Sarah Butcher

Genome Expansion and Gene Loss in Powdery Mildew Fungi Reveal Tradeoffs in Extreme Parasitism

From Blight to Powdery Mildew Pathogenic effects of microbes on plants have widespread consequences. Witness, for example, the cultural upheavals driven by potato blight in the 1800s. A variety of microbial pathogens continue to afflict crop plants today, driving both loss of yield and incurring the increased costs of control mechanisms. Now, four reports analyze microbial genomes in order to understand better how plant pathogens function (see the Perspective by Dodds). Raffaele et al. (p. 1540) describe how the genome of the potato blight pathogen accommodates transfer to different hosts. Spanu et al. (p. 1543) analyze what it takes to be an obligate biotroph in barley powdery mildew, and Baxter et al. (p. 1549) ask a similar question for a natural pathogen of Arabidopsis. Schirawski et al. (p. 1546) compared genomes of maize pathogens to identify virulence determinants. Better knowledge of what in a genome makes a pathogen efficient and deadly is likely to be useful for improving agricultural crop management and breeding. A group of papers analyzes pathogen genomes to find the roots of virulence, opportunism, and life-style determinants. Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.

Comparative whole-genome analyses reveal over 100 putative phase-variable genes in the pathogenic Neisseria spp.

Previously, a complete genome analysis of Neisseria meningitidis strain MC58 revealed the largest repertoire of putative phase-variable genes described in any species to date. Initial comparisons with two incomplete Neisseria spp. genome sequences available at that time revealed differences in the repeats associated with these genes in the form of polymorphisms, the absence of the potentially unstable elements in some alleles, and in the repertoire of the genes that were present. Analyses of the complete genomes of N. meningitidis strain Z2491 and Neisseria gonorrhoeae strain FA1090 have been performed and are combined with a comprehensive comparative analysis between the three available complete genome sequences. This has increased the sensitivity of these searches and provided additional contextual information that facilitates the interpretation of the functional consequences of repeat instability. This analysis identified: (i) 68 phase-variable gene candidates in N. meningitidis strain Z2491, rather than the 27 previously reported; (ii) 83 candidates in N. gonorrhoeae strain FA1090; and (iii) 82 candidates in N. meningitidis strain MC58, including an additional 19 identified through cross-comparisons with the other two strains. In addition to the 18 members of the opa gene family, a repertoire of 119 putative phase-variable genes is described, indicating a huge potential for diversification mediated by this mechanism of gene switching in these species that is central to their interactions with the host and environmental transitions. Eighty-two of these are either known (14) or strong (68) candidates for phase variation, which together with the opa genes make a total of 100 identified genes. The repertoires of the genes identified in this analysis diverge from the different species groupings, indicating horizontal exchange that significantly affects the species and strain complements of these genes.

WebACT—an online companion for the Artemis Comparison Tool

UNLABELLED WebACT is an online resource which enables the rapid provision of simultaneous BLAST comparisons between up to five genomic sequences in a format amenable for visualization with the well-known Artemis Comparison Tool (ACT). Comparisons can be generated on-the-fly using sequences directly retrieved via EMBL database queries, or by entering or uploading user sequences. Furthermore, pre-computed comparisons are available between all publicly available, completed prokaryotic genomes and plasmids currently contained within the Genome Reviews database (372 sequences, representing 175 different species). The system is designed to minimize the volume of downloaded data and maximize performance. Genome sequences, annotation and pre-computed comparisons are stored in a relational database allowing flexible querying based on user-defined sequence regions, from whole genome to a defined region flanking a specified gene. Comparison and sequence files, whether computed online or retrieved from the database of pre-computed genome comparisons, can be viewed online using ACT and are available for download. AVAILABILITY Freely accessible at http://www.webact.org. SUPPLEMENTARY INFORMATION User guide and worked examples are available at http://www.webact.org/WebACT/docs.

Identification of genes selectively disallowed in the pancreatic islet

We have previously identified two genes, encoding lactate dehydrogenase (Ldha) and the monocarboxylate carrier, MCT1 (Slc16a1) whose expression is remarkably low in pancreatic β-cells and islets. We sought here to determine whether these may be part of a larger family of genes selectively repressed (“disallowed”) in the pancreatic islet. Using new and publicly available microarray data, we undertook a bioinformatic analysis of gene expression in islets and a range of other murine tissues. We compared data sets from three sources of mouse pancreatic islets with a total of 30 datasets from nine tissues, to identify genes with at least five-fold down-regulation in islets. 39 genes were revealed as being specifically repressed in islets. These included Ldha and Slc16a1 as expected but also genes involved in several other metabolic pathways which could affect glucose stimulated insulin secretion. Of these, adenylate kinase 3 (AK3) is a mitochondrial enzyme which acts on GTP, and ornithine aminotransferase (OAT) lies on the pathway converting glutamate to ornithine. The removal of an enzyme which could dissipate mitochondrial GTP levels in beta cells provides support for the theory that mitochondrial GTP may be an important for regulating insulin secretion, whilst blocking an alternative metabolic fate for glutamate is consistent with a signalling role for glutamate. The identification of these genes should inform efforts to generate fully functional β-cells from stem cell sources, and may provide new targets in type 2 diabetes.

The South Asian genome.

The genetic sequence variation of people from the Indian subcontinent who comprise one-quarter of the worlds population, is not well described. We carried out whole genome sequencing of 168 South Asians, along with whole-exome sequencing of 147 South Asians to provide deeper characterisation of coding regions. We identify 12,962,155 autosomal sequence variants, including 2,946,861 new SNPs and 312,738 novel indels. This catalogue of SNPs and indels amongst South Asians provides the first comprehensive map of genetic variation in this major human population, and reveals evidence for selective pressures on genes involved in skin biology, metabolism, infection and immunity. Our results will accelerate the search for the genetic variants underlying susceptibility to disorders such as type-2 diabetes and cardiovascular disease which are highly prevalent amongst South Asians.

