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Background: Plastid-encoded eubacterial-type RNA polymerase (PEP) plays a critical role in the transcription of photosynthesis genes in chloroplasts. Notably, some of the reaction center genes, including psaA, psaB, psbA, and psbD genes, are differentially transcribed by PEP in mature chloroplasts. However, the molecular mechanism of promoter selection in the reaction center gene transcription by PEP is not well understood. Objective: Sigma factor proteins direct promoter selection by a core PEP in chloroplasts as well as bacteria. AtSIG5 is a unique chloroplast sigma factor essential for psbD light-responsive promoter (psbD LRP) activity. To analyze the role of AtSIG5 in chloroplast transcription in more detail, we assessed the effect of AtSIG5 hyper-expression on the transcription of plastid-encoded genes in chloroplast transgenic plants. Results: The chloroplast transgenic tobacco (CpOX-AtSIG5) accumulates AtSIG5 protein at extremely high levels in chloroplasts. Due to the extremely high-level expression of recombinant AtSIG5, most PEP holoenzymes are most likely to include the recombinant AtSIG5 in the CpOXAtSIG5 chloroplasts. Thus, we can assess the promoter preference of AtSIG5 in vivo. The overexpression of AtSIG5 significantly increased the expression of psbD LRP transcripts encoding PSII reaction center D2 protein and psaA/B operon transcripts encoding PSI core proteins. Furthermore, run-on transcription analyses revealed that AtSIG5 preferentially recognizes the psaA/B promoter, as well as the psbD LRP. Moreover, we found that psbD LRP is constitutively active in CpOX-AtSIG5 plants irrespective of light and dark. Conclusion: AtSIG5 probably plays a significant role in differential transcription of reaction center genes in mature chloroplasts.

International audience; The major histocompatibility complex (MHC) is central to the innate and adaptive immune responses of jawed vertebrates. Characteristic of the MHC are high gene density, gene copy number variation, and allelic polymorphism. Because apes and monkeys are the closest living relatives of humans, the MHCs of these non-human primates (NHP) are studied in depth in the context of evolution, biomedicine, and conservation biology. The Immuno Polymorphism Database (IPD)-MHC NHP Database (IPD-MHC NHP), which curates MHC data of great and small apes, as well as Old and New World monkeys, has been upgraded. The curators of the database are responsible for providing official designations for newly discovered alleles. This nomenclature report updates the 2012 report, and summarizes important nomenclature issues and relevant novel features of the IPD-MHC NHP Database.

The association between gene polymorphisms and plasma virus load at the set point (SP-PVL) was investigated in Mauritian macaques inoculated with SIV. Among 44 macaques inoculated with 50 AID50, six individuals were selected: three with SP-PVL among the highest and three with SP-PVL among the lowest. The exons of 390 candidate genes of these six animals were sequenced. Twelve non-synonymous single nucleotide polymorphisms (NS-SNPs) lying in nine genes potentially associated with PVL were genotyped in 23 animals. Three NS-SNPs with probabilities of association with PVL less than 0.05 were genotyped in a total of 44 animals. One NS-SNP lying in exon 1 of the IL37 gene displayed a significant association (p = 3.33 × 10−4) and a strong odds ratio (19.52). Multiple linear regression modeling revealed three significant predictors of SP-PVL, including the IL37 exon 1 NS-SNP (p = 0.0004) and the MHC Class IB haplotypes M2 (p = 0.0007) and M6 (p = 0.0013). These three factors in conjunction explained 48% of the PVL variance (p = 4.8 × 10−6). The potential role of IL37 in the control of SIV infection is discussed.

Female human pluripotent stem cells (hPSCs) regularly show erosion of X chromosome inactivation featured by the loss of the long non-coding (lnc) RNA XIST and the accumulation of lncXACT. Here, we report that a common mechanism for the initiation of erosion depends on XIST loss but not XACT accumulation on inactive X chromosomes. We further demonstrate that XACT deletion does not affect X-linked gene dosage in eroded hPSCs and that aberrant XIST RNA diffusion induced by the CRISPR activation system is independent of the presence of XACT RNA. In contrast, the deletion of XACT results in the upregulation of neuron-related genes, facilitating neural differentiation in both male and eroded female hPSCs. XACT RNA repression by CRIPSR inhibition results in the same phenotype. Our study finds that XACT is dispensable for maintaining the erosion of X-lined gene repression on inactive X chromosomes but affects neural differentiation in hPSCs.

The highly polymorphic human major histocompatibility complex (MHC) also known as the human leukocyte antigen (HLA) encodes class I and II genes that are the cornerstone of the adaptive immune system. Their unique diversity (>25,000 alleles) might affect the outcome of any transplant, infection, and susceptibility to autoimmune diseases. The recent rapid development of new next-generation sequencing (NGS) methods provides the opportunity to study the influence/correlation of this high level of HLA diversity on allele expression levels in health and disease. Here, we describe the NGS capture RNA-Seq method that we developed for genotyping all 12 classical HLA loci (HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5) and assessing their allelic imbalance by quantifying their allele RNA levels. This is a target enrichment method where total RNA is converted to a sequencing-ready complementary DNA (cDNA) library and hybridized to a complex pool of RNA-specific HLA biotinylated oligonucleotide capture probes, prior to NGS. This method was applied to 161 peripheral blood mononuclear cells and 48 umbilical cord blood cells of healthy donors. The differential allelic expression of 10 HLA loci (except for HLA-DRA and HLA-DPA1) showed strong significant differences (P < 2.1 × 10−15). The results were corroborated by independent methods. This newly developed NGS method could be applied to a wide range of biological and medical questions including graft rejections and HLA-related diseases.

Female human pluripotent stem cells (hPSCs) regularly show erosion of X-chromosome inactivation (XCI) featured by loss of the long non-coding (lnc) RNA XIST and accumulation of XACT. Although both lncRNAs are expressed from X-chromosome, XACT is mainly expressed in pluripotent cells, and the expression pattern is reciprocal to XIST, suggesting an important role in dosage compensation and differentiation. In this study, we aimed to dissect the role of XACT in the erosion of dosage compensation and differentiation potential in female hPSCs. We found that XACT RNA accumulation on inactive X-chromosomes is hPSC line dependent. XIST RNA overlapped with H3K27me3 but not H3K9me3, and XIST/H3K27me3 loss occurred during erosion in all female hPSC lines analyzed. These results indicate that a common mechanism for initiation of erosion depends on XIST loss but not XACT accumulation on inactive X-chromosomes. We further demonstrated that XACT deletion does not affect X-linked gene dosage in eroded hPSCs. Additionally, aberrant XIST RNA diffusion induced by the CRISPR activation system was independent of the presence of XACT RNA. In contrast, genetic ablation of XACT in female hPSCs resulted in the up-regulation of neuron-related genes to facilitate neural differentiation. Finally, we confirmed that XACT repression by the CRISPR inhibition system leads to dysregulation of the genes and affects neural differentiation, indicating that XACT RNA is involved in gene regulation affecting neural differentiation. Our study demonstrates that XACT is dispensable for the maintenance of dosage of X-linked genes, but impacts neural differentiation in female hPSCs, implying that XACT might play important roles in the regulation of spatiotemporal development rather than X-chromosome dosage compensation in human pluripotent cells.

