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D C Ward

Publications and source records attributed to D C Ward.

At least 217 records · Page 12Linked to original sources
PubMed1984

Chromosomal and nuclear distribution of the HindIII 1.9-kb human DNA repeat segment.

A human interspersed repetitive DNA cloned in pBR322, the HindIII 1.9-kb (kilobase pair) sequence, was labeled with biotinylated dUTP and hybridized to acid-fixed chromosomes and paraformaldehyde-fixed whole cells in situ. Using our most sensitive detection techniques this probe highlighted on the order of 200 discrete loci, in punctate or banded arrays, that resembled a Giemsa-dark band pattern on chromosome arms. Interphase cells also displayed many discrete punctate spots of hybridization along chromosome fibers. The ubiquitous Alu sequence repeat also appeared to be concentrated in specific regions of the chromosome and predominantly highlighted Giemsa-light bands. Centromeric or ribosomal spacer DNA repeats used as controls in all studies gave the expected hybridization profiles and showed no non-specific labeling of chromosome arms. Cohesive groups of centromeric DNA arrays and rDNA clusters were observed in interphase nuclei. Refinements in methods for detecting biotin-labeled probes in situ were developed during these studies and calculations indicated that about 20 kb or more of the 1.9-kb repeat were present at each hybridization site. The chromosomal distribution of the 1.9-kb repeat suggests that this sequence may reflect, or participate in defining, ordered structural domains along the chromosome.

Base Composition↗
PubMed1984

Widespread presence of histologically occult cytomegalovirus.

Disseminated cytomegalovirus (CMV) has been investigated by in situ hybridization in formalin-fixed paraffin-embedded tissue sections with biotinylated DNA probes. Two cases of disseminated CMV infection were studied at autopsy by this highly specific technique. The presence of CMV in cytomegalic cells is readily shown. In addition, CMV has been detected and localized in many normal-appearing cells. This occult infection occurs in cardiac myocytes, hepatocytes, spleen and lymph node reticular cells, endometrial stromal and glandular cells, and breast stromal cells, as well as in cells in the renal glomerulus, tubule, and interstitium, adrenal cortex and medulla, fallopian tube submucosa, myometrium, and anterior pituitary. Cytomegalovirus infection of endothelial cells has been further documented by immunohistochemical methods utilizing antibody to Factor VIII. These findings suggest that CMV disseminates hematogenously throughout the body, initiating necrotizing foci of infection. The appearance of many diffuse foci suggests that local viral spread occurs via endothelial cell infection. Surprisingly , lymphocyte involvement was not observed.

Adult↗
PubMed1984

Differentiation of Neisseria gonorrhoeae from other Neisseria species by use of the restriction endonuclease HaeIII.

We used the restriction endonuclease HaeIII to differentiate Neisseria gonorrhoeae from other Neisseria species and Branhamella catarrhalis. A total of 16 clinical isolates and four American Type Culture Collection strains of N. gonorrhoeae were resistant to HaeIII digestion, whereas 17 isolates and four American Type Culture Collection strains from eight different bacterial species were susceptible. This resistance was not caused by an enzyme inhibitor. We propose that protection of the HaeIII recognition sequence by methylation is the mechanism of resistance since N. gonorrhoeae DNA became susceptible to digestion when passed in Escherichia coli as part of a plasmid clone.

Base Sequence↗
PubMed1984

Expression of minute virus of mice structural proteins in murine cell lines transformed by bovine papillomavirus-minute virus of mice plasmid chimera.

Recombinant plasmids containing the genomes of both bovine papillomavirus type I and minute virus of mice (MVM) were constructed and used to transform mouse C127 cells. Transformed lines that express MVM gene products with high efficiency were isolated and characterized. These transformants synthesize large amounts of MVM structural polypeptides and spontaneously assemble them into empty virion particles that are released into the culture medium. These lines were, however, genetically unstable; they slowly generated subpopulations that failed to express MVM-specific proteins, and they possessed episomal DNA in which both MVM and bovine papillomavirus sequences were deleted or rearranged, or both. Clonal isolates of these transformants were also superinfectible by infectious MVM virus. Therefore, in spite of their instability, they should be useful host cell lines for transcomplementing mutations introduced into the MVM genome and for growing defective viruses as virions.

Animals↗
PubMed1983

Effect of nucleotide analogs on the cleavage of DNA by the restriction enzymes AluI, DdeI, HinfI, RsaI, and TaqI.

