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.