CLONING OF BACTERIOPHAGE-PHI29 GENE 15; ISOLATION, OVERPRODUCTION AND PURIFICATION OF PHI29 LYTIC ENZYME.

Persistent Link:
http://hdl.handle.net/10150/184008
Title:
CLONING OF BACTERIOPHAGE-PHI29 GENE 15; ISOLATION, OVERPRODUCTION AND PURIFICATION OF PHI29 LYTIC ENZYME.
Author:
SAEDI, MOHAMMAD SAEED.
Issue Date:
1987
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
A spontaneous deletion mutant of Bacillus phage φ29 (φ29Δ1) is characterized in the first part of this study. This mutant has a 1,112 base pair deletion, which covers the entire coding sequence of genes 14 and 15 including the early promoter, B2. While lysis is very delayed, the phage DNA synthesis and internal phage development appears to be normal in the cells infected with this deletion mutant. These results indicate that the early functions are intact in φ29Δ1. Results also suggest that genes 14 and 15 are dispensable for bacteriophage φ29 growth, and that the B2 promoter may also be despensable for the early functions of φ29. To further explore the function of gene 15, a DNA fragment of φ29 chromosome, encoding the entire sequence of this gene, has been cloned into the Escherichia coli expression vector pPLc245, under the control of the phage lambda major early leftward promoter, PL. Upon heat induction, a protein with an apparent size of 26 kdal was over-produced. This protein has been purified to near homogeneity and confirmed to be the product of gene 15 by amino acid sequence analysis of its N terminus. The purified product of gene 15 has a lysozyme activity similar to the other phage-type lysozymes: products of phage T4 gene e and of phage P22 gene 19. This is the first lysozyme to be cloned and purified from a gram positive system. Bacteriophage φ29 lysozyme has been characterized in the last part of this study. Results show that this enzyme seems to more active than hen egg-white lysozyme against B. subtilis, E. coli, and M. lysodeikticus cells. Most of the characteristics of φ29 lysozyme appears to be similar to the P22 and T4 lysozymes, however, φ29 lysozyme seems to be about 2 times more thermostable than the other two lysozymes.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Bacteriophages.; Molecular cloning.; Lysozyme.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Molecular and Cellular Biology; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Ito, Junetsu

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleCLONING OF BACTERIOPHAGE-PHI29 GENE 15; ISOLATION, OVERPRODUCTION AND PURIFICATION OF PHI29 LYTIC ENZYME.en_US
dc.creatorSAEDI, MOHAMMAD SAEED.en_US
dc.contributor.authorSAEDI, MOHAMMAD SAEED.en_US
dc.date.issued1987en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractA spontaneous deletion mutant of Bacillus phage φ29 (φ29Δ1) is characterized in the first part of this study. This mutant has a 1,112 base pair deletion, which covers the entire coding sequence of genes 14 and 15 including the early promoter, B2. While lysis is very delayed, the phage DNA synthesis and internal phage development appears to be normal in the cells infected with this deletion mutant. These results indicate that the early functions are intact in φ29Δ1. Results also suggest that genes 14 and 15 are dispensable for bacteriophage φ29 growth, and that the B2 promoter may also be despensable for the early functions of φ29. To further explore the function of gene 15, a DNA fragment of φ29 chromosome, encoding the entire sequence of this gene, has been cloned into the Escherichia coli expression vector pPLc245, under the control of the phage lambda major early leftward promoter, PL. Upon heat induction, a protein with an apparent size of 26 kdal was over-produced. This protein has been purified to near homogeneity and confirmed to be the product of gene 15 by amino acid sequence analysis of its N terminus. The purified product of gene 15 has a lysozyme activity similar to the other phage-type lysozymes: products of phage T4 gene e and of phage P22 gene 19. This is the first lysozyme to be cloned and purified from a gram positive system. Bacteriophage φ29 lysozyme has been characterized in the last part of this study. Results show that this enzyme seems to more active than hen egg-white lysozyme against B. subtilis, E. coli, and M. lysodeikticus cells. Most of the characteristics of φ29 lysozyme appears to be similar to the P22 and T4 lysozymes, however, φ29 lysozyme seems to be about 2 times more thermostable than the other two lysozymes.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBacteriophages.en_US
dc.subjectMolecular cloning.en_US
dc.subjectLysozyme.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineMolecular and Cellular Biologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorIto, Junetsuen_US
dc.contributor.committeememberBernstein, Carolen_US
dc.contributor.committeememberSpizizen, Johnen_US
dc.contributor.committeememberLindell, Tomen_US
dc.contributor.committeememberMount, Daviden_US
dc.identifier.proquest8709898en_US
dc.identifier.oclc698371500en_US
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