Mutational analysis of a conserved sequence motif on the BPV-1 E1 protein and reconstitution of a minimal functional replication origin /
The BPV- I genome is maintained in transfected cultured cells and infected host basal cells as a circular episome complexed with host-derived histone proteins as a minichromosome. Otherwise dependent on the host cell replication machinery, BPV-1 provides two replication proteins, El and E2, which ar...
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| Format: | Thesis Book |
| Language: | English |
| Published: |
[Place of publication not identified] :
[publisher not identified] ;
1996.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=739669521&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | The BPV- I genome is maintained in transfected cultured cells and infected host basal cells as a circular episome complexed with host-derived histone proteins as a minichromosome. Otherwise dependent on the host cell replication machinery, BPV-1 provides two replication proteins, El and E2, which are absolutely required for replication of DNA containing the viral origin in vivo. The 605 amino acid El protein is the major viral replication protein and the transcriptional transactivator E2 enhances BPV-1 DNA replication. The experiments in this study were intended to yield information about the trans and cis functional requirements of BPV- I for DNA replication and transformation. Sequence analysis of all known papillomavirus El sequences for conserved sequence motifs revealed the conservation, within the first 25-60 amino terminal residues, of a serine followed by a stretch of acidic amino acids. This high degree of conservation indicates a potential biological role for the motif. Mutational analysis was used to investigate the biological relevance of the corresponding site in the El protein of BPV-1, serine 48. Reduced transformation capacity and DNA replication ability were observed for the mutant BPV- 1 genome. The mutant El open reading frame was negative for replication competency when heterologously expressed with the E2 protein in trans to an origin-containing plasmid DNA. The results described above indicate that Ser-48, directly or indirectly, has a role in the normal functioning of the El protein. The BPV-1 replication origin has an AT-rich region, an initiation protein binding site (E1BS), and a site for binding by a transcriptional transactivator (E2BS). The involvement of cis-regulatory elements and their organizational requirements in the regulation of BPV-1 DNA replication was investigated by the reconstitution of a functional minimal BPV-1 origin. An absolute requirement was found for the AT-rich region and at least one copy of the E2 protein binding site. Additional copies of the E2BS were found to "rescue" the replication competency of the reconstituted origin to the level seen for the native BPV-1 replication origin in the plasmid background. |
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| Item Description: | Vita. "Major Subject: Medical Sciences". |
| Physical Description: | x, 107 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references: pages 88-105. |