Transcription of the mouse dihydrofolate reductase gene proceeds unabated through seven polyadenylation sites and terminates near a region of repeated DNA.

Abstract

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).

Analysis of the mouse dhfr promoter region: existence of a divergently transcribed gene.

“…Previous work has shown that transcription terminates approximately 6 kb from 3' of the dhfr termination codon (12). The data from the UV mapping experiments suggested that all of the transcribed RNA was due to initiation in the same region and that there was no significant transcription initiation within the transcribed DNA.…”

“…The work presented here combined with our previous results (12) serve to define the boundaries of the dhfr transcription unit. Hybridization of pulse-labeled RNA to filter-bound DNA showed that there was no transcription on the dhfr strand further than 1 kb 5' of the dhfr ATG, and the level of hybridization to the probe mDRT10 suggested that transcription initiated within several hundred nucleotides of the ATG.…”

“…The mouse S180-M500 cell line contains approximately 1,000 copies of the dhfr gene, and due to this gene amplification, it is possible to isolate enough pulse-labeled RNA to measure transcriptional activity throughout the dhfr gene region. We used this technique, which had been used previously to define the 3' boundary of the dhfr transcription unit (12), in an effort to identify the 5' end of the dhfr transcription unit. Pulse-labeled total RNA from the S180-M500 cells was hybridized to unique sequences throughout the dhfr structural gene and 5'-flanking region (Fig.…”

“…The 3' ends of these mRNAs correspond to the utilization of seven polyadenylation sites distributed over a 5-kb region of genomic DNA at the 3' end of the dhfr structural gene. The use of dhfr gene amplification mutants (2,33) (12).…”

The Transcription Termination Region of the Adenovirus 2 Major Late Transcript Contains Multiple Functional Elements

“…For the majority of eukaryotic genes studied to date, transcription appears to terminate in large regions of DNA, which may span hundreds of nucleotides (Hofer & Darnell, 1981 ;LeMeur et al, 1984;Hagenbuchle et al, 1984;Frayne et al, 1984;Amara et al, 1984). A specific transcription terminator, with homology to a yeast termination signal, has been mapped in the human gastrin gene Baek et al, 1986).…”