An improved method for typing of plasma α1-protease inhibitor (PI1) variants in sheep

Summary
Sheep plasma α₁-protease inhibitor (PI1) variants were typed by analysing neuraminidase treated samples by isoelectric focusing in ultra-thin polyacrylamide gels followed by staining for trypsin inhibition. The 10 different PI1 pheno-types observed were shown to be controlled by four codominant alleles. The probability of sire exclusion provided by PI1 polymorphism was about 0.45 in both of the studied milk sheep breeds (Latxa and Karrantzar) of the Basque Country.     

Applicability of SSCP analysis for MHC genotyping: fingerprinting of Ovar-DRB1 exon 2 alleles from Finnish and Russian breeds

The applicability of single strand conformation polymorphism (SSCP) analysis for major histocompatibility complex (MHC) genotyping in sheep was studied. A panel of Ovar-DRB1 exon 2 'allele fingerprints' was defined. The panel could accelerate DRB1 genotyping of new breeds when already existing sequences are used as references in SSCP analysis. In this study, seven new exon 2 sequences and 19 different alleles in total were detected from 31 animals of Finnish and Russian sheep breeds. Ovar-DRB1 * 0201 was detected in all the six grey Finnsheep animals included in this study, suggesting reduced MHC diversity within these animals.     

HLA-DR polymorphism in a Senegalese Mandenka population: DNA oligotyping and population genetics of DRB1 specificities.

HLA class II loci are useful markers in human population genetics, because they are extremely variable and because new molecular techniques allow large-scale analysis of DNA allele frequencies. Direct DNA typing by hybridization with sequence-specific oligonucleotide probes (HLA oligotyping) after enzymatic in vitro PCR amplification detects HLA allelic polymorphisms for all class II loci. A detailed HLA-DR oligotyping analysis of 191 individuals from a geographically, culturally, and genetically well-defined western African population, the Mandenkalu, reveals a high degree of polymorphism, with at least 24 alleles and a heterozygosity level of .884 for the DRB1 locus. The allele DRB1*1304, defined by DNA sequencing of the DRB1 first-domain exon, is the most frequent allele (27.1%). It accounts for an unusually high DR13 frequency, which is nevertheless within the neutral frequency range. The next most frequent specificities are DR11, DR3, and DR8. Among DRB3-encoded alleles, DR52b (DRB3*02) represents as much as 80.7% of all DR52 haplotypes. A survey of HLA-DR specificities in populations from different continents shows a significant positive correlation between genetic and geographic differentiation patterns. A homozygosity test for selective neutrality of DR specificities is not significant for the Mandenka population but is rejected for 20 of 24 populations. Observed high heterozygosity levels in tested populations are compatible with an overdominant model with a small selective advantage for heterozygotes.     

Sequence-based genotyping of the sheep MHC class II DRB1 locus

The immunopolymorphism database (IPD) provides a single nomenclature for alleles at the major histocompatibility complex (MHC) loci for a range of different species. The minimum requirements for inclusion of a sheep class II DRB1 sequence is a submission that includes all polymorphic sites within the second exon from at least two independent polymerase chain reactions (PCR). In order to meet these requirements, we have developed a DNA-based genotyping method for the rapid analysis of allelic diversity at the DRB1 locus in domestic sheep, Ovis aries. Using a series of primers located within introns flanking exon 2 and genomic DNA from a cohort of 214 sheep representing 15 different breeds and crossbreeds, the complete exon 2 sequences of 38 Ovar-DRB1 alleles were obtained. This sequence resource allowed the development of a generic set of locus-specific primers which amplify a fragment that includes all polymorphic sites within the second exon. Bidirectional sequence analysis of the PCR product provides a composite sequence where each polymorphic site is represented by the corresponding International Union of Biochemistry nucleotide code. A Basic Local Alignment Search Tool search of alleles held within the IPD or National Center for Biotechnology Information databases allows individual allele sequences to be identified. Low levels of homozygosity (7.48%) within the cohort and verification of previously genotyped samples confirmed the broad allelic specificity of this method. It improves on currently available methods and is broadly applicable to the analysis of MHC diversity in studies investigating linkages with resistance or susceptibility to disease.     

