Biochemical transformation of thymidine kinase (TK)-deficient mouse cells by herpes simplex virus type 1 DNA fragments purified from hybrid plasmids.
The thymidine kinase (TK) gene of HSV-1 has been cloned in Escherichia coli K12 plasmids, pMH1, pMH1A, and pMH4. These plasmids contain a 1,92Obp HSV-1 TK DNA sequence, which replaces a 2,067 bp EcoR I to Pvu II sequence of plasmid pBR322 DNA. Superhelical DNAs of plasmids pMH1, pMH1A, and pMH4 as well as plasmid DNAs cleaved by EcoR I, Hinc II, Bg1 II, Sma I, and Pvu II transformed TK-deficient LM(TK-) cells to the TK+ phenotype. A 1,230bp EcoR I-Sma I fragment purified from pMH1 DNA (and from plasmid pAGO, DNA, the parent of pMH1) also transformed LM(TK-) cells. Serological and disc PAGE studies demonstrated that the TK activity expressed in biochemically transformed cells were HSV-1-specific. The experiments suggest that the HSV-1 TK coding region may be contained within a l.1kbp DNA sequence extending from about the Hinc II (or Bgl II) cleavage site to the Sma I site. 35S-methionine labeling experiments carried out on cell lines transformed by Hinc II-cleaved pMH1 DNA and by the EcoR I-Sma I fragment showed that the TKs purified from the transformed cells consisted of about 39-40,000 dalton polypeptides.
D000818:Animals; D001483:Base Sequence; D002472:Cell Transformation, Viral; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005796:Genes; D007739:L Cells / Q000201:enzymology; D051379:Mice; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D018139:Simplexvirus / Q000201:enzymology*; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000172:deficiency*
D004274:DNA, Recombinant; D004279:DNA, Viral; D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes