f Thymidine Kinase Deletion Mutants of Herpes Simplex Virus Type 1
- Authors: Peter G. Sanders†, Neil M. Wilkie‡, Andrew J. Davison
- J. Gen. Virol., December 1982 63: 277-295, doi: 10.1099/0022-1317-63-2-277
- Subject: Animal
- Published Online:
Deletions in the cloned thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-1), strain 17 syn +, were produced by two methods. Removal of a 506 base pair fragment from between the unique SstI and BglII restriction endonuclease sites of pTK1 (HSV-1 BamHI p cloned in pAT153) and subsequent transformation of Escherichia coli resulted in the isolation of 50 deleted plasmids. Sequential digestion of pTK1 with BglII and nuclease BAL 31 followed by ligation and recleavage with BglII resulted in the isolation of 31 deleted plasmids. Three clones, pTK2, pTK3 and pTK4, obtained following BglII and SstI treatment of pTK1 were recombined with wild-type (wt) HSV-1 (17) syn + DNA in baby hamster kidney (BHK) cells to produce TK- deletion mutants HSV-1 (17) TK 1301, HSV-1 (17) TK 1302 and HSV-1 (17) TK 1303 respectively. 5-Bromo-2′-deoxyuridine, 5-bromo-2′-deoxycytidine and 9-(2-hydroxyethoxymethyl)guanine were used to reduce the background of TK+ virus in heterogeneous recombinant stocks analysed for the presence of TK- recombinants. All recombinant clones isolated produced a small syncytial plaque morphology in BHK cells. The mutants HSV-1 (17) TK 1301 and HSV-1 (17) TK 1302 were TK-, failed to produce polypeptides of molecular weights 43000 and 19000 found in wt-infected cells and demonstrated one-step growth curves different from wt virus and the TK- mutant HSV-1 (17) dPyk-7. Superinfection studies with HSV-1 (17) TK 1301, HSV-1 (17) TK 1302, HSV-1 (MDK) and HSV-1 (17) dPyk-7 indicated that all TK- mutants except dPyK-7 produce a trans-acting gene product which can switch on the transforming HSV-1 TK gene.
Present address: Institute of Genetics, University of Glasgow, Church Street, Glasgow G11 5JS, U.K.
Present address: The Beatson Institute for Cancer Research, Wolfson Laboratory for Molecular Pathology, Garscube Estate, Switchback Road, Glasgow G61 1BD, U.K.
© Society for General Microbiology 1982 | Published by the Microbiology Society
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