Biomedical subjects

T Haaf

Publications and source records attributed to T Haaf.

At least 109 records · Page 6Linked to original sources

PubMed1992

Organization, polymorphism, and molecular cytogenetics of chromosome-specific alpha-satellite DNA from the centromere of chromosome 2.

The general usefulness of alpha-satellite DNA probes for the molecular, genetic, and cytogenetic analysis of the human genome is enhanced by their being chromosome specific. Here, we describe the isolation and characterization of an alpha-satellite subset specific for human chromosome 2. Three clones, p2-7, p2-8, and p2-11, obtained from an EcoRI-digested lambda phage library from flow-sorted chromosome 2, are specific for the centromere of chromosome 2 by somatic cell hybrid mapping and chromosomal in situ hybridization. Nucleotide sequence analysis identifies the chromosome 2-specific alpha-satellite subset D2Z1 as a member of the suprachromosomal subfamily II, which is based on a characteristic two-monomer repeat. The D2Z1 subset is further organized as a series of diverged 680-bp tetramers, revealed after digestion of genomic DNA with HaeIII, HindIII, HinfI, StuI, and XbaI. Using pulsed-field gel electrophoresis (PFGE), probes p2-7, p2-8, and p2-11 detect polymorphic restriction patterns within the alpha-satellite array. Among 15 different chromosomes 2 (in two two-generation families and one three-generation family), the length of the D2Z1 alpha-satellite array varied between 1050 and 2900 kb (mean = 1850 kb, SD = 550 kb). The inheritance of the chromosome 2 alpha-satellite arrays and their associated polymorphisms was strictly Mendelian.

Aneuploidy↗

PubMed1992

A microchromosome derived from chromosome 11 in a patient with the CREST syndrome of scleroderma.

A patient with the CREST syndrome of scleroderma was found to carry a mosaicism for a supernumerary microchromosome. The microchromosome was approximately 1 micron in size and present in over half of the lymphocyte metaphases examined. It bound centromeric proteins specifically recognized by CREST autoimmune sera (including the patient's serum). In situ hybridization with a panel of chromosome-specific alpha-satellite probes showed that the microchromosome was derived from chromosome 11, most or all of its chromatin consisting of the chromosome 11 subset of alpha-satellite DNA. It had no detectable telomeric sequences. Microchromosomes observed by electron microscopy had no visible free ends. The chromatin looked exactly the same as it did in normal chromosomes. Although we have no direct evidence for a circular structure, we conclude that the microchromosome originated by an interstitial deletion including the alpha-satellite DNA sequences and subsequent ring formation. The newly formed chromosomal element proved to be relatively stable somatically and was transmitted through meiosis. Since it possesses at least some structural and functional features of a centromeric region, the microchromosome can be thought of as an isolated centromere.

Centromere↗

PubMed1991

Opitz trigonocephaly syndrome.

We report on a patient with Opitz trigonocephaly syndrome. The girl was the first-born child of consanguineous parents and had trigonocephaly, apparent hypertelorism, upslanted palpebral fissures, strabismus, small nose with broad root, abnormally modeled ears, high palate, short neck with loose skin, polysyndactyly, and prominent clitoris and labia majora. In addition, a complex cardiovascular defect (Eisenmenger disease) was observed. The patient was mentally retarded.

Abnormalities, Multiple↗

PubMed1991

Chromosome topology in mammalian interphase nuclei.

Since 1968, when Comings published the pioneering paper on "the rationale for an ordered arrangement of chromatin in the interphase nucleus," technical methods have progressed tremendously and improved our understanding of interphase organization. The existence of highly ordered organizational patterns of the cell nucleus appears to be beyond any doubt and it is difficult to escape the conclusion that interphase chromosome topology is important for the complex regulation of the many varied and interrelated nuclear processes. However, it is worth emphasizing that a universally valid principle of chromosome arrangement does not exist and, therefore, any generalization of interphase patterns can be misleading. The factors of order according to which the chromosomes are arranged inside the nucleus are manifold: (1) Individual chromosomes remain in spatially separated domains throughout interphase, preventing an intermingling of the decondensed euchromatin. (2) Chromosome regions that contain constitutive heterochromatin associate into larger chromocenters. (3) In most cell types direct associations between interphase domains of homologous chromosomes are not observed. In others homologous heterochromatic regions tend to be paired preferentially. (4) Interphase chromosomes do not float freely in the nucleoplasm; they are associated to varying degrees with the nuclear membrane and other components of the nuclear scaffold. The number of attachment sites for each chromosome to the nuclear membrane is relatively low. (5) The positions of centromeres (and pericentromeric heterochromatin) are nonrandom and characteristic of each cell type. Specific centromere movements occur during the cell cycle, during differentiation, and under certain pathophysiological conditions. (6) The telomeric chromosome ends are particularly prone to associate in certain somatic cell types and in meiotic prophase cells. (7) The arrangement of repetitive DNA families appears to determine a structural framework of the interphase nucleus. Different cell types of one organism can exhibit marked differences in their repetitive DNA framework, whereas cells that are in an identical differentiated state or an identical phase of the cell cycle often show comparable interphase patterns even in evolutionarily distant species. (8) The various steps of ribosome biogenesis take place in a precise fashion within a separate nuclear domain, the nucleolus. The topologically well-defined nucleolar substructures are required for rDNA transcription and pre-rRNA processing. (9) A compartmentalization of transcriptional and processing events is also evident in the rest of the nucleus. However, it is not yet known if the in situ sites of transcription and RNA processing for a particular (nonribosomal) gene or gene family are actually adjacent. (10) DNA replication is precisely spatiotemporally regulated within the nucleus. The replication domains are immobilized on the nuclear matrix.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Quantitative determination of rDNA transcription units in vertebrate cells.

The adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) unravels the compact nucleoli to necklace-like structures when applied to living cells. The nucleolar beads contain RNA polymerase I (RPI) and argyrophilic proteins, both properties considered to be characteristic of ribosomal gene activity. Each granule is supposed to represent a single transcription unit consisting of an actively transcribing gene and its RPI complex. Indirect immunofluorescence with anti-RPI antibodies was used to determine the number of transcription units in DRB-treated cells of some representative mammals, marsupials, birds, and amphibians. We estimate that 45 to 145 rRNA genes are transcriptionally active in vertebrate fibroblasts, depending on the species. Nucleolar transcriptional activity does not correlate with the total number of rRNA genes. During in vitro aging of fibroblasts, the number of transcription units appears to remain unchanged. Different cell types of one same organism show varying numbers of transcription units, reflecting their differential metabolic activity. A particular situation exists in phytohemagglutinin-stimulated lymphocytes. In the course of nucleolar activation, the number of transcription units is increased considerably, implying that formerly inactive rRNA genes are recruited for transcription. The opposite phenomenon is observed during spermatogenesis. With the diploid spermatocytes developing into haploid spermatids, the transcriptionally active rRNA genes decrease in number until rRNA synthesis is completely blocked.

Animals↗

PubMed1991

PCR amplification of chromosome-specific alpha satellite DNA: definition of centromeric STS markers and polymorphic analysis.

Alpha satellite DNA is a tandemly repetitive DNA family found at the centromere of every human chromosome. Chromosome-specific subsets have been isolated for over half the chromosomes and have prove useful as markers for both genetic and physical mapping. We have developed specific oligonucleotide primer sets for polymerase chain reaction (PCR) amplification of alpha satellite DNA from chromosomes 3, 7, 13/21, 17, X, and Y. For each set of primers, PCR products amplified from human genomic DNA are specific for the centromere of the target chromosome(s), as shown by somatic cell hybrid mapping and by fluorescence in situ hybridization. These six subsets represent several evolutionarily related alpha satellite subfamilies, suggesting that specific primer pairs can be designed for most or all chromosomal subsets in the genome. The PCR products from chromosome 17 directly reveal the polymorphic nature of this subset, and a new DraI polymorphism is described. The PCR products from chromosome 13 are also polymorphic, allowing in informative cases genetic analysis of this centromeric subset distinguished from the highly homologous chromosome 21 subset. These primer sets should allow placement of individual centromeres on the proposed STS map of the human genome and may be useful for somatic cell hybrid characterization and for making in situ probes. In addition, the ability to amplify chromosome-specific repetitive DNA families directly will contribute to the structural and functional analysis of these abundant classes of DNA.

Animals↗

PubMed1990

Y isochromosome associated with a mosaic karyotype and inactivation of the centromere.

A patient with azoospermia and a Y isochromosome is described. The breakpoint producing this i(Y) was within the terminal short arm of the Y chromosome. Lymphocyte cultures from peripheral blood contained a high proportion of 45,X cells and cells with different Y-chromosome rearrangements. The i(Y) had either a monocentric or dicentric appearance. In dicentrics, anti-kinetochore immunofluorescence was present at both centromeres. However, this was also true for most of the functional monocentrics (pseudodicentrics). Kinetochore staining was generally positive at the site of the inactive centromeres; only a minority of the suppressed centromeres had lost their antigenic properties. Permanently growing lymphoblasts consistently showed a monocentric i(Y) with only one fluorescing kinetochore; the immunonegative Y centromere did not recover antigenicity.

Adult↗

PubMed1990

Kinetochore formation in experimentally undercondensed chromosomes.

