p82H identifies sequences at every human centromere

Summary A cloned alphoid sequence, p82H, hybridizes in situ to the centromere of every human chromosome. After washing under stringent conditions, no more than 8% of the grains are located on any specific chromosome. p82H thus differs from other centromeric sequences which are reported to be chromosome specific, because it detects sequences that are conserved among the chromosomes. Two experimental approaches show that the p82H sequences are closely associated with the centromere. First, p82H remains with the relocated centromeres in an inv(19) and an inv(6) chromosome. Second, p82H hybridizes at the centromere but not to the centromeric heterochromatin of chromosomes 1, 9 and 16 that have elongated 1qh, 9qh and 16qh regions produced by short growth in 5-azacytidine. The only noncentromeric site of hybridization is at the distal end of the 9qh region.     

Organization and genomic distribution of “82H” alpha satellite DNA

Summary We have investigated the organization and genomic distribution of sequences homologous to p82H, a cloned human alpha satellite sequence purported, based on previous in situ hybridization experiments, to exist at the centromere of each human chromosome. We report here that, using Southern blotting analysis under conditions of high stringency, p82H hybridizes solely to a low-copy or single-copy alphoid domain located at or near the centromeric region of human chromosome 14. In contrast, conditions of reduced hybridization stringency permit extensive cross-hybridization with nonidentical, chromosome-specific alpha satellite subsets found elsewhere in the human genome. Thus, the previously described ubiquity of 82H human centromeric sequences reflects the existence of diverse alpha satellite subsets located at the centromeric region of each human chromosome.     

Chromosome-specific α-satellite DNA from the centromere of chimpanzee chromosome 4

The centromeric regions of human and primate chromosomes are characterized by diverged subsets of tandemly repeated α-satellite DNA. Comparison of the α-satellites on known homologous chromosomes in human and chimpanzee provides insight into the very rapid evolution of satellite DNA sequences and the mechanisms that shape complex genomes. By using oligonucleotide primers specific for a conserved region of human α-satellite DNA, we have amplified a chromosome-specific α-satellite subset from the chimpanzee genome by the polymerase chain reaction. Fluorescence in situ hybridization showed that clones pαPTR4N and pαPTR4H are homologous to sequences at the centromere of the chimpanzee chromosome 4. This α-satellite subset is organized as a series of pentameric (higher-order) repeats, operationally defined by digestion of genomic DNA with HaeIII, MboI, RsaI, SstI, and XbaI. The lengths of four independent centromeric arrays measured by pulsed-field gel electrophoresis varied between 800 and 3,500 kb (mean = 1,850 kb, SD = 1,000 kb). Nucleotide sequence analysis demonstrated that chimpanzee chromosome 4 α-satellite is most closely related to the suprachromosomal subfamily II, which is evolutionarily different from the subfamily I to which the α-satellite on the homologous human chromosome 5 belongs. This implies that the human-chimpanzee sequence divergence has not arisen from a common ancestral α-satellite repeat(s) but instead represents concerted evolution of distinct repeats on homologous chromosomes. Received: 21 February 1997; in revised form: 26 February 1997 / Accepted: 27 February 1997     

A chimpanzee-derived chromosome-specific alpha satellite DNA sequence conserved between chimpanzee and human

We describe a cloned 2.7 kb alpha satellite sequence, Pan-3, from the pygmy chimpanzee (Pan paniscus) that specifically hybridizes in situ to chromosome 19 in the pygmy chimpanzee and to the homeologous human chromosome, no. 17. Using high stringency conditions of hybridization on Southern blots, this sequence hybridized to DNA from both species of chimpanzee (P. paniscus and P. troglodytes) and from human but not to DNA from gorilla (Gorilla gorilla) or orangutan (Pongo pygmaeus). Partial sequence analysis showed that Pan-3 and a previously described human chromosome 17-specific clone have up to 91% sequence identity. To our knowledge this is the highest sequence similarity reported between alphoid subsets from human and any other primate.by T.C. Hsu     

Molecular hybridization of iodinated 4S, 5S, and 18S + 28S RNA to salamander chromosomes

4S, 5S, AND 18S + 28S RNA from the newt Taricha granulosa granulosa were iodinated in vitro with carrier-free 125I and hybridized to the denatured chromosomes of Taricha granulosa and Batrachoseps weighti. Iodinated 18S + 28S RNA hybridizes to the telomeric region on the shorter arm of chromosome 2 and close to the centromere on the shorter arm of chromosome 9 from T. granulosa. On this same salamander the label produced by the 5S RNA is located close to or on the centromere of chromosome 7 and the iodinated 4S RNA labels the distal end of the longer arm of chromosome 5. On the chromosomes of B. wrighti, 18S + 28S RNA hybridizes close to the centromeric region on the longer arm of the largest chromosome. Two centromeric sites are hybridized by the iodinated 5S RNA. After hybridization with iodinated 4S RNA, label is found near the end of the shorter arm of chromosome 3. It is concluded that both ribosomal and transfer RNA genes are clustered in the genome of these two salamanders.     

