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1.
Exp Parasitol ; 91(2): 126-32, 1999 Feb.
Article in English | MEDLINE | ID: mdl-9990340

ABSTRACT

The expression and activity of topoisomerase I (PfTopoI) has been examined during the intraerythrocytic stages of the Plasmodium falciparum life cycle. The promoter is inactive during the early ring stage and becomes active only during the later trophozoite and schizont stages. The PfTOP1 transcript starts to accumulate in the trophozoite stage parasite, decreasing again in the schizont stage. Using both stage-specific Western analysis and immunofluorescent assays we show that PfTopoI is present at low levels in rings and accumulates to approximately equal levels in the trophozoite and schizont stages. Experiments to determine the activity of PfTopoI, using a topoisomerase I relaxation assay, show that there is a low level of PfTopoI activity in both ring and trophozoite stages, but activity increases dramatically in the schizont stage. The PfTopoI activity can be inhibited by treatment with specific antiserum and by the type I topoisomerase-specific inhibitor camptothecin.


Subject(s)
DNA Topoisomerases, Type I/genetics , Gene Expression Regulation, Enzymologic , Plasmodium falciparum/genetics , Animals , Antibodies, Protozoan/chemistry , Blotting, Northern , Blotting, Western , Camptothecin/pharmacology , DNA Topoisomerases, Type I/metabolism , DNA, Protozoan/chemistry , Electrophoresis, Agar Gel , Electrophoresis, Polyacrylamide Gel , Enzyme Inhibitors/pharmacology , Fluorescent Antibody Technique , Malaria, Falciparum/enzymology , Malaria, Falciparum/parasitology , Nucleic Acid Hybridization , Plasmodium falciparum/drug effects , Plasmodium falciparum/enzymology , Promoter Regions, Genetic/physiology , Rabbits
2.
Mol Biochem Parasitol ; 92(1): 39-46, 1998 Apr 01.
Article in English | MEDLINE | ID: mdl-9574908

ABSTRACT

The stage-specific relationship between promoter activity, transcript production, protein expression and enzyme activity has been investigated for the gene encoding Plasmodium falciparum topoisomerase II (PfTopoII). Nuclear run-on experiments have shown that the P. falciparum topoisomerase II gene (PfTOP2) promoter is active at low levels in ring stage parasites, but reaches high levels of activity as the parasites progress into trophozoite/schizont asexual stages. Steady-state PfTOP2 transcripts are present at low levels in rings, accumulate in trophozoites, but are completely undetectable in schizonts. An antiserum raised against the species-divergent carboxy-terminus of PfTopoII, which neutralised the decatenation activity in parasite extracts, was used to probe Western blots of ring, trophozoite and schizont stage parasite extracts. Relatively low levels of PfTopoII were seen in rings compared with those in trophozoite and schizont preparations. Parasite extracts were also used to compare the patterns of protein accumulation and enzyme activity at these stages. Complete decatenation of kinetoplast substrate DNA (KDNA) was found in schizont stages, very low levels of activity were observed in rings and trophozoites showed intermediate levels. These finding show that, as parasites progress towards the stages where DNA replication occurs, there is a concomitant increase in both topoisomerase II production and activity.


Subject(s)
DNA Topoisomerases, Type II/biosynthesis , Erythrocytes/parasitology , Plasmodium falciparum/enzymology , Animals , Cell Differentiation , Crithidia fasciculata/genetics , DNA Topoisomerases, Type II/genetics , DNA, Circular/metabolism , Gene Expression Regulation , Humans , Plasmodium falciparum/cytology , Plasmodium falciparum/genetics , Promoter Regions, Genetic , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Protozoan/analysis , RNA, Protozoan/genetics , Time Factors
3.
Methods ; 13(2): 190-207, 1997 Oct.
Article in English | MEDLINE | ID: mdl-9405202

ABSTRACT

Biochemical and genetic analyses are required to identify potential drug targets in apicomplexan parasites, but these studies have proved difficult in most parasite systems. We have developed methods based on expression of parasite proteins in the budding yeast, Saccharomyces cerevisiae, to rapidly screen drugs directed against particular parasite targets, to study the structure and function of these target molecules, and to identify mutations in the parasite genes that alter enzyme specificity or drug sensitivity. In this paper we outline the parameters that need to be considered to design yeast strains that function efficiently to assay function of parasite proteins. Basic protocols and methods are included. We detail some problems that might be encountered in the engineering of these yeast strains and suggest possible solutions.


