Browsing School, Graduate by Subject "Bacteriophage T4--genetics"
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Mechanism of gene amplification of genes 17 and 18 in bacteriophage T4Bacteriophage T4 gene amplification mutants (Hp17s) carrying two to more than six copies of genes 17 to 18 in tandem were isolated by plating gene 17 amber mutant on an ochre suppressor containing host. Almost all (190/191) of the independently isolated Hp mutants arise from rearrangement in a 5 bp sequence, GCTCA, in two G-C rich regions of 24 bp partial homology (4 mismatches) located within genes 16 and 19. Analysis of several dispensable mutants showed that phage T4 topoisomerase (39, 52 and 60), T4 DNA primase (61), and T4 uvsX gene product are required for gene amplification, whereas the denV and the host recA gene products are not. The absence of the internal protein Alt is essential to isolate gene amplification mutants (Hp17) because more DNA can be packaged into the alt head which compensates the selection pressure against gene duplication. However, similar gene amplification mutants can not be found in genes 16, 18, 23, 32, 37, and 43 from the alt mutant. Site-directed mutations in the 24 bp recombination box in the phage T4 genome either reduced or eliminated the gene amplification mutants in T4 phage. This evidence suggests that sequence specificity rather than homology is important for the gene amplification event. DNAs isolated from T4D wild-type phage, plasmid pCBR2 which contains genes 16-67, and Hp17 phage had recombined in the GCTCA sequence in the 24 bp box as shown by the polymerase chain reaction (PCR) and direct sequencing of the PCR products. Mutations in E. coli genes recA, recB, recC, recE, recF, and recN did not eliminate the 200 bp recombination PCR signal in the plasmid pCBR2 DNA preparations indicating that their gene products are not required for the initial rearrangement event. Two E. coli proteins, of 69 kd and 30 kd, which bind a DNA fragment containing the recombination box in gene 16, were co-purified through DEAE-cellulose, MonoQ, and single-stranded DNA cellulose columns. However, protein preparations containing these two proteins did not exhibit sequence specific DNA binding activity in a competition assay. Although T4 late gene in the plasmid may not express well with the host RNA polymerase, removal of the N-terminal region of gene 16 from the plasmid eliminated the recombination signal suggesting that gp16 is involved in catalyzing the initial recombination event. Possible mechanisms of the gene amplification in mutant Hp17s are discussed.
The mechanism of the phage T4 terminase complex,gp16 andgp17, in DNA packagingIn T4, earlier genetic studies showed that terminase complex is composed of gp16 and gp17 and three cs20 (cold sensitive) mutants have the same phenotype as that of gene 17 amber mutants, accumulation of empty proheads which can be packaged in vitro. One cs20 mutation could be reversed by a second site ts17 suppressor mutation (Ile364 Phe364). These observations suggested that the terminase interaction with the T4 connector, composed of twelve copies of gp20, is located within the large terminase subunit, gp (gene product) 17. The cs mutations are all clustered within a 30 amino acid short segment of gp20. The clustering of these cs mutations in the small region within the 524 amino acid long protein suggests that this region plays a role in forming a terminase docking site. A 15 amino acid oligopeptide corresponding to residues 287 to 340 (with the addition of Cys at its C-terminus) of the gp20 segment was synthesized and polymerized to raise anti-peptide antibodies. The polymerized oligopeptide inhibited in vitro DNA packaging. Overexpressed radioactive gp17 produced from a T7 expression plasmid could be cross-linked to the polymerized oligopeptide and was coimmunoprecipitated by the anti-peptide antibodies. In addition, purified overexpressed gp17 was able to bind to an oligopeptide-conjugated epoxy-activated Sepharose CL 6B affinity column. Both lines of evidence suggest that this segment is part of the terminase interaction site. However the anti-peptide antibodies could not precipitate the proheads, which suggests that the docking site may be hidden within the connector. In addition to the large subunit, T4 terminase complex contains the small subunit, gp16. We developed a new purification procedure to obtain large quantities of purified gp16 from an overexpression vector. The pure protein is found in two molecular weight forms, due to specific C-terminal truncation, displays in vitro packaging activity, and binds but does not hydrolyze ATP. Gp16 forms specific oligomers, rings and side-by-side double rings. The single ring contains about 8 monomers, and the rings have a diameter of about 8 nm with a central hole of about 2 nm. A DNA binding helix-turn-helix (HTH) motif close to the N-terminus of gp16 is predicted. The oligomers do not bind to DNA, but following denaturation and renaturation in the presence or DNA, binding can be demonstrated by gel shift and filter binding assays. gp16 binds to dsDNA but not ssDNA, and appears to bind preferentially to a gene 16 DNA sequence. T4 packages headfuls of DNA from a concatemer but its mechanism of DNA recognition remains to be determined. Phage T4 sequences were introduced into prophage lambda i (= imm)434 and plasmids in order to assess their effect on packaging as measured by transduction frequency and DNA content of T4 transducing particles. Multiple copies of plasmid or lambda i434 genes were transduced at high frequencies compared to single copy chromosomal genes. T4 gene 16 containing inserts could enhance plasmid transducing particle formation to ca. 10%. Deletions of the gene 16 3' end depressed these elevated plasmid transduction frequencies, suggesting that this is a preferred T4 pac site. We conclude that both DNA sequences and their copy numbers are important for packaging, consistent with a synapsis model for regulation of terminase cutting and packaging in phage T4. (Abstract shortened by UMI.)