Many significant gene mutations in E. coli have contributed to the development of genetics. Among these, a commonly used recA mutation, Δ(srl-recA)306 has been sequenced by a next-generation sequencer combined with a long PCR. An original report described that Δ(srl-recA)306 cells were deleted from srlR to recA genes in their genome. The next-generation sequencer enables more accurate details to be determined. We ask whether both surrounding genes from hypF to norV for srlR and alaS for recA is there first. The long PCR was carried out with primers, norR and alaS, and amplified DNA fragments differed in length from wild to Δ(srl-recA)306 cells, suggesting that an entire Δ(srl-recA)306 mutation was included. Sequences of those DNA fragments indicated that 9147 bp, from srlR to recA including 10 genes, were replaced by a Tn10 DNA sequence. Junction points at both srlR-Tn10 and Tn10-recA were determined precisely. The results indicate that the first 97% of recA gene sequences were lost with a downstream recX gene remaining intact. The phenotypic difference between Δ(srl-recA)306 and ΔrecA::Km is discussed.

The enteric protozoa Entamoeba histolytica is the causative agent of amebiasis, which is one of the most common parasitic diseases in developed and developing countries. Entamoeba nuttalli is the genetically closest species to E. histolytica in current phylogenetic analyses of Entamoeba species, and is prevalent in wild macaques. Therefore, E. nuttalli may be a key organism in which to investigate molecules required for infection of human or non-human primates. To explore the molecular signatures of host-parasite interactions, we conducted de novo assembly of the E. nuttalli genome, utilizing self-correction of PacBio long reads and polishing corrected reads using Illumina short reads, followed by comparative genomic analysis with two other mammalian and a reptilian Entamoeba species. The final draft assembly of E. nuttalli included 395 contigs with a total length of approximately 23 Mb, and 9,647 predicted genes, of which 6,940 were conserved with E. histolytica. In addition, we found an E. histolytica-specific repeat known as ERE2 in the E. nuttalli genome. GO-term enrichment analysis of mammalian host-related molecules indicated diversification of transmembrane proteins, including AIG1 family and BspA-like proteins that may be involved in the host-parasite interaction. Furthermore, we identified an E. nuttalli-specific protein that contained 42 repeats of an octapeptide ([G,E]KPTDTPS). This protein was shown to be localized on the cell surface using immunofluorescence. Since many repeat-containing proteins in parasites play important roles in interactions with host cells, this unique octapeptide repeat-containing protein may be involved in colonization of E. nuttalli in the intestine of macaques. Overall, our draft assembly provides a valuable resource for studying Entamoeba evolution and host-parasite selection., Author summary Determination of host specificity is one of the most significant themes in the field of infectious diseases. Identification of molecules related to the host specificity of pathogens can lead to new treatment and prevention methods, and prediction of potential host shifts. Entamoeba histolytica is a human parasite that causes hemorrhagic diarrhea, amebic colitis, and liver abscess, which may result in death in severe cases. Entamoeba nuttalli is the closest species to E. histolytica and infects various wild macaques as natural hosts. E. nuttalli might also be a pathogen with a zoonotic hazard because severe inflammatory reactions in hamster livers and an asymptomatic case of human infection have been recorded. Here, we report E. nuttalli genomic data with a quality that allows comparison with genomes from other Entamoeba species. Comparative genomics revealed common proteins shared with other mammalian Entamoeba species, as well as E. nuttalli-specific proteins. Therefore, this study identifies candidate molecules required for host specificity and subsequent pathogenicity of Entamoeba species.

The original version of this article contained a spelling error in the Acknowledgments regarding the name of the funding organisation supporting GM and JAH. UKRI-BBSCR should have been UKRI-BBSRC, as is now indicated correctly below.

The human Major Histocompatibility Complex (MHC) genes are part of the supra‐locus onchromosome 6p21 known as the human leukocyte antigen (HLA) system [...]

In bacteria, the hyper-phosphorylated nucleotides, guanosine 5’-diphosphate 3’-diphosphate (ppGpp) and guanosine 5’-triphosphate 3’-diphosphate (pppGpp), function as secondary messengers in the regulation of various metabolic processes of the cell, including transcription, translation, and enzymatic activities, especially under nutrient deficiency. The activity carried out by these nucleotide messengers is known as the stringent response. (p)ppGpp levels are controlled by two distinct enzymes, namely, RelA and SpoT, in Escherichia coli. RelA-SpoT homologs (RSHs) are also conserved in plants and algae where they function in the plastids. The model plant Arabidopsis thaliana contains four RSHs: RSH1, RSH2, RSH3, and Ca2+-dependent RSH (CRSH). Genetic characterizations of RSH1, RSH2, and RSH3 were undertaken, which showed that the (p)ppGpp-dependent plastidial stringent response significantly influences plant growth and stress acclimation. However, the physiological significance of CRSH-dependent (p)ppGpp synthesis remains unclear, as no crsh-null mutant has been available. Here to investigate the function of CRSH, a crsh-knockout mutant of Arabidopsis was constructed using a site-specific gene-editing technique, and its phenotype was characterized. A transient increase of ppGpp was observed for 30 min in the wild type (WT) after light-to-dark transition, but this increase was not observed in the crsh mutant. Similar analyzes were performed with the rsh2rsh3 double and rsh1rsh2rsh3 triple mutants of Arabidopsis and showed that the transient increments of ppGpp in the mutants were higher than those in the WT. The increase of (p)ppGpp in the WT and rsh2rsh3 accompanied decrements in the mRNA levels of psbD transcribed by the plastid-encoded plastid RNA polymerase. These results indicated that the transient increase of intracellular ppGpp at night is due to CRSH-dependent ppGpp synthesis and the (p)ppGpp level is maintained by the hydrolytic activities of RSH1, RSH2, and RSH3 to accustom plastidial gene expression to darkness.