Interdependent MHC-DRB exon-plus-intron evolution in artiodactyls

Exon 2 sequences of an expressed MHC-DRB locus from sheep were examined for polymorphisms in both the antigen-binding regions and the adjacent intronic mixed simple tandem repeat. Twenty-one novel exon 2 Ovar-DRB alleles were identified. Short nucleotide motifs are extensively shared between certain exon 2 regions of Ovar-DRB alleles. The simple repeat variations, the number of different amino acids at usually polymorphic sites, and the number of silent substitutions were reduced in the intraspecies analyses of sheep DRB sequences, compared with those of cattle and goats. It was paradoxical that the abundance of different sheep alleles was similar to that of cattle and goats. This paradox may be explained by postulating a relatively small number of "ancient" alleles, with the present-day Ovar-DRB alleles being generated by reciprocal exchange of nucleotide motifs. At the antigen-binding sites, new combinations of amino acids were maintained in Ovar-DRB alleles by strong positive selection. In sheep--and less pronounced in goats and cattle--the DRB alleles can be divided into two groups. In one group, silent substitutions are increased when compared with the other. This suggests separate evolutionary pathways for certain groups of DRB alleles within a species. The simple repetitive sequences are also discussed with respect to the evolution of DRB alleles.      

梅花鹿DRB 第二外显子等位基因鉴定的一种新方法

A new method was found which integrated Motif specific-PCR, SSCP and direct sequencing to identify the exon 2 of alleles of DRB genes in sika deer. Using this method, the detected 15 sequences which included no artificial sequences from mutation or recombination in the process all met the criterion to distinguish a new allele. The criterion is that there have to be identical sequences identified from at least two different individuals by PCR and direct sequencing. Compared with PCR-SSCP-cloning sequencing previously, this method can efficiently remove artificial sequences. Combined with the above criterion, this method provided a new approach for investigating DRB gene polymorphism of sika deer and other deer species.     

Evolution of MHC class II SLA‐DRB1 locus in the Croatian wild boar (Sus scrofa) implies duplication and weak signals of positive selection

The wild boar is an ancestor of the domestic pig and an important game species with the widest geographical range of all ungulates. Although a large amount of data are available on major histocompatibility complex (MHC) variability in domestic pigs, only a few studies have been performed on wild boars. Due to their crucial role in appropriate immune responses and extreme polymorphism, MHC genes represent some of the best candidates for studying the processes of adaptive evolution. Here, we present the results on the variability and evolution of the entire MHC class II SLA‐DRB1 locus exon 2 in 133 wild boars from Croatia. Using direct sequencing and cloning methods, we identified 20 SLA‐DRB1 alleles, including eight new variants, with notable divergence. In some individuals, we documented functional locus duplication, and SLA‐DRB1*04:10 was identified as the allele involved in the duplication. The expression of a duplicated locus was confirmed by cloning and sequencing cDNA‐derived amplicons. Based on individual genotypes, we were able to assume that alleles SLA‐DRB1*04:10 and SLA‐DRB1*06:07 are linked as an allelic combination that co‐evolves as a two‐locus haplotype. Our investigation of evolutionary processes at the SLA‐DRB1 locus confirmed the role of intralocus recombination in generating allelic variability, whereas tests of positive selection based on the dN/dS (non‐synonymous/synonymous substitution rate ratio) test revealed atypically weak and ambiguous signals.     

