TLR4 and MD2 variation among horses with differential TNFα baseline concentrations and response to intravenous lipopolysaccharide infusion.

Gram-negative bacterial septicemia is mediated through binding of lipopolysaccharide (LPS) to mammalian toll-like receptor protein 4 (TLR4). TLR4 and its cognate protein, myeloid differentiation factor 2 (MD2) form a heterodimeric complex after binding LPS. This complex induces a cascade of reactions that results in increased proinflammatory cytokine gene expression, including TNFα, which leads to activation of innate immunity. In horses, the immune response to LPS varies widely. To determine if this variation is due to differences in TLR4 or MD2, DNA from 15 healthy adult horses with different TNFα dynamics after experimental intravenous LPS infusion was sequenced across exons of TLR4 and MD2. Haplotypes were constructed for both genes using all identified variants. Four haplotypes were observed for each gene. No significant associations were found between either TNFα baseline concentrations or response to LPS and haplotype; however, there was a significant association (P value = 0.0460) between the baseline TNFα concentration and one MD2 missense variant. Three-dimensional structures of the equine TLR4-MD2-LPS complex were built according to haplotype combinations observed in the study horses, and the implications of missense variants on LPS binding were modeled. Although the sample size was small, there was no evidence that variation in TLR4 or MD2 explains the variability in TNFα response observed after LPS exposure in horses.

High-level, constitutive expression of the mgtC gene confers increased thermotolerance on Salmonella enterica serovar Typhimurium.

We found that mutations that increased the transcription of the mgtCBR (Mg2+ transport-related) operon conferred increased thermotolerance on this organism. The 5' leader of the mgtCBR mRNA contains two short open reading frames (ORFs), mgtM and mgtP, whose translation regulates the expression of the mgtCBR operon by a mechanism that is similar to attenuation in amino acid biosynthetic operons. We obtained two types of mutations that resulted in elevated transcription of the operon: defects in the mgtM ribosome-binding site, impairing the translation of this ORF and deletions encompassing the stop codon of mgtM that extend the translation of this ORF across a downstream Rho termination site. These mgtM mutations give further insights into the mechanism of the transcriptional control of the mgtCBR operon that we discuss in this work. We show that the increased thermotolerance requires elevated expression of the mgtC gene, but functional mgtB and mgtR, which respectively encode an Mg2+ transporter and a regulatory protein, are dispensable for this response. MgtC has been shown to have complex functions, including a requirement for virulence, flagella-independent motility and synthesis of cellulose and we now found that it has a role in the regulation of thermotolerance.

New insights into the phylogenetics and population structure of the prairie falcon (Falco mexicanus).

BACKGROUND: Management requires a robust understanding of between- and within-species genetic variability, however such data are still lacking in many species. For example, although multiple population genetics studies of the peregrine falcon (Falco peregrinus) have been conducted, no similar studies have been done of the closely-related prairie falcon (F. mexicanus) and it is unclear how much genetic variation and population structure exists across the species' range. Furthermore, the phylogenetic relationship of F. mexicanus relative to other falcon species is contested. We utilized a genomics approach (i.e., genome sequencing and assembly followed by single nucleotide polymorphism genotyping) to rapidly address these gaps in knowledge. RESULTS: We sequenced the genome of a single female prairie falcon and generated a 1.17 Gb (gigabases) draft genome assembly. We generated maximum likelihood phylogenetic trees using complete mitochondrial genomes as well as nuclear protein-coding genes. This process provided evidence that F. mexicanus is an outgroup to the clade that includes the peregrine falcon and members of the subgenus Hierofalco. We annotated > 16,000 genes and almost 600,000 high-quality single nucleotide polymorphisms (SNPs) in the nuclear genome, providing the raw material for a SNP assay design featuring > 140 gene-associated markers and a molecular-sexing marker. We subsequently genotyped ~ 100 individuals from California (including the San Francisco East Bay Area, Pinnacles National Park and the Mojave Desert) and Idaho (Snake River Birds of Prey National Conservation Area). We tested for population structure and found evidence that individuals sampled in California and Idaho represent a single panmictic population. CONCLUSIONS: Our study illustrates how genomic resources can rapidly shed light on genetic variability in understudied species and resolve phylogenetic relationships. Furthermore, we found evidence of a single, randomly mating population of prairie falcons across our sampling locations. Prairie falcons are highly mobile and relatively rare long-distance dispersal events may promote gene flow throughout the range. As such, California's prairie falcons might be managed as a single population, indicating that management actions undertaken to benefit the species at the local level have the potential to influence the species as a whole.

