Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene.

AUTOR(ES)

Mitchell, P J

RESUMO

Transcription of the 26-kilobase (kb) dihydrofolate reductase (dhfr) gene in CHO cells is initiated at two sites: a major site (approximately 85% of the dhfr mRNA) at -63 relative to the translation start and a minor site (approximately 15%) at -107. Transcription also occurs from the opposite DNA strand in the dhfr 5' region, with a probable initiation site at approximately -195 relative to the dhfr translation start. A 4-kb polyadenylated RNA that is derived from the opposite-strand transcription increases threefold in abundance after serum starvation of CHO cells for 24 h. dhfr mRNA levels do not change during this time. The first dhfr exon lies within a 1-kb genomic region marked by exceptionally high G + C content and lack of DNA methylation. This region also includes a 214-base-pair (bp) exon for the opposite-strand transcript and five of the six DNase I-hypersensitive sites identified at the dhfr locus. Analysis of the DNA sequences of hamster, human (M. Chen, T. Shimada, A. D. Moulton, A. Cline, R. K. Humphries, J. Maizel, and A. W. Nienhuis, J. Biol. Chem. 259:3933-3943, 1984), and mouse (M. McGrogan, C. C. Simonsen, D. T. Smouse, P. J. Farnham, and R. T. Schimke, J. Biol. Chem. 260:2307-2314, 1985) dhfr genes reveals the presence of a 29-bp unit that is conserved 45 to 49 bp upstream of major and minor dhfr transcription start sites. This unit follows the consensus: GRGGCGGTGGCCTNNNNTGTCRCAARTRGGTR. The 5' part of the 29-bp unit contains a GC box that agrees with the GGGCGG consensus-binding site for the RNA polymerase II transcription factor Sp1 (D. Gidoni, W. A. Dynan, and R. Tjian, Nature (London) 312:409-413, 1984). Each of the three mammalian dhfr genes has several G-rich GC boxes proximal to the major dhfr transcription start site and several GC boxes of the opposite orientation (C rich) in a distal region about 500 bp upstream.

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