The cleavage of specific DNA sequences by the restriction endonucleases AluI, DdeI, HinfI, RsaI, and TaqI has been studied by monitoring the effect of various nucleotide modifications on the rate of DNA digestion. Bacteriophage fd DNA was completely substituted in one strand with a single nucleotide analog, using an in vitro primed DNA synthesis reaction on a single-stranded viral DNA template. Twelve deoxynucleotide analogs were incorporated into these DNA substrates: 2-aminopurine, 2,6-diaminopurine, deoxytubercidin, deoxyuridine, 5-bromodeoxyuridine, 5-allylamine deoxyuridine, 5-biotinyl deoxyuridine, deoxypseudouridine, deoxyinosine, 8-azadeoxyguanosine, 5-iododeoxycytidine, and 5-bromodeoxycytidine. The restriction enzymes tested varied considerably in their ability to digest hemi-substituted DNAs containing these modified nucleotides. Structural alterations in the base pairs immediately adjacent to the phosphodiester bonds cleaved by the enzyme reduced the rate of enzyme activity most dramatically, and in most cases more than a single determinant on each base pair altered activity. Interactions with nucleotides outside the recognition site seem to have little importance in the binding or catalytic activity of these enzymes.

Base Sequence↗
PubMed1983

Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes.

A method of in situ cytohybridization is described for the detection of specific viral genomes in infected cell cultures or paraffin-embedded tissue sections without the use of radioisotopes. Biotin-labeled analogs of TTP are incorporated into viral DNA in vitro by nick translation and the resultant DNA probes hybridized to cytologic samples. Cells containing viral genetic material are then revealed by standard immunofluorescence, immunoperoxidase, or affinity cytochemical techniques that are based on the specific interaction between biotin and antibiotin IgG or avidin. Hybridization probes containing nucleotides that have an 11- or 16-atom spacer arm between the biotin molecule and the pyrimidine ring interact with these detector proteins more efficiently than probes containing biotin-nucleotides with a 4-atom spacer arm. The total procedure can be performed fairly rapidly (24 hr or less) and numerous samples can be processed simultaneously. Although the detection methods employed to date are not as sensitive as autoradiographic procedures with high specific activity probes, more sensitive protein detector complexes are currently being constructed. The speed, specificity, and resolving power of this technique should be of general utility in screening for the presence of infectious agents in cell or tissue samples. Here we report the visualization of parvovirus, polyomavirus, herpes simplex virus, adenovirus, and retrovirus genetic material in infected cell cultures and herpes simplex and adenovirus DNA in paraffin-embedded autopsy tissues.

Adenoviruses, Human↗
PubMed1983

The genome of minute virus of mice, an autonomous parvovirus, encodes two overlapping transcription units.

Four virus-specific transcripts have been identified in murine cells infected with Minute-Virus-of-Mice (MVM). These RNAs, 4.8, 3.3, 3.0 and 1.8 kilobases in length, designated R1 to R4 respectively, are all transcribed from the virion (-) strand of DNA and they are all polyadenylated and spliced. The R1 transcript is derived from sequences that reside on the genome between 4.0 and 95 map units (mu). Transcript R2 is composed of exon sequences derived from mu coordinates 4.0-10.0, 40-46 and 48-95. The most abundant RNA, R3, is transcribed from sequences mapping between 40 and 95 mu. All three of these RNAs have a short intron sequence between 46-48 mu removed. The least abundant transcript, R4, has not been mapped precisely, however it hybridizes with all three EcoRI fragments which span the entire 5 kb genome. In vitro transcription of cloned restriction fragments of MVM DNA confirm the existence of functional promoters at map coordinates 4.0 and 39 and sequence analysis of these regions of the viral DNA reveal the characteristic features of RNA polymerase II promoters. These results indicate that MVM DNA encodes two overlapping transcription units with separate promoters near the left end (4.0 mu) and middle (39 mu) of the genome.

Animals↗
PubMed1983

The complete DNA sequence of minute virus of mice, an autonomous parvovirus.

We have determined the complete nucleotide sequence of the genome of Minute Virus of Mice, an autonomous parvovirus. This single-stranded DNA is 5081 nucleotides long. The 3'-and 5'-ends of the viral strand contain imperfect palindromic sequences which consist, respectively, of 115 and 206 nucleotides. The 3'-terminal palindrome is composed of a unique sequence, whereas the 5'-terminal palindrome contains two sequences in equimolar amounts; these are related in that one is the inverted complement of the other. The DNA strand complementary to that which is encapsidated into virions contains two large open reading frames which together span almost the entire genome. Transcriptional and translational signals within the sequence have been identified and related to the known map coordinates of the viral transcripts. In this report we summarize some of the salient structural and organizational features of the MVM genome.

