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Exploring the mialome of ticks: An annotated catalogue of midgut transcripts from the hard tick, Dermacentor variabilis (Acari: Ixodidae)
Background:
Ticks are obligate blood feeders. The midgut is the first major region of the body where blood and microbes ingested with the blood meal come in contact with the tick's internal tissues. Little is known about protein expression in the digestive tract of ticks. In this study, for analysis of global gene expression during tick attachment and feeding, we generated and sequenced 1,679 random transcripts (ESTs) from cDNA libraries from the midguts of female ticks at varying stages of feeding.
Results:
Sequence analysis of the 1,679 ESTs resulted in the identification of 835 distinct transcripts, from these, a total of 82 transcripts were identified as proteins putatively directly involved in blood meal digestion, including enzymes involved in oxidative stress reduction/antimicrobial activity/detoxification, peptidase inhibitors, protein digestion (cysteine-, aspartic-, serine-, and metallo- peptidases), cell, protein and lipid binding including mucins and iron/heme metabolism and transport. A lectin-like protein with a high match to lectins in other tick species, allergen-like proteins and surface antigens important in pathogen recognition and/or antimicrobial activity were also found. Furthermore, midguts collected from the 6-day-fed ticks expressed twice as many transcripts involved in bloodmeal processing as midguts from unfed/2-day-fed ticks.
Conclusion:
This tissue-specific transcriptome analysis provides an opportunity to examine the global expression of transcripts in the tick midgut and to compare the gut response to host attachment versus blood feeding and digestion. In contrast to those in salivary glands of other Ixodid ticks, most proteins in the D. variabilis midgut cDNA library were intracellular. Of the total ESTs associated with a function, an unusually large number of transcripts were associated with peptidases, cell, lipid and protein binding, and oxidative stress or detoxification. Presumably, this is consistent with their role in intracellular processing of the blood meal and response to microbial infections. The presence of many proteins with similar functions is consistent with the hypothesis that gene duplication contributed to the successful adaptation of ticks to hematophagy. Furthermore, these transcripts may be useful to scientists investigating the role of the tick midgut in blood-meal digestion, antimicrobial activity or the transmission of tick-borne pathogens.
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Prediction of alternatively skipped exons and splicing enhancers from exon junction arrays
Background:
Alternative splicing of exons in a pre-mRNA transcript is an important mechanism which contributes to protein diversity in human. Arrays for detecting alternative splicing are available using several different probe designs, including those based on exon-junctions. In this work, we introduce a new method for predicting alternatively skipped exons from exon-junction arrays. Predictions based on our method are compared against controls and their sequences are analyzed to identify motifs important for regulating alternative splicing.
Results:
Our comparison of several alternative methods shows that an exon-skipping score based on neighboring junctions best discriminates between positive and negative controls. Sequence analysis of our predicted exons confirms the presence of known splicing regulatory sequences. In addition, we also derive a set of development-related alternatively spliced genes based on fetal versus adult tissue comparisons and find that our predictions are consistent with their functional annotations. Ab initio motif finding algorithms are applied to identify several motifs that may be relevant for splicing during development.
Conclusions:
This work describes a new method for analyzing exon-junction arrays, identifies sequence motifs that are specific for alternative and constitutive splicing and suggests a role for several known splicing factors and their motifs in developmental regulation.
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Genome-wide and expression analysis of protein phosphatase 2C in rice and Arabidopsis
Background:
The protein phosphatase 2Cs (PP2Cs) from various organisms have been implicated to act as negative modulators of protein kinase pathways involved in diverse environmental stress responses and developmental processes. A genome-wide overview of the PP2C gene family in plants is not yet available.
Results:
A comprehensive computational analysis identified 80 and 78 PP2C genes in Arabidopsis thaliana (AtPP2Cs) and Oryza sativa (OsPP2Cs), respectively, which denotes the PP2C gene family as one of the largest families identified in plants. Phylogenic analysis divided PP2Cs in Arabidopsis and rice into 13 and 11 subfamilies, respectively, which are supported by the analyses of gene structures and protein motifs. Comparative analysis between the PP2C genes in Arabidopsis and rice identified common and lineage-specific subfamilies and potential 'gene birth-and-death' events. Gene duplication analysis reveals that whole genome and chromosomal segment duplications mainly contributed to the expansion of both OsPP2Cs and AtPP2Cs, but tandem or local duplication occurred less frequently in Arabidopsis than rice. Some protein motifs are widespread among the PP2C proteins, whereas some other motifs are specific to only one or two subfamilies. Expression pattern analysis suggests that 1) most PP2C genes play functional roles in multiple tissues in both species, 2) the induced expression of most genes in subfamily A by diverse stimuli indicates their primary role in stress tolerance, especially ABA response, and 3) the expression pattern of subfamily D members suggests that they may constitute positive regulators in ABA-mediated signaling pathways. The analyses of putative upstream regulatory elements by two approaches further support the functions of subfamily A in ABA signaling, and provide insights into the shared and different transcriptional regulation machineries in dicots and monocots.
