Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 identified a component of the eukaryotic RNA polymerase II transcriptional machinery that is more heat-labile than TFIID. DHFR transcriptional activity was severely reduced in 40 degrees C heat-treated extracts in which TFIID was fully active. This heat-labile activity was required for the transcription of both TATA box and non-TATA box promoters that are activated by the transcription factor Sp1. Gel mobility shifts indicated that Sp1 DNA binding activity was heat-labile, and the addition of purified Sp1 to 40 degrees C heat-treated extracts fully restored DHFR transcriptional activity. In contrast, the addition of Sp1 to 47 degrees C heat-treated extract did not result in transcriptional activity from the DHFR promoter. We conclude that reduction in Sp1 DNA binding activity is partially responsible for the heat-sensitive loss of DHFR transcriptional activity, but that a second essential activity is also inactivated by 47 degrees C heat-treatment. The discovery of this heat-labile component of Sp1 activation has two important implications in the analysis of transcriptional regulation. First, it demonstrates that heat-treated extracts are not appropriate for examination of the involvement of TFIID in the transcription of Sp1-activated promoters. Second, it explains the previously reported low-temperature optima for transcription from the DHFR promoter and demonstrates that transcriptional studies of Sp1-activated promoters should not be performed at 30 degrees C.
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PMID:Sp1 activation of RNA polymerase II transcription complexes involves a heat-labile DNA-binding component. 182 Feb 11

A new gene transfer protocol has been developed that introduces up to 800 copies of an expression vector into Chinese hamster ovary cells in a single step by electroporation. The DNA typically integrates in tandem repeats so that the restriction endonuclease site used to linearize the input DNA remains intact. This is likely due to ligation of vector DNA via cohesive ends prior to integration. This high-copy-number procedure is far more rapid than the conventional stepwise gene amplification method used to generate stable eukaryotic protein production cell lines. By employing the expression vector pJODtPA, in which the selectable marker dihydrofolate reductase (DHFR) and the human tissue plasminogen activator (tPA) casettes are separated by a spacer and an RNA polymerase II terminator, cell lines secreting as much as 24 pg/cell.day tPA were isolated following electroporation and a single methotrexate selection. Gene copies and expression levels are stable over long periods of growth. A single round of gene amplification was performed following the high-copy-number procedure to yield a clone having a tPA production level of 45 pg/cell.day.
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PMID:Introduction of stable high-copy-number DNA into Chinese hamster ovary cells by electroporation. 211 95

We have identified a sequence element that specifies the position of transcription initiation for the dihydrofolate reductase gene. Unlike the functionally analogous TATA box that directs RNA polymerase II to initiate transcription 30 nucleotides downstream, the positioning element of the dihydrofolate reductase promoter is located directly at the site of transcription initiation. By using DNase I footprint analysis, we have shown that a protein binds to this initiator element. Transcription initiated at the dihydrofolate reductase initiator element when 28 nucleotides were inserted between it and all other upstream sequences, or when it was placed on either side of the DNA helix, suggesting that there is no strict spatial requirement between the initiator and an upstream element. Although neither a single Sp1-binding site nor a single initiator element was sufficient for transcriptional activity, the combination of one Sp1-binding site and the dihydrofolate reductase initiator element cloned into a plasmid vector resulted in transcription starting at the initiator element. We have also shown that the simian virus 40 late major initiation site has striking sequence homology to the dihydrofolate reductase initiation site and that the same, or a similar, protein binds to both sites. Examination of the sequences at other RNA polymerase II initiation sites suggests that we have identified an element that is important in the transcription of other housekeeping genes. We have thus named the protein that binds to the initiator element HIP1 (Housekeeping Initiator Protein 1).
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PMID:Transcription initiation from the dihydrofolate reductase promoter is positioned by HIP1 binding at the initiation site. 230 58

The presence of specific promoter elements, notably the TATA and GC boxes, has been useful for categorizing genes transcribed by RNA polymerase II. The gene for the murine ribosomal protein (r-protein) L32 lacks both of these elements, although it has GC-rich regions. The conditions required for its optimal synthesis in vitro, however, resemble the properties of promoters containing TATA (adenovirus major late promoter) rather than GC boxes (dihydrofolate reductase). To further investigate the relationship of the r-protein gene to different promoter elements, transcription competition analyses were used to distinguish the presence of common protein-binding sequences. The low levels of competition observed by either the adenovirus major late promoter or dihydrofolate reductase promoter with the r-protein gene promoter resulted from general transcription factors present in each initiation complex. Competition by factors binding to common sequence elements was not observed, indicating the r-protein L32 gene possesses elements distinct from those present in the other genes examined.
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PMID:Characterization of a mammalian ribosomal protein gene promoter. 239 98

