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T Haaf

Publications and source records attributed to T Haaf.

At least 91 records · Page 5Linked to original sources
PubMed1996

Mammalian ubiquitin-conjugating enzyme Ubc9 interacts with Rad51 recombination protein and localizes in synaptonemal complexes.

Hsubc9, a human gene encoding a ubiquitin-conjugating enzyme, has been cloned. The 18-kDa HsUbc9 protein is homologous to the ubiquitin-conjugating enzymes Hus5 of Schizosaccharomyces pombe and Ubc9 of Saccharomyces cerevisiae. The Hsubc9 gene complements a ubc9 mutation of S. cerevisiae. It has been mapped to chromosome 16p13.3 and is expressed in many human tissues, with the highest levels in testis and thymus. According to the Ga14 two-hybrid system analysis, HsUbc9 protein interacts with human recombination protein Rad51. A mouse homolog, Mmubc9, encodes an amino acid sequence that is identical to the human protein. In mouse spermatocytes, MmUbc9 protein, like Rad51 protein, localizes in synaptonemal complexes, which suggests that Ubc9 protein plays a regulatory role in meiosis.

Amino Acid Sequence↗
PubMed1996

Region-specific YAC banding and painting probes for comparative genome mapping: implications for the evolution of human chromosome 2.

To date, several hundred nonchimeric yeast artificial chromosomes (YACs) from the Centre d'Etude du Polymorphisme Humain containing polymorphic sequence-tagged sites have been mapped by fluoresence in situ hybridization (FISH) on human metaphase chromosomes. Because they carry an average of 1 Mb of human genomic DNA, CEPH YACs generate high-intensity in situ hybridization signals. The available set of cytogenetically and genetically anchored YACs, approximately one every 5-10 cM evenly spaced over almost the entire human genome, provides complex region-specific probes for molecular cytogenetics. YAC probes can be adapted with unlimited flexibility to specific FISH applications such as the study of chromosomal evolution. We have generated representational probes for YAC banding and painting of human chromosome 2 and its great ape homologs. Convergent inversions were found in the pericentric region of the gorilla and orangutan homologs of chromosome 2p.

Animals↗
PubMed1996

Chromosomal localization of long trinucleotide repeats in the human genome by fluorescence in situ hybridization.

Trinucleotide microsatellites are widespread in the human and other mammalian genomes. Expansions of unstable trinucleotide repeats have been associated so far with a number of different genetic diseases including fragile X, myotonic dystrophy (DM) and Huntington disease. While ten possible trinucleotides can occur at the DNA level, only CTG and CCG repeats are involved in the disorders described so far. However, the repeat expansion detection (RED) technique has identified additional large repeats of ATG, CCT, CTT, and TGG of potentially pathological significance in the human genome. We now show that conclusive information about the chromosomal localization of long trinucleotide repeats can be achieved in a relatively short time using fluorescence in situ hybridization (FISH) with biotin-labelled trinucleotide polymers. Large CTG expansions (> 1 kb) in DM and an unstable (CTG)306 repeat in a patient with schizophrenia were detected by eye through the microscope without electronic enhancement. Digital imaging was used to analyse the chromosomal distribution of long CCA and AGG repeats. Our results suggest that long trinucleotide repeats occur in the normal human genome and that the size of individual repeat loci may be polymorphic.

Base Sequence↗
PubMed1996

High-resolution analysis of DNA replication in released chromatin fibers containing 5-bromodeoxyuridine.

A strategy has been devised to physically map replication sites in released chromatin of mammalian cells. When added to the culture medium, 5-bromodeoxyuridine (BrdU) is incorporated into replicating DNA, partially replacing thymidine. BrdU pulses as short as one minute can be visualized on preparations of straightened chromatin fibers from protein-extracted nuclei by means of monoclonal anti-BrdU antibody. Short BrdU pulses (< 10 min) appear as strings of fluorescent signals that are 50-300 kb in length. This corresponds to the estimated size of individual replication units. Pulse chase experiments reveal that replicating DNA is tightly associated with the residual nuclear matrix, whereas newly replicated DNA is positioned on the released loop chromatin of nuclear halo preparations. Simultaneous fluorescence in situ hybridization (FISH) on BrdU-substituted released chromatin fibers suggests that replication can initiate at multiple sites anywhere within an alpha-satellite array of several Mb.

Bromodeoxyuridine↗
PubMed1995

Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes.

