Hybridization of banteng (Bos javanicus) and zebu (Bos indicus) revealed by mitochondrial DNA, satellite DNA, AFLP and microsatellites.

Hybridization between wild and domestic bovine species occurs worldwide either spontaneously or by organized crossing. We have analysed hybridization of banteng (Bos javanicus) and zebu (Bos indicus) in south-east Asian cattle using mitochondrial DNA (PCR-RFLP and sequencing), AFLP, satellite fragment length polymorphisms (SFLP or PCR-RFLP of satellite DNA) and microsatellite genotyping. The Indonesian Madura zebu breed is reputed to be of hybrid zebu-banteng origin, but this has never been documented and Bali cattle are considered to be a domesticated form of banteng. The banteng mitochondrial type was found in all animals sampled on the isle of Bali, Indonesia, but only in 35% of the animals from a Malaysian Bali-cattle population. The Madura animals also carried mitochondrial DNA of either zebu and banteng origin. In both populations, zebu introgression was confirmed by AFLP and SFLP. Microsatellite analysis of the Malaysian Bali population revealed for 12 out of 15 loci screened, Bali-cattle-specific alleles, several of which were also found in wild banteng animals. The tools we have described are suitable for the detection of species in introgression studies, which are essential for the genetic description of local breeds and the preservation of their economic and cultural value.

Phylogeny of bovine species based on AFLP fingerprinting.

The Bovini species comprise both domestic and wild cattle species. Published phylogenies of this tribe based on mitochondrial DNA contain anomalies, while nuclear sequences show only low variation. We have used amplified fragment length polymorphism (AFLP) fingerprinting in order to detect variation in loci distributed over the nuclear genome. Computer-assisted scoring of electrophoretic fingerprinting patterns yielded 361 markers, which provided sufficient redundancy to suppress stochastic effects of intraspecies polymorphisms and length homoplasies (comigration of non-homologous fragments). Tree reconstructions reveal three clusters: African buffalo with water buffalo, ox with zebu, and bison with wisent. Similarity values suggest a clustering of gaur and banteng, but bifurcating clustering algorithms did not assign consistent positions to these species and yak. We propose that because of shared polymorphisms and reticulations, tree topologies are only partially adequate to represent the phylogeny of the Bovini. Principal-coordinate analysis positions zebu between a gaur/banteng cluster and taurine cattle. This correlates with the region of origin of these species and suggests that genomic distances between the cattle species have been influenced by genetic exchange between neighbouring ancestral populations.

Amplified fragment length polymorphism analysis of genetic diversity of Haemonchus contortus during selection for drug resistance.

For the first time we used amplified fragment length polymorphism on individual nematode parasites to analyse the genetic diversity between and within isolates during consecutive stages of increased benzimidazole resistance and of increased levamisole resistance of Haemonchus contortus. The genetic diversity of the H. contortus genome turned out to be unusually high, within and between the isolates. The difference between individuals of an isolate could be as high as between individuals of two different mammalian species that do not interbreed. During benzimidazole selection the genetic constitution of the population was changed, but did not lead to a decrease in the genetic diversity. The selection for levamisole resistance resulted in a limited reduction of the genetic diversity only after the first selection step. The extensive genetic diversity apparently has allowed a fast and flexible response of H. contortus to drug selection as shown by the appearance of drug resistant isolates. This selection however has little or no effect on the extent of the genetic diversity of these resistant isolates. Implications for more sustainable control methods are discussed.

EST sequencing of the parasitic nematode Haemonchus contortus suggests a shift in gene expression during transition to the parasitic stages.

Expressed sequence tags from the parasitic nematode Haemonchus contortus were generated in order to identify anchor loci for comparative mapping between nematode genomes and candidate targets for future control measures. In total, 370 SL1 trans-spliced cDNAs from different developmental stages representing 195 different genes were partially sequenced. From these expressed sequence tags 50% were similar to genes with a known or predicted function and 19% were similar to nematode sequences with no ascribed function. From the first, free-living L1 and L3 stages relatively many cDNAs matched to housekeeping genes, and 11% (L1) or 23% (L3) of the encoded proteins were predicted to contain signal peptides. In contrast, no function could be ascribed to most of the cDNAs from the early L5 and adult parasitic stages, but for 30% (L5) or 55% (adult) of the encoded proteins a signal sequence was predicted. This limited analysis suggests that during the transition from the free-living to parasitic stages gene expression shifts towards the synthesis of less conserved extracellular proteins. These proteins offer the best perspectives for vaccine development and the development of anthelmintic drugs. In contrast, cDNAs from the first larval stages may be most suitable for comparative mapping with the free-living nematode Caenorhabditis elegans.

