University of Illinois Urbana-Champaign

Genetic analysis of the proteins required for site-specific recombination of bacteriophage lambda: Interactions with each other and their recognition sites

Numrych, Thomas Edward

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Description

Title
Genetic analysis of the proteins required for site-specific recombination of bacteriophage lambda: Interactions with each other and their recognition sites
Author(s)
Numrych, Thomas Edward
Issue Date
1992
Doctoral Committee Chair(s)
Gardner, Jeffrey F.
Department of Study
  • Biology, Molecular
  • Biology, Microbiology
Discipline
  • Biology, Molecular
  • Biology, Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
  • Biology, Molecular
  • Biology, Microbiology
Language
eng
Abstract
  • In order to study the importance of the individual Int arm-type binding sites in $\lambda$ site-specific recombination, triple-base changes were made in each of the five Int arm-type binding sites, and then characterized through in vitro and in vivo recombination assays. These mutants demonstrated that the P1, P$\sp\prime$2, P$\sp\prime$3, and possibly the P$\sp\prime$1 sites are required for integration, while the P2, P$\sp\prime$1, and P$\sp\prime$2 sites are important for excision.
  • The bacteriophage P22-based challenge-phage system was used to study the binding of Xis, Int, and FIS to their sites in attP of $\lambda$. Challenge phages were constructed that contained the region encompassing the X1, X2, and F sites. These phages exhibited lysogenization dependent on the presence of both Xis and FIS. Thirty-eight unique mutations in the X1, X2, and F sites were isolated that inhibited Xis and FIS from binding. These mutants verified the importance of certain bases within the proposed consensus sequences for Xis and FIS, and provided evidence that the DNA sequence outside of the proposed binding sites may affect the binding of the individual proteins or the cooperativity between them. An additional challenge phage was constructed that contained the region encompassing the P2, X1, X2, and F sites. This new phage was dependent upon the presence of Xis, Int, and FIS, to form lysogens. Furthermore, lysogenization by this phage required Xis and Int to bind cooperatively, and thus demonstrated that Xis and Int bind cooperatively in vivo.
  • A mutational analysis of the xis gene of bacteriophage $\lambda$ was performed. Thirty-one unique Xis mutants were isolated and characterized by an excision assay and challange-phage assays with the forementioned challenge phages. The results of these assays demonstrated that only the first fifty amino acids of Xis are required for the protein to bind the DNA, interact cooperatively with FIS, and promote $\lambda$ excision. In addition, the mutants revealed that the carboxyl portion of Xis is required for cooperative interactions between Xis and Int. This cooperativity requires the presence of FIS, but not the Int core-type binding sites. The Xis mutants also demonstrated the importance of FIS-Xis-Int cooperativity in $\lambda$ excision.
Type of Resource
text
Permalink
http://hdl.handle.net/2142/23534
Copyright and License Information
Copyright 1992 Numrych, Thomas Edward

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Graduate Dissertations and Theses at Illinois PRIMARY
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