Conclusion:
This comparative genome-wide overview of the PP2C family in Arabidopsis and rice provides insights into the functions and regulatory mechanisms, as well as the evolution and divergence of the PP2C genes in dicots and monocots. Bioinformatics analyses suggest that plant PP2C proteins from different subfamilies participate in distinct signaling pathways. Our results have established a solid foundation for future studies on the functional divergence in different PP2C subfamilies.
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Genome-wide survey of prokaryotic serine proteases: Analysis of distribution and domain architectures of five serine protease families in prokaryotes
Background:
Serine proteases are one of the most abundant groups of proteolytic enzymes found in all the kingdoms of life. While studies have established significant roles for many prokaryotic serine proteases in several physiological processes, such as those associated with metabolism, cell signalling, defense response and development, functional associations for a large number of prokaryotic serine proteases are relatively unknown. Current analysis is aimed at understanding the distribution and probable biological functions of the select serine proteases encoded in representative prokaryotic organisms.
Results:
A total of 966 putative serine proteases, belonging to five families, were identified in the 91 prokaryotic genomes using various sensitive sequence search techniques. Phylogenetic analysis reveals several species-specific clusters of serine proteases suggesting their possible involvement in organism-specific functions. Atypical phylogenetic associations suggest an important role for lateral gene transfer events in facilitating the widespread distribution of the serine proteases in the prokaryotes. Domain organisations of the gene products were analysed, employing sensitive sequence search methods, to infer their probable biological functions. Trypsin, subtilisin and Lon protease families account for a significant proportion of the multi-domain representatives, while the D-Ala-D-Ala carboxypeptidase and the Clp protease families are mostly single-domain polypeptides in prokaryotes. Regulatory domains for protein interaction, signalling, pathogenesis, cell adhesion etc. were found tethered to the serine protease domains. Some domain combinations (such as S1-PDZ; LON-AAA-S16 etc.) were found to be widespread in the prokaryotic lineages suggesting a critical role in prokaryotes.
Conclusions:
Domain architectures of many serine proteases and their homologues identified in prokaryotes are very different from those observed in eukaryotes, suggesting distinct roles for serine proteases in prokaryotes. Many domain combinations were found unique to specific prokaryotic species, suggesting functional specialisation in various cellular and physiological processes.
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A genome survey of Moniliophthora perniciosa gives new insights into Witches' Broom Disease of cacao
Background:
The basidiomycete fungus Moniliophthora perniciosa is the causal agent of Witches' Broom Disease (WBD) in cacao (Theobroma cacao). It is a hemibiotrophic pathogen that colonizes the apoplast of cacao's meristematic tissues as a biotrophic pathogen, switching to a saprotrophic lifestyle during later stages of infection. M. perniciosa, together with the related species M. roreri, are pathogens of aerial parts of the plant, an uncommon characteristic in the order Agaricales. A genome survey (1.9X coverage) of M. perniciosa was analyzed to evaluate the overall gene content of this phytopathogen.
Results:
Genes encoding proteins involved in retrotransposition, reactive oxygen species (ROS) resistance, drug efflux transport and cell wall degradation were identified. The great number of genes encoding cytochrome P450 monooxygenases (1.15% of gene models) indicates that M. perniciosa has a great potential for detoxification, production of toxins and hormones; which may confer a high adaptive ability to the fungus. We have also discovered new genes encoding putative secreted polypeptides rich in cysteine, as well as genes related to methylotrophy and plant hormone biosynthesis (gibberellin and auxin). Analysis of gene families indicated that M. perniciosa have similar amounts of carboxylesterases and repertoires of plant cell wall degrading enzymes as other hemibiotrophic fungi. In addition, an approach for normalization of gene family data using incomplete genome data was developed and applied in M. perniciosa genome survey.
