Kei Hanzawa

Comparative Genomic Analysis of Two Avian (Quail and Chicken) MHC Regions

We mapped two different quail Mhc haplotypes and sequenced one of them (haplotype A) for comparative genomic analysis with a previously sequenced haplotype of the chicken Mhc. The quail haplotype A spans 180 kb of genomic sequence, encoding a total of 41 genes compared with only 19 genes within the 92-kb chicken Mhc. Except for two gene families (B30 and tRNA), both species have the same basic set of gene family members that were previously described in the chicken “minimal essential” Mhc. The two Mhc regions have a similar overall organization but differ markedly in that the quail has an expanded number of duplicated genes with 7 class I, 10 class IIB, 4 NK, 6 lectin, and 8 B-G genes. Comparisons between the quail and chicken Mhc class I and class II gene sequences by phylogenetic analysis showed that they were more closely related within species than between species, suggesting that the quail Mhc genes were duplicated after the separation of these two species from their common ancestor. The proteins encoded by the NK and class I genes are known to interact as ligands and receptors, but unlike in the quail and the chicken, the genes encoding these proteins in mammals are found on different chromosomes. The finding of NK-like genes in the quail Mhc strongly suggests an evolutionary connection between the NK C-type lectin-like superfamily and the Mhc, providing support for future studies on the NK, lectin, class I, and class II interaction in birds.

Contribution of mutation, recombination, and gene conversion to chicken MHC-B haplotype diversity.

The Mhc is a highly conserved gene region especially interesting to geneticists because of the rapid evolution of gene families found within it. High levels of Mhc genetic diversity often exist within populations. The chicken Mhc is the focus of considerable interest because of the strong, reproducible infectious disease associations found with particular Mhc-B haplotypes. Sequence data for Mhc-B haplotypes have been lacking thereby hampering efforts to systematically resolve which genes within the Mhc-B region contribute to well-defined Mhc-B-associated disease responses. To better understand the genetic factors that generate and maintain genomic diversity in the Mhc-B region, we determined the complete genomic sequence for 14 Mhc-B haplotypes across a region of 59 kb that encompasses 14 gene loci ranging from BG1 to BF2. We compared the sequences using alignment, phylogenetic, and genome profiling methods. We identified gene structural changes, synonymous and non-synonymous polymorphisms, insertions and deletions, and allelic gene rearrangements or exchanges that contribute to haplotype diversity. Mhc-B haplotype diversity appears to be generated by a number of mutational events. We found evidence that some Mhc-B haplotypes are derived by whole- and partial-allelic gene conversion and homologous reciprocal recombination, in addition to nucleotide mutations. These data provide a framework for further analyses of disease associations found among these 14 haplotypes and additional haplotypes segregating and evolving in wild and domesticated populations of chickens.

The major histocompatibility complex (Mhc) class IIB region has greater genomic structural flexibility and diversity in the quail than the chicken.

BackgroundThe quail and chicken major histocompatibility complex (Mhc) genomic regions have a similar overall organization but differ markedly in that the quail has an expanded number of duplicated class I, class IIB, natural killer (NK)-receptor-like, lectin-like and BG genes. Therefore, the elucidation of genetic factors that contribute to the greater Mhc diversity in the quail would help to establish it as a model experimental animal in the investigation of avian Mhc associated diseases.Aims and approachesThe main aim here was to characterize the genetic and genomic features of the transcribed major quail MhcIIB (CojaIIB) region that is located between the Tapasin and BRD2 genes, and to compare our findings to the available information for the chicken MhcIIB (BLB). We used four approaches in the study of the quail MhcIIB region, (1) haplotype analyses with polymorphic loci, (2) cloning and sequencing of the RT-PCR CojaIIB products from individuals with different haplotypes, (3) genomic sequencing of the CojaIIB region from the individuals with the different haplotypes, and (4) phylogenetic and duplication analysis to explain the variability of the region between the quail and the chicken.ResultsOur results show that the Tapasin-BRD2 segment of the quail Mhc is highly variable in length and in gene transcription intensity and content. Haplotypic sequences were found to vary in length between 4 to 11 kb. Tapasin-BRD2 segments contain one or two major transcribed CojaIIBs that were probably generated by segmental duplications involving c-type lectin-like genes and NK receptor-like genes, gene fusions between two CojaIIBs and transpositions between the major and minor CojaIIB segments. The relative evolutionary speed for generating the MhcIIBs genomic structures from the ancestral BLB2 was estimated to be two times faster in the quail than in the chicken after their separation from a common ancestor. Four types of genomic rearrangement elements (GRE), composed of simple tandem repeats (STR), were identified in the MhcIIB genomic segment located between the Tapasin-BRD2 genes. The GREs have many more STR numbers in the quail than in the chicken that displays strong linkage disequilibrium.ConclusionThis study suggests that the Mhc classIIB region has a flexible genomic structure generated by rearrangement elements and rapid SNP accumulation probably as a consequence of the quail adapting to environmental conditions and pathogens during its migratory history after its divergence from the chicken.