In the Mauritian macaque experimentally inoculated with SIV, gene polymorphisms potentially associated with the plasma virus load at a set point, approximately 100 days post inoculation, were investigated. Among the 42 animals inoculated with 50 AID50 of the same strain of SIV, none of which received any preventive or curative treatment, nine individuals were selected: three with a plasma virus load (PVL) among the lowest, three with intermediate PVL values and three among the highest PVL values. The complete genomes of these nine animals were then analyzed. Initially, attention was focused on variants with a potential functional impact on protein encoding genes (non-synonymous SNPs (NS-SNPs) and splicing variants). Thus, 424 NS-SNPs possibly associated with PVL were detected. The 424 candidates SNPs were genotyped in these 42 SIV experimentally infected animals (including the nine animals subjected to whole genome sequencing). The genes containing variants most probably associated with PVL at a set time point are analyzed herein. This work was supported by the “Investissements d’Avenir” programs managed by the ANR under reference ANR-11-INBS-0008 funding the Infectious Disease Models and Innovative Therapies (IDMIT, Fontenay-aux-Roses, France) infrastructure, and ANR-10-EQPX-02-01 funding the FlowCyTech facility (IDMIT, Fontenay-aux-Roses, France). We warmly thank all members of ASW and L2I core lab facility from IDMIT center. TMB is supported by MINECO BFU2014-55090-P (FEDER), U01 MH106874 grant, Howard Hughes International Early Career, Obra Social “La Caixa” and Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya.

Sequences of long-interspersed elements (LINE-1, L1) make up ∼17% of the human genome. De novo insertions of retrotransposition-active L1s can result in genetic diseases. It has been recently shown that the homozygous inactivation of two adjacent genes SLCO1B1 and SLCO1B3 encoding organic anion transporting polypeptides OATP1B1 and OATP1B3 causes a benign recessive disease presenting with conjugated hyperbilirubinemia, Rotor syndrome. Here, we examined SLCO1B1 and SLCO1B3 genes in six Japanese diagnosed with Rotor syndrome on the basis of laboratory data and laparoscopy. All six Japanese patients were homozygous for the c.1738C>T nonsense mutation in SLCO1B1 and homozygous for the insertion of a ∼6.1-kbp L1 retrotransposon in intron 5 of SLCO1B3, which altogether make up a Japanese-specific haplotype. RNA analysis revealed that the L1 insertion induced deleterious splicing resulting in SLCO1B3 transcripts lacking exon 5 or exons 5-7 and containing premature stop codons. The expression of OATP1B1 and OATP1B3 proteins was not detected in liver tissues. This is the first documented case of a population-specific polymorphic intronic L1 transposon insertion contributing to molecular etiology of recessive genetic disease. Since L1 activity in human genomes is currently seen as a major source of individual genetic variation, further investigations are warranted to determine whether this phenomenon results in other autosomal-recessive diseases.

HLA-DPB1 T-cell epitope (TCE) mismatching algorithm and rs9277534 SNP at the 3' untranslated region (3'UTR) in the HLA-DPB1 gene are key factors for transplant-related events in unrelated hematopoietic cell transplantation (UR-HCT). However, the association of these 2 mechanisms has not been elucidated. We analyzed 19 frequent HLA-DPB1 alleles derived from Japanese healthy subjects by next-generation sequencing of the entire HLA-DPB1 gene region and multi-SNP data of the HLA region in 1589 UR-HCT pairs. The risk of acute graft-versus-host disease (aGVHD) was analyzed in 1286 patients with single HLA-DPB1 mismatch UR-HCT. The phylogenetic tree constructed using the entire gene region demonstrated that HLA-DPB1 alleles were divided into 2 groups, HLA-DP2 and HLA-DP5. Although a phylogenetic relationship in the genomic region from exon 3 to 3'UTR (Ex3-3'UTR) obviously supported the division of HLA-DP2 and HLA-DP5 groups, which in exon 2 showed intermingling of HLA-DPB1 alleles in a non-HLA-DP2 and non-HLA-DP5-group manner. Multi-SNP data also showed 2 discriminative HLA-DPB1 groups according to Ex3-3'UTR. Risk of grade 2-4 aGVHD was significantly higher in patient HLA-DP5 group mismatch than patient HLA-DP2 group mismatch (hazard ratio, 1.28; P = .005), regardless of donor mismatch HLA-DP group. Regarding TCE mismatch, increasing risk of aGVHD in patient HLA-DP5 group mismatch and TCE-nonpermissive mismatch were observed only in patients with TCE-permissive mismatch and patient HLA-DP2 group mismatch, respectively. Evolutionary analysis revealed that rs9277534 represented a highly conserved HLA-DPB1 Ex3-3'UTR region and may provoke aGVHD differently to TCE mismatching algorithm, reflecting exon 2 polymorphisms. These findings enrich our understanding of the mechanism of aGVHD in HLA-DPB1 mismatch UR-HCT.

The transcription of photosynthesis genes encoded by the plastid genome is mainly mediated by a prokaryotic-type RNA polymerase called plastid-encoded plastid RNA polymerase (PEP). Standard PEP-dependent promoters resemble bacterial sigma-70-type promoters containing the so-called -10 and -35 elements. On the other hand, an unusual light- and stress-responsive promoter (psbD LRP) that is regulated by a 19-bp AAG-box immediately upstream of the -35 element has been mapped upstream of the psbD-psbC operon in some angiosperms. However, the occurrence of the AAG-box containing psbD LRP in plant evolution remains elusive. We have mapped the psbD promoters in eleven embryophytes at different evolutionary stages from liverworts to angiosperms. The psbD promoters were mostly mapped around 500–900 bp upstream of the psbD translational start sites, indicating that the psbD mRNAs have unusually long 5′-UTR extensions in common. The -10 elements of the psbD promoter are well-conserved in all embryophytes, but not the -35 elements. We found that the AAG-box sequences are highly conserved in angiosperms and gymnosperms except for gnetaceae plants. Furthermore, partial AAG-box-like sequences have been identified in the psbD promoters of some basal embryophytes such as moss, hornwort, and lycophyte, whereas liverwort has the standard PEP promoter without the AAG-box. These results suggest that the AAG-box sequences of the psbD LRP may have evolved from a primitive type of AAG-box of basal embryophytes. On the other hand, monilophytes (ferns) use another type of psbD promoter composed of a distinct cis-element upstream of the potential -35 element. Furthermore, we found that psbD expression is not regulated by light in gymnosperms or basal angiosperms, although they have the well-conserved AAG-box sequences. Thus, it is unlikely that acquisition of the AAG-box containing psbD promoter is directly associated with light-induced transcription of the psbD-psbC operon. Light- and stress-induced transcription may have evolved independently and multiple times during terrestrial plant evolution.

Large-scale phenotyping of multicellular organisms is one of the current challenges in biology. We present a comprehensive and scalable pipeline that allows for the efficient phenotyping of root growth traits on a large scale. This includes a high-resolution, low-cost acquisition setup as well as the automated image processing software BRAT. We assess the performance of this pipeline in Arabidopsis thaliana under multiple growth conditions and show its utility by performing genome-wide association studies on 16 root growth traits quantified by BRAT each day during a 5-d time-course experiment. The most significantly associated genome region for root growth rate is a locus encoding a calcium sensing receptor. We find that loss of function and overexpression of this gene can significantly alter root growth in a growth condition dependent manner and that the minor natural allele of the Calcium Sensor Receptor locus is highly significantly enriched in populations in coastal areas, demonstrating the power of our approach to identify regulators of root growth that might have adaptive relevance.

