Transcriptional regulation of hepatitis B virus by sequence-specific DNA-binding proteins.
Item
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Title
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Transcriptional regulation of hepatitis B virus by sequence-specific DNA-binding proteins.
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Identifier
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AAI8820873
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identifier
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8820873
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Creator
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Karpen, Saul Joseph.
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Contributor
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Adviser: George Acs
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Date
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1988
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Molecular
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Abstract
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Two major messenger RNAs (RNAs) are transcribed from the Hepatitis B Virus (HBV) genome. Although both are involved in viral gene expression, one of these, the 3.5 kilobase (kb) mRNA, also plays a central role in HBV replication. In addition, published reports have repeatedly demonstrated that 3.5 kb mRNA expression is highly cell-specific: limited to cell lines derived from human hepatocytes (the natural host of HBV replication). In this thesis, studies exploring the factors controlling the cell-specific expression of the 3.5 kb mRNA were undertaken, with a particular emphasis placed on the possible role of sequence-specific DNA-binding proteins.;An EcoRI-Alul restriction fragment (HBV subtype ayw; nucleotides (nt) 1-1878, including the start site of 3.5 kb mRNA transcription and {dollar}\approx{dollar}1.8 kb of 5{dollar}\sp\prime{dollar} flanking sequences) was inserted 5{dollar}\sp\prime{dollar} to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene of pSVoCAT, to create the recombinant plasmid pEcoAluCAT. Transfection experiments showed that pEcoAluCAT-driven CAT expression paralleled the human hepatocyte-specific expression of the 3.5 kb mRNA. In vivo CAT competition assays (in the human hepatoblastoma-derived cell line HepG2) demonstrated that pEcoAluCAT expression was primarily dependent on two cis-acting DNA elements; within the previously defined HBV enhancer (ENH; nt 1082-1378), and core promoter (CP; nt 1645-1888) regions. Furthermore, pEcoAluCAT expression was shown to involve the interaction of trans-acting HepG2 cellular factors, with sequences within these two DNA elements. These data were corroborated with in vitro binding experiments, using gel mobility shift assays. Appropriate DNA competition experiments demonstrated the presence of distinct HepG2 nuclear proteins, containing high affinity DNA-binding domains specific for sequences within ENH and CP elements. Assays performed with extracts from non-human and non-hepatocyte nuclei revealed shift patterns different from those obtained from HepG2 nuclear extract. Methylation interference assays detailed the protein binding regions within the CP (between nt 1655-1684), and ENH (nt 1089-1170) DNA elements. Both binding sites contained palindromes, and homologies were seen with a limited number of sequenced genes.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
