Random mutagenesis of the thymidine kinase gene of varicella-zoster virus
| Autor: | K. L. Powell, D. J. M. Purifoy, R. W. Honess, Simon F. Lacey, T. Suzutani |
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| Rok vydání: | 1992 |
| Předmět: |
Herpesvirus 3
Human Immunology Mutant DNA Mutational Analysis Molecular Sequence Data Thymidylate kinase activity Biology medicine.disease_cause Microbiology Thymidylate kinase Thymidine Kinase Virology medicine Amino Acid Sequence Cloning Molecular Phosphorylation Peptide sequence Genetics chemistry.chemical_classification Mutation Binding Sites Base Sequence Nucleic acid sequence Drug Resistance Microbial Molecular biology Amino acid Phenotype chemistry Thymidine kinase Mutagenesis Insect Science DNA Viral Research Article |
| Zdroj: | Journal of virology. 66(4) |
| ISSN: | 0022-538X |
| Popis: | To understand the relationship between the primary structure and function of varicella-zoster virus thymidine kinase (VZV TK; EC 2.7.1.21), we established rapid screening and phenotypic selection of mutant VZV TK genes in TK-deficient Escherichia coli C600 by using a constitutive pKK223-3 expression plasmid. In this screening system, mutant TK genes generated by random mutagenesis were identified by the sensitivity of E. coli-expressing VZV TKs to 5-bromo-2'-deoxyuridine and 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl) uracil. Twenty-four mutant clones with amino acid substitutions were isolated, and their nucleotide sequence and enzymatic activities were determined. Of the 24 clones, 20 had single amino acid substitutions, 2 clones had double amino acid substitutions, and 1 clone had triple amino acid substitutions. In 17 cases of single amino acid substitution, six mutations led to lost enzyme activity, and four of these six mutations centered in the ATP-binding site. The other 11 mutations resulted in reduction of both TK and thymidylate kinase activities or only thymidylate kinase activity and were located in scattered positions in the VZV TK gene, although 5 mutations showed a tendency to cluster in the region between positions 251 and 260. |
| Databáze: | OpenAIRE |
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