Analysis of the Mouse dhfr Promoter Region: Existence of a Divergently Transcribed Gene

Abstract

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.

Nucleotide sequence and nuclease hypersensitivity of the Chinese hamster dihydrofolate reductase gene promoter region

“…An extensive region of partial homology between the hamster and murine genes extends from approximately -900 to the ATG codon (Figures 3 and 5A). This homology includes the sequences upstream from the symmetrically arranged G/C boxes, in a region that may encode a second message in the mouse and hamster that is transcribed in the opposite direction (Crouse et al, 1985;Mitchell et al, 1986) (see Discussion).…”

“…The inverted and symmetrical arrangment of the G/C box clusters in the three genes suggests that the upstream elements may serve a similar role in the transcription of a second gene transcribed in a direction opposite to the DHFR gene. However, in the hamster and murine DHFR genes, divergent transcripts have been detected that initiate at -195 and -245 to -305, respectively, relative to the ATG codon (Mitchell et al, 1986;Crouse et al, 1985). These observations argue against the presence of a promoter in the region upstream from the 5' G/C box cluster and suggest that the 29-bp-repeated sequences (elements 3) act as bidirectional promoters for both the DHFR gene and the divergent gene (Mitchell et Crouse et al, 1985).…”

Proximal and Long-Range Alterations in Chromatin Structure Surrounding the Chinese Hamster Dihydrofolate Reductase Promoter

“…The gene encoding this enzyme is constitutively expressed in all tissues. The DHFR genes from mouse, human, and Chinese hamster have been cloned, mapped, and partially sequenced (Azizkhan et al, 1986;Carothers et al, 1983;Chen et al, 1984;Crouse et al, 1985; Hamlin & Ma, 1990;McGrogan et al, 1985;Milbrandt et al, 1983;Mitchell et al, 1986; Nunberg et al, 1980;Yang et al, 1984). Each gene has six exons, and the sequences of the coding regions and the 3' untranslated region up to the first polyadenylation site, as well as the positions of intron-exon boundaries, are highly conserved.…”

“…The DHFR promoter is considered to be bidirectional, since a divergently transcribed gene has been identified in murine, Chinese hamster, and human systems (Crouse et al, 1985;Fujii et al, 1992;Linton et al, 1989;Mitchell et al, 1986;Shimada et al, 1989;Smith et al, 1990). In the murine and Chinese hamster genes, these transcripts initiate at ~-300 and -195 bp, respectively, relative to the start codon of the DHFR gene.…”

“…In the murine and Chinese hamster genes, these transcripts initiate at ~-300 and -195 bp, respectively, relative to the start codon of the DHFR gene. The distal clusters of GC boxes mapping between -700 and -400 relative to the start codons of the DHFR genes are therefore incorporated into the bodies of the divergent genes (Crouse et al, 1985;Famham & Schimke, 1986;Mitchell et al, 1986).…”