The impact of a His-tag on DNA binding by RNA polymerase alpha-C-terminal domain from Helicobacter pylori.

Polyhistidine tags (His-tags) are commonly employed in protein purification strategies due to the high affinity and specificity for metal-NTA columns, the relative simplicity of such protocols, and the assumption that His-tags do not affect the native activities of proteins. However, there is a growing body of evidence that such tags can modulate protein structure and function. In this study, we demonstrate that a His-tag impacts DNA complex formation by the C-terminal domain of the α-subunit (αCTD) of Helicobacter pylori RNA polymerase in a metal-dependent fashion. The αCTD was purified with a cleavable His-tag, and complex formation between αCTD, the nickel-responsive metalloregulator HpNikR, and DNA was investigated using electrophoretic mobility shift assays. An interaction between His-tagged αCTD (HisαCTD) and the HpNikR-DNA complex was observed; however, this interaction was not observed upon removal of the His-tag. Further analysis revealed that complex formation between HisαCTD and DNA is non-specific and dependent on the type of metal ions present. Overall, the results indicate that a histidine tag is able to modulate DNA-binding activity and suggests that the impact of metal affinity tags should be considered when analyzing the in vitro biomolecular interactions of metalloproteins.

Molecular identification of an insulin growth factor binding protein (IGFBP) and its potential role in an insulin-like peptide system of the pearl oyster, Pinctada fucata.

Insulin-like growth factors (IGFs) play critical roles in regulating metabolism, growth, and reproduction in invertebrates. IGF binding proteins (IGFBPs) serve as major regulators of IGF activity and regulate endocrine system. In the present study, the full-length cDNA of an igfbp was identified from the pearl oyster, Pinctada fucata, using expressed sequence tag (EST) sequence. The 1124bp Pfigfbp cDNA contains a 465bp open reading frame (ORF) encoding a putative protein of 154 amino acids, a 5'-untranslated region (UTR) of 238bp, and a 3'-UTR of 394bp (not including polyA+). Multiple sequence alignment of the deduced IB domain sequences revealed that twelve conserved Cys and ILP binding site in PfIGFBP were well aligned with human IGFBPs1-7, Mizuhopecten yessoensis IGFBP5 and Eriocheir sinensis IGFBP7. Gene expression analysis indicated that Pfigfbp mRNA was expressed in all the tissues and developmental stages examined, with a higher level in the foot than in other tissues and a higher level in the polar body stage and 32-cell stage than in the other stages. Pfigfbp and PfILP (insulin-like peptide) mRNA levels significantly increased in the digestive gland after feeding, while levels were dramatically reduced during a week of food deprivation and increased upon refeeding. In vitro experiments indicated that Pfigfbp mRNA expression in mantle cells was affected by insulin/IGFs (IGF-I, IGF-II). Our data suggests that Pfigfbp may be involved in endocrine signaling in P. fucata via the regulation of insulin-like peptide signaling.

High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG.

Adenylate kinase (AK) from Escherichia coli was used as both solubility and affinity tag for recombinant protein production. When fused to the N-terminus of a target protein, an AK fusion protein could be expressed in soluble form and purified to near homogeneity in a single step from Blue-Sepherose via affinity elution with micromolar concentration of P1, P5- di (adenosine-5') pentaphosphate (Ap5A), a transition-state substrate analog of AK. Unlike any other affinity tags, the level of a recombinant protein expression in soluble form and its yield of recovery during each purification step could be readily assessed by AK enzyme activity in near real time. Coupled to a His-Tag installed at the N-terminus and a thrombin cleavage site at the C terminus of AK, the streamlined method, here we dubbed AK-TAG, could also allow convenient expression and retrieval of a cleaved recombinant protein in high yield and purity via dual affinity purification steps. Thus AK-TAG is a new addition to the arsenal of existing affinity tags for recombinant protein expression and purification, and is particularly useful where soluble expression and high degree of purification are at stake.

Multiplex pairwise assembly of array-derived DNA oligonucleotides.

While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192-252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131-250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput 'Dial-Out PCR' protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens.

Molecular characterization and knock-down of salmon louse (Lepeophtheirus salmonis) prostaglandin E synthase.

