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)

The TFDP genes encode a family of transcription factors that can form heterodimers with E2F family proteins in vivo. The E2F-TFDP transcription factors are major regulators of genes that are required for the progression of S-phase, such as DHFR and DNA polymerase alpha, and they play a critical role in cell cycle regulation and differentiation. The retinoblastoma tumor suppressor protein has been shown to induce growth arrest by binding to E2F-TFDP and repressing its activity. Two human TFDP genes have been cloned, namely TFDP1 and TFDP2 (or DP1 and DP2). In the present study, we identified genomic clones of TFDP1, its pseudogene TFDP1P and TFDP2, and we mapped them to chromosome 13q34, 1q32.3, and 3q23, respectively. Chromosomal abnormalities involving regions 13q34 and 3q23 have been reported in certain lymphomas and other diseases associated with loss of cell cycle regulation, and the involvement of the TFDP transcription factors remains to be elucidated.
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PMID:Genomic cloning and chromosomal assignment of the E2F dimerization partner TFDP gene family. 902 91

To elucidate the detailed gene organization of the human leukocyte antigen (HLA) class I region on chromosome 6, seven contiguous cosmid genomic clones covering the 237-kb segment around the HLA-B and -C loci were subjected to DNA sequencing by the shotgun strategy to give a single contig of 236,822 bp from the MICA gene (58.2 kb centromeric of HLA-B) to 90.8 kb telomeric of HLA-C. This region was confirmed to contain four known genes, MICA, HLA-17, HLA-B, and HLA-C, from centromere to telomere. Further, a new member of the P5 multicopy genes was found to be about 1.3 kb upstream of the HLA-17 gene and designated P5.8. Five novel genes designated NOB1-5 were identified by RT-PCR and Northern blot hybridization. In addition, two pseudogenes, dihydrofolate reductase pseudogene (DHFRP) and ribosomal protein L3 homologous gene (RPL3-Hom), were also found in the vicinity of the HLA-B and -C genes, respectively. The two segments (about 40 kb) downstream of the HLA-B and HLA-C genes showed high sequence homology to each other, suggesting that segmental genome duplication including the major histocompatibility complex (MHC) class I gene must have occurred during the evolution of the MHC.
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PMID:Nucleotide sequence analysis of the HLA class I region spanning the 237-kb segment around the HLA-B and -C genes. 917 76

We have previously isolated and characterized a mouse cDNA orthologous to the human synovial sarcoma associated SS18 (formerly named SSXT and SYT) cDNA. Here, we report the characterization of the genomic structure of the mouse Ss18 gene. Through in silico methods with sequence information contained in the public databases, we did the same for the human SS18 gene and two human SS18 homologous genes, SS18L1 and SS18L2. In addition, we identified a mouse Ss18 processed pseudogene and mapped it to chromosome 1, band A2-3. The mouse Ss18 gene, which is subject to extensive alternative splicing, is made up of 11 exons, spread out over approximately 45 kb of genomic sequence. The human SS18 gene is also composed of 11 exons with similar intron-exon boundaries, spreading out over about 70 kb of genomic sequence. One alternatively spliced exon, which is not included in the published SS18 cDNA, corresponds to a stretch of sequence which we previously identified in the mouse Ss18 cDNA. The human SS18L1 gene, which is also made up of 11 exons with similar intron-exon boundaries, was mapped to chromosome 20 band q13.3. The smaller SS18L2 gene, which is composed of three exons with similar boundaries as the first three exons of the other three genes, was mapped to chromosome 3 band p21. Through sequence and mutation analyses this gene could be excluded as a candidate gene for 3p21-associated renal cell cancer. In addition, we created a detailed BAC map around the human SS18 gene, placing it unequivocally between the CA-repeat marker AFMc014wf9 and the dihydrofolate reductase pseudogene DHFRP1. The next gene in this map, located distal to SS18, was found to be the TBP associated factor TAFII-105 (TAF2C2). Further analogies between the mouse Ss18 gene, the human SS18 gene and its two homologous genes were found in the putative promoter fragments. All four promoters resemble the promoters of housekeeping genes in that they are TATA-less and embedded in canonical CpG islands, thus explaining the high and widespread expression of the SS18 genes.
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PMID:Mapping and characterization of the mouse and human SS18 genes, two human SS18-like genes and a mouse Ss18 pseudogene. 1143 5

Hypermutagenic PCR has been used to simulate pseudogene evolution of the Escherichia coli R67 dihydrofolate reductase gene. Each time the most divergent clone was used as template for another round of hypermutagenesis. After six rounds, with an average mutation rate of 0.05 per base per round, up to a 46% nucleic acid sequence variation was achieved. For a few clones the protein information content could be annihilated. As the intermediates were cloned and sequenced, it was possible to establish the real lineage and compute the true number of mutations. Not surprisingly the true number of forward and back mutations as well as variable sites exceeded those based on comparing any single intermediate to the initial sequence. However, the true number of forward and backward mutations, as well as the number of variable sites, increased linearly with sequence divergence from the original sequence, suggesting an empirical means to correct for branch lengths.
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PMID:Simulating pseudogene evolution in vitro: determining the true number of mutations in a lineage. 1168 9

