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Background: Sulfate-reducing bacteria (SRB) are key players of the carbon- and sulfur-cycles in the sediments of the world's oceans. Habitat relevant SRBs are often members of the Desulfosarcina-Desulfococcus clade belonging to the deltaproteobacterial family of Desulfobacteraceae. Despite this environmental recognition, their molecular (genome-based) physiology and their potential to contribute to organic carbon mineralization as well as to adapt to changing environmental conditions have been scarcely investigated. A metabolically versatile representative of this family is Desulfococcus multivorans that is able to completely oxidize (to CO 2 ) a variety of organic acids, including fatty acids up to C 14, as well as aromatic compounds., Results: In this study the complete 4.46 Mbp and manually annotated genome of metabolically versatile Desulfococcus multivorans DSM 2059 is presented with particular emphasis on a proteomics-driven metabolic reconstruction. Proteomic profiling covered 17 substrate adaptation conditions (6 aromatic and 11 aliphatic compounds) and comprised 2D DIGE, shotgun proteomics and analysis of the membrane protein-enriched fractions. This comprehensive proteogenomic dataset allowed for reconstructing a metabolic network of degradation pathways and energy metabolism that consists of 170 proteins (154 detected; ~91 % coverage). Peripheral degradation routes feed via central benzoyl-CoA, (modified) β-oxidation or methylmalonyl-CoA pathways into the Wood-Ljungdahl pathway for complete oxidation of acetyl-CoA to CO 2 . Dissimilatory sulfate reduction is fueled by a complex electron transfer network composed of cytoplasmic components (e.g., electron transfer flavoproteins) and diverse membrane redox complexes (Dsr, Qmo, Hmc, Tmc, Qrc, Nuo and Rnf). Overall, a high degree of substrate-specific formation of catabolic enzymes was observed, while most complexes involved in electron transfer appeared to be constitutively formed., Conclusions: A highly dynamic genome structure in combination with substrate-specifically formed catabolic subproteomes and a constitutive subproteome for energy metabolism and electron transfer appears to be a common trait of Desulfobacteraceae members.

Background: Acholeplasma oculi belongs to the Acholeplasmataceae family, comprising the genera Acholeplasma and 'Candidatus Phytoplasma'. Acholeplasmas are ubiquitous saprophytic bacteria. Several isolates are derived from plants or animals, whereas phytoplasmas are characterised as intracellular parasitic pathogens of plant phloem and depend on insect vectors for their spread. The complete genome sequences for eight strains of this family have been resolved so far, all of which were determined depending on clone-based sequencing., Results: The A. oculi strain 19L chromosome was sequenced using two independent approaches. The first approach comprised sequencing by synthesis (Illumina) in combination with Sanger sequencing, while single molecule real time sequencing (PacBio) was used in the second. The genome was determined to be 1,587,120 bp in size. Sequencing by synthesis resulted in six large genome fragments, while the single molecule real time sequencing approach yielded one circular chromosome sequence. High-quality sequences were obtained by both strategies differing in six positions, which are interpreted as reliable variations present in the culture population. Our genome analysis revealed 1,471 protein-coding genes and highlighted the absence of the F1FO-type Na+ ATPase system and GroEL/ES chaperone. Comparison of the four available Acholeplasma sequences revealed a core-genome encoding 703 proteins and a pan-genome of 2,867 proteins., Conclusions: The application of two state-of-the-art sequencing technologies highlights the potential of single molecule real time sequencing for complete genome determination. Comparative genome analyses revealed that the process of losing particular basic genetic features during genome reduction occurs in both genera, as indicated for several phytoplasma strains and at least A. oculi. The loss of the F1FO-type Na+ ATPase system may separate Acholeplasmataceae from other Mollicutes, while the loss of those genes encoding the chaperone GroEL/ES is not a rare exception in this bacterial class.