Misregulation of Scm3p/HJURP causes chromosome instability in Saccharomyces cerevisiae and human cells

The kinetochore (centromeric DNA and associated proteins) is a key determinant for high fidelity chromosome transmission. Evolutionarily conserved Scm3p is an essential component of centromeric chromatin and is required for assembly and function of kinetochores in humans, fission yeast, and budding yeast. Overexpression of HJURP, the mammalian homolog of budding yeast Scm3p, has been observed in lung and breast cancers and is associated with poor prognosis; however, the physiological relevance of these observations is not well understood. We overexpressed SCM3 and HJURP in Saccharomyces cerevisiae and HJURP in human cells and defined domains within Scm3p that mediate its chromosome loss phenotype. Our results showed that the overexpression of SCM3 (GALSCM3) or HJURP (GALHJURP) caused chromosome loss in a wild-type yeast strain, and overexpression of HJURP led to mitotic defects in human cells. GALSCM3 resulted in reduced viability in kinetochore mutants, premature separation of sister chromatids, and reduction in Cse4p and histone H4 at centromeres. Overexpression of CSE4 or histone H4 suppressed chromosome loss and restored levels of Cse4p at centromeres in GALSCM3 strains. Using mutant alleles of scm3, we identified a domain in the N-terminus of Scm3p that mediates its interaction with CEN DNA and determined that the chromosome loss phenotype of GALSCM3 is due to centromeric association of Scm3p devoid of Cse4p/H4. Furthermore, we determined that similar to other systems the centromeric association of Scm3p is cell cycle regulated. Our results show that altered stoichiometry of Scm3p/HJURP, Cse4p, and histone H4 lead to defects in chromosome segregation. We conclude that stringent regulation of HJURP and SCM3 expression are critical for genome stability.     

Characterization of constitutive heterochromatin in Cebus apella (Cebidae, Primates) and Pan troglodytes (Hominidae, Primates): comparison to human chromosomes.

Using G bands, some homologies between the chromosomes of Cebus apella (CAP) and human chromosomes are difficult to establish. To solve this problem, we analyzed these homologies by fluorescence in situ hybridization using human whole chromosome probes (ZOO-FISH). The results indicated that 1) the human probe for chromosome 2 partially hybridizes with CAP chromosomes 13 and 5, 2) the human probe for chromosome 3 partially hybridizes with CAP chromosomes 18 and 20, 3) the human probe for chromosome 9 partially hybridizes with CAP chromosome 19, and 4) the human probe for chromosome 14 hybridizes with the p-terminal and q-terminal regions of CAP chromosome 6. However, none of the human probes employed hybridized with the heterochromatic regions of CAP chromosomes. For this reason, we characterized the heterochromatic regions of CAP chromosomes and of the chromosomes of Pan troglodytes (PTR), to allow comparison between CAP, PTR, and human chromosomes using in situ digestion of fixed chromosomes with the restriction enzymes AluI, HaeIII, and RsaI and by fluorescent staining with DA/DAPI. The results show that 1) centromeric heterochromatin is heterogeneous in the three species studied and 2) noncentromeric heterochromatin is homogeneous within each of the three species, but is different for each species. Thus, centromeric heterochromatin undergoes a higher degree of variability than noncentromeric heterochromatin.     

Chromosome-specific alpha satellite DNA from human chromosome 1: hierarchical structure and genomic organization of a polymorphic domain spanning several hundred kilobase pairs of centromeric DNA

The human alpha satellite repetitive DNA family is organized as distinct chromosome-specific subsets localized to the centromeric region of each chromosome. Here, we report he isolation and characterization of cloned repeat units which define a hierarchical subset of alpha satellite on human chromosome 1. This subset is characterized by a 1.9-kb higher-order repeat unit which consists of 11 tandem approximately 171-bp alpha satellite monomer repeat units. The higher-order repeat unit is itself tandemly repeated, present in at least 100 copies at the centromeric region of chromosome 1. Using pulsed-field gel electrophoresis we estimate the total array length of these tandem sequences at the centromere of chromosome 1 to be several hundred kilobase pairs. Under conditions of high stringency, the higher-order repeat probe hybridizes specifically to chromosome 1 and can be used to detect several associated restriction fragment length DNA polymorphisms. As such, this probe may be useful for molecular and genetic analyses of the centromeric region of human chromosome 1.     

