Abstract
DNA polymerase beta (beta-pol) is a housekeeping enzyme considered to be involved in DNA repair in vertebrate cells. We cloned a fragment of genomic DNA spanning the first two exons of the human beta-pol gene and approximately 11 kilobases of the flanking region. The segment just 5' of the transcription start site can direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene in HeLa cells. A sequence containing only 113 base pairs of flanking DNA has promoter activity, and various constructs containing up to 4.8 kilobases of flanking sequence are expressed at a similar level, indicating that with this assay the important regulatory elements are located within or proximal to the approximately 100-bp core promoter. S1 nuclease mapping was used to show that transcription of the transfected genes is initiated at the same position as the endogenous beta-pol gene. The region upstream of the transcription start site is G + C rich and contains neither CAAT nor TATA boxes, but does have three decanucleotide elements matching high affinity binding sites for the RNA polymerase II transcription factor Sp1. Extending 5' from position -39 and surrounded by Sp1 consensus binding elements, there is a 10-nucleotide sequence with perfect dyad symmetry, GTGACGTCAC. Similar sequences are found in a number of cellular and viral promoters, including several adenovirus promoters. Experiments to test whether the core beta-pol promoter is activated by the adenovirus early region products showed that cotransfection with an adenovirus expression plasmid strongly activates expression of the beta-pol promoter.
Highlights
A doublet of about 326-330 nucleotides is predicted for correctly initiated transcripts. This pattern is observed with both plasmids (Fig. 6), indicating correct initiation of transcription. These results show that both plasmids express similar levels of RNA, and this is consistent with our observation of similar CAT activity with each plasmid (Fig. 6)
Two plasmids with deletions of 3 and 44 bp showed reduced activity (Table 111).These results demonstrate that thedecanucleotide element is required for full promoter activity
The gene for DNA polymerase @ is considered among the group of cellular housekeeping genes that typically are expressed in most if not all tissues at relatively low levels
Summary
Vol 263,No 32,Issue of November 15,pp. 1699-16998,1988 Printed in U.S.A. From theLaboratory of Biochemistry, NationalCancer Institute, National Institutesof Health, Bethesda, Maryland20892. Multiple determinations were made The probe used for S1 mapping of the RNA expressed from the of each nucleotide, and both strands were sequenced except for about transfected plasmids was made by labeling ppP2 at the EcoRI site 200 bp at the5' end of the BglII-KpnI fragment. The junction of the 8- This primer is complementary to positions +173 to +188 of the first pol and CAT fragments was sequenced to confirm the structure of exon, and a labeled probe was prepared by extending this to thKe pnI the plasmid. Plasmid pop was made by reaction using the above primer to sequence a segment of genomic cutting pap with HindIII and MluI (position -51) and religating DNA by the chain termination method [19]. Cutting with HindIII and AatII (position -41) and treatment with T4 DNA polymerase
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