Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.2.1.7 (BAL)
 1,977 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Deletion analysis of the 5' flank of the Chinese hamster dihydrofolate reductase (dhfr) gene reveals a promoter region starting 48 base pairs upstream of the major transcriptional start site. A dhfr minigene containing approximately 900 base pairs of 5' flank and one small intron was used as a wild-type standard. Seven deletions were created with BAL-31. Promoter activity was measured in three ways: 1) transient expression of the dhfr gene; 2) frequence of transfection of dhfr- Chinese hamster cells to a dhfr+ phenotype; and 3) RNase protection analysis of dhfr transcripts in pooled populations of permanently transfected cells. The transient expression assay was developed in this work for the rapid analysis of dhfr promoter mutants; this assay could be of general use for analyzing constructs carrying dhfr as a reporter gene. Two of the deletions define a requirement for part or all of the sequence GGGCGT located 48 base pairs upstream of the major transcriptional start site. This site has been shown to bind transcription factor Sp1 in the mouse dhfr gene. The function of the major promoter is independent of the function of the minor promoter. These minigene constructs also contain cryptic promoters located upstream of the natural start sites, probably in the plasmid vector. Transcripts originating from these upstream sites are inefficiently spliced, but do result in messenger RNA molecules that are translated into active dihydrofolate reductase.
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PMID:Deletion analysis of the Chinese hamster dihydrofolate reductase gene promoter. 318 92

Resistant strains for trimethoprim, a potent inhibitor of dihydrofolate reductase, were obtained by transforming the ligated products of Escherichia coli K12 DNA and plasmid pBR322 BamH I fragments. The strains carry a 13.6 kbp plasmid, pTP1, which contains the trimethoprim- and ampicillin-resistance determinant genes. The trimethoprim-resistance determinant gene was estimated to consist of more than 500 nucleotides and less than 1,500 nucleotides and was restricted by EcoR I and Sal I. Trimethoprim-, ampicillin-, and tetracycline-resistant plasmids were made in the following way, and the resultant plasmids contained a unique EcoR I "insertional inactivation" site for trimethoprim resistance: the DNA sequences extraneous to the determinant gene of the trimethoprim resistance on BamH I fragment of pTP 1 were eliminated by digestion with a double-strand-specific exonuclease BAL 31, and the resultant fragments were ligated with pBR 322 which had been digested by EcoR I and a single-strand-specific nuclease S1. The strains carrying pTP 1 or trimethoprim-resistant plasmids produced about 10 times more dihydrofolate reductase than control strains. The enhancement of the enzyme production, which is due to an increase in the copy number of the enzyme gene, seems to be responsible for the trimethoprim resistance of the transformed cells.
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PMID:Cloning of dihydrofolate reductase gene of Escherichia coli K12. 704 10