Treatment of human and mouse cell cultures with the cytidine analogue 5-azadeoxycytidine and the AT-specific DNA ligand Hoechst 33258 dramatically inhibited condensation of the pericentromeric heterochromatin in several chromosomes. When stained with antikinetochore autoimmune sera, these experimentally undercondensed chromosomes showed kinetochores with preserved antigenicity. The undercondensed and normally condensed chromosomes share the major antigenic determinants of the kinetochore.

Animals↗

PubMed1990

Nucleolar transcriptional activity in mouse Sertoli cells is dependent on centromere arrangement.

Experimental evidence suggests that centromere arrangement is relevant to the expression of ribosomal genes in murine Sertoli cells. Nuclei endowed with a nucleolus inactive in rRNA synthesis presented several clusters, each containing a bunch of individual centromeres. RNA polymerase I was not cytochemically detected in the nucleolar structure, which contained only small amounts of fibrillarin. In the course of nucleolar activation, the centromeres within the separate clusters became fused into larger centromeric bodies. Synthesis of precursor rRNAs and their processing were visualized by strong nucleolar fluorescence signals using antibodies to RNA polymerase I and fibrillarin.

Animals↗

PubMed1990

Paired arrangement of nonhomologous centromeres during vertebrate spermiogenesis.

Indirect immunofluorescence staining with human anti-kinetochore antibodies was used to study the position of centromeres during vertebrate spermiogenesis. Many species of Amphibia have a low chromosome number and very large spermatids and spermatozoa. The number of kinetochore dots correlates exactly with the haploid chromosome number. This implies that kinetochore duplication occurs in the interval between meiosis I and meiosis II. The nonhomologous centromeres are arranged in tandem during the entire course of spermiogenesis and in mature spermatozoa. A higher order centromere arrangement was found in spermiogenic cells of Anura and Urodela. In mammals, immunofluorescence analysis is complicated by the extreme condensation of chromatin during spermiogenesis and the high chromosome numbers. Nevertheless, centromere-centromere associations were observed in mammalian round spermatids and sporadically in testicular spermatozoa. This indicates that pair-wise association of centromeres is a universal principle of centromere arrangement at the postmeiotic stage.

Animals↗

PubMed1990

Immunocytogenetics. IV. Human autoantibodies to heterochromatin-associated proteins.

We report here a novel class of human autoantibodies with immunological affinity for constitutive heterochromatin. Indirect immunofluorescence localized different proteinaceous antigens to the AT-rich paracentromeric heterochromatin of mouse chromosomes, the GC-rich heterochromatic regions of sheep chromosomes, and the large C-banded regions of human chromosomes 1, 9, and 16. Minor amounts of the heterochromatin-associated proteins were uniformly distributed on the chromosomes of all vertebrate species. Their antigenic determinants have been highly conserved during evolution. The chromosomal distribution of the heterochromatin-associated antigens is not altered in interspecific somatic cell hybrids but apparently reflects a stable structural property germane to each chromosome type. The antigenic proteins remain bound to their respective epitopes throughout the entire cell cycle and in meiosis. The heterochromatin-associated antigens represent a major nonhistone component of the mitotic chromosome scaffold and the interphase nuclear matrix. Immunoblotting performed with a human autoimmune serum to mouse heterochromatin revealed a characteristic pattern of polypeptides with molecular weights ranging from 35 to 120 kDal. Drugs that interfere in vivo with the higher-order chromatin structure had no effect on the expression and chromosomal distribution of the heterochromatin-associated antigens. However, the antibody binding sites in chromatin can be completely masked by treating fixed cell preparations with certain DNA-binding ligands.

Animals↗

PubMed1990

Immunocytogenetics. V. A highly conserved NOR antigen (He) is facultatively associated with nucleolar or nucleoplasmic granules.

A new protein antigen of the nucleolus organizer region (NOR), designated He, was recognized by human autoantibodies obtained from a patient with Raynaud phenomenon. In mitotic cells of all vertebrate species tested. He serum selectively immunostained the chromosomal NORs. A completely unexpected characteristic of the He antigen was its location during interphase. In mammalian cell substrates, it was concentrated in numerous nucleoplasmic granules, with minor amounts of the antigen uniformly distributed throughout the entire nucleus. In interphase nuclei of lower vertebrate cells, however, the antigen was preferentially located in the nucleolus. The antigenicity of He is not dependent on RNA or DNA; its cytochemical properties operationally classify it as a nonhistone component of the chromosome scaffold. The He antigen was present in the residual nucleolar structures of cells that were not at all active in rRNA synthesis, such as mammalian late spermatids and amphibian erythrocytes.

Animals↗

PubMed1990

Immunocytogenetics. VI. A nonhistone antigen is cell type-specially associated with constitutive heterochromatin and reveals condensation centers in metaphase chromosomes.