The Cell Wall Arabinose-Deficient Arabidopsis thaliana Mutant murus5 Encodes a Defective Allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2.

Traditional marker-based mapping and next-generation sequencing was used to determine that the Arabidopsis (Arabidopsis thaliana) low cell wall arabinose mutant murus5 (mur5) encodes a defective allele of REVERSIBLY GLYCOSYLATED POLYPEPTIDE2 (RGP2). Marker analysis of 13 F2 confirmed mutant progeny from a recombinant mapping population gave a rough map position on the upper arm of chromosome 5, and deep sequencing of DNA from these 13 lines gave five candidate genes with G→A (C→T) transitions predicted to result in amino acid changes. Of these five, only insertional mutant alleles of RGP2, a gene that encodes a UDP-arabinose mutase that interconverts UDP-arabinopyranose and UDP-arabinofuranose, exhibited the low cell wall arabinose phenotype. The identities of mur5 and two SALK insertional alleles were confirmed by allelism tests and overexpression of wild-type RGP2 complementary DNA placed under the control of the 35S promoter in the three alleles. The mur5 mutation results in the conversion of cysteine-257 to tyrosine-257 within a conserved hydrophobic cluster predicted to be distal to the active site and essential for protein stability and possible heterodimerization with other isoforms of RGP.

Mycoplasma haemocanis--the canine hemoplasma and its feline counterpart in the genomic era.

Mycoplasma haemocanis is a hemotrophic mycoplasma (hemoplasma), blood pathogen that may cause acute disease in immunosuppressed or splenectomized dogs. The genome of the strain Illinois, isolated from blood of a naturally infected dog, has been entirely sequenced and annotated to gain a better understanding of the biology of M. haemocanis. Its single circular chromosome has 919 992 bp and a low G + C content (35%), representing a typical mycoplasmal genome. A gene-by-gene comparison against its feline counterpart, M. haemofelis, reveals a very similar composition and architecture with most of the genes having conserved synteny extending over their entire chromosomes and differing only by a small set of unique protein coding sequences. As in M. haemofelis, M. haemocanis metabolic pathways are reduced and apparently rely heavily on the nutrients afforded by its host environment. The presence of a major percentage of its genome dedicated to paralogous genes (63.7%) suggests that this bacterium might use antigenic variation as a mechanism to evade the host's immune system as also observed in M. haemofelis genome. Phylogenomic comparisons based on average nucleotide identity (ANI) and tetranucleotide signature suggest that these two pathogens are different species of mycoplasmas, with M. haemocanis infecting dogs and M. haemofelis infecting cats.

Complete genome sequence of Mycoplasma wenyonii strain Massachusetts.

Mycoplasma wenyonii is a hemotrophic mycoplasma that causes acute and chronic infections in cattle. Here, we announce the first complete genome sequence of this organism. The genome is a single circular chromosome with 650,228 bp and G+C% of 33.9. Analyses of M. wenyonii genome will provide insights into its biology.

Complete genome sequence of Mycoplasma haemocanis strain Illinois.

Mycoplasma haemocanis is a blood pathogen that may cause acute disease in immunosuppressed or splenectomized dogs. The genome of the strain Illinois is a single circular chromosome with 919,992 bp and a GC content of 35%. Analyses of the M. haemocanis genome will provide insights into its biology and in vitro cultivation requirements.

Genome of Mycoplasma haemofelis, unraveling its strategies for survival and persistence.

