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D C Ward

Publications and source records attributed to D C Ward.

At least 19 recordsLinked to original sources
PubMed1992

In situ hybridization to the Crithidia fasciculata kinetoplast reveals two antipodal sites involved in kinetoplast DNA replication.

Kinetoplast DNA is a network of interlocked minicircles and maxicircles. In situ hybridization, using probes detected by digital fluorescence microscopy, has clarified the in vivo structure and replication mechanism of the network. The probe recognizes only nicked minicircles. Hybridization reveals prereplication kinetoplasts (with closed minicircles), donut-shaped replicating kinetoplasts (with nicked minicircles on the periphery and closed minicircles in the center), and postreplication kinetoplasts (with nicked minicircles). Replicating kinetoplasts are associated with two peripheral structures containing free minicircle replication intermediates and DNA polymerase. Replication may involve release of closed minicircles from the center of the kinetoplast and their migration to the peripheral structures, replication of the free minicircles therein, and then peripheral reattachment of the progeny minicircles to the kinetoplast.

Animals↗
PubMed1992

Joints formed by RecA protein from oligonucleotides and duplex DNA block initiation and elongation of transcription.

In the presence of the non-hydrolyzable analog of ATP, ATP gamma S, RecA protein can polymerize on an oligodeoxy-ribonucleotide to form a stable oligonucleoprotein filament that can find its homologous sequence in double-stranded DNA. The homologous joint formed by the oligonucleotide and duplex DNA is stable only if RecA protein is not removed. Such a nucleoprotein joint, covering a part or all of the promoter region of T3 or T7 phage RNA polymerase, blocked transcription directed by those polymerases. The same kind of joint, located downstream of the RNA polymerase promoter, also inhibited elongation of transcription and caused accumulation of truncated transcripts. These observations suggest that RecA protein can be used to shut off transcription from any promoter of known sequence.

Base Sequence↗
PubMed1992

Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy.

Combinatorial labeling of probes (i.e., with two or more different reporters) increases the number of target sequences that can be detected simultaneously by fluorescence in situ hybridization. We have used an epifluorescence microscope equipped with a digital imaging camera and computer software for pseudocoloring and merging images to distinguish up to seven different probes using only three fluorochromes. Chromosome-specific centromere repeat clones and chromosome-specific "composite" probe sets were generated by PCR in which different mixtures of modified nucleotides, including fluorescein-conjugated dUTP, were incorporated. Cosmid clones were labeled similarly by nick-translation. The technique has been used to delineate the centromeres of seven different human chromosomes, on both 4',6-diamidino-2-phenylindole-stained metaphase spreads and interphase nuclei, to map six cosmid clones in a single hybridization experiment and to detect chromosome translocations by chromosome painting. Multiparameter hybridization analysis should facilitate molecular cytogenetics, probe-based pathogen diagnosis, and gene mapping studies.

Centromere↗
PubMed1992

Two diverged human homeobox genes involved in the differentiation of human hematopoietic progenitors map to chromosome 1, bands q41-42.1.

Proteins encoded by homeobox containing genes are sequence-specific DNA binding proteins implicated in the control of gene expression in both developing and adult tissues. Two recently characterized human homeobox genes, HB9 and HB24, are highly expressed in CD34-positive marrow cells but not in CD34-depleted marrow cells. Their expression is readily down-regulated during the differentiation of hematopoietic progenitors to specific cell lineages. In this study, genomic DNA fragments isolated with HB9 (3 kb) and HB24 (6 kb) cDNAs were used to map their chromosomal location by fluorescence in situ hybridization. Both HB9 and HB24 DNA probes gave specific hybridization signals on chromosome 1. The hybridization loci were identified by combining fluorescence images of the probe signals with fluorescence banding patterns generated by cohybridization in situ with an Alu probe (R-like banding) and by DAPI staining (G-like). The results demonstrate that the loci of the HB24 and HB9 genes are within bands 1q41-q42.1. A cohybridization experiment utilizing both probes with two-color fluorescence imaging could not resolve separate loci for the two genes.

Cell Differentiation↗
PubMed1992

Delineation of DNA replication time zones by fluorescence in situ hybridization.

