Genetic and molecular mapping of chromosome region 85A in Drosophila melanogaster.
(9/21)
Chromosome region 85A contains at least 12 genetic complementation groups, including the genes dhod, pink and hunchback. In order to better understand the organization of this chromosomal segment and to permit molecular studies of these genes, we have carried out a genetic analysis coupled with a chromosome walk to isolate the DNA containing these genes. Complementation tests with chromosomal deficiencies permitted unambiguous ordering of most of the complementation groups identified within the 85A region. Recombinant bacteriophage clones were isolated that collectively span over 120 kb of 85A DNA and these were used to produce a molecular map of the region. The breakpoint sites of a number of 85A chromosome rearrangements were localized on the molecular map, thereby delimiting regions of the DNA that contain the various genetic complementation groups. (+info)
Complete inhibition of dihydro-orotate oxidation and superoxide production by 1,1,1-trifluoro-3-thenoylacetone in rat liver mitochondria.
(10/21)
1,1,1-Trifluoro-3-thenoylacetone was shown to cause complete inhibition of dihydroorotate oxidation in rat liver mitochondria as measured by orotate formation and the rate of dihydro-orotate-dependent reduction of 2,6-dichlorophenol-indophenol or cytochrome c. The inhibition by trifluorothenoylacetone was dose-dependent, and a concentration of 1 mM completely inhibited dihydro-orotate dehydrogenase activity. 1,10-Phenanthroline, another iron-chelating agent, also caused total inhibition of the liver enzyme. Whereas the iron chelators inhibited 100% of dihydro-orotate dehydrogenase activity in liver mitochondria, they inhibited only a maximum of 72% in the case of the brain enzyme. The inhibition by trifluorothenoylacetone was not prevented by addition of phenazine methosulphate or ubiquinone. Dihydro-orotate dehydrogenase-mediated generation of superoxide was abolished when the enzyme was fully inhibited by trifluorothenoylacetone or when the electron-transport system was blocked by antimycin A. These results suggest that the iron component(s) of dihydro-orotate dehydrogenase is of strategic importance for catalytic activity and transfer of reducing equivalents from the primary enzyme to the electron-transport chain. Furthermore, the study indicates that production of superoxide radicals during dihydro-orotate dehydrogenase-catalysed oxidation of dihydro-orotate may be at the cytochrome b-c1 segment of the electron-transport chain (as a consequence of autooxidation of ubisemiquinone) rather than at a site on the primary enzyme. (+info)
Nucleotide sequence of the pyrD gene of Escherichia coli and characterization of the flavoprotein dihydroorotate dehydrogenase.
(11/21)
Dihydroorotate dehydrogenase (EC 1.3.3.1) was purified to near electrophoretic homogeneity from the membranes of a strain of Escherichia coli carrying the pyrD gene on a multicopy plasmid. The preparation had a specific activity of 120 mumol min-1 mg-1 and contained flavin mononucleotide (FMN) in amounts stoichiometric to the dihydroorotate dehydrogenase subunit (Mr = 37000). The flavin group was reduced when dihydroorotate was added in the absence of electron acceptors. The complete sequence of 1357 base pairs of an EcoRI-EcoRI DNA fragment containing the pyrD gene was established. Dihydroorotate dehydrogenase is encoded by a 336-triplets open reading frame. The molecular mass (Mr = 36732), the amino acid composition and the N-terminal sequence of the predicted polypeptide agree well with the data obtained by analysis of the purified protein. A region of the amino acid sequence (residues 292-303, i.e. Ile-Ile-Gly-Val-Gly-Gly-Ile-Asp-Ser-Val-Ile-Ala) shows distinct homology to the cofactor binding site of other flavoproteins. No hydrophobic regions large enough to span the cytoplasmic membrane were observed. By the S1-nuclease technique an mRNA start was mapped 34 +/- 2 nucleotide residues upstream of the beginning of the coding frame of pyrD. The leader region contains no similarity to the attenuators of the pyrB and pyrE genes of E. coli. (+info)
Mechanism of action of the novel anticancer agent 6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinolinecarbo xylic acid sodium salt (NSC 368390): inhibition of de novo pyrimidine nucleotide biosynthesis.
(12/21)
Exposure of cultured clone A human colon tumor cells to 25 to 75 microM of NSC 368390 [6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinolinecarbox yli c acid sodium salt, DuP 785] for 48 to 72 h resulted in a 99.9% cell kill as determined by clonogenic assay. Cells exposed to NSC 368390 became depleted in intracellular pools of uridine 5'-triphosphate and cytidine 5'-triphosphate. Both uridine 5'-triphosphate and cytidine 5'-triphosphate were decreased to 50% of levels in control cells at 3 h and were undetectable at 15 h after addition of 25 microM of NSC 368390 to the cultures. Similar effects were observed in L1210 leukemia cells. Addition of 0.1 mM of uridine or cytidine restored intracellular pools of uridine 5'-triphosphate and cytidine 5'-triphosphate to control levels and rescued clone A cells from NSC 368390 cytotoxicity. Addition of uridine circumvented NSC 368390 cytotoxicity in L1210 cells, but addition of cytidine did not. This result is consistent with the fact that L1210 cells lack cytidine deaminase and thus cannot form uridine or its anabolites from cytidine. These results indicated that NSC 368390 inhibits a step in the de novo biosynthetic pathway leading to uridine 5'-monophosphate. Therefore, the effects of NSC 368390 on the six enzymes that comprise the de novo pathway leading to the formation of uridine 5'-monophosphate were examined. The results showed that NSC 368390 was a potent inhibitor of dihydroorotate dehydrogenase, the fourth enzyme in the pathway; thus, this study demonstrates that NSC 368390 exerts its tumoricidal effect by inhibiting a step in de novo pyrimidine biosynthesis resulting in the depletion of critical precursors for RNA and DNA synthesis. (+info)
Regulation of uridylic acid biosynthesis in the cyanobacterium Anabaena variabilis.