The Obg/Era proteins of P-loop GTPase superclass are conserved in various prokaryotic and eukaryotic organisms. Some of th ese proteins are critical regulators of many aspects of basic cellular processes, including ribosome biogenesis, translation and signal transduction. However, a genome-wide overview of the Obg /EraGTPase genes in plants is not available. Recently studies on comprehensive genomic analyses of Arabidopsis thaliana identified 11 Obg-Hflx like GTPase genes that are divided into nine f amilies/subfamilies: Archaea-related Drg and No g1, and Eubacteria-related O bg, EngB, HflX, Era, TrmE, EngA, and EngD. In this study we found that Arabidopsis has 3 (three) Drg homologue proteins, namely AtDrg 1-1, AtDr g1-2 and AtDrg 1-3. S ubcellular localization analysis of Arabidopsis Obg-Hflx homologues revealed that Archaea-derived Drg proteins are mainly targ eted to cytoplasm, except Drg1-3 was detected not only in cytoplasm but also in nucleus. Furthermore, based on expression p attern profiling indicates that Drg1-1 and Drg1-2 were expressed constitutively trough plant development. On the other hand, Drg 1-3 was detected in dry seed and under stress plant. © 2013 The Authors. Published by Elsevier B.V. Selection and peer-review under responsibility ofthe SustaiN conference committee and supported by Kyoto University; (RISH), (OP IR), (GCOE-ARS) and (GSS) as co-hosts.

High genetic diversity and high similarity on phenotypic performance of local Sulawesi cacao (Theobroma cacao L.) was challenge in elucidating of identification including developing of DNA fingerprint approaches. Identification of nuclear genome polymorphism, based on RAPD and SSR marker are well established. Recently, genetic variation on chloroplast genome was introduced as alternative in this polymorphism identification works. Here we analysis the possibility of application of SSR and chloroplast markers independently, also in combination of both approaches. The combination analysis showed the accuracy in analyzing sub species level on local Sulawesi cacao.

The current information on the polymorphism variation and haplotype structure of the domestic dog leukocyte antigen (DLA) genes is limited in comparison to other experimental animals. In this paper, to better elucidate the degree and types of polymorphisms and genetic differences for DLA-88, DLA-12 and DLA-64, we genotyped four families of 38 beagles and another 404 unrelated dogs representing 49 breeds by RT-PCR based Sanger sequencing. We also sequenced and analyzed the genomic organization of the DLA-88 and DLA-12 gene segments to better define these two-gene DLA haplotypes more precisely. We identified 45 alleles for DLA-88, 15 for DLA-12 and six for DLA-64, of which 20, 14 and six, respectively, were newly described alleles. Therefore, this study shows that the DLA-12 and DLA-64 loci are far more polymorphic than previously reported. Phylogenetic analysis strongly supported that the DLA-88, DLA-12 and DLA-64 alleles were independently generated after the original divergence of the DLA-79 alleles. Two distinct DLA-88 and DLA-12 haplotype structures, tentatively named DLA-88–DLA-12 and DLA-88–DLA-88L, were identified, and the novel haplotype DLA-88–DLA-88L contributed to 32.7% of the unrelated dogs. Quantitative real-time PCR analysis showed that the gene expression levels of DLA-88L and DLA-88 were similar, and that the gene expression level of DLA-12 was significantly lower. In addition, haplotype frequency estimations using frequently occurring alleles revealed 45 different DLA-class I haplotypes (88-88L/12-64) overall, and 22 different DLA-class I haplotypes in homozygous dogs for 18 breeds and mongrels.

Differences in the pathogenicity of genetically closely related H5N1 highly pathogenic avian influenza viruses (HPAIVs) were evaluated in White Leghorn chickens. These viruses varied in the clinical symptoms they induced, including lethality, virus shedding, and replication in host tissues. A comparison of the host responses in the lung, brain, and spleen suggested that the differences in viral replication efficiency were related to the host cytokine response at the early phase of infection, especially variations in the proinflammatory cytokine IL-6. Based on these findings, we inoculated the virus that showed the mildest pathogenicity among the five tested, A/pigeon/Thailand/VSMU-7-NPT/2004, into four breeds of Thai indigenous chicken, Phadu-Hung-Dang (PHD), Chee, Dang, and Luang-Hung-Khao (LHK), to explore effects of genetic background on host response. Among these breeds, Chee, Dang, and LHK showed significantly longer survival times than White Leghorns. Virus shedding from dead Thai indigenous chickens was significantly lower than that from White Leghorns. Although polymorphisms were observed in the Mx and MHC class I genes, there was no significant association between the polymorphisms in these loci and resistance to HPAIV.

The major histocompatibility complex (MHC) is a highly polymorphic genomic region that encodes the transplantation and immune regulatory molecules. It receives special attention for genetic investigation because of its important role in the regulation of innate and adaptive immune responses and its strong association with numerous infectious and/or autoimmune diseases. Recently, genotyping of the polymorphisms of MHC genes using targeted next-generation sequencing (NGS) technologies was developed for humans and some nonhuman species. Most species have numerous highly homologous MHC loci so the NGS technologies are likely to replace traditional genotyping methods in the near future for the investigation of human and animal MHC genes in evolutionary biology, ecology, population genetics, and disease and transplantation studies. In this chapter, we provide a short review of the use of targeted NGS for MHC genotyping in humans and nonhuman species, particularly for the class I and class II regions of the Crab-eating Macaque MHC (Mafa).

Chloroplasts in land plants have a small genome consisting of only 100 genes encoding partial sets of proteins for photosynthesis, transcription and translation. Although it has been thought that chloroplast transcription is mediated by a basically cyanobacterium-derived system, due to the endosymbiotic origin of plastids, recent studies suggest the existence of a hybrid transcription machinery containing non-bacterial proteins that have been newly acquired during plant evolution. Here, we highlight chloroplast-specific non-bacterial transcription mechanisms by which land plant chloroplasts have gained novel functions.

