Micro-colinearity between rice, <Emphasis Type="Italic">Brachypodium</Emphasis>, and <Emphasis Type="Italic">Triticum monococcum</Emphasis> at the wheat domestication locus <Emphasis Type="Italic">Q</Emphasis>

Brachypodium, a wild temperate grass with a small genome, was recently proposed as a new model organism for the large-genome grasses. In this study, we evaluated gene content and microcolinearity between diploid wheat (Triticum monococcum), Brachypodium sylvaticum, and rice at a local genomic region harboring the major wheat domestication gene Q. Gene density was much lower in T. monococcum (one per 41 kb) because of gene duplication and an abundance of transposable elements within intergenic regions as compared to B. sylvaticum (one per 14 kb) and rice (one per 10 kb). For the Q gene region, microcolinearity was more conserved between wheat and rice than between wheat and Brachypodium because B. sylvaticum contained two genes apparently not present within the orthologous regions of T. monococcum and rice. However, phylogenetic analysis of Q and leukotriene A-4 hydrolase-like gene orthologs, which were colinear among the three species, showed that Brachypodium is more closely related to wheat than rice, which agrees with previous studies. We conclude that Brachypodium will be a useful tool for gene discovery, comparative genomics, and the study of evolutionary relationships among the grasses but will not preclude the need to conduct large-scale genomics experiments in the Triticeae.     

Isolation and characterization of a <Emphasis Type="Italic">Bacillus</Emphasis> species capable of producing tetrodotoxin from the puffer fish <Emphasis Type="Italic">Fugu obscurus</Emphasis>

We obtained puffer fish Fugu obscurus from Wudi, China and analyzed the level of tetrodotoxin (TTX) toxictiy by mouse bioassay. The ovary showed the highest potency (125 MU/g), followed by the liver, intestine, and skin. A TTX-producing strain, namely, W-3, was isolated from the ovary of puffer fish F. obscurus. After culturing at 28 °C for 48 h, toxins were extracted from the liquid medium and analyzed by mouse bioassay, high-performance liquid chromatography, and gas chromatography-mass spectrometry. The results showed that strain W-3 produced TTX and related compounds. Physiological and biochemical characterization and 16S rRNA analysis indicated that this strain represents a novel species within the Bacillus genus; we named this strain as Bacillus sp. W-3. Our results suggested that marine bacteria play a role in the production of TTX in puffer fish F. obscurus.     

Evolutionary flux of potentially <Emphasis Type="Italic">bldA</Emphasis>-dependent <Emphasis Type="Italic">Streptomyces</Emphasis> genes containing the rare leucine codon TTA

Previous studies have shown that one of the six leucine codons, UUA, is rare in Streptomyces, and that, while the gene for the UUA-specific tRNA, bldA, can generally be inactivated in diverse streptomycetes without impairing vegetative growth, bldA mutants are typically defective in reproductive aerial growth and in antibiotic production. Here, four complete genome sequences and 143 gene clusters for antibiotic biosynthesis from diverse streptomycetes were analysed in order to evaluate the evolution and function of genes whose possession of TTA codons makes them dependent on bldA. It was deduced that the last common ancestor of the four sequenced genomes, possibly 220 million years ago, already possessed the bldA system, together with perhaps 200 TTA-containing target genes. Some 33 of these genes are retained by the modern descendants, though only three of them retain a TTA in all occurrences. Nearly all of these 33, as well as many of the TTA-containing genes with orthologues in two or three of the four genomes, have the same location on the chromosomes as in their common ancestor. However, the majority of TTA-containing genes (61% overall in the four genomes) are species-specific, and were probably acquired by comparatively recent horizontal gene transfer. Most of these genes are of unknown function, and it is likely that many of them confer specialised ecological benefits. On the other hand, one class of species-specific, functionally recognisable, horizontally acquired genes--the gene clusters for antibiotic production--very often contain TTA codons; and nearly half of them have TTA codons in their pathway-specific regulatory genes.     

<Emphasis Type="Italic">Photobacterium damselae</Emphasis> subsp. <Emphasis Type="Italic">piscicida</Emphasis>: an integrated view of a bacterial fish pathogen

Pasteurellosis, or pseudotuberculosis, is a bacterial septicaemia caused by the halophilic bacterium Photobacterium damselae subsp. piscicida (formerly Pasteurella piscicida). Although this disease was first described in wild populations of white perch and striped bass, currently the natural hosts of the pathogen are a wide variety of marine fish. The disease has great economic impact both in Japan, where it affects mainly yellowtail cultures, and in the Mediterranean area, due to the losses it causes in seabream and seabass farms. This microorganism serves as a perfect model to study a bacterial fish pathogen, either at an applied level, to resolve or to mitigate the high economic losses of fish farmers, or at a basic level, for a better understanding of P. damselae subsp. piscicida biology. This article discusses the methods employed in our laboratory to study the causative agent of pasteurellosis. It reviews important aspects, from the diverse procedures for the detection and isolation of the pathogen to the latest molecular studies that have allowed its correct taxonomic allocation. Characterization of some virulence mechanisms and the available methods to prevent the disease are also presented. Electronic Publication     