Amino Acid Sequence↗
PubMed1983

Early results of subcutaneous mastectomy with immediate silicone prosthetic implant for carcinoma of the breast.

Surgery for carcinoma of the breast is becoming less radical even to the extent of breast conservation. Subcutaneous mastectomy and implant offers itself as an alternative, retaining a breast shape and removing almost all breast tissue. Forty-four consecutive patients who had this procedure for breast carcinoma were studied with a follow-up of 5-44 months (mean 25.3 months). Only 4 patients (9 per cent) had local recurrences, i.e. recurrence in the skin flap or scar. This compares favourably with other series and other forms of treatment. Removal of the prosthesis was necessary in 10 patients due to local factors. The cosmetic result as assessed by the patients was almost universally pleasing. Twenty-six of 27 patients with prostheses in situ who were interviewed in person or by postal questionnaire were pleased with the results of their surgery. The absence of high recurrence rates locally and high patient acceptance makes this a definite surgical alternative.

Adult↗
PubMed1983

Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose: Bio-blots.

Biotin-labelled DNA probes, prepared by nick-translation in the presence of biotinylated analogs of TTP, are hybridized to DNA or RNA immobilized on nitrocellulose filters. After removal of residual probe, the filters are incubated for 2--5 min with a preformed complex made with avidin-DH (or streptavidin) and biotinylated polymers of intestinal alkaline phosphatase. The filters are then incubated with a mixture of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium, which results in the deposition of a purple precipitate at the sites of hybridization. This procedure will detect target sequences in the 1- to 10-pg range after enzyme incubation periods of 1 hr or less. The incubation period can be extended up to 24 hr, if required, to increase the color intensity of the hybridization signal. Furthermore, at high probe concentrations (250--7560 ng/ml), biotin-labeled DNA exhibits lower nonspecific binding to nitrocellulose than does radiolabeled DNA, so hybridization times required for the analysis of unique mammalian gene sequences can be decreased to 1--2 hr. This nonradiographic method of probe detection should be of general utility for genetic studies using Southern, RNA, or dot-blot hybridization protocols.

Alkaline Phosphatase↗
PubMed1983

Construction of an infectious molecular clone of the autonomous parvovirus minute virus of mice.

The linear single-stranded DNA genome of minute virus of mice, an autonomous parvovirus, was cloned in duplex form into the bacterial plasmid pBR322. The recombinant clones of minute virus of mice were infectious when transfected into monolayers of human 324K cells and produced virus plaques with an efficiency of about 6% that obtained with duplex replicative-form DNA purified from cells infected with minute virus of mice. Southern blot analysis of transfected cells indicated that the cloned minute virus of mice genome requires both termini to be intact for excision and replication as a linear duplex molecule.

Cells, Cultured↗
PubMed1983

Proteins tightly bound to HeLa cell DNA at nuclear matrix attachment sites.

DNA-protein complexes have been isolated from HeLa cell nuclei and nuclear matrix preparations. Two proteins, 55 and 66 kilodaltons in size, remain bound to HeLa DNA after treatment at 80 degrees C in 2% sodium dodecyl sulfate and purification by exclusion chromatography on Sepharose 2B-CL in the presence of 0.3% sodium dodecyl sulfate. These proteins appear to be tightly bound but not covalently linked to the DNA, and they are distributed over the DNA with an average spacing of 40 kilobase pairs. This spacing distribution remains essentially constant throughout the cell cycle. The proteins are bound to the residual 2% of HeLa cell DNA which remains attached to the nuclear matrix after extensive nuclease digestion, a condition which reduces the average size of the DNA to approximately 150 base pairs. Our results suggest that these tightly bound proteins are involved in anchoring cellular DNA to the nuclear matrix. These tightly bound proteins are identical by partial peptide mapping to proteins found tightly bound to the DNA of mammalian, plant, and bacterial cells (D. Werner and C. Petzelt, J. Mol. Biol. 150:297-302, 1981), implying that these proteins are involved in the organization of chromosomal domains and are highly conserved in both procaryotic and eucaryotic cells.

Binding Sites↗
PubMed1982

Effect of UV-irradiation on DNA replication of the parvovirus minute-virus-of-mice in mouse fibroblasts.

