Population genetic diversity and geographical differentiation of MHC class II DAB genes in the vulnerable Chinese egret (<Emphasis Type="Italic">Egretta eulophotes</Emphasis>)

Major histocompatibility complex (MHC) genes are excellent markers for the study of adaptive genetic variation occurring over different geographical scales. The Chinese egret (Egretta eulophotes) is a vulnerable ardeid species with an estimated global population of 2600–3400 individuals. In this study, we sampled 172 individuals of this egret (approximately 6 % of the global population) from five natural populations that span the entire distribution range of this species in China. We examined their population genetic diversity and geographical differentiation at three MHC class II DAB genes by identifying eight exon 2 alleles at Egeu-DAB1, eight at Egeu-DAB2 and four at Egeu-DAB3. Allelic distributions at each of these three Egeu-DAB loci varied substantially within the five populations, while levels of genetic diversity varied slightly among the populations. Analysis of molecular variance showed low but significant genetic differentiation among five populations at all three Egeu-DAB loci (haplotype-based ?_ST: 0.029, 0.020 and 0.042; and distance-based ?_ST: 0.036, 0.027 and 0.043, respectively; all P < 0.01). The Mantel test suggested that this significant population genetic differentiation was likely due to an isolation-by-distance pattern of MHC evolution. However, the phylogenetic analyses and the Bayesian clustering analysis based on the three Egeu-DAB loci indicated that there was little geographical structuring of the genetic differentiation among five populations. These results provide fundamental population information for the conservation genetics of the vulnerable Chinese egret.     

Expression pattern of two paralogs of the <Emphasis Type="Italic">PI</Emphasis>/<Emphasis Type="Italic">GLO</Emphasis>-like locus during <Emphasis Type="Italic">Orchis italica</Emphasis> (Orchidaceae,Orchidinae) flower development

The class B MADS-box genes belong to two distinct functional groups: the AP3/DEF-like and the PI/GLO-like sub-families. In orchids, AP3/DEF-like genes are present in four copies, each with a different role in floral organ formation, which is described in the “orchid code” model. Interestingly, the orchid PI/GLO-like genes are present in two copies in Orchidinae, whereas they are described as single copy in the other orchid lineages. The two PI/GLO-like paralogs have site-specific different selective constraints; in addition, they show relaxation of purifying selection when compared to the single-copy lineages. In this study, we present a comparative analysis of the expression patterns of the two PI/GLO-like paralogs, OrcPI and OrcPI2, in floral tissues of Orchis italica in different developmental stages using real-time PCR. The two genes show similar expression profiles in the tissue examined, with differences detectable between immature and mature inflorescence. In all cases, OrcPI2 is expressed at a higher level than OrcPI. Real-time PCR results reveal that the co-expression of the two duplicated loci could have a fully or partially redundant function. The possible evolutionary fate of OrcPI and OrcPI2 is discussed as well as their involvement in ovary development.     

Patterns of variability at the major histocompatibility class I and class II loci in populations of the endangered cyprinid <Emphasis Type="Italic">Ladigesocypris ghigii</Emphasis>

The patterns of MHC diversity were studied at UAA and DAB1 loci and the two domains involved in the recognition of antigenic peptides (α2 and β1, respectively) in eight Ladigesocypris ghigii populations inhabiting streams and a concrete reservoir, in order to understand the significance of these genes in bottlenecked populations of an endemic species and develop conservation rationale. In agreement with previous study employing RAPD and mtDNA markers (Mamuris et al., Freshw Biol 50:1441–1453, 2005), both loci exhibited a very low level of polymorphism with only two and four alleles detected for UAA and DAB1, respectively. The functional MHC diversity was even lower since UAA alleles were distinguished by a single synonymous substitution. The type of habitat did not affect the level of polymorphism. Our data suggest that DAB1 polymorphism might be the outcome of the positive selection, imposed by the temporal and spatial variation of pathogen load, and the genetic drift as a result of successive habitat shrinkage and deterioration by water abstraction year after year. The populations studied were significantly less diverged at MHC loci than expected based on nuclear and mtDNA markers, suggesting that common parasites might act as causative factors to homogenize selection. Sufficient epidemiological data are required for the interpretation of the results and decision-making on suitable conservation actions.     

