Characterization ofcis-acting elements of influenza A virus RNA.
Item
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Title
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Characterization ofcis-acting elements of influenza A virus RNA.
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Identifier
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AAI9417487
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identifier
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9417487
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Creator
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Li, Xingqiang.
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Contributor
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Adviser: Peter Palese
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Date
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1994
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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, Microbiology | Biology, Molecular
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Abstract
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Influenza A virus is an enveloped virus that contains a segmented RNA genome of negative polarity. My work has centered on studying cis-acting elements of influenza virus RNAs. First, an in vitro RNA synthesis system was established to analyze the promoter required for virion RNA (vRNA) synthesis. Substitution mutations were introduced into each of the first 13 positions of the 3{dollar}\sp\prime{dollar} noncoding sequence of a complementary RNA (cRNA) template, and the effects of these mutations on vRNA synthesis were determined. The first 11 nucleotides of the 3{dollar}\sp\prime{dollar} noncoding sequence were found to contain the minimum promoter required for vRNA synthesis. It was also found that addition of extra nucleotides at the 3{dollar}\sp\prime{dollar} end decreased the promoter activity of the templates, indicating that the influenza virus polymerase does not recognize an internal promoter efficiently.;The mutations in the model RNAs were also examined in vivo using a ribonucleoprotein (RNP) transfection system. In contrast to the in vitro system, it was found that the majority of mutations at the 3{dollar}\sp\prime{dollar} terminal sequence significantly decreased or abolished expression of the model RNAs containing the chloramphenicol acetyltransferase (CAT) reporter gene. These results suggest that the cRNA promoter overlaps other essential cis-acting elements required for CAT expression in vivo.;The second part of my studies on cis-acting elements concerned a further analysis of the polyadenylation signal of influenza A virus RNAs. Earlier work had shown that a stretch of uridines near the 5{dollar}\sp\prime{dollar} end of the virion RNAs and the RNA duplex made up of the 3{dollar}\sp\prime{dollar} and 5{dollar}\sp\prime{dollar} terminal sequences adjacent to the U stretch are involved in polyadenylation. I have further characterized the polyadenylation signal of influenza virus RNAs using the RNP transfection system. I found that (1) the optimal length of the U stretch is 5 to 7 uridine residues; (2) the sequence upstream of the U stretch at the 5{dollar}\sp\prime{dollar} end is not involved in polyadenylation; and (3) the optimal distance between the 5{dollar}\sp\prime{dollar} end and the U stretch is 16 nucleotides. The combination of these specific features defines the polyadenylation site of influenza virus RNAs.
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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.
