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dc.contributor.advisorAnthony Poteete
dc.contributor.authorRennell, Dale
dc.date2022-08-11T08:08:40.000
dc.date.accessioned2022-08-23T16:03:46Z
dc.date.available2022-08-23T16:03:46Z
dc.date.issued1988-02-01
dc.date.submitted2007-01-22
dc.identifier.doi10.13028/q282-a318
dc.identifier.urihttp://hdl.handle.net/20.500.14038/31545
dc.description<p>Pages appear misnumbered. Page C101 is missing.</p>
dc.description.abstractP22 lysozyme, encoded by gene 19, is an essential phage protein responsible for hydrolyzing the bacterial cell wall during lytic infection. P22 lysozyme is related to T4 lysozymein its mode of action, substrate specificities, and in its structure. Gene 19 was located on the phage genome, subcloned, and then sequenced. lysozyme was produced in large quantities and purified for biochemical characterization and for crystallograpic studies. Gene 19consists of 146 codons, and encodes a protein with a molecular weight of 16,117. Amber mutations were created in gene 19 by in vitro primer-directed mutagenesis. The mutations were crossed by homologous recombination onto the phage genome. The phages bearing the amber mutations in gene 19 were screened for the ability to grow on six different amber suppressor strains. Amino acid substitutions that resulted in nonfunctional or less functional lysozyme were determined. Of 60 possible amino acid substitutions at 11 different sites in P22 lysozyme, 20 are deleterious. The phage bearing amber mutations in gene 19that failed to grow on given suppressor strains were reverted and second site intragenic revertants were obtained. The mutations were sequenced. A substitution of serine for glutamine at residue 82 is compensated for by changing residue 46 from serine to leucine. This single change enables the phage to form a plaque at 300C but not at 400C. When the triple change asn42->lys; ser46->leu; and ser43->pro is present the lysozyme produced is no longer temperature sensitive. The crystal structure of P22 lysozyme is not yet solved. Assuming that the structures of T4 lysozyme and P22 lysozyme are similar, one can examine the positions of equivalent residues in the T4 lysozyme structure. The spatial arrangement of the residues changed by the secondary site mutations and the original substitution can then be visualized. The mutations discussed above all map far from the original mutation on the T4 three dimensional model. A substitution of leucine for tyrosine at position 22 is compensated for by the double mutation of arg18->ser and ser23->lys. When the equivalent residues are mapped on the T4 three dimensional model the changes map in close proximity to the original mutation.
dc.language.isoen_US
dc.rightsCopyright is held by the author, with all rights reserved.
dc.subjectMuramidase
dc.subjectViral Core Proteins
dc.subjectAmino Acids, Peptides, and Proteins
dc.subjectEnzymes and Coenzymes
dc.subjectGenetic Phenomena
dc.titleA Genetic and Structural Analysis of P22 Lysozyme: A Thesis
dc.typeDoctoral Dissertation
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=1238&amp;context=gsbs_diss&amp;unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/gsbs_diss/238
dc.legacy.embargo2017-04-24T00:00:00-07:00
dc.identifier.contextkey244133
refterms.dateFOA2022-08-23T16:03:47Z
html.description.abstract<p>P22 lysozyme, encoded by gene <em>19</em>, is an essential phage protein responsible for hydrolyzing the bacterial cell wall during lytic infection. P22 lysozyme is related to T4 lysozymein its mode of action, substrate specificities, and in its structure. Gene <em>19</em> was located on the phage genome, subcloned, and then sequenced. lysozyme was produced in large quantities and purified for biochemical characterization and for crystallograpic studies. Gene <em>19</em>consists of 146 codons, and encodes a protein with a molecular weight of 16,117.</p> <p>Amber mutations were created in gene <em>19</em> by in vitro primer-directed mutagenesis. The mutations were crossed by homologous recombination onto the phage genome. The phages bearing the amber mutations in gene <em>19</em> were screened for the ability to grow on six different amber suppressor strains. Amino acid substitutions that resulted in nonfunctional or less functional lysozyme were determined. Of 60 possible amino acid substitutions at 11 different sites in P22 lysozyme, 20 are deleterious. The phage bearing amber mutations in gene <em>19</em>that failed to grow on given suppressor strains were reverted and second site intragenic revertants were obtained. The mutations were sequenced.</p> <p>A substitution of serine for glutamine at residue 82 is compensated for by changing residue 46 from serine to leucine. This single change enables the phage to form a plaque at 30<sup>0</sup>C but not at 40<sup>0</sup>C. When the triple change asn42->lys; ser46->leu; and ser43->pro is present the lysozyme produced is no longer temperature sensitive. The crystal structure of P22 lysozyme is not yet solved. Assuming that the structures of T4 lysozyme and P22 lysozyme are similar, one can examine the positions of equivalent residues in the T4 lysozyme structure. The spatial arrangement of the residues changed by the secondary site mutations and the original substitution can then be visualized. The mutations discussed above all map far from the original mutation on the T4 three dimensional model.</p> <p>A substitution of leucine for tyrosine at position 22 is compensated for by the double mutation of arg18->ser and ser23->lys. When the equivalent residues are mapped on the T4 three dimensional model the changes map in close proximity to the original mutation.</p>
dc.identifier.submissionpathgsbs_diss/238
dc.contributor.departmentMolecular Genetics & Microbiology
dc.description.thesisprogramMolecular Genetics and Microbiology


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