A cloned sequence,p82H,of the alphoid repeated DNA family found at the centromeres of all human chromosomes

Clone p82H is a human DNA sequence which hybridises in situ exclusively to the centromeric regions of all human chromosomes. It is composed of approximately 14 tandemly repeated variants of a basic 172 bp sequence, and is related to the alphoid family. The organisation of the family of cross-hybridising sequences, detected by the clone p82H, is described both in the human genome and on certain chromosomes, and its relationship to known sequence families is discussed.     

The yeast RSC chromatin-remodeling complex is required for kinetochore function in chromosome segregation

The accurate segregation of chromosomes requires the kinetochore, a complex protein machine that assembles onto centromeric DNA to mediate attachment of replicated sister chromatids to the mitotic spindle apparatus. This study reveals an important role for the yeast RSC ATP-dependent chromatin-remodeling complex at the kinetochore in chromosome transmission. Mutations in genes encoding two core subunits of RSC, the ATPase Sth1p and the Snf5p homolog Sfh1p, interact genetically with mutations in genes encoding kinetochore proteins and with a mutation in centromeric DNA. RSC also interacts genetically and physically with the histone and histone variant components of centromeric chromatin. Importantly, RSC is localized to centromeric and centromere-proximal chromosomal regions, and its association with these loci is dependent on Sth1p. Both sth1 and sfh1 mutants exhibit altered centromeric and centromere-proximal chromatin structure and increased missegregation of authentic chromosomes. Finally, RSC is not required for centromeric deposition of the histone H3 variant Cse4p, suggesting that RSC plays a role in reconfiguring centromeric and flanking nucleosomes following Cse4p recruitment for proper chromosome transmission.     

Prenatal molecular cytogenetic diagnosis of partial tetrasomy 10p due to neocentromere formation in an inversion duplication analphoid marker chromosome

Neocentromeres are fully functional centromeres found on rearranged or marker chromosomes that have separated from endogenous centromeres. Neocentromeres often result in partial tri- or tetrasomy because their formation confers mitotic stability to acentric chromosome fragments that would normally be lost. We describe the prenatal identification and characterization of a de novo supernumerary marker chromosome (SMC) containing a neocentromere in a 20-wk fetus by the combined use of comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). GTG-banding of fetal metaphases revealed a 47,XY,+mar karyotype in 100% of cultured amniocytes; parental karyotypes were both normal. Although sequential tricolor FISH using chromosome-specific painting probes identified a chromosome 10 origin of the marker, a complete panel of chromosome-specific centromeric satellite DNA probes failed to hybridize to any portion of the marker. The presence of a neocentromere on the marker chromosome was confirmed by the absence of hybridization of an all-human-centromere alpha-satellite DNA probe, which hybridizes to all normal centromeres, and the presence of centromere protein (CENP)-C, which is associated specifically with active kinetochores. Based on CGH analysis and FISH with a chromosome 10p subtelomeric probe, the marker was found to be an inversion duplication of the distal portion of chromosome 10p. Thus, the proband's karyotype was 47,XY,+inv dup(10)(pter-->p14 approximately 15::p14 approximately 15-->neo-->pter), which is the first report of partial tetrasomy 10p resulting from an analphoid marker chromosome with a neocentromere. This study illustrates the use of several molecular strategies in distinguishing centric alphoid markers from neocentric analphoid markers.     

Chromosome-specific alpha satellite DNA: isolation and mapping of a polymorphic alphoid repeat from human chromosome 10

Distinct subsets of the human alpha satellite repetitive DNA family can be found in the centromeric region of each chromosome. Here we described the isolation and mapping of an alpha satellite repeat unit specific for human chromosome 10, using a somatic cell hybrid in which the only human centromere derives from chromosome 10. A hierarchical higher-order repeat unit, consisting of eight tandem approximately 171-bp alphoid monomer units, is defined by six restriction endonucleases. Under high-stringency conditions, a cloned representative of this 8-mer repeat family hybridizes to chromosome 10 only, both by Southern blot analysis of a somatic cell hybrid panel and by in situ hybridization. The probe furthermore detects a polymorphic restriction pattern of the alpha satellite array on chromosome 10. These features will make this probe a valuable genetic marker for studies of the centromeric region of chromosome 10.     

Karyotype of the gibbons hylobates lar and h. moloch inversion in chromosome 7.

A karyotype of the gibbon, Hylobates, has been prepared based on the chromosome banding patterns produced by quinacrine, trypsin-Giemsa, and centromeric heterochromatin stains. The banding patterns of H. lar and H. moloch are virtually identical. No brilliant quinacrine-fluorescent areas are present. The banding pattern of most of the gibbon chromosomes show less resemblance to those of the human, chimpanzee, gorilla, or orangutan than the chromosomes of the higher primates do to each other, suggesting a relatively large evolutionary separation of the gibbon from the higher primates. A pericentric inversion of chromosome 7 is present in one gibbon.     

A human chromosome 9-specific alphoid DNA repeat spatially resolvable from satellite 3 DNA by fluorescent in situ hybridization.