Background: Most molecular studies of plant stress tolerance have been performed with Arabidopsis thaliana, although it is not particularly stress tolerant and may lack protective mechanisms required to survive extreme environmental conditions. Thellungiella salsuginea has attracted interest as an alternative plant model species with high tolerance of various abiotic stresses. While the T. salsuginea genome has recently been sequenced, its annotation is still incomplete and transcriptomic information is scarce. In addition, functional genomics investigations in this species are severely hampered by a lack of affordable tools for genome-wide gene expression studies., Results: Here, we report the results of Thellungiella de novo transcriptome assembly and annotation based on 454 pyrosequencing and development and validation of a T. salsuginea microarray. ESTs were generated from a non-normalized and a normalized library synthesized from RNA pooled from samples covering different tissues and abiotic stress conditions. Both libraries yielded partially unique sequences, indicating their necessity to obtain comprehensive transcriptome coverage. More than 1 million sequence reads were assembled into 42,810 unigenes, approximately 50% of which could be functionally annotated. These unigenes were compared to all available Thellungiella genome sequence information. In addition, the groups of Late Embryogenesis Abundant (LEA) proteins, Mitogen Activated Protein (MAP) kinases and protein phosphatases were annotated in detail. We also predicted the target genes for 384 putative miRNAs. From the sequence information, we constructed a 44 k Agilent oligonucleotide microarray. Comparison of same-species and cross-species hybridization results showed superior performance of the newly designed array for T. salsuginea samples. The developed microarrays were used to investigate transcriptional responses of T. salsuginea and Arabidopsis during cold acclimation using the MapMan software., Conclusions: This study provides the first comprehensive transcriptome information for the extremophile Arabidopsis relative T. salsuginea. The data constitute a more than three-fold increase in the number of publicly available unigene sequences and will greatly facilitate genome annotation. In addition, we have designed and validated the first genome-wide microarray for T. salsuginea, which will be commercially available. Together with the publicly available MapMan software this will become an important tool for functional genomics of plant stress tolerance.

Background: The Manila clam, Ruditapes philippinarum, is one of the major aquaculture species in the world and a potential sentinel organism for monitoring the status of marine ecosystems. However, genomic resources for R. philippinarum are still extremely limited. Global analysis of gene expression profiles is increasingly used to evaluate the biological effects of various environmental stressors on aquatic animals under either artificial conditions or in the wild. Here, we report on the development of a transcriptomic platform for global gene expression profiling in the Manila clam., Results: A normalized cDNA library representing a mixture of adult tissues was sequenced using a ultra high-throughput sequencing technology (Roche 454). A database consisting of 32,606 unique transcripts was constructed, 9,747 (30%) of which could be annotated by similarity. An oligo-DNA microarray platform was designed and applied to profile gene expression of digestive gland and gills. Functional annotation of differentially expressed genes between different tissues was performed by enrichment analysis. Expression of Natural Antisense Transcripts (NAT) analysis was also performed and bi-directional transcription appears a common phenomenon in the R. philippinarum transcriptome. A preliminary study on clam samples collected in a highly polluted area of the Venice Lagoon demonstrated the applicability of genomic tools to environmental monitoring., Conclusions: The transcriptomic platform developed for the Manila clam confirmed the high level of reproducibility of current microarray technology. Next-generation sequencing provided a good representation of the clam transcriptome. Despite the known limitations in transcript annotation and sequence coverage for non model species, sufficient information was obtained to identify a large set of genes potentially involved in cellular response to environmental stress.

Background: The genus Erwinia includes plant-associated pathogenic and non-pathogenic Enterobacteria. Important pathogens such as Erwinia amylovora, the causative agent of fire blight and E. pyrifoliae causing bacterial shoot blight of pear in Asia belong to this genus. The species E. tasmaniensis and E. billingiae are epiphytic bacteria and may represent antagonists for biocontrol of fire blight. The presence of genes that are putatively involved in virulence in E. amylovora and E. pyrifoliae is of special interest for these species in consequence., Results: Here we provide the complete genome sequences of the pathogenic E. pyrifoliae strain Ep1/96 with a size of 4.1 Mb and of the non-pathogenic species E. billingiae strain Eb661 with a size of 5.4 Mb, de novo determined by conventional Sanger sequencing and next generation sequencing techniques. Genome comparison reveals large inversions resulting from homologous recombination events. Furthermore, comparison of deduced proteins highlights a relation of E. billingiae strain Eb661 to E. tasmaniensis strain Et1/99 and a distance to E. pyrifoliae for the overall gene content as well as for the presence of encoded proteins representing virulence factors for the pathogenic species. Pathogenicity of E. pyrifoliae is supposed to have evolved by accumulation of potential virulence factors. E. pyrifoliae carries factors for type III secretion and cell invasion. Other genes described as virulence factors for E. amylovora are involved in the production of exopolysaccharides, the utilization of plant metabolites such as sorbitol and sucrose. Some virulence-associated genes of the pathogenic species are present in E. tasmaniensis but mostly absent in E. billingiae., Conclusion: The data of the genome analyses correspond to the pathogenic lifestyle of E. pyrifoliae and underlines the epiphytic localization of E. tasmaniensis and E. billingiae as a saprophyte.

