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D C Ward

Publications and source records attributed to D C Ward.

At least 91 records · Page 5Linked to original sources
PubMed1994

Structure and chromosomal localization of the gene (BDKRB2) encoding human bradykinin B2 receptor.

The bradykinin B2 receptor (BDKRB2) has high affinity for the intact kinins, which mediate a wide spectrum of biological effects, including pain, inflammation, vasodilation, and smooth muscle contraction and relaxation. In the present study, we have cloned and sequenced the gene encoding human bradykinin B2 receptor from a human genomic library. The B2 receptor gene contains three exons separated by two introns. The first and second exons are noncoding, while the third exon contains the full-length coding region, which encodes a protein of 364 amino acids forming 7 transmembrane domains. The human B2 gene shares high sequence identity with rat and mouse B2 receptor genes and significant similarity with the gene encoding the angiotensin II type I receptor in the nucleotide sequence and exon-intron arrangement. In the 5' flanking region, a consensus TATA box and several putative transcription factor-binding sites have been identified. Genomic Southern blot analysis showed that the B2 receptor is encoded by a single-copy gene that was localized to chromosome 14q32 by in situ hybridization. In a Southern blot analysis following reverse transcription and polymerase chain reaction, the human B2 receptor was found to be expressed in most human tissues.

Amino Acid Sequence↗
PubMed1994

Molecular cloning, sequence analysis, and chromosomal localization of the human protease inhibitor 4 (kallistatin) gene (PI4).

The gene encoding human protease inhibitor 4 (kallistatin; gene symbol PI4), a novel serine proteinase inhibitor (serpin), has been isolated and completely sequenced. The kallistatin gene is 9618 bp in length and contains five exons and four introns. The structure and organization of the kallistatin gene are similar to those of the genes encoding alpha 1-antichymotrypsin, protein C inhibitor, and alpha 1-antitrypsin. The kallistatin gene is also similar to the genes encoding rat and mouse kallikrein-binding proteins. The first exon of the kallistatin gene is a noncoding 89-bp fragment, as determined by primer extension. The fifth exon, which contains 308 bp of noncoding sequence, encodes the reactive center of kallistatin. In the 5'-flanking region of the kallistatin gene, 1125 bp have been sequenced and a consensus promoter segment with potential transcription regulatory sites, including CAAT and TATA boxes, an AP-2 binding site, a GC-rich region, a cAMP response element, and an AP-1 binding site, has been identified within this region. The kallistatin gene was localized by in situ hybridization to human chromosome 14q31-q32.1, close to the serpin genes encoding alpha 1-antichymotrypsin, protein C inhibitor, alpha 1-antitrypsin, and corticosteroid-binding globulin. In a genomic DNA Southern blot, kallistatin-related genes were identified in monkey, mouse, rat, bovine, dog, cat, and a ground mole. The patterns of hybridization revealed clues of human serpin evolution.

Amino Acid Sequence↗
PubMed1994

A YAC contig spanning a cluster of human type III receptor protein tyrosine kinase genes (PDGFRA-KIT-KDR) in chromosome segment 4q12.

We have mapped five genes encoding protein tyrosine kinases (PTKs) to the pericentromeric region of human chromosome 4. PTK4 and TYRO4, which encode nonreceptor intracellular PTKs, are located at 4p12 and 4q13, respectively. The other three genes, PDGFRA, KIT, and KDR, encode type III transmembrane receptor PTKs for known ligands. We have developed a contig of 29 yeast artificial chromosomes (YACs) spanning approximately 2 Mb of DNA at 4q12 that includes PDGFRA, KIT, and KDR, and we have used this YAC contig to map 12 different sequence-tagged sites in this region. PDGFRA, KIT, and KDR thus constitute a cluster of genes at 4q12 encoding closely related type III receptor PTKs. Mutations of the human KIT gene result in piebaldism, an autosomal dominant disorder of melanocyte development.

Base Sequence↗
PubMed1994

The Epstein-Barr virus (EBV) small RNA EBER1 binds and relocalizes ribosomal protein L22 in EBV-infected human B lymphocytes.