Rad51 protein of Saccharomyces cerevisiae is a structural homolog of the Escherichia coli recombination enzyme RecA. In yeast, the Rad51 protein is required for mitotic and meiotic recombination and for repair of double-strand breaks in DNA. We have used antibodies raised against the homologous human protein, HsRad51, expressed in E. coli, to visualize the spatial distribution of the protein in mammalian somatic and meiotic cells. In cultured human cells, the HsRad51 protein is concentrated in multiple discrete foci in the nucleoplasm; it is largely absent from cytoplasm and nucleoli. After treatment of cells with methyl methanesulfonate, ultraviolet irradiation, or 137Cs irradiation, the percentage of cells with HsRad51 protein immunofluorescence increases; the same cells show unscheduled DNA synthesis. Induction of Rad51 foci is blocked by inhibitors of transcription. In mouse pachytene spermatocytes, the mouse homolog of Rad51 protein is highly enriched in synaptonemal complexes that are formed between the paired homologous chromosomes during meiotic prophase. We conclude that the mammalian proteins homologous to yeast Rad51 are involved in repair of DNA damage and recombinational repair during meiosis.

Animals↗
PubMed1995

Higher order nuclear structure in mammalian sperm revealed by in situ hybridization and extended chromatin fibers.

The higher order organization of chromosomes in human and mouse sperm-cell nuclei has been visualized by fluorescence in situ hybridization. In mouse testicular sperm, centromeres form several linear higher order structures that are attached to a heterochromatic chromocenter at the center of the nucleus. Telomeres of the acrocentric mouse chromosomes are associated with either the heterochromatic center or the nuclear membrane. Whole chromosome domains are arranged parallel to the heterochromatic chromocenter and occasionally wrapped around it. We propose that constitutive heterochromatin serves as the nucleation point for a cell-type-specific organization of mouse sperm chromatin. In human mature sperm, individual chromosomes also appear as elongated territories. When human sperm nuclei are lysed with high salt and detergent, the normally condensed sperm chromatin unravels into linear arrays that exhibit a discrete nodular substructure (of < 300 nm diameter). These beads may represent a basic packaging unit of sperm chromatin. Larger superbead-like structures are also seen along the extended chromatin fibers and these could contain multiples of the basic packaging unit. These observations indicate that mammalian sperm nuclei have a highly defined nuclear architecture. The implications of an ordered organization of DNA in sperm are unknown, but it is possible that functional compartmentalization of the sperm-cell nucleus influences the initiation and regulation of paternal gene activity in the early embryo.

Animals↗
PubMed1995

Presence and abundance of CENP-B box sequences in great ape subsets of primate-specific alpha-satellite DNA.

CENP-B, a highly conserved centromere-associated protein, binds to alpha-satellite DNA, the centromeric satellite of primate chromosomes, at a 17-bp sequence, the CENP-B box. By fluorescence in situ hybridization (FISH) with an oligomer specific for the CENP-B box sequence, we have demonstrated the abundance of CENP-B boxes on all chromosomes (except the Y) of humans, chimpanzee, pygmy chimpanzee, gorilla, and orangutan. This sequence motif was not detected in the genomes of other primates, including gibbons, Old and New World monkeys, and prosimians. Our results indicate that the CENP-B box containing subtype of alpha-satellite DNA may have emerged recently in the evolution of the large-bodied hominoids, after divergence of the phylogenetic lines leading to gibbons and apes; the box is thus on the order of 15-25 million years of age. The rapid process of dispersal and fixation of the CENP-B box sequence throughout the human and great ape genomes is thought to be a consequence of concerted evolution of alpha-satellite subsets on both homologous and nonhomologous chromosomes.

Animals↗
PubMed1995

The effects of 5-azacytidine and 5-azadeoxycytidine on chromosome structure and function: implications for methylation-associated cellular processes.

5-Azacytidine (5-aza-C) analogs demonstrate a remarkable ability to induce heritable changes in gene and phenotypic expression. These cellular processes are associated with the demethylation of specific DNA sequences. On the other hand, 5-aza-C analogs have dramatic effects on chromosomes, leading to decondensation of chromatin structure, chromosomal instability and an advance in replication timing. Condensation inhibition of genetically inactive chromatin occurs when the DNA is still hemimethylated or fully methylated. In cell cultures prolonged for several replication cycles, chromosomal rearrangements and instability affect the 5-aza-C-sensitive regions. Moreover, the normally late-replicating inactive chromatin undergoes a transient temporal shift to an earlier DNA replication, characteristic of activatable chromatin. zThe induced alterations of chromosome structure and behavior may trigger the 5-aza-C-dependent process of cellular reprogramming. Apart from their differentiating and gene-modifying effects, 5-aza-C analogs can tumorigenically transform cells and modulate their metastatic potential. High doses of 5-aza-C analogs have cytotoxic and antineoplastic activities.

