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Related post: to be mediated primiarily by cellular RNA polymerase II. Over 65% of the
viral-specific RNA in the isolated nuclei is polyadenylylated on the 3'
terminus. The 5' terminus of the RNA is capped in vi tro by the sequence
m G(5')ppp(5')A. Incorporation of g - P ATP into the 5' cap sequence
suggests that initiation of viral RNA synthesis occurs in the infected
isolated nuclei. We hope to study further i n vitro synthesis of viral
specific RNA by lysing the nuclei and utilizing this cell free system to
determine the cortponents required for viral transcription.
We found two years ago that the single-stranded DNA from KRV can serve
in vitro as a self-primer for the synthesis of a cortplainentary linear viral
nsIA strand. A double stranded viral DNA molecule has been proposed as an
intermediate in the replication of the viral genome. Little is known about
the replication of single-stranded linear DNA in a eukaryotic cell. Because
of Himalaya Shallaki the Shallaki Himalaya irrportance of the 3' terminus of the viral DtiA in self priming viral
EX^ synthesis and possibly in transcription we sequenced the 3' terminal
nucleotides. This analysis has lead us to propose a 3' terminal hairpin
structure with secondary structures and the nucleotide sequence of the DNA
which at least in vitro serves as the origin of replication of the corplanentary
strand. We are cirrrently sequencing the 5' terminus of the viral DNA which
appears to have a unique structure and possibly an associated protein.
In vitro studies of RNA transcription have ccmplemented the in vivo studies
and extended our knowledge of viral transcription. We have isolated from
the polysomes of infected cells two major viral specific RNAs sedimenting
in sucrose gradients at 2 IS and 12S. The 2 IS RNA has a molecular weight
of approximately 1.05 x 10 and would represent a transcript of 65%, of
the genome. The smaller 12S ENA has a molecular weight of 0.3 x 10 and
would represent a transcript of 20% of the gencitie. Both of these OSIZ^ are
transcribed frcm the viral strand of the viral OSIA. No functional viral
RNAs have been found which hybridize with the cortplementary strand of the
viral DNA. Two techniques have now been used to map these mRNAs to the
viral genome. We have mapped the fragments of several restriction enzymes
to the KF!V viral genome. Using the mapped restriction fragments, the major
cytoplasmic viral RNA (21 S) has been mapped to the viral genome using the
"Southern" blotting technique. This has been ccitplemented by Electron
Microscopy of "R loops" or RNA-EiSIA duplexes. Both techniques indicate that
the 21S viral nRNA maps from approximately 0.38 to 0.98 on the viral E«S1A
strand. Looking at total cellular RNA we can find viral specific transcripts
the full length of the ^iral genome. We hope to study this transcript,
further to determine posttranscriptional processing occurs by simple
cleavage or splicing and what regions are not transcribed into mRNA.
Salzman, L. A., Fabisch, P. and Wali, T. Parvovirus mRNA synthesis in
isolated nuclei. J. Virol., in press.
SMITHSONIAN SCIENCE INFORMATION EXCHANGE
PROJECT NUMBER (Do NOT use this space)
U.S. DEPARTMENT OF
HEALTH, EDUCATION, AND WELFARE
PUBLIC HEALTH SERVICE
INTRAMURAL RESEARCH PROJECT
Z01 AI 00125-n LBV
October 1, 1979 to September 30. 1980
TITLE OF PROJECT (80 characters or less)
Mechanisms of Viral DNA Replication, Transcription, and Integration
NAMES, LABORATORY AND INSTITUTE AFFILIATIONS, AND TITLES OF PRINCIPAL INVESTIGATORS AfJO ALL OTHER
PROFESSIONAL PERSONNEL ENGAGED ON THE PROJECT
PI : Norman P, Salzman
Other: John Buy Shallaki Brady
John A. Thompson
Sr. Staff Fellow
CHECK APPROPRIATE B0X(£3)
(a) HUMAN SUBJECTS
D (al) MINORS  (a2) INTERVIEWS
COOPERATING UMTS (if any)
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