The salmon louse (Lepeophtheirus salmonis) is a major parasite of salmonid fish in the marine environment. The interaction between the parasite and the host upon infection is not completely understood. However, it is clear that the parasite influences the host and its immune system. Prostaglandins produced by parasites such as flatworms, roundworms and ticks are documented or assumed to play a role in immunomodulation of the host. In the salmon louse, the effect of prostaglandins on the host is assumed, but remains to be documented. In this study, a salmon louse prostaglandin E2 synthase (LsPGES2) is characterized. Ontogenetic analysis showed that LsPGES2 is relatively stable expressed during development. The highest level of expression was seen in the free living stages, although elevated levels of LsPGES2 were also found in adult females. In copepodids, LsPGES2 is found around muscle cells, while it is observed in the reproductive organs of adult female lice. LsPGES2 expression was knocked-down by RNA interference in nauplii, but emerging copepodids did not display any changes in morphology nor ability to infect and develop to adult stages on fish. Additional knock-down of LsPGES2 in adult female lice did not produce any characteristic changes in phenotype nor reproductive output. It is concluded that under these experimental conditions, knock-down of LsPGES2 did not affect any essential functions of the salmon louse, neither in the free-living nor the parasitic stages.

N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes.

Comparative genomics of grass EST libraries reveals previously uncharacterized splicing events in crop plants.

BACKGROUND: Crop plants such as rice, maize and sorghum play economically-important roles as main sources of food, fuel, and animal feed. However, current genome annotations of crop plants still suffer false-positive predictions; a more comprehensive registry of alternative splicing (AS) events is also in demand. Comparative genomics of crop plants is largely unexplored. RESULTS: We performed a large-scale comparative analysis (ExonFinder) of the expressed sequence tag (EST) library from nine grass plants against three crop genomes (rice, maize, and sorghum) and identified 2,879 previously-unannotated exons (i.e., novel exons) in the three crops. We validated 81% of the tested exons by RT-PCR-sequencing, supporting the effectiveness of our in silico strategy. Evolutionary analysis reveals that the novel exons, comparing with their flanking annotated ones, are generally under weaker selection pressure at the protein level, but under stronger pressure at the RNA level, suggesting that most of the novel exons also represent novel alternatively spliced variants (ASVs). However, we also observed the consistency of evolutionary rates between certain novel exons and their flanking exons, which provided further evidence of their co-occurrence in the transcripts, suggesting that previously-annotated isoforms might be subject to erroneous predictions. Our validation showed that 54% of the tested genes expressed the newly-identified isoforms that contained the novel exons, rather than the previously-annotated isoforms that excluded them. The consistent results were steadily observed across cultivated (Oryza sativa and O. glaberrima) and wild (O. rufipogon and O. nivara) rice species, asserting the necessity of our curation of the crop genome annotations. Our comparative analyses also inferred the common ancestral transcriptome of grass plants and gain- and loss-of-ASV events. CONCLUSIONS: We have reannotated the rice, maize, and sorghum genomes, and showed that evolutionary rates might serve as an indicator for determining whether the identified exons were alternatively spliced. This study not only presents an effective in silico strategy for the improvement of plant annotations, but also provides further insights into the role of AS events in the evolution and domestication of crop plants. ExonFinder and the novel exons/ASVs identified are publicly accessible at http://exonfinder.sourceforge.net/ .

New insights into the wheat chromosome 4D structure and virtual gene order, revealed by survey pyrosequencing.

Survey sequencing of the bread wheat (Triticum aestivum L.) genome (AABBDD) has been approached through different strategies delivering important information. However, the current wheat sequence knowledge is not complete. The aim of our study is to provide different and complementary set of data for chromosome 4D. A survey sequence was obtained by pyrosequencing of flow-sorted 4DS (7.2×) and 4DL (4.1×) arms. Single ends (SE) and long mate pairs (LMP) reads were assembled into contigs (223Mb) and scaffolds (65Mb) that were aligned to Aegilops tauschii draft genome (DD), anchoring 34Mb to chromosome 4. Scaffolds annotation rendered 822 gene models. A virtual gene order comprising 1973 wheat orthologous gene loci and 381 wheat gene models was built. This order was largely consistent with the scaffold order determined based on a published high density map from the Ae. tauschii chromosome 4, using bin-mapped 4D ESTs as a common reference. The virtual order showed a higher collinearity with homeologous 4B compared to 4A. Additionally, a virtual map was constructed and ∼5700 genes (∼2200 on 4DS and ∼3500 on 4DL) predicted. The sequence and virtual order obtained here using the 454 platform were compared with the Illumina one used by the IWGSC, giving complementary information.