Conclusion:
This genome survey gives an overview of the M. perniciosa genome, and reveals that a significant portion is involved in stress adaptation and plant necrosis, two necessary characteristics for a hemibiotrophic fungus to fulfill its infection cycle. Our analysis provides new evidence revealing potential adaptive traits that may play major roles in the mechanisms of pathogenicity in the M. perniciosa/cacao pathosystem.
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Architecture of thermal adaptation in an Exiguobacterium sibiricum strain isolated from 3 million year old permafrost: A genome and transcriptome approach
Background:
Many microorganisms have a wide temperature growth range and versatility to tolerate large thermal fluctuations in diverse environments, however not many have been fully explored over their entire growth temperature range through a holistic view of its physiology, genome, and transcriptome. We used Exiguobacterium sibiricum strain 255-15, a psychrotrophic bacterium from 3 million year old Siberian permafrost that grows from -5oC to 39oC to study its thermal adaptation.
Results:
The E. sibiricum genome has one chromosome and two small plasmids with a total of 3,015 protein-encoding genes (CDS), and a GC content of 47.7%. The genome and transcriptome analysis along with the organism's known physiology was used to better understand its thermal adaptation. A total of 27%, 3.2%, and 5.2% of E. sibiricum CDS spotted on the DNA microarray detected differentially expressed genes in cells grown at -2.5oC, 10oC, and 39oC, respectively, when compared to cells grown at 28oC. The hypothetical and unknown genes represented 10.6%, 0.89%, and 2.3% of the CDS differentially expressed when grown at -2.5oC, 10oC, and 39oC versus 28oC, respectively.
Conclusions:
The results show that E. sibiricum is constitutively adapted to cold temperatures stressful to mesophiles since little differential gene expression was observed between 4oC and 28oC, but at the extremities of its Arrhenius growth profile, namely -2.5oC and 39oC, several physiological and metabolic adaptations associated with stress responses were observed.
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Transcriptional Response of Rat Frontal Cortex following Acute In Vivo Exposure to the Pyrethroid Insecticides Permethrin and Deltamethrin
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Background:
Pyrethroids are neurotoxic pesticides that interact with membrane bound ion channels in neurons and disrupt nerve function. The purpose of this study was to characterize and explore changes in gene expression that occur in the rat frontal cortex, an area of CNS affected by pyrethroids, following an acute low-dose exposure.
Results:
Rats were acutely exposed to either deltamethrin (0.3 - 3 mg/kg) or permethrin (1 - 100 mg/kg) followed by collection of cortical tissue at 6 hours. The doses used range from those that cause minimal signs of intoxication at the behavioral level to doses well below apparent no effect levels in the whole animal. A statistical framework based on parallel linear (SAM) and isotonic regression (PIR) methods identified 95 and 53 probe sets as dose-responsive. The PIR analysis was most sensitive for detecting transcripts with changes in expression at the NOAEL dose. A sub-set of genes (Camk1g, Ddc, Gpd3, c-fos and Egr1) was then confirmed by qRT-PCR and examined in a time course study. Changes in mRNA levels were typically less than 3-fold in magnitude across all components of the study. The responses observed are consistent with pyrethroids producing increased neuronal excitation in the cortex following a low-dose in vivo exposure. In addition, Significance Analysis of Function and Expression (SAFE) identified significantly enriched gene categories common for both pyrethroids, including some relating to branching morphogenesis. Exposure of primary cortical cell cultures to both compounds resulted in an increase (~25%) in the number of neurite branch points, supporting the results of the SAFE analysis.
Conclusions:
In the present study, pyrethroids induced changes in gene expression in the frontal cortex near the threshold for decreases in ambulatory motor activity in vivo. The penalized regression methods performed similarly in detecting dose-dependent changes in gene transcription. Finally, SAFE analysis of gene expression data identified branching morphogenesis as a biological process sensitive to pyrethroids and subsequent in vitro experiments confirmed this predicted effect. The novel findings regarding pyrethroid effects on branching morphogenesis indicate these compounds may act as developmental neurotoxicants that affect normal neuronal morphology.
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A salmonid EST genomic study: genes, duplications, phylogeny and microarrays
Background:
Salmonids are of interest because of their relatively recent genome duplication, and their extensive use in wild fisheries and aquaculture. A comprehensive gene list and a comparison of genes in some of the different species provide valuable genomic information for one of the most widely studied groups of fish.
