Bacteriophage T7 is a relatively simple, well characterized virus that infects E. coli. It contains a single molecule of linear DNA almost 40,000 base pair long whose complete nucleotide sequence is known. The nucleotide sequence predicts that there will be 56 T7 proteins, and mutations affecting almost all of them are available. Individual genes and genetic signals have been cloned and are being genetic and biochemical analysis and for manipulation of the virus. The T7 system is an excellent one in which to study the molecular details of DNA replication and related processes.
Our aim i s to understand all aspects of DNA metabolism during T7 infection, including entry of T7 DNA into the cell; protection from host nucleases; selective degradation of host DNA; replication and repair of T7 DNA; genetic recombination; and maturation and packaging of the DNA into virions. These processes are studied in normal and mutant phage infections, using techniques that include radioactive labeling, sedimentation, gel electrophoresis, restriction analysis, filter hybridization, and cloning. T7 genes are expressed individually from clones, both to determine their effects on the host cell and to obtain large amounts of protein for biochemcial and structural analysis. A protein central to both RNA and DNA metabolism during T7 infection is T7 RNA polymerase, which recognizes a large promoter sequence not found in host DNA. This highly selective promoter recognition is used by T7 to direct both transcription and replication to its own DNA, and to ensure that its own DNA is packaged into phage particles. T7 RNA polymerase also forms a specific complex with T7 lysozyme, and interaction that shuts off late transcription and may have a role in replication, packaging and/or lysis. Genetic selections are being applied to the cloned gene in order to analyze further the functions and interactions of T7 RNA polymerase. Other aspects of T7 DNA metabolism currently being studied include the role of the T7 single-stranded DNA binding protein, the early steps in phage assembly, and DNA packaging.
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