Using the bacteria-2-hybrid system to determine the role of S2 in the equine infectious anemia virus (EIAV) life cycle
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After in vivo and in vitro experiments comparing wild type and S2 knockout strains of EIAV were conducted, the role of one small accessory protein still eluted discovery, S2. S2, is a small protein that has a nuclear localization sequence, SH3 binding domain ligand, and is only 19 kD in size. It was used in a bacterial two-hybrid to discover interacting host cell partner proteins from a mouse lung cDNA library via transcriptional activation. The experiment used a genetically modified strain of E. coli to test interactions between fusion products made by fusing S2 with lambda repressor, and random cellular proteins to RNA polymerase (RNAP). Since a reporter strain cassette is contained within the genetically altered bacteria, the lambda repressor can bind to the altered lambda operator, freeing the S2 particle for interaction. If a single bacteria has the plasmid that codes for S2 and the interacting partner, then the interacting partner can bind to S2, guiding RNAP to transcribe the reporter gene, carbenicillin. The experiment used plates with differing carbenicillin concentrations to test for stronger protein-protein interactions, and has resulted in about thirty different putative positive partners.
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Includes bibliographical references (leaves 26-27).
Weidner, Jason Lee (2003). Using the bacteria-2-hybrid system to determine the role of S2 in the equine infectious anemia virus (EIAV) life cycle. Texas A&M University. Available electronically from