Evolution of MHC-<Emphasis Type="Italic">DRB</Emphasis> class II polymorphism in the genus <Emphasis Type="Italic">Apodemus</Emphasis> and a comparison of <Emphasis Type="Italic">DRB</Emphasis> sequences within the family Muridae (Mammalia: Rodentia)

Allelic diversity at major histocompatibility complex (MHC) genes is thought to be maintained by balancing selection over long periods of time, even across multiple speciation events. Trans-species sharing of MHC alleles among genera has been supported by many studies on mammals and fish, but in rodents, the results are ambiguous. We investigated natural levels of MHC-DRB variability and evolutionary processes in the wood mouse (Apodemus sylvaticus) and the yellow-necked mouse (Apodemus flavicollis), which are common, sympatric murid rodents in European forests. Using single-strand conformation polymorphism analysis and DNA sequencing, 38 DRB exon 2 alleles were detected among 162 A. sylvaticus from nine different locations in Germany and Switzerland, and 15 DRB exon 2 alleles were detected among 60 A. flavicollis from three different locations in northern Germany. There was evidence for balancing selection in both species. Phylogenetic analysis, including additional murid taxa, showed that the DRB exon 2 sequences did not separate according to species, consistent with trans-species evolution of the MHC in these taxa.     

2004 Nomenclature for the chicken major histocompatibility (<Emphasis Type="Italic">B</Emphasis> and <Emphasis Type="Italic">Y</Emphasis> ) complex

The first standard nomenclature for the chicken (Gallus gallus) major histocompatibility (B) complex published in 1982 describing chicken major histocompatibility complex (MHC) variability is being revised to include subsequent findings. Considerable progress has been made in identifying the genes that define this polymorphic region. Allelic sequences for MHC genes are accumulating at an increasing rate without a standard system of nomenclature in place. The recommendations presented here were derived in workshops held during International Society of Animal Genetics and Avian Immunology Research Group meetings. A nomenclature for B and Y (Rfp-Y) loci and alleles has been developed that can be applied to existing and newly defined haplotypes including recombinants. A list of the current standard B haplotypes is provided with reference stock, allele designations, and GenBank numbers for corresponding MHC class I and class II sequences. An updated list of proposed names for B recombinant haplotypes is included, as well as a list of over 17 Y haplotypes designated to date.     

Extended sequence of the turkey MHC <Emphasis Type="Italic">B</Emphasis>-locus and sequence variation in the highly polymorphic B-G loci

Genetic variation in the major histocompatibility complex (MHC) is directly correlated to differences in disease resistance. Immunity is greatly dependent on highly polymorphic genes in the MHC, such as class I, class II, and class III complement genes. Preliminary studies of wild turkey populations show extreme polymorphisms in a family of genes exclusive to the avian MHC, the class IV or B-G genes. Significance of this variation is unclear as there are few and conflicting studies of the expression of these genes. Confounding understanding of B-G variation is the lack of a complete delineation of the number of loci in the turkey genome. Direct 454 sequencing of a clone from the CHORI-260 BAC library was used to extend the turkey MHC B-locus sequence, identifying five additional complete B-locus genes including two B-G loci. Sequences of the new B-G genes were compared with those of other turkey gene (BG1–3) and sequences available for other galliformes. Phylogenetic analysis shows species-specific gene evolution supporting a birth–death model of evolution for the B-G gene family. Analysis of variation within the signal peptide sequence (exon 1) found two clusters of polymorphism among the turkey B-G genes. Resequencing of exon 1 in a diverse sample including wild, heritage, and commercial turkeys confirmed multiple alleles at each B-G gene. Future studies aim to correlate B-G variation with group and individual immunological differences.     

