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R Hand

Publications and source records attributed to R Hand.

At least 55 records · Page 3Linked to original sources
PubMed1980

Decreased initiation of DNA synthesis in a temperature-sensitive mutant of hamster cells.

We have analyzed ongoing DNA replication in ts BN-2, a dna- mutant of BHK-21 cells (Nishimoto et al. '78). At the non-permissive temperature of 39.5 degrees C, inhibition of 3H-thymidine into acid-precipitable material begins 1 to 2 h after the cells are released from a block at the start of the S-phase. The fraction of nuclei incorporating 3H-thymidine is similar to that of wild-type cells through the synchronized S-phase of 8 h. Alkaline sucrose gradient analysis shows that pulse-labeled DNA from mutant cells is incorporated into high molecular weight material after 3 h at either the permissive or non-permissive temperature. DNA fiber autoradiograms reveal that, at 39.5 degrees C, the rate of replication fork movement is about 30% increased in the mutant as compared to wild-type cells. In the mutant cells, however, the interval between adjacent initiation sites is increased and the relative frequency of initiation events is decreased at the restrictive temperature. The results indicate that there is a block to ongoing replication in ts BN-2 at the level of initiation of synthesis on individual replication units; elongation of nascent chains is not inhibited.

Animals↗
PubMed1980

Inhibition of DNA chain elongation in a purine-auxotrophic mutant of Chinese hamster.

DNA synthesis has been examined in a purine-auxotrophic mutant cell line of Chinese hamster (V79 pur 1) under conditions of purine deprivation. At 6 h after the removal of purines from the growth medium, there is a decrease in semiconservative DNA replication. Alkaline velocity centrifugation of the DNA synthesized during a 1-min pulse under conditions of purine deprivation shows that approximately 50% of the newly replicated DNA is the size of Okazaki pieces. These are not incorporated into bulk DNA during a 1-h chase. If the purine supply is restored to the growth medium, these short DNA pieces are jointed to full-sized DNA within 1 h. DNA fiber autoradiolgraphy reveals a retardation in the rate of DNA replication fork movement but no apparent inhibition of initiation of synthesis on replication units within clusters actively engaged in replication. Our results indicate that purine deprivation specifically inhibits elongation of nascent dna chains.

Animals↗
PubMed1979

DNA synthesis: evaluation of a hydrodynamic method for measuring the rate of replication fork movement.

When replicating DNA is labeled sequentially with radioactive and density tracers and analyzed by equilibrium centrifugation, the fraction banding at heavier than normal density is inversely proportional to the rate of replication fork movement if there is a sharp transition from one tracer to the other on the newly synthesized chains (Painter and Schaefer, '69). Primate CV-1 DNA labeled for 5 to 30 minutes with 3H-dThd and then for three hours with BrdUrd in the presence of FdUrd bands in a bimodal distribution in alkaline CsCl, rather than in a continuous distribution with a skew toward heavier density seen when FdUrd is omitted and centrifugation is in neutral CsCl. The heavy density peak represents interspersion of both tracers in the DNA and is caused by slow transition from dThd to BrdUrd incorporation when the tracers are switched in the labeling medium. This may result from preferential uptake and incorporation of dThd over BrdUrd. Because of the interspersion, calculation of the rate of replication fork movement is inaccurate. Reversal of the labeling sequence with administration of the long density pulse before the radioactive pulse reduces the problem of interspersion. Using this sequence of labeling, estimates of the rate of fork movement of 0.36-0.38 micrometer/min are obtained when the 3H pulse time is long enough to allow accurate measurement of the fraction of heavy DNA. Analysis by fiber autoradiography yields a rate of 0.56 micrometer/min in the same cell line. If appropriate precautions are taken to minimize mixing of the two tracers in the precursor pool and to ensure that the fraction of heavy DNA is measured accurately, the hydrodynamic technique provides an objective method of measuring rate of fork movement that gives values only slightly lower than those obtained by autoradiography.

