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D C Ward

Publications and source records attributed to D C Ward.

At least 163 records · Page 9Linked to original sources
PubMed1992

Localization of the gene for MEN 2A.

The search for the gene that causes the multiple endocrine neoplasia type 2A (MEN 2A) syndrome is entering a new phase. Genetic linkage studies have localized the gene to the pericentromeric region of chromosome 10. The statistical portion of mapping the gene for MEN 2A is nearly complete and now classical molecular biological/gene mapping techniques will be employed. We have used fluorescence in situ hybridization to estimate the size of the MEN2A region to be about 2 to 5 mb, using some liberal assumptions; at worst the region should contain no more than about 10 mb of non-alphoid DNA. Our mapping panels (meiotic recombinant and radiation reduced hybrid) give consistent orders of markers in this small region. We describe our initial attempts to clone the region using yeast artificial chromosomes.

Chromosome Mapping↗
PubMed1992

Preimplantation genetic diagnosis. In situ hybridization as a tool for analysis.

In situ hybridization allows one to directly visualize DNA sequences of interest for which probes are available. Fluorescent signals can be detected in single cells in either metaphase or interphase nuclei, thus obviating the need for cell synchronization. The potential to use this technology in conjunction with in vitro fertilization techniques to genetically analyze an embryo before transfer could offer patients at genetic risk an alternative. Currently, these patients wait until 9 to 16 weeks' gestation to find out about the genetic makeup of their pregnancy. As such, sexing the embryos of patients at risk for transmitting sex-linked disorders or for performing numerical chromosome analysis could be performed before embryo transfer. Techniques for applying in situ hybridization to single cells obtained from embryos are presented. Hybridization of specific probes to blastomeres of mouse and human embryos as well as sperm is demonstrated.

Cell Nucleus↗
PubMed1992

The gene for human erythrocyte protein 4.2 maps to chromosome 15q15.

Protein 4.2 (P4.2), one of the major components of the red-blood-cell membrane, is located on the interior surface, where it binds with high affinity to the cytoplasmic domain of band 3. Individuals whose red blood cells are deficient in P4.2 have osmotically fragile, abnormally shaped cells and moderate hemolytic anemia. cDNA clones from both the 5' and the 3' coding regions of the P4.2 gene were used to map its chromosomal location by fluorescence in situ hybridization. The probes, individually or in combination, gave specific hybridization signals on chromosome 15. The hybridization locus was identified by combining fluorescence images of the probe signals with fluorescence banding patterns generated by Alu-PCR (R-like) probe and by DAPI staining (G-like). Our results demonstrate that the locus of the P4.2 gene is located within 15q15.

Biotin↗
PubMed1991

Human dopamine D5 receptor pseudogenes.

Molecular cloning studies have now identified five structurally homologous genes encoding the biosynthesis of the human dopamine receptors, DRD1, DRD2, DRD3, DRD4, and DRD5. Two of these dopamine receptors (DRD1 and DRD5) are encoded by intronless genes. To ascertain whether there are other intronless genes that share identity with the gene (DRD5) encoding the DRD5 receptor, we used a cloning method based on the polymerase chain reaction (PCR). Human genomic DNA was amplified by PCR with oligodeoxyribonucleotides (oligos) based on the DRD5 nucleotide (nt) sequence. Amplification of nt sequences between these oligos allowed the isolation of two independent intronless genes that share identity with DRD5. The full-length clones have also been isolated by screening human genomic libraries. The deduced amino acid sequences for these genes, PG-1 and PG-2, share 91% and 92% identity to DRD5, respectively. However, each of the genes contains differences in the coding regions that would render these genes incapable of encoding functional receptors. Thus, the human genome contains at least two DRD5 pseudogenes, consistent with in situ human chromosomal hybridization analysis which reveals the presence of two pseudogenes.

Amino Acid Sequence↗
PubMed1991

Characterization and mapping of human genes encoding zinc finger proteins.

The zinc finger motif, exemplified by a segment of the Drosophila gap gene Krüppel, is a nucleic acid-binding domain present in many transcription factors. To investigate the gene family encoding this motif in the human genome, a placental genomic library was screened at moderate stringency with a degenerate oligodeoxynucleotide probe designed to hybridize to the His/Cys (H/C) link region between adjoining zinc fingers. Over 200 phage clones were obtained and are being sorted into groups by partial sequencing, cross-hybridization with oligodeoxynucleotide probes, and PCR amplification. Further, the genomic clones were cross-hybridized with a set of 30 zinc finger-encoding cDNAs (Kox1-Kox30) isolated from a human T-cell cDNA library. Four cDNAs (Kox4, Kox7, Kox12, and Kox15) were identified that match one or more genomic clones; these matches were confirmed by nucleotide sequence analysis. One or more clones from each locus were mapped onto human metaphase chromosomes by chromosomal in situ suppression hybridization with fluorescent probe detection. We mapped ZNF7/Kox4 to chromosome 8qter, ZNF19/Kox12 to 16q22, ZNF22/Kox15 to 10q11, and ZNF44/Kox7 to 16p11. The results of these analyses support the conclusion that the human genome contains many, probably several hundred, zinc finger genes with consensus H/C link regions.

