Molecular analysis of the Sendai virus genome.
Item
-
Title
-
Molecular analysis of the Sendai virus genome.
-
Identifier
-
AAI9304716
-
identifier
-
9304716
-
Creator
-
Park, Kyeong Hoon.
-
Contributor
-
Adviser: Mark Krystal
-
Date
-
1992
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Biology, Molecular | Biology, Microbiology
-
Abstract
-
In the first part of this thesis, an artificial mini-genome of Sendai virus, a prototype paramyxovirus, was developed as an initial step to get around the problems imposed by the viral genome. The mini-genome RNA, called Send-CAT, was synthesized via in vitro T7 phage RNA polymerase transcription from a cDNA containing the antisense coding region of chloramphenicol acetyltransferase (CAT) gene flanked by 5{dollar}\sp\prime{dollar} 145 and 3{dollar}\sp\prime{dollar} 119 terminal sequences of the viral genome. When introduced into cells and followed by Sendai virus infection, the RNA was transcribed and translated, as detected by CAT activity. The efficiency of CAT expression was markedly increased by pre-incubating the RNA with cytoplasmic cell extract derived from either Sendai virus-infected or uninfected cells, suggesting that a host cell component(s) may enhance viral gene expression. Furthermore, the Send-CAT RNA was also readily replicated and packaged into progeny virions. Once packaged, the Send-CAT RNA could be stably passaged and even be grown to a very high titer. These studies provided a well-suited system for examining various cis-acting signals of the viral genome.;In the second part of the thesis, an in vivo model for pseudo-templated transcription of Sendai virus was developed. During transcription of the P/C gene, the Sendai virus transcriptase inserts one or more nontemplated guanine nucleotides at position 1053. Based on the Send-CAT mini-genome, a derivative construct was made that contained a stretch of nucleotide sequence spanning the editing site of the P/C gene. When the resulting construct, called PC-CAT, was transfected into cells which were then infected with Sendai virus, 6.5% of its mRNAs contained a nontemplated G nucleotide at the expected position, indicating that the sequence normally responsible for promoting RNA editing in Sendai virus genome was now functionally residing in the PC-CAT construct. By progressively deleting the P/C gene sequence from the 5{dollar}\sp\prime{dollar} end (in the genome sense), 24 bases were shown to be sufficient for the pseudo-templated transcription in the model system. In addition, mutational analysis of the consensus sequence showed that the three C residues preceding the insertion site were essential and could not be substituted by G residues. This system will now permit precise delineation of the cis-acting element(s) required for various aspects of RNA editing. (Abstract shortened by UMI.).
-
Type
-
dissertation
-
Source
-
PQT Legacy CUNY.xlsx
-
degree
-
Ph.D.
