Benzimidazole resistance in Haemonchus contortus is correlated with a conserved mutation at amino acid 200 in beta-tubulin isotype 1.
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Alleles↗
Biomedical subjects
M H Roos
Publications and source records attributed to M H Roos.
Differential expression of two succinate dehydrogenase subunit-B genes and a transition in energy metabolism during the development of the parasitic nematode Haemonchus contortus.
The carbohydrate metabolism of free-living and parasitic stages of the sheep nematode Haemonchus contortus was studied, and it was demonstrated that during development a switch occurred from Krebs-cycle activity towards a more fermentative metabolism. During this switch a transition might take place in complex II of the respiratory chain. In the free-living (L3) and early parasitic (XL3) stages, complex II catalyses the oxidation of succinate to fumarate via the Krebs cycle, whereas in adults complex II functions in the reverse reaction, the reduction of fumarate to succinate. L3 and XL3 were shown to already possess a large anaerobic capacity. They survived well in the absence of oxygen or in the presence of cyanide, which completely blocked respiration. Krebs-cycle activity, however, was only partially inhibited by cyanide; the XL3s in particular produced in the presence of cyanide large amounts of propanol, the production of which probably functions as an alternative electron sink. For further investigation of the observed metabolic switch, complex II of the respiratory chain, a key enzyme involved in this switch, was studied. The B subunit of complex II was cloned and sequenced. These clones all showed sequences similar to the B subunit of succinate dehydrogenase from other species, and included the amino-terminal signal sequence for importation into mitochondria. Two genes were identified, types 1 and 2, based on the DNA and amino acid sequences and on the lack of cross-reaction to each other when used as probes on Southern blots. On Northern blots, the two genes showed a different expression pattern during the development of the parasite.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Effect of selection for benzimidazole resistance in Haemonchus contortus on beta-tubulin isotype 1 and isotype 2 genes.
In vitro selection for benzimidazole (BZ) resistance in the nematode Haemonchus contortus, a parasite of sheep, suggested the involvement of beta-tubilin genes. Two distinct beta-tubulin isotype classes were analyzed. Selection with stepwise increased concentrations of BZ showed for the first time that the increasing degrees of resistance correlate to a dual phase adaptation mechanism. At lower degrees of developing resistance, selection for a single beta-tubulin isotype 1 variant takes place. Subsequently, at higher degrees of drug resistance, an abrupt elimination of individuals carrying beta-tubulin isotype 2 genes in their genome is seen. To evaluate the in vitro experiments we carried out a survey of resistant field populations and found that in the most resistant population (RNO) apparently the same mechanism had happened.
Animals↗
Species-specific PCR for the parasitic nematodes Haemonchus contortus and Trichostrongylus colubriformis.
Reliable species determination of gastrointestinal nematode parasites from sheep is usually carried out on third-stage larvae (L3) by visual differentiation. The culturing of L3 takes about 1 week so a reliable method that could use eggs for the determination would accelerate the procedure. We developed a polymerase chain reaction (PCR), using very small amounts of parasite DNA and an oligonucleotide set according to parts of the DNA sequence of a beta-tubulin gene from Haemonchus contortus, that can discriminate H. contortus DNA and Trichostrongylus colubriformis DNA from each other and from several other sheep nematode parasites. This method can be used for every stage of the parasite, including eggs. For the first time a PCR method is reported to discriminate between sheep nematode parasites.
Animals↗
Molecular aspects of drug resistance in parasitic helminths.
Parasitic helminths (worms) cause serious infectious diseases in humans and animals. As control of these infections relies mostly on chemotherapeutics, the anthelmintics, resistance has developed against most of these drugs in several parasite species. These resistant parasites are being used to elucidate the molecular mechanisms of drug action.
Animals↗
Molecular characterisation of beta-tubulin genes present in benzimidazole-resistant populations of Haemonchus contortus.
