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1994-10-25
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Document 3313
DOCN M94A3313
TI Study of differential RNA expression during HIV infection using
arbitrarily primed PCR.
DT 9412
AU Boyer V; Ferre F; Pezzoli P; Jensen FC; Carlo DJ; Immune Response
Corporation, Carlsbad CA.
SO Int Conf AIDS. 1994 Aug 7-12;10(1):104 (abstract no. PA0036). Unique
Identifier : AIDSLINE ICA10/94369262
AB OBJECTIVE: To identify differential RNA expression during HIV infection
of a T cell line (HUT78 cell line), we used the RNA arbitrarily primed
PCR (RAP) fingerprinting method. METHODS: Total RNA was isolated from
HUT78 cells and HIV-1 (HIVZ321 strain)-infected HUT78 cells. cDNA was
generated using an arbitrarily selected primer. The first PCR cycle was
run at low stringency (40 degrees C), using an arbitrary primer
different from that of the cDNA step and three RNA concentrations for
each assay, followed by 40 cycles at high stringency (60 degrees C). A
total of 30 arbitrary primers were screened using this methodology.
RESULTS: A reproducible pattern for 3 PCR products revealed differential
expression between the control uninfected and the infected cell line.
Different patterns of differential expression were observed for all 3
PCR products. Two genes were either up-regulated in the HIV-infected
cells or specifically expressed in HUT78 + HIV cells. Finally, the third
gene was down-regulated in the non-infected cells. The DNA sequences of
these bands were obtained by excising them from the polyacrylamide gel,
re-amplifying them by PCR using the same arbitrary primers that first
generated them and cloning them into a vector. The up-regulated gene is
gamma actin. The specific gene expressed only in the HUT78 + HIV cells
in the HIV Nef gene. Finally, the down-regulated product is the unknown
gene. The differential expression of these three products identified by
RAP-PCR was confirmed, by Northern, and then by RT-PCR using specific
primers designed from the sequenced gene. CONCLUSIONS: Thus, the rapid
and efficient RAP fingerprinting method allowed us to detect genes 1)
only expressed in a cellular type (for Nef). 2) up-regulated by HIV (for
gamma actin). 3) unknown, modulated by HIV, and expressed at a low
level. The relevance of the down regulation of the unknown gene is under
investigation.
DE Actins/GENETICS Down-Regulation (Physiology) DNA Fingerprinting DNA
Primers/GENETICS DNA, Viral/GENETICS/ISOLATION & PURIF Gene Expression
Regulation, Viral Genes, nef Human HIV
Infections/*GENETICS/*MICROBIOLOGY HIV-1/*GENETICS Polymerase Chain
Reaction/METHODS RNA, Viral/*GENETICS Up-Regulation (Physiology)
MEETING ABSTRACT
SOURCE: National Library of Medicine. NOTICE: This material may be
protected by Copyright Law (Title 17, U.S.Code).