Fluorescence in situ hybridization has been used to visualize specific genomic DNA sequences in interphase nuclei. In normal diploid cells, unreplicated DNA segments give singlet hybridization signals while replicated loci are characterized by doublets. The distribution of these two patterns in unsynchronized cell populations can be used to determine the S phase replication time of any DNA sequence. The validity of this approach was established by analyzing genes whose replication profiles in expressing and non-expressing cells had been determined previously by conventional methods. Using this technique it has been possible to map the replication timing topography of the DNA within and flanking the cystic fibrosis (CF) gene locus on chromosome 7. The gene itself is located within a defined time zone which is approximately 500 kb in length and is under developmental control. It is early replicating in cells which express CF but late replicating in other cell types. These time zones probably represent basic units of chromosome structure.

Bromodeoxyuridine↗
PubMed1992

Morphometry of gastric carcinoma: its association with patient survival, tumour stage, and DNA ploidy.

Morphometric image analysis of nuclear features was performed on tissue from 46 patients who had had curative resections for gastric cancer. Clinical, pathological, flow cytometric, and follow-up data were available for these patients, which were drawn from a larger, previously reported series. The morphometric data were compared with patient survival, clinico-pathological status, and DNA ploidy. Univariate survival analysis revealed that morphometric parameters were not significantly related to survival, but examination of clinico-pathological data showed lymph node involvement, involvement of the resection margin, and lymphatic invasion to be significantly associated (P < 0.01) with patient prognosis. Multivariate survival analysis using the Cox model found only lymph node and resection margin involvement to be independently related to survival. Comparison of morphometric results with the clinico-pathological parameters showed various features, relating to nuclear size, and its variation to be significantly associated (P < 0.01) with the presence of lymphatic invasion, resection margin involvement, and tumour pattern (intestinal/diffuse). A comparison of morphometry with flow cytometric analysis in these cases showed that nuclear size was not significantly related to either DNA aneuploidy or the DNA proliferative index.

Aged↗
PubMed1992

Confirmation of the human cathepsin B gene (CTSB) assignment to chromosome 8.

Human cathepsin B gene (CTSB) has been mapped to two locations: 8p22 and 13q14. Here we confirm the chromosome 8 assignment by three independent methods: (1) analysis of human-hamster somatic cell hybrid DNA by polymerase chain reaction; (2) comparison of hybridization signals to cathepsin B in interphase nuclei of normal fibroblasts and fibroblasts with a chromosome 8 deletion; and (3) fluorescence in situ hybridization to metaphase spreads using cathepsin B cosmid clones. Our results indicate that human CTSB is located at 8p22-p23.1.

Base Sequence↗
PubMed1992

Cell cycle dependent chromosomal movement in pre-mitotic human T-lymphocyte nuclei.

Fluorescent in situ hybridization with chromosome specific probes was used in conjunction with laser scanning confocal microscopy to assess the three-dimensional distribution of chromosomes in human T-lymphocyte nuclei. Cells in the G1-phase of the cell cycle exhibit a distinctly non-random chromosome organization:centromeric regions of the ten chromosomes examined are localized on the nuclear periphery, often making contact with the nuclear membrane, while telomeric domains are consistently localized within the interior 50% of the nuclear volume. Chromosome homolog pairing is not observed. Transition from the G1 to G2 cell cycle phase is accompanied by extensive chromosome movement, with centromeres assuming a more interior location. Chromosome condensation and chromatin depleted areas are observed in a small subset of G2 nuclei approaching mitosis. These results demonstrate that dynamic chromosome rearrangements occur in non-mitotic nuclei during the cell cycle.

Cell Cycle↗
PubMed1992

Generation and characterization of a human chromosome 9 cosmid library.

A cosmid library has been constructed from the hamster-human hybrid cell line PK-87-9, which contains chromosome 9 as its sole known human component. Ten thousand colonies were produced, of which approximately 200, or 2%, contain human material. Fifty of these 200 were regionally mapped by an Alu-primed PCR product hybridization procedure. These cosmids were localized to all regions of chromosome 9, but were especially concentrated in the distal portion of 9q. The map location derived by the Alu-primed PCR product hybridization procedure was compared to the map location derived by fluorescent in situ hybridization. Assignment of chromosomal location by the two methods was correspondent in all but a few cases. The presumptive presence of HTF islands was investigated for 130 cosmids by digestion with the restriction enzyme NotI. Twenty percent of cosmids contained at least one NotI site. A number of simple sequence repeat polymorphisms identified from the cosmid set were characterized and will provide a link between the genetic and physical maps for this chromosome.

