Transcriptional repression of the mouse dihydrofolate reductase gene during muscle cell commitment.
A differentiation-competent mouse muscle cell line containing 50-100-times the diploid number of dihydrofolate reductase (DHFR) genes was used to study regulation of DHFR mRNA levels during myogenic withdrawal from the cell cycle. Quantitative RNase protection assays showed DHFR mRNA levels decreased 15-fold during commitment; DHFR pre-mRNA levels decreased 7-fold. Concomitantly, transcription products were analyzed by hybridization to Southern blots of dhfr-containing plasmids. Control run-on assays performed on nonamplified parental cells indicated that run-on signals measured in amplified cells were dhfr amplicon-specific. Run-on signals were sensitive to alpha-amanitin, indicating RNA polymerase 2 specificity, and did not hybridize to pBR322 sequences, demonstrating hybridization stringency. Comparison of run-on signals hybridizing to DNA fragments representing either the 5' end of the gene or the entire gene showed that transcriptional repression occurred within the first 660 bases of the 30-kilobase gene, consistent with regulation at the level of either initiation or early pretermination. In contrast to the DHFR gene, DNA 5' to all but the first few bases of the DHFR coding region (between -1000 and +60 base pairs from the preferred cap site) showed strong run-on transcription in both proliferative and committed cells. Northern blot analysis using a probe complementary both to the dhfr coding region and the upstream region showed a uniform decrease in all detectable transcripts. No commitment-dependent changes in dhfr cap site usage, splicing, or polyadenylylation site usage were detected. Our results support a transcriptional model for regulation of DHFR mRNA levels.
Schmidt
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Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331.
Merrill
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GF
D000818:Animals; D015152:Blotting, Northern; D015139:Blotting, Southern; D002454:Cell Differentiation; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D004794:Enzyme Repression; D015971:Gene Expression Regulation, Enzymologic; D051379:Mice; D009132:Muscles / Q000166:cytology / Q000201:enzymology*; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D015347:RNA Probes / Q000138:chemical synthesis; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D014158:Transcription, Genetic
D015347:RNA Probes; D012333:RNA, Messenger; D013762:Tetrahydrofolate Dehydrogenase