Mycoplasma haemofelis is a mycoplasmal pathogen (hemoplasma) that attaches to the host's erythrocytes. Distributed worldwide, it has a significant impact on the health of cats causing acute disease and, despite treatment, establishing chronic infection. It might also have a role as a zoonotic agent, especially in immunocompromised patients. Whole genome sequencing and analyses of M. haemofelis strain Ohio2 was undertaken as a step toward understanding its survival and persistence. Metabolic pathways are reduced, relying on the host to supply many of the nutrients and metabolites needed for survival. M. haemofelis must import glucose for ATP generation and ribose derivates for RNA/DNA synthesis. Hypoxanthine, adenine, guanine, uracil and CMP are scavenged from the environment to support purine and pyrimidine synthesis. In addition, nicotinamide, amino acids and any vitamins needed for growth, must be acquired from its environment. The core proteome of M. haemofelis contains an abundance of paralogous gene families, corresponding to 70.6% of all the CDSs. This "paralog pool" is a rich source of different antigenic epitopes that can be varied to elude the host's immune system and establish chronic infection. M. haemofelis also appears to be capable of phase variation, which is particularly relevant to the cyclic bacteremia and persistence, characteristics of the infection in the cat. The data generated herein should be of great use for understanding the mechanisms of M. haemofelis infection. Further, it will provide new insights into its pathogenicity and clues needed to formulate media to support the in vitro cultivation of M. haemofelis.

Complete genome sequence of Mycoplasma suis and insights into its biology and adaption to an erythrocyte niche.

Mycoplasma suis, the causative agent of porcine infectious anemia, has never been cultured in vitro and mechanisms by which it causes disease are poorly understood. Thus, the objective herein was to use whole genome sequencing and analysis of M. suis to define pathogenicity mechanisms and biochemical pathways. M. suis was harvested from the blood of an experimentally infected pig. Following DNA extraction and construction of a paired end library, whole-genome sequencing was performed using GS-FLX (454) and Titanium chemistry. Reads on paired-end constructs were assembled using GS De Novo Assembler and gaps closed by primer walking; assembly was validated by PFGE. Glimmer and Manatee Annotation Engine were used to predict and annotate protein-coding sequences (CDS). The M. suis genome consists of a single, 742,431 bp chromosome with low G+C content of 31.1%. A total of 844 CDS, 3 single copies, unlinked rRNA genes and 32 tRNAs were identified. Gene homologies and GC skew graph show that M. suis has a typical Mollicutes oriC. The predicted metabolic pathway is concise, showing evidence of adaptation to blood environment. M. suis is a glycolytic species, obtaining energy through sugars fermentation and ATP-synthase. The pentose-phosphate pathway, metabolism of cofactors and vitamins, pyruvate dehydrogenase and NAD(+) kinase are missing. Thus, ribose, NADH, NADPH and coenzyme A are possibly essential for its growth. M. suis can generate purines from hypoxanthine, which is secreted by RBCs, and cytidine nucleotides from uracil. Toxins orthologs were not identified. We suggest that M. suis may cause disease by scavenging and competing for host' nutrients, leading to decreased life-span of RBCs. In summary, genome analysis shows that M. suis is dependent on host cell metabolism and this characteristic is likely to be linked to its pathogenicity. The prediction of essential nutrients will aid the development of in vitro cultivation systems.

Next-generation sequencing and potential applications in fungal genomics.

Since the first fungal genome was sequenced in 1996, sequencing technologies have advanced dramatically. In recent years, it has become possible to cost-effectively generate vast amounts of DNA sequence data using a number of cell- and electrophoresis-free sequencing technologies, commonly known as "next" or "second" generation. In this chapter, we present a brief overview of next-generation sequencers that are commercially available now. Their potential applications in fungal genomics studies are discussed.

Exceptional lability of a genomic complex in rice and its close relatives revealed by interspecific and intraspecific comparison and population analysis.

