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D J Kemp

Publications and source records attributed to D J Kemp.

At least 73 records · Page 4Linked to original sources
PubMed1990

Simplified colorimetric analysis of polymerase chain reactions: detection of HIV sequences in AIDS patients.

We have previously described a colorimetric test, designated an amplified DNA assay (ADA), for specific segments of DNA amplified by polymerase chain reactions (PCRs), suited to diagnostic applications. This relied on binding the amplified DNA via a sequence in one oligodeoxyribonucleotide (oligo) to the DNA-binding protein GCN4 coated on the wells of a microtiter dish. Avidin-peroxidase was then bound to biotin at the 5' end of the other oligo and detected colorimetrically. Two successive PCRs with nested oligos were utilized. We describe here several modifications that greatly simplify the ADA. First, we bind the DNA to a glutathione S-transferase-GCN4 fused polypeptide (GST-GCN4) and avidin-peroxidase simultaneously, rather than successively. Second, we carry out the two successive PCRs in the one reaction mixture, using the thermal stabilities of oligos of differing lengths to separate the two reactions. Third, PCRs can be performed in the wells of a microtiter dish and the amplified DNA captured and detected via GST-GCN4 immobilized on beads attached to the lid of the microtiter dish. Hence it is only necessary to pipette the DNA sample once, and up to 96 samples can then be handled simultaneously.

Acquired Immunodeficiency Syndrome↗
PubMed1990

Several alleles of the multidrug-resistance gene are closely linked to chloroquine resistance in Plasmodium falciparum.

The lethal form of human malaria caused by Plasmodium falciparum is virtually uncontrollable in many areas because of the development of drug resistance, in particular chloroquine resistance (CQR). CQR is biologically similar to the multiple drug resistance phenotype (MDR) of mammalian tumour cells, as both involve expulsion of drug from the cell and both can be reversed by calcium channel antagonists. A homologue (pfmdr1) of the mammalian multidrug resistance gene has been implicated in CQR because it is amplified in some CQR isolates of P. falciparum as is an mdr gene in MDR tumour cells. We show here that the complete sequences of pfmdr1 genes from 2 CQ sensitive (CQS) P. falciparum isolates are identical. In 5 CQR isolates, 1-4 key nucleotide differences resulted in amino acid substitutions. On the basis of these substitutions, we have correctly predicted the CQS/CQR status of a further 34 out of 36 isolates. This is a paradox as CQR arises much less frequently than would be predicted if single point mutations were sufficient. We conclude that a mutated pfmdr1 gene is one of at least two mutated genes required for CQR.

Alleles↗
PubMed1990

A molecular karyotype of Eimeria tenella as revealed by contour-clamped homogeneous electric field gel electrophoresis.

DNA from sporozoites of Eimeria tenella was resolved by pulsed field gel electrophoresis into nine chromosomal bands. Some bands of this molecular karyotype contained more than one chromosome as determined by the relative intensity of both staining with ethidium bromide and hybridisation to an E. tenella telomeric probe. Haploid forms of the parasite must be presumed to contain at least 12 chromosomes. The two smallest chromosomes were about 1.1 and 1.4 megabases. Most chromosomes were in excess of 3 Mb with the largest over 5 Mb as determined by comparison with the co-migration of chromosomes from Schizosaccharomyces pombe. A 5S ribosomal gene probe hybridised to a single chromosomal band.

Animals↗
PubMed1990

Detection of DNA from infectious laryngotracheitis virus by colourimetric analyses of polymerase chain reactions.

A combination of the polymerase chain reaction and a novel ELISA-type DNA colourimetric assay (developed from studies with a retrovirus from man) was used in a preliminary study to detect DNA from avian infectious laryngotracheitis virus. The method is sensitive, specific and easy to perform. Since it can be readily adapted for the detection of DNA from other sources it could be useful for the identification of a variety of pathogens from other species of veterinary importance.

Animals↗
PubMed1990

Structural diversity in the 45-kilodalton merozoite surface antigen of Plasmodium falciparum.

An integral membrane protein associated with the merozoite surface of Plasmodium falciparum termed merozoite surface antigen 2 (the 45-kDa merozoite surface antigen), occurs in antigenically diverse forms. Here we report the sequences of the MSA 2 gene from two other isolates of P. falciparum. The 43 N-terminal residues and the 74 C-terminal residues of all three MSA 2 sequences are highly conserved, but between these conserved regions there are dramatic differences among the alleles. Instead of the two copies of a 32-amino-acid repeat present in the MSA 2 of isolate FC27, MSA 2 from clone 3D7 and isolate Indochina 1 contain 5 and 12 copies respectively of the four amino acid sequence Gly Gly Ser Ala. The sequences flanking the repeats also differ among the three antigens. The repeats in MSA 2 appear to be immunodominant during natural infection, and antibodies to the repeat regions of different alleles react with a restricted number of parasite isolates.

