Abstract
Z-DNA affinity adsorbtion 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 inretention 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 511-516 |
| Number of pages | 6 |
| Journal | Journal of Molecular Biology |
| Volume | 203 |
| Issue number | 2 |
| DOIs | |
| State | Published - Sep 20 1988 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Virology
Cite this
Z-DNA-binding proteins in Escherichia coli purification, generation of monoclonal antibodies and gene isolation. / Lafer, Eileen M; Sousa, Rui J; Rich, Alexander.
In: Journal of Molecular Biology, Vol. 203, No. 2, 20.09.1988, p. 511-516.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Z-DNA-binding proteins in Escherichia coli purification, generation of monoclonal antibodies and gene isolation
AU - Lafer, Eileen M
AU - Sousa, Rui J
AU - Rich, Alexander
PY - 1988/9/20
Y1 - 1988/9/20
N2 - Z-DNA affinity adsorbtion 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 inretention 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.
AB - Z-DNA affinity adsorbtion 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 inretention 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=0024293263&partnerID=8YFLogxK
U2 - 10.1016/0022-2836(88)90017-4
DO - 10.1016/0022-2836(88)90017-4
M3 - Article
C2 - 3058987
AN - SCOPUS:0024293263
VL - 203
SP - 511
EP - 516
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 2
ER -