Amino Acid Sequence↗
PubMed1991

Origin of human chromosome 2: an ancestral telomere-telomere fusion.

We have identified two allelic genomic cosmids from human chromosome 2, c8.1 and c29B, each containing two inverted arrays of the vertebrate telomeric repeat in a head-to-head arrangement, 5'(TTAGGG)n-(CCCTAA)m3'. Sequences flanking this telomeric repeat are characteristic of present-day human pretelomeres. BAL-31 nuclease experiments with yeast artificial chromosome clones of human telomeres and fluorescence in situ hybridization reveal that sequences flanking these inverted repeats hybridize both to band 2q13 and to different, but overlapping, subsets of human chromosome ends. We conclude that the locus cloned in cosmids c8.1 and c29B is the relic of an ancient telomere-telomere fusion and marks the point at which two ancestral ape chromosomes fused to give rise to human chromosome 2.

Base Sequence↗
PubMed1991

Mapping of human microtubule-associated protein 1B in proximity to the spinal muscular atrophy locus at 5q13.

A polyclonal antiserum directed against the C-terminal domain of dystrophin was used to isolate a cDNA clone encoding an antigenically cross-reactive protein, microtubule-associated protein 1B (MAP-1B). Physical mapping of the human MAP-1B locus places its chromosomal location at 5q13, in proximity to the spinal muscular atrophy (SMA) locus. SMA is a degenerative disorder primarily affecting motor neurons. Genetic linkage analysis of SMA families using a human dinucleotide repeat polymorphism just 3' of the MAP-1B gene has shown tight linkage to SMA mutations. These mapping data together with the postulated role of MAP-1B in neuronal morphogenesis and its localization in anterior horn motor neurons suggest a possible association with SMA.

Animals↗
PubMed1991

Cloning of a human cDNA expressing a high voltage-activating, TEA-sensitive, type-A K+ channel which maps to chromosome 1 band p21.

Over ten different mammalian genes related to the Drosophila Shaker gene (the Sh gene family) have been identified recently. These genes encode subunits of voltage-dependent K+ channels. The family consists of four subfamilies: ShI genes are homologues of Shaker; ShII, ShIII, and ShIV are homologues of three other Shaker-like genes in Drosophila, Shab, Shaw, and Shal, respectively. We report here the cloning of a human K+ channel ShIII cDNA (HKShIIIC) obtained from a brain stem cDNA library. HKShIIIC transcripts express an atypical voltage-dependent transient (A-type) K+ current in Xenopus oocytes. This current is activated by large membrane depolarizations and is extremely sensitive to the K+ channel blocker TEA unlike most A-type currents. The gene encoding HKShIIIC maps to chromosome 1p21.

Amino Acid Sequence↗
PubMed1991

Comparative mapping of a gorilla-derived alpha satellite DNA clone on great ape and human chromosomes.

We have isolated an alpha satellite DNA clone, pG3.9, from gorilla DNA. Fluorescence in situ hybridization on banded chromosomes under high stringency conditions revealed that pG3.9 identifies homologous sequences at the centromeric region of ten gorilla chromosomes, and, with few exceptions, also recognizes the homologous chromosomes in human. A pG3.9-like alphoid DNA is present on a larger number of orangutan chromosomes, but, in contrast, is present on only two chromosomes in the chimpanzee. These results show that the chromosomal subsets of related alpha satellite DNA sequences may undergo different patterns of evolution.

Animals↗
PubMed1991

Generation and characterization of irradiation hybrids of human chromosome 4.

In recent years investigators have attempted to develop more rapid and precise methods to isolate specific chromosomal DNA regions. In this paper we demonstrate a modification of the method first developed by Goss and Harris for generation of irradiation hybrids. The gene encoding the dominant selectable marker for resistance to neomycin was introduced into human chromosome 4 using retroviral insertion into human fibroblasts. Transfer of these chromosomes via microcells into the mouse cell line NIH3T6 produced a somatic cell line containing chromosome 4 as the only human chromosome. Irradiation of this cell line followed by fusion with the hamster cell line CHTG49 generated hybrids containing only small portions of chromosome 4p on a hamster background. The use of selection produced stable hybrids that retained chromosome 4 fragments over long periods of tissue culture passage. To obtain new polymorphic markers for Huntington's disease, one of these hybrids was to isolate new genomic fragments. We identified 41 single-copy fragments, of which 27 have been mapped to specific regions of chromosome 4; 52% of these fragments map to the region of chromosome 4 containing the HD gene.

Animals↗
PubMed1991

Isolation and chromosomal localization of a novel nonerythroid ankyrin gene.