We report a nonhistone antigen to be cell type-specifically associated with constitutive heterochromatin. Human autoantibodies were used to analyze by indirect immunofluorescence the pattern of association of the antigenic protein with the heterochromatin of murine chromosomes, as well as those of other representative vertebrate species. The evolutionary stability of its cell type-specific distribution pattern suggests that this nonhistone antigen plays an important role in the structure and/or function of constitutive heterochromatin. In mitotic chromosomes, the antigen was localized to discrete granules scattered throughout the entire chromatin. These structural elements may function as condensation centers, with each granule representing an aggregation of anchoring complexes for the chromatin loops.

Animals↗

PubMed1990

Human autoantibodies to spermatogenic antigens and Sertoli cells.

Immunofluorescence staining using human autoantibodies is a simple and reliable method for investigation of meiotic and post-meiotic cells. Patients suffering from autoimmune diseases often produce circulating autoantibodies to antigens of germ cells and Sertoli cells. Four hundred human autoimmune sera were screened by indirect immunofluorescence on mouse seminiferous tubule cells. Autoantibodies of several specificities were found: one group reacted with organelles of meiotic prophase spermatocytes or spermatozoa. Included in this group were autoantibodies to synaptonemal complexes, sex vesicle, acrosome, and sperm tail. A second group of autoantibodies was found to stain different spermatogenic cell types uniformly, such as round spermatids or Sertoli cells.

Animals↗

PubMed1990

The minor-groove binding DNA-ligands netropsin, distamycin A and berenil cause polyploidisation via impairment of the G2 phase of the cell cycle.

Distamycin A, netropsin and berenil are known to cause undercondensation of heterochromatic regions of metaphase chromosomes. These ligands interfere with DNA curvature by binding to the minor groove of the DNA. Whereas the effects of these ligands upon chromatin structure are well established, little is known about their possible interference with cell cycle progression. We show that the presence of these DNA-ligands causes protracted cell growth consisting of a prolongation of the G1 phase of the cell cycle along with arrest in the G2 compartment. Concomitant with these cell kinetic disturbances the DNA ligands cause increased polyploidisation. These observations suggest that the DNA-minor groove may play an important role in progression through the G2 phase and proper mitotic transit.

Amidines↗

PubMed1989

5-Azadeoxycytidine induced undercondensation in the giant X chromosomes of Microtus agrestis.

Fibroblasts of female Microtus agrestis were treated with 5-azadeoxycytidine (5-aza-dCyd) at a final concentration of 10(-5) M during the last 2 h of culture. This cytidine analogue induces distinct undercondensation of the constitutive heterochromatin in the giant X chromosomes. The undercondensed heterochromatic thread exhibits longitudinal segmentation reminiscent of a chromomere pattern. In the late-replicating X chromosome, 5-aza-dCyd also inhibits condensation of the genetically inactivated euchromatin (facultative heterochromatin). The described effects of 5-aza-dCyd on the X chromosome structure appear to be incorporation independent.

Animals↗

PubMed1989

Centromeric association of a microchromosome. A new category of non-random arrangement of metaphase chromosomes.

A supernumerary microchromosome measuring 0.5-1 microns found in over half of the metaphases of a CREST scleroderma patient and his daughter has been characterized by various cytogenetic techniques. The microchromosome consisted of constitutive heterochromatin and contained nuclear antigens reacting with specific anti-kinetochore antibodies. The most remarkable property of the microchromosome was its non-random position: it was closely associated with the centromere of any of the normal chromosomes in the majority of the metaphases. Furthermore, an inordinately high rate of Y chromosome aneuploidy was found in the CREST scleroderma patient. The origin and structure of the microchromosome, its possible connection with the CREST variant of scleroderma, and the phenomenon of centromeric association are discussed.

Aged↗

PubMed1989

Centromeric association and non-random distribution of centromeres in human tumour cells.

Centromere arrangement in interphase and metaphase cells of two human tumour cell lines was analysed using anti-kinetochore antibodies as immunofluorescent probes. In GLC1 interphase nuclei, kinetochores were non-randomly positioned around the nucleolus and close to the nuclear membrane. During S and early G2 phase, necklace-like strands of kinetochores were formed in the centre of the nucleus. The duplication of sister kinetochores during the G2 phase was not synchronized. At late G2 phase, a relatively random topological distribution of centromeres was observed with short linear arrays of sister kinetochores. Carefully spread metaphase plates of MDA-MB231 cells generally exhibited a linear alignment of centromeres and large centromeric clusters. In completely pulverized MDA-MB231 cells, centromeres showed a strong tendency to associate with each other.

Breast Neoplasms↗