A beta-tubulin isotype 1 gene, gru-1, from a benzimidazole (BZ)-resistant population of the nematode parasite, Haemonchus contortus, was cloned and sequenced. The predicted gene organisation showed 10 exons and 9 introns, one of which was H. contortus specific. Using probes and restriction sites selected from this sequence, restriction maps were constructed from and around beta-tubulin genes of 3 BZ-susceptible and 7 BZ-resistant populations. There was a reduction in beta-tubulin isotype 1 genes to usually one, in BZ-resistant populations. So, our previously reported reduction of beta-tubulin probe-reactive RFLP fragments in resistant populations correlated with the reduction of beta-tubulin isotype 1 genes. The beta-tubulin isotype 1 gene present on the apparently selected fragment and was not always the same, and the geographical origin of the resistant populations indicated independent development rather than geographical spread of the resistant populations. The beta-tubulin genes on the apparently selected fragments were transcribed and processed to mRNA using the nematode-specific trans-spliced leader (SL1). Comparison of the derived amino acid sequence of gru-1, with known sequences from a susceptible population, identified 3 mutations that could be involved in BZ resistance.
Amino Acid Sequence↗
Molecular analysis of selection for benzimidazole resistance in the sheep parasite Haemonchus contortus.
The molecular basis for the resistance of the sheep parasitic nematode Haemonchus contortus to the benzimidazole (BZ) group of anthelmintics was investigated. Three BZ-susceptible and three resistant populations from different geographical locations were characterized with respect to the egg-hatch assay with thiabendazole (TBZ), mebendazole (MBZ) binding tests and restriction fragment length polymorphism (RFLP) after Southern blotting. Cloned H. contortus alpha- and beta-tubulin genes were used as probes to analyze the RFLPs of genomic DNA prepared from mixtures of infectious larvae (L3) or adults. The susceptible populations showed, with both alpha- and beta-tubulin probes, 2 to 6 different fragments, depending on the restriction enzyme used. The three resistant populations showed as many fragments with the alpha-tubulin probe as the susceptible populations, but when probed with beta-tubulin only 1 or 2 fragments were visible, but always less than in the susceptible populations. An in vitro selection experiment was carried out using a susceptible population that was isolated in the laboratory before BZ came on the market. The results showed that after two selections with increasing amounts of TBZ, the population had become resistant, according to the egg-hatch assay values and MBZ binding assay. Using RFPL, the number of beta-tubulin probe reactive DNA fragments was reduced from 5 to 1. Analysis of the DNA of individual male adults of susceptible populations indicated a heterogeneity among the individual worms regarding the number of beta-tubulin probe reactive fragments (1 to 4) and frequency of the specific fragments. Usually, only one specific fragment (9 kb) was found in the resistant individuals. This 9-kb fragment was already present in some individuals in the susceptible population although it was in combination with other fragments. This would imply that genes conferring BZ resistance were present in H. contortus populations before BZ came on the market, and could explain the fast selection for BZ resistance in the field.
Animals↗
The molecular nature of benzimidazole resistance in helminths.
The economic importance of benzimidazole (BZ) resistance has resulted in the isolation of resistant populations of helminths and their study (see pp 127-129 this issue). Recent research indicates that BZs act by binding to free beta-tubulin in the cell and inhibiting the formation of microtubules. The effects of BZs on other processes in the cell, such as transport and anaerobic metabolism, probably result from the inhibition of one or more of the functions of tubulin (see pp 112-115, this issue). In this article, Marleen Roos examines the evidence for changes in the beta-tubulin structure and the rate of its synthesis in BZ-resistant parasitic nematodes.
Journal Article↗
A feline class II alpha gene with striking similarity to the HLA-DPA pseudogene.