Background: Bisulfite sequencing is a popular method to analyze DNA methylation patterns at high resolution. A region of interest is targeted by PCR and about 20-50 subcloned DNA molecules are usually analyzed, to determine the methylation status at single CpG sites and molecule resolution., Results: The BISMA (Bisulfite Sequencing DNA Methylation Analysis) software for analysis of primary bisulfite sequencing data implements sequencing data extraction and enhanced data processing, quality controls, analysis and presentation of the methylation state. It uses an improved strategy for detection of clonal molecules and accurate CpG site detection and it supports for the first time analysis of repetitive sequences., Conclusions: BISMA works highly automated but still provides the user full control over all steps of the analysis. The BISMA software is freely available as an online tool for academic purposes for the analysis of bisulfite sequencing data from both unique and repetitive sequences http://biochem.jacobs-university.de/BDPC/BISMA/.

Background: Differential DNA methylation between alleles is well established in imprinted genes and the X chromosomes in females but has rarely been reported at non-imprinted loci on autosomes., Results: We studied DNA methylation of cytosine-guanine dinucleotide (CpG) islands on chromosome 21 in leukocytes from several healthy individuals and observed novel cases of pronounced differential methylation of alleles. Allele-specific methylation affected complete CpG islands with methylation differences between alleles of up to 85%. The methylation differences between alleles were strongly correlated with the genotypes, excluding a connection to imprinting. We show that allele-specific methylation can lead to allelic repression of the methylated gene copy. Based on our results, allele-specific methylation is likely to affect about 10% of all human genes and to contribute to allele-specific expression and monoallelic gene silencing. Therefore, allele-specific methylation represents an epigenetic pathway of how genetic polymorphisms may lead to phenotypic variability. In most cases, we observed that some, but not all, heterozygous individuals showed allele-specific methylation, suggesting that allele-specific methylation is the outcome of an epigenetic drift, the direction of which is determined by the genetic differences between the alleles. We could show that the tendency to acquire hypermethylation in one allele was inherited., Conclusions: We observed that larger differences in methylation levels between individuals were often coupled to allele-specific methylation and genetic polymorphisms, suggesting that the inter-individual variability of DNA methylation is strongly influenced by genetic differences. Therefore, genetic differences must be taken into account in future comparative DNA methylation studies.

During bisulfite genomic sequencing projects large amount of data are generated. The Bisulfite sequencing Data Presentation and Compilation (BDPC) web interface (http://biochem.jacobs-university.de/BDPC/) automatically analyzes bisulfite datasets prepared using the BiQ Analyzer. BDPC provides the following output: (i) MS-Excel compatible files compiling for each PCR product (a) the average methylation level, the number of clones analyzed and the percentage of CG sites analyzed (which is an indicator of data quality), (b) the methylation level observed at each CG site and (c) the methylation level of each clone. (ii) A methylation overview table compiling the methylation of all amplicons in all tissues. (iii) Publication grade figures in PNG format showing the methylation pattern for each PCR product embedded in an HMTL file summarizing the methylation data, the DNA sequence and some basic statistics. (iv) A summary file compiling the methylation pattern of different tissues, which is linked to the individual HTML result files, and can be directly used for presentation of the data in the internet. (v) A condensed file, containing all primary data in simplified format for further downstream data analysis and (vi) a custom track file for display of the results in the UCSC genome browser.

The generation of EST information is an essential step in the genomic characterisation of species. In the context of the European Network Marine Genomics, a common goal was to significantly increase the amount of ESTs in commercial marine mollusk species and more specifically in the less studied but ecologically and commercially important groups, such as mussel and clam genera. Normalized cDNA libraries were constructed for four different relevant bivalves species (Crassostrea gigas, Mytilus edulis, Ruditapes decussatus and Bathymodiolus azoricus), using numerous tissues and physiological conditions. In this paper, we present the analysis of the 13,013 expressed sequence tags (ESTs) generated. Each EST library was independently assembled and 1300-3000 unique sequences were identified in each species. For the different species, functional categories could be assigned to only about 16 to 27% of ESTs using the GO annotation tool. All sequences have been incorporated into a publicly available database and form the basis for subsequent microarray design, SNP detection and polymorphism analysis, and the placement of novel markers on genetic linkage maps.