Cloned human rRNA gene fragments that included the promoter region were introduced into Chinese hamster dihydrofolate reductase-deficient (dhfr-) cells by cotransformation with a dhfr minigene and amplified by selection for methotrexate resistance. The human ribosomal DNA was transcribed by RNA polymerase II, not RNA polymerase I or III. The metaphase chromosome regions containing the transcriptionally active human ribosomal DNA failed to show silver staining.
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PMID:Human ribosomal DNA fragments amplified in hamster cells are transcribed only by RNA polymerase II and are not silver stained. 243 41

Wheat germ RNA polymerase II was used to raise monoclonal antibodies (mAbs) that cross-react with the largest subunit of calf thymus RNA polymerase II. Most of these mAbs were of the IgM isotype and were shown to react with a synthetic peptide containing the consensus sequence for the C-terminal heptapeptide repeat that has been found on the largest subunit of RNA polymerase II from a variety of eukaryotic organisms. A representative mAb (3WG2) was tested for its effect on transcription in both in vitro and in vivo systems. Antibody 3WG2 did not affect the transcription (elongation) of wheat germ RNA polymerase II on denatured calf thymus DNA. When HeLa cell nuclear extracts were preincubated with the mAb, run-off transcription from a promoter that contains a TATA box (the adenovirus-2 major late promoter) and from a promoter that does not contain a TATA box (the murine dihydrofolate reductase gene promoter = dhfr) was inhibited. Transcription from these promoters was also inhibited by the synthetic peptide containing the consensus sequence when it was conjugated to bovine serum albumin. HeLa cell nuclear extract in which the endogenous RNA polymerase II had been inhibited by the specific mAb was used to examine the ability of added mammalian RNA polymerase II that lacks the C-terminal domain to accurately transcribe specific genes. When calf thymus RNA polymerase II that lacked the C-terminal domain was added back to the inhibited extract, a discrete transcript that was initiated correctly was obtained with the adenovirus-2 major late promoter; however, no discrete transcript was observed from the mouse dhfr gene promoter. When injected into Xenopus laevis oocytes, antibody 3WG2 inhibited transcription of the human histone H2b gene (contains a TATA box) and the human U1 small nuclear RNA gene (does not contain a TATA box), but did not inhibit transcription from RNA polymerase I or RNA polymerase III promoters. These results indicate that the C-terminal heptapeptide repeat plays a critical role in promoter-directed transcription, although enzyme that lacks this domain can initiate from some promoters in vitro.
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PMID:Inhibition of in vivo and in vitro transcription by monoclonal antibodies prepared against wheat germ RNA polymerase II that react with the heptapeptide repeat of eukaryotic RNA polymerase II. 247 98

The complete nucleotide sequence of a 1957 bp DNA fragment containing the dihydrofolate reductase gene (DFR1) of the yeast Saccharomyces cerevisiae is presented. Within this region a single open reading frame of 633 base pairs was found which is capable of encoding a 211 amino acid residue protein with a calculated Mr of 24,233. The amino acid sequence derived from the yeast DFR1 gene shows limited homology with sequences from both eukaryotic and non-eukaryotic DHFR enzymes. Northern blot hybridization reveals that the mRNA from this gene is a 900 base polyadenylated transcript. Yeast strains containing the cloned DFR1 gene on multicopy number shuttle vector plasmids show dramatically enhanced methotrexate resistance. Consensus DNA sequences responsible for RNA polymerase II interaction and general amino acid control in S. cerevisiae are located within the 5'-noncoding region with respect to the open reading frame. The DNA fragment containing these sequences has been shown to be necessary for DFR1 gene expression in both S. cerevisiae and E. coli.
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PMID:Molecular characterization of the Saccharomyces cerevisiae dihydrofolate reductase gene (DFR1). 282 21