The swine is an important animal model for allo- and xeno-transplantation donor studies, which necessitates an extensive characterization of the expression and sequence variations within the highly polygenic and polymorphic swine leukocyte antigen (SLA) region. Massively parallel pyrosequencing is potentially an effective new 2ndGen method for simultaneous high-throughput genotyping and detection of SLA class I gene expression levels. In this study, we compared the 2ndGen method using the Roche Genome Sequencer 454 FLX with the conventional method using sub-cloning and Sanger sequencing to genotype SLA class I genes in five pigs of the Clawn breed and four pigs of the Landrace breed. We obtained an average of 10.4 SLA class I sequences per pig by the 2ndGen method, consistent with the inheritance data, and an average of only 6.0 sequences by the conventional method. We also performed a correlation analysis between the sequence read numbers obtained by the 2ndGen method and the relative expression values obtained by quantitative real-time PCR analysis at the allele level. A significant correlation coefficient (r = 0.899, P

β2-Microglobulin (β2M) is believed to have arisen in a basal jawed vertebrate (gnathostome) and is the essential L chain that associates with most MHC class I molecules. It contains a distinctive molecular structure called a constant-1 Ig superfamily domain, which is shared with other adaptive immune molecules including MHC class I and class II. Despite its structural similarity to class I and class II and its conserved function, β2M is encoded outside the MHC in all examined species from bony fish to mammals, but it is assumed to have translocated from its original location within the MHC early in gnathostome evolution. We screened a nurse shark bacterial artificial chromosome library and isolated clones containing β2M genes. A gene present in the MHC of all other vertebrates (ring3) was found in the bacterial artificial chromosome clone, and the close linkage of ring3 and β2M to MHC class I and class II genes was determined by single-strand conformational polymorphism and allele-specific PCR. This study satisfies the long-held conjecture that β2M was linked to the primordial MHC (Ur MHC); furthermore, the apparent stability of the shark genome may yield other genes predicted to have had a primordial association with the MHC specifically and with immunity in general.

In the current study, we describe four novel members of the 90 kDa heat shock protein (HSP90) family expressed in Japanese quail, Coturnix japonica. The coding regions of the genes, CjHSP90AA1, CjHSP90AB1, CjHSP90B1 and CjTRAP1, exhibited more than 94% similarity to their related genes in chicken. The putative proteins encoded by these quail genes contained motifs considered essential for HSP90 gene function. In addition, the predicted proteins were more similar to HSP90AA1, HSP90AB1, HSP90B1 and TRAP1 proteins expressed in vertebrates than they were to other members of the HSP90 family. Exon numbers of CjHSP90AA1 (11), CjHSP90AB1 (12) or CjTRAP1 (18) are the same as the chicken and mammalian orthologs. Furthermore, gene order in the regions surrounding CjHSP90AB1 and CjTRAP1 has been preserved, providing evidence that the genomic regions were orthologous to HSP90-containing regions in the chicken genome. The promoter regions of the genes also contained conserved motifs identified in related genes of chicken. However, the nucleotide sequences of the 5'-flanking region of these genes were highly polymorphic. We also found that CjHSP90AA1 exhibited a robust response to heat shock treatment. Taken together, the data suggest that CjHSP90AA1, CjHSP90AB1, CjHSP90B1 and CjTRAP1 encode orthologs of HSP90AA1, HSP90AB1, HSP90B1 and TRAP1, respectively.

The frequency and HLA-A allelic associations of a HERVK9 DNA structural polymorphism located in close proximity to the highly polymorphic HLA-A gene within the major histocompatibility complex (MHC) genomic region were determined in Japanese, African Americans, and Australian Caucasians to better understand its human population evolutionary history. The HERVK9 insertion or deletion was detected as a 3′ LTR or a solo LTR, respectively, by separate PCR assays. The average insertion frequency of the HERVK9.HG was significantly different (P < 1.083e−6) between the Japanese (0.59) and the African Americans (0.34) or Australian Caucasians (0.37). LD analysis predicted a highly significant (P < 1.0e−5) linkage between the HLA-A and HERVK9 alleles, probably as a result of hitchhiking (linkage). Evolutionary time estimates of the solo, 5′ and 3′ LTR nucleotide sequence divergences suggest that the HERVK9 was inserted 17.3 MYA with the first structural deletion occurring 15.1 MYA. The LTR/HLA-A haplotypes appear to have been formed mostly during the past 3.9 MY. The HERVK9 insertion and deletion, detected by a simple and economical PCR method, is an informative genetic and evolutionary marker for the study of HLA-A haplotype variations, human migration, the origins of contemporary populations, and the possibility of disease associations.

We have identified three members of the heat shock protein (HSP) 70 family from the Japanese quail, Coturnix japonica. The coding regions of the genes, CjHSPA2, CjHSPA5 and CjHSPA8, exhibit more than 96% similarity to related genes in the chicken, GgHSP70, GgHSPA5 and GgHSC70. The putative proteins encoded by the quail genes contain motifs considered essential for HSP70 family members. In addition, the predicted proteins are more similar to HSPA2, HSPA5, and HSPA8 proteins from vertebrates than they are to other members of the HSP70 family. As is true for orthologs from chicken and mammals, the quail genomic loci consist of a single exon (CjHSPA2) or eight exons (CjHSPA5 and CjHSPA8). The promoter regions of the genes also contain conserved motifs and are similar in structure to promoter regions of related genes in other species. Furthermore, gene order in the regions surrounding the quail HSP70 has been preserved, providing evidence that the genomic regions are orthologous to HSP70-containing regions in the chicken genome. One of the quail HSP70-related genes, CjHSPA2, exhibited a robust response to heat shock treatment and transcription of CjHSPA8 mRNA was weakly induced by heat treatment. Taken together, the data suggest that CjHSPA2, CjHSPA5 and CjHSPA8 encode orthologs of HSPA2, HSPA5 and HSPA8 with the notable difference that CjHSPA2 and CjHSPA8 are heat shock-inducible genes, whereas HSPA2 and HSPA8 are constitutively expressed in a heat shock-independent manner.

MHC haplotypes have a remarkable influence on whether tumors form following infection of chickens with oncogenic Marek’s disease herpesvirus. Although resistance to tumor formation has been mapped to a subregion of the chicken MHC-B region, the gene or genes responsible have not been identified. A full gene map of the subregion has been lacking. We have expanded the MHC-B region gene map beyond the 92-kb core previously reported for another haplotype revealing the presence of 46 genes within 242 kb in the Red Jungle Fowl haplotype. Even though MHC-B is structured differently, many of the newly revealed genes are related to loci typical of the MHC in other species. Other MHC-B loci are homologs of genes found within MHC paralogous regions (regions thought to be derived from ancient duplications of a primordial immune defense complex where genes have undergone differential silencing over evolutionary time) on other chromosomes. Still others are similar to genes that define the NK complex in mammals. Many of the newly mapped genes display allelic variability and fall within the MHC-B subregion previously shown to affect the formation of Marek’s disease tumors and hence are candidates for genes conferring resistance.

