Richard Machanoff

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Escherichia coli plasmid pRR36, which expresses Rhodospirillum rubrum ribulose bisphosphate carboxylase/oxygenase (EC 4.1.1.39) as a fusion protein [Nargang et al., Mol. Gen. Genet. 193 (1984) 220-224], was used to construct a new clone of the carboxylase gene (rbc) whose expression product is the wild-type enzyme. This construction entailed removing all(More)
Prostaglandins E1, E2, and F2alpha (PGE1, PGE2, and PGF2alpha) were shown to inhibit the growth of mouse leukaemia lymphoblasts L5178Y in culture. The effects of PGE1 and PGE2 were greater than that of PGF2alpha. PGE1 and PGE2, at the concentration of 100 mug per ml showed significant inhibitory effects on the rates of incorporation of tritiated thymidine,(More)
The induction of mutation by a variety of mutagens has been measured utilizing the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells (CHO/HGPRT) system). These mutagens include physical agents such as UV light and X-rays, and chemicals such as alkylating agents, ICR-191, and metallic compounds. This system(More)
Host-mediated assays have been developed to allow determination of the mutagenic potential of promutagens and procarcinogens which require metabolic activation to exert their effects on indicator organisms. We report here the development of the host-(mouse)-mediated CHO/HGPRT system using the procarcinogen dimethylnitrosamine (DMN) as a model agent. Using a(More)
Chemical modification of spinach ribulosebisphosphate carboxylase/oxygenase by diethyl pyrocarbonate led to the conclusion that His-298 is an essential active-site residue (Igarashi, Y., McFadden, B. A., and El-Gul, T. (1985) Biochemistry 24, 3957-3962). From the pH dependence of inactivation, the pKa of His-298 was observed to be approximately 6.8, and it(More)
We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient CHO cells that have been transformed by the plasmid vector pSV2gpt. One isolated transformant, designated AS52, carries a single copy of the Escherichia coli gpt gene stably(More)
Affinity labeling and comparative sequence analyses have placed Lys-166 of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum at the active site. The unusual nucleophilicity and acidity of the epsilon-amino group of Lys 166 (pKa = 7.9) suggest its involvement in catalysis, perhaps as the base that enolizes ribulosebisphosphate (Hartman,(More)
We have developed a system to study mutations which affect expression of the E. coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) in hypoxanthine-guanine phosphoribosyl transferase-deficient (HPRT-) Chinese hamster ovary (CHO) cells that have been transformed by the plasmid pSV2gpt. Several gpt-transformed cell lines have been isolated and(More)
Previous reports provide indirect evidence for the presence of Glu-48 at the active site of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillum rubrum. This possibility has been examined directly by replacement of Glu-48 with glutamine via site-directed mutagenesis. This single amino acid substitution does not prevent subunit association or(More)
An assay is described for the measurement of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells utilizing resistance to 6-thioguanine (TG). Optimal selection conditions are defined for such parameters as phenotypic expression time prior to selection, and TG concentration and cell(More)