The effect of UV-irradiation on the conversion of the single-stranded DNA of the parvovirus Minute-Virus-of-Mice (MVM) to duplex Replicative Forms (RF) was studied after infection of mouse A9 fibroblasts. UV-irradiation of the virus prior to infection of unirradiated cells resulted in a dose-dependent, single-hit, inhibition of RF formation. Restriction fragment analysis indicated that this inhibition could be ascribed to the introduction of absolute blocks which prevent elongation of the newly synthesized complementary strand. Cell exposure to UV-light prior to infection with UV-irradiated MVM enhanced the fraction of input viral DNA which was converted to RF. This enhancement required de novo protein synthesis during the interval between cell irradiation and virus infection. These results suggest that DNA replication constitutes a target in the viral life cycle that leads to the UV-enhanced Reactivation of virus survival, however, they do not permit us to identify the step of RF formation which is enhanced in UV-pretreated cells.

Animals↗
PubMed1982

Immunological method for mapping genes on Drosophila polytene chromosomes.

A method is described for localizing DNA sequences hybridized in situ to Drosophila polytene chromosomes. This procedure utilizes a biotin-labeled analog of TTP that can be incorporated enzymatically into DNA probes by nick-translation. After hybridization in situ, the biotin molecules in the probe serve as antigens which bind affinity-purified rabbit antibiotin antibodies. The site of hybridization is then detected either fluorimetrically, by using fluorescein-labeled goat anti-rabbit IgG, or cytochemically, by using an anti-rabbit IgG antibody conjugated to horseradish peroxidase. When combined with Giemsa staining, the immunoperoxidase detection method provides a permanent record that is suitable for detailed cytogenetic analysis. This immunological approach offers four advantages over conventional autoradiographic procedures for detecting in situ hybrids: (i) the time required to determine the site of hybridization is decreased markedly, (ii) biotin-labeled probes are chemically stable and give reproducible results for many months; (iii) biotin-labeled probes appear to produce less background noise than do radiolabeled probes; and (iv) the resolving power is equal to and often greater than that achieved autoradiographically.

Animals↗
PubMed1982

Actin gene expression visualized in chicken muscle tissue culture by using in situ hybridization with a biotinated nucleotide analog.

The chicken muscle tissue culture system has been used for visualizing actin gene expression after in situ hybridization. Cell differentiation is morphologically distinguishable in this system as the myoblasts fuse into myotubes. This differentiation involves the production of large amounts of actin required for myofibrils. The presence of actin mRNA has been observed in cells preserved with ethanol and paraformaldehyde by hybridizing a recombinant plasmid into which a biotinated analog of dUTP was incorporated by nick-translation. The biotin was then detected by using an anti-biotin antibody and a rhodamine-conjugated second antibody. Alternatively, avidin conjugated to rhodamine or avidin complexed to biotinated peroxidase has been used for mRNA detection. The procedure described preserved morphological detail yet is compatible with hybridization conditions and reveals the disposition of actin mRNA during gene expression.

Actins↗
PubMed1982

31P nuclear magnetic resonance evidence for the regulation of intracellular pH by Ehrlich ascites tumor cells.

The phenomenon of intracellular pH (pHin) regulation in cultured Ehrlich ascites cells was investigated using 31P nuclear magnetic resonance (NMR) spectroscopy. Measurements were made with a Bruker WH 360 wide bore NMR spectrometer at a 31P frequency of 145.78 MHz. Samples at a density of 10(8) cells ml-1 were suspended in a final volume of 2 ml of growth medium in 10 mm diameter NMR tubes. Intracellular pH was calculated from the chemical shifts of either intracellular inorganic phosphate (Piin) or intracellular 2-deoxyglucose-6-phosphate (2dG6Pin). The sugar phosphate was used as a pH probe to supplement the Piin measurements, which could not always be observed. When available, the pHin calculated from the Piin peak was identical within experimental error to the pHin calculated from the 2dG6Pin peak. Intracellular pH was measured to be more alkaline than the medium at an external pH (pHex) below 7.1. Typical values were pHin = 7.00 for pHex = 6.50. These measurements were constant for times up to 165 min using well-energized, respiring cells. This pH gradient was seen to collapse immediately upon onset of anaerobic shock. Above a pHex of 7.2 there was no significant difference between pHin and pHex. These results unequivocally demonstrate the steady state nature of the pH regulation and its dependence upon energization.

Animals↗
PubMed1982

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold.

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.

Animals↗