Sugarcane giant borer transcriptome analysis and identification of genes related to digestion.

Sugarcane is a widely cultivated plant that serves primarily as a source of sugar and ethanol. Its annual yield can be significantly reduced by the action of several insect pests including the sugarcane giant borer (Telchin licus licus), a lepidopteran that presents a long life cycle and which efforts to control it using pesticides have been inefficient. Although its economical relevance, only a few DNA sequences are available for this species in the GenBank. Pyrosequencing technology was used to investigate the transcriptome of several developmental stages of the insect. To maximize transcript diversity, a pool of total RNA was extracted from whole body insects and used to construct a normalized cDNA database. Sequencing produced over 650,000 reads, which were de novo assembled to generate a reference library of 23,824 contigs. After quality score and annotation, 43% of the contigs had at least one BLAST hit against the NCBI non-redundant database, and 40% showed similarities with the lepidopteran Bombyx mori. In a further analysis, we conducted a comparison with Manduca sexta midgut sequences to identify transcripts of genes involved in digestion. Of these transcripts, many presented an expansion or depletion in gene number, compared to B. mori genome. From the sugarcane giant borer (SGB) transcriptome, a number of aminopeptidase N (APN) cDNAs were characterized based on homology to those reported as Cry toxin receptors. This is the first report that provides a large-scale EST database for the species. Transcriptome analysis will certainly be useful to identify novel developmental genes, to better understand the insect's biology and to guide the development of new strategies for insect-pest control.

Analysis of expressed sequence tags (ESTs) from a normalized cDNA library and isolation of EST simple sequence repeats from the invasive cotton mealybug Phenacoccus solenopsis.

The cotton mealybug, Phenacoccus solenopsis Tinsley, is a serious and invasive pest. At present, genetic resources for studying P. solenopsis are limited, and this negatively affects genetic research on the organism and, consequently, translational work to improve management of this pest. In the present study, expressed sequence tags (ESTs) were analyzed from a normalized complementary DNA library of P. solenopsis. In addition, EST-derived microsatellite loci (also known as simple sequence repeats or SSRs) were isolated and characterized. A total of 1107 high-quality ESTs were acquired from the library. Clustering and assembly analysis resulted in 785 unigenes, which were classified functionally into 23 categories according to the Gene Ontology database. Seven EST-based SSR markers were developed in this study and are expected to be useful in characterizing how this invasive species was introduced, as well as providing insights into its genetic microevolution.

Antennal uridine diphosphate (UDP)-glycosyltransferases in a pest insect: diversity and putative function in odorant and xenobiotics clearance.

Uridine diphosphate UDP-glycosyltransferases (UGTs) are detoxification enzymes widely distributed within living organisms. They are involved in the biotransformation of various lipophilic endogenous compounds and xenobiotics, including odorants. Several UGTs have been reported in the olfactory organs of mammals and involved in olfactory processing and detoxification within the olfactory mucosa but, in insects, this enzyme family is still poorly studied. Despite recent transcriptomic analyses, the diversity of antennal UGTs in insects has not been investigated. To date, only three UGT cDNAs have been shown to be expressed in insect olfactory organs. In the present study, we report the identification of eleven putative UGTs expressed in the antennae of the model pest insect Spodoptera littoralis. Phylogenetic analysis revealed that these UGTs belong to five different families, highlighting their structural diversity. In addition, two genes, UGT40R3 and UGT46A6, were either specifically expressed or overexpressed in the antennae, suggesting specific roles in this sensory organ. Exposure of male moths to the sex pheromone and to a plant odorant differentially downregulated the transcription levels of these two genes, revealing for the first time the regulation of insect UGTs by odorant exposure. Moreover, the specific antennal gene UGT46A6 was upregulated by insecticide topical application on antennae, suggesting its role in the protection of the olfactory organ towards xenobiotics. This work highlights the structural and functional diversity of UGTs within this highly specialized tissue.