Animals↗
PubMed1979

Protein kinase activity associated with the D2 hybrid protein related to simian virus 40 T antigen: some characteristics of the reaction products.

Protein kinase activity (ATP:protein phosphotransferase, EC 2.7.1.37) has been found associated with the D2 hybrid protein, a highly purified protein of 107,000 daltons specified by the adenovirus-simian virus 40 (SV40) hybrid Ad2(+)D2, which has many properties associated with authentic SV40 T antigen [Tjian, R. & Robbins, A. (1979) Proc. Natl. Acad. Sci. USA 76, 610-614]. We have now examined some of the biochemical characteristics of the reaction products. Acceptors for the terminal phosphoryl group of [gamma-(32)P]ATP are the purified protein itself and at least four proteins extracted from nuclei of uninfected cells. Purified histones do not serve as substrate for the enzyme. Phosphorylation is markedly reduced by heating the D2 hybrid protein to 50 degrees C for 30 min. The products of phosphorylation are stable to treatment with ethanol/ether, DNase, and RNase, but completely degraded by digestion with Pronase, demonstrating their protein nature. The phosphate bonds are liable to hot alkali and sensitive to digestion with alkaline phosphatase but stable to treatment with hot acid or hydroxylamine. These results provide evidence that (32)P is incorporated into O-phosphoserine or O-phosphothreonine residues of acceptor proteins, indicating that the enzymatic activity is characteristic for protein kinase, and that cell-specified nuclear proteins other than histones may serve as substrates for the enzyme.

Adenoviruses, Human↗
PubMed1979

Replication of mammalian DNA in vitro. Evidence for initiation from fiber autoradiography.

We have used fiber autoradiography to examine the DNA product made in vitro in a lysed cell system. CHO cells were treated with 0.01% Brij-58 and the lysates were incubated at 30 degrees C in a complete reaction mixture for in vitro DNA synthesis with [3H]thymidine triphosphate ([3H]TTP) as the radioactive tracer. Fiber autoradiograms prepared from the DNA showed that it was synthesized on tandemly arranged replication units that were of average size of 20 micrometers, very similar to the size of units found in vivo. The rate of replication fork movement was 25--50% of the in vivo rate. More than 80% of forks stopped functioning by 15 min, and 95% stopped by 60 min. This suggests that synthesis is halted by premature terminations. Evidence for new initiations was provided by replication units with labeled origins in DNA synthesized in an in vitro reaction in which radioactivity was omitted for the first 10 min of incubation. This, plus the observations that the distance between initiation points (replication unit size) is not increased and that premature termination accounts largely for the cessation of synthesis, suggest that significant initiation takes place in this in vitro replication system.

Animals↗
PubMed1978

DNA and histone synthesis in reovirus-infected cells.

Reovirus infection inhibits the incorporation of 3H-thymidine into cellular DNA. We have now investigated several aspects of this inhibition in L-929 cells early (8 h) after infection at high multiplicity (200 to 250 p.f.u./cell). Using equilibrium sedimentation analysis of DNA sequentially labelled with density and radioactive analogues of thymidine, we find a 52% reduction in the amount of DNA synthesized with no change in rate of replication fork movement in infected cells. Gel electrophoresis of histones labelled with 3H-lysine shows that infection inhibits their synthesis by 76% several hours before overall cellular protein synthesis is inhibited. There is also a reduction of nearly 50% in the size of the thymidine triphosphate pool, as measured by enzymic assay. The proportion of exogenous nucleotide in the pool is the same as in uninfected cells since there is no change in the buoyant density of DNA labelled during a short pulse with 3H-bromodeoxyuridine. The uptake of thymidine is reduced, but its phosphorylation to thymidine triphosphate is normal. The findings provide direct evidence that DNA synthesis is inhibited early in infection. This inhibition is accompanied by other derangements of thymidine and chromatin metabolism suggesting that there is an early and specific attack by reovirus on nuclear function in infected cells.

