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2.  Recent advances in the study of chloroplast gene expression and its evolution 
Chloroplasts are semiautonomous organelles which possess their own genome and gene expression system. However, extant chloroplasts contain only limited coding information, and are dependent on a large number of nucleus-encoded proteins. During plant evolution, chloroplasts have lost most of the prokaryotic DNA-binding proteins and transcription regulators that were present in the original endosymbiont. Thus, chloroplasts have a unique hybrid transcription system composed of the remaining prokaryotic components, such as a prokaryotic RNA polymerase as well as nucleus-encoded eukaryotic components. Recent proteomic and transcriptomic analyses have provided insights into chloroplast transcription systems and their evolution. Here, we review chloroplast-specific transcription systems, focusing on the multiple RNA polymerases, eukaryotic transcription regulators in chloroplasts, chloroplast promoters, and the dynamics of chloroplast nucleoids.
PMCID: PMC3933795  PMID: 24611069
chloroplast; transcription; PEP; NEP; pTAC; nucleoid
3.  Light-dependent expression of flg22-induced defense genes in Arabidopsis 
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.
PMCID: PMC4191550  PMID: 25346742
photosynthesis; flg22; defense gene; DBMIB; DCMU; retrograde signaling; salicylic acid; CAS
4.  Evidence for lateral gene transfer (LGT) in the evolution of eubacteria-derived small GTPases in plant organelles 
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 30 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.
PMCID: PMC4263083  PMID: 25566271
endosymbiotic gene transfer; genomic analysis; lateral gene transfer; small GTPase; evolution of organelle
5.  Primordial Linkage of β2-Microglobulin to the MHC 
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.
PMCID: PMC3805034  PMID: 21321107
6.  Direct Comparison of Autologous and Allogeneic Transplantation of iPSC-Derived Neural Cells in the Brain of a Nonhuman Primate 
Stem Cell Reports  2013;1(4):283-292.
Induced pluripotent stem cells (iPSCs) provide the potential for autologous transplantation using cells derived from a patient’s own cells. However, the immunogenicity of iPSCs or their derivatives has been a matter of controversy, and up to now there has been no direct comparison of autologous and allogeneic transplantation in the brains of humans or nonhuman primates. Here, using nonhuman primates, we found that the autologous transplantation of iPSC-derived neurons elicited only a minimal immune response in the brain. In contrast, the allografts caused an acquired immune response with the activation of microglia (IBA-1+/MHC class II+) and the infiltration of leukocytes (CD45+/CD3+). Consequently, a higher number of dopaminergic neurons survived in the autografts. Our results suggest that the autologous transplantation of iPSC-derived neural cells is advantageous for minimizing the immune response in the brain compared with allogeneic grafts.
Graphical Abstract
•iPSC-derived autografts cause only a minimal immune response in the primate brain•Autografts have advantages over allografts even at an immune-privileged site•Dopamine neurons survive even in allografts without immunosuppression
The autologous transplantation of iPSC-derived neurons elicited only a minimal immune response in the brain. In contrast, the allografts caused an acquired immune response with the activation of microglia (IBA-1+/MHC class II+) and the infiltration of leukocytes (CD45+/CD3+). Autologous transplantation is advantageous for minimizing the immune response compared with allogeneic grafts.
PMCID: PMC3849265  PMID: 24319664
7.  Evolutionary Relations of Hexanchiformes Deep-Sea Sharks Elucidated by Whole Mitochondrial Genome Sequences 
BioMed Research International  2013;2013:147064.
Hexanchiformes is regarded as a monophyletic taxon, but the morphological and genetic relationships between the five extant species within the order are still uncertain. In this study, we determined the whole mitochondrial DNA (mtDNA) sequences of seven sharks including representatives of the five Hexanchiformes, one squaliform, and one carcharhiniform and inferred the phylogenetic relationships among those species and 12 other Chondrichthyes (cartilaginous fishes) species for which the complete mitogenome is available. The monophyly of Hexanchiformes and its close relation with all other Squaliformes sharks were strongly supported by likelihood and Bayesian phylogenetic analysis of 13,749 aligned nucleotides of 13 protein coding genes and two rRNA genes that were derived from the whole mDNA sequences of the 19 species. The phylogeny suggested that Hexanchiformes is in the superorder Squalomorphi, Chlamydoselachus anguineus (frilled shark) is the sister species to all other Hexanchiformes, and the relations within Hexanchiformes are well resolved as Chlamydoselachus, (Notorynchus, (Heptranchias, (Hexanchus griseus, H. nakamurai))). Based on our phylogeny, we discussed evolutionary scenarios of the jaw suspension mechanism and gill slit numbers that are significant features in the sharks.
