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1.
S Cheesman S McAleese M Goman D Johnson P Horrocks R G Ridley B J Kilbey 《Nucleic acids research》1994,22(13):2547-2551
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DNA polymerase delta: gene sequences from Plasmodium falciparum indicate that this enzyme is more highly conserved than DNA polymerase alpha. 总被引:7,自引:4,他引:3
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R G Ridley J H White S M McAleese M Goman P Alano E de Vries B J Kilbey 《Nucleic acids research》1991,19(24):6731-6736
Genes encoding proteins homologous to the catalytic subunits of DNA polymerase alpha and delta have been cloned from the human malaria parasite Plasmodium falciparum. These are among the first cellular replicative DNA polymerase genes to be cloned and their sequences allow us to make new statements about the relative degrees of conservation of these two enzymes. The most important finding was that P. falciparum Pol delta showed considerable homology to the only other Pol delta enzyme for which published sequence is available, that of S. cerevisiae, displaying an overall amino acid identity of 45% and identity over a highly conserved central region of 59%. In contrast, the level of identity shown over the equivalent central region of Pol alpha between the P. falciparum and S. cerevisiae sequences is only 32%. The sequence data also allowed us to examine the degree of conservation in putative exonuclease domains of Pol delta. The Pol delta gene of P. falciparum maps to chromosome 10 and evidence is presented for the presence of different sized Pol delta mRNA's in the asexual and sexual erythrocytic stages of parasite development. 相似文献
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Partial purification and characterization of mitochondrial DNA polymerase from Plasmodium falciparum 总被引:1,自引:0,他引:1
Chavalitshewinkoon-Petmitr P Chawprom S Naesens L Balzarini J Wilairat P 《Parasitology international》2000,49(4):279-288
Mitochondria of chloroquine-resistant Plasmodium falciparum (K1 strain) were isolated from mature trophozoites by differential centrifugation. The mitochondrial marker enzyme cytochrome c reductase was employed to monitor the steps of mitochondria isolation. Partial purification of DNA polymerase from P. falciparum mitochondria was performed using fast protein liquid chromatography (FPLC). DNA polymerase of P. falciparum mitochondria was characterized as a gamma-like DNA polymerase based on its sensitivity to the inhibitors aphidicolin, N-ethylmaleimide and 9-beta-D-arabinofuranosyladenine-5'-triphosphate. In contrast, the enzyme was found to be strongly resistant to 2',3'-dideoxythymidine-5'-triphosphate (IC(50)>400 microM) and differed in this aspect from the human homologue, possibly indicating structural differences between human and P. falciparum DNA polymerase gamma. In addition, the DNA polymerase of parasite mitochondria was shown to be resistant (IC(50)>1 mM) to the nucleotide analogue (S)-1-[3-hydroxy-2-phosphonylmethoxypropyl]adenine diphosphate (HPMPApp). 相似文献
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The germline gene encoding DNA polymerase alpha in the hypotrichous ciliate Oxytricha nova is extremely scrambled. 总被引:2,自引:1,他引:1
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We report the structure of the micronuclear (germline) gene encoding the large catalytic subunit of DNA polymerase alpha (DNA pol alpha) in the ciliate Oxytricha nova. It contains 44 internal eliminated segments (IESs) that divide the gene into 45 macronuclear-destined segments (MDSs) that are in a non-randomly scrambled order with an inversion near the gene center. Odd numbered MDSs 29-43, containing 230 bp out of a total of 4938 bp of macronuclear sequence, are missing from the 14 kb cloned gene. The missing MDSs have not been located but are at least several kilobases from the main body of the gene. The remarkably scrambled DNA pol alpha gene must be extensively cut, re-ordered and spliced and an inversion must occur to produce an unscrambled, functional version of the gene during development of a new macronucleus. Unscrambling is hypothesized to occur by a homologous recombination mechanism guided by repeat sequences at MDS ends. 相似文献
