Transformation of human 143 tk- cells with plasmids containing the gene encoding the adenovirus DNA-binding protein.
Human cell lines that contain and express the gene encoding the adenovirus type 5 DNA-binding protein (Ad5 DBP) are very useful for the isolation of adenovirus mutants with an altered DBP. In order to obtain these cells, human 143 tk- cells were transfected, using the calcium phosphate technique, with plasmids containing the Ad5 DBP gene and the herpes simplex virus thymidine kinase (HSV tk) gene as a selectable marker. Characterization of several tk+ transformants revealed that these cells did contain the HSV tk gene, but in none of these cells could Ad5 DBP DNA sequences be detected. However, when 143 tk- cells were co-transfected with a plasmid containing the Ad5 DBP gene and another plasmid carrying early region E1, integration of the Ad5 DBP gene in chromosomal DNA could be detected. Integration of Ad5 DNA sequences was also observed when transfection was performed with plasmids containing the Ad5 DBP gene and the long terminal repeat of Moloney murine leukaemia virus. By employing a radioimmunoassay it could be shown that DBP-related proteins were synthesized in two of the cell lines containing the Ad5 DBP gene. Since both cell lines support the growth of the temperature-sensitive viral DBP mutant, H5ts125, at the non-permissive temperature, the DBP-related proteins expressed in these cells must be functional.
D000260:Adenoviruses, Human / Q000235:genetics* / Q000254:growth & development; D002460:Cell Line; D004262:DNA Restriction Enzymes; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*; D015247:Deoxyribonuclease HindIII; D005814:Genes, Viral; D006801:Humans; D008979:Moloney murine leukemia virus / Q000235:genetics; D009154:Mutation; D010957:Plasmids; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D018139:Simplexvirus / Q000201:enzymology / Q000235:genetics; D013937:Thymidine Kinase / Q000096:biosynthesis / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics*
D004268:DNA-Binding Proteins; D014764:Viral Proteins; D013937:Thymidine Kinase; D004262:DNA Restriction Enzymes; D015247:Deoxyribonuclease HindIII