(13/21)
The pathway of uridylic acid biosynthesis established by Leiberman, Kornberg, and Simms has been shown to be operative in the filamentous cyanobacterium Anabaena variabilis. The only enzyme of uridylic acid biosynthesis found to be lacking in two uracil-requiring strains of A. variabilis was aspartate transcarbamylase, the first enzyme in the pathway of de novo biosynthesis of uridvlic acid. Neither uracil-limited growth of a uracil-requiring mutant nor growth of the wild type in high concentrations of uracil resulted in substantial changes in the specific activities of enzymes of uridylic acid biosynthesis. It is therefore concluded that A. variabilis does not regulate all enzymes of this pathway by means of repression. However, control of the flow of intermediates through this pathway is possible by feedback inhibition of aspartate transcarbamylase by a variety of nucleotides. (+info)
Effect of 6-azauridine on de novo pyrimidine biosynthesis in cultured Ehrlich ascites cells. Orotate inhibition of dihydroorotase and dihydroorotate dehydrogenase.
(14/21)
The inhibition of dihydro-orotase (E 3.5.2.3) and dihydroorotate (DHO) dehydrogenase (dihydro-orotate oxidase, EC 1.3.3.1) by cellular orotate (OA) in Ehrlich ascites cells was studied by measuring the accumulation of the intermediates of de novo pyrimidine biosynthesis at various times after the addition of 6-azauridine to the culture medium. The addition of 6-azauridine resulted in the accumulation of orotidine, OA, DHO, and carbamyl aspartate (CAA). The use of the observed ratios of [CCA]/[OA] and [DHO]/[OA] and other known constants allowed us to calculate that the increased cellular OA concentration caused primarily an inhibition of DHO dehydrogenase rather than an inhibition of dihydroorotase. A constant ratio of [CAA]/[DHO] was observed which probably indicates that the interconversion of these two intermediates catalyzed by dihydroorotase is near equilibrium in these cells as has been observed in vitro (Christopherson, R.I., Matsuura, T., and Jones, M.E. (1978) Anal. Biochem. 89, 225-234). It is suggested that the probable intracellular accumulation of CAA in patients with oroticaciduria may have significant secondary effects. (+info)
On auxotrophy for pyrimidines of respiration-deficient chick embryo cells.
(15/21)
Chick embryo cells treated with chloramphenicol are inherently resistant to the growth-inhibitory effect of the drug when cultured in the presence of tryptose phosphate broth. The cells were found to be auxotrophic for pyrimidines and the presence in the broth of compounds of pyrimidine origin is demonstrated by chromatographic procedures and mass spectral analyses. They are in the form of ribonucleosides, ribonucleotides and pyrimidine-containing oligoribonucleotides. To understand the mechanism responsible for pyrimidine auxotrophy, the activity of enzymes involved in the pyrimidine biosynthetic pathway was determined. Measurement of the conversion of dihydroorotic acid to orotic acid in cell-free extracts revealed that chloramphenicol-treated chick embryo cells are deficient in dihydroorotate dehydrogenase activity. The data in vitro are supported by studies on the nutritional requirements of the respiration-deficient cells and by the incorporation in vivo of labelled dihydroorotic acid into the acid-insoluble fraction of the cells. Although the activity of the dehydrogenase in vitro is decreased by 95%, the enzyme is present in chloramphenicol-treated cells and its activity is unmasked by the artificial electron acceptor menadione. A study of the activity of other enzymes of the pyrimidine biosynthetic pathway demonstrated that their activity is comparable to that in control cells. The present results indicate that auxotrophy for pyrimidines results from the inhibition of the flow of electrons along the mitochondrial electron transport chain. (+info)
Repression and derepression of the enzymes of the pyrimidine biosynthetic pathway in Salmonella typhimurium.
(16/21)
Activities of five enzymes of the pyrimidine biosynthetic pathway and one enzyme involved in arginine synthesis were measured during batch culture of Salmonella typhimurium. Aspartate carbamoyltransferase, dihydroorotase, and the arginine pathway enzyme, ornithine carbamoyltransferase, remained constant during the growth cycle but showed a sharp decrease in activity after entering the stationary phase. Dihydroorotate dehydrogenase, orotate phosphoribosyltransferase and orotidine-5'-monophosphate (OMP) decarboxylase showed peaks of activity corresponding to the mid-point of the exponential phase of growth while remaining comparatively stable in the stationary phase. Derepression studies carried out by starving individual pyrimidine (Pyr-) deletion mutants for uracil showed that the extent of derepression obtained for aspartate carbamoyltransferase, dihydroorotase and dihydroorotate dehydrogenase depended on the location of the pyr gene mutation. Orotate phosphoribosyltransferase and OMP decarboxylase derepression levels were independent of the location of the pyr mutation. Aspartate carbamoyltransferase showed the greatest degree of derepression of the six enzymes studied, with pyrA strains (blocked in the first step of the pathway) showing about twice as much derepression as pyrF strains (blocked in the sixth step of the pathway). A study of the kinetics of repression on derepressed levels of the pyrimidine enzymes produced data that were compatible with dilution of specific activity by cell division when repressive amounts of uracil were added to the derepression medium. (+info)