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 Title: An Analysis of Bacteriophage Lambda Site-Specific Recombination Using IHF Binding Site Mutants Author(s): Waechter-Brulla, Daryle A. Department / Program: Microbiology Discipline: Microbiology Degree Granting Institution: University of Illinois at Urbana-Champaign Degree: Ph.D. Genre: Dissertation Subject(s): Biology, General Abstract: Integration Host Factor (IHF) is an Escherichia coli protein required for integration and excision of bacteriophage lambda. Mutations of the three sites to which this protein binds within the lambda attachment site were constructed using oligonucleotide-directed site-specific mutagenesis and were ananlysed for their effects on site-specific recombination. One group of mutations had alterations across one site; in a second group, a pattern of changes was made in each of the three sites. Certain base changes affected some sites more severely than others; some changes had more effect in vitro than in vivo. All mutations depress integrative recombination in vivo and depress or eliminate it in vitro. In excision, mutations of the H1 site raise the frequency of excisive recombination, while mutations of the H2 or H$\sp\prime$ sites lower the frequency of excisive recombination. These results indicate an involvement of all three sites in both integration and excision; occupancy by IHF of the H2 and H$\sp\prime$ sites is required for either reaction, while the presence of IHF at H1 favors integration and its absence favors excision.Two other projects concerning site-specific recombination between lambda and its host are presented. The isolation and analysis of a novel secondary attachment site for lambda is described. The DNA sequence of this site, located within the deoD gene of E. coli, is compared to other published primary and secondary attachment site sequences to identify the point of exchange and regions resembling protein binding sites. The structure of secondary attachment sites is discussed in terms of current models for lambda site-specific recombination.A new protocol for the purification of gpxis is detailed. This procedure yields abundant protein in fewer, easier steps than previously published procedures and removes a Mg-dependent endonuclease activity which co-purified with Xis activity in one procedure. The amino acid length (72 amino acids) of the Xis protein agrees with the value predicted from the DNA sequence. The predominant species ($>$90%) of protein present is gpxis, according to Northern blot analysis. Issue Date: 1988 Type: Text Description: 213 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988. URI: http://hdl.handle.net/2142/72521 Other Identifier(s): (UMI)AAI8908882 Date Available in IDEALS: 2014-12-17 Date Deposited: 1988
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