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T Haaf

Publications and source records attributed to T Haaf.

At least 73 records · Page 4Linked to original sources

Systematic characterisation of disease associated balanced chromosome rearrangements by FISH: cytogenetically and genetically anchored YACs identify microdeletions and candidate regions for mental retardation genes.

Disease associated balanced chromosome rearrangements (DBCRs) have been instrumental in the isolation of many disease genes. To facilitate the molecular cytogenetic characterisation of DBCRs, we have generated a set of >1200 non-chimeric, cytogenetically and genetically anchored CEPH YACs, on average one per 3 cM, spaced over the entire human genome. By fluorescence in situ hybridisation (FISH), we have performed a systematic search for YACs spanning translocation breakpoints. Patients with DBCRs and either syndromic or non-syndromic mental retardation (MR) were ascertained through the Mendelian Cytogenetics Network (MCN), a collaborative effort of, at present, 270 cytogenetic laboratories throughout the world. In this pilot study, we have characterised 10 different MR associated chromosome regions delineating candidate regions for MR. Five of these regions are narrowed to breakpoint spanning YACs, three of which are located on chromosomes 13q21, 13q22, and 13q32, respectively, one on chromosome 4p14, and one on 6q25. In two out of six DBCRs, we found cytogenetically cryptic deletions of 3-5 Mb on one or both translocation chromosomes. Thus, cryptic deletions may be an important cause of disease in seemingly balanced chromosome rearrangements that are associated with a disease phenotype. Our region specific FISH probes, which are available to MCN members, can be a powerful tool in clinical cytogenetics and positional cloning.

Adolescent↗

Ectopic NORs on human chromosomes 4qter and 8q11: rare chromosomal variants detected in two families.

Two different NOR bearing non-acrocentric chromosomes were detected during prenatal diagnosis performed on two probands because of advanced maternal age. In the first case, a chromosome 4 carried a NOR in the telomeric region of the long arm (4qs), while in the second case a NOR was inserted into chromosome 8q11. Family analysis showed the variant chromosomes to be transmitted through at least three generations in each family. There were no reports of reproductive problems or phenotypic effects in the carriers of these chromosomes, indicating the benign character of the aberrant chromosomes. In order to characterise the chromosomal variants more precisely, various differential banding techniques were applied.

Chromosomes, Human, Pair 4↗

Characterization of the mouse Src homology 3 domain gene Sh3d2c on Chr 7 demonstrates coexpression with huntingtin in the brain and identifies the processed pseudogene Sh3d2c-ps1 on Chr 2.

Formation of intracellular protein complexes is often mediated by Src homology 3 domain-containing proteins interacting with proline-rich target sequences on other proteins. The Sh3d2c gene or its rat/human orthologs have been implicated in synaptic vesicle recycling due to interaction with dynamin I and synaptojanin in nerve terminals. In a yeast two-hybrid system, association with a huntingtin fragment containing an elongated stretch of polyglutamines was observed recently. By genetic mapping and fluorescence in situ hybridization we demonstrate the localization of Sh3d2c on mouse chromosome 7. A processed pseudogene of Sh3d2c, Sh3d2c-ps1, was identified and mapped to mouse chromosome 2. Using RNA in situ hybridization, we show that Sh3d2c is transcribed in various regions of the brain. The striatum, hippocampus, cortex, basal hypothalamus, brain stem, and cerebellum are the most prominent sites of expression. Because huntingtin and Sh3d2c are coexpressed in most regions of the brain, it can be speculated that there is a link between the association of huntingtin/Sh3d2c and the pathogenesis of Huntington disease.

Animals↗

Interaction of human rad51 recombination protein with single-stranded DNA binding protein, RPA.