Molecular evolution of paclitaxel biosynthetic genes <Emphasis Type="Italic">TS</Emphasis> and <Emphasis Type="Italic">DBAT</Emphasis> of <Emphasis Type="Italic">Taxus</Emphasis> species

Evolutionary patterns of sequence divergence were analyzed in genes from the conifer genus Taxus (yew), encoding paclitaxel biosynthetic enzymes taxadiene synthase (TS) and 10-deacetylbaccatin III-10β-O-acetyltransferase (DBAT). N-terminal fragments of TS, full-length DBAT and internal transcribed spacer (ITS) were amplified from 15 closely related Taxus species and sequenced. Premature stop codons were not found in TS and DBAT sequences. Codon usage bias was not found, suggesting that synonymous mutations are selectively neutral. TS and DBAT gene trees are not consistent with the ITS tree, where species formed monophyletic clades. In fact, for both genes, alleles were sometimes shared across species and parallel amino acid substitutions were identified. While both TS and DBAT are, overall, under purifying selection, we identified a number of amino acids of TS under positive selection based on inference using maximum likelihood models. Positively selected amino acids in the N-terminal region of TS suggest that this region might be more important for enzyme function than previously thought. Moreover, we identify lineages with significantly elevated rates of amino acid substitution using a genetic algorithm. These findings demonstrate that the pattern of adaptive paclitaxel biosynthetic enzyme evolution can be documented between closely related Taxus species, where species-specific taxane metabolism has evolved recently.     

Reduced MHC class II diversity in island compared to mainland populations of the black-footed rock-wallaby (<Emphasis Type="Italic">Petrogale lateralis lateralis</Emphasis>)

Many animal populations that are endangered in mainland areas exist in stable island populations, which have the potential to act as an “ark” in case of mainland population declines. Previous studies have found neutral genetic variation in such species to be up to an order of magnitude lower in island compared to mainland populations. If low genetic variation is prevalent across fitness-related loci, this would reduce the effectiveness of island populations as a source of individuals to supplement declining mainland populations or re-establish extinct mainland populations. One such species, the black-footed rock-wallaby (Petrogale lateralis lateralis), exists within fragmented mainland populations and small island populations off Western Australia. We examined sequence variation in this species within a fitness-related locus under positive selection, the MHC class II DAB β1 locus. The mainland populations displayed greater levels of allelic diversity (4–7 alleles) than the island population, despite being small and isolated, and contained at least two DAB gene copies. The island population displayed low allelic diversity (2 alleles) and fewer alleles per individual in comparison to mainland populations, and probably possesses only one DAB gene copy. The patterns of DAB diversity suggested that the island population has a markedly lower level of genetic variation than the mainland populations, in concordance with results from microsatellites (genotyped in a previous study), but preserved unique alleles which were not found in mainland populations. Where possible, conservation actions should pool individuals from multiple populations, not only island populations, for translocation programs, and focus on preventing further declines in mainland populations.     

Phylogeny and divergence of basal angiosperms inferred from<Emphasis Type="Italic"> APETALA3</Emphasis>- and<Emphasis Type="Italic"> PISTILLATA</Emphasis>-like MADS-box genes

The B-class MADS-box genes composed of APETALA3 (AP3) and PISTILLATA (PI) lineages play an important role in petal and stamen identity in previously studied flowering plants. We investigated the diversification of the AP3-like and PI-like MADS-box genes of eight species in five basal angiosperm families: Amborella trichopoda (Amborellaceae); Brasenia schreberi and Cabomba caroliniana (Cabombaceae); Euryale ferox, Nuphar japonicum, and Nymphaea tetragona (Nymphaeaceae); Illicium anisatum (Illiciaceae); and Kadsura japonica (Schisandraceae). Sequence analysis showed that a four amino acid deletion in the K domain, which was found in all previously reported angiosperm PI genes, exists in a PI homologue of Schisandraceae, but not in six PI homologues of the Amborellaceae, Cabombaceae, and Nymphaeaceae, suggesting that the Amborellaceae, Cabombaceae, and Nymphaeaceae are basalmost lineages in angiosperms. The results of molecular phylogenetic analyses were not inconsistent with this hypothesis. The AP3 and PI homologues from Amborella share a sequence of five amino acids in the 5 region of exon 7. Using the linearized tree and likelihood methods, the divergence time between the AP3 and PI lineages was estimated as somewhere between immediately after to several tens of millions of years after the split between angiosperms and extant gymnosperms. Estimates of the age of the most recent common ancestor of all extant angiosperms range from ~140–210 Ma, depending on the trees used and assumptions made.     