The functional analysis of genes encoded by the chloroplast genome of tobacco by reverse genetics is routine. Nevertheless, for a small number of genes their deletion generates heteroplasmic genotypes, complicating their analysis. There is thus the need for additional strategies to develop deletion mutants for these genes. We have developed a homologous copy correction-based strategy for deleting/mutating genes encoded on the chloroplast genome. This system was used to produce psbA knockouts. The resulting plants are homoplasmic and lack photosystem II (PSII) activity. Further, the deletion mutants exhibit a distinct phenotype; young leaves are green, whereas older leaves are bleached, irrespective of light conditions. This suggests that senescence is promoted by the absence of psbA. Analysis of the transcript levels indicates that NEP (nuclear-encoded plastid RNA polymerase)-dependent plastid genes are up regulated in the psbA deletion mutants, whereas the bleached leaves retain plastid-encoded plastid RNA polymerase activity. Hence, the expression of NEP-dependent plastid genes may be regulated by photosynthesis, either directly or indirectly.

A plausible explanation for many MHC-linked diseases is lacking. Sequencing of the MHC class I region (coding units or full contigs) in several human and nonhuman primate haplotypes allowed an analysis of single nucleotide variations (SNV) across this entire segment. This diversity was not evenly distributed. It was rather concentrated within two gene-rich clusters. These were each centered, but importantly not limited to, the antigen-presenting HLA-A and HLA-B/-C loci. Rapid evolution of MHC-I alleles, as evidenced by an unusually high number of haplotype-specific (hs) and hypervariable (hv) (which could not be traced to a single species or haplotype) SNVs within the classical MHC-I, seems to have not only hitchhiked alleles within nearby genes, but also hitchhiked deleterious mutations in these same unrelated loci. The overrepresentation of a fraction of these hvSNV (hv1SNV) along with hsSNV, as compared to those that appear to have been maintained throughout primate evolution (trans-species diversity; tsSNV; included within hv2SNV) tends to establish that the majority of the MHC polymorphism is de novo (species specific). This is most likely reminiscent of the fact that these hsSNV and hv1SNV have been selected in adaptation to the constantly evolving microbial antigenic repertoire.

We describe the generation and analysis of an integrated sequence map of a 2.4-Mb region of pig chromosome 7, comprising the classical class I region, the extended and classical class II regions, and the class III region of the major histocompatibility complex (MHC), also known as swine leukocyte antigen (SLA) complex. We have identified and manually annotated 151 loci, of which 121 are known genes (predicted to be functional), 18 are pseudogenes, 8 are novel CDS loci, 3 are novel transcripts, and 1 is a putative gene. Nearly all of these loci have homologues in other mammalian genomes but orthologues could be identified with confidence for only 123 genes. The 28 genes (including all the SLA class I genes) for which unambiguous orthology to genes within the human reference MHC could not be established are of particular interest with respect to porcine-specific MHC function and evolution. We have compared the porcine MHC to other mammalian MHC regions and identified the differences between them. In comparison to the human MHC, the main differences include the absence of HLA-A and other class I-like loci, the absence of HLA-DP-like loci, and the separation of the extended and classical class II regions from the rest of the MHC by insertion of the centromere. We show that the centromere insertion has occurred within a cluster of BTNL genes located at the boundary of the class II and III regions, which might have resulted in the loss of an orthologue to human C6orf10 from this region.

The application of a new gene-based strategy for sequencing the wheat mitochondrial genome shows its structure to be a 452 528 bp circular molecule, and provides nucleotide-level evidence of intra-molecular recombination. Single, reciprocal and double recombinant products, and the nucleotide sequences of the repeats that mediate their formation have been identified. The genome has 55 genes with exons, including 35 protein-coding, 3 rRNA and 17 tRNA genes. Nucleotide sequences of seven wheat genes have been determined here for the first time. Nine genes have an exon–intron structure. Gene amplification responsible for the production of multicopy mitochondrial genes, in general, is species-specific, suggesting the recent origin of these genes. About 16, 17, 15, 3.0 and 0.2% of wheat mitochondrial DNA (mtDNA) may be of genic (including introns), open reading frame, repetitive sequence, chloroplast and retro-element origin, respectively. The gene order of the wheat mitochondrial gene map shows little synteny to the rice and maize maps, indicative that thorough gene shuffling occurred during speciation. Almost all unique mtDNA sequences of wheat, as compared with rice and maize mtDNAs, are redundant DNA. Features of the gene-based strategy are discussed, and a mechanistic model of mitochondrial gene amplification is proposed.

To elucidate the origins of the MHC-B-MHC-C pair and the MHC class I chain-related molecule (MIC)A-MICB pair, we sequenced an MHC class I genomic region of humans, chimpanzees, and rhesus monkeys and analyzed the regions from an evolutionary stand-point, focusing first on LINE sequences that are paralogous within each of the first two species and orthologous between them. Because all the long interspersed nuclear element (LINE) sequences were fragmented and nonfunctional, they were suitable for conducting phylogenetic study and, in particular, for estimating evolutionary time. Our study has revealed that MHC-B and MHC-C duplicated 22.3 million years (Myr) ago, and the ape MICA and MICB duplicated 14.1 Myr ago. We then estimated the divergence time of the rhesus monkey by using other orthologous LINE sequences in the class I regions of the three primate species. The result indicates that rhesus monkeys, and possibly the Old World monkeys in general, diverged from humans 27-30 Myr ago. Interestingly, rhesus monkeys were found to have not the pair of MHC-B and MHC-C but many repeated genes similar to MHC-B. These results support our inference that MHC-B and MHC-C duplicated after the divergence between apes and Old World monkeys.

Summary Eubacterial-type multi-subunit plastid RNA polymerase (PEP) is responsible for the principal transcription activity in chloroplasts. PEP is composed of plastid-encoded core subunits and one of multiple nuclear-encoded sigma factors that confer promoter specificity on PEP. Thus, the replacement of sigma factors associated with PEP has been assumed to be a major mechanism for the switching of transcription patterns during chloroplast development. The null mutant (sig6-1) of plastid sigma factor gene AtSIG6 exhibited a cotyledon-specific pale green phenotype. Light-dependent chloroplast development was significantly delayed in the sig6-1 mutant. Genetic complementation of the mutant phenotype by the AtSIG6 cDNA demonstrated that AtSIG6 plays a key role in light-dependent chloroplast development. Northern and array-based global analyses for plastid transcripts revealed that the transcript levels of most PEP-dependent genes were greatly reduced in the sig6-1 mutant, but that the accumulation of nuclear-encoded RNA polymerase (NEP)-dependent transcripts generally increased. As the PEP α subunit and PEP-dependent trnV accumulated at normal levels in the sig6-1 mutant, the AtSIG6 knockout mutant probably retained functional PEP, and the transcriptional defects are likely to have been directly caused by AtSIG6 deficiency. Most of the AtSIG6-dependent genes are preceded by σ70-type promoters comprised of conserved −35/−10 elements. Thus, AtSIG6 may act as a major general sigma factor in chloroplasts during early plant development. On the other hand, the mutant phenotype was restored in older seedlings. Arabidopsis probably contains another late general sigma factor, the promoter specificity of which widely overlaps with that of AtSIG6.