The common marmoset monkey (Callithrix jacchus) is a New World primate that is increasingly used in biomedical research as a model organism. Due to the occurrence of natural bone marrow chimerism, it represents a particularly useful primate model in immunological research. In this study, we describe the genomic organization of the CD94, NKG2, and LY49L genes in the NK complex (NKC) of the common marmoset based on complete sequencing of a bacterial artificial chromosome clonal contig. This region of the marmoset NKC is 1.5 times smaller than its human counterpart, but the genes are colinear and orthologous. One exception is the activating NKG2CE gene, which is probably an ancestral form of the NKG2C- and NKG2E-activating receptor genes of humans and great apes. The two completely sequenced marmoset bacterial artificial chromosome clones are derived from distinct haplotypes, which differ by 200 sites in the overlapping sequence. Analyses of NKC genes in nine additional marmoset individuals revealed a moderate degree of polymorphism of the CD94, NKG2A, NKG2CE, and NKG2D genes. Furthermore, expression analyses identified several alternatively spliced transcripts, particularly of the CD94 gene. Several products of alternative splicing of NKC genes are highly conserved among primates. Alternative transcriptional start sites were found, but these probably do not lead to a change of the translational start site or result in longer or shorter cytoplasmic regions of these type II membrane receptors.

The human X chromosome has a unique biology that was shaped by its evolution as the sex chromosome shared by males and females. We have determined 99.3% of the euchromatic sequence of the X chromosome. Our analysis illustrates the autosomal origin of the mammalian sex chromosomes, the stepwise process that led to the progressive loss of recombination between X and Y, and the extent of subsequent degradation of the Y chromosome. LINE1 repeat elements cover one-third of the X chromosome, with a distribution that is consistent with their proposed role as way stations in the process of X-chromosome inactivation. We found 1,098 genes in the sequence, of which 99 encode proteins expressed in testis and in various tumour types. A disproportionately high number of mendelian diseases are documented for the X chromosome. Of this number, 168 have been explained by mutations in 113 X-linked genes, which in many cases were characterized with the aid of the DNA sequence.

In addition to the BAC-based reference sequence of the accession Columbia-0 from the year 2000, several short read assemblies of THE plant model organism Arabidopsis thaliana were published during the last years. Also, a SMRT-based assembly of Landsberg erecta has been generated that identified translocation and inversion polymorphisms between two genotypes of the species. Here we provide a chromosome-arm level assembly of the A. thaliana accession Niederzenz-1 (AthNd-1_v2c) based on SMRT sequencing data. The best assembly comprises 69 nucleome sequences and displays a contig length of up to 16 Mbp. Compared to an earlier Illumina short read-based NGS assembly (AthNd-1_v1), a 75 fold increase in contiguity was observed for AthNd-1_v2c. To assign contig locations independent from the Col-0 gold standard reference sequence, we used genetic anchoring to generate a de novo assembly. In addition, we assembled the chondrome and plastome sequences. Detailed analyses of AthNd-1_v2c allowed reliable identification of large genomic rearrangements between A. thaliana accessions contributing to differences in the gene sets that distinguish the genotypes. One of the differences detected identified a gene that is lacking from the Col-0 gold standard sequence. This de novo assembly extends the known proportion of the A. thaliana pan-genome. [ABSTRACT FROM AUTHOR]

Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection. [ABSTRACT FROM AUTHOR]

The utility of genome assemblies does not only rely on the quality of the assembled genome sequence, but also on the quality of the gene annotations. The Pacific Biosciences Iso-Seq technology is a powerful support for accurate eukaryotic gene model annotation as it allows for direct readout of full-length cDNA sequences without the need for noisy short read-based transcript assembly. We propose the implementation of the TeloPrime Full Length cDNA Amplification kit to the Pacific Biosciences Iso-Seq technology in order to enrich for genuine full-length transcripts in the cDNA libraries. We provide evidence that TeloPrime outperforms the commonly used SMARTer PCR cDNA Synthesis Kit in identifying transcription start and end sites in Arabidopsis thaliana. Furthermore, we show that TeloPrime-based Pacific Biosciences Iso-Seq can be successfully applied to the polyploid genome of bread wheat (Triticum aestivum) not only to efficiently annotate gene models, but also to identify novel transcription sites, gene homeologs, splicing isoforms and previously unidentified gene loci. [ABSTRACT FROM AUTHOR]

