Abstract
A factor present in nuclear extracts of NIH-3T3 fibroblasts was identified which binds to a region between -315 and -295 with respect to the start of transcription in the mouse alpha 2(I) collagen promoter. This factor was assayed using three different DNA binding assays. Evidence that this factor is nuclear factor 1 or a related protein is based on competition experiments using fragments of another promoter that contains a nuclear factor 1 binding site as well as a fragment containing a consensus sequence for nuclear factor 1 binding sites. The nucleotide sequence of the binding site in the alpha 2(I) promoter. TCGN5GCCAA, presents similarities to the sequence published previously as a consensus recognition sequence for nuclear factor 1. Methylation interference experiments indicate that the factor interacts at least with two successive G residues in the major groove complementary to the two successive C residues in the GCCAA motif. This factor is different from a factor that binds to a CCAAT element between -84 and -80 in the mouse alpha 2(I) collagen promoter. This was shown by competition experiments using both crude NIH-3T3 fibroblast nuclear extracts and a partially purified CCAAT binding factor. These results suggest that in the alpha 2(I) collagen promoter nuclear factor 1 and a CCAAT binding factor bind to separate sites, despite analogies in their recognition sequences.
Highlights
A factor present in nuclear extracts of NIH-3T3 fibroblasts was identified which binds to a region between -315 and -295 with respect to the start of transcription in the mouse aZ(I)collagen promoter
Around 80 bp’ upstream of the startof transcription has been identified (2, 11).This CCAAT sequence is present at the same distance from the start of transcription in several cellular and viral promoters
The analogies in sequences between the nuclear factor l binding sites and CCAAT sites raise the possibility that these two sites arerecognized by one and the same factor
Summary
Several eukaryotic factors that bind to specific DNA sequences have been identified recently. The nuclei were resuspended in an extraction buffer in a volume corresponding to between 2.5 and 4 times the initial volume of packed cells so that thefinal NaCl concentration was 0.35M. This suspension was slowly stirred for 30 min at 4 “C. Plasmid pBSla was constructed by cloning a double-stranded oligonucleotide that corresponds to the putative nuclear factor 1 binding site in the a2(I)collagen promoter between the BarnHI and HindIIIsites of pUC19. Plasmids were cut by one restriction enzyme, treated by alkaline phosphatase, extracted with phenol/chloroform/isoamyl alcohol, precipitated by ethanol, labeled at the5’ ends usingT4 polynucleotide kinase, cleaved with a second restriction enzyme, and the appropriate fragments separated by electrophoresis on a 6% polyacrylamide gel. 5 pl of a 2,000-fold dilution of the enzyme (1 mg/ml) was used for naked DNA and up to 50 times more for reactions containing pAZ IO09
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