Length Of Open Reading Frame Research Articles

Molecular and genetic characterization of peste des petits ruminants virus Kurdistan 2011 strain based on the haemagglutinin and fusion protein genes sequences

Severe outbreaks of peste des petits ruminants virus (PPRV) occurred during 2011 in wild goats in the Kurdistan region in Iraq. Based on a partial sequence of the nucleoprotein (N) gene the causative virus, named PPRV Kurdistan 2011, was classified as PPRV lineage IV genotype. For further characterization, the open reading frames (ORFs) coding for the haemagglutinin (H) and fusion (F) protein of PPRV Kurdistan 2011 were amplified from infected-cell RNA by RT-PCR. Sequencing of the ORFs revealed sizes of 1830 nucleotides (nt) for the H-ORF and 1641 nt for the F-ORF. This study represents the first report describing the full length ORFs of H and F for a PPRV strain from Kurdistan, Iraq.Comparative sequence analysis performed for the entire H- and F- ORFs of Kurdistan 2011 with respective sequences of PPRV isolates showed the highest homology with PPRV lineage IV isolates which is also reflected by comparative amino acid analysis. Phylogenetic analysis performed based on the partial and complete nucleotide sequences of both H- and F-ORFs clustered Kurdistan 2011 within PPRV lineage IV genotype. H-gene based phylogenetic trees aligned Kurdistan 2011 strain along with Turkey/TR/AFYON/2011-35H and Turkey/2000 strains whereas F-gene based phylogenetic trees aligned Kurdistan 2011 strain along with Qatar/37/10-02, Kurdistan/2012 and Egypt/2009 strains.

Systematic Analysis of Long Non-Coding RNAs and mRNAs in the Ovaries of Duroc Pigs During Different Follicular Stages Using RNA Sequencing.

The dynamic process involving the selection and maturation of follicles is regulated and controlled by a highly synchronized and exquisitely timed cascade of gene expression. Studies have shown that long non-coding RNA (lncRNA) is essential for the normal maintenance of animal reproductive function and has an important regulatory function in ovarian development and hormone secretion. In this study, a total of 2076 lncRNAs (1362 known lncRNAs and 714 new lncRNAs) and 25,491 mRNAs were identified in libraries constructed from Duroc ovaries on days 0, 2 and 4 of follicle development. lncRNAs were shorter, had fewer exons, exhibited a shorter ORF (Open Reading Frame) length and lower expression levels, and were less conserved than mRNAs. Furthermore, 1694 transcripts (140 lncRNAs and 1554 mRNAs) were found to be differentially expressed in pairwise comparisons. A total of 6945 co-localized mRNAs were detected in cis in 2076 lncRNAs. The most enriched GO (Gene Ontology) terms were related to developmental processes. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis revealed that the differentially expressed lncRNAs targeted mRNAs, and the differentially expressed mRNAs were related to the TGF-β signaling pathway, the PI3K-Akt signaling pathway, the Retinol metabolic pathway and the Wnt signaling pathway. This study deepened our understanding of the genetic basis and molecular mechanisms of follicular development in pigs.

Cloning and characterization of two glutathione S-transferases cDNAs in Foc4 and expression under exogenous oxidative stress

In order to identify two putative glutathione S-transferase (GSTs) genes in Fusarium oxysporum f. sp. cubense race 4 (Foc4), cDNA sequences of the entire coding regions of the two genes were cloned from Foc4 using RT-PCR method. Subsequently, the two genes were named Fogst1 and Fogst2 respectively. The length of open reading frame of Fogst1 was 609 bp and encoded a protein including 202 amino acid residues, Fogst2 possessed an open reading frame with 693 bp which encoded a 230-amino acid protein. Phylogenetic analysis showed that Fogst1 belonged to sigma (σ) subtype members of the GSTs superfamily, and Fogst2 was a new member of an unknown subfamily in the GSTs superfamily. To verify the expression of Fogst1 and Fogst2, the recombinant prokaryotic expression vector pET28a-Fogst1 and pET28a-Fogst2 were constructed and transformed into Escherichia coli expression strain BL21(DE3). The soluble recombinant proteins Fogst1 and Fogst2 were obtained after being induced by IPTG. GSTs activity assays showed that both of the two recombinant proteins had specific activity with CDNB. For real time RT- PCR analysis, the mycelium samples of Foc4 were collected after treatment by H₂O₂ for 1, 5, 12, 24 hours. The results showed that the expression of Fogst1 and Fogst2 were significantly up-regulated in the first 5 hours, and then decreased and returned to normal level. These results suggested that Fogst1 and Fogst2 may be involved in the process of Foc4 resistance to exogenous oxidative stress.

