Genetic system for analyzing Escherichia coli thymidylate synthase

Area of Science:

• Molecular Biology
• Enzymology
• Bacterial Genetics

Background:

• The thyA gene encodes thymidylate synthase, a critical enzyme in DNA synthesis.
• Understanding thyA's regulatory elements and functional domains is essential for various biological processes.

Purpose of the Study:

• To delineate the regulatory elements of the thyA gene.
• To map the functional domains of thymidylate synthase.
• To characterize mutations affecting thyA gene function and regulation.

Main Methods:

• Random in vitro mutagenesis of the thyA gene.
• Positive selection for synthase-deficient Escherichia coli mutants.
• In situ 5-fluorodeoxyuridylate binding assays.
• Genetic mapping using pBR322- or M13-borne deletion derivatives.

Main Results:

• Isolation and categorization of 400 thyA mutants based on phenotypic and genetic criteria.
• Development of rapid assays for probing substrate binding domains.
• Localization of mutations using deletion mapping techniques.
• Sequencing of mutations, including an amber mutation and one affecting catalysis but not binding.

Conclusions:

• Established streamlined procedures for mutant isolation and characterization.
• Identified specific mutations affecting thymidylate synthase activity and substrate binding.
• Determined the orientation of the thyA gene on the E. coli chromosome.