Results:
298,304 expressed sequence tags (ESTs) from Atlantic salmon (69% of the total), 11,664 chinook, 10,813 sockeye, 10,051 brook trout, 10,975 grayling, 8,630 lake whitefish, and 3,624 northern pike ESTs were obtained in this study and have been deposited into the public databases. Contigs were built and putative full-length Atlantic salmon clones have been identified. A database containing ESTs, assemblies, consensus sequences, open reading frames, gene predictions and putative annotation is available. The overall similarity between Atlantic salmon ESTs and those of rainbow trout, chinook, sockeye, brook trout, grayling, lake whitefish, northern pike and rainbow smelt is 93.4, 94.2, 94.6, 94.4, 92.5, 91.7, 89.6, and 86.2% respectively. An analysis of 78 transcript sets show Salmo as a sister group to Oncorhynchus and Salvelinus within Salmoninae, and Thymallinae as a sister group to Salmoninae and Coregoninae within Salmonidae. Extensive gene duplication is consistent with a genome duplication in the common ancestor of salmonids. Using all of the available EST data, a new expanded salmonid cDNA microarray of 32,000 features was created. Cross-species hybridizations to this cDNA microarray indicate that this resource will be useful for studies of all 68 salmonid species.
Conclusion:
An extensive collection and analysis of salmonid RNA putative transcripts indicate that Pacific salmon, Atlantic salmon and charr are 94-96% similar while the more distant whitefish, grayling, pike and smelt are 93, 92, 89 and 86% similar to salmon. The salmonid transcriptome reveals a complex history of gene duplication that is consistent with an ancestral salmonid genome duplication hypothesis. Genome resources, including a new 32K microarray, provide valuable new tools to study salmonids.
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Application of Dissociation Curve Analysis to Radiation Hybrid Panel Marker Scoring: Generation of a Map of River Buffalo (B. bubalis) Chromosome 20
Background:
Fluorescence of dyes bound to double-stranded PCR products has been utilized extensively in various real-time quantitative PCR applications, including post-amplification dissociation curve analysis, or differentiation of amplicon length or sequence composition. Despite the current era of whole-genome sequencing, mapping tools such as radiation hybrid DNA panels remain useful aids for sequence assembly, focused resequencing efforts, and for building physical maps of species that have not yet been sequenced. For placement of specific, individual genes or markers on a map, low-throughput methods remain commonplace. Typically, PCR amplification of DNA from each panel cell line is followed by gel electrophoresis and scoring of each clone for the presence or absence of PCR product. To improve sensitivity and efficiency of radiation hybrid panel analysis in comparison to gel-based methods, we adapted fluorescence-based real-time PCR and dissociation curve analysis for use as a novel scoring method.
Results:
As proof of principle for this dissociation curve method, we generated new maps of river buffalo (Bubalus bubalis) chromosome 20 by both dissociation curve analysis and conventional marker scoring. We also obtained sequence data to augment dissociation curve results. Few genes have been previously mapped to buffalo chromosome 20, and sequence detail is limited, so 65 markers were screened from the orthologous chromosome of domestic cattle. Thirty bovine markers (46%) were suitable as cross-species markers for dissociation curve analysis in the buffalo radiation hybrid panel under a standard protocol, compared to 25 markers suitable for conventional typing. Computational analysis placed 27 markers on a chromosome map generated by the new method, while the gel-based approach produced only 20 mapped markers. Among 19 markers common to both maps, the marker order on the map was maintained perfectly.
Conclusions:
Dissociation curve analysis is reliable and efficient for radiation hybrid panel scoring, and is more sensitive and robust than conventional gel-based typing methods. Several markers could be scored only by the new method, and ambiguous scores were reduced. PCR-based dissociation curve analysis decreases both time and resources needed for construction of radiation hybrid panel marker maps and represents a significant improvement over gel-based methods in any species.
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Genomic characterization of putative allergen genes in peach/almond and their synteny with apple
Background:
Fruits from several species of the Rosaceae family are reported to cause allergic reactions in certain populations. The allergens identified belong to mainly four protein families: pathogenesis related 10 proteins, thaumatin-like proteins, lipid transfer proteins and profilins. These families of putative allergen genes in apple (Mal d 1 to 4) have been mapped on linkage maps and subsequent genetic study on allelic diversity and hypoallergenic traits has been carried out recently. In peach (Prunus persica), these allergen gene families are denoted as Pru p 1 to 4 and for almond (Prunus dulcis) Pru du 1 to 4. Genetic analysis using current molecular tools may be helpful to establish the cause of allergenicity differences observed among different peach cultivars. This study was to characterize putative peach allergen genes for their genomic sequences and linkage map positions, and to compare them with previously characterized homologous genes in apple (Malus domestica).