Animals↗
PubMed1992

Clustering of C2-H2 zinc finger motif sequences within telomeric and fragile site regions of human chromosomes.

Ninety-three phage clones identified by hybridization with a C2-H2 zinc finger sequence probe have been grouped into 23 genetic loci. Partial sequencing verified that each locus belonged to the zinc finger family. Oligonucleotide primer pairs were developed from these sequences to serve as STS markers for these loci. One or more clones from each locus was mapped onto human metaphase chromosomes by fluorescence in situ hybridization. Several loci map to identical chromosomal regions, indicating the possible presence of multigene clusters. Zinc finger loci were found to reside predominantly either in telomeric regions or in chromosomal bands known to exhibit chromosome fragility. Chromosome 19 carries a disproportionate fraction (10 of 23) of the mapped zinc finger loci.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and chromosomal mapping of CCND genes encoding human D-type cyclins.

A human D-type cyclin gene (CCND1/cyclin D1/PRAD1) was previously isolated by virtue of its ability to complement a triple G1 cyclin (Cln) deficiency of Saccharomyces cerevisiae and was also identified as a candidate BCL1 oncogene. We now report the molecular cloning of two additional human D-type cyclin genes, CCND2 (cyclin D2) and CCND3 (cyclin D3). All three human D-type cyclin genes encode small (33-34 kDa) proteins that share an average of 57% identity over the entire coding region and 78% in the cyclin box. The D-type cyclins are most closely related to cyclin A (39% identity) and cyclin E (36%), followed by cyclin B (29%) and cyclin C (21%). Isolation and characterization of genomic clones revealed two pseudogenes corresponding to CCND2 and CCND3, respectively. All three cyclin D genes are interrupted by an intron at the same position. CCND2 has been mapped to chromosome 12p13, and CCND3 has been mapped to chromosome 6p21.

Amino Acid Sequence↗
PubMed1992

Cosmid linking clones localized to the long arm of human chromosome 11.

Molecular probes that contain DNA flanking CpG-rich restriction sites are extremely valuable in the construction of physical maps of chromosomes and in the identification of genes associated with hypomethylated HTF (HpaII tiny fragment) islands. We describe a new approach to the isolation and characterization of linking clones in arrayed chromosome-specific cosmid libraries through the large-scale semiautomated restriction mapping of cosmid clones. We utilized a cosmid library representing human chromosome 11q12-11qter and carried out automated restriction enzyme analysis, followed by regional localization to chromosome 11q using high-resolution in situ suppression hybridization. Using this approach, 165 cosmid linking clones containing one or more NotI, BssHII, SfiI, or SacII sites were identified among 960 chromosome-specific cosmids. Furthermore, this analysis allowed clones containing a single site to be distinguished from those containing clusters of two or more rare sites. This analysis demonstrated that more than 75% of cosmids containing a rare restriction site also contained a second rare restriction site, suggesting a high degree of CpG-rich restriction site clustering. Thirty chromosome 11q-specific cosmids containing rare CpG-rich restriction sites were regionally localized by high-resolution fluorescence in situ suppression hybridization, demonstrating that all of the CpG-rich sites detected by this method were located in bands 11q13 and 11q23. In addition, the distribution of (CA)n repetitive sequences was determined by hybridization of the arrayed cosmid library with oligonucleotide probes, confirming a random distribution of microsatellites among CpG-rich cosmid clones. This set of reagent cosmid clones will be useful for physical linking of large restriction fragments detected by pulsed-field gel electrophoresis and will provide a new and highly efficient approach to the construction of a physical map of human chromosome 11q.

Chromosomes, Human, Pair 11↗
PubMed1992

Chromosome 16-specific repetitive DNA sequences that map to chromosomal regions known to undergo breakage/rearrangement in leukemia cells.