Animals↗
PubMed1995

C-band-positive double minutes have kinetochore proteins.

Double minutes (DMs) have been shown generally to lack centromeres. In the present paper we report on C-band-positive DMs in Colcemid-resistant SEWA cells. These DMs were heavily labeled after in situ hybridization with mouse major satellite DNA and, furthermore, reacted positively with antikinetochore antibodies. Upon antikinetochore antibody labeling, the immunofluorescent signals of some DMs were strong, indicating that they had functional centromeres.

Animals↗
PubMed1995

Rabl orientation of CENP-B box sequences in Tupaia belangeri fibroblasts.

The chromosomes of the tree shrew Tupaia belangeri exhibit highly localized CENP-B box sequences in the centromeric regions of most chromosomes. Telomeric sequences are present at the ends of all chromosomes and, in addition, at specific interstitial chromosomal sites that likely represent remnants of ancestral telomeres. This suggests that Robertsonian and tandem chromosome fusion events have occurred in the karyotypic evolution of Tupaiidae. In Tupaia skin fibroblasts CENP-B boxes are almost always clustered together at one pole of the interphase nucleus, whereas the telomeric domains are relatively evenly distributed throughout the whole nuclear volume. The observed orientation of the centromeres is reminiscent of the Rabl polarization of chromosomes; this is the first mammalian cell substrate in which such an higher-order chromosomal organization has been observed. CENP-B box sequences are found in several other mammalian species. The implications for recent parallel evolution of CENP-B binding motifs and concerted evolution of these sequences are discussed.

Animals↗
PubMed1994

High resolution ordering of YAC contigs using extended chromatin and chromosomes.

The general usefulness of fluorescence in situ hybridization (FISH) for the physical mapping of the human genome is greatly enhanced by improved DNA resolution. Several techniques have been described for decondensing or stretching interphase chromatin in a linear fashion, allowing long-range FISH mapping in finer detail. Highly extended linear chromatin can be hybridized over physical distances of at least several megabases, possibly over the whole length of a chromosome. By multi-color FISH, we have determined the order and overlaps of YACs from a 5q34-35 contig. Cosmids can be localized within larger YAC clones. Extended chromatin mapping can be applied as an adjunct ordering technique in genome studies.

Amsacrine↗
PubMed1994

Structural analysis of alpha-satellite DNA and centromere proteins using extended chromatin and chromosomes.

Human centromeres are characterized by distinct subsets of alpha-satellite DNA and by a number of centromeric proteins (CENPs) at least one of which, CENP-B, binds specifically to alpha-satellite DNA sequences. When the centromeres of metaphase chromosomes are mechanically stretched to five to 20 times their normal length, CENPs specifically recognized by CREST autoantibodies extend over the entire length of the linear alpha-satellite array. For higher resolution analysis we spread interphase chromatin across a slide resulting in highly extended chromatin fibers. By fluorescence in situ hybridization (FISH) with human alpha-satellite DNA and an oligomer specific for the CENP-B box sequence, the regular spacing of CENP-B binding motifs within arrays of alpha-satellite DNA was visualized directly. FISH with elongated chromatin structures released from interphase nuclei with the drug N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulfonamide shows that D7Z1 and D7Z2, two distinct alpha-satellite arrays on chromosome 7, are not interspersed with each other but are separated by as little as several hundred kilobases, consistent with previous long-range mapping data. The D7Z2 array, which does not bind detectable amounts of CENPs, can be assigned to the short arm side of the D7Z1 array using artificially stretched chromosomes. In interphase nuclei unreplicated segments give a singlet hybridization signal, whereas fully replicated loci appear as doublets. Although D7Z1 is replicated prior to D7Z2 in the majority of cells, the replication timing of one array relative to the other is variable. The replication of alpha-satellite arrays on homologous chromosomes is highly asynchronous. The newly replicated alpha-satellite lacks the CENP component.

Amsacrine↗
PubMed1993

Organization and molecular cytogenetics of a satellite DNA family from Hoplias malabaricus (Pisces, Erythrinidae).