Nucleotide variation at the dopa decarboxylase (<Emphasis Type="Italic">Ddc</Emphasis>) gene in natural populations of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We studied nucleotide sequence variation at the gene coding for dopa decarboxylase (Ddc) in seven populations of Drosophila melanogaster. Strength and pattern of linkage disequilibrium are somewhat distinct in the extensively sampled Spanish and Raleigh populations. In the Spanish population, a few sites are in strong positive association, whereas a large number of sites in the Raleigh population are associated nonrandomly but the association is not strong. Linkage disequilibrium analysis shows presence of two groups of haplotypes in the populations, each of which is fairly diverged, suggesting epistasis or inversion polymorphism. There is evidence of two forms of natural selection acting on Ddc. The McDonald-Kreitman test indicates a deficit of fixed amino acid differences between D. melanogaster and D. simulans, which may be due to negative selection. An excess of derived alleles at high frequency, significant according to the H-test, is consistent with the effect of hitchhiking. The hitchhiking may have been caused by directional selection downstream of the locus studied, as suggested by a gradual decrease of the polymorphism-to-divergence ratio. Altogether, the Ddc locus exhibits a complicated pattern of variation apparently due to several evolutionary forces. Such a complex pattern may be a result of an unusually high density of functionally important genes.     

The Flow of Antimicrobial Peptide Genes Through a Genetic Barrier Between <Emphasis Type="Italic">Mytilus edulis</Emphasis> and <Emphasis Type="Italic">M. galloprovincialis</Emphasis>

We studied the population genetics of two antimicrobial peptide (AMP) loci, called Mytilin B and Mytilus galloprovincialis defensin 2 (MGD2), in the secondary contact mosaic hybrid zone between Mytilus edulis and M. galloprovincialis. The isolation period between the two species was estimated to be ~1 million years (range, 0.5 million to 2 million years) long. During this period, coevolution between microbes and the immune system has likely occurred. The secondary contact, which would date back to ~25,000 (0–200,000) years, recently allowed these coadaptations to be rearranged through hybridization. Distinctive polymorphisms were uncovered in coding sequences of the two AMP loci such as insertion/deletion of codons or bisubstituted codons. Very low levels of differentiation were observed between populations of the two species at both loci, while other nuclear loci often showed marked structure among the same samples. The absence of population differentiation proved to be the consequence of secondary introgression of highly divergent alleles. While only a few recombinants were observed at the Mytilin B locus, the MGD2 locus showed a high intragenic recombination rate, which increased in the exon coding for the mature peptide. In addition, standard neutrality tests revealed significant deviations from the mutation-drift equilibrium at both loci. These results suggest that either balancing or directional selection is likely to play a role in the evolution of the two AMPs and introgression would be adaptive. However, evidence accumulated at the Mytilin B locus allows neither for identification of the direction of selection nor for any conclusions on whether selection acted directly on the antimicrobial peptide itself. At the MGD2 locus, a spatial variation of polymorphism patterns along the sequence suggests that selection was direct, although the precise nature of the selection (directional vs. balancing) remains unclear. This study concurs with previous reports of an effect of slight selection on AMP genes evolution in other invertebrates, although selection does not necessarily act on the mature peptides. E. Boon and M. F. Faure contributed equally to this work.     

