Artificial mutants generated by the insertion of random oligonucleotides into the putative nucleoside binding site of the HSV-1 thymidine kinase gene

Journal Article · Tue Dec 24 04:00:00 EST 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00115a004· OSTI ID:5488303

Dube, D K; Parker, J D; French, D C; Cahill, D S; Dube, S; Horwitz, M S.Z.; Munir, K M; Loeb, L A ^[1]

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1. Univ. of Washington, Seattle (United States)

The authors have obtained 42 active artificial mutants of HSV-1 thymidine kinase by replacing codons 166 and 167 with random nucleotide sequences. Codons 166 and 167 are within the putative nucleoside binding site in the HSV-1 tk gene. The spectrum of active mutations indicates that neither Ile{sup 166} nor Ala{sup 167} is absolutely required for thymidine kinase activity. Each of these amino acids can be replaced by some but not all of the 19 other amino acids. The active mutants can be classified as high activity or low activity on two bases: (1) growth of Escherichia coli KY895 in the presence of thymidine and (2) uptake of thymidine by this strain, when harboring plasmids with the random insertions. E. coli KY895 harboring high-activity plasmids or wild-type plasmids can grow in the presence of low amounts of thymidine but are unable to grow in the presence of high amounts of thymidine. The high-activity plasmids also have an enhanced ({sup 3}H)dT uptake. The amounts of thymidine kinase activity in vitro in unfractionated extracts do not correlate with either growth at low thymidine concentration or the rate of thymidine uptake. Heat inactivation studies indicate that the mutant enzymes are without exception more temperature-sensitive than the wild-type enzyme. This thermolability could account for the less than expected thymidine kinase activity in the extracts and suggests that amino acid substitutions at Ile{sup 166} and Ala{sup 167} have produced major changes in protein stability.

• OSTI ID: 5488303
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 30:51; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ALANINES
AMINO ACIDS
AZINES
BACTERIA
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CODONS
DISEASES
ENZYME ACTIVITY
ENZYMES
ESCHERICHIA COLI
GENES
HERPES SIMPLEX
HETEROCYCLIC COMPOUNDS
HYDROGEN COMPOUNDS
INACTIVATION
INFECTIOUS DISEASES
LEUCINE
MICROORGANISMS
MUTANTS
NUCLEOSIDES
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
PARASITES
PHOSPHORUS-GROUP TRANSFERASES
PHOSPHOTRANSFERASES
PLASMIDS
PROTEINS
PYRIMIDINES
RIBOSIDES
SKIN DISEASES
THERMAL DEGRADATION
THYMIDINE
TRANSFERASES
TRITIUM COMPOUNDS
UPTAKE
VIRAL DISEASES
VIRUSES