Mutagenesis of the bovine papillomavirus type 1 E1 open reading frame : analysis of replication and transformation /
A mutagenesis system for creating translational termination mutations and in-frame insertion and deletion mutations was developed utilizing a vector that expressed a recA-E1-lacZ tribrid fusion gene. Translational termination linkers were randomly inserted into the plasmid and mapped by restriction...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1995.
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| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=742162241&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | A mutagenesis system for creating translational termination mutations and in-frame insertion and deletion mutations was developed utilizing a vector that expressed a recA-E1-lacZ tribrid fusion gene. Translational termination linkers were randomly inserted into the plasmid and mapped by restriction endonuclease digestion. Linkers inserted within the El ORF were digested with Exonuclease III and S1 nuclease to created in-frame deletions. The majority of deletion mutations obtained were in a region of the El protein homologous to the ATP binding/ATPase domain of the SV40 large T antigen. All of the El mutants were analyzed for transient DNA replication at 320C, 370C, and 390C. None of the El mutants could replicate transiently, at any of the tested temperatures, in the context of the viral genome. All of the El mutants could be complemented with an E2 mutant, E2TTL, proving that the viral mutants were defective for a trans-acting factor encoded by the El ORF. The El ORF mutants created in this study were analyzed for their efficiency of inducing foci formation in mouse C127 cells. All El mutants had a reduced transformation efficiency when compared to wildtype. However, two levels of transformation were observed. The mutations in the ATP binding/ATPase domain were reduced 50% and mutations in the DNA binding domain were reduced 80%. The El ORF mutants were created in a bacterial vector that expresses a RecA-El fusion protein that retains specific binding to the viral origin of replication. This protein was shown to be specifically labeled by the covalent affinity label, ox-ATP. The El ORF mutants were expressed as RecA-El fusion proteins and analyzed for the ability to be labeled by ox-ATP. All of the mutants posessed the ability to be labeled by ox-ATP revealing that some other function has been affected resulting in the defect for viral DNA replication. Further analysis of El protein activities such as E2TA binding and DNA binding by the El mutants will allow a better understanding of the El functions which influence viral transformation and replication. |
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| Item Description: | Vita. "Major Subject: Genetics". In title, numerals are used. |
| Physical Description: | x, 105 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references. |