Analysis of the completely determined genomes of the plant-derived Acholeplasma brassicae strain O502 and A. palmae strain J233 revealed that the circular chromosomes are 1,877,792 and 1,554,229 bp in size, have a G + C content of 36 and 29%, and encode 1,690 and 1,439 proteins, respectively. Comparative analysis of these sequences and previously published genomes of A. laidlawii strain PG-8, 'Candidatus Phytoplasma asteris' strains, 'Ca. P. australiense' and 'Ca. P. mali' show a limited shared basic genetic repertoire. The acholeplasma genomes are characterized by a low number of rearrangements, duplication and integration events. Exceptions are the unusual duplication of rRNA operons in A. brassicae and an independently introduced second gene for a single-stranded binding protein in both genera. In contrast to phytoplasmas, the acholeplasma genomes differ by encoding the cell division protein FtsZ, a wide variety of ABC transporters, the F0F1 ATP synthase, the Rnf-complex, SecG of the Sec-dependent secretion system, a richly equipped repertoire for carbohydrate metabolism, fatty acid, isoprenoid and partial amino acid metabolism. Conserved metabolic proteins encoded in phytoplasma genomes such as the malate dehydrogenase SfcA, several transporters and proteins involved in host-interaction, and virulence-associated effectors were not predicted for the acholeplasmas. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Delayed hatching is a form of dormancy evolved in some amphibian and fish embryos to cope with environmental conditions transiently hostile to the survival of hatchlings or larvae. While diapause and cryptobiosis have been extensively studied in several animals, very little is known concerning the molecular mechanisms involved in the sensing and response of fish embryos to environmental cues. Embryos of the euryhaline killifish Fundulus heteroclitus advance dvelopment when exposed to air but hatching is suspended until flooding with seawater. Here, we investigated how transcriptome regulation underpins this adaptive response by examining changes in gene expression profiles of aerially incubated killifish embryos at ∼100% relative humidity, compared to embryos continuously flooded in water. The results confirm that mid-gastrula embryos are able to stimulate development in response to aerial incubation, which is accompanied by the differential expression of at least 806 distinct genes during a 24 h period. Most of these genes (∼70%) appear to be differentially expressed within 3 h of aerial exposure, suggesting a broad and rapid transcriptomic response. This response seems to include an early sensing phase, which overlaps with a tissue remodeling and activation of embryonic development phase involving many regulatory and metabolic pathways. Interestingly, we found fast (0.5–1 h) transcriptional differences in representatives of classical “stress” proteins, such as some molecular chaperones, members of signalling pathways typically involved in the transduction of sensor signals to stress response genes, and oxidative stress-related proteins, similar to that described in other animals undergoing dormancy, diapause or desiccation. To our knowledge, these data represent the first transcriptional profiling of molecular processes associated with desiccation resistance during delayed hatching in non-mammalian vertebrates. The exceptional transcriptomic plasticity observed in killifish embryos provides an important insight as to how the embryos are able to rapidly adapt to non-lethal desiccation conditions. [ABSTRACT FROM AUTHOR]

Campylobacter jejuni is currently the leading cause of bacterial gastroenteritis in humans. Comparison of multiple Campylobacter strains revealed a high genetic and phenotypic diversity. However, little is known about differences in transcriptome organization, gene expression, and small RNA (sRNA) repertoires. Here we present the first comparative primary transcriptome analysis based on the differential RNA–seq (dRNA–seq) of four C. jejuni isolates. Our approach includes a novel, generic method for the automated annotation of transcriptional start sites (TSS), which allowed us to provide genome-wide promoter maps in the analyzed strains. These global TSS maps are refined through the integration of a SuperGenome approach that allows for a comparative TSS annotation by mapping RNA–seq data of multiple strains into a common coordinate system derived from a whole-genome alignment. Considering the steadily increasing amount of RNA–seq studies, our automated TSS annotation will not only facilitate transcriptome annotation for a wider range of pro- and eukaryotes but can also be adapted for the analysis among different growth or stress conditions. Our comparative dRNA–seq analysis revealed conservation of most TSS, but also single-nucleotide-polymorphisms (SNP) in promoter regions, which lead to strain-specific transcriptional output. Furthermore, we identified strain-specific sRNA repertoires that could contribute to differential gene regulation among strains. In addition, we identified a novel minimal CRISPR-system in Campylobacter of the type-II CRISPR subtype, which relies on the host factor RNase III and a trans-encoded sRNA for maturation of crRNAs. This minimal system of Campylobacter, which seems active in only some strains, employs a unique maturation pathway, since the crRNAs are transcribed from individual promoters in the upstream repeats and thereby minimize the requirements for the maturation machinery. Overall, our study provides new insights into strain-specific transcriptome organization and sRNAs, and reveals genes that could modulate phenotypic variation among strains despite high conservation at the DNA level. [ABSTRACT FROM AUTHOR]