Subject(s)
Antiprotozoal Agents/pharmacology , Apicomplexa/drug effects , Apicomplexa/genetics , DNA Topoisomerases, Type II/genetics , Protozoan Proteins/biosynthesis , Pyrimethamine/pharmacology , Saccharomyces cerevisiae/genetics , Animals , Apicomplexa/metabolism , Base Sequence , DNA Topoisomerases, Type II/biosynthesis , Drug Design , Drug Evaluation, Preclinical/methods , Drug Resistance , Enzymes/biosynthesis , Enzymes/genetics , Genes, Protozoan , Genetic Complementation Test , Molecular Sequence Data , Mutagenesis , Protozoan Proteins/genetics , RNA, Messenger/biosynthesis , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/growth & development , Transcription, Genetic
4.
Mol Biochem Parasitol ; 82(2): 207-15, 1996 Nov 25.
Article in English | MEDLINE | ID: mdl-8946386

ABSTRACT

RNase protection assays and primer extension analysis have been used to locate a major transcription start site 960 bp upstream from the translational start of the PfPCNA coding sequence. A second, minor, site is situated a further 40 bp upstream. Intraerythrocytic parasite stages were transiently transfected with constructs containing a firefly luciferase reporter gene under the transcriptional control of variously modified elements of the PfPCNA 5' flanking sequence. These experiments identified a 470 bp region essential for promoter activity, which contains the physically mapped transcriptional start sites. In addition, a region between 290 and 620 bp upstream of the transcriptional start sites is required for efficient promoter activity.


Subject(s)
Plasmodium falciparum/genetics , Proliferating Cell Nuclear Antigen/genetics , Protozoan Proteins/genetics , Transcription, Genetic , Animals , Base Sequence , DNA Mutational Analysis , DNA, Protozoan , DNA, Recombinant , Gene Expression Regulation , Genes, Protozoan , Molecular Sequence Data , Promoter Regions, Genetic , Transfection
5.
Mol Biochem Parasitol ; 79(2): 177-82, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8855554

ABSTRACT

Antisera raised against proliferating cell nuclear antigen (PfPCNA) and DNA polymerase delta (PfDNA Pol delta) have been used against extracts from synchronised parasites to show that both proteins accumulate in trophozoites and persist in schizonts. The steady-state transcripts from both PfPCNA and PfDNA Pol delta also accumulate at the trophozoite stage. However, nuclear run on analysis shows that, whereas PfDNA Pol delta promoter activity is absent in rings but present in trophozoites and schizonts, the PfPCNA promoter is active throughout the intraerythrocytic cycle. This suggests that mechanisms regulating the expression of these two genes may be different although their coordinated activity is required for DNA replication.


Subject(s)
DNA-Directed DNA Polymerase/biosynthesis , Plasmodium falciparum/growth & development , Proliferating Cell Nuclear Antigen/biosynthesis , Animals , Blotting, Northern , Blotting, Western , DNA Polymerase III , Plasmodium falciparum/enzymology , Plasmodium falciparum/metabolism
6.
Parasitol Today ; 12(4): 151-5, 1996 Apr.
Article in English | MEDLINE | ID: mdl-15275224

ABSTRACT

Malaria is increasing as a global problem. Many of the drugs that were effective earlier in this century are now becoming obsolete as the parasite develops resistance to them and, despite earlier hopes, an affordable and effective vaccine remains elusive. It is hoped that a deeper understanding of the parasite's cell and molecular biology will give us a resource for the future and help us to achieve effective control. One aspect of parasite metabolism that has been the subject of recent studies is DNA replication: its timing during parasite development, the enzymes involved and the genes encoding them. In this review John White and Brian Kilbey report on the present status of these studies.

7.
Genetics ; 141(1): 1-5, 1995 Sep.
Article in English | MEDLINE | ID: mdl-8536959
8.
Gene ; 163(1): 151-4, 1995 Sep 22.
Article in English | MEDLINE | ID: mdl-7557466

ABSTRACT

Part of the topoisomerase I (TopoI)-encoding gene from Plasmodium falciparum (Pf) was isolated by PCR from cDNA using oligodeoxyribonucleotides modelled on the highly conserved regions of sequence from other species. The entire TopoI gene was obtained by screening a Pf K1 HindIII-EcoRI genomic library in lambda NM1149 with a random-labeled heterologous probe from the Saccharomyces cerevisiae TopoI gene. DNA sequence analysis revealed an open reading frame of 2520 nt encoding a deduced protein of 839 amino acids (aa) with no detectable introns. The Pf TopoI aa sequence has about 40% identity with most eukaryotic TopoI homologues. The gene is located as a single copy on chromosome 5 and Northern analysis identified a transcript of 3.8 kb.