Microsatellite diversity of isolates of the parasitic nematode Haemonchus contortus.

The alarming development of anthelmintic resistance in important gastrointestinal nematode parasites of man and live-stock is caused by selection for specific genotypes. In order to provide genetic tools to study the nematode populations and the consequences of anthelmintic treatment, we isolated and sequenced 59 microsatellites of the sheep and goat parasite Haemonchus contortus. These microsatellites consist typically of 2-10 tandems CA/GT repeats that are interrupted by sequences of 1-10 bp. A predominant cause of the imperfect structure of the microsatellites appeared mutations of G/C bp in the tandem repeat. About 44% of the microsatellites were associated with the HcREP1 direct repeat, and it was demonstrated that a generic HcREP1 primer could be used to amplify HcREP1-associated microsatellites. Thirty microsatellites could be typed by polymerase chain reaction (PCR) of which 27 were polymorphic. A number of these markers were used to detect genetic contamination of an experimental inbred population. The microsatellites may also contribute to the genetic mapping of drug resistance genes.

Transposon associated markers for the parasitic nematode Haemonchus contortus.

We have previously characterized a Tcl-like transposable element Hctcl, from the parasitic nematode Haemonchus contortus. Here we describe the genetic variation of Hctcl insertion sites in H. contortus populations differing in geographical origin, resistance to chemotherapeutics and level of inbreeding. Clear differences between populations were observed on Southern blots with a Hctcl-specific probe. Sequencing the 5'- or 3'-flanks of individual Hctcl integration sites allowed the design of PCR reactions between a Hctcl-specific primer and the flanking regions. This revealed a considerable variation of integration sites of Hctcl both within and between populations, although several integrations were shared by populations of different geographical origin. For four of the eight markers allele frequencies were shifted during selection for resistance to chemotherapeutics and/or inbreeding. For two positions both the 5' and 3' regions flanking Hctcl were isolated and PCR showed that for these two positions the variation of transposon associated markers between populations were indeed caused by variation in integration of Hctcl. For these two positions co-dominant markers were developed. These results indicate that Hctcl insertions may serve as genetic markers for H. contortus.

Satellite DNA polymorphisms and AFLP correlate with Bos indicus-taurus hybridization.

We describe satellite DNA variation that detects hybridization of Bos indicus (zebu or indicine cattle) and Bos taurus (taurine cattle) in African cattle populations. On Southern blots hybridized to a satellite III probe, relative intensities of Hinfl fragments correlated with the taurine-zebu composition in hybrid animals as deduced from AFLP genotyping of the same animals and previous data on microsatellite allele frequencies. Similar results were obtained by PCR-RFLP analysis of a zebu-specific mutation in the repeat unit of satellite 1.711b. Analysis of individuals from 20 African cattle breeds indicate that the centromeric satellites of the sanga breeds are of the taurine type and that several East-African zebu breeds are hybrids between taurine and zebu. These satellite RFLP, or SFLP, markers provide a fast method to screen the genetic makeup of African cattle.

Characterisation of a polymorphic Tc1-like transposable element of the parasitic nematode Haemonchus contortus.

Hctc1, a member of the Tc1-family of transposable elements was isolated from the parasitic nematode Haemonchus contortus. Hctc1 is 1590 bp long, is flanked by 55 bp inverted repeats and carries a single open reading frame of a 340 amino acid transposase-like protein. Hctc1 is similar to Tc1 of Caenorhabditis elegans and elements Tcb1 and Tcb2 of Caenorhabditis briggsae in the inverted terminal repeats, the open reading frame, as well as the target insertion sequence. Furthermore, the copy number of Hctc1 is comparable with the Tc1 copy number in low copy strains of C. elegans. The sequence of Hctc1 is highly variable in H. contortus due to deletions, insertions and point mutations, with at least five distinct length variants of Hctc1. Most of the Hctc1 variation was within rather than between H. contortus populations. The high level of sequence variation is probably due to variation generally found for members of the Tc1-family, as well as a high background level of genetic variation of H. contortus.

Polymorphic DNA markers in the genome of parasitic nematodes.