Gene organization of the quail major histocompatibility complex (MhcCoja) class I gene region

Abstract Class I genomic clones of the quail (Coturnix japonica) major histocompatibility complex (MhcCoja) were isolated and characterized. Two clusters spanning the 90.8 kilobase (kb) and 78.2 kb class I gene regions were defined by overlapping cosmid clones and found to contain at least twelve class I loci. However, unlike in the chicken Mhc, no evidence for the existence of any Coja class II gene was obtained in these two clusters. Based on comparative analysis of the genomic sequences with those of the cDNA clones, Coja-A, Coja-B, Coja-C, and Coja-D (Shiina et al. 1999), these twelve loci were assigned to represent one Coja-A gene, two Coja-B genes (Coja-B1 and -B2), four Coja-C genes (Coja-C1-C4), four Coja-D genes (Coja-D1-D4), and one new Coja-E gene. A class I gene-rich segment of 24.6 kb in which five of these genes (Coja-B1, -B2, -D1, -D2 and -E) are densely packed were sequenced by the shotgun strategy. All of these five class I genes are very compact in size [2089 base pairs (bp)–2732 bp] and contain no apparent genetic defect for functional expression. A transporter associated with the antigen processing (TAP) gene was identified in this class I gene-rich segment. These results suggest that the quail class I region is physically separated from the class II region and characterized by a large number of the expressible class I loci (at least seven) in contrast to the chicken Mhc, where the class I and class II regions are not clearly differentiated and only at most three expressed class I loci so far have been recognized.

Isolation and characterization of cDNA clones for Japanese quail (Coturnix japonica) major histocompatibility complex (MhcCoja) class I molecules.

The chicken major histocompatibility complex [(MHC) (B complex)] includes not only the genes encoding the class I and class II antigens (B-F and B-L, respectively), but also the genes coding for the class IV (B-G) antigens, which have so far been described only in this species as well as, mouse and human (Danielle et al. 1993). Three chicken class I B-F cDNA clones from different haplotypes probably representing three different alleles in a single locus have been isolated and characterized: B-F 10 from the B 12 haplotype, B-F 19 from B 19, and B-FL 1-1 from B b~ (blank; Guillemot et al. 1988; Kaufman et al. 1992; Pharr et al. 1994). The relationship between these cDNAs and six B-F genes so far isolated by gene cloning from the chicken MHC region remains unclear (Guillemot et al. 1988)_ Domestic chicken has been until now the only bird species whose MHC had been studied in detail by molecular cloning techniques, although the class II gene has been recently cloned from the ringnecked pheasant (Wittzell et al. 1994)_ The Japanese quail (Coturnixjaponica) is a bird allied to partridge, inhabiting all of the islands of Japan. The resistant trait to Newcastle disease virus in this species is believed to be conferred by certain alleles of the quail MHC gene(s) (Takahashi et al. 1989). In order to elucidate the molecular structure of the Japanese quail MhcCoja which is

Basic characterization of avian β-defensin genes in the Japanese quail, Coturnix japonica.

In this study, we identified a cluster of 14 avian β-defensins (AvBD; approximately 66 kbp) in the Japanese quail, Coturnix japonica. Except for AvBD12 (CjAvBD12) and -13, the CjAvBDs coding sequences exhibited greater than 78.0% similarity to the respective orthologous chicken AvBD genes (GgAvBD). The putative amino acid sequence encoded by each CjAvBD contained six cysteine residues and the GXC (X1-2) motif considered essential for the β-defensin family. Each CjAvBDs also formed a sub-group with the respective orthologous genes of various bird species in a phylogenetic tree analysis. Synteny between the CjAvBD cluster and GgAvBD cluster was confirmed. The CjAvBD cluster was mapped on the long-arm end of chromosome 3 by linkage analysis based on single nucleotide polymorphisms (SNPs) of CjAvBD1 and CjAvBD12 (approximately 46 kbp), as well as GgAvBD cluster. We also confirmed that CjAvBD1, -4, -5, -9, and -10 are transcribed in 20 tissues, including immune and digestive tissues. However, our experimental data indicated that the CjAvBD cluster lacks the AvBD3 and -7 loci, whereas the CjAvBD101α, -101β, and -101θ loci arose from gene duplication of the AvBD6 orthologous locus in the CjAvBD cluster after differentiation between Coturnix - Gallus.