Transcription of the 26-kilobase (kb) dihydrofolate reductase (dhfr) gene in CHO cells is initiated at two sites: a major site (approximately 85% of the dhfr mRNA) at -63 relative to the translation start and a minor site (approximately 15%) at -107. Transcription also occurs from the opposite DNA strand in the dhfr 5' region, with a probable initiation site at approximately -195 relative to the dhfr translation start. A 4-kb polyadenylated RNA that is derived from the opposite-strand transcription increases threefold in abundance after serum starvation of CHO cells for 24 h. dhfr mRNA levels do not change during this time. The first dhfr exon lies within a 1-kb genomic region marked by exceptionally high G + C content and lack of DNA methylation. This region also includes a 214-base-pair (bp) exon for the opposite-strand transcript and five of the six DNase I-hypersensitive sites identified at the dhfr locus. Analysis of the DNA sequences of hamster, human (M. Chen, T. Shimada, A. D. Moulton, A. Cline, R. K. Humphries, J. Maizel, and A. W. Nienhuis, J. Biol. Chem. 259:3933-3943, 1984), and mouse (M. McGrogan, C. C. Simonsen, D. T. Smouse, P. J. Farnham, and R. T. Schimke, J. Biol. Chem. 260:2307-2314, 1985) dhfr genes reveals the presence of a 29-bp unit that is conserved 45 to 49 bp upstream of major and minor dhfr transcription start sites. This unit follows the consensus: GRGGCGGTGGCCTNNNNTGTCRCAARTRGGTR. The 5' part of the 29-bp unit contains a GC box that agrees with the GGGCGG consensus-binding site for the RNA polymerase II transcription factor Sp1 (D. Gidoni, W. A. Dynan, and R. Tjian, Nature (London) 312:409-413, 1984). Each of the three mammalian dhfr genes has several G-rich GC boxes proximal to the major dhfr transcription start site and several GC boxes of the opposite orientation (C rich) in a distal region about 500 bp upstream.
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PMID:Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene. 302 46

We have previously shown by affinity chromatography that RAP30 and RAP74 are the mammalian proteins that have the highest affinity for RNA polymerase II. Here we show that RAP30 binds to RAP74 and that the RAP30-RAP74 complex (RAP30/74) is required for accurate initiation by RNA polymerase II. RAP30/74 is required for accurate transcription from the following promoters: the adenovirus major late promoter, the long terminal repeat of human immunodeficiency virus, P2 of the human c-myc gene, the mouse beta maj-globin promoter (all of which have TATA boxes), and the mouse dihydrofolate reductase promoter (which lacks a TATA box). RAP30/74 is not required for initiation by RNA polymerase III at the adenovirus virus-associated RNA promoters. Therefore, RAP30/74 is a general initiation factor that binds to RNA polymerase II.
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PMID:RAP30/74: a general initiation factor that binds to RNA polymerase II. 338 90

The expression of a number of genes was measured in P1798 cells treated for various periods of time with 0.1 microM dexamethasone. Thymidine kinase (TK) activity decreased under these conditions with 50% inhibition achieved within approximately 8 h. Decreased TK activity was associated with reduced abundance of TK mRNA. Analysis of nuclear transcription indicated that this was attributable to a decrease in the number of RNA polymerase II molecules engaged in transcription of the TK gene. With respect to TK, there was an overall correlation between enzyme activity, mRNA, and nuclear transcription. The data are consistent with the hypothesis that glucocorticoid inhibition of expression of TK is primarily due to inhibition of transcription. Transcription of the TK gene was also reduced by greater than 90% after inhibition of protein synthesis for 6 h. This suggests that transcription of this gene requires a protein of short biological half-life. It is proposed that this hypothetical transcription factor is regulated by glucocorticoids. The amount of thymidylate synthase and dihydrofolate reductase remained constant for at least 24 h in dexamethasone-treated P1798 cells. Dihydrofolate reductase mRNA likewise remained constant. However, the mRNA encoding thymidylate synthase decreased 80-90% within 24 h. The mRNA encoding ornithine decarboxylase also decreased. In neither case did this appear to be primarily due to inhibition of transcription of the respective genes. The abundance of the mRNAs encoding hypozanthine-guanine phosphoribosyl transferase and phosphoglycerate kinase did not decrease in dexamethasone-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Glucocorticoid regulation of the genes encoding thymidine kinase, thymidylate synthase, and ornithine decarboxylase in P1798 cells. 339 44


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