Z-DNA-binding proteins in Escherichia coli purification, generation of monoclonal antibodies and gene isolation.
Z-DNA affinity adsorption of an Escherichia coli lysate in the presence of excess B-DNA results in a 1000-fold enrichment for three proteins with apparent molecular weights, on SDS/polyacrylamide gel electrophoresis, of 50,000, 90,000 and 100,000. When retention of these proteins on resins constructed with Z-DNA (Br-poly(dG-dC).poly(dG-dC)) was compared with retention on resins constructed with B-DNA or Br-B-DNA, it was found that approximately 100-fold more of the 50,000 Mr protein, 1000-fold more of the 90,000 Mr protein, and greater than 1000-fold more of the 100,000 Mr protein was retained on the Z-DNA resin. No difference in retention on the B-DNA versus brominated B-DNA resin was found, indicating that the increased retention on the Z-DNA resin was not due to bromination of the Z-DNA. This demonstration of Z-DNA-specific binding in vitro makes these proteins candidates for binding to Z-DNA in vivo. In an effort to determine the function of these proteins we have prepared monoclonal antibodies against each protein and isolated its respective gene. Western blot analysis of lysogens carrying these genes confirms their identity and shows that the complete coding region and promoter for each gene has been cloned.
Lafer
E M
EM
Dept. of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Sousa
R J
RJ
Rich
A
A
D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D001426:Bacterial Proteins / Q000276:immunology / Q000302:isolation & purification*; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000276:immunology / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D005798:Genes, Bacterial
D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D004247:DNA