<Emphasis Type="Italic"> Fugu rubripes</Emphasis> possesses genes for the entire set of the ITAM-bearing transmembrane signal subunits

The transmembrane signaling subunits (TSSs) bearing the immunoreceptor tyrosine-based activation motif (ITAM) play a crucial role in triggering the effector functions of mammalian leukocytes. The involvement in key immune reactions and obvious extension through duplication events make TSSs valuable markers of the evolution of the immune system. We surveyed the genomic sequences of the teleostean fish Fugu rubripes for the presence of genes encoding these accessory molecules. Automatic gene prediction was not efficient because of the poor ability of the programs used to recognize the short exons encoding the intracellular regions of TSSs. However, the unique compactness of the Fugu genome and the conservation of the exon/intron arrangements of the TSS genes facilitated their recognition by visual inspection of the candidate genomic sequences. Evidence for the presence of the CD3, CD3/, CD79a, CD79b, TCR, FcR, DAP12 and DAP10 genes in the Fugu genome was obtained. Furthermore, conserved synteny for the short regions including the TSS genes was revealed by comparison of the Fugu and human genomes. The data demonstrate that the set of TSSs arose before the teleost–tetrapod split and provide a starting point for experimental investigation of the molecular evolution of the leukocyte-activating receptor complexes from fish species to mammals.Abbreviations TSS Transmembrane signal subunit - ITAM Immunoreceptor tyrosine-based activation motif     

Cloning and expression of <Emphasis Type="Italic">Tenebrio molitor</Emphasis> antifreeze protein in <Emphasis Type="Italic">Escherichia coli</Emphasis>

A novel antifreeze protein cDNA was cloned by RT-PCR from the larva of the yellow mealworm Tenebrio molitor. The coding fragment of 339 bp encodes a protein of 112 amino acid residues and was fused to the expression vectors pET32a and pTWIN1. The resulted expression plasmids were transformed into Escherischia coli strains BL21 (DE3), ER2566, and Origami B (DE3), respectively. Several strategies were used for expression of the highly disulfide-bonded β-helix-contained protein with the activity of antifreeze in different expression systems. A protocol for production of refolded and active T. molitor antifreeze protein in bacteria was obtained.     

<Emphasis Type="Italic">In silico</Emphasis> physical mapping software for the <Emphasis Type="Italic">Triticum aestivum</Emphasis> genome

The large size of the Triticum aestivum genome makes it unlikely that a complete genome sequence for wheat will be available in the near future. Exploiting the conserved genome organization between wheat and rice and existing genomic resources, we have constructed in silico physical mapping software for wheat, assigning a gross physical location(s) into chromosome bins to 22,626 representative wheat gene sequences. To validate the predictions from the software we compared the predicted locations of ten ESTs to their positions experimentally determined by SNP marker analysis. Six of the sequences were correctly positioned on the map including four that demonstrated a high level of colinearity with their orthologous rice genomic region. This tool will facilitate the development of molecular markers for regions of interest and the creation of map-based cloning strategies in areas demonstrating high levels of sequence conservation and organization between wheat and rice.     

Comparative genomic analysis of CIPK gene family in <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">Populus</Emphasis>

Calcium serves as a second messenger in various signal transduction pathways in plants. CBL-interacting protein kinases (CIPKs), which have a variety of functions, are involved in calcium signal transduction. Previous, the studies on CIPK family members focused on Arabidopsis and rice. Here, we present a comparative genomic analysis of the CIPK gene family in Arabidopsis and poplar, a model tree species. Twenty-seven potential CIPKs were identified from poplar using genome-wide analysis. Like the CIPK gene family from Arabidopsis, CIPK genes from poplar were also divided into intron-free and intron-harboring groups. In the intron-harboring group, the intron distribution of CIPKs is rather conserved during the genome evolutionary process. Many homologous gene pairs were found in the CIPK gene family, indicating duplication events might contribute to the amplification of this gene family. The phylogenetic comparison of CIPKs in combination with intron distribution analysis revealed that CIPK genes from both Arabidopsis and poplar might have an ancient origin, which formed earlier than the separation of these two eudicot species. Our genomic and bioinformatic analysis will provide an important foundation for further functional dissection of the CBL-CIPK signaling network in poplars. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular cloning and expression analysis of <Emphasis Type="Italic">Bmrbp1</Emphasis>, the <Emphasis Type="Italic">Bombyx mori</Emphasis> homologue of the <Emphasis Type="Italic">Drosophila</Emphasis> gene <Emphasis Type="Italic">rbp1</Emphasis>