Immunoreactive isoforms of erythrocyte ankyrin have been shown to be present in a variety of nonerythroid tissues. Isolation of the genes that encode these isoforms will clarify their relationship to erythrocyte ankyrin. Using an erythrocyte ankyrin cDNA clone as a hybridization probe, we screened a human genomic library and isolated a clone that hybridizes with the probe at low stringency but not at high stringency. Partial nucleotide sequence of the clone revealed the presence of a 99-bp segment that is homologous to an exon of the erythrocyte ankyrin gene. Northern analysis showed that a labeled fragment of the clone hybridized to a 7-kb message in RNA of fetal brain but not of erythroid cells, suggesting that this clone is part of a novel gene that is expressed predominantly in nonerythroid tissue. Comparison of the sequence of the genomic clone with that of a recently isolated cDNA clone for brain ankyrin (Otto et al., 1989) showed identity of 96 of 99 bp between the putative exon and a segment of the cDNA clone (V. Bennett, personal communication, 1991), suggesting that the genomic clone is part of a gene for nonerythroid ankyrin, which we have designated ANK2. By analysis of somatic cell hybrids and fluorescence in situ hybridization, we assigned ANK2 to human chromosome 4 at a position equivalent to bands 4q25-q27.

Amino Acid Sequence↗
PubMed1991

Isolation, characterization, and localization of human genomic DNA encoding the beta 1 subunit of the GABAA receptor (GABRB1).

Genomic DNA that encodes the beta 1 subunit of the human gamma-aminobutyric acidA (GABAA) receptor was cloned and mapped. Exons and flanking introns (greater than 14 kb) were sequenced to determine the structural organization of the gene. The gene was localized on human chromosome 4, in bands p12-13. The beta 1 subunit is encoded by a relatively large gene (greater than 65 kb) on nine exons. In contrast to other conserved regions of the subunit polypeptide, the proposed channel-forming domain (M2) is derived from more than one exon. The organization of exons was compared with that of the genes that code for subunits of nicotinic acetylcholine receptors. There is no evidence for conservation of gene structure between these two members of the proposed gene superfamily. However, intron-exon junctions were found to be conserved precisely between subtypes of GABAA receptor subunits.

Amino Acid Sequence↗
PubMed1991

In situ hybridization banding of human chromosomes with Alu-PCR products: a simultaneous karyotype for gene mapping studies.

Polymerase chain reaction products generated from a single Alu primer and human genomic DNA produce a distinct and highly reproducible R-banding pattern when hybridized to metaphase chromosome spreads. Individual chromosomes can be readily identified and karyotyped. Compared to conventional fluorescence banding on heat-denatured chromosomes, the in situ hybridization banding (ISHB) shows high contrast and definition. We demonstrate that this banding method can be employed effectively in double-labeling experiments for the rapid and simultaneous assignment of probes to specific chromosomal bands. Since virtually any fluorochrome can be used to delineate chromosomal bands, ISHB should provide added flexibility for multicolor mapping strategies.

Chromosome Banding↗
PubMed1991

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.

Aneuploidy↗
PubMed1991

Analysis of genes and chromosomes by nonisotopic in situ hybridization.

Nonisotopic in situ hybridization is a powerful tool to analyze the organization of complex genomes. Current approaches utilizing this technique for the analysis of linear and spatial genome organizations are presented. Clinical applications of these approaches, which open new avenues for diagnosis of disease-related chromosomal changes, are also discussed.

Chromosome Mapping↗
PubMed1991

Determination of time-varying 222Rn concentrations using flow-through scintillation flasks.

This paper describes a method to obtain time-varying 222Rn concentrations from the series of gross alpha measurements recorded with a flow-through scintillation detector. The continuous input of 222Rn into the detector is treated as a series of independent pulses. The response of the detector to a single pulse containing 222Rn can be measured in the laboratory and is used to determine a normalized detector response function (NDRF). The NDRF contains information on the combined effects of detector design, operating parameters, and the presence of 222Rn progeny. The estimation of individual plate-out or counting efficiencies is not needed, as they are appropriately included in the NDRF. The equation describing the response function can be inverted to yield the actual 222Rn concentration in terms of detector output. The technique was applied to actual indoor monitoring data and the results presented.

Air Pollutants, Radioactive↗
PubMed1991

Unusual Sp1-GC box interaction in a parvovirus promoter.

The P4 promoter of the parvovirus minute virus of mice contains a single degenerate GC box sequence which binds the transcription factor Sp1 with high affinity. The two promoters of murine Sp1 were affinity purified, and their interactions with the P4 promoter were examined. Several unusual features were observed. Methylation interference experiments demonstrated that Sp1 makes contacts with both DNA strands, including the central guanine as well as an adenine residue on the cytidine-rich strand of the GC box. UV photocross-linking revealed that the 95- and the 105-kDa promoters cross-link exclusively to opposite strands of the GC box. These results suggest that the phosphorylation of the 95-kDa Sp1 promoter results in a change in the way Sp1 is positioned on the P4 GC box and identifies a high-affinity GC box motif.

Animals↗