Heterogeneity at the 5' termini of mouse dihydrofolate reductase mRNAs. Evidence for multiple promoter regions

• Published in: The Journal of biological chemistry Vol. 260; no. 4; pp. 2307 - 2314
• Main Authors: McGrogan, M, Simonsen, C C, Smouse, D T, Farnham, P J, Schimke, R T
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25-02-1985; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Base Sequence; Cell Line; Cricetinae; DNA; DNA Restriction Enzymes; Endonucleases; Female; Gene Amplification; Mice; Nucleic Acid Hybridization; Ovary; Plasmids; Promoter Regions, Genetic; Repetitive Sequences, Nucleic Acid; RNA, Messenger - genetics; Single-Strand Specific DNA and RNA Endonucleases; Tetrahydrofolate Dehydrogenase - genetics; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)89555-2

We have determined the sequence of the 1000 base pairs of DNA preceding the coding region of the mouse dihydrofolate reductase gene. 700 base pairs upstream of the translation start codon (position + 1) is the sequence CAACT, separated by 50 base pairs from the sequence TAATAA; these sequences resemble controlling elements responsible for accurate and efficient transcription initiation in a variety of eukaryotic genes. The region between -244 and -101 consists of a 3-fold tandem repeat of a 48-base pair sequence. S1 mapping results indicate that the 5' termini of the multiple dihydrofolate reductase mRNAs are heterogeneous, with the major terminus at -115 and with minor termini in the regions of -275 and -450. In addition, a portion of the 5'-ward region of the gene from -543 to -405 is represented differentially in some, but not all, mouse dihydrofolate reductase mRNAs. The above findings are consistent with the presence of two transcription promoter elements in the 5' end of the mouse dihydrofolate reductase gene. This has been substantiated by inserting portions of the 5'-ward 1000 base pairs of the gene into modular dihydrofolate reductase plasmids. When such constructs are transfected into Chinese hamster ovary cells lacking dihydrofolate reductase, function can be restored with equal transfection frequency when sequences surrounding the 5' CAACT-TAATAA region at -700 or a complete 48-base pair repeat from the region of -148 to -101 are present in the plasmid construct.