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B J Kilbey

Publications and source records attributed to B J Kilbey.

At least 19 recordsLinked to original sources
PubMed1996

Physical and functional mapping of the transcriptional start sites of Plasmodium falciparum proliferating cell nuclear antigen.

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.

Animals↗
PubMed1996

Stage specific expression of proliferating cell nuclear antigen and DNA polymerase delta from Plasmodium falciparum.

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.

Animals↗
PubMed1996

DNA replication in the malaria parasite.

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.

Journal Article↗
PubMed1994

The gene encoding topoisomerase II from Plasmodium falciparum.

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.

Amino Acid Sequence↗
PubMed1993

The gene encoding DNA polymerase alpha from Plasmodium falciparum.

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.

Amino Acid Sequence↗
PubMed1993

Molecular characterisation and stage-specific expression of proliferating cell nuclear antigen (PCNA) from the malarial parasite, Plasmodium falciparum.

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.

Amino Acid Sequence↗
PubMed1993

Mutagenesis induced by single UV photoproducts in E. coli and yeast.

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.

Bacteriophage M13↗
PubMed1993

The frequency and accuracy of replication past a thymine-thymine cyclobutane dimer are very different in Saccharomyces cerevisiae and Escherichia coli.

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.

Base Sequence↗
PubMed1991

DNA polymerase delta: gene sequences from Plasmodium falciparum indicate that this enzyme is more highly conserved than DNA polymerase alpha.

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.

Amino Acid Sequence↗
PubMed1986

Sequencing studies of ICR-170 mutagenic specificity in the am (NADP-specific glutamate dehydrogenase) gene of Neurospora crassa.

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.

Amino Acid Sequence↗
PubMed1986

cdc7 alleles and the control of induced mutagenesis in yeast.

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.

Arginine↗
PubMed1984

Mutagenesis in yeast-misreplication or misrepair?

Evidence from the phenotype of mutants which partially block mutagenesis and from experiments made to time induced mutagenesis relative to cell division in yeast is used to question whether mutagenesis in yeast should be regarded as an error-prone repair phenomenon.

4-Nitroquinoline-1-oxide↗
PubMed1982

cdc7-1 a temperature sensitive cell-cycle mutant which interferes with induced mutagenesis in Saccharomyces cerevisiae.

The mutant cdc7-1 is shown here to block UV induced reversion of six different auxotrophic mutations and forward mutations at several genes concerned with adenine biosynthesis in Saccharomyces cerevisiae. Chemical mutagenesis is also drastically reduced. In its effect on mutagenesis cdc7-1 resembles rad6-1. However, in contrast to rad6-1, cdc7-1 does not affect sporulation or mitotic recombination neither is it sensitive to the antifolate drug trimethoprim. It appears to fall in the same epistatic group as rad6-1. Possible explanations for its action are briefly considered.

Cell Cycle↗
PubMed1982

Mutagenesis in cdc7 strains of yeast. The fate of premutational lesions induced by ultraviolet light.

cdc7-1 cells bearing UV-revertible mutations are virtually immutable by all means so far tested. By mating UV treated cdc7-1 cells with untreated cdc7+ cells carrying the same revertible alleles it is possible to rescue premutational lesions as revertants and to study their fate in cdc7-1 cells. If storage intervenes between treatment and mating there is a rapid decline in revertants rescued. This is not related to the death of cdc7-1 cells with storage nor does it reflect a progressive loss in their ability to mate.

Alleles↗