Bacteriophage T7 is a relatively simple virus that has been well characterized physiologically and genetically. The nucleotide sequence of the T7 DNA molecule, 39,926 base pairs, is now completely known. The nucleotide sequence predicts 50 genes and mutations affecting all but six of them have been found. About 99% of the molecule has been cloned as specific, well defined fragments, which are being used for genetic and biochemical characterization of the virus. Thus, 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 the DNA into the bacterial cell, protection from host nucleases, selective degradation of host DNA, replication and repair of T7 DNA, genetic recomination, and maturation and packaging into virions. Almost all of the proteins involved in these processes appear to be specified by T7 itself. We study the normal processes, and the changes induced in them by specific mutations, using techniques including radioactive labeling, sedimentation, gel electrophoresis, restriction analysis, filter hybridization and cloning. The interaction of infecting phages with cloned fragments of T7 DNA resident in the host is proving very informative. Examples of projects currently underway include the following: We have found that the mode of entry of T7 DNA into the cell plays an important part in overcoming host restriction, and we are analyzing how the entry process might be controlled and whether other aspects of DNA metabolism are coupled to DNA entry. We find that cloned origins of replication of T7 DNA serve as origins of replication in plasmids during T7 infection, and we are using this as an assay to define what elements of the nucleotide sequence are needed for initiation of replication. We have isolated conditional-defective mutants of the T7 single-stranded DNA-binding protein and are using them to analyze the role of this protein in DNA metabolism. We are exploring the possibility of using cloned fragments of T7 DNA to produce large amounts of specific T7 replication proteins.
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