Subject(s)
DNA Topoisomerases, Type I/genetics , Genes, Protozoan , Plasmodium falciparum/enzymology , Plasmodium falciparum/genetics , Amino Acid Sequence , Animals , Base Sequence , Conserved Sequence , Drosophila/genetics , Genes, Fungal , Humans , Introns , Mice , Molecular Sequence Data , Open Reading Frames , Polymerase Chain Reaction , Restriction Mapping , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Schizosaccharomyces/enzymology , Schizosaccharomyces/genetics , Sequence Homology, Amino Acid
10.
Nucleic Acids Res ; 22(13): 2547-51, 1994 Jul 11.
Article in English | MEDLINE | ID: mdl-8041616

ABSTRACT

The gene for topoisomerase II has been isolated from genomic libraries of strain K1 of the human malarial parasite, Plasmodium falciparum. The sequence reveals an open reading frame of 4194 nucleotides which predicts a polypeptide of 1398 amino acids. There are apparently no introns. The sequence is present as a single copy which has an identity of 47.4% and a similarity of 65.4% with its human homologue. Sequences conserved in topoisomerase II from other species are present in Pftopoisomerase II but in addition it has two adjacent asparagine-rich insertions which are unique to it. We have also detected asparagine-rich regions in the gene for PfDNA polymerase alpha. The gene for Pftopoisomerase II has been localised to chromosome 14 and northern analysis reveals a transcript of 5.8 kb. Two independent antisera raised in mice against glutathione-S-transferase fusion proteins containing the amino terminal portion of the malarial protein detect a weak band on western blots at about 160kDa, the expected size of the protein. Use of the same antisera for immunofluorescence analysis suggests that the protein is present at all stages of intraerythrocytic growth of the parasite.


Subject(s)
DNA Topoisomerases, Type II/genetics , Plasmodium falciparum/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Western , Chromosome Mapping , DNA Replication , DNA Topoisomerases, Type II/metabolism , DNA, Protozoan/biosynthesis , Genes, Protozoan , Humans , Molecular Sequence Data , Open Reading Frames , Plasmodium falciparum/enzymology , Sequence Homology, Amino Acid , Transcription, Genetic
11.
Nucleic Acids Res ; 21(16): 3643-6, 1993 Aug 11.
Article in English | MEDLINE | ID: mdl-8367280

ABSTRACT

The gene encoding DNA polymerase alpha from the human malaria parasite Plasmodium falciparum has been sequenced and characterised. The deduced amino acid sequence possesses the seven sequence motifs which characterise eukaryotic replicative DNA polymerases (I-VII) and four of five motifs (A-E) identified in alpha DNA polymerases. The predicted protein also contains sequences which are reminiscent of Plasmodium proteins but absent from other DNA polymerases. These include four blocks of additional amino acids interspersed with the conserved motifs of the DNA polymerases, four asparagine rich sequences and a novel carboxy-terminal extension. Repetitive sequences similar to those found in other malarial proteins are also present. cDNA-directed PCR was used to establish the presence of these features in the approximately 7kb mRNA. The coding sequence contains a single intron. The gene for DNAPol alpha is located on chromosome 4 and is transcribed in both asexual and sexual erythrocytic stages of the parasite.


Subject(s)
DNA Polymerase II/genetics , Genes, Protozoan , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Amino Acid Sequence , Animals , Humans , Molecular Sequence Data , Plasmodium falciparum/enzymology , RNA, Protozoan/genetics , Repetitive Sequences, Nucleic Acid , Sequence Homology, Amino Acid
12.
J Bacteriol ; 175(9): 2607-12, 1993 May.
Article in English | MEDLINE | ID: mdl-8478326