The chromosomes of the primitive South American teleost fish Hoplias malabaricus have been analyzed by classical cytogenetic (C-, AgNOR-, Hoechst 33258-, and Q-banding) techniques. A highly repetitive DNA family has been cloned and sequenced. It is a tandemly repeated sequence of about 355 bp, yielding an overall base pair composition of 67% AT with long runs of > 50% As and 70% Ts. Analysis of sequence variation has allowed the further categorization of Hoplias satellite DNA into two evolutionary related subfamilies A and B, distinguishable by characteristic insertions and deletions within this 355-bp monomer. Subfamily A satellite is found (in diverged form) at the centromeres of most H. malabaricus chromosomes. Sequence variants are clustered on specific chromosomal subsets. Subfamily B satellite is highly specific for the paracentromeric heterochromatin on one particular chromosome pair by fluorescence in situ hybridization. These results indicate that the Hoplias satellite DNA family has evolved in a concerted manner predominantly via recombination events involving homologous, rather than non-homologous chromosome regions. The clones isolated here may be useful for the molecular, genetic, and cytological analysis of the genus Hoplias.

Animals↗
PubMed1993

5-Azadeoxycytidine distinguishes between active and inactive X chromosome condensation.

Treatment with 5-azadeoxycytidine (5-aza-dC) causes dramatic inhibition of mitotic condensation in the inactive X chromosome, without affecting the active X chromosome and autosomes. The undercondensed chromatin structure is most prominent in the late-replicating regions of the inactive X chromosome, whereas specific earlier-replicating segments on the inactive X probably associated with large blocks of genes that escape inactivation remain normally condensed. This segmentation of inactive X chromosomes has been evolutionarily conserved and is unique to mammals. It suggests temporal or mechanistic separation in condensation of inactive X chromatin. Condensation of chromatin structure is a novel and general feature of inactive X chromosomes that may be involved, directly or indirectly, in the maintenance and stabilization of X inactivation.

Animals↗
PubMed1992

Integration of human alpha-satellite DNA into simian chromosomes: centromere protein binding and disruption of normal chromosome segregation.

Centromeres of mammalian and other complex eukaryotic chromosomes are dominated by one or more classes of satellite DNA. To test the hypothesis that alpha-satellite DNA, the major centromeric satellite of primate chromosomes, is involved in centromere structure and/or function, human alpha-satellite DNA was introduced into African green monkey (AGM) cells. Centromere protein binding was apparent at the sites of integrated human alpha-satellite DNA. In the presence of an AGM centromere on the same chromosome, human alpha-satellite was associated with bridges between the separating sets of chromatids at anaphase and an increased number of lagging chromosomes at metaphase, both features consistent with the integrated alpha-satellite disrupting normal chromosome segregation. These experiments suggest that alpha-satellite DNA provides the primary sequence information for centromere protein binding and for at least some functional aspect(s) of a mammalian centromere, playing a role either in kinetochore formation or in sister chromatid apposition.

Animals↗
PubMed1992

Characterization of centromere arrangements and test for random distribution in G0, G1, S, G2, G1, and early S' phase in human lymphocytes.

The arrangement of centromeres, cluster formation and association with the nucleolus and the nuclear membrane were characterized in human lymphocytes during the course of interphase in a cell-phase-dependent manner. We evaluated 3,893 cell nuclei categorized by five parameters. The centromeres were visualized by means of indirect immunofluorescent labeling with anti-centromere antibodies (ACA) contained in serum of patients with CREST syndrome. The cell nuclei were classified as G0, G1, S, G2, G1' and early S' phase by comparing microscopically identified groups of cell nuclei with flow cytometric determination of cell cycle stage of synchronized and unsynchronized lymphocyte cell cultures. Based on a discrimination analysis, a program was devised that calculated the probability for any cell nucleus belonging to the G0, G1, S, G2, G1' and early S' phase using only two microscopic parameters. Various characteristics were determined in the G0, S, and G2 stages. A transition stage to S phase within G1 was detected. This stage shows centromere arrangements not repeated in later cell cycles and which develop from the dissolution of centromere clusters in the periphery of the nucleus during G0 and G1. S phase exhibits various non-random centromere arrangements and associations of centromeres with the nucleolus. G1' and early S' phase of the second cell cycle display no characteristic centromere arrangement. The duplication of centromeres in G2 is asynchronous in two phases. For all cell phases a test for random distribution of the centromeres in the cell nucleus was performed. There is a distinct tendency for centromeres to be in a peripheral position during G0 and G1; this tendency becomes weaker in S phase. Although the visual impression is a seemingly random distribution of centromeres in G2 and G1', statistical analysis still demonstrates a significant deviation from random distribution in favor of a peripheral location. Only the early S phase of the second cell cycle shows no significant deviation from a random distribution.

Cell Cycle↗