Genetic divergence of mitochondrial DNA in white char <Emphasis Type="Italic">Salvelinus albus</Emphasis> and northern Dolly Varden char <Emphasis Type="Italic">Salvelinus malma malma</Emphasis>

Comparative analysis of mitochondrial DNA variation was performed in white char Salvelinus albus Glubokovsky and in its putative ancestor species, northern Dolly Varden char Salvelinus malma malma Walbaum. Highly statistically significant differentiation of S. albus and S. m. malma in the areas of sympatric (Kamchatka River basin) and allopatric (Kronotskoe Lake and Kronotskaya River) residence was demonstrated. The mtDNA divergence between S. albus and S. m. malma did not exceed the range of intraspecific variation in the populations of northern Dolly Varden char. At the same time, clusterization pattern of the Salvelinus chars provides hypothesis on the common origin of two allopatric populations of white char. Genealogical analysis of haplotypes indicates that S. albus and S. m. malma currently demonstrate incomplete divergence of mitochondrial lineages. The low nucleotide divergence estimates between S. albus and S. m. malma reflect the short time period since the beginning of the divergence of ancestral lineages. These estimates are determined by ancestral polymorphism and haplotype exchange between the diverged phylogenetic groups as a result of introgressive hybridization.     

Identification and expression analysis of rainbow trout <Emphasis Type="Italic">pumilio</Emphasis>-1 and <Emphasis Type="Italic">pumilio</Emphasis>-2

Pumilio is a sequence-specific RNA-binding protein that regulates translation from the relevant mRNA. The PUF-domain, the RNA-binding motif of Pumilio, is highly conserved across species. In the present study, we have identified two pumilio genes (pumilio-1 and pumilio-2) in rainbow trout and analyzed their expression patterns in its tissues. Pumilio-1 mRNA and pumilio-2A mRNA code for typical full length Pumilio proteins that contain a PUF-domain, whereas pumilio-2B mRNA is a splice variant of pumilio-2 and encodes a protein that lacks the PUF-domain. We have also identified a novel 72-bp exon that has not been reported in other animal species but is conserved in fish species. The insertion of this novel exon leads to the expression of an isoform of the Pumilio-2 protein with a slightly altered conformation of the PUF-domain. Pumilio-1 mRNA and pumilio-2A mRNA (irrespective of the presence of the 72-bp exon) are expressed in both the brain and ovaries at high levels, whereas pumilio-2B mRNA is expressed at low levels in all the rainbow trout tissues examined. Western blot analysis also indicates that the full length Pumilio proteins are expressed predominantly in the brain and ovaries. These data suggest that the Pumilio proteins have physiological roles and are involved in regulatory mechanisms in rainbow trout.This work was in part supported by a grant from the Akiyama Foundation to E.I. Nucleotide sequence data for rainbow trout pumilio-1 and pumilio-2 have been deposited in the DDBJ/EMBL/GenBank databases.     

Evolutionary dynamics of the human <Emphasis Type="Italic">ABO</Emphasis> gene

The ABO polymorphism has long been suspected to be under balancing selection. To explore this possibility, we analyzed two datasets: (1) a set of 94 23-Kb sequences in European- and African-Americans produced by the Seattle SNPs project, and (2) a set of 814 2-Kb sequences in O alleles from seven worldwide populations. A phylogenetic analysis of the Seattle sequences showed a complex pattern in which the action of recombination and gene conversion are evident, and in which four main lineages could be individuated. The sequence patterns could be linked to the expected blood group phenotype; in particular, the main mutation giving rise to the null O allele is likely to have appeared at least three times in human evolution, giving rise to allele lineages O02, O01, and O09. However, the genealogy changes along the gene and variations of both numbers of branches and of their time depth were observed, which could result from a combined action of recombination and selection. Several neutrality tests clearly demonstrated deviations compatible with balancing selection, peaking at several locations along the gene. The time depth of the genealogy was also incompatible with neutral evolution, particularly in the region from exons 6 to 7, which codes for most of the catalytic domain. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mutation analysis of the different<Emphasis Type="Italic"> tfd</Emphasis> genes for degradation of chloroaromatic compounds in<Emphasis Type="Italic"> Ralstonia eutropha</Emphasis> JMP134