Characterization of EST-SSR markers in durum wheat EST library and functional analysis of SSR-containing EST fragments.

The goal of this study is to identify characterization of expressed sequence tag (EST)-simple sequence repeats (SSR) markers from EST library of durum wheat and functional analysis of SSR-containing EST sequences for application in comparative genomics and breeding. 19,141 sequences were analyzed among which 18,937 ESTs were selected. Consistent with MISA results, 313 EST-SSRs were yielded. The final EST-SSRs were compared to the GenBank non-redundant database using BLASTX and classified based on these functions. Results indicated that the perfect EST-SSRs are the most frequent. The TTG/CTG imperfect EST-SSR had gamma-gliadin putative function that can be appropriate for durum wheat. Also, the mononucleotides and trinucleotides were the most frequent. Findings suggested that the identified EST-SSRs could be categorized into 83 types. Motifs TTG in trinucleotides and TC in dinucleotides had the highest frequency. TTG is the new motif in durum wheat identified in this study. We identified new EST-SSRs with more than trinucleotide and detected motifs that have potential to code amino acids. Arginine was the most frequent amino acid. Enzymes had the highest frequency among predicted functions. EST-SSRs have been identified in this study can be used for developing ESS-SSR-based detection tool for durum wheat in future studies and will be a useful resource for molecular breeding, genetics, genomics, and environmental stress studies. Motifs coding amino acids could be used as a new source of functional markers and biological study. In addition to, designed new PCR primer pairs are new resources for to identify useful alleles in transcription factors, storage proteins, and enzymes which incorporated them again into the cultivated material.

Characterization of conserved microRNAs from five different cucurbit species using computational and experimental analysis.

MicroRNAs (miRNAs) are ∼21 nt non-coding small RNAs which regulate gene expression at the post-transcriptional level in plants and animals. Until recently, only limited numbers of miRNAs were identified in Cucurbitaceae, a large flowering plant family. In this study, 220 potential miRNA candidates were identified from five species of Cucurbitaceae family using a comparative genome-based computational analysis. A comprehensive bioinformatic analysis of EST (expressed sequence tag) and GSS (genomic survey sequence) data of five cucurbit species showed that at least 41, 108, 21, 17 and 33 miRNAs existed in Cucumis sativus, Cucumis melo, Citrullus lanatus, Siraitia grosvenorii and Cucurbita pepo, respectively. Quantitative real-time PCR (qRT-PCR) analysis revealed the differentially expression levels of miRNAs in the four tissues of cucumber and melon. These identified miRNAs in the five species potentially targeted 578 protein-coding genes and one target of the C. melo miRNA cme-miR160a-5p was verified by 5' RLM-RACE. GO and KEGG analysis suggested that many melon miRNAs might involve in nucleotide metabolism, oxidative phosphorylation, cell redox homeostasis and signal transduction.

Possible formation of mitochondrial-RNA containing chimeric or trimeric RNA implies a post-transcriptional and post-splicing mechanism for RNA fusion.

Human cells are known to express many chimeric RNAs, i.e. RNAs containing two genes' sequences. Wondering whether there also is trimeric RNA, i.e. an RNA containing three genes' sequences, we wrote simple computer code to screen human expression sequence tags (ESTs) deposited in different public databases, and obtained hundreds of putative trimeric ESTs. We then used NCBI Blast and UCSC Blat browsers to further analyze their sequences, and identified 61 trimeric and two tetrameric ESTs (one EST containing four different sequences). We also identified 57 chimeric, trimeric or teterameric ESTs that contained both mitochondrial (mt) RNA and nuclear RNA (nRNA), i.e. were mtRNA-nRNA fusions. In some trimeric ESTs, the downstream partner was fused to the poly-A tail of the upstream partner, which, together with the mtRNA-nRNA fusions, suggests a possible new mechanism for RNA fusion that occurs after both transcription and splicing have been terminated, and possibly outside the nucleus, in contrast to the two current hypothetical mechanisms, trans-splicing and transcriptional-slippage, that occur in the nucleus. The mt-sequences in the mtRNA-nRNA fusions had pseudogenes in the nucleus but, surprisingly, localized mainly in chromosomes 1 and 5. In some mtRNA-nRNA fusions, as well as in some ESTs that were derived only from mtRNA, the mt-sequences might be cis- or trans-spliced. Actually, we cloned a new cis-spliced mtRNA, coined as 16SrRNA-s. Hence, mtDNA may not always be intron-less. Fusion of three or more RNAs to one, fusion of nRNA to mtRNA, and cis- or trans-splicing of mtRNA should all enlarge the cellular RNA repertoire, in turn enlarging the cellular functions. Therefore, future experimental verification of the existence of these novel classes of fusion RNAs and spliced mtRNAs in human cells should significantly advance our understanding of biology and medicine.