沙田柚谷胱甘肽转移酶基因(<i>CmGST</i>)的生物信息学及表达分析

利用高通量测序对沙田柚自交和异交花柱进行转录组测序，通过差异分析得到了沙田柚谷胱甘肽转移酶基因(CmGST)序列。CmGST全长为872 bp (GenBank登录号为KY612456)，开放阅读框为657 bp，编码219个氨基酸，推测蛋白质分子量为25.00 kDa，理论等电点pI为5.48。利用生物信息分析软件对CmGST基因进行同源性比对和系统进化分析表，结果显示该基因编码的氨基酸具有谷胱甘肽S转移酶典型的C端与 N端双结构域，属于GST Tau家族蛋白；与克莱门柚(Citrus maxima)的相似度约为98%。这些分析结果可为今后深入研究该蛋白的结构特征和功能提供参考。 In this paper, the transcriptome of the self-pollinated style and cross-pollinated style of Citrus maxima cv. Shatian Yu were sequences by high-throughput sequencing technology. The glutathione S-transferase gene (CmGST) sequence of Citrus maxima cv. Shatian Yu was obtained through differential analysis method. The fu11 length of CmGST gene was 872 bp (GenBank accession number: KY612456), and the ORF (Open Reading Frame) length was 657 bp, encoding a protein polypeptide of 219 amino acids with a predicted molecular weight of 25.00 kDa and pI of 5.48. Bioinformatics analysis showed that the CmGST protein contained the Tau GST-specific N-terminal domain (G site) and the C-terminal domain (H site). The homology analysis of amino acid sequence indicated that the glutathione S-transferase protein shared high homology with glutathione S- transferase protein of Citrus clementina (98%). This work provides a systemic sequence analysis of the glutathione S-transferase protein of Citrus maxima cv. Shatian Yu. It will provide the useful reference for further investigation of its structure and function.

Long noncoding RNAs in skin diseases

Long noncoding RNAs (LncRNAs) , a group of endogenous RNA molecules longer than 200 nucleotides in length, lack complete and specific open reading frames and have little or no protein-coding capability. They can regulate gene expressions at epigenetic, transcriptional and post-transcriptional levels, and are extensively involved in physiological and pathological processes in the body. Recent researches have indicated that LncRNAs play important roles in occurrence and development of many skin diseases, such as melanoma, non-melanoma skin cancers, psoriasis, and so on. Studies on LncRNAs may provide valuable information for prediction, diagnosis, treatment and assessment of prognosis of these diseases. Key words: Skin diseases; Psoriasis; Gene expression regulation; Biological processes; LncRNA

Cloning of the ω-secalin gene family in a wheat 1BL/1RS translocation line using BAC clone sequencing

Background: Wheat 1BL/1RS translocation lines are planted around the world for their disease resistance and high yield. Most of them are poor in bread making, which is partially caused by ω-secalins that are encoded by the ω-secalin gene family, which is located on the short arm of rye chromosome 1R (1RS). However, information on the structure and evolution of the ω-secalin gene family is still limited. Results: We first generated a physical map of the ω-secalin gene family covering 195 kb of the Sec- 1 locus based on sequencing three bacterial artificial chromosome (BAC) clones of the 1BL/1RS translocation wheat cultivar Shimai 15. A BAC contig was constructed spanning 168 kb of the Sec -1 locus on 1RS. Twelve ω-secalin genes were arranged in a head-to-tail fashion, separated by 8.2-21.6 kb spacers on the contig, whereas six other ω-secalin genes were arranged head-to-tail, separated by 8.2-8.4 kb of spacers on clone BAC125. The 18 ω-secalin genes can be classified into six types among which eight ω-secalin genes were expressed during seed development. The ω-secalin genes with the 1,074-bp open reading frame (ORF) represented the main population. Except for two pseudogenes, the N-terminal of the ω-secalin gene was conserved, whereas variations in the C-terminal led to a change in ORF length. The spacers can be sorted into two classes. Class-1 spacers contained conserved and non-conservative sequences. Conclusion: The ω-secalin gene family consisted of at least 18 members in the 1BL/1RS translocation line cv. Shimai 15. Eight ω-secalin genes were expressed during seed development. Eighteen members may originate from a progenitor with a 1,074-bp ORF. The spacers differed in length and sequence conservation.

Trans-spliced Cas9 allows cleavage of HBB and CCR5 genes in human cells using compact expression cassettes.

CRISPR/Cas9 systems have been used in a wide variety of biological studies; however, the large size of CRISPR/Cas9 presents challenges in packaging it within adeno-associated viruses (AAVs) for clinical applications. We identified a two-cassette system expressing pieces of the S. pyogenes Cas9 (SpCas9) protein which splice together in cellula to form a functional protein capable of site-specific DNA cleavage. With specific CRISPR guide strands, we demonstrated the efficacy of this system in cleaving the HBB and CCR5 genes in human HEK-293T cells as a single Cas9 and as a pair of Cas9 nickases. The trans-spliced SpCas9 (tsSpCas9) displayed ~35% of the nuclease activity compared with the wild-type SpCas9 (wtSpCas9) at standard transfection doses, but had substantially decreased activity at lower dosing levels. The greatly reduced open reading frame length of the tsSpCas9 relative to wtSpCas9 potentially allows for more complex and longer genetic elements to be packaged into an AAV vector including tissue-specific promoters, multiplexed guide RNA expression, and effector domain fusions to SpCas9. For unknown reasons, the tsSpCas9 system did not work in all cell types tested. The use of protein trans-splicing may help facilitate exciting new avenues of research and therapeutic applications through AAV-based delivery of CRISPR/Cas9 systems.

