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Biomedical subjects

R M Watson

Publications and source records attributed to R M Watson.

At least 127 records · Page 7Linked to original sources
PubMed1977

Mapping of closed circular DNAs by cleavage with restriction endonucleases and calibration by agarose gel electrophoresis.

The cleavage of DNA by restriction endonucleases can be limited by the addition of ethidium bromide. When closed circular DNA is used as a substrate, DNA with one-site cleavages of one or both strands can be made by adding appropriate amounts of dye. The singly cleaved DNA is a complete set of full-length permuted linear molecules. Fractionation of the products of a digestion of the permuted linears with a single-hitting restriction endonuclease by gel electrophoresis yields a series of bands that can be used to determine relative molecular weights of the DNA fragments in the gel without the introduction of standards. It is possible to determine the relative molecular weight of a fragment to within +/-2.5%. These molecular weights immediately allow the determination of the HindIII and Hpa I maps of simian virus 40. The HindIII map of bacteriophage PM2 was determined by this method with one ambiguity that was resolved by using traditional techniques.

Bacteriophages↗
PubMed1977

Detection of a contaminant cell culture line by restriction endonuclease cleavage patterns of mitochondrial DNA.

A putative HeLa cell culture line was discovered to be contaminated with mouse cells by examination of agarose gel profiles of restriction endonuclease digests of mitochondrial DNA. The contamination was confirmed by karyotypic analysis, and by observation of the mouse satellite band in an analytical buoyant density centrifugation of total cellular DNA. Restriction endonuclease analysis of mitochondrial DNA is suggested as a useful method for monitoring the species of cells in culture.

Animals↗
PubMed1977

Restriction endonuclease cleavage maps of rat and mouse mitochondrial DNAs.

Mitochondrial DNA from an Old World mouse, Mus musculus, and from an Old World rat, Rattus norvegicus, contain 19 and 22 distinct sites, respectively, for the 8 restriction endonucleases, BamHI, EcoRI, HaeII, HhaI, HincII, HindIII, HpaI and PstI. The relative positions of the sites have been mapped by the study of partial and double enzyme digests. Some sites may been conserverd between the mouse and rat mitochondrial genomes.

Animals↗
PubMed1976

The structures and fidelity of replication of mouse mitochondrial DNA-pSC 101 EcoRI recombinant plasmids grown in E. coli K12.

Recombinant DNAs containing the E. coli plasmid pSC101 and mouse cell (La9) mitochondrial DNA (mtDNA) were formed in vitro via ligation of DNA fragments from limit EcoRI endonuclease digests and were used to transform E. coli K12. Four structurally different recombinant plasmid DNAs from transformed clones were characterized. Two of these were analyzed extensively and the mtDNA portions compared with mtDNA from LA9 cells. No differences were detected in the physical or chemical properties examined, except that the E. coli mtDNA lacked the alkali lability characteristic of animal mtDNAs. Heteroduplexes between the LA9 portions of the recombinant plasmids and LA9 mtDNA were analyzed by absorbance melting. The melting temperatures were indistinguishable from reannealed LA9 mtDNA homoduplexes, indicating that single-base replication errors occur at a frequency of fewer than 1 nucleotide in 300. Electron microscopic analyses of plasmid-LA9 mtDNA heteroduplexes and a comparison of agarose gel electrophoresis of restriction endonuclease fragments also indicated no differences. These results were independent of the order or the relative orientation of the pSC101 and mtDNA fragments. A third EcoRI fragment in LA9 mtDNA, not found in an earlier study (Brown and Vinograd, 1974), has been positioned in the LA9, EcoRI map. This fragment contains 165+/-10 nucleotide pairs.

Cell Line↗
PubMed1973

Regulation of aspartokinase activity in Clostridium perfringens.

Cells of Clostridium perfringens type D apparently possess only a single species of aspartokinase. This enzyme has been partially purified and shown to be feedback inhibited by meso-diaminopimelate in an allosteric manner. The inhibitor exerts its action noncompetitively with respect to both substrates. The kinetic analysis further indicates that no homotropic cooperative interactions occur between either multiple substrate or inhibitor sites. Like aspartokinases from other bacteria, the clostridial enzyme is stimulated by the presence of either potassium or ammonium cations. A molecular weight of 102,000 was estimated for the enzyme following gel-filtration chromatography. Enzyme activity remains relatively constant throughout the growth cycle of the organism even well into the stationary growth phase. These results are discussed in terms of the role of the enzyme in the growth of the organism.

Aspartic Acid↗