Epstein-Barr virus (EBV), an oncogenic herpesvirus, encodes two small RNAs (EBERs) that are expressed at high levels during latent transformation of human B lymphocytes. Here we report that a 15-kDa cellular protein called EAP (for EBER associated protein), previously shown to bind EBER1, is in fact the ribosomal protein L22. Approximately half of the L22 in EBV-positive cells is contained within the EBER1 ribonucleoprotein (RNP) particle, whereas the other half residues in monoribosomes and polysomes. Immunofluorescence with anti-L22 antibodies demonstrates that L22 is localized in the cytoplasm and the nucleoli of uninfected human cells, as expected, whereas EBV-positive lymphocytes also show strong nucleoplasmic staining. In situ hybridization indicates that the EBER RNPs are predominantly nucleoplasmic, suggesting that L22 relocalization correlates with binding to EBER1 in vivo. Since incubation of uninfected cell extracts with excess EBER1 RNA does not remove L22 from preexisting ribosomes, in vivo binding of L22 by EBER1 may precede ribosome assembly. The gene encoding L22 has recently been identified as the target of a chromosomal translocation in certain patients with leukemia, suggesting that L22 levels may be a determinant in cell transformation.

B-Lymphocytes↗
PubMed1994

Detection of nondisjunction and recombination in meiotic and postmeiotic cells from XYSxr [XY,Tp(Y)1Ct] mice using multicolor fluorescence in situ hybridization.

Current meiotic dogma holds that synapsis is required for recombination and that recombination is required for proper disjunction. The mouse chromosome aberration XYSxr [sex reversal; redesignated XY,Tp(Y)1Ct] appears to challenge this assumption, for although chromosomes X and Y often fail to synapse and recombine, there is no dramatic increase in aneuploid progeny. An explanation of this conundrum might be that X-Y univalent spermatocytes do not survive. The phenotype of sex reversal is generated by the "obligatory" crossover between the X and Y chromosomes, which always occurs proximal to a duplicated copy of the testis-determining gene Sry and transfers one copy from one chromatid of the Y chromosome to one chromatid of the X. Animals that inherit an X chromosome with the Sry gene are chromosomally female but phenotypically male. We have used fluorescence in situ hybridization (FISH) to visualize probes for the X and Y chromosomes and for the Sry sequence and chromosome 8 to track the fate of both recombinant and nonrecombinant chromosomes through metaphases I and II into spermatids and sperm. In the 219 gametes examined by multicolor FISH, the frequency of aneuploid products (XY or "O") was low (3.7%) despite a high frequency (66%) of X-Y separation at metaphase I. In balanced gametes, X and Y recombinant chromosomes slightly exceeded nonrecombinants. Both of these observations support the earlier proposal that asynapsis and nondisjunction in primary spermatocytes lead to their developmental arrest and degeneration.

Aneuploidy↗
PubMed1994

Twenty-one polymorphic markers from human chromosome 12 for integration of genetic and physical maps.

Twenty-one physically mapped, polymorphic markers have been developed from a chromosome 12-specific cosmid library. The markers consist of CA repeat-containing sequence-tagged sites (STSs) derived from cosmid clones mapped by fluorescence in situ hybridization (FISH). Three methods for determining the sequence flanking CA microsatellites were used, including one using degenerate primer sets for direct sequence analysis. Oligonucleotide primer pairs suitable for use in polymerase chain reaction (PCR) were selected from the sequences flanking the CA microsatellite and were tested for their ability to generate unique PCR products. The informativeness of these STSs as genetic markers was determined by typing 10 unrelated individuals who are part of the Centre d'Etude du Polymorphisme Humaine (EPH) pedigrees. Eleven of the 21 FISH-mapped, polymorphic STSs are heterozygous in 7 or more of the individuals tested. Since these markers are derived from physically mapped cosmids, genetic linkage analysis with them will facilitate the integration of the developing physical and genetic maps of chromosome 12.

Alleles↗
PubMed1994

Numerical chromosomal aberrations in thyroid tumors detected by double fluorescence in situ hybridization.