Adult Opisthorchis felineus major protein fractions deduced from transcripts: comparison with liver flukes Opisthorchis viverrini and Clonorchis sinensis.

The epidemiologically important liver flukes Opisthorchis felineus, Opisthorchis viverrini, and Clonorchis sinensis are of interest to health professionals, epidemiologists, pharmacologists, and molecular biologists. Recently the transcriptomes of the latter two species were intensively investigated. However our knowledge on molecular biology of O. felineus is scarce. We report the first results of the O. felineus transcriptome analysis. We isolated and annotated a total of 2560 expressed sequence tag (EST) sequences from adult O. felineus (deposited within the database of expressed sequence tags (dbEST), under accession numbers GenBank: JK624271-JK626790, JK006511-JK006547, JK649790-JK649792). Clustering and analysis resulted in the detection of 267 contigs. Of the protein sequences deduced from these, 82% had homologs in the NCBI (nr) protein database and 63% contained conserved domains, allowing the functions to be interpreted using the Gene Ontology terms. Comprehensive analysis of Opisthorchiidae- and Trematoda-specific substitutions within amino acid sequences deduced for the proteins myoglobin, vitelline precursor protein, cathepsin F, and 28kDa glutathione transferase was carried out. The gene set of the 32 ribosomal proteins for the three Opisthorchiidae species with the addition of available Schistosoma and Fasciola orthologs was created and is provided in the supplementary. The orthologous gene set created was used for inferring phylogeny within the Trematoda with special attention to interrelations within the Opisthorchiidae. The phylogenetic analysis revealed a closer relationship between C. sinensis and O. viverrini and some divergence of O. felineus from either O. viverrini or C. sinensis.

Combining gene expression and genetic analyses to identify candidate genes involved in cold responses in pea.

Cold stress affects plant growth and development. In order to better understand the responses to cold (chilling or freezing tolerance), we used two contrasted pea lines. Following a chilling period, the Champagne line becomes tolerant to frost whereas the Terese line remains sensitive. Four suppression subtractive hybridisation libraries were obtained using mRNAs isolated from pea genotypes Champagne and Terese. Using quantitative polymerase chain reaction (qPCR) performed on 159 genes, 43 and 54 genes were identified as differentially expressed at the initial time point and during the time course study, respectively. Molecular markers were developed from the differentially expressed genes and were genotyped on a population of 164 RILs derived from a cross between Champagne and Terese. We identified 5 candidate genes colocalizing with 3 different frost damage quantitative trait loci (QTL) intervals and a protein quantity locus (PQL) rich region previously reported. This investigation revealed the role of constitutive differences between both genotypes in the cold responses, in particular with genes related to glycine degradation pathway that could confer to Champagne a better frost tolerance. We showed that freezing tolerance involves a decrease of expression of genes related to photosynthesis and the expression of a gene involved in the production of cysteine and methionine that could act as cryoprotectant molecules. Although it remains to be confirmed, this study could also reveal the involvement of the jasmonate pathway in the cold responses, since we observed that two genes related to this pathway were mapped in a frost damage QTL interval and in a PQL rich region interval, respectively.

Gene expression analysis by ESTs sequencing of the Brazilian frog Phyllomedusa nordestina skin glands.

