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Biomedical subjects

R Bestwick

Publications and source records attributed to R Bestwick.

4 recordsLinked to original sources
PubMed1987

A hypervariable repeated sequence on human chromosome 1p36.

When used to probe Southern blots of TaqI-digested DNAs from unrelated individuals, p1-79, a 900 bp subclone of a random human cosmid, revealed at least 50 fragments, many of which were polymorphic. Each of 27 unrelated individuals tested with p1-79 displayed a distinct band pattern. Similar variation was seen with several other enzymes, including HaeIII, MspI, PstI and PvuII, whereas other enzymes yielded primarily large fragments of greater than 40 kb. In situ hybridization of p1-79 showed that the loci of hybridization are clustered on human chromosome band 1p36; localization of all TaqI fragments to chromosome 1 was confirmed with a human-rodent somatic cell hybrid panel. DNA sequencing of p1-79 revealed several copies of a 39 bp repeat whose variation in copy number might be the basis of the observed length polymorphisms. Studies of 3-generation Utah families suggest that the numerous restriction fragments homologous to p1-79 are inherited as haplotypes, implying that recombination within this cluster of loci is rare and allowing the cluster to serve as a useful marker for human gene mapping.

Animals↗
PubMed1983

A rapid method for preparation of bacterial plasmids.

A method for isolating plasmids from Escherichia coli which requires less than 8 h from cell pellet to purified plasmid essentially free of protein, RNA, and chromosomal DNA is presented. By this procedure, amplified plasmid pBR322 was isolated from E. coli strain RR1. The final product had no detectable protein or RNA, and plasmid comprised approximately 99% of the total DNA. The procedure includes lysozyme treatment in hypertonic solution followed by lysis with a mild detergent in the presence of high salt and an RNase inhibitor--conditions which prevent unfolding of the bacterial nucleoid. After centrifuging out the nucleoid and cell debris, the nucleic acids are selectively precipitated with a neutral solution of sodium trichloroacetate and ethanol. RNA is degraded with RNase and the degradation products and RNase are eliminated through a second trichloroacetate/ethanol precipitation. Finally, the plasmid is resuspended and passed through a nitrocellulose filter to remove aggregates and any residual protein and single-stranded DNA--giving a plasmid preparation suitable for electrophoretic fractionation or cleavage with restriction nucleases.

Bacteria↗
PubMed1983

Loss of leukemogenicity caused by mutations in the membrane glycoprotein structural gene of Friend spleen focus-forming virus.

Friend virus infection of mice causes progressive leukemogenesis--a rapid splenic erythroblastosis that develops weeks later into a disseminating erythroleukemia. Furthermore, the replication-defective Friend spleen focus-forming virus (F-SFFV) encodes a membrane glycoprotein with an apparent Mr of 55,000 (designated gp55), which is structurally and immunologically related to the membrane envelope glycoproteins of dual tropic murine leukemia viruses. We now have isolated three spontaneous F-SFFV mutants that encode abnormally sized gp55-related glycoproteins with apparent Mrs of 40,000, 54,000, and 58,000, respectively. RNA blot and Southern blot analyses indicate that the mutant nucleic acids do not have substantial deletions or insertions in their glycoprotein gene regions. Protein fragmentation patterns indicate that the mutations affect nonoverlapping domains of the glycoprotein. Furthermore, these mutant glycoproteins seem to be defective in their processing to the plasma membranes. Although transmitted efficiently between cultured cells, the mutants have dramatically reduced leukemogenicities compared with the same titers of wild-type F-SFFV. We conclude that the gp55 structural gene is necessary for initiating the erythroblast proliferative phase of Friend disease and that changes in membranes can be primary causes rather than only secondary consequences of tumor progression.

Animals↗