Discrete human dihydrofolate reductase gene transcripts present in polysomal RNA map with their 5' ends several hundred nucleotides upstream of the main mRNA start site.

Abstract

The 5' ends of dihydrofolate reductase (DHFR)-specific transcripts have been mapped in the 5'-flanking region of the amplified DEHFR gene of the human methotrexate-resistant cell line 6A3 by primer extension and Si protection experiments. The main 5' end, at position -71 relative to the first nucleotide of the DHFR reading frame, corresponds to the recently identified main transcription initiation site for the DHFR gene and pertains to transcripts representing approximately 99% of the DHFR-specific polysomal polyadenylic acid-containing RNA, and including the previously described DHFR mRNAs with sizes of 3.8, 1.0, and 0.8 kilobases. At least six other minor 5' ends have been mapped to nucleotide positions -449 to -480 upstream of the DHFR gene and pertain to approximately 1% of the DHFR-specific polysomal polyadenylic acid-containing RNA. These upstream initiating transcripts appear to include five major discrete species with sizes of 4.3, 3.8, 3.1, 2.1, and 1.0 kilobases and four minor ones with sizes of 7.3, 5.0, 1.4, and 0.8 kilobases. These species, with the exception of those of 3.1-and 2.1-kilobase sizes, also have been found in VA2-B cells, the parental line of 6A3, and in HeLa cells. The upstream initiating transcripts present in all three cell lines are increased in amount in 6A3 cells as compared with the other cell lines, in about the same proportion as the three identified DHFR mRNAs.The analysis of the mode of expression of the dihydrofolate reductase (DHFR) gene in mammalian cells, which has been greatly facilitated by the availability of cell lines with amplified genes, has revealed an unsuspected complexity both in these and the parental cell lines, in the form of the existence of multiple species of DHFR mRNAs. Thus, four major polyadenylated [poly(A)+] species of DHFR mRNA in mouse cells (32), three species in Chinese hamster cells (22), and three main species in human cells (27) have been identified. These multiple mRNA species differ mainly in the length of the 3'-untranslated tail (10,25,32), which can be up to severalfold longer than the reading frame, as is the case for the major human DHFR mRNA (3.8-kilobase [kb] mRNA). Furthermore, there is good evidence indicating that the 3'-end tails of the multiple mRNAs are colinear (25,32). These observations would be compatible with the idea that the same transcript could produce different forms of mRNA by processing and polyadenylation at alternative sites.In the present work, an extensive mapping study of the 5' ends of DHFR-specific transcripts in human cell lines with an amplified or normal DHFR gene complement has unexpectedly revealed the presence, besides a main transcription initiation point at position -71 relative to the first nucleotide (nt) of the DHFR reading frame (11), of at least six additional minor sites several hundred nt's upstream of the DHFR gene, which correspond to 5' ends of DHFR-specific transcripts. These upstream initiating transcripts are found both in polysomal RNA, where they include several discrete species in the...

Novel promoter upstream of the human c-myc gene and regulation of c-myc expression in B-cell lymphomas.

“…6,1986 minor RNA initiation sites similar to P0 have been observed upstream of the major promoters for the human e-globin gene (2) and the human and mouse dihydrofolate reductase genes (16,30). The function of such upstream promoters is unknown.…”

c-Ha-ras gene bidirectional promoter expressed in vitro: location and regulation.

“…These so-called GC boxes were first discovered in the 21-bp repeats of the SV40 early promoter, where they have been shown to bind transcription factor SP1 (26,27). GC boxes have since been discovered in a number of cellular housekeeping genes, including those encoding HMG coenzyme A reductase (52), human adenosine deaminase (62), mouse and human dihydrofolate reductase (41,45), and mouse and human hypoxanthine phosphoribosyltransferase (38,47). A 350-bp region containing the c-Ha-ras exon -1 contains 10 GC boxes, 6 of which bind SP1 (35).…”

Nucleotide sequence of the Drosophila glucose-6-phosphate dehydrogenase gene and comparison with the homologous human gene

“…Although most genes containing PolII promoters possess a 'TATA' box, there are PolII promoters that lack the TATA box (Nevins, 1983). In particular, many housekeeping genes, i.e., genes which are fairly uniformly expressed in most tissue types throughout the life cycle of the organism, do not possess a TATA box: the hydroxymethyl glutaryl CoA reductase gene (Reynolds et al, 1984), the hypoxanthine phosphoribosyltransferase gene (Pate1 et al, 1986;Melton et al, 1984), the adenosine deaminase gene (Valerio, 1985), the DHFR gene (Masters and Attardi, 1985;Mitchell et al, 1986), one of the two glyceraldehyde-3-phosphate dehydrogenase genes in Drosophila (Tso et al, 1985) as well as the PrP 27-30 gene (Basler et al, 1986) and the Ul RNA gene (Roebuck and Stump, 1985). These genes do, however, contain one or more copies of the sequence GGGCGG or its inverse complement CCGCCC upstream from their transcription start point.…”