BACKGROUND: Extensive DNA rearrangement of genic colinearity, as revealed by comparison of orthologous genomic regions, has been shown to be a general concept describing evolutionary dynamics of plant genomes. However, the nature, timing, lineages and adaptation of local genomic rearrangement in closely related species (e.g., within a genus) and haplotype variation of genomic rearrangement within populations have not been well documented. RESULTS: We previously identified a hotspot for genic rearrangement and transposon accumulation in the Orp region of Asian rice (Oryza sativa, AA) by comparison with its orthologous region in sorghum. Here, we report the comparative analysis of this region with its orthologous regions in the wild progenitor species (O. nivara, AA) of Asian rice and African rice (O. glaberrima) using the BB genome Oryza species (O. punctata) as an outgroup, and investigation of transposon insertion sites and a segmental inversion event in the AA genomes at the population level. We found that Orp region was primarily and recently expanded in the Asian rice species O. sativa and O. nivara. LTR-retrotransposons shared by the three AA-genomic regions have been fixed in all the 94 varieties that represent different populations of the AA-genome species/subspecies, indicating their adaptive role in genome differentiation. However, LTR-retrotransposons unique to either O. nivara or O. sativa regions exhibited dramatic haplotype variation regarding their presence or absence between or within populations/subpopulations. CONCLUSIONS: The LTR-retrotransposon insertion hotspot in the Orp region was formed recently, independently and concurrently in different AA-genome species, and that the genic rearrangements detected in different species appear to be differentially triggered by transposable elements. This region is located near the end of the short arm of chromosome 8 and contains a high proportion of LTR-retrotransposons similar to observed in the centromeric region of this same chromosome, and thus may represent a genomic region that has recently switched from euchromatic to heterochromatic states. The haplotype variation of LTR-retrotransposon insertions within this region reveals substantial admixture among various subpopulations as established by molecular markers at the whole genome level, and can be used to develop retrotransposon junction markers for simple and rapid classification of O. sativa germplasm.

An examination of targeted gene neighborhoods in strawberry.

BACKGROUND: Strawberry (Fragaria spp.) is the familiar name of a group of economically important crop plants and wild relatives that also represent an emerging system for the study of gene and genome evolution. Its small stature, rapid seed-to-seed cycle, transformability and miniscule basic genome make strawberry an attractive system to study processes related to plant physiology, development and crop production; yet it lacks substantial genomics-level resources. This report addresses this deficiency by characterizing 0.71 Mbp of gene space from a diploid species (F. vesca). The twenty large genomic tracks (30-52 kb) captured as fosmid inserts comprise gene regions with roles in flowering, disease resistance, and metabolism. RESULTS: A detailed description of the studied regions reveals 131 Blastx-supported gene sites and eight additional EST-supported gene sites. Only 15 genes have complete EST coverage, enabling gene modelling, while 76 lack EST support. Instances of microcolinearity with Arabidopsis thaliana were identified in twelve inserts. A relatively high portion (25%) of targeted genes were found in unanticipated tandem duplications. The effectiveness of six FGENESH training models was assessed via comparisons among ab initio predictions and homology-based gene and start/stop codon identifications. Fourteen transposable-element-related sequences and 158 simple sequence repeat loci were delineated. CONCLUSIONS: This report details the structure and content of targeted regions of the strawberry genome. The data indicate that the strawberry genome is gene-dense, with an average of one protein-encoding gene or pseudogene per 5.9 kb. Current overall EST coverage is sparse. The unexpected gene duplications and their differential patterns of EST support suggest possible subfunctionalization or pseudogenization of these sequences. This report provides a high-resolution depiction of targeted gene neighborhoods that will aid whole-genome sequence assembly, provide valuable tools for plant breeders and advance the understanding of strawberry genome evolution.

Exceptional diversity, non-random distribution, and rapid evolution of retroelements in the B73 maize genome.

Recent comprehensive sequence analysis of the maize genome now permits detailed discovery and description of all transposable elements (TEs) in this complex nuclear environment. Reiteratively optimized structural and homology criteria were used in the computer-assisted search for retroelements, TEs that transpose by reverse transcription of an RNA intermediate, with the final results verified by manual inspection. Retroelements were found to occupy the majority (>75%) of the nuclear genome in maize inbred B73. Unprecedented genetic diversity was discovered in the long terminal repeat (LTR) retrotransposon class of retroelements, with >400 families (>350 newly discovered) contributing >31,000 intact elements. The two other classes of retroelements, SINEs (four families) and LINEs (at least 30 families), were observed to contribute 1,991 and approximately 35,000 copies, respectively, or a combined approximately 1% of the B73 nuclear genome. With regard to fully intact elements, median copy numbers for all retroelement families in maize was 2 because >250 LTR retrotransposon families contained only one or two intact members that could be detected in the B73 draft sequence. The majority, perhaps all, of the investigated retroelement families exhibited non-random dispersal across the maize genome, with LINEs, SINEs, and many low-copy-number LTR retrotransposons exhibiting a bias for accumulation in gene-rich regions. In contrast, most (but not all) medium- and high-copy-number LTR retrotransposons were found to preferentially accumulate in gene-poor regions like pericentromeric heterochromatin, while a few high-copy-number families exhibited the opposite bias. Regions of the genome with the highest LTR retrotransposon density contained the lowest LTR retrotransposon diversity. These results indicate that the maize genome provides a great number of different niches for the survival and procreation of a great variety of retroelements that have evolved to differentially occupy and exploit this genomic diversity.

A lineage-specific centromere retrotransposon in Oryza brachyantha.

Most eukaryotic centromeres contain large quantities of repetitive DNA, such as satellite repeats and retrotransposons. Unlike most transposons in plant genomes, the centromeric retrotransposon (CR) family is conserved over long evolutionary periods among a majority of the grass species. CR elements are highly concentrated in centromeres, and are likely to play a role in centromere function. In order to study centromere evolution in the Oryza (rice) genus, we sequenced the orthologous region to centromere 8 of Oryza sativa from a related species, Oryza brachyantha. We found that O. brachyantha does not have the canonical CRR (CR of rice) found in the centromeres of all other Oryza species. Instead, a new Ty3-gypsy (Metaviridae) retroelement (FRetro3) was found to colonize the centromeres of this species. This retroelement is found in high copy numbers in the O. brachyantha genome, but not in other Oryza genomes, and based on the dating of long terminal repeats (LTRs) of FRetro3 it was amplified in the genome in the last few million years. Interestingly, there is a high level of removal of FRetro3 based on solo-LTRs to full-length elements, and this rapid turnover may have played a role in the replacement of the canonical CRR with the new element by active deletion. Comparison with previously described ChIP cloning data revealed that FRetro3 is found in CENH3-associated chromatin sequences. Thus, within a single lineage of the Oryza genus, the canonical component of grass centromeres has been replaced with a new retrotransposon that has all the hallmarks of a centromeric retroelement.

Sequence analysis of Escherichia coli O157:H7 bacteriophage PhiV10 and identification of a phage-encoded immunity protein that modifies the O157 antigen.

Bacteriophage PhiV10 is a temperate phage, which specifically infects Escherichia coli O157:H7. The nucleotide sequence of the PhiV10 genome is 39 104 bp long and contains 55 predicted genes. PhiV10 is closely related to two previously sequenced phages, the Salmonella enterica serovar Anatum (Group E1) phage epsilon15 and a prophage from E. coli APEC O1. The attachment site of PhiV10, like those of its two closest relatives, overlaps the 3' end of guaA in the host chromosome. PhiV10 encodes an O-acetyltransferase, which modifies the O157 antigen. This modification is sufficient to block PhiV10 superinfection, indicating that the O157 antigen is most likely the PhiV10 receptor.

Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains.