Human dihydrofolate reductase (DHFR) was previously thought to be the only enzyme capable of the reduction of dihydrofolate to tetrahydrofolate; an essential reaction necessary to ensure a continuous supply of biologically active folate. DHFR has been studied extensively from a number of perspectives because of its role in health and disease. Although the presence of a number of intronless DHFR pseudogenes has been known since the 1980s, it was assumed that none of these were expressed or functional. We show that humans do have a second dihydrofolate reductase enzyme encoded by the former pseudogene DHFRP4, located on chromosome 3. We demonstrate that the DHFRP4, or dihydrofolate reductase-like 1 (DHFRL1), gene is expressed and shares some commonalities with DHFR. Recombinant DHFRL1 can complement a DHFR-negative phenotype in bacterial and mammalian cells but has a lower specific activity than DHFR. The K(m) for NADPH is similar for both enzymes but DHFRL1 has a higher K(m) for dihydrofolate when compared to DHFR. The need for a second reductase with lowered affinity for its substrate may fulfill a specific cellular requirement. The localization of DHFRL1 to the mitochondria, as demonstrated by confocal microscopy, indicates that mitochondrial dihydrofolate reductase activity may be optimal with a lowered affinity for dihydrofolate. We also found that DHFRL1 is capable of the same translational autoregulation as DHFR by binding to its own mRNA; with each enzyme also capable of replacing the other. The identification of DHFRL1 will have implications for previous research involving DHFR.
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PMID:The former annotated human pseudogene dihydrofolate reductase-like 1 (DHFRL1) is expressed and functional. 2187 84

The de novo and salvage dTTP pathways are essential for maintaining cellular dTTP pools to ensure the faithful replication of both mitochondrial and nuclear DNA. Disregulation of dTTP pools results in mitochondrial dysfunction and nuclear genome instability due to an increase in uracil misincorporation. In this study, we identified a de novo dTMP synthesis pathway in mammalian mitochondria. Mitochondria purified from wild-type Chinese hamster ovary (CHO) cells and HepG2 cells converted dUMP to dTMP in the presence of NADPH and serine, through the activities of mitochondrial serine hydroxymethyltransferase (SHMT2), thymidylate synthase (TYMS), and a novel human mitochondrial dihydrofolate reductase (DHFR) previously thought to be a pseudogene known as dihydrofolate reductase-like protein 1 (DHFRL1). Human DHFRL1, SHMT2, and TYMS were localized to mitochondrial matrix and inner membrane, confirming the presence of this pathway in mitochondria. Knockdown of DHFRL1 using siRNA eliminated DHFR activity in mitochondria. DHFRL1 expression in CHO glyC, a previously uncharacterized mutant glycine auxotrophic cell line, rescued the glycine auxotrophy. De novo thymidylate synthesis activity was diminished in mitochondria isolated from glyA CHO cells that lack SHMT2 activity, as well as mitochondria isolated from wild-type CHO cells treated with methotrexate, a DHFR inhibitor. De novo thymidylate synthesis in mitochondria prevents uracil accumulation in mitochondrial DNA (mtDNA), as uracil levels in mtDNA isolated from glyA CHO cells was 40% higher than observed in mtDNA isolated from wild-type CHO cells. These data indicate that unlike other nucleotides, de novo dTMP synthesis occurs within mitochondria and is essential for mtDNA integrity.
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PMID:Identification of a de novo thymidylate biosynthesis pathway in mammalian mitochondria. 2187 88

A novel single-nucleotide polymorphism (SNP) in the 3'-untranslated region of the human dihydrofolate reductase (DHFR) gene with enhanced expression was identified in 2001. In 2007, it was reported that this SNP, DHFR C829T, was located close to a microRNA binding site and contributed to the stability of mRNA. Many researchers have analyzed this SNP in several races including Asians and Caucasians. However, the mutation allele is not yet confirmed in most populations. In this study, we reinvestigated the frequency of this SNP using three methods. First, this SNP in genomic DNA was analyzed by a PCR-restriction fragment length polymorphism method. Second, this SNP in mRNA was analyzed by a single nucleotide extension method following a reverse transcription reaction. Third, the mRNA expression level was analyzed by a real-time PCR method. The findings in our study, regarding the discovery of this SNP, suggest that the SNP is an artifact caused by contamination by the genomic DNA of the pseudogene DHFRP1. This study is a reinvestigation of a newly discovered genetic polymorphism.
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PMID:An artifact derived from a pseudogene led to the discovery of microRNA binding site polymorphism in the 3'-untranslated region of the human dihydrofolate reductase gene. 2220 Nov 19


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