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Query: UNIPROT:P06889 (Mol)
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Vestigial (vg) mutants of Drosophila melanogaster are characterized by atrophied wings. In this paper we show that: (1) aminopterin an inhibitor of dihydrofolate reductase (DHFR) and fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase induce nicks in the wings of wild-type flies and phenocopies of the vg mutant phenotype when vg/+ and vgB/+ flies are reared on these substances (vgB is a deficiency of the vg locus). Only thymidine and thymidylate can rescue the flies from the effect of aminopterin. We propose that the vg phenotype is due to a decrease in the dTMP pool in the wings. (2) Mutant vg strains yield more offspring on medium containing aminopterin than on normal medium. The resistance of vg larvae to the inhibitor seems specific to the gene. This is the first case of aminopterin resistance in living eucaryotes. In contrast sensitivity of the vg larvae to FUdR is observed. (3) An increase in the activity and amount of DHFR is observed in mutant strains as compared with the wild-type flies. Our data suggest that the vg+ gene is a regulatory gene acting on the DHFR gene or a structural gene involved in the same metabolic pathway.
Mol Gen Genet 1989 Sep
PMID:Vestigial mutants of Drosophila melanogaster live better in the presence of aminopterin: increased level of dihydrofolate reductase in a mutant. 253 Dec 71

The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene from the monogenetic kinetoplastid protozoan, Crithidia fasciculata, was isolated and characterized. The gene is located on a single chromosome of approximately one megabase, and shows significant sequence similarity to other eukaryotic and prokaryotic DHFR and TS genes. There is a single low-abundance polyadenylated DHFR-TS transcript of approximately 3100 nt. One major miniexon splice site was identified by primer extension analysis. The 5' flanking region of the gene is divergently transcribed and shows strong similarities to a consensus DHFR promoter as well as to other eukaryotic 'housekeeping' gene promoter regions. A sequence downstream of the DHFR promoter consensus region is complementary to the 3' end of the C. fasciculata miniexon-derived RNA. This suggests a means by which the two separately transcribed RNAs may be juxtaposed for trans-splicing. In the 3' flanking region of the DHFR-TS gene, there is a sequence that is present in all of the chromosomes from this species and also from Leishmania tarentolae.
Mol Biochem Parasitol 1989 May 01
PMID:Structure, genomic organization and transcription of the bifunctional dihydrofolate reductase-thymidylate synthase gene from Crithidia fasciculata. 254 Apr 36

Trimethoprim resistance mediated by the Staphylococcus aureus multi-resistance plasmid pSK1 is encoded by a structure with characteristics of a composite transposon which we have designated Tn4003. Nucleotide sequence analysis of Tn4003 revealed it to be 4717 bp in length and to contain three copies of the insertion element IS257 (789-790 bp), the outside two of which are flanked by directly repeated 8-bp target sequences. IS257 has imperfect terminal inverted repeats of 27-28 bp and encodes for a putative transposase with two potential alpha-helix-turn-alpha-helix DNA recognition motifs. IS257 shares sequence similarities with members of the IS15 family of insertion sequences from Gram-negative bacteria and with ISS1 from Streptococcus lactis. The central region of the transposon contains the dfrA gene that specifies the S1 dihydrofolate reductase (DHFR) responsible for trimethoprim resistance. The S1 enzyme shows sequence homology with type I and V trimethoprim-resistant DHFRs from Gram-negative bacteria and with chromosomally encoded DHFRs from Gram-positive and Gram-negative bacteria. 5' to dfrA is a thymidylate synthetase gene, designated thyE.
Mol Microbiol 1989 Feb
PMID:Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257. 254 57