The plastid genome is transcribed by nucleus-encoded (NEP) and plastid encoded (PEP) RNA polymerases. NEP transcribes housekeeping genes as well as genes coding for PEP core subunits and its activity is replaced by PEP in chloroplasts resulting in differential expression of genes in a developmental context. PEP is a prokaryotic-type enzyme in which nuclear-encoded σ factors function as promoter recognition subunit. A phylogenetic analysis for σ factors identified so far in plants shows that plant σ factors are members of bacterial σ70 family and divided into six groups, Sig1 through Sig6, which are integrated into four clusters consisting of Sig1 and Sig4, Sig2 and Sig3, Sig5 and Sig6. All plastid σ factors recognize bacterial σ70-type promoters, but they differ in promoter preference and the tissue-, developmental stage- and environmental-dependent expression. Sig5 is distinct from the other σ factors in its structure, function, and expression in response to light and stress. A promoter of the p...

We have determined the sequence of a 4-Mb interval on rat chromosome 20p12 that encompasses the rat major histocompatibility complex (MHC). This is the first report of a finished sequence for a segment of the rat genome and constitutes one of the largest contiguous sequences thus far for rodent genomes in general. The rat MHC is, next to the human MHC, the second mammalian MHC sequenced to completion. Our analysis has resulted in the identification of at least 220 genes located within the sequenced interval. Although gene content and order are well conserved in the class II and class III gene intervals as well as the framework gene regions, profound rat-specific features were encountered within the class I gene regions, in comparison to human and mouse. Class I region-associated differences were found both at the structural level, the number, and organization of class I genes and gene families, and, in a more global context, in the way that evolution worked to shape the present-day rat MHC.

Light is one of the most important environmental factors regulating expression of photosynthesis genes. The plastid psbD gene encoding the photosystem II reaction center protein D2 is under the control of a unique blue light responsive promoter (BLRP) that is transcribed by a bacterial-type plastid RNA polymerase (PEP). Promoter recognition of PEP is mediated by one of the six nuclear-encoded σ factors in Arabidopsis . The replacement of the plastid σ factor associated with PEP may be the major mechanism for switching of plastid transcription pattern in response to environmental and developmental signals. This study demonstrates that AtSig5 is a unique σ factor that is essential for psbD BLRP activity. A T-DNA insertional mutant with reduced AtSIG5 expression resulted in loss of primary transcripts from the psbD BLRP. Furthermore, transient overexpression of AtSig5 in dark-adapted protoplasts specifically elevated psbD and psbA transcription activities. On the other hand, overproduction of AtSig2 enhanced the transcription of psbA gene and trnE operon, but not psbD transcription. The AtSIG5 gene is phylogenetically distinct from other plastid σ factors, and its expression is induced exclusively by blue light. We propose that AtSig5 acts as a mediator of blue light signaling that specifically activates the psbD BLRP in response to blue light in Arabidopsis .

The major histocompatibility complex (MHC) genomic region is composed of a group of linked genes involved functionally with the adaptive and innate immune systems. The class I and class II genes are intrinsic features of the MHC and have been found in all the jawed vertebrates studied so far. The MHC genomic regions of the human and the chicken (B locus) have been fully sequenced and mapped, and the mouse MHC sequence is almost finished. Information on the MHC genomic structures (size, complexity, genic and intergenic composition and organization, gene order and number) of other vertebrates is largely limited or nonexistent. Therefore, we are mapping, sequencing and analyzing the MHC genomic regions of different human haplotypes and at least eight nonhuman species. Here, we review our progress with these sequences and compare the human MHC structure with that of the nonhuman primates (chimpanzee and rhesus macaque), other mammals (pigs, mice and rats) and nonmammalian vertebrates such as birds (chicken and quail), bony fish (medaka, pufferfish and zebrafish) and cartilaginous fish (nurse shark). This comparison reveals a complex MHC structure for mammals and a relatively simpler design for nonmammalian animals with a hypothetical prototypic structure for the shark. In the mammalian MHC, there are two to five different class I duplication blocks embedded within a framework of conserved nonclass I and/or nonclass II genes. With a few exceptions, the class I framework genes are absent from the MHC of birds, bony fish and sharks. Comparative genomics of the MHC reveal a highly plastic region with major structural differences between the mammalian and nonmammalian vertebrates. Additional genomic data are needed on animals of the reptilia, crocodilia and marsupial classes to find the origins of the class I framework genes and examples of structures that may be intermediate between the simple and complex MHC organizations of birds and mammals, respectively.

It has been 30 years since it was first proposed that the vertebrate genome evolved through several rounds of genome-wide duplications (polyploidizations)1. Despite rapid advances in genetics, including sequencing of the complete genomes of several divergent species, this hypothesis has not been tested rigorously and is still a matter of debate2. If polyploidizations occurred during chordate evolution, there should be a network of paralogous regions in the present-day jawed vertebrate (Gnathostomata) genomes3. Here we present an investigation of the major histocompatibility complex (MHC) paralogous regions, which we accomplished by characterizing the corresponding region in amphioxus by identifying nine anchor genes and sequencing both the anchor genes and the regions that flank them (a total of 400 kb). Phylogenetic analysis of 31 genes (including the anchor genes) in these regions shows that duplications occurred after the divergence of cephalochordates and vertebrates but before the Gnathostomata radiation. The distribution of human and amphioxus orthologs in their respective genomes and the relationship between these distributions support the en bloc duplication events. Our analysis represents the first step towards demonstrating that the human ancestral genome has undergone polyploidization. Moreover, reconstruction of the pre-duplicated region indicates that one of the duplicated regions retains the ancestral organization.

Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. The wheat plastome consists of a 134,545-bp circular molecule with 20,703-bp inverted repeats and the same gene content as the rice and maize plastomes. However, some structural divergence was found even in the coding regions of genes. These alterations are due to illegitimate recombination between two short direct repeats and/or replication slippage. Overall comparison of chloroplast DNAs among the three cereals indicated the presence of some hot-spot regions for length mutations. Whereas the region with clustered tRNA genes and that downstream of rbcL showed divergence in a species-specific manner, the deletion patterns of ORFs in the inverted-repeat regions and the borders between the inverted repeats and the small single-copy region support the notion that wheat and rice are related more closely to each other than to maize.