DNA↗
PubMed1977

Bloom's syndrome: DNA replication in cultured fibroblasts and lymphocytes.

Analysis of DNA fiber autoradiograms from Bloom's syndrome skin fibroblasts and blood lymphocytes shows a retarded rate of replication fork movement compared to normal adult controls. Other measurements from the autoradiograms--replication unit length, incidence of bidirectional replication, and degree of initiation synchrony--are normal in Bloom's syndrome cells. These results suggest that a slow rate of fork movement is a specific manifestation of defective DNA synthesis in all Bloom's syndrome cells.

Abnormalities, Multiple↗
PubMed1977

Human DNA replication: fiber autoradiographic analysis of diploid cells from normal adults and from Fanconi's anemia and ataxia telangiectasia.

Fiber autoradiograms prepared from radioactive DNA of diploid skin fibroblasts and lymphocytes from normal adult humans show patterns of replication of chromosomal DNA that are closely similar to those from other mammalian cells. The rate of replication fork movement is 0.64 micrometer/min and 0.42 micrometer/min for fibroblasts and lymphocytes respectively. In both types of cells, replication units show a median length of about 70 micrometer. Replication proceeds bidirectionally from a central origin in most units and adjacents units initiate synthesis synchronously. In skin fibroblasts from individuals with Fanconi's anemia or ataxia telangiectasia, the autoradiographic patterns of DNA replication are the same as in controls. This suggests that S phase DNA synthesis is normal in these disorders.

Anemia, Aplastic↗
PubMed1977

Hospital-acquired bacterial meningitis in neurosurgical patients.

The authors review 23 cases of hospital-acquired meningitis occurring over a 15 year period in neurosurgical patients. Factors associated with the development of meningitis include recent craniotomy, cerebrospinal fluid leak, the presence of ventricular or lumbar drainage tubes, and skull fracture. Four cases were caused by Staphylococcus epidermidis; one of these patients died. In 19 cases, Gram-negative enteric bacteria were the etiologic agents, most commonly members of the Klebsiella-Enterobacter-Serratia group. Eleven of these patients died. The particular antibiotic or group of antibiotics used and the route of administration made no difference in the outcome of Gram-negative bacillary meningitis.

Adolescent↗
PubMed1976

Action of dichlorobenzimidazole riboside on RNA synthesis in L-929 and HeLa cells.

5,6-Dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) inhibits RNA synthesis in L-929 cells (mouse fibroblast line) and HeLa cells (human epitheloid carcinoma line) within 2 min of addition of the compound to the medium. By removing DRB from the medium, the inhibition is promptly and completely reversed after treatment of cells for as long as 1 h or even longer. The inhibitory effect of DRB on the overall rate of RNA synthesis is similar in L and HeLa cells and is markedly concentration-dependent in the low dose range (5-20 muM or 1.6-6.4 mug/ml), but not as higher concentrations of DRB. At a concentration of 12 muM, DRB has a highly selective inhibitory effect on the synthesis of nuclear heterogenous RNA in L cells. At higher concentrations, there is also inhibition of 45 S ribosomal precursor RNA synthesis, but at all concentrations the effect on heterogeneous RNA synthesis in L cells in considerably greater than that on preribosomal RNA synthesis. In HeLa cells, too, DRB has a selective effect on heterogeneous RNA synthesis, but quantitatively the selectivity of action is somewhat less pronounced. In both L and HeLa cells, the inhibition of synthesis of nuclear heterogeneous RNA is incomplete even at very high concentrations of DRB (150 muM). Thus, while DRB is a selective inhibitor of nuclear heterogeneous RNA synthesis, not all such RNA synthesis is sensitive to inhibition. It is proposed that messenger precursor RNA synthesis may largely be sensitive to inhibition by DRB. In short-term experiments, DRB has no effect on protein synthesis in L or HeLa cells. DRB has a slight to moderate inhibitory effect on uridine uptake into L cells and a moderate to marked effect on uptake of uridine into HeLa cells.

Benzimidazoles↗