The metabolism of 5-fluoro-2'-deoxycytidine (FdC) with and without tetrahydrouridine (H4U) or 2'-deoxytetrahydrouridine (dH4U) was examined in log phase HEp-2 cells using HPLC and TLC methods which quantified: the incorporation of FdC-related antimetabolites into RNA and DNA and pool size levels of FdC-related antimetabolites. [3H]-FdC administered to log phase HEp-2 cells at a concentration of 0.01 microM for 24 hr resulted in the incorporation of 5.22 X 10(-8) mol of FdC/mol of DNA phosphate, a 0.021% substitution of FdC for dC. Coadministration of 1.0 mM H4U or dH4U resulted in 2- and 25-fold increases in the incorporation of FdC, respectively. No detectable incorporation of 5-fluoro-2'-deoxyuridine (FdU) into HEp-2 DNA resulted (detection limit, approximately 5 fmol). In contrast, treatment of HEp-2 cells with 0.1 microM FdU resulted in the incorporation of 1.83 X 10(-9) mol of FdU (74.7 fmol detected)/mol of DNA phosphate. A linear incorporation of FdC into the DNA of HEp-2 cells was found with increasing concentrations of FdC and 1.0 mM dH4U . 0.1 microM FdC resulted in the incorporation of 2.39 X 10(-6) mol of FUMP/mol of cytoplasmic RNA phosphate and 2.23 X 10(-5) mol of FUMP/mol of nuclear RNA phosphate. Similarly, HEp-2 cells treated with 0.1 microM FdU resulted in the incorporation of 1.10 X 10(-5) mol of FUMP/mol of nuclear RNA phosphate and 9.44 X 10(-7) mol of FUMP/mol of cytoplasmic RNA phosphate. In contrast, no detectable FUMP incorporation into either nuclear or cytoplasmic RNAs of HEp-2 cells resulted when H4U or dH4U was coadministered with 0.1 microM FdC. Pool size analyses of log phase HEp-2 cells following a 30-min exposure to FdU or FdC with and without H4U or dH4U were also performed; 0.1 microM FdC treatment resulted in the formation of 169 fmol of FUMP/1.0 X 10(6) viable HEp-2 cells. Treatment with 0.1 microM FdU produced 253 fmol of FUMP/1.0 X 10(6) viable HEp-2 cells. In contrast, no detectable FUMP pools were formed when H4U or dH4U was coadministered with 0.1 microM FdC (detection limit, approximately 5 fmol). Pool levels of FdUMP, the inhibitor of thymidylate synthetase, were also assayed; 36.9 fmol of FdUMP/1.0 X 10(6) viable HEp-2 cells were detected upon administration of 0.1 microM FdC.(ABSTRACT TRUNCATED AT 400 WORDS)
Mol Pharmacol 1985 May
PMID:Metabolic channeling of 5-fluoro-2'-deoxycytidine utilizing inhibitors of its deamination in cell culture. 258 Nov 25

The beta-sandwich in thymidylate synthase comprises two six-stranded mixed beta-sheets, each contributed by one subunit of the dimeric molecule. In contrast to other proteins of known structure in which beta-sheets stack face to face, the central beta-sheets in the thymidylate synthase dimer are related by a right-handed rather than a left-handed twist. Using a highly refined model of an Escherichia coli thymidylate synthase ternary complex, we show that the individual beta-sheets in each subunit are severely distorted by an unusual series of stacked beta-bulges, which partitions each larger sheet into two smaller beta-sheets approximately orthogonal to one another. These stacked beta-bulges are locally stabilized by hydrogen bonding involving eight conserved residues. This extended structure anchors the phosphate of bound dUMP and controls the precise orientation of the catalytically essential active site cysteine. Stereochemical factors associated with the pronounced crease caused by these stacked bulges account for the right-handed twist of opposing beta-sheets.
J Mol Biol 1989 Jan 20
PMID:Stacked beta-bulges in thymidylate synthase account for a novel right-handed rotation between opposing beta-sheets. 264 2

The dihydrofolate reductase-thymidylate synthase (DHFR-TS) bifunctional complex from pyrimethamine-sensitive (3D7) and drug-resistant (HB3 and 7G8) clones from Plasmodium falciparum was purified to homogeneity. A modified sequence of purification steps with a 10-formylfolate affinity column at its center, allows the isolation of the enzyme complex with a 10-fold higher yield than previously reported, irrespective of the pyrimethamine resistance of the parasites. Titration of the homogenous DHFR-TS complex with the inhibitor revealed a 500-fold lower affinity of the enzyme from clone 7G8 for the drug than found with the enzyme from clone 3D7. Direct comparison of the homogenous enzyme preparations on SDS-PAGE revealed no difference in the molecular mass of the DHFR-TS from the 3 clones, nor could a reproducible difference be detected in the peptide patterns obtained after digesting the DHFR-TS complex with various proteases. The amplification of segments from the DHFR-TS coding region of the 3 clones and 7 isolates of P. falciparum by polymerase chain reaction resulted in fragments of the predicted length without any size heterogeneity. The DNA sequence of the DHFR coding region from FCR-3, 3D7, HB3 and 7G8 differs in a total of 4 nucleotides. One point mutation changes amino acid residue 108 from threonine (FCR-3) or serine (3D7) to asparagine (HB3 and 7G8). The presence of asparagine-108 appears to be the molecular basis of pyrimethamine resistance of HB3 and 7G8. The degree of resistance is associated with a point mutation affecting the codon for amino acid 51 in 7G8.
Mol Biochem Parasitol 1989 Oct
PMID:Point mutations in the dihydrofolate reductase-thymidylate synthase gene as the molecular basis for pyrimethamine resistance in Plasmodium falciparum. 267 19

The promoter region of the mouse thymidylate synthase gene was analyzed by deletion and site-directed mutagenesis. Elimination of an upstream Sp1 element reduced expression threefold, whereas elimination of an adenovirus upstream stimulatory factor element had little effect. All of the upstream elements that are essential for promoter activity are located within 22 nucleotides of the first transcriptional initiation site.
Mol Cell Biol 1989 Sep
PMID:The mouse thymidylate synthase promoter: essential elements are in close proximity to the transcriptional initiation sites. 277 79