Ralstonia eutropha JMP134 possesses two sets of similar genes for degradation of chloroaromatic compounds, tfdCDEFB (in short: tfd _I cluster) and tfdD _II C _II E _II F _II B _II (tfd _II cluster). The significance of two sets of tfd genes for the organism has long been elusive. Here, each of the tfd genes in the two clusters on the original plasmid pJP4 was replaced by double recombination with a gene fragment in which a kanamycin resistance gene was inserted into the respective tfd genes reading frame. The insertion mutants were all tested for growth on 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), and 3-chlorobenzoate (3-CBA). None of the tfdD _II C _II E _II F _II B _II genes appeared to be essential for growth on 2,4-D or on 3-CBA. Mutations in tfdC, tfdD and tfdF also did not abolish but only retarded growth on 2,4-D, indicating that they were redundant to some extent as well. Of all tfd genes tested, only tfdE and tfdB were absolutely essential, and interruption of those two reading frames abolished growth on 2,4-D, 3-CBA (tfdE only), and MCPA completely. Interestingly, strains with insertion mutations in the tfd _I cluster and those in tfdD _II , tfdC _II , tfdE _II and tfdB _II were severely effected in their growth on MCPA, compared to the wild-type. This indicated that not only the tfd _I cluster but also the tfd _II cluster has an essential function for R. eutropha during growth on MCPA. In contrast, insertion mutation of tfdD _II resulted in better growth of R. eutropha JMP134 on 3-CBA, which is most likely due to the prevention of toxic metabolite production in the absence of TfdD_II activity.     

Characterization of expressed class II MHC sequences in the banner-tailed kangaroo rat (<Emphasis Type="Italic">Dipodomys spectabilis</Emphasis>) reveals multiple <Emphasis Type="Italic">DRB</Emphasis> loci

Genes of the major histocompatibility complex (MHC) are exceptionally polymorphic due to the combined effects of natural and sexual selection. Most research in wild populations has focused on the second exon of a single class II locus (DRB), but complete gene sequences can provide an illuminating backdrop for studies of intragenic selection, recombination, and organization. To this end, we characterized class II loci in the banner-tailed kangaroo rat (Dipodomys spectabilis). Seven DRB-like sequences (provisionally named MhcDisp-DRB*01 through *07) were isolated from spleen cDNA and most likely comprise ≥5 loci; this multiformity is quite unlike the situation in muroid rodents such as Mus, Rattus, and Peromyscus. In silico translation revealed the presence of important structural residues for glycosylation sites, salt bonds, and CD4+ T-cell recognition. Amino-acid distances varied widely among the seven sequences (2–34%). Nuclear DNA sequences from the Disp-DRB*07 locus (∼10 kb) revealed a conventional exon/intron structure as well as a number of microsatellites and short interspersed nuclear elements (B4, Alu, and IDL-Geo subfamilies). Rates of nucleotide substitution at Disp-DRB*07 are similar in both exons and introns (π = 0.015 and 0.012, respectively), which suggests relaxed selection and may indicate that this locus is an expressed pseudogene. Finally, we performed BLASTn searches against Dipodomys ordii genomic sequences (unassembled reads) and find 90–97% nucleotide similarity between the two kangaroo rat species. Collectively, these data suggest that class II diversity in heteromyid rodents is based on polylocism and departs from the muroid architecture. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers EU817477–EU817485.     

Characterization of the horse (<Emphasis Type="Italic">Equus caballus</Emphasis>)<Emphasis Type="Italic"> IGHA</Emphasis> gene

