Persistent Link:
http://hdl.handle.net/10150/186223
Title:
Genetic and biochemical studies on PRD1 terminal protein.
Author:
Chang, Stella Shihyi Shiue.
Issue Date:
1993
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:
DNA replication of PRD1, a lipid-containing phage, is initiated by a protein-priming mechanism. The terminal protein encoded by gene 8 acts as a protein primer in DNA synthesis by forming an initiation complex with the 5'-terminal nucleotide, dGMP. The linkage between the terminal protein and the 5'-terminal nucleotide is a tyrosylphosphodiester bond. The PRD1 terminal protein contains 13 tyrosine residues in a total of 259 amino acids. By site-directed mutagenesis of cloned PRD1 gene 8, we replaced 12 of the 13 tyrosine residues in the terminal protein with phenylalanine and the other tyrosine residue with asparagine. Functional analysis of these mutant terminal proteins suggested that tyrosine-190 is the linking amino acid that forms a covalent bond with dGMP. Cyanogen bromide cleavage studies also implicated tyrosine-190 as the DNA-linking amino acid residue of the PRD1 terminal protein. The results further show that tyrosine residues at both the amino-terminal and the carboxyl-terminal regions are important for the initiation complex forming activity. Predicted secondary structures for the regions around the DNA linking amino acid residues were compared in three terminal proteins (φ29, adenovirus-2, and PRD1). While the linking amino acids serine-232 (φ29) and serine-577 (adenovirus-2) are found in β-turns in hydrophilic regions, the linking tyrosine-190 of the PRD1 terminal protein is found in a β-sheet in a hydrophobic region. A 3 kb DNA fragment of phage PRD1 containing the terminal protein and DNA polymerase genes has been cloned into phagemid pEMBLex3. This phagemid expression vector cannot produce enough quantities of soluble terminal protein. We have tried different methods for solubilization of protein aggregates to obtain a renatured form. However, the recovery of protein activity is too low for further terminal protein (TP) purification. To increase the gene 8 expression, we applied PCR methodology. Two primers were designed to contain sequence of two restriction sites for amplifying the TP gene. The PRD1 TP gene was then cloned into pKK223-3. The expression vector pKK223-3 contains a strong tac promoter which is regulated by the lac repressor and induced by the addition of isopropyl-β-D-thiogalactoside (IPTG) to the medium. The recombinant clone was then used for overexpression of terminal protein. TP was purified to near homogeneity and used for functional studies.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Dissertations, Academic.; Microbiology.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Microbiology and Immunology; Graduate College
Degree Grantor:
University of Arizona
Committee Chair:
Junetsu, Ito

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleGenetic and biochemical studies on PRD1 terminal protein.en_US
dc.creatorChang, Stella Shihyi Shiue.en_US
dc.contributor.authorChang, Stella Shihyi Shiue.en_US
dc.date.issued1993en_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.abstractDNA replication of PRD1, a lipid-containing phage, is initiated by a protein-priming mechanism. The terminal protein encoded by gene 8 acts as a protein primer in DNA synthesis by forming an initiation complex with the 5'-terminal nucleotide, dGMP. The linkage between the terminal protein and the 5'-terminal nucleotide is a tyrosylphosphodiester bond. The PRD1 terminal protein contains 13 tyrosine residues in a total of 259 amino acids. By site-directed mutagenesis of cloned PRD1 gene 8, we replaced 12 of the 13 tyrosine residues in the terminal protein with phenylalanine and the other tyrosine residue with asparagine. Functional analysis of these mutant terminal proteins suggested that tyrosine-190 is the linking amino acid that forms a covalent bond with dGMP. Cyanogen bromide cleavage studies also implicated tyrosine-190 as the DNA-linking amino acid residue of the PRD1 terminal protein. The results further show that tyrosine residues at both the amino-terminal and the carboxyl-terminal regions are important for the initiation complex forming activity. Predicted secondary structures for the regions around the DNA linking amino acid residues were compared in three terminal proteins (φ29, adenovirus-2, and PRD1). While the linking amino acids serine-232 (φ29) and serine-577 (adenovirus-2) are found in β-turns in hydrophilic regions, the linking tyrosine-190 of the PRD1 terminal protein is found in a β-sheet in a hydrophobic region. A 3 kb DNA fragment of phage PRD1 containing the terminal protein and DNA polymerase genes has been cloned into phagemid pEMBLex3. This phagemid expression vector cannot produce enough quantities of soluble terminal protein. We have tried different methods for solubilization of protein aggregates to obtain a renatured form. However, the recovery of protein activity is too low for further terminal protein (TP) purification. To increase the gene 8 expression, we applied PCR methodology. Two primers were designed to contain sequence of two restriction sites for amplifying the TP gene. The PRD1 TP gene was then cloned into pKK223-3. The expression vector pKK223-3 contains a strong tac promoter which is regulated by the lac repressor and induced by the addition of isopropyl-β-D-thiogalactoside (IPTG) to the medium. The recombinant clone was then used for overexpression of terminal protein. TP was purified to near homogeneity and used for functional studies.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectDissertations, Academic.en_US
dc.subjectMicrobiology.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineMicrobiology and Immunologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.chairJunetsu, Itoen_US
dc.contributor.committeememberFriedman, Richard L.en_US
dc.contributor.committeememberCharles, Gerbaen_US
dc.contributor.committeememberAdam, Rodney D.en_US
dc.identifier.proquest9322733en_US
dc.identifier.oclc716272481en_US
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.