Murine mammary carcinoma FM3A cells, deficient in cytosol thymidine (dThd) kinase (TK) activity and transformed by the herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2) TK gene (designated FM3A TK-/HSV-1 TK+ and FM3A TK-/HSV-2 TK+, respectively) proved extremely sensitive to the cytostatic action of the potent antiherpetic drugs (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) and (E)-5-(2-iodovinyl)-2'-deoxyuridine (IVDU). The fact that FM3A TK-/HSV-2 TK+ cells were 5-fold more sensitive to the cytostatic action of BVDU and IVDU but incorporated [125I]IVDU to a 20-fold lower extent into their DNA than did FM3A TK-/HSV-1 TK+ cells led us to conclude that incorporation of these compounds into DNA of HSV TK gene-transformed cell lines is not directly related to their cytostatic action. In attempts to unravel the mechanism of the cytostatic effects of BVDU and IVDU on HSV TK gene-transformed FM3A cells, both compounds were submitted to an intensive biochemical study. Thymidylate synthase was identified as the principal target enzyme for the cytostatic action of BVDU and IVDU since (i) both compounds were far more inhibitory to 2(1)-deoxyuridine (dUrd) than to dThd incorporation into HSV TK gene-transformed FM3A cell DNA, (ii) the cytostatic action of BVDU and IVDU was more readily reversed by dThd than by dUrd, (iii) both compounds strongly inhibited the metabolic pathway leading to the incorporation of 2'-deoxycytidine (dCyd) into DNA thymidylate, (iv) BVDU and IVDU strongly inhibited tritium release from [5-3H]dCyd and [5-3H]dUrd in intact HSV TK gene-transformed FM3A cells, and (v) [125I]IVDU accumulated intracellularly as its 5'-monophosphate to concentration levels considerably higher than those required to inhibit partially purified thymidylate synthase. The inhibitory effects mentioned under (i) to (iv) were not observed with the parental FM3A/0 and FM3A/TK- cells; they were more pronounced for FM3A TK-/HSV-2 TK+ cells than for FM3A TK-/HSV-1 TK+ cells, which correlates with the differential cytostatic effects of BVDU and IVDU on these cells.
Mol Pharmacol 1987 Sep
PMID:Thymidylate synthase is the principal target enzyme for the cytostatic activity of (E)-5-(2-bromovinyl)-2'-deoxyuridine against murine mammary carcinoma (FM3A) cells transformed with the herpes simplex virus type 1 or type 2 thymidine kinase gene. 282 92

The molecular karyotypes of five species of Leishmania were studied by pulsed field gradient gel electrophoresis (PFGGE) of chromosome-sized DNA bands. Each species exhibits a unique pattern of 22-28 bands in the size range approximately 200-2200 kb whereas strains of one species exhibit similar karyotypes. Analysis of the behaviour of kinetoplast DNA during PFGGE showed that minicircle DNA remains confined to the gel slot but a proportion of the maxicircle DNA fractionates as a low molecular weight band below band 1. The band location of genes for alpha and beta tubulin, the 5' spliced leader sequence (5'SL), heat shock proteins 70 (hsp 70) and 83 (hsp 83) and thymidylate synthase-dihydrofolate reductase (TS-DHFR) were analysed. Housekeeping genes are not clustered in Leishmania but are found on at least 7 bands in L. major. The hsp 83 gene is linked to the tandemly repeated beta tubulin allele on band 21 in L. major. Among different species, the location of the unlinked hsp 83 and hsp 70 genes is conserved whereas the TS-DHFR and 5'SL sequences are found on bands of varying size. The 5'SL gene may be rearranged in L. enriettii and two 5'SL loci were identified in L. donovani and L. tropica. The conservation of loci in strains of L. major suggests that the chromosomal genetic linkage map should be a reliable marker for identifying unknown isolates of Leishmania. Sequences on one band in L. mexicana sp. were shared among several bands and distributed on homologous and non-homologous bands in other species showing that DNA sequences are rearranged during speciation in Leishmania.
Mol Biochem Parasitol 1987 Oct
PMID:Molecular karyotype of five species of Leishmania and analysis of gene locations and chromosomal rearrangements. 282 21

We have analyzed the cell cycle effects that different domains of the adenovirus E1A proteins have on quiescent primary BRK cells. Studies with deletion mutants that in combination removed all but the N-terminal 85 amino acids common to both the 12S and 13S proteins suggest that this region may be sufficient for the induction of synthesis of proliferating cell nuclear antigen and the stimulation of DNA synthesis. A second domain also common to the N-terminal exon of the 12S and 13S proteins was required for the induction of mitosis and stimulation of proliferation of primary BRK cells. A virus containing a mutation in this region was still able to stimulate DNA synthesis efficiently. A third domain, unique to the 13S protein, was required for the accelerated activation of the cellular thymidylate synthase gene in a manner similar to the 13S-dependent stimulation of adenovirus early region genes.
Mol Cell Biol 1987 Feb
PMID:Different functional domains of the adenovirus E1A gene are involved in regulation of host cell cycle products. 288 Nov 97


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