Polymorphic molecular markers are being identified to characterize the genomes of parasitic nematodes. The aim is to construct a map with markers evenly spread over the six chromosomes. With such a map, regions can be identified that are under selection pressure when attempts are being made to eradicate worms, be it by drugs, vaccines or genetic resistance in the sheep. Several types of markers have been developed, microsatellites, transposon-associated markers, amplified fragment length polymorphism (AFLP) and expressed sequence tag (EST) markers. Linkage groups can be constructed using several genetic crosses between inbred and drug resistant strains. EST markers will be especially important for comparative mapping with the genome of Caenorhabditis elegans, and therefore localization of the linkage group on a chromosome. It will then be possible to identify functional genes close to markers that have changed allele frequencies under selection pressure and identify the mechanisms of resistance to parasite control.

Use of AFLP markers for gene mapping and QTL detection in the rat.

The AFLP technique is a new DNA marker technology based on the selective amplification of restriction fragments. Multiple polymorphic markers are simultaneously produced and can be tested in one PCR. No prior information on genomic DNA sequences is needed. In the current study, we contribute 18 AFLP markers to the linkage map of the rat. Seven AFLP markers were assigned to specific chromosomes by analysis of a (BN x ACI)F1 x ACI backcross progeny. Another 11 AFLP markers were mapped by using a panel of the H x B/B x H recombinant inbred (RI) strains. Genotypes of these AFLP markers were also tested for correlations with some blood pressure phenotypes in the RI strains. Suggestive correlation was found between the mean arterial pressure and two closely linked AFLP markers located on chromosome 20. The current study illustrates the value of AFLP markers for the construction of linkage maps and the detection of quantitative trait loci.

Use of simple sequence length polymorphisms for genetic characterization of rat inbred strains.

Genetic monitoring is an essential component of colony management and for the rat has been accomplished primarily by using immunological and biochemical markers. Here, we report that simple sequence length polymorphisms (SSLPs) are a faster and more economical way of monitoring inbred strains of rats. We characterized 61 inbred strains of rats, using primer pairs for 37 SSLPs. Each of these loci appeared to be highly polymorphic, with the number of alleles per locus ranging between 3 and 14 and, as a result, all the 61 inbred strains tested in this study could be provided with a unique strain profile. These strain profiles are also used for estimating the degree of similarity between strains. This information may provide the rationale in selecting strains for genetic crosses or for other specific purposes.

Linkage of the gene for uncoupling protein to esterase-1,2 and haptoglobin in the rat.

Genetic analysis of a (BN x ACI)ACI backcross for the biochemical markers Es-1 and Es-2 (linkage group V) and a microsatellite marker of Ucp (chromosome 19) revealed linkage between these three loci. This linkage relationship confirms that in the rat linkage group V genes are located on chromosome 19. Genetic analysis of a (BN x LEW)LEW backcross showed that Hp is linked to Ucp, thus indicating that the Hp locus is also located on chromosome 19. These results substantiate the linkage homology of RNO 19, MMU 8, and HSA 16.

Enhancement of the antibody-dependent cellular cytotoxicity of human peripheral blood lymphocytes with interleukin-2 and interferon alpha.

Antibody-dependent cellular cytotoxicity (ADCC) is regarded as an important mechanism by which monoclonal antibodies (mAb) can exert an antitumour effect in vivo. It may be possible, therefore, to enhance the therapeutic efficacy of mAb by cytokines that are able to enhance the ADCC of human CD3-, CD56+, CD16+ natural killer (NK) cells. We investigated in vitro the effects of recombinant interferon alpha (rIFN alpha) and recombinant interleukin 2 (rIL-2), alone or in combination, on the ADCC of human peripheral blood NK cells. Both cytokines enhanced the ADCC of the human effector cells. rIFN alpha induced a maximally increased ADCC after an exposure of human effector cells to 20 IU/ml for 15-30 min, while rIL-2 induced optimal ADCC after incubation of the cells for 2 days in 20-50 U/ml. We now show that activation of the NK cells with a combination of rIL-2 and rIFN alpha induced significantly higher levels of ADCC than either cytokine alone. The highest ADCC was induced if the cells were first exposed to rIL-2 before rIFN alpha was added to the culture. Culture of NK cells in medium or rIL-2 decreased the expression of Fc gamma RIII (CD16), indicating that intensity of CD16 expression and level of ADCC are not directly correlated, although blocking experiments with a mAb directed against CD16 showed that this Fc gamma R was essential for ADCC of the human effector cells.