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Supreet Kaur Nicholas S. Nieto Peter McDonald Josh R. Beck Richard B. Honzatko Anuradha Roy Scott W. Nelson 《Journal of enzyme inhibition and medicinal chemistry》2022,37(1):1320
Malaria is caused by infection with protozoan parasites of the Plasmodium genus, which is part of the phylum Apicomplexa. Most organisms in this phylum contain a relic plastid called the apicoplast. The apicoplast genome is replicated by a single DNA polymerase (apPOL), which is an attractive target for anti-malarial drugs. We screened small-molecule libraries (206,504 compounds) using a fluorescence-based high-throughput DNA polymerase assay. Dose/response analysis and counter-screening identified 186 specific apPOL inhibitors. Toxicity screening against human HepaRG human cells removed 84 compounds and the remaining were subjected to parasite killing assays using chloroquine resistant P. falciparum parasites. Nine compounds were potent inhibitors of parasite growth and may serve as lead compounds in efforts to discover novel malaria drugs. 相似文献
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tRNA genes transcribed from the plastid-like DNA of Plasmodium falciparum. 总被引:2,自引:0,他引:2
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Besides their mitochondrial genome, malarial parasites contain a second organellar DNA. This 35 kb circular molecule has a number of features reminiscent of plastid DNAs. Sequence analysis shows that along with other genes the circle codes for 25 different tRNAs all of which are transcribed. Six of the tRNAs have some unusual features, and one has an intron, the only one found so far on the circle. Comparison of codon and anticodon usage indicates that the 25 tRNAs are sufficient to decode all the protein genes present on the circle. The maintenance of such a parsimonious but complete translation system is further evidence for the functionality of the circle. 相似文献
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The evolutionary conservation of DNA polymerase alpha was assessed by immunological and molecular genetic approaches. Four anti-human KB cell DNA polymerase alpha monoclonal antibodies were tested for their ability to recognize a phylogenetically broad array of eukaryotic DNA polymerases. While the single non-neutralizing antibody used in this study recognizes higher mammalian (human, simian, canine, and bovine) polymerases only, three neutralizing antibodies exhibit greater, but variable, extents of cross-reactivity among vertebrate species. The most highly cross-reactive antibody recognizes a unique epitope on a 165-180 kDa catalytic polypeptide in cell lysates from several eukaryotic sources, as distant from man as the amphibians. Genomic Southern hybridization studies with the cDNA of the human DNA polymerase alpha catalytic polypeptide identify the existence of many consensus DNA sequences within the DNA polymerase genes of vertebrate, invertebrate, plant and unicellular organisms. These findings illustrate the differential evolutionary conservation of four unique epitopes on DNA polymerase alpha among vertebrates and the conservation of specific genetic sequences, presumably reflective of critical functional domains, in the DNA polymerase genes from a broad diversity of living forms. 相似文献
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Isolation of the gene encoding yeast DNA polymerase I 总被引:52,自引:0,他引:52
A yeast genomic DNA expression library in lambda gt11 antibody prepared against yeast DNA polymerase I were used to isolate the gene encoding DNA polymerase I. The identity of the DNA polymerase I gene was determined by several criteria. First, the clone-encoded protein is immunologically related to DNA polymerase I. Second, cells containing the gene cloned in the high copy number plasmid YEp24 overproduce the polymerase activity 4- to 5-fold as measured in yeast extracts. Finally, insertion of the gene downstream from a bacteriophage T7 promoter allows synthesis of yeast DNA polymerase I in Escherichia coli. Gene disruption and Southern hybridization experiments show that the polymerase is encoded by an essential, single copy gene. Examination of the germinated spores containing the disrupted gene reveals a defect in nuclear division and a terminal phenotype typical of replication mutants. 相似文献
11.
