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D C Ward

Publications and source records attributed to D C Ward.

At least 181 records · Page 10Linked to original sources
PubMed1990

Selective enrichment of a large size genomic DNA fragment by affinity capture: an approach for genome mapping.

A method to enrich large size DNA fragments obtained by digestion with rare cutting restriction endonucleases was developed and applied for the isolation of a 150 kb SfiI fragment containing the beta-globin gene cluster. The digested DNA is rendered single stranded at the ends by diffusing a strand specific exonuclease into an agarose plug containing DNA. The plug is melted and solution hybridization is then performed with a bridge RNA containing specific sequences from the end of a desired fragment linked to a common probe sequence. The common probe sequence is annealed to a biotinylated RNA and the resulting tripartite hybrid is retained onto a solid matrix containing avidin and specifically released by ribonuclease action. Enrichments of greater than 350 fold have been achieved consistently. Such directed purification of large DNA fragments without cloning can considerably expedite mapping and gene localization in a complex genome and facilitate the construction of sublibraries from defined regions of the genome.

Base Sequence↗
PubMed1990

High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones.

Cosmid clones containing human DNA inserts have been mapped on chromosome 11 by fluorescence in situ hybridization under conditions that suppress signal from repetitive DNA sequences. Thirteen known genes, one chromosome 11-specific DNA repeat, and 36 random clones were analyzed. High-resolution mapping was facilitated by using digital imaging microscopy and by analyzing extended (prometaphase) chromosomes. The map coordinates established by in situ hybridization showed a one to one correspondence with those determined by Southern (DNA) blot analysis of hybrid cell lines containing fragments of chromosome 11. Furthermore, by hybridizing three or more cosmids simultaneously, gene order on the chromosome could be established unequivocally. These results demonstrate the feasibility of rapidly producing high-resolution maps of human chromosomes by in situ hybridization.

Blotting, Southern↗
PubMed1990

Preembryo biopsy and analysis of blastomeres by in situ hybridization.

We developed a method for the biopsy of preimplantation mouse embryos (preembryos) at the four- to eight-cell stage, which uses partial zona pellucida dissection. The preembryos were collected in calcium- and magnesium-free phosphate-buffered saline solution with 0.01% ethylenediaminetetraacetic acid, 0.1 mol/L sucrose, and 4 mg/ml of bovine serum albumin to facilitate removal of blastomeres. This allows entry of a fine micropipette into the perivitelline cavity with subsequent removal of a single blastomere by gentle suction. The majority of embryos (75%) from which biopsy specimens were obtained in this fashion developed to the blastocyst stage. The blastomeres obtained were mainly intact and they were fixed to glass slides. After permeabilization, in situ hybridization was performed with chromosome X- and chromosome 3-specific probes. Human unfertilized eggs and blastomeres from human polyspermic embryos also have been analyzed by in situ hybridization with chromosome specific probes. The combination of nondestructive embryo biopsy and in situ hybridization is a possible approach for preimplantation genetic diagnosis.

Animals↗
PubMed1990

Genetic and physical mapping and population studies of a fibronectin receptor beta-subunit-like sequence on human chromosome 19.

A cDNA clone of the beta subunit of human fibronectin receptor (FNRB) detects two different polymorphic loci: (a) a codominant system previously mapped to the pericentromeric region of chromosome 10, the site of the functional FNRB gene; and (b) a dominant system not linked to the first one or to any chromosome 10 marker tested. This second polymorphism is characterized by the presence or absence of a band (or a set of bands). We have used linkage analysis and biotin-labeled in situ hybridization to map this dominant polymorphism to the short arm of chromosome 19; we hypothesize that it may be due to the insertion of part of the cDNA from the chromosome 10 gene into chromosome 19. This "insertion" is polymorphic in all populations studied.

Chromosome Mapping↗
PubMed1990

Rapid analysis of mouse-hamster hybrid cell lines by in situ hybridization.

In situ hybridization techniques for analyzing the murine DNA complement of mouse-hamster hybrid cells are described. Total genomic mouse DNA is labeled with biotin and hybridized without suppression to metaphase spreads from a mouse-hamster hybrid line containing the mouse fusion chromosome X12. Detection via fluorochrome-conjugated avidin reveals mouse chromosomal DNA with high sensitivity and permits the identification of both normal and aberrant murine chromosomes. Conversely, biotinylated total genomic DNA from a hybrid line can be used as a probe on normal mouse metaphase spreads if suppression techniques are employed, facilitating the analysis of mouse chromosomes present in the hybrid line.

