title: Insertion and deletion mutants of vaccinia virus.
abstract: Thirteen viable insertion mutants of vaccinia virus have been constructed. These mutants, containing coding sequences of the herpes simplex virus thymidine kinase (HSV-TK) gene, were generated by marker transfer via in vivo recombination. The mutants were identified using a replica filter plating technique by in situ hybridization using 32P-nick translated HSV-TK sequences and obtained as pure cultures by repeated plaque purification. Some of these insertion mutants were in turn used as substrates to generate viable deletion mutants of vaccinia virus in the presence of 5'-bromodeoxyuridine. An example of this approach resulting in a vaccinia virus deleted of approximately 1.5 kb of nonessential DNA is presented. Furthermore, the analysis of spontaneously occurring viable deletion mutants of vaccinia lacking approximately 21.4 kb of nonessential DNA is described.
mesh_terms: D000818:Animals; D001426:Bacterial Proteins; D001483:Base Sequence; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002642:Chick Embryo; D002872:Chromosome Deletion; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004251:DNA Transposable Elements; D015245:Deoxyribonuclease BamHI; D015247:Deoxyribonuclease HindIII; D015252:Deoxyribonucleases, Type II Site-Specific; D009154:Mutation; D011995:Recombination, Genetic; D018139:Simplexvirus / Q000201:enzymology; D013937:Thymidine Kinase / Q000235:genetics; D014616:Vaccinia virus / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; D004251:DNA Transposable Elements; D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes; D015245:Deoxyribonuclease BamHI; D015247:Deoxyribonuclease HindIII; C024404:BglII endonuclease; D015252:Deoxyribonucleases, Type II Site-Specific; D001973:Bromodeoxyuridine