A Study of Site-specific Recombination in Bacteriophage Lamda Using the Electron Microscope PDF Download

Author: Jeffrey A. Engler
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ISBN:
Category : Bacteriophage lambda
Languages : en
Pages : 492

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Author: Daryle Anne Waechter-Brulla
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Category :
Languages : en
Pages : 410

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Author: Gabriel Guarneros-Peña
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ISBN:
Category :
Languages : en
Pages : 220

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Author: Geoffrey Cassell
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ISBN:
Category : Bacteriophage lambda
Languages : en
Pages : 320

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Author: Thomas Edward Numrych
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ISBN:
Category :
Languages : en
Pages : 288

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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$spprime$2, P$spprime$3, and possibly the P$spprime$1 sites are required for integration, while the P2, P$spprime$1, and P$spprime$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.

Author: Stephen Sum-man Chung
Publisher:
ISBN:
Category :
Languages : en
Pages : 218

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Author: Lea Jessop
Publisher:
ISBN:
Category : Bacteriophage lambda
Languages : en
Pages : 330

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Site-specific recombination occurs in many cells types, providing a variety of functions ranging from controlling gene expression to integrating and excising viral and bacteriophage genomes into and out of host chromosomes. We have focused on the 4 site-specific recombination pathways mediated by the bacteriophage Lambda Int protein. In order to better understand the determinants of directionality and efficiency in these recombination pathways, we have developed two assays to determine the architecture of the synaptic intermediates. Crystal structures of these large nucleoprotein complexes are unavailable, consequently we have taken a biochemical approach to address 4 specific questions: (1) What is the multimeric state of Int during recombination? (2) Where do the 4 cysteines of Int map relative to multimerization interfaces? (3) What effect, if any, does tethering through these interfaces have on recombination? (4) Which Int binding sites are bridged by a single Int monomer within the recombination intermediates? Using protein-protein crosslinking we have shown that Int forms a tetramer during recombination conditions, independent of the recombination pathway. There are at least two multimerization interfaces within the tetramer; C25 is located at one of these interfaces while C197 and C262 are located at the second. C217 is also present at or near a multimerization interface, but no cysteine from the adjacent monomer is present to allow tethering by sulfhydryl reactive compounds. Modification of C217 affects excision differently than bent-L recombination. Specifically, modification at C217 is not tolerated during excision but is allowed for bent-L recombination, suggesting differences in the architecture of intermediates between these two pathways. We have also developed an assay to identify intra- and intermolecular bridges formed by Int within the nucleoprotein complexes. In the straight-L recombination pathway, Int is delivered to the point of top strand cleavage, the B core, from the P2 arm site of the partner attL site. During excision, the monomer of Int which cleaves the top strand of attL, the B core, is delivered from the P2 arm site of attR. Thus for two pathways, Int is delivered to the same core site from two different arm sites, which may influence the contacts made between Int and the core sites. We suggest that differences in the way in which Int contacts the core sites may control directionality and efficiency of the Int-mediated recombination pathways.

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Category :
Languages : en
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Author: Lina Maria Moitoso de Vargas
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Category :
Languages : en
Pages : 266

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Author: Zhao Wu
Publisher:
ISBN:
Category :
Languages : en
Pages : 318

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