrence in C-negative regions, but abundance was low in the B chromosome. Finally, the first demonstration of the U2 snRNA gene in B chromosomes in A. flavolineata may shed light on its possible origin. These results provide new information regarding chromosomal variability for repetitive DNAs in grasshoppers and the specific molecular composition of B chromosomes.     

The organization of the mouse satellite DNA at centromeres

The mouse genome contains a major and a minor satellite DNA family of repetitive DNA sequences. The use of 5-azacytidine has allowed us to demonstrate that these satellite DNAs are organized in two separate domains at the centromeres of mouse chromosomes. The minor satellite is closer to the short arms of the acrocentric chromosomes than the major satellite. The major satellite is farther away, flanking the minor satellite and adjacent to the euchromatic long arm of each mouse chromosome. At the level of resolution afforded by the in situ hybridization technique it would appear that the organization of the centromeric domain of the mouse is similar to that in man. That is, both contain two repetitive DNA sequence families arranged in major blocks.     

Localization of repetitive DNA sequences on in vitro Xenopus laevis chromosomes by primed in situ labeling (PRINS)

Xenopus laevis is an important reference model organism used in many vertebrate studies. Gene mapping in X. laevis, in comparison to other reference organisms, is in its early stages. Few studies have been conducted to localize DNA sequences on X. laevis chromosomes. Primed in situ labeling (PRINS) is a recently developed innovative tool that has been used to locate specific DNA sequences in various organisms. PRINS has been reported to have increased sensitivity compared to other in situ hybridization techniques. In the present study, PRINS was first used to label the location of telomeres at the ends of in vitro X. laevis chromosomes. The terminal location was as expected from in vivo reports, however, the overall amount seemed to decrease in the in vitro chromosomes. Once the PRINS technique was optimized, this technique was used to determine the chromosomal location of the satellite 1 repetitive sequence, which is an important sequence in X. laevis development. The sequence was observed on the interstitial regions of the majority of the chromosomes similar to the in vivo locations reported. In contrast to the telomeric sequence, the amount of sequence appeared to increase in the satellite 1 sequence. PRINS was found to be useful in the localization of repetitive DNA sequences in the X. laevis genome.     

The distribution of interspersed repetitive DNA sequences in the human genome

The distribution of interspersed repetitive DNA sequences in the human genome has been investigated, using a combination of biochemical, cytological, computational, and recombinant DNA approaches. "Low-resolution" biochemical experiments indicate that the general distribution of repetitive sequences in human DNA can be adequately described by models that assume a random spacing, with an average distance of 3 kb. A detailed "high-resolution" map of the repetitive sequence organization along 400 kb of cloned human DNA, including 150 kb of DNA fragments isolated for this study, is consistent with this general distribution pattern. However, a higher frequency of spacing distances greater than 9.5 kb was observed in this genomic DNA sample. While the overall repetitive sequence distribution is best described by models that assume a random distribution, an analysis of the distribution of Alu repetitive sequences appearing in the GenBank sequence database indicates that there are local domains with varying Alu placement densities. In situ hybridization to human metaphase chromosomes indicates that local density domains for Alu placement can be observed cytologically. Centric heterochromatin regions, in particular, are at least 50-fold underrepresented in Alu sequences. The observed distribution for repetitive sequences in human DNA is the expected result for sequences that transpose throughout the genome, with local regions of "preference" or "exclusion" for integration.     