Comparative analysis of the Saccharomyces cerevisiae and Caenorhabditis elegans protein interaction networks

BackgroundProtein interaction networks aim to summarize the complex interplay of proteins in an organism. Early studies suggested that the position of a protein in the network determines its evolutionary rate but there has been considerable disagreement as to what extent other factors, such as protein abundance, modify this reported dependence.ResultsWe compare the genomes of Saccharomyces cerevisiae and Caenorhabditis elegans with those of closely related species to elucidate the recent evolutionary history of their respective protein interaction networks. Interaction and expression data are studied in the light of a detailed phylogenetic analysis. The underlying network structure is incorporated explicitly into the statistical analysis. The increased phylogenetic resolution, paired with high-quality interaction data, allows us to resolve the way in which protein interaction network structure and abundance of proteins affect the evolutionary rate. We find that expression levels are better predictors of the evolutionary rate than a proteins connectivity. Detailed analysis of the two organisms also shows that the evolutionary rates of interacting proteins are not sufficiently similar to be mutually predictive.ConclusionIt appears that meaningful inferences about the evolution of protein interaction networks require comparative analysis of reasonably closely related species. The signature of protein evolution is shaped by a proteins abundance in the organism and its function and the biological process it is involved in. Its position in the interaction networks and its connectivity may modulate this but they appear to have only minor influence on a proteins evolutionary rate.

Protein identification and quantification by two-dimensional infrared spectroscopy: implications for an all-optical proteomic platform.

Electron-vibration-vibration two-dimensional coherent spectroscopy, a variant of 2DIR, is shown to be a useful tool to differentiate a set of 10 proteins based on their amino acid content. Two-dimensional vibrational signatures of amino acid side chains are identified and the corresponding signal strengths used to quantify their levels by using a methyl vibrational feature as an internal reference. With the current apparatus, effective differentiation can be achieved in four to five minutes per protein, and our results suggest that this can be reduced to <1 min per protein by using the same technology. Finally, we show that absolute quantification of protein levels is relatively straightforward to achieve and discuss the potential of an all-optical high-throughput proteomic platform based on two-dimensional infrared spectroscopic measurements.

Bioinformatic analysis of Entamoeba histolytica SINE1 elements

BackgroundInvasive amoebiasis, caused by infection with the human parasite Entamoeba histolytica remains a major cause of morbidity and mortality in some less-developed countries. Genetically E. histolytica exhibits a number of unusual features including having approximately 20% of its genome comprised of repetitive elements. These include a number of families of SINEs - non-autonomous elements which can, however, move with the help of partner LINEs. In many eukaryotes SINE mobility has had a profound effect on gene expression; in this study we concentrated on one such element - EhSINE1, looking in particular for evidence of recent transposition.ResultsEhSINE1s were detected in the newly reassembled E. histolytica genome by searching with a Hidden Markov Model developed to encapsulate the key features of this element; 393 were detected. Examination of their sequences revealed that some had an internal structure showing one to four 26-27 nt repeats. Members of the different classes differ in a number of ways and in particular those with two internal repeats show the properties expected of fairly recently transposed SINEs - they are the most homogeneous in length and sequence, they have the longest (i.e. the least decayed) target site duplications and are the most likely to show evidence (in a cDNA library) of active transcription. Furthermore we were able to identify 15 EhSINE1s (6 pairs and one triplet) which appeared to be identical or very nearly so but inserted into different sites in the genome; these provide good evidence that if mobility has now ceased it has only done so very recently.ConclusionsOf the many families of repetitive elements present in the genome of E. histolytica we have examined in detail just one - EhSINE1. We have shown that there is evidence for waves of transposition at different points in the past and no evidence that mobility has entirely ceased. There are many aspects of the biology of this parasite which are not understood, in particular why it is pathogenic while the closely related species E. dispar is not, the great genetic diversity found amongst patient isolates and the fact, which may be related, that only a small proportion of those infected develop clinical invasive amoebiasis. Mobile genetic elements, with their ability to alter gene expression may well be important in unravelling these puzzles.

Development of a PCR-based method for the detection of enteroviruses and hepatitis A virus in molluscan shellfish and its application to polluted field samples

The use of the polymerase chain reaction (PCR) for detection of low levels of enteric viruses in bivalve shellfish is hindered by the presence of potent amplification inhibitors. A procedure previously developed for removing the majority of these amplification inhibitors is applied to the detection of enteroviruses and hepatitis A virus in naturally polluted field samples. Quantification of PCR inhibition showed that PCR sample tolerance ranged from 2 to 4.7g shellfish for highly polluted samples. These results indicate the need for adequate controls for PCR inhibition, particularly for negative samples. Reverse transcription (RT)-PCR results were compared with conventional enterovirus isolation for a range of naturally contaminated shellfish. All enterovirus isolation positive samples were also positive by enterovirus RT-PCR. At one field site shellfish were positive by enterovirus RT-PCR but negative for virus isolation. All shellfish tested were negative for hepatitis A by RT-PCR. The procedure for removal of PCR amplification inhibitors should be equally applicable to the detection of Norwalk and related Small Round Structured Viruses (SRSVs) in shellfish.

The evolution of standards and data management practices in systems biology

A recent community survey conducted by Infrastructure for Systems Biology Europe (ISBE) informs requirements for developing an efficient infrastructure for systems biology standards, data and model management.