A re-examination of the 5' termini of mouse dihydrofolate reductase RNA.
Using primer extension and nuclease S1-mapping techniques we have re-examined the 5' termini of RNA transcribed from the mouse dihydrofolate reductase gene. We characterize a previously undescribed transcription initiation site at position -55 relative to the AUG codon, in addition to the previously identified start site at position -115. Differences in the 5' noncoding regions of these two transcripts with respect to their length and relative G + C content result in their differential ability to form stable hybrids with the DNA probe used in previous analyses of these transcripts and thus precluded the detection of transcripts initiated at -55. We show that changes in the temperature of the hybridization reaction result in the ability to detect the RNA having a shorter noncoding region and a lower G + C content. That position -55 represents an authentic transcription start site is confirmed by use of a DNA probe with which the two transcripts can form S1-resistant hybrids of equal stability and by primer extension analysis using an oligonucleotide primer that hybridizes near the AUG codon. These analyses also demonstrate that the transcript with a 5' end mapping near position -55 accounts for the majority of cellular dihydrofolate reductase RNA.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D002999:Clone Cells; D004262:DNA Restriction Enzymes; D004720:Endonucleases; D051379:Mice; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000235:genetics*; D015719:Single-Strand Specific DNA and RNA Endonucleases; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014158:Transcription, Genetic
D012333:RNA, Messenger; D013762:Tetrahydrofolate Dehydrogenase; D004720:Endonucleases; D004262:DNA Restriction Enzymes; D015719:Single-Strand Specific DNA and RNA Endonucleases