Replication protein A (RPA), a heterotrimeric single-stranded DNA binding protein, is required for recombination, and stimulates homologous pairing and DNA strand exchange promoted in vitro by human recombination protein HsRad51. Co-immunoprecipitation revealed that purified RPA interacts physically with HsRad51, as well as with HsDmc1, the homolog that is expressed specifically in meiosis. The interaction with HsRad51 was mediated by the 70 kDa subunit of RPA, and according to experiments with deletion mutants, this interaction required amino acid residues 169-326. In exponentially growing mammalian cells, 22% of nuclei showed foci of RPA protein and 1-2% showed foci of Rad51. After gamma-irradiation, the percentage of cells with RPA foci increased to approximately 50%, and those with Rad51 foci to 30%. All of the cells with foci of Rad51 had foci of RPA, and in those cells the two proteins co-localized in a high fraction of foci. The interactions of human RPA with Rad51, replication proteins and DNA are suited to the linking of recombination to replication.

Animals↗

Identification of a novel Ran binding protein 2 related gene (RANBP2L1) and detection of a gene cluster on human chromosome 2q11-q12.

The giant 358-kDa protein Ran binding protein 2 (RanBP2/Nup358) is localized at the cytoplasmic side of the nuclear pore complex and likely constitutes the Ran-GTP binding site at the cytoplasmic face of the complex. RanBP2/Nup358 furthermore acts as a chaperone for red/green opsin molecules. Here, we report on the physical mapping of human RanBP2 between markers D2S340 and D2S1893. A duplication of the 5'-end sequence of RanBP2 occurs within 3 Mb distal to RanBP2. Detailed sequence analysis resulted in primers specific for this distal duplication. Polymerase chain reaction-based screening of cDNA libraries indicates that this transcript, called RanBP2alpha (HGMW-approved symbol RANBP2L1), is expressed in several tissues. Screening of a fetal brain cDNA library yielded a 4057-bp partial cDNA clone for RanBP2alpha. Its 5'-end is almost identical to RanBP2, whereas its 3'-part is distinct from RanBP2. Northern blot analysis using a probe of the 3'-untranslated sequence of RanBP2alpha detected in several tissues an 8-kb transcript representing the full length of the transcript. In pancreas and placenta, an additional transcript of 14 kb was detected. PAC clones containing the bona fide RanBP2 sequences were localized to 2q11-q12 by FISH analysis, and a region of high similarity was detected on 2p11-p12. In summary, we have identified a RanBP2 gene cluster on 2q11-q12 together with a novel gene termed RanBP2alpha, with high sequence similarity to RanBP2.

Amino Acid Sequence↗

IRS-PCR-based genetic mapping of the huntingtin interacting protein gene (HIP1) on mouse chromosome 5.

Huntington's disease (HD) is a devastating central nervous system disorder. Even though the gene responsible has been positionally cloned recently, its etiology has remained largely unclear. To investigate potential disease mechanisms, we conducted a search for binding partners of the HD-protein huntingtin. With the yeast two-hybrid system, one such interacting factor, the huntingtin interacting protein-1 (HIP-1), was identified (Wanker et al. 1997; Kalchman et al. 1997) and the human gene mapped to 7q11.2. In this paper we demonstrate the localization of the HIP1 mouse homologue (Hip1) into a previously identified region of human-mouse synteny on distal mouse Chromosome (Chr) 5, both employing an IRS-PCR-based mapping strategy and traditional fluorescent in situ hybridization (FISH) mapping.

Animals↗

Orangutan alpha-satellite monomers are closely related to the human consensus sequence.

Alpha-satellite is a family of tandemly repeated DNA found at the centromeric regions of all human and primate chromosomes. Human alpha-satellite subsets are largely chromosome-specific and have been further grouped into four suprachromosomal families (SFs), each characterized by a unique set of monomeric types. Although chimpanzee and gorilla alpha-satellites share sufficient sequence similarity to fit the established SFs, the assumption that the derived human alpha-satellite consensus and monomeric types represent the sequence of ancestral repeats remains unestablished. By using oligonucleotide primers specific for a conserved region of human alpha-satellite DNA, we have PCR amplified, cloned, and characterized alpha-satellite sequences from the orangutan genome. Nucleotide sequence analysis demonstrated that orangutan alpha-satellite is formed by a single monomeric type that is significantly closer in percentage of sequence identity (mean = 92%, range = 89-96%) to the overall consensus of human alpha-satellite than to the monomeric types corresponding to the four SFs. Use of cloned sequences as hybridization probes to orangutan genomic DNA digested with a panel of restriction enzymes showed that most orangutan alpha-satellite subsets are characterized by a monomeric construction. The subset homologous to clone PPY2-5 is organized in distinct higher-order repeat structures consisting of 18 adjacent monomers. By FISH two clones, PPY3-4 and PPY3-5, proved to be specific for the alpha-satellite on the orangutan homologs of human Chromosomes (Chrs) 10 and 8, respectively. Our data indicate that there was an ancestral monomeric type displaying high sequence similarity to the overall human consensus from which the different great ape and human subsets and SFs may have originated.

