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Construction and properties of a recombinant plasmid containing gene 32 of bacteriophage T4D

1981; Elsevier BV; Volume: 148; Issue: 3 Linguagem: Inglês

10.1016/0022-2836(81)90535-0

1089-8638

Henry Krisch, Gerald Selzer,

Protein purification and stability

Abstract This paper describes the construction and characterization of a chimeric plasmid that encodes the single-stranded DNA-binding protein of bacteriophage T4D (the product of gene 32). The plasmid contains a 2·6 × 10 3 base Hin dIII segment of T4 DNA that includes genes 59 and 32 as well as a portion of gene 33. Isolation of bacteria carrying the recombinant plasmid became possible when the segment of phage DNA contained an amber mutation in gene 32. This suggests that a functional gene 32 is deleterious to the cell. Using antibody to gene 32 protein, we have been able to demonstrate expression of the plasmid-borne gene 32 in uninfected bacteria. Deletion variants of the gene 32 plasmid have been constructed in vitro . These have been used to align the genetic map of the region with the restriction map and to study phage gene expression from the plasmid in both infected and uninfected cells. In phage-infected cells the level of functional gene 32 product regulates the efficiency of translation of its own messenger RNA. We also observe such self-regulation for gene 32 present on the plasmid.