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).