A genomic library was constructed from DNA of a domestic cat and screened with a human HLA-DR alpha probe at low stringency. Several positive clones were isolated, and the DNA sequence of one of these clones was determined. Comparison with class II alpha gene sequences from other species suggested that the feline gene is a DPA homologue (FLA-DPA) showing 84% similarity with HLA-DP alpha 1 in the exon encoding the second domain. The FLA-DPA gene that was isolated is a pseudogene, as two frame-shift mutations are present: one in the exon encoding the second domain, causing premature termination of translation, and one in the exon encoding the transmembrane region. The latter mutation and the further deletion of two codons in the transmembrane exon show a remarkable resemblance to the same exon of the human pseudogene, HLA-DPA2. Hence, both pseudogenes evolved from the same ancestral gene. The inactivation of this DPA gene could therefore have occurred prior to the major mammalian divergence.
Amino Acid Sequence↗
DNA sequence of the coding region of the HLA-B44 gene.
An HLA-B44 cDNA clone was identified in a cDNA library constructed from an HLA-B44 homozygous cell line. The DNA sequence was determined and was found to contain the complete coding sequence but for (probably) the three N-terminal codons. Comparisons of the derived amino acid sequence with other HLA-A and -B locus amino acid sequences revealed four HLA-B44-specific substitutions including a new polymorphic site. Regions of strong sequence conservation for HLA-B-locus products were found at the nucleotide and amino acid levels.
Amino Acid Sequence↗
Genetics of human C4 polymorphism: detection and segregation of rare and duplicated haplotypes.
Applying a combined technology for the detection of allotypic variation of the fourth component of human complement (C4), including immunofixation with anti-C4 and C4-dependent lysis after agarose electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of C4 to separate the C4A and B alpha-chains, and the determination of Rodgers (Rg) and Chido (Ch) determinants of C4 in serum and at the blotted C4 alpha-chains, we detected rare human C4 allotypes and studied the genetic linkage. Partial inhibitors (p.i.) of anti-Rg and anti-Ch sera were found; the C4A51 allotype characterized as Rg p.i. and the C4A1 and C4B51 allotypes as Ch p.i. were genetically inherited. The C4A1 allotype has a unique Rg- Ch+ C4A alpha-chain. Duplicated C4A loci, A*3, A*2, and A*5, A*2 were both associated with a C4BQO and the HLA haplotype A3-Cw4-Bw35-DR1. These additions to the already known extensive C4 polymorphism may help to sort out their significance for the biological functions of human C4.
Complement C4↗
Analysis of factor B in European mouse strains; a polymorphism not due to variation of carbohydrate content.
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Animals↗
Subdivision of the S region of the mouse major histocompatibility complex by identification of genomic polymorphisms of the class III genes.
The S region of the mouse major histocompatibility complex (MHC) encodes the class III proteins, the second (C2) and fourth (C4) components of complement, and factor B. Previously, the assignment of S-region haplotypes was based on analysis of protein polymorphisms. The recent availability of C2, C4, and factor B cDNA probes prompted a search for restriction fragment length polymorphisms which would serve as additional genetic markers for these loci. DNA was isolated from livers of mice of all standard inbred H-2 haplotypes and of haplotypes pz and bs. These DNA samples were digested with restriction endonucleases and analyzed by Southern blot. By the pattern of restriction fragment length polymorphism observed, specific markers have been identified in factor B of haplotypes f, u, z, bs, r, and v, and in C4 of haplotypes b,q,f,j,p,s,pz,r, and v. These genetic markers were used in the analysis of S-region composition in strains B10.TFR5(H-2ap5) and C3H.LG(H-2dx), and a possible intra-S-region recombinant was revealed in the H-2dx haplotype. The genetic markers identified here subdivide the S region and will be of value in defining further the composition of the complement gene complex of the mouse MHC.
Alleles↗
An international reference typing for Ch and Rg determinants on rare human C4 allotypes.