Animals↗

Inhibition of condensation in human chromosomes induced by the thymidine analogue 5-iododeoxyuridine.

Human lymphocyte cultures were treated with iododeoxyuridine. This 5-halogenated thymidine analogue induces distinct undercondensations of the heterochromatin of human chromosome 9. The condensation of the other heterochromatic regions on chromosomes 1, 15, 16 and Y is also inhibited, but to a lesser extent. Optimum cell culture conditions required for the induction of undercondensations were determined. Up to 90% of mitotic cells reveal chromosome 9 heterochromatin decondensations when the substance is present in quantities of 1 x 10(-4) M during the last 7 h before cell fixation. In addition, examples of the usefulness of 5-IUDR in the analysis of chromosome aberrations involving chromosome 9 are presented. The interaction between 5-IUDR and chromosomal DNA, the modification of chromosomal proteins and factors inducing chromosomal decondensations are discussed.

Cells, Cultured↗

DNA nicks and increased sensitivity of DNA to fluorescence in situ end labeling during functional spermiogenesis.

Terminal transferase can be used to quantitate DNA strand breaks in situ by labeling free 3'-hydroxyl ends with exogenous nucleotides. Endogenous nicks in DNA temporally appear and disappear during functionally significant structural rearrangements of chromatin. Fluorescence in situ end labeling of mouse and rat testicular cells demonstrated that functional spermiogenesis is associated with abundant DNA nicks that occur in elongating spermatids, most likely as a result of nucleoprotein changes during terminal differentiation. Detectable DNA breaks were not observed in round spermatids and epididymal sperm.

Animals↗

Construction and characterization of a 10-genome equivalent yeast artificial chromosome library for the laboratory rat, Rattus norvegicus.

Increasing attention has been focused in recent years on the rat as a model organism for genetic studies, in particular for the investigation of complex traits, but progress has been limited by the lack of availability of large-insert genomic libraries. Here, we report the construction and characterization of an arrayed yeast artificial chromosome (YAC) library for the rat genome containing approximately 40,000 clones in the AB1380 host using the pCGS966 vector. An average size of 736 kb was estimated from 166 randomly chosen clones; thus the library provides 10-fold coverage of the genome, with a 99.99% probability of containing a unique sequence. Eight of 39 YACs analyzed by fluorescence in situ hybridization were found to be chimeric, indicating a proportion of about 20-30% of chimeric clones. The library was spotted on high-density filters to allow the identification of YAC clones by hybridization and was pooled using a 3-dimensional scheme for screening by PCR. Among 48 probes used to screen the library, an average of 9.3 positive clones were found, consistent with the calculated 10-fold genomic coverage of the library. This YAC library represents the first large-insert genomic library for the rat. It will be made available to the research community at large as an important new resource for complex genome analysis in this species.

Animals↗

Chromosome-specific alpha-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 alpha-satellite DNA. Comparison of the alpha-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 alpha-satellite DNA, we have amplified a chromosome-specific alpha-satellite subset from the chimpanzee genome by the polymerase chain reaction. Fluorescence in situ hybridization showed that clones palphaPTR4N and palphaPTR4H are homologous to sequences at the centromere of the chimpanzee chromosome 4. This alpha-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 alpha-satellite is most closely related to the suprachromosomal subfamily II, which is evolutionarily different from the subfamily I to which the alpha-satellite on the homologous human chromosome 5 belongs. This implies that the human-chimpanzee sequence divergence has not arisen from a common ancestral alpha-satellite repeat(s) but instead represents concerted evolution of distinct repeats on homologous chromosomes.