Amino Acid Sequence↗
PubMed1990

Chromosome 9 from independent clones and isolates of Plasmodium falciparum undergoes subtelomeric deletions with similar breakpoints in vitro.

We show that chromosome 9 in all isolates and clones of Plasmodium falciparum examined so far exists as one of two distinctly different forms, a large form about 1.9 megabases long or a smaller form about 25% shorter. Physical maps of chromosome 9 from independent clones with large and small forms of chromosome 9, and from an isolate with the large form and 3 derived clones with the small form reveal the underlying structural basis of this size polymorphism. The small form differs from the large only in that there are subtelomeric deletions at each end, one of these deletions involving about 0.45 megabases. Remarkably, the breakpoints map within about +/- 1% of the total chromosome length for each of these populations. We discuss some possible mechanisms for this.

Animals↗
PubMed1989

Amplification of the multidrug resistance gene in some chloroquine-resistant isolates of P. falciparum.

Resistance of Plasmodium falciparum to chloroquine shares features with the multidrug resistance (MDR) phenotype of mammalian tumor cells. We report here the sequence of pfmdr, the P. falciparum homolog of mdr. We show that pfmdr is amplified in some chloroquine-resistant parasites but not in any of the sensitive isolates examined and that pfmdr transcript levels are increased. The gene is located on chromosome 5, and in one chloroquine-resistant line with an amplified pfmdr gene, chromosome 5 is greatly enlarged. The chromosome heterogeneity is due to varying copy numbers of different-sized pfmdr-containing amplicons. The existence of an mdr gene in P. falciparum and its amplification in some chloroquine-resistant lines greatly adds to the circumstantial evidence that pfmdr mediates chloroquine resistance in these lines.

Amino Acid Sequence↗
PubMed1989

Plasmodium falciparum: cytoadherence of a knobless clone.

Sequestration of Plasmodium falciparum-infected erythrocytes is crucial to parasite survival as it prevents destruction in the liver and spleen. Knobs have been considered necessary but not sufficient for cytoadherence to vascular endothelial cells in vivo and to melanoma or umbilical vein endothelial cells in vitro. We describe here a knobless clone that cytoadheres strongly to C32 melanoma cells. This clone cannot express the knob-associated histidine-rich protein (KAHRP) due to the deletion of the KAHRP gene. Our results raise the possibility of an alternative mechanism for in vitro cytoadherence and suggest that the use of long term cultured isolates and melanoma cells as a model for cytoadherence in vivo may be misleading.

Animals↗
PubMed1989

Molecular karyotyping of the rodent malarias Plasmodium chabaudi, Plasmodium berghei and Plasmodium vinckei.

The molecular karyotypes of four isolates of Plasmodium chabaudi, three of the subspecies P. chabaudi adami and one P. chabaudi chabaudi, as well as P. berghei and P. vinckei were studied by means of pulsed field gradient (PFG) gel electrophoresis. Each species appears to have 14 chromosomes, ranging in size from approximately 730 kb to greater than 2000 kb. The three P. chabaudi adami isolates did not appear any more similar to each other than to the P. c. chabaudi isolate. The chromosome locations of genes for a heat shock protein (hsp) 70, ribosomal RNA (rRNA), the precursor to the major merozoite surface proteins, dihydrofolate reductase and P. falciparum antigen 352 as well as four cloned DNA markers and a telomere probe were determined. However, a number of probes representing cloned P. falciparum antigens failed to hybridize to P. chabaudi DNA. Hence genes for malaria antigens appear to be much more divergent than genes for housekeeping functions.

Animals↗
PubMed1989

Chromosomes of malaria parasites.

The advent of pulsed field gradient electrophoresis has proved remarkably useful for studying chromosomes of the genetically intractable malaria parasite Plasmodium falciparum. Advances include determination of the karyotype, a linkage map and restriction maps of individual chromosomes that enable the ordering of genes. The structural basis underlying a frequently occurring form of chromosome size polymorphism is now understood and other polymorphisms are providing tantalizing clues to the mechanisms underlying drug resistance.

Animals↗
PubMed1989

A protective monoclonal antibody recognizes a linear epitope in the precursor to the major merozoite antigens of Plasmodium chabaudi adami.

The monoclonal antibody 5C10/66 was shown to afford strong protection in mice against fulminating Plasmodium chabaudi adami infection. This was remarkable, as immunity to this organism is regarded to be mainly T-cell mediated. This antibody identified a 250-kDa molecule in schizonts and an 83-kDa fragment in merozoites. A cDNA clone selected by 5C10/66 was the homologue of the Plasmodium falciparum precursor to the major merozoite surface antigen (PMMSA). Comparison with the P. falciparum sequence showed that the P. chabaudi adami clone encoded the middle portion of the gene and that it can also be divided into variable and conserved blocks. Screening of a set of all possible octamer peptides predicted by the cDNA clone revealed that the core epitope of 5C10/66 was Glu-Thr-Thr-Glu-Thr. This region resides in a variable block of PMMSA.

Amino Acid Sequence↗