The kinetochore is an enhancer of pericentric cohesin binding

The recruitment of cohesins to pericentric chromatin in some organisms appears to require heterochromatin associated with repetitive DNA. However, neocentromeres and budding yeast centromeres lack flanking repetitive DNA, indicating that cohesin recruitment occurs through an alternative pathway. Here, we demonstrate that all budding yeast chromosomes assemble cohesin domains that extend over 20–50 kb of unique pericentric sequences flanking the conserved 120-bp centromeric DNA. The assembly of these cohesin domains requires the presence of a functional kinetochore in every cell cycle. A similar enhancement of cohesin binding was also observed in regions flanking an ectopic centromere. At both endogenous and ectopic locations, the centromeric enhancer amplified the inherent levels of cohesin binding that are unique to each region. Thus, kinetochores are enhancers of cohesin association that act over tens of kilobases to assemble pericentric cohesin domains. These domains are larger than the pericentric regions stretched by microtubule attachments, and thus are likely to counter microtubule-dependent forces. Kinetochores mediate two essential segregation functions: chromosome movement through microtubule attachment and biorientation of sister chromatids through the recruitment of high levels of cohesin to pericentric regions. We suggest that the coordination of chromosome movement and biorientation makes the kinetochore an autonomous segregation unit.     

CENP-A associated complex satellite DNA in the kinetochore of the Indian muntjac

The centromere/kinetochore complex is a chromosomal assembly that mediates chromosome motility and mitotic regulation by interacting with microtubules of the mitotic spindle apparatus. Centromere protein A (CENP-A) is a histone H3 homolog that is concentrated in the chromatin of the inner kinetochore plate of human chromosomes. To identify DNA sequences associated with the inner kinetochore plate, we used anticentromere autoantibodies to immunoprecipitate CENP-A associated chromatin selectively from Indian muntjac fibroblasts. DNA was cloned from immunoprecipitated CENP-A- associated chromatin and characterized by DNA sequence and hybridization analyses. A novel centromeric satellite DNA sequence was identified and shown by fluorescence in situ hybridization analysis to be present at all centromeres of the Indian muntjac. This satellite DNA constitutes a 972 bp monomer repeat and shows partial homology with satellite II DNA of the white-tailed deer. Southern blot analysis of muntjac genomic DNA suggests that this satellite DNA is present in repetitive tandem arrays and contains complex internal arrangements. In conjunction with previous work showing the association of CENP-A with human α-satellite DNA, we conclude that the mammalian inner kinetochore plate contains a unique form of chromatin that contains CENP-A in association with complex satellite DNA. Received: 18 May 1999; in revised form: 5 July 1999 / Accepted: 20 July 1999     

Mapping of repeated DNA sequences in plant chromosomes by PRINS and C-PRINS

 The primed in situ DNA labelling (PRINS) procedure was optimised for the rapid physical mapping of several types of repetitive DNA sequences on the mitotic chromosomes of Vicia faba, Pisum sativum and Secale cereale. A localization of the highly repeated FokI sequence on V. faba chromosomes was achieved after a 7-min total reaction time. In addition, we report a procedure for direct cycling-PRINS (C-PRINS), a variation of PRINS which involves a sequence of thermal cycles analogous to the polymerase chain reaction. Compared to PRINS, C-PRINS was more sensitive. Further work is needed to improve the sensitivity of the reaction to allow for the reliable detection of low-copy DNA sequences. Received: 17 September 1996 / Accepted: 18 October 1996     

Pac-Man does not resolve the enduring problem of anaphase chromosome movement

Summary The Pac-Man hypothesis suggests that poleward movement of chromosomes during anaphase A is brought about by: disassembly of kinetochore microtubules (MTs) at the kinetochore; generation of the poleward force exclusively at or very close to the kinetochore; and the required energy coming from coupled disassembly of these MTs. This model has become widely accepted and cited as the sole or major mechanism of anaphase A. Rarely acknowledged are several significant phenomena that refute some or all of these postulates. We summarise these anomalies as follows: poleward movement of chromosomes occurring without insertion of any MTs at the kinetochore; anaphase shortening of kinetochore fibres in spindles entirely devoid of chromosomes and, presumably, kinetochores; continued movement of chromosomes while their severed kinetochore stub elongated poleward after treatment with UV microbeams; and fluxing of tubulin subunits through kinetochore MTs during anaphase A, indicating that during anaphase, kinetochore MTs disassemble partly or solely at the poles.Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

The distribution of repetitive DNAs between regular and supernumerary chromosomes in Species of Glossina (Tsetse): a two-step process in the origin of supernumeraries