Human chromosome 16-specific low-abundance repetitive (CH16LAR) DNA sequences have been identified during the course of constructing a physical map of this chromosome. At least three CH16LAR sequences exist and they are interspersed, in small clusters, over four regions that constitute more than 5% of the chromosome. CH16LAR sequences were observed in one unusually large cosmid contig (number 55), where the ordering of clones was difficult because these sequences led to false overlaps between noncontiguous clones. Contig 55 contains 78 clones, or approximately 2% of all the clones contained within the present cosmid contig physical map. Fluorescent in situ hybridization of multiple clones, including cosmid and YAC contig 55 clones, mapped the four CH16LAR-rich regions to bands p13, p12, p11, and q22. These regions are of biological interest since the pericentric inversion and the interhomologue translocation breakpoints commonly found in acute nonlymphocytic leukemia (ANLL) subtype M4 fall within these bands. Sequence analysis of a 2.2-kb HindIII fragment from a cosmid containing a CH16LAR sequence indicated that one of the CH16LAR elements is similar to a minisatellite sequence in that the core repeat is only 40 bp in length. Additional characterization of other repetitive elements is in progress.

Animals↗
PubMed1992

FRA2B is distinct from inverted telomere repeat arrays at 2q13.

Human chromosome 2 was formed by a telomere-to-telomere fusion of two ancestral ape chromosomes. The fusion point is localized in chromosomal band 2q13, which also contains the rare, folate-sensitive fragile site FRA2B. It has been hypothesized that this fragile site may be related to the presence of interstitial telomeric and subtelomeric sequences, which have come to lie in an inverted repeat arrangement as a result of the fusion event. Fluorescence in situ hybridization of a genomic cosmid c8.1, which spans the fusion point, was carried out on metaphase spreads of an individual who expressed the fragile site at 2q13. We show that the fusion point maps distal to this fragile site. Therefore, we conclude that the inverted arrays of telomeric and subtelomeric sequences found at this fusion point are unlikely to correspond to the rare fragile site at 2q13.

Base Sequence↗
PubMed1992

Rapid physical mapping of cloned DNA on banded mouse chromosomes by fluorescence in situ hybridization.

Physical mapping of DNA clones by nonisotopic in situ hybridization has greatly facilitated the human genome mapping effort. Here we combine a variety of in situ hybridization techniques that make the physical mapping of DNA clones to mouse chromosomes much easier. Hybridization of probes containing the mouse long interspersed repetitive element to metaphase chromosomes produces a Giemsa-like banding pattern which can be used to identify individual Mus musculus, Mus spretus, and Mus castaneus chromosomes. The DNA binding fluorophore, DAPI, gives quinacrine-like bands that can complement the hybridization banding data. Simultaneous hybridization of a differentially labeled clone of interest with the banding probe allows the assignment of a mouse clone to a specific cytogenetic band. These methods were validated by first mapping four known genes, Cpa, Ly-2, Cck, and Igh-6, on banded chromosomes. Twenty-seven additional clones, including twenty anonymous cosmids, were then mapped in a similar fashion. Known marker clones and fractional length measurements can also provide information about chromosome assignment and clone order without the necessity of recognizing banding patterns. Clones hybridizing to each murine chromosome have been identified, thus providing a panel of marker probes to assist in chromosome identification.

Animals↗
PubMed1992

Isolation and initial characterization of a large repeat sequence element specific to mouse chromosome 8.

A clone containing 15.6 kb of mouse genomic DNA was specifically localized to murine chromosome 8 by fluorescence in situ hybridization. The major signal, mapping just below the centromeric heterochromatin, was much too intense for a single-copy probe. Two additional weak hybridization signals were detected in or near distal bands 8B3 and 8D. Six subclones spanning the entire 15.6-kb insert gave strong centromere proximal signals; however, none of these clones cross-hybridized with each other, suggesting that the repeat unit was quite large. Sequence data support this interpretation. An analysis of over 4 kb of sequence, including two subclones in their entirety, did not reveal any common sequence motif. Copy number reconstruction and Southern blotting experiments indicate that between 60 and 80 copies of the sequence (approximately 0.9-1.2 Mb in total) reside on each chromosome 8, most likely organized in a clustered but not tandemly duplicated fashion. Although the probe hybridizes to Mus spretus and Mus castaneus as well as to Mus musculus, it is not detectable in the rat, Chinese hamster, Armenian hamster, or human genomes.

Animals↗