Extensive polymorphism of the BOLA-DRB3 gene distinguished by PCR-RFLP

A polymerase chain reaction (PCR)-based method is described for typing of alleles of the bovine lymphocyte antigen (BoLA)-DRB3 gene. A total of 30 DRB3 alleles were distinguished by digestion of PCR amplification products of BoLA-DRB3 exon 2 with RsaI, BstYI and HaeIII (PCR-RFLP). All restriction fragment patterns, with the exception of one HaeIII pattern, were consistent with restriction sites that were found among 14 previously sequenced DRB3 alleles. The PCR-RFLP typing method was evaluated on 168 genomic DNA samples collected from animals of 10 cattle breeds, 48 of which were typed in the Fourth International BoLA Workshop for BoLA-DRB and -DQ by conventional restriction fragment length polymorphism (RFLP) analysis using heterologous and homologous DNA probes. Thirty-one DRB/DQ haplotypes containing 23 DRB3 alleles were identified among the 48 workshop animals analysed. Using PCR-RFLP, 11 DRB3 alleles were identified in 18 workshop animals for which DRB RFLPs were not informative. PCR-RFLP typing of additional animals revealed five new DRB3 alleles, of which three contained a putatively located three basepair deletion in the identical position as found for the sequenced allele DRB*2A. PCR-RFLP was shown to be a rapid and sensitive method for the detection of polymorphism in a functionally relevant domain of the BoLA-DRB3 gene and should be useful for studying the evolution of DRB polymorphism in cattle and other Bovidae.     

Ovar-MHC Polymorphism in Malpura and Avikalin Sheep Vaccinated for Peste des Petits Ruminants (PPR) Virus

India harbors a vast diversity of sheep (40 breeds). The study was carried out to assess the genetic diversity of DRB1 and DQA2 locus of the ovar-MHC and their possible association with Peste des petits ruminants (PPR) virus vaccine response in Malpura and Avikalin sheep breeds maintained at an organized institute flock in the semi-arid region of India. Genetic analysis revealed the rich diversity of DRB1 locus with 23 alleles in Malpura and 21 alleles in Avikalin sheep that included 9 new alleles. DQA2 locus also had rich diversity with 19 alleles in Malpura and 20 alleles in Avikalin sheep that included 7 new alleles. At the protein level, high variability alike at the nucleotide level was observed. A marker for footrot susceptibility, DQA2*1101 was absent in both breeds. Genotypic association of DRB1 and DQA2 with PPR vaccine response was statistically non-significant. Vaccine response being a multifactorial (polygenic and influenced by environment) variable, could not show statistically significant association with MHC genotypes in the present study. However, rich genetic diversity of DRB1 and DQA2 gene reflects the importance of this locus for future selection programs.     

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

MHC-DRB exon 2 allele polymorphism in Indian river buffalo (Bubalus bubalis)

The polymorphism of the major histocompatibility complex (MHC) class II DRB gene of riverine buffalo (Bubalus bubalis) was studied. Second exon sequences from the buffalo DRB locus, homologous to the cattle DRB3 gene, were amplified and characterized. A combination of single strand conformation polymorphism (SSCP) and heteroduplex analysis (HA) in a non-denaturing gel was used to identify new DRB second exon sequences. SSCP, HA and finally sequencing allowed the identification of 22 MHC-DRB exon 2 alleles from 25 unrelated Indian river buffalo. These are the first river buffalo DRB second exon sequences reported. A high degree of polymorphism in the sequences encoding the peptide binding regions was observed and some amino acid substitutions were found unique to the river buffalo.     