Animals↗
PubMed1990

Fluorescence in situ hybridization with Alu and L1 polymerase chain reaction probes for rapid characterization of human chromosomes in hybrid cell lines.

Human-rodent hybrid cell lines have been analyzed with regard to their human DNA content by using various DNA probe sets, derived from the hybrids, for in situ hybridization to normal human metaphase chromosome spreads. Total genomic hybrid DNA was compared with probe sets of hybrid DNA that were highly enriched in human sequences. The latter probes were obtained by amplification through the polymerase chain reaction (PCR) using oligonucleotide primers directed to human specific subsequences of the interspersed repetitive sequences Alu and L1. Previously unidentified chromosomal material within hybrid lines was characterized with speed and precision. It is demonstrated that the complete human complement of hybrid lines can be rapidly assessed by comparing the data obtained with the Alu-PCR products with the results from the L1-PCR products or from the genomic hybrid DNA. This approach using interspersed repetitive sequence-PCR products is simple and fast and also provides an alternative way of generating complex DNA probe sets for the specific delineation of entire chromosomes or subchromosomal regions by in situ hybridization.

Animals↗
PubMed1990

Differential distribution of long and short interspersed element sequences in the mouse genome: chromosome karyotyping by fluorescence in situ hybridization.

Fluorescence in situ hybridization has been used to demonstrate the differential distribution of interspersed repetitive elements in the genome of Mus musculus domesticus. Hybridization with a mouse long interspersed element sequence results in a sharp, highly reproducible banding pattern on metaphase chromosomes, which is quite similar to Giemsa banding for all chromosomes except 7 and X. The families of short interspersed elements, B1 and B2, preferentially cluster in the R, or reverse, bands. There is no evidence of any interspersed repeat present in the centromeric heterochromatic regions. Both the long interspersed element and B2 probes give banding patterns suitable for karyotype analysis. Simultaneous hybridization of the biotinylated long interspersed element probe and a digoxigenin-labeled cosmid to metaphase spreads allows rapid localization of a probe of interest to a particular cytogenetic band on a chromosome.

Animals↗
PubMed1990

Positive and negative regulation of the minute virus of mice P38 promoter.

5' deletion mutants of the minute virus of mice P38 promoter were constructed and analyzed for transcriptional activity in vitro and in vivo. In uninfected mouse A9 cell extracts, 107 base pairs upstream of the RNA start sites are required for optimal activity. Within this region, the only readily recognizable cis-acting control elements are a GC box and a TATA box. However, in infected cell extracts, deletion of a sequence between -167 and -121, which shares homology with the 30-base-pair trans-activation region (TAR) of H-1 virus (S. L. Rhode and S. M. Richard, J. Virol. 61:2807-2815, 1987), results in a three- to fourfold decrease in transcriptional activity. Interestingly, in vivo transfection experiments demonstrate a three- to eightfold increase in transcription relative to the wild-type promoter when the TAR element homology region is deleted and reveal a functional role for a CCAAT motif which lies immediately downstream of the TAR element. These results indicate both positive and negative regulation of the P38 promoter.

Animals↗
PubMed1989

Comparison of histopathological and flow cytometric parameters in prediction of prognosis in gastric cancer.

Flow cytometric analysis was performed retrospectively on material from 76 patients having potentially curative resection for gastric carcinoma between 1968 and 1984. The prognostic significance of DNA aneuploidy was compared with that of conventional histological grading and staging of the tumour. The presence of DNA aneuploidy was associated with a significantly poorer prognosis when compared with diploid tumours (P less than 0.02), but was not found to be predictive of survival when the presence of lymph node metastases (P less than 0.0001) and resection margin involvement (P less than 0.003) were allowed for using multiple regression analysis. When intestinal and diffuse types of gastric carcinoma were analysed separately, DNA aneuploidy was associated with a significantly shorter survival only in patients with intestinal type tumours.

Adult↗
PubMed1989

c-src and other proto-oncogenes implicated in neuronal differentiation.

