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Mol Cell Biol. 1988 January; 8(1): 35-41

Both upstream and intron sequence elements are required for elevated expression of the rat somatic cytochrome c gene in COS-1 cells.

Department of Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611.

ABSTRACT

To investigate the transcriptional control of nuclear-encoded respiratory genes in mammals, we have performed a deletional analysis of cis-acting regulatory sequences in the rat somatic cytochrome c gene. Three major regions are required for maximal expression of the transfected gene in kidney cell lines CV-1 and COS-1. One of these, region III (+71 to +115 from the transcription initiation site), is an unusual intragenic controlling element found in the 5' end of the first intron, while the other two, region I (-191 to -165) and region II (-139 to -84), define the upstream promoter. Region II contains two consensus CCAAT boxes and mediates a constitutive level of expression in both cell lines. In contrast, regions I and III are both required for the increased promoter activity observed in COS-1 cells compared with promoter activity observed in CV-1 cells, and the regions function individually as competitors with the full promoter for trans-acting factors or complexes. Region III contains a perfect octanucleotide homology with region I in addition to a consensus Sp1-transcription-factor-binding site. Promoter stimulation in COS-1 cells can be duplicated in CV-1 cells by cotransfecting with a T-antigen-producing vector, but purified T antigen does not bind anywhere in the cytochrome c promoter. A control promoter from the mouse metallothionein I gene is similarly activated in T-antigen-producing cells only in the presence of zinc, which activates its upstream regulatory sites. We conclude that T antigen stimulates these cellular promoters through the activation or induction of cellular factors or complexes that mediate their effects through promoter-specific regulatory elements. Cytochrome c promoter regions activated in this system may play a physiological role in controlling gene expression.

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