The subfamily Phyllomedusinae has attracted a great interest of many researchers mainly due to the high diversity of these frog species and plethora of pharmacological activities frequently observed for their skin secretions. Despite of this fact, mainly for new species, limited information is available regarding the molecular composition of these skin secretions and the cellular components involved in their production. Phyllomedusa nordestina is a recently described Brazilian frog species also popularly known as 'tree-frogs'. Aiming at contributing to the biological knowledge of this species, we show here the gene expression profile of this frog skin secretion using a global ESTs analysis of a cDNA library. The marked aspect of this analysis revealed a significant higher transcriptional level of the opioid peptide dermorphins in P. nordestina skin secretion than in Phyllomedusa hypochondrialis, which is its closest related species, belonging both to the same phylogenetic group. Precursors of bioactive peptides as dermaseptins, phylloseptins, tryptophyllins, and bradykinin-like peptideswere also found in this library. Transcripts encoding proteins related to ordinary cellular functions and pathways were also described. Some of them are chiefly involved in the production of the skin secretion. Taken together, the data reported here constitute a contribution to the characterization of the molecular diversity of gene-encoded polypeptides with potential possibility of pharmacological exploitation. The transcriptional composition of the skin secretion may also help to give the necessary support for the definition of P. nordestina as a new species, which actually relies basically on frog morphological characteristics and geographical distribution.

Assessment of technical protocols for novel embryonic stem cell tests with molecular markers (Hand1- and Cmya1-ESTs): a preliminary cross-laboratory performance analysis.

The Hand1- and Cmya1-ESTs are novel short-term tests for embryotoxic chemicals using genetically engineering mouse ES cells for luciferase reporter gene assays. These ESTs allow convenient determination of differentiation toxicity and cell viability in a short duration with high throughput 96-well microplates for prediction of embryotoxicity of chemicals. To assess the Hand1-EST technical protocol, we firstly compared reporter gene assay and cytotoxicity test data for a representative compound (hydroxyurea) from four different laboratories with tests carried out under the same experimental conditions. Extensive investigations of the Hand1- and Cmya1-ESTs were then performed to explore reproducibility by comparing a set of 6 well-known test chemicals, including hydroxyurea, across the laboratories. The results gave good correspondence in all four laboratories, indicating that transferability, intra-laboratory variability and inter-laboratory variability of the present technical protocols of the ESTs were sufficient to conduct further validation studies.

Insect phylogenomics: results, problems and the impact of matrix composition.

In this study, we investigated the relationships among insect orders with a main focus on Polyneoptera (lower Neoptera: roaches, mantids, earwigs, grasshoppers, etc.), and Paraneoptera (thrips, lice, bugs in the wide sense). The relationships between and within these groups of insects are difficult to resolve because only few informative molecular and morphological characters are available. Here, we provide the first phylogenomic expressed sequence tags data ('EST': short sub-sequences from a c(opy) DNA sequence encoding for proteins) for stick insects (Phasmatodea) and webspinners (Embioptera) to complete published EST data. As recent EST datasets are characterized by a heterogeneous distribution of available genes across taxa, we use different rationales to optimize the data matrix composition. Our results suggest a monophyletic origin of Polyneoptera and Eumetabola (Paraneoptera + Holometabola). However, we identified artefacts of tree reconstruction (human louse Pediculus humanus assigned to Odonata (damselflies and dragonflies) or Holometabola (insects with a complete metamorphosis); mayfly genus Baetis nested within Neoptera), which were most probably rooted in a data matrix composition bias due to the inclusion of sequence data of entire proteomes. Until entire proteomes are available for each species in phylogenomic analyses, this potential pitfall should be carefully considered.

Molecular cloning of large alternative transcripts based on comparative phylogenetic analysis and exploration of an EST database.

In animals, a gene may be 50 kb or over and contain multiple alternative transcripts with sequences that are not experimentally validated. Under these special circumstances, PCR-based cloning may become very difficult. Here a simple cloning strategy is described using the mNLRC5 gene as an example. We performed comparative phylogenetic analysis between murine and human NLR protein families to anchor the translation start codon, searched an EST database with the 3' end of the genomic DNA sequence to obtain ESTs from the farthest 3' end of the gene, and isolated the full-length CDS of the mNLRC5 of about 6 kb through conventional RT-PCR and 3' RACE.