The use of murine dihydrofolate reductase (dhfr) gene amplification mutants enabled us to identify important structural and functional features of the dhfr promoter region. We found another transcription unit, at least 14 kilobases in size, which initiates within 130 base pairs of the major dhfr transcript and is transcribed divergently. The 5' ends of both transcripts were analyzed and found to have multiple initiation sites. The major dhfr transcript and the divergent transcript appear to share the same promoter region; the longer transcripts of the dhfr gene overlap with the divergent transcripts and use a different promoter region. The divergent transcript appears to code for a protein; an homologous sequence to its first exon is found in the corresponding location near the human dhfr gene.
We observed equimolar transcription throughout the 35-kilobase mouse dihydrofolate reductase structural gene. Transcription termination occurred within a discrete region (900 base pairs) located 1 kilobase beyond the last of seven functional polyadenylation sites and near a repetitive DNA sequence element. The results imply that a distinct genetic signal may be associated with the process of transcription termination.The 5' ends of mature mRNAs in higher eucaryotes correspond to sites of transcription initiation (7,20 (23,24). The seven poly(A) sites used in the formation of these mRNAs are distributed throughout a 5-kb region of genomic DNA located at the 3' end of the structural gene. Therefore, to determine the number, location, and efficiency of transcription termination sites relative to the seven poly(A) sites within the dhfr transcription unit, we have measured the level of transcriptional activity throughout this region. These studies were made possible by the use of dhfr gene amplification mutants which permitted the detection of transcriptional activity from a gene expressed at a level too low to measure accurately in normal cells. In the S180-500R cell line (1, 11) used in the present study, the amplified unit extends over 10 kb 3' of the dhfr gene and is unaltered in this region relative to the S180 parental cell line; therefore, these cells serve as a suitable model for studying the dhfr transcription unit (6,8,22).
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