McConkey GA 《Experimental parasitology》2000,94(1):23-32
The final step in guanylate nucleotide biosynthesis is catalysed by GMP synthase. This paper presents the first isolation of a gene encoding a protozoan GMP synthase. The deduced amino acid sequence from Plasmodium falciparum shares 40% identity with yeast GMP synthase and contains motifs conserved in catalysis. Expression of the gene is regulated through the parasite's development in human red blood cells with maximal expression during the point of DNA replication. Psicofuranine, which inhibits GMP synthase, interrupts parasite growth, supporting the role of this enzyme. These findings will aid development of inhibitors of purine salvage in malaria parasites. 相似文献
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Some structural properties of the DNA of Plasmodium falciparum were studied thoroughly using several techniques. Its G+C content was found to be extremely low (17-19%), the lowest reported for a living organism. The DNA seems to be composed only of the four major bases as no methylated bases were detected. This DNA had a Tm value of 62.5 degrees C and its denaturation profile showed no marked intramolecular heterogeneity. 相似文献
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DNA polymerase alpha 总被引:16,自引:0,他引:16
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An alpha-like DNA polymerase has been identified and characterized in the extracts from the malarial parasite Plasmodium falciparum. The enzyme is sensitive to the specific inhibitors of alpha-DNA polymerase, N-ethylmaleimide and aphidicolin, and is cell-cycle specific. High activity has been found in the schizont, is lower in trophozoites, and has only negligible activity in the ring form. The enzyme has a molecular weight of about Mr 100,000-103,000 estimated by detecting activity in SDS-polyacrylamide electrophoresis and by Bio-Gel filtration. Another active band of a molecular Mr 68,000 was detected by SDS electrophoresis when the enzyme was stored for 2 months at -20 degrees C. The catalytic activity of parasite enzyme was optimal between pH 8 and pH 9. The apparent Michaelis constant for dTTP was 4.3 microM. 相似文献
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Plasmodium falciparum, the major causative agent of human malaria, contains three separate genomes. The apicoplast (an intracellular organelle) contains an ∼ 35-kb circular DNA genome of unusually high A/T content (> 86%) that is replicated by the nuclear-encoded replication complex Pfprex. Herein, we have expressed and purified the DNA polymerase domain of Pfprex [KPom1 (Klenow-like polymerase of malaria 1)] and measured its fidelity using a LacZ-based forward mutation assay. In addition, we analyzed the kinetic parameters for the incorporation of both complementary and noncomplementary nucleotides using Kpom1 lacking 3′ → 5′ exonucleolytic activity. KPom1 exhibits a strongly biased mutational spectrum in which T → C is the most frequent single-base substitution and differs significantly from the closely related Escherichia coli DNA polymerase I. Using E. coli harboring a temperature-sensitive polymerase I allele, we established that KPom1 can complement the growth-defective phenotype at an elevated temperature. We propose that the error bias of KPom1 may be exploited in the complementation assay to identify nucleoside analogs that mimic this base-mispairing and preferentially inhibit apicoplast DNA replication. 相似文献
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Evolution of internal eliminated segments and scrambling in the micronuclear gene encoding DNA polymerase alpha in two Oxytricha species. 总被引:1,自引:0,他引:1
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To learn about the evolution of internal eliminated segments (IESs) and gene scrambling in hypotrichous ciliates we determined the structure of the micronuclear (germline) gene encoding DNA polymerasealpha(DNA polalpha) in Oxytricha trifallax and compared it to the previously published structure of the germline DNA polalphagene in Oxytricha nova . The DNA polalphagene of O.trifallax contains 51 macronuclear-destined segments (MDSs) separated by 50 IESs, compared to 45 MDSs and 44 IESs in the O.nova gene. This means that IESs and MDSs have been gained and/or lost during evolutionary divergence of the two species. Most of the MDSs are highly scrambled in a similar non-random pattern in the two species. We present a model to explain how IESs, non-scrambled MDSs and scrambled MDSs may be added and/or eliminated during evolution. Corresponding IESs in the two species differ totally in sequence, and junctions between MDSs and IESs are shifted by 1-18 bp in O.trifallax compared to the O.nova gene. In both species a short region of the gene is distantly separated from the main part of the gene. Comparison of the gene in the two species shows that IESs and scrambling are highly malleable over evolutionary time. 相似文献
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The homogeneous DNA polymerase alpha from early embryos of Drosophila melanogaster contains four polypeptides designated alpha, beta, gamma, and delta, with molecular weights of 148,000, 58,000, 46,000, and 43,000, respectively (Banks, G. R., Boezi, J. A., and Lehman, I. R. (1979) J. Biol. Chem. 254, 9886-9892). The four polypeptides are structurally distinct from one another, as indicated by their different peptide patterns following limited proteolysis with Staphylococcus aureus protease. Furthermore, the inclusion of the protease inhibitors, leupeptin and pepstatin, in addition to phenpylmethylsulfonyl fluoride and sodium metabisulfite, which are used routinely during the purification, does not alter the pattern of polypeptides in the purified polymerase, suggesting that the four polypeptides are not a consequence of nonspecific proteolysis during purification. Thus, the alpha, beta, gamma, and delta polypeptides appear to be distinct subunits of the alpha-DNA polymerase of D. melanogaster. The alpha subunit is required for DNA polymerase activity. However, the specific activity of the isolated subunit is substantially lower than when it is associated with the beta, gamma, and delta subunits. 相似文献