Gene 1.7 of bacteriophage T7 confers sensitivity of phage growth to dideoxythymidine.
Bacteriophage T7 DNA polymerase efficiently incorporates dideoxynucleotides into DNA, resulting in chain termination. Dideoxythymidine (ddT) present in the medium at levels not toxic to Escherichia coli inhibits phage T7. We isolated 95 T7 phage mutants that were resistant to ddT. All contained a mutation in T7 gene 1.7, a nonessential gene of unknown function. When gene 1.7 was expressed from a plasmid, T7 phage resistant to ddT still arose; analysis of 36 of these mutants revealed that all had a single mutation in gene 5, which encodes T7 DNA polymerase. This mutation changes tyrosine-526 to phenylalanine, which is known to increase dramatically the ability of T7 DNA polymerase to discriminate against dideoxynucleotides. DNA synthesis in cells infected with wild-type T7 phage was inhibited by ddT, suggesting that it resulted in chain termination of DNA synthesis in the presence of gene 1.7 protein. Overexpression of gene 1.7 from a plasmid rendered E. coli cells sensitive to ddT, indicating that no other T7 proteins are required to confer sensitivity to ddT.
Tran
Ngoc Q
NQ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Rezende
Lisa F
LF
Qimron
Udi
U
Richardson
Charles C
CC
Tabor
Stanley
S
D017123:Bacteriophage T7 / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000096:biosynthesis; D024882:Drug Resistance, Viral / Q000187:drug effects*; D004926:Escherichia coli / Q000201:enzymology / Q000821:virology; D017353:Gene Deletion; D005814:Genes, Viral; D009154:Mutation / Q000235:genetics; D017422:Sequence Analysis, DNA; D013936:Thymidine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D013937:Thymidine Kinase / Q000378:metabolism
D004279:DNA, Viral; D013937:Thymidine Kinase; D013936:Thymidine