PMCID: PMC3780621  PMID: 24089661
9.  Immune-Related Gene Expression Profile in Laboratory Common Marmosets Assessed by an Accurate Quantitative Real-Time PCR Using Selected Reference Genes 
PLoS ONE  2013;8(2):e56296.
The common marmoset (Callithrix jacchus) is considered a novel experimental animal model of non-human primates. However, due to antibody unavailability, immunological and pathological studies have not been adequately conducted in various disease models of common marmoset. Quantitative real-time PCR (qPCR) is a powerful tool to examine gene expression levels. Recent reports have shown that selection of internal reference housekeeping genes are required for accurate normalization of gene expression. To develop a reliable qPCR method in common marmoset, we used geNorm applets to evaluate the expression stability of eight candidate reference genes (GAPDH, ACTB, rRNA, B2M, UBC, HPRT, SDHA and TBP) in various tissues from laboratory common marmosets. geNorm analysis showed that GAPDH, ACTB, SDHA and TBP were generally ranked high in stability followed by UBC. In contrast, HPRT, rRNA and B2M exhibited lower expression stability than other genes in most tissues analyzed. Furthermore, by using the improved qPCR with selected reference genes, we analyzed the expression levels of CD antigens (CD3ε, CD4, CD8α and CD20) and cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12β, IL-13, IFN-γ and TNF-α) in peripheral blood leukocytes and compared them between common marmosets and humans. The expression levels of CD4 and IL-4 were lower in common marmosets than in humans whereas those of IL-10, IL-12β and IFN-γ were higher in the common marmoset. The ratio of Th1-related gene expression level to that of Th2-related genes was inverted in common marmosets. We confirmed the inverted ratio of CD4 to CD8 in common marmosets by flow cytometric analysis. Therefore, the difference in Th1/Th2 balance between common marmosets and humans may affect host defense and/or disease susceptibility, which should be carefully considered when using common marmoset as an experimental model for biomedical research.
PMCID: PMC3581525  PMID: 23451040
10.  Evolutionary aspects of plastid proteins involved in transcription: The transcription of a tiny genome is mediated by a complicated machinery 
Transcription  2012;3(6):290-294.
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.
PMCID: PMC3630183  PMID: 22889841
chloroplast; nucleoid; PEP; NEP; pTAC
11.  Memory Immune Responses against Pandemic (H1N1) 2009 Influenza Virus Induced by a Whole Particle Vaccine in Cynomolgus Monkeys Carrying Mafa-A1*052∶02 
PLoS ONE  2012;7(5):e37220.
We made an H1N1 vaccine candidate from a virus library consisting of 144 ( = 16 HA×9 NA) non-pathogenic influenza A viruses and examined its protective effects against a pandemic (2009) H1N1 strain using immunologically naïve cynomolgus macaques to exclude preexisting immunity and to employ a preclinical study since preexisting immunity in humans previously vaccinated or infected with influenza virus might make comparison of vaccine efficacy difficult. Furthermore, macaques carrying a major histocompatibility complex class I molecule, Mafa-A1*052∶02, were used to analyze peptide-specific CD8+ T cell responses. Sera of macaques immunized with an inactivated whole particle formulation without addition of an adjuvant showed higher neutralization titers against the vaccine strain A/Hokkaido/2/1981 (H1N1) than did sera of macaques immunized with a split formulation. Neutralization activities against the pandemic strain A/Narita/1/2009 (H1N1) in sera of macaques immunized twice with the split vaccine reached levels similar to those in sera of macaques immunized once with the whole particle vaccine. After inoculation with the pandemic virus, the virus was detected in nasal samples of unvaccinated macaques for 6 days after infection and for 2.67 days and 5.33 days on average in macaques vaccinated with the whole particle vaccine and the split vaccine, respectively. After the challenge infection, recall neutralizing antibody responses against the pandemic virus and CD8+ T cell responses specific for nucleoprotein peptide NP262-270 bound to Mafa-A1*052∶02 in macaques vaccinated with the whole particle vaccine were observed more promptly or more vigorously than those in macaques vaccinated with the split vaccine. These findings demonstrated that the vaccine derived from our virus library was effective for pandemic virus infection in macaques and that the whole particle vaccine conferred more effective memory and broader cross-reactive immune responses to macaques against pandemic influenza virus infection than did the split vaccine.
PMCID: PMC3356377  PMID: 22623997
12.  A Novel System of Polymorphic and Diverse NK Cell Receptors in Primates 
PLoS Genetics  2009;5(10):e1000688.
There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in “higher” primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire.