ABSTRACT

We have compared the mutagenic properties of a T-T cyclobutane dimer in baker's yeast, Saccharomyces cerevisiae, with those in Escherichia coli by transforming each of these species with the same single-stranded shuttle vector carrying either the cis-syn or the trans-syn isomer of this UV photoproduct at a unique site. The mutagenic properties investigated were the frequency of replicational bypass of the photoproduct, the error rate of bypass, and the mutation spectrum. In SOS-induced E. coli, the cis-syn dimer was bypassed in approximately 16% of the vector molecules, and 7.6% of the bypass products had targeted mutations. In S. cerevisiae, however, bypass occurred in about 80% of these molecules, and the bypass was at least 19-fold more accurate (approximately 0.4% targeted mutations). Each of these yeast mutations was a single unique event, and none were like those in E. coli, suggesting that in fact the difference in error rate is much greater. Bypass of the trans-syn dimer occurred in about 17% of the vector molecules in both species, but with this isomer the error rate was higher in S. cerevisiae (21 to 36% targeted mutations) than in E. coli (13%). However, the spectra of mutations induced by the latter photoproduct were virtually identical in the two organisms. We conclude that bypass and error frequencies are determined both by the structure of the photoproduct-containing template and by the particular replication proteins concerned but that the types of mutations induced depend predominantly on the structure of the template. Unlike E. coli, bypass in S. cerevisiae did not require UV-induced functions.


Subject(s)
DNA Replication , Escherichia coli/genetics , Mutagenesis/genetics , Pyrimidine Dimers , Saccharomyces cerevisiae/genetics , Base Sequence , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA, Bacterial/radiation effects , DNA, Fungal/genetics , DNA, Fungal/metabolism , DNA, Fungal/radiation effects , Enzyme Induction , Escherichia coli/metabolism , Escherichia coli/radiation effects , Genetic Vectors , Molecular Sequence Data , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/radiation effects , Species Specificity , Transformation, Genetic
13.
Mutat Res ; 299(3-4): 157-63, 1993 May.
Article in English | MEDLINE | ID: mdl-7683084

ABSTRACT

Data from experiments with single-stranded vectors that carry a site-specific cyclobutane dimer, pyrimidine (6-4) pyrimidone adduct, or abasic lesion, replicated in either E. coli or, in some cases, bakers' yeast, Saccharomyces cerevisiae, are used to examine two questions: (i) what factors are responsible for the lesion's mutagenicity? and (ii) what are the relative contributions of different photoproducts to the spectrum of UV-induced mutations? With respect to the first question, we suggest that the structure of the mutagen-modified template itself largely determines the kinds of mutations induced, but the relative frequencies of these mutations, the error frequency, and the bypass frequency are strongly dependent on the particular organism studied. With respect to the second question, we suggest that cyclobutane dimers may be responsible for most of the mutations in slowly replicating genomes because of the deamination of cytosine, and that the T-T, and to a lesser extent the T-C, (6-4) adducts play a greater role in the UV mutagenesis of quickly replicating viruses, such as M13 and lambda phage.


Subject(s)
DNA Damage , Escherichia coli/genetics , Mutagenesis , Pyrimidine Dimers , Saccharomyces cerevisiae/genetics , Ultraviolet Rays , Bacteriophage M13/genetics , Bacteriophage M13/radiation effects , Bacteriophage lambda/genetics , Bacteriophage lambda/radiation effects , DNA, Bacterial/radiation effects , DNA, Fungal/radiation effects , Escherichia coli/radiation effects , Saccharomyces cerevisiae/radiation effects
14.
Nucleic Acids Res ; 21(2): 239-43, 1993 Jan 25.
Article in English | MEDLINE | ID: mdl-8095096

ABSTRACT

The gene encoding the malarial homologue of proliferating cell nuclear antigen, PCNA, has been identified and characterised. It is located on chromosome 13. The coding sequence of 825 nucleotides predicts a protein of 30,586 Da. There are no introns and northern analysis reveals a transcript of approximately 1.6kb. The conserved residues which characterise the PCNAs of human, Drosophila, Saccharomyces and Xenopus are present in PfPCNA but the overall identity of PfPCNA with human and yeast PCNAs is low; 34% and 31% respectively. PfPCNA is longer than the PCNAs of these other species by about 16 amino acids, most of which are present in a block near the carboxy terminus. Antibodies against a purified PfPCNA-glutathione-S-transferase fusion protein recognise a single band in western blots of parasite extracts at 32kDa. The same antiserum has been used to demonstrate that the expression of PfPCNA is regulated during the intraerythrocytic development of the parasite. Expression increases dramatically in late trophozoites and is maintained during the subsequent nuclear divisions which produce schizonts.


