In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage
- PMID: 3061875
- DOI: 10.1101/gad.2.7.801
In situ detection of sequence-specific DNA binding activity specified by a recombinant bacteriophage
Abstract
We have used a recombinant bacteriophage that expresses the DNA-binding domain of C/EBP to optimize conditions for a screening technique that may facilitate the cloning of genes that encode sequence-specific DNA-binding proteins. The method relies on the expression of cDNA inserts in bacteriophage lambda gt11. Fusion protein adsorbed onto nitrocellulose filters is probed with radioactive, double-stranded DNA as a ligand. Two procedures greatly increase the level of binding between ligand and recombinant fusion protein. First, nitrocellulose filters are processed through a denaturation/renaturation regimen using 6 M guanidine hydrochloride. Second, synthetic DNA corresponding to the specific binding site is catenated extensively using DNA ligase. The combination of these procedures leads to remarkably strong detection signals. Specific DNA-binding signals can be detected on duplicate filters, and filters can be washed and reused by repeating the cycle of denaturation/renaturation.
Similar articles
-
A nonradioactive screening method for cloning genes encoding sequence-specific DNA binding proteins.Anal Biochem. 1991 Jan;192(1):17-22. doi: 10.1016/0003-2697(91)90176-t. Anal Biochem. 1991. PMID: 1828652
-
Detection, purification, and characterization of cDNA clones encoding DNA-binding proteins.Curr Protoc Mol Biol. 2001 May;Chapter 12:Unit 12.7. doi: 10.1002/0471142727.mb1207s13. Curr Protoc Mol Biol. 2001. PMID: 18265090
-
Determination of recognition-sequences for DNA-binding proteins by a polymerase chain reaction assisted binding site selection method (BSS) using nitrocellulose immobilized DNA binding protein.Nucleic Acids Res. 1992 Dec 11;20(23):6317-21. doi: 10.1093/nar/20.23.6317. Nucleic Acids Res. 1992. PMID: 1475193 Free PMC article.
-
A positive selection vector for cloning high molecular weight DNA by the bacteriophage P1 system: improved cloning efficacy.Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2056-60. doi: 10.1073/pnas.89.6.2056. Proc Natl Acad Sci U S A. 1992. PMID: 1549564 Free PMC article.
-
Genetic selection for genes encoding sequence-specific DNA-binding proteins.Proc Natl Acad Sci U S A. 1989 May;86(10):3689-93. doi: 10.1073/pnas.86.10.3689. Proc Natl Acad Sci U S A. 1989. PMID: 2657725 Free PMC article.
Cited by
-
H-2RIIBP, a member of the nuclear hormone receptor superfamily that binds to both the regulatory element of major histocompatibility class I genes and the estrogen response element.Proc Natl Acad Sci U S A. 1989 Nov;86(21):8289-93. doi: 10.1073/pnas.86.21.8289. Proc Natl Acad Sci U S A. 1989. PMID: 2554307 Free PMC article.
-
OCSBF-1, a maize ocs enhancer binding factor: isolation and expression during development.Plant Cell. 1990 Sep;2(9):891-903. doi: 10.1105/tpc.2.9.891. Plant Cell. 1990. PMID: 2152133 Free PMC article.
-
Novel member of the zinc finger superfamily: A C2-HC finger that recognizes a glia-specific gene.Mol Cell Biol. 1992 Dec;12(12):5632-9. doi: 10.1128/mcb.12.12.5632-5639.1992. Mol Cell Biol. 1992. PMID: 1280325 Free PMC article.
-
Murine helix-loop-helix transcriptional activator proteins binding to the E-box motif of the Akv murine leukemia virus enhancer identified by cDNA cloning.Mol Cell Biol. 1992 Aug;12(8):3449-59. doi: 10.1128/mcb.12.8.3449-3459.1992. Mol Cell Biol. 1992. PMID: 1321336 Free PMC article.
-
Chicken vitellogenin gene-binding protein, a leucine zipper transcription factor that binds to an important control element in the chicken vitellogenin II promoter, is related to rat DBP.Mol Cell Biol. 1991 Oct;11(10):4863-75. doi: 10.1128/mcb.11.10.4863-4875.1991. Mol Cell Biol. 1991. PMID: 1922023 Free PMC article.
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