Nucleotide sequences of the immunoglobulin constant heavy chain genes of the horse have been described for IGHM, IGHG and IGHE genes, but not for IGHA. Here, we provide the nucleotide sequence of the genomic IGHA gene of the horse (Equus caballus), including its secretion region and the transmembrane exon. The equine IGHA gene shows the typical structure of a mammalian IGHA gene, with only three exons, separated by two introns of similar size. The hinge exon is located at the 5 end of the CH2 exon and encodes a hinge region of 11 amino acids, which contains five proline residues. The coding nucleotide sequence of the secreted form of the equine IGHA gene shares around 72% identity with the human IGHA1 and IGHA2 genes, as well as the bovine, ovine, porcine and canine IGHA genes, without distinct preference for any of these species. The same species also cluster together in a phylogenetic tree of the IGHA coding regions of various mammals, whereas rodent, rabbit, marsupial and monotreme IGHA genes each build a separate cluster.The nucleotide sequences reported in this paper have been assigned the EMBL/GenBank accession numbers AY247966 and AY351982     

Comparative genomic analysis of the major histocompatibility complex class I region in the teleost genus <Emphasis Type="Italic">Oryzias</Emphasis>

The major histocompatibility complex (MHC) class I region of teleosts harbors a tight cluster of the class IA genes and several other genes directly involved in class I antigen presentation. Moreover, the dichotomous haplotypic lineages (termed d- and N- lineages) of the proteasome subunit beta genes, PSMB8 and PSMB10, are present in this region of the medaka, Oryzias latipes. To understand the evolution of the Oryzias MHC class I region at the nucleotide sequence level, we analyzed bacterial artificial chromosome clones covering the MHC class I region containing the d- lineage of Oryzias luzonensis and the d- and N- lineages of Oryzias dancena. Comparison among these three elucidated sequences and the published sequences of the d- and N- lineages of O. latipes indicated that the order and orientation of the encoded genes were completely conserved among these five genomic regions, except for the class IA genes, which showed species-specific variation in copy number. The PSMB8 and PSMB10 genes showed trans-species dimorphism. The remaining regions flanking the PSMB10, PSMB8, and class IA genes showed high degrees of sequence conservation at interspecies as well as intraspecies levels. Thus, the three independent evolutionary patterns under apparently distinctive selective pressures are recognized in the Oryzias MHC class I region. Electronic Supplementary Material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Isolation of the <Emphasis Type="Italic">B</Emphasis> incompatibility factor mutants in <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis>

 B incompatibility factor mutants (Bmut) in Pleurotus ostreatus were recovered from common-B mating heterokaryons resulted from matings between wild-type monokaryons with different A but the same B factors (A1B2 and A2B2) after NTG mutagenesis. The mutant monokaryons such as A1B2mut and A2B2mut were observed to have regularly uninucleated hyphal cells and to be compatible with each other. Matings between A1B2mut and A2B2mut monokaryons produced stable heterokaryons (A1B2mut + A2B2mut) that had binucleated hyphal cells with true clamp connections and formed normal fruit-bodies. Mating tests using basidiospore progeny from each of these heterokaryons revealed the bipolar mating pattern. Genetic analysis suggested that the mutation of B factor in P. ostreatus might occur in the B incompatibility factor genes. Received: August 3, 2001 / Accepted: January 18, 2002     

High variability in the MHC class II DA beta chain of the brushtail possum (<Emphasis Type="Italic">Trichosurus vulpecula</Emphasis>)

The diversity of class II major histocompatibility complex (MHC) loci was investigated in the brushtail possum, an important marsupial pest species in New Zealand. Immunocontraception, a form of fertility control that generates an autoimmune response, is being developed as a population control method for the possum. Because the immune response is partly under genetic control, an understanding of immunogenetics in possum will be crucial to the development of immunocontraceptive vaccines. MHC molecules are critical in the vertebrate immune response. Class II MHC molecules bind and present exogenously derived peptides to T lymphocytes and may be important in the presentation of immunocontraceptives. We used polymerase chain reaction primers designed to amplify the peptide binding region of possum class II MHC genes to isolate sequences from 49 animals. We have previously described 19 novel alleles from the DAB locus in the possum (Holland et al., Immunogenetics 60:449–460, 2008). Here, we report on another 11 novel alleles isolated from possum DAB, making this the most diverse marsupial locus described so far. This high level of diversity indicates that DAB is an important MHC locus in the possum and will need to be taken into account in the design of immunocontraceptive vaccines.