Thesis (M.S.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed on June 25, 2009). Includes bibliographical references (p. 52-56).

The superinfection exclusion gene (sieB) of Salmonella phage P22 was mapped using phage deletion mutants. The DNA sequence in the region was re-examined in order to find an open reading frame consistent with the deletion mapping. Several discrepancies from the previously published sequence were discovered. The revised sequence revealed a single open reading frame of 242 codons with six likely translation initiation codons. On the basis of deletion and amber mutant phenotypes the second of these six sites was inferred to be the translation initiation site of the sieB gene. The sieB gene encodes a polypeptide with 192 amino acid residues with a calculated molecular weight of 22,442, which is in reasonable agreement with that estimated from polyacrylamide gels. The transcription start-site of sieB was identified by the use of an RNAase protection assay. The sieB promoter thus identified was inactivated by a two-base substitution in its -10 hexamer. The sieB gene of coliphage λ was also identified. The promoter for λ sieB was identified by homology to that of P22 sieB. sieB aborts the lytic development of some phages. P22 itself is insensitive to the lethal effect of SieB because it harbours a determinant called esc. It was found that the sieB gene encodes two polypeptides-SieB, which is the exclusion protein, and Esc, which is a truncated version of SieB that inhibits its action. Superinfecting P22 synthesizes an antisense RNA, sas, that inhibits synthesis of SieB but allows continued synthesis of Esc, thus allowing P22 to by-pass SieB-mediated exclusion. This translational switch induced by sas RNA is essential to vegetatively developing P22; a mutation that prevents this switch causes P22 to commit SieB-mediated suicide. It was also found that P22's Esc allows it to circumvent the SieB-mediated exclusion system of bacteriophage λ.

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The sequence of 1360 base pairs of the P22 PL operon was determined, linking a continuous sequence from PL through abc2. P22 mutants bearing deletions in the sequenced region were constructed and tested for their phenotypes. Plasmids were constructed to express PL operon genes singly and in combinations from PlacUV5. Two previously known genes, 17 and c3, are located within this sequence. In addition, three new genes have been identified: ral, kil and arf. Genes ral and c3 are homologous, as well as functionally analogous, to λ ral and cIII, respectively. P22 kil, like λ kil, kills the host cell when it is expressed. The two kil genes, although analogous in cell killing and map location, have no apparent sequence homology. The functions of the P22 and λ kil genes are unknown; however, P22 kil is essential for lytic growth in the absence of abc. Gene arf (accessory recombination function) is located just upstream of erf; it is essential for P22 growth in the absence of kil or other genes upstream in PL. The growth defect of P22 bearing a deletion that removes arf is complemented by expression of either arf or the λ red genes from plasmids. P22 sequences that include the stop codon for 17 potentially form a small stem-loop structure; these sequences are nearly identical to λ sequences that contain the stop codon for ssb. In λ this potential stem-loop structure occupies a map position near the terminator tL2b. Plasmids that include the potential P22 structure negatively regulate kil gene expression in cis.