Red cells, serum and plasma samples of 20 individuals, selected for their C4 allotypes, were distributed from Bonn to five laboratories, for investigation of their Chido (Ch) and Rodgers (Rg) determinants. One anti-Ch (M.H.) and one anti-Rg(Prest.) were distributed, but the individual laboratories also used their own reagents and their own typing methods. There was general agreement in interpretation of the majority of samples. Partial inhibition for Ch and Rg was detected. Two samples gave anomalous results; one sample with C4 A1,3 BQO, QO had Ch determinants on the red cells and in plasma (partial inhibition), and another sample with C4 A3,4 B5, QO apparently lacked Ch determinants on the red cells and in plasma. Heterogeneity of anti-Ch and anti-Rg was suggested in testing red cells, perhaps, reflecting a quantitative effect. This heterogeneity was confirmed by inhibition studies. The capacity of some reagents to detect partial inhibition probably reflects qualitative as well as quantitative differences.
Alleles↗
Rodgers (Rg) and Chido (Ch) determinants on human C4: characterization of two C4 B5 subtypes, one of which contains Rg and Ch determinants.
The genetically determined polymorphism of the fourth component of human complement was further extended with the aid of a panel of human allo-anti-C4 sera, anti-Rodgers and anti-Chido. These antisera were found previously to react with the alpha-chains of the C4 molecules controlled by the C4A and C4B loci, respectively. We analyzed a number of new and rare C4 allotypes, and found that they generally followed the expected pattern. Some interesting exceptions, however, were found. The alpha-chain of the allotype C4A1 was found to react with anti-Chido, unlike all other C4A allotypes. Also the C4B5 allotype could be subdivided into two subtypes on the basis of their reaction with anti-Rodgers. They were tentatively named B5Rg+ and B5Rg-. Moreover, the B5Rg+ subtype reacted not only with anti-Rodgers but also with some anti-Chido sera, indicating for the first time that Chido and Rodgers determinants are present on the same allotype.
Antigen-Antibody Reactions↗
Statement on the nomenclature of human C4 allotypes.
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Complement C4↗
A molecular basis for the two locus model of human complement component C4.
The major histocompatibility complex(MHC)-linked fourth component of complement (C4) shows a high degree of polymorphism in several animal species. In man C4 polymorphism was detected by distinct charge differences of the variants. O'Neill et al. showed that this C4 polymorphism was controlled by two closely linked genetic loci, F (C4A) and S (C4B) and these results were extended by Awdeh et al. with an improved typing method. Biochemical analysis of human C4 has revealed that it consists of three polypeptide chains, alpha, beta and gamma. In all reports so far on the molecular analysis of human C4, no molecular weight differences between the A and B locus-encoded molecules have been noticed. Here we demonstrate that the C4A and C4B locus-encoded alpha-chains have a molecular weight (MW) of 96,000 and 94,000, respectively, presenting for the first time a molecular basis for the difference between all C4A and C4B variants tested. Even rare variants that are difficult to allocate to the A or B locus on the basis of charge differences could be identified as C4A or C4B variants in this way, thereby providing new insights into the relationships between the C4A and C4B loci.
Complement C4↗
Molecular heterogeneity of D-end products detected by anti-H-2.28 sera. I. A. molecule similar to Qa-2, detected in the BALB/cBy but not in the BALB/c-H-2dm2 mutant.
Immunoprecipitation of NP-40 lysates of 125I-labeled lymph-node cells with different anti-H-2 sera and with anti-Qa-2 serum has shown that the BALB/cByA strain (H-2d, Qa-2-negative) expresses, besides H-2Ld, another molecule that is not detectable in the BALB/c-H-2dm2 strain. Electrophoresis in SDS polyacrylamide gels indicated that this molecule, provisionally designated Lq, has an apparent molecular weight of 41000 daltons, in contrast to approximately 49000 daltons for H-2Kd and H-2Ld, and 47000 daltons for H-2Dd molecules. The anti-Qa-2 serum precipitated from the Qa-2-positive strains BALB/cHeA but not from the Qa-2-negative strains BALB/cByA and BALB/c-H-2dm2 a protein that gave a very strong band corresponding to the molecular weight 41000 daltons in the gel electrophoresis. The biochemical characteristics of the Lq molecule are thus more similar to those of Qa-2 than of H-2 antigens.
Animals↗