Abstract: This study assessed the relationship between the occurrence and function of intronless or single exon genes (SEG) in the genome of five teleost species and their phylogenetic distance. The results revealed that Takifugu rubripes, Tetraodon nigroviridis, Oryzias latipes, Gasterosteus aculeatus and Danio rerio genomes are respectively comprised of 2.83%, 3.42%, 4.49%, 4.35% and 4.02% SEGs. These SEGs encode for a variety of family proteins including claudins, olfactory receptors and histones that are essential for various biological functions. Subsequently, we predicted and annotated SEGs in three European sea bass, Dicentrarchus labrax chromosomes that we have sequenced, and compared results with those of stickleback (G. aculeatus) homologous chromosomes. While the annotation features of three D. labrax chromosomes revealed 78 (5.30%) intronless genes, comparisons with G. aculeatus showed that SEG composition and their order varied significantly among corresponding chromosomes, even for those with nearly complete synteny. More than half of SEGs identified in most of the species have at least one ortholog multiple exon gene in the same genome, which provides insight to their possible origin by retrotransposition. In spite of the fact that they belong to the same lineage, the fraction of predicted SEGs varied significantly between the genomes analyzed, and only a low fraction of proteins (4.1%) is conserved between all five species. Furthermore, the inter-specific distribution of SEGs as well as the functional categories shared by species did not reflect their phylogenetic relationships. These results indicate that new SEGs are continuously and independently generated after species divergence over evolutionary time as evidenced by the phylogenetic results of single exon claudins genes. Although the origin of SEGs cannot be inferred directly from the phylogeny, our results provide strong support for the idea that retrotransposition followed by tandem duplications is the most probable event that can explain the expansion of SEGs in eukaryotic organisms. [Copyright &y& Elsevier]

High genetic diversity is a hallmark of the gastric pathogen Helicobacter pylori. We used 454 sequencing technology to perform whole-genome comparisons for five sets of H. pylori strains that had been sequentially cultured from four chronically infected Colombians (isolation intervals = 3-16 y) and one human volunteer experimentally infected with H. pylori as part of a vaccine trial. The four sets of genomes from Colombian H. pylori differed by 27-232 isolated SNPs and 16-441 imported clusters of polymorphisms resulting from recombination. Imports (mean length = 394 bp) were distributed nonrandomly over the chromosome and frequently occurred in groups, suggesting that H. pylori first takes up long DNA fragments, which subsequently become partially integrated in multiple shorter pieces. Imports were present at significantly increased frequency in members of the hop family of outer membrane gene paralogues, some of which are involved in bacterial adhesion, suggesting diversifying selection. No evidence of recombination and few other differences were identified in the strain pair from an infected volunteer, indicating that the H. pylori genome is stable in the absence of mixed infection. Among these few differences was an OFF/ON switch in the phase-variable adhesin gene hopZ, suggesting strong in vivo selection for this putative adhesin during early colonization. [ABSTRACT FROM AUTHOR]

Abstract: Single nucleotide polymorphisms (SNPs) are believed to contain relevant information and have been therefore extensively used as genetic markers in population and conservation genetics, and molecular ecology studies. This study reports on the identification of potential SNPs in a diploid European sea bass Dicentrarchus labrax genome by using reference sequences from three assembled chromosomes and mapping all WGS datasets onto them (3× Sanger, 3× 454 and 20× SOLEXA). A total of 20,779 SNPs were identified over the 1469 gene loci and intergenic space analysed. Within chromosomes the occurrence of SNPs was the lowest in exons and higher in introns and intergenic regions, which may be explained by the fact, that coding regions are under strong selective pressure to maintain their biological function. The ratio of nonsynonymous to synonymous mutations was smaller than one for all the chromosomes, suggesting that most of deleterious nonsynonymous mutations were eliminated by negative selection. SNPs were not uniformly distributed over the chromosomes. Two chromosomes exhibited large regions with extremely low SNP density, which might represent homozygous regions in the diploid genome. The results of this study show how SNP detection can take profit from sequencing a single diploid individual, but also uncover the limits of such an approach. SNPs that have been identified will support marker development for genetic linkage mapping, population genetics and aquaculture related questions in general. [ABSTRACT FROM AUTHOR]

Background: The SLC11A1/Nramp1 and SLC11A2/Nramp2 genes belong to the SLC11/Nramp family of transmembrane divalent metal transporters, with SLC11A1 being associated with resistance to pathogens and SLC11A2 involved in intestinal iron uptake and transferrin-bound iron transport. Both members of the SLC11 gene family have been clearly identified in tetrapods; however SLC11A1 has never been documented in teleost fish and is believed to have been lost in this lineage during early vertebrate evolution. In the present work we characterized the SLC11 genes in teleosts and evaluated if the roles attributed to mammalian SLC11 genes are assured by other fish specific SLC11 gene members. Results: Two different SLC11 genes were isolated in the European sea bass (Dicentrarchus. labrax), and named slc11a2-a and slc11a2-b, since both were found to be evolutionary closer to tetrapods SLC11A2, through phylogenetic analysis and comparative genomics. Induction of slc11a2-a and slc11a2-b in sea bass, upon iron modulation or exposure to Photobacterium damselae spp. piscicida, was evaluated in in vivo or in vitro experimental models. Overall, slc11a2-a was found to respond only to iron deficiency in the intestine, whereas slc11a2-b was found to respond to iron overload and bacterial infection in several tissues and also in the leukocytes. Conclusions: Our data suggests that despite the absence of slc11a1, its functions have been undertaken by one of the slc11a2 duplicated paralogs in teleost fish in a case of synfunctionalization, being involved in both iron metabolism and response to bacterial infection. This study provides, to our knowledge, the first example of this type of sub-functionalization in iron metabolism genes, illustrating how conserving the various functions of the SLC11 gene family is of crucial evolutionary importance. [ABSTRACT FROM AUTHOR]

