Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.5.1.3 (dihydrofolate reductase)
 5,819 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have shown previously that 48 base pairs (bp) of 5'-flanking sequence are necessary for correct initiation at the major transcriptional start site of the Chinese hamster dihydrofolate reductase (dhfr) gene (Ciudad et al., 1988). As an upstream element, this sequence alone confers 25% of maximum promoter activity. The 5' half of this sequence is particularly well conserved among mammalian species; it contains one Sp1 binding site (GC box) and one CAA element. In the present work, we have analyzed the role of this region by extensive point mutational analysis. Twenty-three dhfr minigene constructs containing 1- or 2-base substitutions in this region of the promoter were tested by measuring their ability to transfect DHFR-deficient Chinese hamster ovary cells to a DHFR+ growth phenotype. Eight mutants, all in or near the GC box, exhibited reduced transfection efficiency. Promoter disfunction in these mutants was confirmed by RNase protection analysis of stable transfectants. Gel retardation experiments showed that mutants affected in the consensus sequence for Sp1 binding were deficient in binding a protein found in nuclear extracts of Chinese hamster ovary cells. Purified human transcription factor Sp1 was also unable to bind a promoter sequence bearing one of these single base substitutions, suggesting that Sp1 itself is involved in dhfr transcription in vivo. We conclude that most single base mutations in the GC box severely cripple or eliminate promoter function by inhibiting binding of transcription factors to this regulatory sequence and that Sp1 is likely to be involved in dhfr transcription in vivo. We also found that the well conserved CAA element is not absolutely necessary for transcription.
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PMID:Point mutational analysis of the hamster dihydrofolate reductase minimum promoter. 174 Apr 17

Multiple-site-specific incorporation of a noncanonical amino acid into a recombinant protein would be a very useful technique to generate multiple chemical handles for bioconjugation and multivalent binding sites for the enhanced interaction. Previously combination of a mutant yeast phenylalanyl-tRNA synthetase variant and the yeast phenylalanyl-tRNA containing the AAA anticodon was used to incorporate a noncanonical amino acid into multiple UUU phenylalanine (Phe) codons in a site-specific manner. However, due to the less selective codon recognition of the AAA anticodon, there was significant misincorporation of a noncanonical amino acid into unwanted UUC Phe codons. To enhance codon selectivity, we explored degenerate leucine (Leu) codons instead of Phe degenerate codons. Combined use of the mutant yeast phenylalanyl-tRNA containing the CAA anticodon and the yPheRS_naph variant allowed incorporation of a phenylalanine analog, 2-naphthylalanine, into murine dihydrofolate reductase in response to multiple UUG Leu codons, but not to other Leu codon sites. Despite the moderate UUG codon occupancy by 2-naphthylalaine, these results successfully demonstrated that the concept of forced ambiguity of the genetic code can be achieved for the Leu codons, available for multiple-site-specific incorporation.
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PMID:Forced Ambiguity of the Leucine Codons for Multiple-Site-Specific Incorporation of a Noncanonical Amino Acid. 2702 6