Subject(s)
Nuclear Proteins/genetics , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Amino Acid Sequence , Animals , Antibodies, Protozoan , Base Sequence , Blotting, Northern , Blotting, Western , Cloning, Molecular , DNA, Single-Stranded , Erythrocytes/parasitology , Humans , Molecular Sequence Data , Nuclear Proteins/metabolism , Plasmodium falciparum/growth & development , Plasmodium falciparum/immunology , Proliferating Cell Nuclear Antigen , Protozoan Proteins/immunology , Protozoan Proteins/metabolism , Restriction Mapping , Sequence Homology, Amino Acid
15.
Nucleic Acids Res ; 19(24): 6731-6, 1991 Dec 25.
Article in English | MEDLINE | ID: mdl-1762904

ABSTRACT

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.


Subject(s)
DNA Polymerase II/genetics , DNA-Directed DNA Polymerase/genetics , Plasmodium falciparum/enzymology , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Blotting, Southern , Cloning, Molecular , DNA Polymerase III , Electrophoresis, Gel, Pulsed-Field , Molecular Sequence Data , Plasmodium falciparum/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Sequence Homology, Nucleic Acid
17.
Curr Genet ; 12(8): 591-7, 1987.
Article in English | MEDLINE | ID: mdl-3502942

ABSTRACT

The ade1 gene of the fission yeast Schizosaccharomyces pombe encodes a bifunctional polypeptide with glycinamide ribotide synthetase (GARSase) and aminoimidazole ribotide synthetase (AIRSase) enzyme activities. These enzyme activities carry out the 2nd and 5th steps, respectively, of the purine synthetic pathway. We report the cloning of the ade1 gene on a 4.4 kb Sau3A insert in the yeast shuttle vector pWH5. Integration of this genomic insert at or near the ade1 locus and its ability to complement, by transformation, three different types of ade1 mutants proved that it contains the ade1 chromosomal gene. Analysis of the nucleotide sequence of this insert revealed the presence of an uninterrupted open reading frame of 2,367 pb. This sequence, and the predicted 789 amino acid sequence encoded, both show a high degree of homology with the functionally equivalent ade5,7 gene sequence of Saccharomyces cerevisiae (approx. 60% overall in both cases) and Gart gene sequences of Drosophila melanogaster. The size of the ade1 RNA transcript is about 2.7 kb.


Subject(s)
Carbon-Nitrogen Ligases , Genes, Fungal , Purines/biosynthesis , Amino Acid Sequence , Base Sequence , Blotting, Northern , Cloning, Molecular , Ligases/genetics , Molecular Sequence Data , Restriction Mapping , Schizosaccharomyces/genetics , Transcription, Genetic , Transformation, Genetic
18.
Genetics ; 113(1): 45-51, 1986 May.
Article in English | MEDLINE | ID: mdl-2423414

ABSTRACT

The acridine half-mustard ICR-170-induced reversion of the mutant am15, which has a single base-pair deletion, at a frequency of between 9 and 28 X 10(-6). In each of three classes of revertants, the mutagen had induced the insertion of a -G- -C- base pair at a -G-G- -C-C- site. The mutant am6, which has a single base pair insertion, is known to be revertible, with UV light, by deletion of a -G- -C- base pair at a -G-G-G- -C-C-C- site. This mutant reverted with ICR-170 at a frequency of 0.1 X 10(-6). These results show that ICR-170 is able to induce addition frameshifts in Neurospora crassa within short, monotonous runs of G:C base pairs, but indicate a lack of deletion activity at such sequences.


Subject(s)
Aminoacridines , Genes, Fungal , Genes , Glutamate Dehydrogenase/genetics , Mutation , Neurospora crassa/genetics , Neurospora/genetics , Nitrogen Mustard Compounds/pharmacology , Amino Acid Sequence , Base Composition , Base Sequence , Neurospora crassa/enzymology
19.
Mutagenesis ; 1(1): 29-31, 1986 Jan.
Article in English | MEDLINE | ID: mdl-3325731

ABSTRACT

Four cdc7 alleles have been tested for their effects on the u.v.-induced reversion of arg4-17, a highly u.v.-revertible ochre mutant. The extent to which mutability is suppressed parallels the temperature sensitivity of growth of the mutants. The implication is that the pleiotropic consequences of the cdc7 mutations are different manifestations of a single function activity.


Subject(s)
Cell Cycle , Genes, Fungal , Mutation , Saccharomyces cerevisiae/genetics , Arginine , DNA Replication , Saccharomyces cerevisiae/radiation effects , Temperature , Ultraviolet Rays
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