Animals↗

Cytogenetics of the genus Leporinus (Pisces, Anostomidae). 1. Karyotype analysis, heterochromatin distribution and sex chromosomes.

Cytogenetic analyses (Giemsa staining, C-banding, AgNO3 labelling of nucleolus organizer regions (NORs) and staining with base-specific fluorochromes) were performed on the South American fish species Leporinus friderici, L. obtusidens and L. elongatus. The overall karyotypic structure, position of NORs, as well as the amount, distribution and composition of constitutive heterochromatin were determined. Particular attention was given to the highly differentiated ZZ/ZW sex chromosome system of L. obtusidens and L. elongatus. Sharing the apparently ancient macroscopic karyotype of Anostomidae, all three species have 2n = 54 meta- or submetacentric chromosomes. NORs were found exclusively on chromosome pair 2, which may represent the ancestral NOR-bearing chromosome of the anostomid karyotype. Observed differences in the relative position of NORs along chromosome 2 and variations in the amount and distribution of constitutive heterochromatin throughout the karyotype were most probably caused by heterochromatin-mediated chromosome rearrangements. Detailed analysis of the morphologically similar heteromorphic ZZ/ZW sex chromosomes of L. obtusidens and L. elongatus allowed detection of differences in the DNA composition of the largely heterochromatic W chromosomes. However, since these and the W chromosomes of three other Leporinus species exhibit homologies with respect to their relative size, centromere position and amount and distribution of heterochromatin, it is concluded that they evolved from the same ancestral W chromosome.

Animals↗

Cytogenetics of the genus Leporinus (Pisces, Anostomidae). II. Molecular cytogenetics, organization and evolutionary conservation of a chromosome-specific satellite DNA from Leporinus obtusidens.

A chromosome-specific satellite DNA from the South American fish species Leporinus obtusidens has been isolated and characterized. Sequence analysis and Southern hybridization studies indicate that the cloned 483-bp fragment is 60% AT rich and appears to comprise two diverged monomers. A highly variable low-copy number polymorphism was detected and, thus, this satellite DNA may serve as a valuable genetic marker. Using a Southern blot approach, the cloned satellite DNA cross-hybridized strongly to the DNA of Leporinus elongatus but failed to detect homologous sequences in the genomes of other closely related Leporinus species and higher vertebrates. Using fluorescence in situ hybridization to mitotic metaphase spreads of L. obtusidens and L. elongatus, this satellite DNA was located to the (peri)centromeric region of one single chromosome pair in both species. As the cloned satellite DNA sequence clearly evolved along a chromosomal lineage and is highly variable, it may serve as a very useful marker in further genetic, molecular and cytogenetic studies of the genus Leporinus.

Animals↗

Analysis of replication timing of ribosomal RNA genes by fluorescence in situ hybridization.

Fluorescence in situ hybridization has been used to study the replication timing of various repeat DNA families in the short arms of human acrocentric chromosomes. In interphase nuclei, unreplicated DNA segments show singlet hybridization signals whereas replicated loci have doublet signals. The distribution of these two patterns in unsynchronized cell cultures revealed that the rRNA gene clusters replicate earlier than the closely juxtaposed alpha- and beta-satellite DNA sequences. Within the rDNA repeat unit, replication of the intergenic spacer appears to precede that of the transcribed rDNA.

DNA Replication↗

Complex FISH probes for the subtelomeric regions of all human chromosomes: comparative hybridization of CEPH YACs to chromosomes of the Old World monkey Presbytis cristata and great apes.

We have generated a human subtelomere probe panel, utilizing well characterized CEPH YACs, for the investigation of human chromosome pathology and evolution through fluorescent in situ hybridization (FISH). Region-specific FISH probes will be extremely valuable for detecting cytogenetically cryptic telomere abnormalities. Here, we present the first comparative mapping study (with 29 subtelomere probes and 6 chromosome paints) to the Old World monkey Presbytis cristata, followed by hybridizations to the great apes, gorilla and orangutan, when rearrangements were detected. We observed that the position of telomere-associated genomic sequences has been only moderately conserved during primate evolution. YAC 364f9, specific for the subtelomeric long arm of human chromosome 3, contains an evolutionary inversion breakpoint that was involved in independent chromosome rearrangements in P. cristata and gorilla.