Double fluorescence in situ hybridization with DNA probes specific for the (peri)centromeric regions of chromosomes 3, 7, 9, 11, 12, 18, and X was performed on fresh isolated nuclei and frozen tissue sections prepared from 2 nodular hyperplasias, 2 adenomas, and 7 papillary carcinomas of the thyroid in order to detect numerical chromosomal changes. Numerical chromosomal aberrations were found in all malignant specimens examined. A consistent presence of at least two trisomies was detected in most cases, especially in the follicular variant specimens; the highest degree of trisomy was observed for chromosome 12. Isolated monosomies of moderate degree for different chromosomes were found in 1 adenoma and 2 papillary carcinomas. Severe monosomy of chromosome 9 was the only significant feature observed in the single metastatic papillary carcinoma.

Adult↗
PubMed1994

Kinetoplast DNA replication: mechanistic differences between Trypanosoma brucei and Crithidia fasciculata.

Kinetoplast DNA, the mitochondrial DNA of trypanosomatid parasites, is a network containing several thousand minicircles and a few dozen maxicircles. We compared kinetoplast DNA replication in Trypanosoma brucei and Crithidia fasciculata using fluorescence in situ hybridization and electron microscopy of isolated networks. One difference is in the location of maxicircles in situ. In C. fasciculata, maxicircles are concentrated in discrete foci embedded in the kinetoplast disk; during replication the foci increase in number but remain scattered throughout the disk. In contrast, T. brucei maxicircles generally fill the entire disk. Unlike those in C. fasciculata, T. brucei maxicircles become highly concentrated in the central region of the kinetoplast after replication; then during segregation they redistribute throughout the daughter kinetoplasts. T. brucei and C. fasciculata also differ in the pattern of attachment of newly synthesized minicircles to the network. In C. fasciculata it was known that minicircles are attached at two antipodal sites but subsequently are found uniformly distributed around the network periphery, possibly due to a relative movement of the kinetoplast disk and two protein complexes responsible for minicircle synthesis and attachment. In T. brucei, minicircles appear to be attached at two antipodal sites but then remain concentrated in these two regions. Therefore, the relative movement of the kinetoplast and the two protein complexes may not occur in T. brucei.

Animals↗
PubMed1994

Organization of small nucleolar ribonucleoproteins (snoRNPs) by fluorescence in situ hybridization and immunocytochemistry.

The organization of the U3, U8, and U13 small nucleolar ribonucleoproteins (snoRNPs) has been investigated in HeLa cells using antisense DNA and 2'-OMe RNA oligonucleotides. Oligomers corresponding to deoxynucleotides that target RNase H degradation of intact RNP particles were synthesized and used for fluorescence in situ hybridization. U3 and U13 are distributed throughout the nucleolus and colocalize with anti-fibrillarin antibodies. U8, however, is organized in discrete ring-like structures near the center of the nucleolus and surround bright punctate regions visualized with anti-RNA polymerase I and anti-UBF/NOR-90 antibodies. In decondensed nucleoli, a necklace of smaller ring-like structures of U8 RNA appear. A model for the recruitment of U8 (and presumably other processing factors) to the sites of rRNA transcription is discussed. Hybridization to mitotic cells showed that unlike pol I and NOR-90, U8 is dispersed into the cytoplasm during mitosis. The subnucleolar organization of U8 is consistent with its demonstrated participation in early intermediate steps in pre-rRNA processing. In contrast, the more dispersed intranucleolar distribution of U3 agrees with its putative involvement in both early and late steps of rRNA maturation. These studies illustrate the feasibility of mapping functional domains within the nucleolus by correlating the in vitro activities of small nuclear RNPs with their in situ locations.

Base Sequence↗
PubMed1994

Construction of a YAC contig and a STS map spanning at least seven megabasepairs in chromosome 5q34-35.

