Functional analysis of GC element binding and transcription in the hamster dihydrofolate reductase gene promoter.
Dihydrofolate reductase (DHFR) gene expression is required for cell growth. The DHFR gene promoter contains several GC elements capable of binding the transcription factor Sp1. In this report we have characterized the effect of protein(s) binding to these sequence elements in the Chinese hamster DHFR promoter on transcription. We have constructed a series of deletions containing from 896 to 103 bp 5' to the start of translation. The protein binding domains have been mapped by DNAse I footprint analysis using HeLa nuclear extract, and the function of the protein-binding elements has been assessed by in vitro transcription and transient CAT expression. Maximal transcription in vitro and CAT expression is obtained with a construct containing 3 GC elements extending to position -184. Removal of GC element binding factor(s), by competition with an oligonucleotide containing an Sp1 binding site, completely abolishes transcription in vitro and significantly diminishes CAT expression. Ten-fold higher molar excess of competitor is required to abolish SV40 early transcription, suggesting that the GC element interactions in the DHFR promoter are different from those in the SV40 early region. Co-transfection of a DHFR CAT construct with an expressor of Sp1 dramatically increased CAT expression in Drosophila cells.
Swick
A G
AG
Lineberger Cancer Research Center, University of North Carolina, Chapel Hill 27599-7295.
Blake
M C
MC
Kahn
J W
JW
Azizkhan
J C
JC
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D006224:Cricetinae; D003596:Cytosine; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D005796:Genes; D006147:Guanine; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009710:Nucleotide Mapping; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D006147:Guanine; D003596:Cytosine; D013762:Tetrahydrofolate Dehydrogenase; D003850:Deoxyribonuclease I