Two divergent routes of evolution gave rise to the DRw13 haplotypes

The HLA class II genes and haplotypes have evolved over a long period of evolutionary time by mechanisms such as gene conversion, reciprocal recombination and point mutation. The extent of the diversity generated is most clearly evident in an analysis of the HLA class II alleles present within DRw13 haplotypes. This study uses cDNA sequencing to examine the first domains of DRB1, DRB3, DQA1, and DQB1 alleles from several American black individuals expressing seven different DRw13 haplotypes, five with undefined HLA-D specificities (i.e., not Dw18 or Dw19). Two new DRw13 alleles described in this study are the first examples of convergent evolution of DR alleles in which gene conversion has apparently combined segments of DRB1 alleles encoding DRw11 and DRw8 to generate two new DRB1 alleles, DRB1*1303 and DRB1*1304, that encode molecules bearing serologic determinants of a third allele, DRw13. These new DRw13 alleles are found embedded in haplotypes of DRw11 origin distinct from haplotypes encoding previously identified DRw13 alleles, DRB1*1301 and DRB1*1302. These data suggest that two evolutionary pathways may have given rise to two subgroups of alleles encoding molecules that share DRw13 serologic determinants yet which possess different structural and, likely, functional motifs. Reciprocal gene recombination events resulting in different DR, DRw52 and DQ allele combinations also appear to have played a crucial role in augmenting the level of diversity found in DRw13 haplotypes. Recombination has resulted in the association of one of the new DRw13 alleles with a DQw2 allele normally found associated with DR7 and the association of the DRw52c-associated DRw13 allele (DRB1*1302) with three different DQw1 alleles. The seven DRw13 haplotypes that have resulted from the effect of recombination on haplotypes formed by the two pathways of DRw13 allelic diversification have resulted in different repertoires of class II molecules and, most likely, different immune response profiles in individuals with these haplotypes.     

应用SSP-PCR/SSO方法进行中国辽宁汉族人HLA-DRB1基因的遗传多态性研究

对１５９名中国辽宁汉族个体的基因组ＤＮＡ进行分析,共检出４２种等位基因,其中以ＤＲＢ１０９０１２（１２．８％）、０７０１（１０．７％）、１５０１（１０．４％）最为常见,其次为ＤＲＢ１１２０１（７９％）、１２０２（７５％）、１１０１（６６％）、０３０１（５．０％）。并发现辽宁汉族人ＤＲＢ１等位基因频率与白种人间存在明显差异,揭示不同人种有其自己的主要等位基因。同时对本技术在ＨＬＡ－ＤＲＢ１分型应用中的优点进行了讨论     

Analysis and frequency of bovine lymphocyte antigen (BoLA-DRB3) alleles in Iranian Holstein cattle

The bovine lymphocyte antigen (BoLA-DRB3) gene encodes cell surface glycoproteins that initiate immune response by presenting processed antigenic peptides to CD4 T helper cells. DRB3 is the most polymorphic bovine MHC class II gene which encodes the peptide-binding groove. DRB3 gene has been extensively evaluated as a candidate marker for association with various bovine diseases and immunological traits. This study describes genetic variability in the BoLA-DRB3 in Iranian Holstein cattle. This is the first study of the DNA polymorphism of the BoLA-DRB3 gene in Iranian Holstein cattle. Hemi-nested PCR-RFLP method is used for identification the frequency of BoLA-DRB3 alleles. The BoLA-DRB3 locus is highly polymorphic in the studied herd (26 alleles). Almost 67% of the alleles were accounted for four alleles (BoLA-DRB3.2*8, *24, *11 and *16) in Iranian Holstein cattle. The DRB3.2*8 allele frequency (26.6%) was higher than the others. The frequencies of the DRB3.2*54, *37, *36, *28, *25, *14, *13, *10, *1 alleles were lower than 1%. Significant distinctions have been found between Iranian Holstein cattle and other cattle breeds studied. In Iranian Holstein cattle the alleles (BoLA-DRB3.2*22, *2 and *16) associated with a lower risk of cystic ovarian disease in Holstein cattle are found. The alleles associated with the resistance to mastitis and to bovine leukemia virus infection BoLA-DRB3.2*11 and *23 are detected with the frequencies 10.4% and 4.4%, respectively. Thus in the Iranian Holstein cows studied are found alleles which are associated with resistance to various diseases. The method of DNA-typing of animals can be used in agricultural practice for BoLA-DRB3 allele genotyping of cattle in order to reduce spreading of alleles providing susceptibility to mastitis or leukemia in cattle herds.     