BACKGROUND: Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR) and methylation spanning linker libraries (MSLL). These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends. RESULTS: A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the SalI MSLL libraries being the most highly enriched (31% align to an EST contig), while the HMPR clones exhibited exceptional depletion of repetitive DNA (to approximately 11%). These two techniques were compared with other gene-enrichment methods, and shown to be complementary. CONCLUSION: MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of epigenetic boundaries are barely understood at this time, MSLL technology flags both approximate boundaries and methylated genes that deserve additional investigation. MSLL and HMPR sequences provide a valuable resource for maize genome annotation, and are a uniquely valuable complement to any plant genome sequencing project. In order to make these results fully accessible to the community, a web display was developed that shows the alignment of MSLL, HMPR, and other gene-rich sequences to the BACs; this display is continually updated with the latest ESTs and BAC sequences.

Construction, alignment and analysis of twelve framework physical maps that represent the ten genome types of the genus Oryza.

We describe the establishment and analysis of a genus-wide comparative framework composed of 12 bacterial artificial chromosome fingerprint and end-sequenced physical maps representing the 10 genome types of Oryza aligned to the O. sativa ssp. japonica reference genome sequence. Over 932 Mb of end sequence was analyzed for repeats, simple sequence repeats, miRNA and single nucleotide variations, providing the most extensive analysis of Oryza sequence to date.

A unified classification system for eukaryotic transposable elements.

Our knowledge of the structure and composition of genomes is rapidly progressing in pace with their sequencing. The emerging data show that a significant portion of eukaryotic genomes is composed of transposable elements (TEs). Given the abundance and diversity of TEs and the speed at which large quantities of sequence data are emerging, identification and annotation of TEs presents a significant challenge. Here we propose the first unified hierarchical classification system, designed on the basis of the transposition mechanism, sequence similarities and structural relationships, that can be easily applied by non-experts. The system and nomenclature is kept up to date at the WikiPoson web site.

Evolutionary dynamics of an ancient retrotransposon family provides insights into evolution of genome size in the genus Oryza.

Long terminal repeat (LTR) retrotransposons constitute a significant portion of most eukaryote genomes and can dramatically change genome size and organization. Although LTR retrotransposon content variation is well documented, the dynamics of genomic flux caused by their activity are poorly understood on an evolutionary time scale. This is primarily because of the lack of an experimental system composed of closely related species whose divergence times are within the limits of the ability to detect ancestrally related retrotransposons. The genus Oryza, with 24 species, ten genome types, different ploidy levels and over threefold genome size variation, constitutes an ideal experimental system to explore genus-level transposon dynamics. Here we present data on the discovery and characterization of an LTR retrotransposon family named RWG in the genus Oryza. Comparative analysis of transposon content (approximately 20 to 27,000 copies) and transpositional history of this family across the genus revealed a broad spectrum of independent and lineage-specific changes that have implications for the evolution of genome size and organization. In particular, we provide evidence that the basal GG genome of Oryza (O. granulata) has expanded by nearly 25% by a burst of the RWG lineage Gran3 subsequent to speciation. Finally we describe the recent evolutionary origin of Dasheng, a large retrotransposon derivative of the RWG family, specifically found in the A, B and C genome lineages of Oryza.

Fine mapping of the Pc locus of Sorghum bicolor, a gene controlling the reaction to a fungal pathogen and its host-selective toxin.

Milo disease in sorghum is caused by isolates of the soil-borne fungus Periconia circinata that produce PC-toxin. Susceptibility to milo disease is conditioned by a single, semi-dominant gene, termed Pc. The susceptible allele (Pc) converts to a resistant form (pc) spontaneously at a gametic frequency of 10(-3) to 10(-4). A high-density genetic map was constructed around the Pc locus using DNA markers, allowing the Pc gene to be delimited to a 0.9 cM region on the short arm of sorghum chromosome 9. Physically, the Pc-region was covered by a single BAC clone. Sequence analysis of this BAC revealed twelve gene candidates. Several of the predicted genes in the region are homologous to disease resistance loci, including one NBS-LRR resistance gene analogue that is present in multiple tandem copies. Analysis of pc isolines derived from Pc/Pc sorghum suggests that one or more members of this NBS-LRR gene family are the Pc genes that condition susceptibility.