RBP1 is an important splicing factor involved in alternative splicing of the pre-mRNA of Drosophila sex-determining gene dsx. In this work, the Bombyx mori homologue of the rbp1 gene, Bmrbp1, was cloned. The pre-mRNA of Bmrbp1 gene is alternatively spliced to produce four mature mRNAs, named Bmrbp1-PA, Bmrbp1-PB, Bmrbp1-PC and Bmrbp1-PD, with nucleotide lengths of 799 nt, 1,316 nt, 894 nt and 724 nt, coding for 142 aa, 159 aa, 91 aa and 117 aa, respectively. BmRBP1-PA and BmRBP1-PD contain a N terminal RNA recognization motif (RRM) and a C terminal arginine/serine-rich domain, while BmRBP1-PB and BmRBP1-PC only share a RRM. Amino acid sequence alignments showed that BmRBP1 is conserved with its homologues in other insects and with other SR family proteins. The RT-PCR showed that Bmrbp1-PA was strongly expressed in all examined tissues and development stages, but Bmrbp1-PB was weakly expressed in these tissues and stages. The expression of both Bmrbp1-PA and Bmrbp1-PB showed no obvious sex difference. While the Bmrbp1-PC and Bmrbp1-PD were beyond detection by RT-PCR very likely due to their tissue/stage specificity. These results suggested that Bmrbp1 should be a member of SR family splicing factors, whether it is involved in the sex-specific splicing of Bmdsx pre-mRNA needs further research.     

Identification and characterization of<Emphasis Type="Italic"> Fugu</Emphasis> orthologues of mammalian interleukin-12 subunits

We have isolated and characterized cDNAs and genes for pufferfish, Fugu rubripes, (Fugu) orthologues of mammalian interleukin (IL)-12 subunits (IL-12 p35 and IL-12 p40). The deduced amino acid sequences of the Fugu IL-12 subunits showed homology with mammalian IL-12 subunits (p35: 50.4–58.0% similarity; p40: 51.2–55.4% similarity). Phylogenetic analysis confirmed that Fugu IL-12 p35 and p40 genes cluster with their mammalian counterpart lineages. The genomic organization of each of the Fugu IL-12 subunit genes is similar to that of the corresponding mouse IL-12 subunit genes, although the Fugu genes are very compact due to small intron size. Comparative genomic analysis showed conserved syntenies within the IL-12 p35 and p40 regions between Fugu and human, indicating that the Fugu IL-12 p35 and p40 genes are orthologues for mammalian IL-12 p35 and p40 encoding genes, respectively. Expression of IL-12 p35 mRNA was observed in lymphoid tissues and several non-lymphoid tissues, while expression of IL-12 p40 mRNA was constitutive and nearly ubiquitous. In the spleen and head kidney, expression of IL-12 p35 was induced by polyriboinosinic polyribocytidylic acid [poly(I:C)] and not by lipopolysaccharide (LPS), while expression of IL-12 p40 was constitutive and unresponsive to both poly(I:C) and LPS. These results indicate that IL-12 levels are regulated by production of IL-12 p35 mRNA and suggest that IL-12 in fish may be involved in antiviral defense. This is the first report of the identification and characterization of IL-12 subunit cDNAs and genes in a non-mammalian vertebrate.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers AB096265, AB096266, AB096267, and AB096268.     

Both the <Emphasis Type="Italic">AOX1</Emphasis> promoter and the <Emphasis Type="Italic">FLD1</Emphasis> promoter work together in a <Emphasis Type="Italic">Pichia pastoris</Emphasis> expression vector

We constructed a Pichia pastoris expression vector with two strongly inducible promoters (an alcohol oxidase 1 promoter and a formaldehyde dehydrogenase 1 promoter) based on pPIC9 k. To test the function of these promoters, the vector was used to co-express two genes that encode for green fluorescent protein (GFP) and a portion of a gelatin gene (an intra- and extracellular protein). The gelatin gene was placed under the control of PAOX1, while the GFP was under the control of PFLD1. The two proteins were simultaneously expressed upon induction with 0.5% (v/v) methanol. The two promoters functioned effectively and their coexistence on one vector did not affect their efficiency in protein expression. Thus, it was possible to simultaneously induce the expression of at least two proteins from one vector, using two different promoters.