Differentiation among Spanish sheep breeds using microsatellites

Genetic variability at 18 microsatellites was analysed on the basis of individual genotypes in five Spanish breeds of sheep – Churra, Latxa, Castellana, Rasa-Aragonesa and Merino -, with Awassi also being studied as a reference breed. The degree of population subdivision calculated between Spanish breeds from F_ST diversity indices was around 7% of total variability. A high degree of reliability was obtained for individual-breed assignment from the 18 loci by using different approaches among which the Bayesian method provided to be the most efficient, with an accuracy for nine microsatellites of over 99%. Analysis of the Bayesian assignment criterion illustrated the divergence between any one breed and the others, which was highest for Awassi sheep, while no great differences were evident among the Spanish breeds. Relationships between individuals were analysed from the proportion of shared alleles. The resulting dendrogram showed a remarkable breed structure, with the highest level of clustering among members of the Spanish breeds in Latxa and the lowest in Merino sheep, the latter breed exhibiting a peculiar pattern of clustering, with animals grouped into several closely set nodes. Analysis of individual genotypes provided valuable information for understanding intra- and inter-population genetic differences and allowed for a discussion with previously reported results using populations as taxonomic units.     

Identification of the amino acids in the Major Histocompatibility Complex class II region of Scottish Blackface sheep that are associated with resistance to nematode infection

Lambs with the Major Histocompatibility Complex DRB1*1101 allele have been shown to produce fewer nematode eggs following natural and deliberate infection. These sheep also possess fewer adult Teladorsagia circumcincta than sheep with alternative alleles at the DRB1 locus. However, it is unclear if this allele is responsible for the reduced egg counts or merely acts as a marker for a linked gene. This study defined the MHC haplotypes in a population of naturally infected Scottish Blackface sheep by PCR amplification and sequencing, and examined the associations between MHC haplotypes and faecal egg counts by generalised linear mixed modelling. The DRB1*1101 allele occurred predominately on one haplotype and a comparison of haplotypes indicated that the causal mutation or mutations occurred in or around this locus. Additional comparisons with another resistant haplotype indicated that mutations in or around the DQB2*GU191460 allele were also responsible for resistance to nematode infections. Further analyses identified six amino acid substitutions in the antigen binding site of DRB1*1101 that were significantly associated with reductions in the numbers of adult T. circumcincta.     

Tracing the origin of HLA-DRB1 alleles by microsatellite polymorphism.

We analyzed the origin of allelic diversity at the class II HLA-DRB1 locus, using a complex microsatellite located in intron 2, close to the polymorphic second exon. A phylogenetic analysis of human, gorilla, and chimpanzee DRB1 sequences indicated that the structure of the microsatellite has evolved, primarily by point mutations, from a putative ancestral (GT)x(GA)y-complex-dinucleotide repeat. In all contemporary DRB1 allelic lineages, with the exception of the human *04 and the gorilla *08 lineages, the (GA)y repeat is interrupted, often by a G-->C substitution. In general, the length of the 3' (GA)y repeat correlates with the allelic lineage and thus evolves more slowly than a middle (GA)z repeat, whose length correlates with specific alleles within the lineage. Comparison of the microsatellite sequence from 30 human DRB1 alleles showed the longer 5' (GT)x to be more variable than the shorter middle (GA)z and 3' (GA)y repeats. Analysis of multiple samples with the same exon sequence, derived from different continents, showed that the 5' (GT)x repeat evolves more rapidly than the middle (GA)z and the 3' (GA)y repeats, which is consistent with findings of a higher mutation rate for longer tracts. The microsatellite-repeat-length variation was used to trace the origin of new DRB1 alleles, such as the new *08 alleles found in the Cayapa people of Ecuador and the Ticuna people of Brazil.