Chloroplasts have been reported to generate retrograde immune signals that activate defense gene expression in the nucleus. However, the roles of light and photosynthesis in plant immunity remain largely elusive. In this study, we evaluated the effects of light on the expression of defense genes induced by flg22, a peptide derived from bacterial flagellins which acts as a potent elicitor in plants. Whole-transcriptome analysis of flg22-treated Arabidopsis thaliana seedlings under light and dark conditions for 30 min revealed that a number of (30%) genes strongly induced by flg22 (>4.0) require light for their rapid expression, whereas flg22-repressed genes include a significant number of genes that are down-regulated by light. Furthermore, light is responsible for the flg22-induced accumulation of salicylic acid (SA), indicating that light is indispensable for basal defense responses in plants. To elucidate the role of photosynthesis in defense, we further examined flg22-induced defense gene expression in the presence of specific inhibitors of photosynthetic electron transport: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB). Light-dependent expression of defense genes was largely suppressed by DBMIB, but only partially suppressed by DCMU. These findings suggest that photosynthetic electron flow plays a role in controlling the light-dependent expression of flg22-inducible defense genes.

The genomes of free-living bacteria frequently exchange genes via lateral gene transfer (LGT), which has played a major role in bacterial evolution. LGT also played a significant role in the acquisition of genes from non-cyanobacterial bacteria to the lineage of ‘primary’ algae and land plants. Small GTPases are widely distributed among prokaryotes and eukaryotes. In this study, we inferred the evolutionary history of organelle-targeted small GTPases in plants. Arabidopsis thaliana contains at least one ortholog in seven subfamilies of OBG-HflX-like and TrmE-Era-EngA-YihA-Septin-like GTPase superfamilies (together referred to as Era-like GTPases). Subcellular localization analysis of all Era-like GTPases in Arabidopsis revealed that all thirteen eubacteria-related GTPases are localized to chloroplasts and/or mitochondria, whereas archaea-related DRG and NOG1 are localized to the cytoplasm and nucleus, respectively, suggesting that chloroplast- and mitochondrion-localized GTPases are derived from the ancestral cyanobacterium and α-proteobacterium, respectively, through endosymbiotic gene transfer (EGT). However, phylogenetic analyses revealed that plant organelle GTPase evolution is rather complex. Among the eubacterium-related GTPases, only four localized to chloroplasts (including one dual targeting GTPase) and two localized to mitochondria were derived from cyanobacteria and α-proteobacteria, respectively. Three other chloroplast-targeted GTPases were related to α-proteobacterial proteins, rather than to cyanobacterial GTPases. Furthermore, we found that four other GTPases showed neither cyanobacterial nor α-proteobacterial affiliation. Instead, these GTPases were closely related to clades from other eubacteria, such as Bacteroides (Era1, EngB-1, and EngB-2) and green non-sulfur bacteria (HflX). This study thus provides novel evidence that LGT significantly contributed to the evolution of organelle-targeted Era-like GTPases in plants.

The human CD1 proteins belong to a lipid-glycolipid antigen-presenting gene family and are related in structure and function to the MHC class I molecules. Previous mapping and DNA hybridization studies have shown that five linked genes located within a cluster on human chromosome 1q22-23 encode the CD1 protein family. We have analyzed the complete genomic sequence of the human CD1 gene cluster and found that the five active genes are distributed over 175,600 nucleotides and separated by four expanded intervening genomic regions (IGRs) ranging in length between 20 and 68 kb. The IGRs are composed mostly of retroelements including five full-length L1 PA sequences and various pseudogenes. Some L1 sequences have acted as receptors for other subtypes or families of retroelements. Alu molecular clocks that have evolved during primate history are found distributed within the HLA class I duplicated segments (duplicons) but not within the duplicons of CD1. Phylogeny of the alpha3 domain of the class I-like superfamily of proteins shows that the CD1 cluster is well separated from HLA class I by a number of superfamily members including MIC (PERB11), HFE, Zn-alpha2-GP, FcRn, and MR1. Phylogenetically, the human CD1 sequences are interspersed by CD1 sequences from other mammalian species, whereas the human HLA class I sequences cluster together and are separated from the other mammalian sequences. Genomic and phylogenetic analyses support the view that the human CD1 gene copies were duplicated prior to the evolution of primates and the bulk of the HLA class I genes found in humans. In contrast to the HLA class I genomic structure, the human CD1 duplicons are smaller in size, they lack Alu clocks, and they are interrupted by IGRs at least 4 to 14 times longer than the CD1 genes themselves. The IGRs seem to have been created as "buffer zones" to protect the CD1 genes from disruption by transposable elements.

Behcet's disease is known to be associated with HLA-B51 in many different populations. Genetic evidence supports that the susceptible gene for Behcet's disease is the HLA-B51 allele at the HLA-B locus. This study was aimed to determine the HLA-B51 nucleotide sequence variation in three Behcet's disease patients and three healthy controls in order to elucidate if any disease specific mutations or polymorphisms may exist in the HLA-B51 gene of patients. Long-range polymerase chain reaction (PCR) was first carried out to give a PCR-amplified product of 9.5 kb which was then used as a template for nested PCR to give a final amplified product of 4.2 kb. This final product containing the 1.3-kb promoter/enhancer region and the entire HLA-B gene except for a 363-bp 3' terminal end segment encoding the 3' untranslated region was subcloned by the BP cloning technique and sequenced. The sequencing results showed that all the patients possessed the HLA-B*51011 allele, and there were no differences in the exonic nucleotide sequences between the three Behcet's disease patients and the three healthy controls. The HLA-B*51011 intronic and promoter/enhancer nucleotide sequences from the three patients had 22 single nucleotide polymorphisms (SNPs), a single insertion of 6 bp and a single deletion of 2 bp. On the other hand, the three healthy controls had 24 SNPs in their intronic and promoter/enhancer regions. However, none of these polymorphisms in the patients were specific for the disease. Therefore, these results clearly demonstrate that the HLA-B exonic sequence that encodes the HLA-B51 allele is the real pathogenic factor in Behcet's disease.

cDNA clones corresponding to the swine histocompatibility complex (SLA: swine leucocyte antigen)-DM alpha chain were isolated using the polymerase chain reaction (PCR) products from the third exon in the human HLA-DMA gene as a probe. Amino acid comparative analysis revealed that these clones were more closely related to the bovine and human DMA genes than to the other swine class II genes alpha chain genes, DRA, DQA and DOA. These results suggest that the SLA-DMA gene is expressed and may function, like HLA-DM, as an important modulator in class II restricted antigen processing in swine. Furthermore, based on the sequences and PCR-restriction fragment length polymorphism (PCR-RFLP) patterns in the SLA-DMA gene, no allelic variation was recognized in the second exon, but five allelic variations were recognized in the third exon in five different breeds of swine. These DMA alleles were defined by variation at four nucleotide positions. Two of these alleles resulted in an amino acid substitution. These results suggest that SLA-DMA has little polymorphism as observed in HLA-DMA and mouse H2-Ma.