Several species of tsetse fly within the Morsitans and Fusca subgenera of Glossina contain supernumerary (B) chromosomes. Previous studies on the meiotic behaviour of chromosomes (Southern and Pell, 1973) and the C-band patterns (Jordan et al., 1977) have indicated a close similarity between the Y chromosome and the supernumeraries. The distributions of the highly abundant families of DNA (satellite DNAs) between the autosomes, sex chromosomes and B chromosomes of G.m. morsitans, G. austeni and G. pallidipes have been examined by in situ hybridisation. In addition, the organisation and sequence homologies of satellite DNAs have been examined by restriction enzymes and heterologous hybridisations in in situ and Southern transfer conditions. The majority of satellite sequences that are homologous between species are distributed in several different arrangements between A and B chromosome telomeres with minority sequences at some centromeric and intercalary locations. There is no extensive satellite DNA similarity between the Y and B chromosomes. We suggest that the Y and B chromosome associations and synchronous allocycly during meiosis are the result of extensive heterochromatinisation of these two chromosome types, that is probably a reflection of two separate stages involved in the generation of the B chromosomes in the genus. The independent evolution of satellites and supernumeraries is discussed.     

Distribution of gamma satellite DNA on the human X and Y chromosomes suggests that it is not required for mitotic centromere function

The bulk of the DNA found at human centromeres is composed of tandemly arranged repeats, the most abundant of which is alpha satellite. Other human centromeric repetitive families have been identified, one of the more recent being gamma satellite. To date, gamma satellite DNAs have been reported at the centromeres of human chromosomes 8 and X. Here, we show that gamma-X satellite DNA is not interspersed with the major DZX1 alpha-X block, but rather is organised as a single array of approximately 40-50 kb on the short-arm side of the alpha satellite domain. This repeat array is absent on two mitotically stable Xq isochromosomes. Furthermore, a related repeat DNA has been identified on the human Y chromosome. Fluorescence in situ hybridisation has localised this satellite DNA to the long arm side of the major DYZ3 alpha-Y domain, outside the region previously defined as that required for mitotic centromere function. Together, these data suggest that while blocks of highly related gamma satellite DNAs are present in the pericentromeric regions of both human sex chromosomes, this repeated DNA is not required for mitotic centromere function.     

Stretching the rules: monocentric chromosomes with multiple centromere domains

The centromere is a functional chromosome domain that is essential for faithful chromosome segregation during cell division and that can be reliably identified by the presence of the centromere-specific histone H3 variant CenH3. In monocentric chromosomes, the centromere is characterized by a single CenH3-containing region within a morphologically distinct primary constriction. This region usually spans up to a few Mbp composed mainly of centromere-specific satellite DNA common to all chromosomes of a given species. In holocentric chromosomes, there is no primary constriction; the centromere is composed of many CenH3 loci distributed along the entire length of a chromosome. Using correlative fluorescence light microscopy and high-resolution electron microscopy, we show that pea (Pisum sativum) chromosomes exhibit remarkably long primary constrictions that contain 3-5 explicit CenH3-containing regions, a novelty in centromere organization. In addition, we estimate that the size of the chromosome segment delimited by two outermost domains varies between 69 Mbp and 107 Mbp, several factors larger than any known centromere length. These domains are almost entirely composed of repetitive DNA sequences belonging to 13 distinct families of satellite DNA and one family of centromeric retrotransposons, all of which are unevenly distributed among pea chromosomes. We present the centromeres of Pisum as novel "meta-polycentric" functional domains. Our results demonstrate that the organization and DNA composition of functional centromere domains can be far more complex than previously thought, do not require single repetitive elements, and do not require single centromere domains in order to segregate properly. Based on these findings, we propose Pisum as a useful model for investigation of centromere architecture and the still poorly understood role of repetitive DNA in centromere evolution, determination, and function.     

