Abstract

Tat and Rev, small proteins encoded by the Human Immunodeficiency Virus type 1 (HIV-1), regulate its pattern of gene expression within infected cells. Tat increases transcription from the integrated pro virus by approximately a thousand-fold; Rev effects a shift in the pattern of splicing of the viral mRNA. Both are likely to interact with cellular proteins in executing their respective functions. Tat has additional activities (including neurotoxicity and inhibition of lymphocyte activation) which may be extraneous to its viral functions.

In order to identify cellular proteins interacting with Tat and Rev, the genes for both were cloned into plasmid vectors of the yeast two-hybrid system. This system enables large-scale screening of the members of a cDNA library for interaction with a protein of interest by the functional reconstitution of the GAL4 transcriptional activator.

The search for Rev-interacting proteins from a mouse embryo cDNA library was conducted with a one-reporter-gene strain of yeast; all of the initial candidate Rev-interacting proteins were later demonstrated to be false-positives. A two-reporter-gene yeast strain was used to identify Tat-interacting proteins from the same library, enabling rapid elimination of false-positives from consideration. A total of twenty-two Tat-interacting proteins were found, one of which was a partial cDNA for a novel brain extracellular matrix protein. A "spurious PCR" strategy was used to clone additional 5' fragments of the cDNA until all 2457 nucleotides of coding sequence (and a small 5' untranslated sequence) were obtained. This protein contains nine epidermal growth factor-like (EGF-like) repeats, cysteine-rich motifs of about forty amino acids which may mediate intra- and inter-protein binding.

The significance of Tat's interaction with this brain protein remains uncertain. The interaction may be "bridged" or mediated by an endogenous yeast protein, probably a transcription factor, since the original fragment of the protein strongly activates transcription when tethered to a promoter. Its function in the developing and mature nervous system remains to be discovered.

LLU Discipline

Microbiology and Molecular Genetics

Department

Microbiology

School

Graduate School

First Advisor

John J. Rossi

Second Advisor

Kelvin Hill

Third Advisor

Donna D. Strong

Fourth Advisor

Barry L. Taylor

Fifth Advisor

Anthony Zuccarelli

Degree Name

Doctor of Philosophy (Medical Science)

Degree Level

Ph.D.

Year Degree Awarded

1997

Date (Title Page)

6-1997

Language

English

Library of Congress/MESH Subject Headings

HIV-1; Gene Expression Regulation; Genes, rev. -- immunology; Genes, tat, -- immunology; Genes, Regulator; Binding Sites; Polymerase Chain Reaction; Sequence Analysis, DNA.

Type

Dissertation

Page Count

x; 169

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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