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)

It is widely accepted that nuclear genes that encode proteins of the oxidative-phosphorylation system are regulated by nuclear factors believed to be specific for such genes. In the present study we show that the promoter for the human cytochrome c1 gene is an exception, in that it involves only conserved Sp1 core elements and an initiator region. Maximal promoter activity within a 1.4-kb 5' flanking region of the cytochrome c1 gene is contained in a fragment (-72 to +18) that lacks TATA and CCAAT elements. The transcriptional start site was mapped to an initiator region by RNase protection of mRNA from human HepG2 cells, and by primer extension of in vitro-generated transcripts, to a sequence that is highly similar to the dihydrofolate reductase family of initiators. Deletion of this region (+1 to +18) severely impairs transcription initiation. Sp1 core elements centered at nucleotides -21 and -39 define the activation domain of the proximal promoter. Only the -39 element is protected from DNase I in the presence of crude nuclear extracts. However, transfection, gel-mobility-shift, supershift and in vitro-transcription experiments show that the -21 element binds Sp1 protein and contributes to transcription activation. No other functional oxidative-phosphorylation-specific response elements have been identified. These data implicate Sp1 as a single activating factor for an oxidative-phosphorylation gene.
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PMID:Expression of the human cytochrome c1 gene is controlled through multiple Sp1-binding sites and an initiator region. 891 68

Cells transduced with either of two human DHFR minigenes express an RNA product which is considerably shorter than what would be predicted from the size of an unspliced transcript expressed from its DNA template. RNA blotting analysis has shown that this short transcript accumulated to exceedingly high levels which were comparable to the levels reached by the highly abundant endogenous actin mRNA, or MoMLV RNA expressed in chronically infected cells. RNA blotting, RNase mapping, primer extension, RT-PCR, and sequencing have shown that this highly expressed transcript, termed TBN, is a spliced RNA product which utilizes cryptic splice signals present in the normally spliced DHFR mRNA. Subcloning experiments have demonstrated that all the information required for the generation and high level accumulation of the TBN transcripts is encoded in the 1.6 kb DHFR DNA minigene. TBN transcripts were generated with comparable efficiency from DNA templates containing either the human ADA or the early SV40 promoters. Since neither the ADA nor the SV40 promoter are considered to be particularly "strong" promoters, this observation argues that initiation of transcription is not the rate limiting step in determining the amount of the TBN transcripts which accumulate in the cell. Insertion of a foreign sequence into the DHFR DNA minigene led to the expression and high level accumulation of a chimeric transcript, suggesting that the unusual properties of this expression system may be used for high level expression of foreign sequences. These observations offer new insights into the mechanisms which control the accumulation of translatable mRNA in the cell, and have potentially important implications for experiments involving optimization of gene expression for gene therapy applications.
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PMID:High level accumulation of an aberrantly spliced human DHFR RNA species. 963 51

The retinoblastoma tumor suppressor gene product (pRb) is involved in controlling cell cycle progression from G1 into S. pRb functions, in part, by regulating the activities of several transcription factors, making pRb involved in the transcriptional control of cellular genes. Transient-transfection assays have implicated pRb in the transcription of several genes, including c-fos, the interleukin-6 gene, c-myc, cdc-2, c-neu, and the transforming growth factor beta2 gene. However, these assays place the promoter in an artificial context and exclude the effects of far 5' upstream regions and chromosomal architecture on gene transcription. In these experiments, we have studied the role of pRb in the control of cell cycle-related genes within a chromosomal context and within the context of the G1 phase of the cell cycle. We have used adenovirus vectors to overexpress pRb in human osteosarcoma cells and breast cells synchronized in early G1. By RNase protection assays, we have assayed the effects of this virus-produced pRb on gene expression in these cells. These results indicate that pRb is involved in the transcriptional downregulation of the E2F-1, E2F-2, dihydrofolate reductase, thymidine kinase, c-myc, proliferating-cell nuclear antigen, p107, and p21/Cip1 genes. However, it has no effect on the transcription of the E2F-3, E2F-4, E2F-5, DP-1, DP-2, or p16/Ink4 genes. The results are consistent with the notion that pRb controls the transcription of genes involved in S-phase promotion. They also suggest that pRb negatively regulates the transcription of two of the transcription factors whose activity it also represses, E2F-1 and E2F-2, and that it plays a role in downregulating the immediate-early gene response to serum stimulation.
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PMID:Regulation of cellular genes in a chromosomal context by the retinoblastoma tumor suppressor protein. 967 66