The completion of genomic sequences of physiologically important genes frequently reveals non-coding genetic elements such as tandem repeats (micro- and minisatellites) that are often more polymorphic than nearby coding sequences. We obtained the complete genomic sequences of three hormone genes in sea bass Dicentrarchus labrax: growth hormone (dlGH), somatolactin (dlSL) and insulin-like growth factor-1 (dlIGF-1), including 5′- and 3′-untranslated regions. Mini- and microsatellites were discovered in both flanking and intron regions. Some were partially conserved across Perciformes. To assess the usefulness and relevance of these gene-associated markers for understanding population structure, an investigation was made on genetic diversity and differentiation at four of them in (i) five wild populations from the North Sea, the Bay of Biscay and the Western Mediterranean, and (ii) two samples of hatchery-bred individuals from a freshwater-acclimation experiment. Gene and allelic diversities were lower in cultured individuals than in wild ones. Significant genetic differentiation was demonstrated between Bay of Biscay + North Sea and Mediterranean populations (Fst > 0.06, p < 0.001), primarily due to dlGH-associated markers. Significant genetic differentiation was also detected among the Atlantic and North Sea samples, but restricted to the locus associated with dlSL. Significant genetic differentiation was also found among experimental individuals before and after a salinity challenge (Fst ≈ 0.05, p < 0.001), but was due to dlSL and dlIGF-1 loci. Gene-associated markers proved to be more efficient than formerly used anonymous microsatellite markers in providing a clear picture of genetic differentiation. [ABSTRACT FROM PUBLISHER]

We characterized two overlapping sugar beet ( Beta vulgaris) bacterial artificial chromosome (BAC) clones representing different haplotypes. A total of 254 kbp of the genomic sequence was determined, of which the two BACs share 92 kbp. Eleven of 15 genes discovered in the sequenced interval locate to the overlap region. The haplotypes differ in exons by 1% (nucleotide level) and in non-coding regions by 9% (6% mismatches, 3% gaps; alignable regions only). Large indels or high sequence divergence comprised 11% of either sequence. Of such indels, 68 and 45%, respectively, could be attributed to haplotype-specific integration of transposable elements. We identified novel repeat candidates by comparing the two BAC sequences to a set of genomic sugar beet sequences. Synteny was found with Arabidopsis chromosome 1 (At1), At2 and At4, Medicago chromosome 7, Vitis chromosome 15 and paralogous regions on poplar chromosomes II and XIV. [ABSTRACT FROM AUTHOR]

Summary: A microbial mat from the Black Sea shelf was analyzed by a metagenomic approach. While the habitat and its microbial community are characterized by anaerobic methane oxidation, a 79kb contiguous DNA sequence obtained from the same mat provided first evidence for the concomitant presence of the capacity for anaerobic benzoate degradation. Benzoyl-CoA is one central intermediate of anaerobic aromatic degradation, among others. Within a stretch of 31kb, all genes required for the complete pathway of anaerobic benzoate degradation (catabolic island) were identified, including the four subunits of the key enzyme benzoyl-CoA reductase (bcrCBAD), which catalyzes the ATP-driven 2-electron reduction of the aromatic ring. Genes for a ketoacid:acceptor oxidoreductase (korABC) and a ferredoxin (fdx), which are required for generation of a suitable electron donor, were also detected. The majority of the identified catabolic gene products are most similar to their respective orthologs from the denitrifying freshwater bacterium Azoarcus evansii, and the genes are also similarly organized. Due to the lack of established markers, the phylogenetic affiliation of the source organism remains unclear. The presented findings indicate that the metabolic diversity of the Black Sea mat is wider than currently known and that probably other bacteria than those of the methane-oxidizing consortia contribute to aromatic degradation in this anoxic habitat. [Copyright &y& Elsevier]