Author Summary
Most receptors of natural killer (NK) cells interact with highly polymorphic major histocompatibility complex (MHC) class I molecules and thereby regulate the activity of NK cells against infected or malignant target cells. Whereas humans, apes, and Old and New World monkeys use the family of killer cell immunoglobulin-like receptors (KIR) as highly diverse NK cell receptors, this function is performed in rodents by the diverse family of lectin-like receptors Ly49. When did this functional separation occur in evolution? We followed this by investigating lemurs, primates that are distantly related to humans. We show here that lemurs employ the CD94/NKG2 family as their highly diversified NK cell receptors. The CD94/NKG2 receptors also belong to the lectin-like receptor family, but are rather conserved in “higher” primates and rodents. We could further demonstrate that lemurs have a single Ly49 gene like other primates but lack functional KIR genes of the KIR3DL lineage and show major deviations in their MHC class I genomic organisation. Thus, lemurs have evolved a “third way” of polymorphic and diverse NK cell receptors. In addition, the multiplied lemur CD94/NKG2 receptors can be freely combined, thereby forming diverse receptors. This is, therefore, the first description of some combinatorial diversity of NK cell receptors.
PMCID: PMC2757895  PMID: 19834558
13.  Contribution of Mutation, Recombination, and Gene Conversion to Chicken Mhc-B Haplotype Diversity1,2 
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, phylo-genetic, 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.
PMCID: PMC2657362  PMID: 18714011
14.  An isoform of Arabidopsis myosin XI interacts with small GTPases in its C-terminal tail region 
Journal of Experimental Botany  2008;59(13):3523-3531.
Myosin XI, a class of myosins expressed in plants is believed to be responsible for cytoplasmic streaming and the translocation of organelles and vesicles. To gain further insight into the translocation of organelles and vesicles by myosin XI, an isoform of Arabidopsis myosin XI, MYA2, was chosen and its role in peroxisome targeting was examined. Using the yeast two-hybrid screening method, two small GTPases, AtRabD1 and AtRabC2a, were identified as factors that interact with the C-terminal tail region of MYA2. Both recombinant AtRabs tagged with His bound to the recombinant C-terminal tail region of MYA2 tagged with GST in a GTP-dependent manner. Furthermore, AtRabC2a was localized on peroxisomes, when its CFP-tagged form was expressed transiently in protoplasts prepared from Arabidopsis leaf tissue. It is suggested that MYA2 targets the peroxisome through an interaction with AtRabC2a.
PMCID: PMC2561144  PMID: 18703495
AtRabC2a; AtRabD1; myosin XI; MYA2; peroxisome
15.  The major histocompatibility complex (Mhc) class IIB region has greater genomic structural flexibility and diversity in the quail than the chicken 
BMC Genomics  2006;7:322.
The 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 approaches
The 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.
Our 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.
This 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.
PMCID: PMC1769493  PMID: 17184537
16.  Construction of a nurse shark (Ginglymostoma cirratum) bacterial artificial chromosome (BAC) library and a preliminary genome survey 
BMC Genomics  2006;7:106.
Sharks are members of the taxonomic class Chondrichthyes, the oldest living jawed vertebrates. Genomic studies of this group, in comparison to representative species in other vertebrate taxa, will allow us to theorize about the fundamental genetic, developmental, and functional characteristics in the common ancestor of all jawed vertebrates.
In order to obtain mapping and sequencing data for comparative genomics, we constructed a bacterial artificial chromosome (BAC) library for the nurse shark, Ginglymostoma cirratum.
The BAC library consists of 313,344 clones with an average insert size of 144 kb, covering ~4.5 × 1010 bp and thus providing an 11-fold coverage of the haploid genome. BAC end sequence analyses revealed, in addition to LINEs and SINEs commonly found in other animal and plant genomes, two new groups of nurse shark-specific repetitive elements, NSRE1 and NSRE2 that seem to be major components of the nurse shark genome. Screening the library with single-copy or multi-copy gene probes showed 6–28 primary positive clones per probe of which 50–90% were true positives, demonstrating that the BAC library is representative of the different regions of the nurse shark genome. Furthermore, some BAC clones contained multiple genes, making physical mapping feasible.
We have constructed a deep-coverage, high-quality, large insert, and publicly available BAC library for a cartilaginous fish. It will be very useful to the scientific community interested in shark genomic structure, comparative genomics, and functional studies. We found two new groups of repetitive elements specific to the nurse shark genome, which may contribute to the architecture and evolution of the nurse shark genome.
PMCID: PMC1513397  PMID: 16672057
17.  Structural dynamics of cereal mitochondrial genomes as revealed by complete nucleotide sequencing of the wheat mitochondrial genome 
Nucleic Acids Research  2005;33(19):6235-6250.
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.
PMCID: PMC1275586  PMID: 16260473

Results 1-17 (17)