Animals↗

Identification of positional candidates for neurological disorders on chromsome 13q14-->q22.

In the course of a research project aimed at the molecular characterization of balanced chromosome rearrangements associated with mental retardation (MR), several YACs spanning MR-associated chromosomal rearrangements in the 13q14-->q22 region were identified. To facilitate the search for relevant candidate genes, we have analyzed a total of 102 EST clones from this region. Sequence comparisons revealed that these 102 clones represent up to 72 distinct transcripts. When no physical mapping data were available, a minimal YAC contig was screened for each unique transcript by the polymerase chain reaction (PCR) or hybridization. Fifty-eight independent ESTs could be localized to YAC clones between the markers D13S1248 and D13S1201. Several ESTs are located on YAC clones detecting chromosomal rearrangements in MR patients. One EST was mapped within the critical region for Rieger syndrome type 2, and three transcripts were identified in the region for the nocturnal enuresis type 1. Some ESTs showed homologies to known genes, including the cadherin-related tumor suppressor gene from Drosophila, the yeast mitotic control protein DIS3, and the human alpha-2-macroglobulin receptor associated protein.

Chromosome Aberrations↗

Characterization of a chromosome-specific chimpanzee alpha satellite subset: evolutionary relationship to subsets on human chromosomes.

Alpha satellite DNA is a tandemly repeated DNA family found at the centromeres of all primate chromosomes examined. The fundamental repeat units of alpha satellite DNA are diverged 169- and 172-bp monomers, often found to be organized in chromosome-specific higher-order repeat units. The chromosomes of human (Homo sapiens (HSA)), chimpanzee (Pan troglodytes (PTR) and Pan paniscus), and gorilla (Gorilla gorilla) share a remarkable similarity and synteny. It is of interest to ask if alpha satellite arrays at centromeres of homologous chromosomes between these species are closely related (evolving in an orthologous manner) or if the evolutionary processes that homogenize and spread these arrays within and between chromosomes result in nonorthologous evolution of arrays. By using PCR primers specific for human chromosome 17-specific alpha satellite DNA, we have amplified, cloned, and characterized a chromosome-specific subset from the PTR chimpanzee genome. Hybridization both on Southern blots and in situ as well as sequence analysis show that this subset is most closely related, as expected, to sequences on HSA 17. However, in situ hybridization reveals that this subset is not found on the homologous chromosome in chimpanzee (PTR 19), but instead on PTR 12, which is homologous to HSA 2p.

Animals↗

Inhibition of RNA polymerase II transcription causes chromatin decondensation, loss of nucleolar structure, and dispersion of chromosomal domains.

Fluorescence in situ hybridization and immunofluorescence have been used to visualize specific genomic DNA sequences and proteins in interphase nuclei treated with transcriptional inhibitors. The adenosine analog 5,6-dichloro-beta-D-ribofuranosylbenzimidazole (DRB) and alpha-amanitin selectively inhibit transcription by RNA polymerase II at low doses. Upon exposure to DRB or alpha-amanitin the fibrillar components of the normally compact nucleolus unravel into necklace-like structures which represent highly extended linear arrays of ribosomal (r)RNA genes. Similarly, blocks of tandemly repeated satellite DNAs dissociate into extended beaded strands. Localized (euchromatic) chromosome domains and even whole chromosome territories disperse throughout the nuclear interior. Treatment of cells with actinomycin D (AMD) at doses that block rRNA synthesis does not cause significant decondensation of nucleolar, heterochromatic, and interphase chromosome domains. Interestingly, both alpha-amanitin and AMD cause coilin to associate with the nucleolar domain. In AMD-treated cells, coilin is enriched in nucleolar caps abutting upon the residual nucleolus. After alpha-amanitin treatment, coilin is concentrated in numerous beads closely associated with individual rDNA transcription units within nucleolar necklaces. The changes in higher-order nuclear structure are reversible in cell cultures exposed to nontoxic doses of transcriptional inhibitors. It therefore may be concluded that nuclear topographic organization is dependent on a continued transcription of nuclear genes, but not of the rRNA genes.

Amanitins↗