Introduction of a premature termination codon (PTC) into an exon of a gene can lead to nonsense-mediated decay of the mRNA, which is best characterized as a cytoplasmic event. However, increasing evidence has suggested that PTCs may also influence the nuclear processing of an RNA transcript, leading to models of nuclear surveillance perhaps involving translating nuclear ribosomes. We used quantitative RT-PCR to measure the in vivo steady-state levels of every exon-intron junction in wild-type, PTC-containing, and missense-containing precursor mRNAs of both the nonrearranging dihydrofolate reductase (DHFR) and the somatically rearranging Ig- micro genes. We find that each exon-intron junction's abundance and, therefore, the rate of intron removal, is not significantly affected by the presence of a PTC in a neighboring exon in either the DHFR or Ig- micro pre-mRNA. Similarly, the abundance of the uncleaved Ig- micro polyadenylation sites does not differ between wild-type and PTC-containing Ig- micro pre-mRNAs. Our Ig- micro data were confirmed by RNase protection analyses, and multiple cell isolates were examined to resolve differences with previously published data on steady-state pre-mRNA levels. We conclude that the presence of a PTC affects the rate of neither splicing nor the cleavage step of 3' end formation during pre-mRNA processing in the nucleus. Our results are discussed with respect to existing evidence for nuclear surveillance mechanisms.
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PMID:Premature termination codons do not affect the rate of splicing of neighboring introns. 1503 75

The fused dihydrofolate reductase/thymidylate synthase gene of Toxoplasma gondii contains ten exons spanning approximately 8 kb of genomic DNA. We have examined the ends of DHFR-TS transcripts within this gene, and find a complex pattern including two discrete 5' termini and multiple polyadenylation sites. No TATAA box or other classical promoter motif is evident in 1.4 kb of genomic DNA upstream of the coding region, but transcript mapping by RNase protection and primer extension reveals two prominent 5' ends at positions -369 and -341 nt relative to the ATG initiation codon. Upstream genomic sequences include GC-rich regions and the (opposite strand) WGAGACG motif previously identified in other T. gondii promoters. Mutagenesis of recombinant reporter plasmids demonstrates that this region is essential for efficient transgene expression. Sequencing the 3' ends from multiple independent mRNA clones demonstrates numerous polyadenylation sites, distributed over >650 nt of genomic sequence beginning approximately 250 nt downstream of the stop codon. Within this region, certain sites seem to be preferred: 14 different positions were found among the 32 polyadenylated transcripts examined, but approximately 40% of the transcripts map to two loci. The 3' noncoding region is rich in A and T nucleotides, and contains an imperfect 50 nt direct repeat, but no obvious poly(A) addition signal was identified.
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PMID:Transcript initiation, polyadenylation, and functional promoter mapping for the dihydrofolate reductase-thymidylate synthase gene of Toxoplasma gondii. 1538 93

Methotrexate (MTX) is a dihydrofolate reductase (DHFR) inhibitor widely used as an anticancer drug in different kinds of human cancers. Here we investigated the anti-tumor mechanism of MTX against non-small cell lung cancer (NSCLC) A549 cells. MTX not only inhibited in vitro cell growth via induction of apoptosis, but also inhibited tumor formation in animal xenograft model. RNase protection assay (RPA) and RT-PCR demonstrated its induction of p53 target genes including DR5, p21, Puma and Noxa. Moreover, MTX promoted p53 phosphorylation at Ser15 and acetylaion at Lys373/382, which increase its stability and expression. The apoptosis and inhibition of cell viability induced by MTX were dependent on p53 and, partially, on p21. In addition, MTX also increased E-cadherin expression through inhibition of histone deacetylase (HDAC) activity and downregulation of polycomb group protein enhancer of zeste homologue 2 (EZH2). Therefore, the anticancer mechanism of MTX acts through initiation of p53-dependent apoptosis and restoration of E-cadherin expression by downregulation of HDAC/EZH2.
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PMID:Methotrexate induces apoptosis through p53/p21-dependent pathway and increases E-cadherin expression through downregulation of HDAC/EZH2. 2111 63

Identification of point mutations has been facilitated by a number of techniques, including transfection assays, oligonucleotide hybridization, electrophoretic migration of heteroduplexes, RNase mismatch analysis, direct sequencing, and DNA-polymerase catalyzed amplification. The large number of available techniques emphasizes the importance of developing rapid and reliable methods to identify molecular changes in genes. To date, we have concentrated on exploiting DNA-polymerase catalyzed amplification methods (1,2) in conjunction with direct manual and automated DNA sequencing to detect point mutations in the dihydrofolate reductase (DHFR) gene of methotrexate-resistant cells.
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PMID:Manual and automated direct sequencing of product generated by the polymerase chain reaction. 2140 Feb 72


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