We have isolated a DNA clone (pMR9A) that identifies an alphoid DNA subset specific for chromosome 9. This alphoid subset is characterized by a dimeric organization as revealed by Southern blot analysis after digestion with HaeIII, HinfI, or StuI. Nonradioactive in situ hybridization demonstrated that pMR9A hybridizes only to the centromeric region of chromosome 9 and reveals chromosome 9 aneuploidies in interphase nuclei. In addition, the probe detects quantitative differences in alpha satellite DNA on chromosome 9, but these quantitative differences are not correlated with the size of the heterochromatic region. Double-labeling experiments, using a chromosome 9-specific satellite 3 clone and pMR9A, enabled us spatially to distinguish the alphoid and satellite 3 domains on metaphase chromosomes after treatment of the cultures with 5-azacytidine.     

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     

Satellite DNA I in chromatin loops of rat pachytene chromosomes and in spermatids

Biotinylated rat satellite DNA I probe p93-50 was used to visualize the chromatin of surface-spread rat pachytene chromosomes. Fluorescein isothiocyanate (FITC)-conjugated avidin produces a beaded fluorescence pattern along the chromatin loops that insert in the centromeric region of the synaptonemal complex (SC), the paired cores of homologous chromosomes. The number of fluorescent beads ranges from zero for centromeres without satellite DNA I homologous to probe p 93-50, to several hundred for satellite-rich centromeric regions. For the chromosomes that can be identified, the relative amount of satellite DNA is chromosome specific. No satellite DNA I was detected at the non-centromeric ends of the chromosomes or interstitially. DNase-digested nuclei or isolated SCs did not have detectable amounts of satellite DNA in the centromeric regions of the chromosomes or in the residual SCs. The fate of the satellite DNA was followed during spermiogenesis. In the round spermatid the centromeric regions, which appear to be attached to the nuclear envelope, are still distinct and have converging loops of fluorescent chromatin. At later stages there are fewer but still bright fluorescent patches. Satellite DNA I is still detectable in the mature sperm head. These results demonstrate the organization of satellite DNA I in the chromatin loops at the centromeric regions, and they forecast the analysis of chromosome organization in unprecedented detail with a variety of probes in surface spreads of meiotic prophase chromosomes.     

Localization and polymorphism of a chromosome 12-specific alpha satellite DNA sequence

The isolation and localization of a chromosome 12-specific alpha satellite DNA sequence, p alpha 12H8, is described. This clone contains a complete copy of the 1.4-kb HindIII higher-order repeat present within the alpha satellite array on chromosome 12. The specificity of p alpha 12H8 was demonstrated by in situ hybridization and Southern blot analysis of a somatic cell hybrid mapping panel, both performed under high-stringency conditions. Polymorphic restriction patterns within the alpha satellite array, revealed by the use of the restriction enzymes BglII and EcoRV, were demonstrated to display Mendelian inheritance. These properties make p alpha 12H8 a valuable genetic marker for the centromeric region of chromosome 12.     

Sequences associated with A chromosome centromeres are present throughout the maize B chromosome

Maize chromosome spreads containing the supernumerary B chromosome were hybridized with probes from various repetitive elements including CentC, CRM, and CentA, which have been localized to centromeric regions on the A chromosomes. Repetitive elements that are enriched or found exclusively near the centromeres of A chromosomes hybridized to many sites distinct from the centromere on the B chromosome. To examine whether these elements recruit kinetochore proteins at locations other than the canonical B centromere, cells were labeled with antibodies against CENH3, a key kinetochore protein. No labeling was detected outside the normal centromere and no evidence of B chromosome holocentromeric activity was observed. This finding suggests that, as in other higher eukaryotes, DNA sequence alone is insufficient to dictate kinetochore location in plants. Additionally, examination of the B centromere region in pachytene chromosomes revealed that the B-specific element ZmBs hybridizes to a much larger region than the site of hybridization of CentC, CRM, and CentA and the labeling by anti-CENH3 antibodies.This revised version was published online in December 2004 with corrections to Table 1.     

Species specific variant of human centromeric DNA repeats: localization on chromosome 18 and recent amplification in human ancestral line

Plasmid library of rapidly renaturating fraction of human DNA was constructed. The library was used for isolation of primate-specific DNA repeats. A clone (1hsp-4) which was intensively hybridizable with human DNA exclusively and produced no signals when hybridized with animal DNAs including the ones from orangoutan and chimpanzee was isolated. The cloned sequences 1hsp-4 have been found to be highly specific to centromeric heterochromatin of the 18 chromosome. Primary structure of a short 1hsp-4 fragment has been determined. The obtained data suggest the emergence of a DNA family homologous to the 1hsp-4 probe to be due to the thousandfold leapwise amplification occuring less than 5-8 millions years ago.