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. 1984 Oct;160(1):371–378. doi: 10.1128/jb.160.1.371-378.1984

Genetic system for analyzing Escherichia coli thymidylate synthase.

M Belfort, J Pedersen-Lane
PMCID: PMC214727  PMID: 6090424

Abstract

Random in vitro mutagenesis of the thyA gene is being used to delineate its regulatory elements as well as the functional domains of its product, thymidylate synthase (EC 2.1.1.45). Streamlined procedures have been developed for the isolation and characterization of the mutants. Positive selection for synthase-deficient thyA Escherichia coli permitted the isolation of 400 mutants, which are being categorized by phenotypic and genetic criteria. An in situ 5-fluorodeoxyuridylate binding assay was devised to rapidly probe the substrate binding domain, whereas facile mapping procedures, based on pBR322- or M13-borne thyA deletion derivatives, were developed to localize mutations. The sequence changes of one amber mutation and another mutation that abolishes catalysis while maintaining substrate binding activity are presented. The orientation of the thyA gene on the E. coli chromosome was established.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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