Satellite DNA properties of the germ line limited DNA and the organization of the somatic genomes in the nematodesAscaris suum andParascaris equorum

During the early cleavage divisions in some Ascarids, parts of the chromosomes are eliminated from the somatic blastomeres (chromatin diminution, Boveri, 1887) while the chromosomes in the germ line cells maintain their integrity. To characterize the germ line and soma genome, DNA was isolated from gametes and embryonic somatic cells of two Ascarid species,Parascaris equorum var. univalens andAscaris suum. It was shown that the germ line limited DNAs of these species have the same density and almost identical reassociation kinetics: in CsCl the predominant component of the germ line limited DNA ofP. equorum andA. suum has the buoyant density of 1.697g/cm³, while soma DNA of both species bands at 1.700 g/cm³. InP. equorum there is a small additional germ line limited satellite DNA component with the density of 1.690 g/cm³, identical to that of mitochondrial DNA of both organisms. Comparison of the reassociation kinetics of germ line and soma DNA demonstrates for both species that the eliminated DNA sequences are highly repetitive. In contrast to these similarities between the germ line limited DNAs ofP. equorum andA. suum the analysis of their base composition revealed differences (40% guanine plus cytosine inP. equorum and 36% inA. suum). The only very fast reassociating DNA sequences which we could isolate from soma DNA was demonstrated to be foldback DNA. The reassociation kinetics of totalA. suum soma DNA was investigated by hydroxylapatite chromatography. Least squares analysis of the data revealed about 10% of intermediate repetitive DNA sequences. Their interspersion between single copy DNA sequences was analyzed by comparing the reassociation kinetics of DNA fragments 0.35 and 7.2 kilobases long. Thus the DNA sequence arrangement ofAscaris does not follow the short period interspersion pattern observed in most organism.     

Sex chromosome positions in human interphase nuclei as studied by in situ hybridization with chromosome specific DNA probes

Summary Two cloned repetitive DNA probes, pXBR and CY1, which bind preferentially to specific regions of the human X and Y chromosome, respectively, were used to study the distribution of the sex chromosomes in human lymphocyte nuclei by in situ hybridization experiments. Our data indicate a large variability of the distances between the sex chromosomes in male and female interphase nuclei. However, the mean distance observed between the X and Y chromosome was significantly smaller than the mean distance observed between the two X-chromosomes. The distribution of distances determined experimentally is compared with three model distributions of distances, and the question of a non-random distribution of sex chromosomes is discussed. Mathematical details of these model distributions are provided in an Appendix to this paper. In the case of a human translocation chromosome (XqterXp22.2::Yq11Y qter) contained in the Chinese hamster x human hybrid cell line 445 x 393, the binding sites of pXBR and CY1 were found close to each other in most interphase nuclei. These data demonstrate the potential use of chromosome-specific repetitive DNA probes to study the problem of interphase chromosome topography.     

A new family of satellite DNA sequences as a major component of centromeric heterochromatin in owls (Strigiformes)

We isolated a new family of satellite DNA sequences from Hae III- and Eco RI-digested genomic DNA of the Blakistons fish owl ( Ketupa blakistoni). The repetitive sequences were organized in tandem arrays of the 174 bp element, and localized to the centromeric regions of all macrochromosomes, including the Z and W chromosomes, and microchromosomes. This hybridization pattern was consistent with the distribution of C-band-positive centromeric heterochromatin, and the satellite DNA sequences occupied 10% of the total genome as a major component of centromeric heterochromatin. The sequences were homogenized between macro- and microchromosomes in this species, and therefore intraspecific divergence of the nucleotide sequences was low. The 174 bp element cross-hybridized to the genomic DNA of six other Strigidae species, but not to that of the Tytonidae, suggesting that the satellite DNA sequences are conserved in the same family but fairly divergent between the different families in the Strigiformes. Secondly, the centromeric satellite DNAs were cloned from eight Strigidae species, and the nucleotide sequences of 41 monomer fragments were compared within and between species. Molecular phylogenetic relationships of the nucleotide sequences were highly correlated with both the taxonomy based on morphological traits and the phylogenetic tree constructed by DNA-DNA hybridization. These results suggest that the satellite DNA sequence has evolved by concerted evolution in the Strigidae and that it is a good taxonomic and phylogenetic marker to examine genetic diversity between Strigiformes species.An erratum to this article can be found at Communicated by Y. Hiraoka