Biomedical subjects

W R Folk

Publications and source records attributed to W R Folk.

75 records · Page 5Linked to original sources

PubMed1970

Isolation and partial characterization of Escherichia coli mutants with altered glycyl transfer ribonucleic acid synthetases.

Isolates with mutations in glyS, the structural gene for glycyl-transfer ribonucleic acid (tRNA) synthetase (GRS) in Escherichia coli, are frequently found among glycine auxotrophs. Extracts of glyS mutants have altered GRS activities. The mutants grow with normal growth rates in minimal media when high levels of glycine are provided. No other metabolite of a variety tested is capable of restoring normal growth. The glyS mutants fail to make ribonucleic acid (RNA) when depleted of exogenous glycine in strains which are RC(str) but do so when the cells are RC(rel). In contrast, biosynthetic mutants which are unable to synthesize glycine (glyA mutants) do not make RNA when deprived of glycine even if they are RC(rel); in this case, RNA is synthesized upon glycine deprivation only when the nucleic acid precursors made from glycine are provided in the medium. The level of serine transhydroxymethylase is unaltered in extracts of any of the glyS mutants, even though the level of charged tRNA(Gly) is at least 20-fold lower than that found in a prototrophic parent; this indicates that, if there is control over the synthesis of serine transhydroxymethylase, it is not modified by reduced levels of charging of the major species of tRNA(Gly).

Carbon Isotopes↗

PubMed1970

Characterization of altered forms of glycyl transfer ribonucleic acid synthetase and the effects of such alterations on aminoacyl transfer ribonucleic acid synthesis in vivo.

The glycyl transfer ribonucleic acid (tRNA) synthetase (GRS) activities of several Escherichia coli glyS mutants have been partially characterized; the K(m) for glycine and the apparent V(max) of several of the altered GRS differ significantly from the parental GRS. Paradoxically, some of the altered forms exhibit more activity in vitro than the GRS from a prototrophic strain (GRS(L)); several parameters of these activities have been studied in an attempt to resolve this problem. The amount of acylated tRNA(Gly) in vivo was examined to assess the GRS activities inside the cells. During exponential growth in media containing glycine, moderate amounts of acylated tRNA(Gly) occur in the glyS mutants; glycine deprivation leads to a dramatic drop in the amount of acylated tRNA(Gly). An alternative measure of the in vivo activities of the altered enzymes is the efficiency of suppression of the trpA36 locus by su(36) (+); glyS mutants grown with added glycine exhibit one-third to one-fourth the suppression efficiency of the prototrophic glyS(H) parent, presumably because they are less efficient, even in the presence of high levels of glycine, in charging the tRNA(Gly) species which functions as the translational suppressor.

Amino Acids↗

PubMedDate not supplied

An improved procedure for measuring DNA replication with transient assays in eukaryotic cells.

We describe an improved method for quantifying covalently closed circular DNAs replicated in eukaryotic cells. Normally, for such assays, plasmid DNAs transfected into animal cells must be extensively digested with restriction enzymes such as Dpn1 and the products of digestion of the input DNA separated from the Dpn1-resistant (replicated) DNAs by electrophoresis. In the procedure described, the input, unreplicated DNA is nicked by light digestion with Dpn1 and subsequently denatured and eliminated by S1 nuclease digestion. The replicated Dpn1-resistant DNA is detected by slot blot hybridization. Multiple samples can be rapidly assayed, with a considerable reduction in expense and labor.

Animals↗