The sequence-based map of a part of the rhesus macaque major histocompatibility complex (MHC) extended class II region is presented. The sequenced region encompasses 67,401 bp and contains the SACM2L, RING1, FABGL and KE4 genes, as well as the HTATSF1-like and ZNF-like pseudogenes. Similar to human, but different from rat and mouse, no class I genes are found in the SACM2L-RING1 interval. The rhesus macaque extended MHC class II region shows a high degree of conservation of exonic as well as intronic and intergenic sequences compared with the respective human region. It is concluded that this particular genomic organization of the extended class II region—i.e., the absence of class I genes and the presence of the HTATSF1-like and ZNF-like pseudogenes—can be traced back to a common ancestor of humans and rhesus macaques about 23 million years ago. [ABSTRACT FROM AUTHOR]

Genes involved in anaerobic degradation of the petroleum hydrocarbon ethylbenzene in the denitrifying Azoarcus-like strain EbN1 were identified on a 56-kb DNA contig obtained from shotgun sequencing. Ethylbenzene is first oxidized via ethylbenzene dehydrogenase to (S)-1-phenylethanol; this is converted by (S)-1-phenylethanol dehydrogenase to acetophenone. Further degradation probably involves acetophenone carboxylase forming benzoylacetate, a ligase forming benzoylacetyl-CoA, and a thiolase forming acetyl-CoA and benzoyl-CoA. Genes of this pathway were identified via N-terminal sequences of proteins isolated from strain EbN1 and by sequence similarities to proteins from other bacteria. Ethylbenzene dehydrogenase is encoded by three genes (ebdABC), in accordance with the heterotrimeric enzyme structure. Binding domains for a molybdenum cofactor (in subunit EbdA) and iron/sulfur-clusters (in subunits EbdA and EbdB) were identified. The previously observed periplasmic location of the enzyme was corroborated by the presence of a twin-arginine leader peptide characteristic of the Tat system for protein export. A fourth gene (ebdD) was identified, the product of which may act as an enzyme-specific chaperone in the maturation of the molybdenum-containing subunit. A distinct gene (ped) coding for (S)-1-phenylethanol dehydrogenase apparently forms an operon with the ebdABCD genes. The ped gene product with its characteristic NAD(P)-binding motif in the N-terminal domain belongs to the short-chain dehydrogenase/reductase (SDR) superfamily. A further operon apparently contains five genes (apc1–5) suggested to code for subunits of acetophenone carboxylase. Four of the five gene products are similar to subunits of acetone carboxylase from Xanthobacter autotrophicus. Upstream of the apc genes, a single gene (bal) was identified which possibly codes for a benzoylacetate CoA-ligase and which is co-transcribed with the apc genes. In addition, an apparent operon containing almost all genes required for β-oxidation of fatty acids was detected; one of the gene products may be involved in thiolytic cleavage of benzoylacetyl-CoA. The DNA fragment also included genes for regulatory systems; these were two sets of two-component systems, two LysR homologs, and a TetR homolog. Some of these proteins may be involved in ethylbenzene-dependent gene expression. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Discusses the generation, annotation, evolutionary analysis and database integration of unique sea urchin expressed sequence tag clusters. Comparison of the number of gene copies in sea urchins in those of chordates and vertebrates; Expression differences between developmental stages based on oligonucleotide fingerprinting counts.

Describes clone-based systematic haplotyping, a methodology to determine the phase of single-nucleotide polymorphisms on a chromosome. Procedures involved in the method; Suitability of the method for constructing maps of ancestral haplotypes, analysis of complex diseases and for diagnosis of rare defects in which the molecular haplotype is crucial.

Homeodomain transcription factors are involved in many developmental processes and have been intensely studied in a few model organisms, such as mouse, Drosophila and Caenorhabditis elegans. Homeobox genes fall into 10 classes (ANTP, PRD, POU, LIM, TALE, SIX, Cut, ZFH, HNF1, Prox) and 89 different families/groups, all of which are present in vertebrates. Additional groups may be uncovered by further genome annotation, particularly of complex vertebrate genomes. Eight of these groups have been found only in vertebrates, but not in the genome of the tunicate Ciona intestinalis. The other 81 groups of homeobox gene that have been detected in vertebrates so far probably appeared during the early evolution of bilaterians or earlier, as they are also present outside the chordates. How the homeobox genes evolved during and after the main radiation of the bilaterians remains poorly understood, as only a few animal genomes have been sequenced completely. However, drastic changes have occurred at least in the lineage of C. elegans , such as loss of several Hox genes and Hox cluster fragmentation . Here we report considerable alterations of the homeobox gene complement in the tunicate lineage. [Copyright &y& Elsevier]