Results:
Eight Pru p/du 1 genes were identified, four of which were new. All the Pru p/du 1 genes were mapped in a single bin on the top of linkage group 1 (G1). Five Pru p/du 2 genes were mapped on four different linkage groups, two very similar Pru p/du 2.01 genes (A and B) were on G3, Pru p/du 2.02 on G7, Pru p/du 2.03 on G8 and Pru p/du 2.04 on G1. There were differences in the intron and exon structure in these Pru p/du 2 genes and in their amino acid composition. Three Pru p/du 3 genes (3.01-3.03) containing an intron and a mini exon of 10 nt were mapped in a cluster on G6. Two Pru p/du 4 genes (Pru p/du 4.01 and 4.02) were located on G1 and G7, respectively. The Pru p/du 1 cluster on G1 aligned to the Mal d 1 clusters on LG16; Pru p/du 2.01A and B on G3 to Mal d 2.01A and B on LG9; the Pru p/du 3 cluster on G6 to Mal d 3.01 on LG12; Pru p/du 4.01 on G1 to Mal d 4.03 on LG2; and Pru p/du 4.02 on G7 to Mal d 4.02 on LG2.
Conclusions:
A total of 18 putative peach/almond allergen genes have been mapped on five linkage groups. Their positions confirm the high macro-synteny between peach/almond and apple. The insight gained will help to identify key genes causing differences in allergenicity among different cultivars of peach and other Prunus species.
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Analysis of the Pythium ultimum transcriptome using Sanger and Pyrosequencing approaches
Background:
Pythium species are an agriculturally important genus of plant pathogens, yet are not understood well at the molecular, genetic, or genomic level. They are closely related to other oomycete plant pathogens such as Phytophthora species and are ubiquitous in their geographic distribution and host rage. To gain a better understanding of its gene complement, we generated Expressed Sequence Tags (ESTs) from the transcriptome of Pythium ultimum DAOM BR144 (=ATCC 200006 = CBS 805.95) using two high throughput sequencing methods, Sanger-based chain termination sequencing and pyrosequencing-based sequencing-by-synthesis.
Results:
A single half-plate pyrosequencing (454 FLX) run on adapter-ligated cDNA from a normalized cDNA library generated 90,664 reads with an average read length of 190 nucleotides following cleaning and removal of sequences shorter than 100 base pairs. After clustering and assembly, a total of 35,507 unique sequences were generated. In parallel, 9,578 reads were generated from the same cDNA library using dideoxy chain termination Sanger sequencing which upon clustering and assembly generated 4,689 unique sequences. A hybrid assembly of both Sanger- and pyrosequencing-derived ESTs resulted in 34,495 unique sequences with 1,110 sequences (3.2 %) that were solely derived from Sanger sequencing alone. A high degree of similarity was seen between P. ultimum sequences and other sequenced plant pathogenic oomycetes with 91% of the hybrid assembly derived sequences > 500 bp having similarity to sequences from plant pathogenic Phytophthora species. An analysis of Gene Ontology assignments revealed a similar representation of molecular function ontologies in the hybrid assembly in comparison to the predicted proteomes of three Phytophthora species, suggesting a broad representation of the P. ultimum transcriptome was present in the normalized cDNA population. P. ultimum sequences with similarity to oomycete RXLR and Crinkler effectors, Kazal-like and cystatin-like protease inhibitors, and elicitins were identified. Sequences with similarity to thiamine biosynthesis enzymes that are lacking in the genome sequences of three Phytophthora species and one downy mildew were identified and could serve as useful phylogenetic markers. Furthermore, we identified 179 candidate simple sequence repeats that can be used for genotyping strains of P. ultimum.
Conclusions:
Through these two technologies, we were able to generate a robust set (~10 Mb) of transcribed sequences for P. ultimum. We were able to identify known sequences present in oomycetes as well as identify novel sequences. An ample number of candidate polymorphic markers were identified in the dataset providing resources for phylogenetic and diagnostic marker development for this species. On a technical level, in spite of the depth possible with 454 FLX platform, the Sanger and pyro-based sequencing methodologies were complementary as each method generated sequences unique to each platform.
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