The proto-oncogene c-src has been implicated in the development and mature function of the nervous system. pp60c-src, the protein product of the c-src gene, is a tyrosine protein kinase that is highly enriched in fetal neural tissue. pp60c-src appears in two phases of neuronal development. Neuroectodermal cells of gastrulating embryos first express pp60c-src around the time of commitment to neuronal or glial pathways. Later, committed neuroepithelial cells express pp60c-src near the onset of terminal neuronal differentiation. Immunocytochemical analyses of pp60c-src in developing chick retina, telencephalon, and cerebellum show immunoreactivity concentrated in regions rich in growth cones and neurites. Moreover, pp60c-src is concentrated approximately 10-fold in a biochemical fraction from fetal rat brain that is enriched in nerve growth cone membranes. These results point toward a function for pp60c-src in neurite outgrowth. A functional role for other proto-oncogenes in the development of the nervous system was indicated from a study of the expression of a battery of proto-oncogenes during the retinoic acid-induced differentiation of the mouse embryonal carcinoma cell line P19 to a neuronal phenotype. Nuclear runoff transcription of the proto-oncogenes c-src, c-fms, c-sis, N-ras, c-myc, and c-fos was observed in proliferating and retinoic acid-treated cells.

Animals↗
PubMed1989

The terminal regions of adenovirus and minute virus of mice DNAs are preferentially associated with the nuclear matrix in infected cells.

The interaction of viral genomes with the cellular nuclear matrix was studied by using adenovirus-infected HeLa cells and minute virus of mice (MVM)-infected A-9 cells. Adenovirus DNA was associated with the nuclear matrix both early and late in infection, the tightest interaction being with DNA fragments that contain the covalently bound 5'-terminal protein. Replicative forms of MVM DNA were also found to be exclusively matrix associated during the first 16 to 20 h of infection; at later times viral DNA species accumulated in the soluble nuclear fraction at different rates, suggesting a saturation of nuclear matrix-binding sites. MVM DNA fragments enriched in the matrix fraction were also derived from the terminal regions of the viral genome. However, only the subset of fragments which possess a covalently bound 5'-terminal protein (i.e., DNA fragments in which the 5' palindromic DNA sequences are in the extended duplex rather than the hairpin conformation) were matrix associated. These observations suggest that the DNA-matrix interactions are, at least in part, mediated by the viral terminal proteins. Since these proteins have previously been shown to be intimately involved in viral DNA replication, our results further indicate that an association with the nuclear matrix may be important for viral genome replication and possibly also for efficient gene transcription.

Adenoviruses, Human↗
PubMed1989

Transcriptional analysis of minute virus of mice P4 promoter mutants.

A series of 5' deletion, internal deletion, and linker-scanning mutants of the minute virus of mice P4 promoter were constructed and analyzed for transcriptional activity in nuclear extracts of mouse A92L fibroblasts. A GC box and a TATA box essential for in vitro transcription from the P4 promoter were localized between nucleotides 150 and 180 (-55 to -25 relative to the primary RNA start site). Although this region also exhibited homologies to other transcriptional control elements, the simian virus 40 enhancer, and the adenovirus E1A enhancer, only the GC box and TATA box appear functional. These two motifs also play an essential role in vivo, although additional upstream sequences (between -139 and -55) are required for optimal transcription. DNase I footprinting, competitive gel retardation assays, and UV-photocrosslinking were used to identify Sp1-like proteins of 95 and 120 kilodaltons in A92L extracts that interact with the GC box of the minute virus of mice P4 promoter.

Base Sequence↗
PubMed1989

Detection of La Crosse and snowshoe hare viral nucleic acids by in situ hybridization.

A molecular hybridization technique was developed to detect bunyavirus RNA in cells. Complementary DNAs (cDNAs) to the small (S) RNA segment of La Crosse (LAC) virus and to a portion of the middle (M) RNA segment of snowshoe hare (SSH) virus were used as probes to detect LAC or SSH viral RNA by in situ hybridization. Protocols were developed and standardized using radiolabeled DNA probes, and adapted for use with biotin labeled probes. The in situ hybridization procedure detected an estimated 3,600 copies of viral S RNA/cell at 24 hr postinfection. In growth curve studies, LAC nucleocapsid antigen was detectable slightly before S RNA. LAC S RNA synthesis was first seen about the nucleus. By 12 hr postinfection, hybridization signal was detected throughout the cytoplasm of the cell. The LAC S RNA probe was group-specific and cross-hybridized to 5 other California group viruses. The SSH M RNA probe was type-specific.

Animals↗