We have constructed a YAC contig containing 54 clones and a minimum of 7 Mbp of human DNA, that maps to bands q34-35 on chromosome 5. The contig was nucleated using FISH mapped cosmid clones shown to flank the t(2;5)(p23;q35) translocation breakpoint in a CD30-positive large cell lymphoma cell line. Thirty of the 54 YAC clones are non-chimeric and six span the translocation breakpoint, as determined by FISH analysis. A total of 28 YAC clone end fragments, 14 non-polymorphic YAC end STS probes and 13 polymorphic microsatellite STS markers have been used to order clones within the contig. The most distal genetic markers (D5S498 and D5S619) are separated by 15 cM based on multipoint linkage analysis. This map of overlapping clones and the set of densely spaced physical markers will promote our understanding of the 5q34-35 region and its associated genes.

Base Composition↗
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↗
PubMed1994

Localization of the human alpha-fodrin gene (SPTAN1) to 9q33-->q34 by fluorescence in situ hybridization.

Using fluorescence in situ hybridization, the human alpha-fodrin gene (nonerythroid alpha spectrin, SPTAN1), a member of the spectrin gene superfamily, was mapped to 9q33-->q34. This locus is near the translocation breakpoint region in the Philadelphia (Ph1) chromosome, t(9;22)(q34;q11). In a human chronic myelogenous leukemia cell line with the Ph1 chromosome, K562, the alpha-fodrin gene mapped centromeric to the translocation breakpoint, indicating that the alpha-fodrin gene is not translocated to the Ph1 chromosome in this cell line.

Carrier Proteins↗
PubMed1994

In situ hybridization mapping of human chromosome 16: evidence for a high frequency of repetitive DNA sequences.

Fluorescence in situ hybridization (FISH) provides a rapid approach to regional localization of overlapping clone sets (contigs) developed by various fingerprinting approaches. We have used 70 cosmid clones derived from 48 different contigs, part of the developing contig map of chromosome 16 (Stallings et al., 1990, 1992a), to cytogenetically map an estimated 8.6 million base pairs (Mbp) of chromosome 16 DNA (approximately 8-9% total coverage). Although the majority of cosmid contigs hybridized to single sites on chromosome 16, a significant fraction (23%) hybridized to multiple regions on chromosome 16; a subset of these also hybridized to other human chromosomes. In most instances, clones that mapped to multiple locations were found to contain low-abundance repetitive DNA sequences. The FISH data presented here, coupled with published mapping data from somatic cell hybrids (Callen et al., 1992), permits independent verification of the integrity of chromosome 16 cosmid contigs. The order of clones derived by FISH agrees closely with the cell hybrid mapping data and can be correlated with chromosome bands and specific chromosomal translocation breakpoints.

Chromosome Mapping↗
PubMed1993

Inhibition of RNA polymerase II transcription by oligonucleotide-RecA protein filaments targeted to promoter sequences.

In the presence of RecA protein, which plays a major role in genetic recombination in Escherichia coli, an oligodeoxyribonucleotide can find its homologous counterpart in double-stranded DNA and form triple-stranded structures. A triple-stranded structure formed by an oligonucleotide with a sequence overlapping essential regulatory elements of a viral promoter, such as TATA or GC boxes, inhibited in vitro transcription driven by RNA polymerase II. An oligonucleotide with eight nucleotides homologous to its target suppressed RNA polymerase II activity in HeLa cell extracts. This procedure offers a potential alternative to the usual mutational analysis of transcriptional promoters.

Base Sequence↗
PubMed1993

Cell cycle-dependent distribution of telomeres, centromeres, and chromosome-specific subsatellite domains in the interphase nucleus of mouse lymphocytes.

Fluorescence in situ hybridization and 3-D image analysis were combined to study the distribution of specific chromosome subdomains through the course of the cell cycle in cultured mouse lymphocytes. DNA probes specific for major satellite DNA, minor satellite DNA, telomeric DNA and to chromosome X-, 8-, and 14-specific subsatellite DNA sequences were used. We demonstrate that a redistribution of the chromatin occurs during the cell cycle in the interphase nucleus, and that the profile of the rearrangement is highly dependent on the nature of the domain considered (centromeres, telomeres, or subsatellite regions). However, the relative arrangement of chromosome homologs to each other does not appear to be spatially defined or regulated.

Animals↗