Primary analysis of DNA polymorphisms in the TRIM region (MHC subregion) of the Japanese quail, Coturnix japonica

Based on sequences of two cosmid clones from Japanese quail (Coturnix japonica, Coja), we confirmed that the syntenic cluster, GNB2L1∼BTN1∼BTN2, is located in the quail TRIM subregion of the quail major histocompatibility complex (MHC Coja) region. These cosmids also included four CjBG loci and one CjLEC locus; therefore, the quail TRIM subregion was thought to be adjacent to the BG/LEC subregion. We then identified three polymorphic markers - CjHEP21, CjTRIM39.2 and CjBTN2 - in the TRIM subregion that may be useful for the functional analysis of the MHC-Coja region. We examined MHC-Coja sequences from 321 individual quails sampled from 11 inbred strains, and we found eight alleles for each of the three genes - CjHEP21, CjTRIM39.2 and CjBTN2. These polymorphisms represent the first avian DNA markers in the TRIM subregion. Additionally, we discovered a quail-specific VNTR (variable number of long tandem repeats, 133-137 bp) in intron 7 of CjBTN2. We identified 25 haplotypes in the sample of 321 quail; these haplotypes comprised combinations of all 24 alleles of the three polymorphic genes. We suggest that there are two recombination hotspots, one between each pair of adjacent loci. All strains, except AMRP, contained multiple haplotypes; the AMRP strain contained a single, apparently fixed haplotype.

IARS mutation causes prenatal death in Japanese Black cattle

Isoleucyl-tRNA synthetase (IARS) c.235G > C (p.V79L) is a causative mutation for a recessive disease called IARS disorder in Japanese black cattle. The disease is involved in weak calf syndrome and is characterized by low birth weight, weakness and poor suckling. The gestation period is often slightly extended, implying that intrauterine growth is retarded. In a previous analysis of 2597 artificial insemination (AI) procedures, we suggested that the IARS mutation might contribute toward an increase in the incidence of prenatal death. In this study, we extended this analysis to better clarify the association between the IARS mutation and prenatal death. The IARS genotypes of 92 animals resulting from crosses between carrier (G/C) × G/C were 27 normal (G/G), 55 G/C and 10 affected animals (C/C) (expected numbers: 23, 46 and 23, respectively). Compared to the expected numbers, there were significantly fewer affected animals in this population (P < 0.05), suggesting that more than half of the affected embryos died prenatally. When the number of AI procedures examined was increased to 11 580, the frequency of re-insemination after G/C × G/C insemination was significantly higher at 61-140 days (P < 0.001). The findings suggested that the homozygous IARS mutation not only causes calf death, but also embryonic or fetal death.

Effect of a single polymorphism in the Japanese quail NK‐lysin gene on antimicrobial activity

NK-lysins are cationic peptides that play important roles in host protection, and are an important constituent of innate immunity. We identified nine single-nucleotide polymorphisms (SNPs) in the NK-lysin open reading frame (ORF) from 32 Japanese quails in six strains: A, B, ND, K, P, and Y. The G to A substitution at nucleotide position 272 in the ORF resulted in a Gly (G) to Asp (D) amino acid substitution (Cj31G and Cj31D alleles). The Cj31D allele was detected in P (frequency 0.76) and Y (frequency 0.03) strains. We compared the antimicrobial activities of four synthetic peptides from the helix 2-loop-helix 3 region of avian NK-lysins against Escherichia coli: Cj31G and Cj31D from quail and Gg29N and Gg29D from chicken. The antimicrobial activities of the four peptides decreased in the following order: Gg29N > Cj31G > Gg29D > Cj31D (P < 0.05). Although there were no differences in the predicted secondary structure of the Cj31G and Cj31D, the net charge of the Cj31G was higher than that of Cj31D. These data indicated that the antimicrobial activity of CjNKL is influenced by net charge, similar to that which has been observed in chicken.

First molecular data on Bornean banteng Bos javanicus lowi (Cetartiodactyla, Bovidae) from Sabah, Malaysian Borneo

Abstract Phylogenetic relationships among three subspecies of banteng, Burma banteng Bos javanicus birmanicus in mainland Southeast Asia, Javan banteng Bos javanicus javanicus in Java, and Bornean banteng Bos javanicus lowi in Borneo, and the presence/absence of interbreeding between wild Bornean banteng and domestic cattle in Sabah, Malaysia, were investigated by partial sequences of cytochrome b and D-loop of mitochondrial DNA. The results show that genetic distance of the Bornean banteng are relatively close to the gaur Bos gaurus/gayal Bos frontalis (the cytochrome b, 0.004–0.025; the D-loop, 0.012–0.021) followed by Burma banteng (the cytochrome b, 0.027–0.035; the D-loop, 0.040–0.045), and kouprey Bos sauveli (the cytochrome b, 0.031–0.035; the D-loop, 0.037–0.042). There are much greater distances between Bornean banteng and domestic cattle, Bos taurus and Bos indicus (the cytochrome b, 0.059–0.076; the D-loop, 0.081–0.090). These results suggest that the Bornean banteng diverged genetically from other banteng subspecies and that the wild Bornean banteng from this study are pure strain and have high conservation value.