Functional domain structure analyses of bovine papillomavirus type-1 E1 protein : determination of the nuclear localization signal sequences and mapping of the DNA-binding domain and E1-E2 interaction region /
El is a 605 amino acid, multifunctional regulatory protein for bovine papillomavirus type-I (BPV-1) DNA replication. It has been demonstrated that El is a nuclear protin that specifically binds to the BPV-1 origin of replication and interacts with another early viral protein, E2. This study was in...
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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=742744461&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | El is a 605 amino acid, multifunctional regulatory protein for bovine papillomavirus type-I (BPV-1) DNA replication. It has been demonstrated that El is a nuclear protin that specifically binds to the BPV-1 origin of replication and interacts with another early viral protein, E2. This study was intended to analyze the domain structure for these activities. Using El/p-galactosidase fusion constructs, only the full-length El and the subfragment from amino acids 84-166 guided the fusion proteins to enter the nucleus. Point mutations of three closely spaced clusters of basic residues in the subfragment diminished the nuclear localization of the corresponding fusion protein. These results indicate that these short amino acid sequences are not only necessary but also sufficient for the nuclear transport of the El/p-galactosidase fusion protein. To determine the domain structure for the activities of DNA binding and El-E2 interaction, a series of amino-terminal and carboxy-terminal deletion mutations and small internal mutations of El were constructed in the context of RecA/El fusions. Each of these El mutant fusion proteins was tested for DNA-binding and E2 binding activities in a modified McKay immunoprecipitation assay. The results showed that the El DNA binding domain was located between amino acids 121 and 286. The El sequences from residues 121 to 311 were not only necessary but also sufficient for specific DNA binding and could not be disrupted without functional impairment. The El-E2 interaction region was mapped between residues 121 and 531 of El. This region was found to have two separate domains: the amino-terminal domain, whose function was observed to be cold-sensitive, resided between residues 121 and 286; the carboxy-terminal domain, whose function was not cold-sensitive, resided between amino acids 273 and 531. These two domains overlapped with each other in a region from residues 273 to 286. The sequences within this overlapping region were critical for the function of both domains. Furthermore, competition experiments showed that these two domains competed against each other for their E2 binding activity, suggesting that E2 has only one or closely related binding site(s) for El protein. |
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| Item Description: | Vita. "Major Subject: Medical Sciences". In title, numerals are used. |
| Physical Description: | xiii, 151 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references. |