Genome-wide identification and comparative analysis of EPSPS (aroA) genes in different plant species

Shikimate pathway produces aromatic amino acids such as phenylalanine, tyrosine and tryptophan, which is essential for plant life. EPSPS (5-enolpyruvylshikimate-3-phosphate synthase, EC 2.5.1.1.9) (aroA) that observed in plants and bacteria plays an important role in shikimate pathway as being the sixth crucial enzyme. In this study, genome-wide comparative analyses of EPSPS genes were performed in different plant species, including Glycine max, Medicago truncatula, Brachypodium disctahyon, Zea mays, Chlamydomonas reinhardtii (green alga), and Physcomitrella patens (moss). One EPSPS gene was identified in M. truncatula, B. distachyon, Z. mays, and C. reinhardtii while two EPSPS genes identified in G. max and P. patens. Based on nucleotide diversity analyses of EPSPS genes, nucleotide diversity was found to be π: 0.29 and θ: 0.34, respectively. Gene structure analysis revealed that 6 of 8 EPSPS genes (75 %) contained seven introns while the length of open reading frame (ORF) ranged from 1173 to 1611 bp. Two highly conserved motifs (LPGSKSLSNRILLLAAL and LFLGNAGTAMRPL) were detected in all plant species. All putative EPSPSs were predicted to be localized in chloroplast while 7 of 8 EPSPSs (87.5 %) contained N-glycosylation sites except for Chlamydomonas. Phylogenetic analyses showed that monocots, dicots, and moss were clustered in group A whereas green alga (C. reinhardtii) was located alone in group B. 3D structure analyses indicated that EPSP synthases contained N-terminal and C-terminal domains while electrostatic potential of EPSPSs were found to exhibit various patterns. In addition, ligand molecule glyphosate was docked with EPSPS of B. disctahyon and G. max. The result showed that there were strong favorable bonds between the proteins and the ligand with favorable conformation.

Reproducibility and reliability assays of the gene expression-measurements.

BackgroundReliability and reproducibility are key metrics for gene expression assays. This report assesses the utility of the correlation coefficient in the analysis of reproducibility and reliability of gene expression data.ResultsThe correlation coefficient alone is not sufficient to assess equality among sample replicates but when coupled with slope and scatter plots expression data equality can be better assessed. Narrow-intervals of scatter plots should be shown as a tool to inspect the actual level of noise within the data. Here we propose a method to examine expression data reproducibility, which is based on the ratios of both the means and the standard deviations for the inter-treatment expression ratios of genes. In addition, we introduce a fold-change threshold with an inter-replicate occurrence likelihood lower than 5% to perform analysis even when reproducibility is not acceptable. There is no possibility to find a perfect correlation between transcript and protein levels even when there is not any post-transcriptional regulatory mechanism. We therefore propose an adjustment for protein abundance with that of transcript abundance based on open reading frame length.ConclusionsHere, we introduce a very efficient reproducibility approach. Our method detects very small changes in large datasets which was not possible through regular correlation analysis. We also introduce a correction on protein quantities which allows us to examine the post-transcriptional regulatory effects with a higher accuracy.Electronic supplementary materialThe online version of this article (doi:10.1186/2241-5793-21-3) contains supplementary material, which is available to authorized users.

Long Open Reading Frame Transcripts Escape Nonsense-Mediated mRNA Decay in Yeast

Nonsense-mediated mRNA decay (NMD) destabilizes eukaryotic transcripts with long 3' UTRs. To investigate whether other transcript features affect NMD, we generated yeast strains expressing chromosomal-derived mRNAs with 979 different promoter and open reading frame (ORF) regions and with the same long, destabilizing 3' UTR. We developed a barcode-based DNA microarray strategy to compare the levels of each reporter mRNA in strains with or without active NMD. The size of the coding region had a significant negative effect on NMD efficiency. This effect was not specific to the tested 3' UTR because two other different NMD reporters became less sensitive to NMD when ORF length was increased. Inefficient NMD was not due to a lack of association of Upf1 to long ORF transcripts. In conclusion, in addition to a long 3' UTR, short translation length is an important feature of NMD substrates in yeast.

Long non-coding RNAs: Insights into the biological property

Long non-coding RNAs (lncRNAs) draw a widespread attention on their study because of their specific properties and complex functions. Here we analyze the biological properties of lncRNAs, describing their identification, properties of their data and their roles in cells, and the key problems in the study of lncRNAs are brought forward. First, the methods applied into the identification of lncRNAs are analysed and compared carefully according to the two aspects, including the biological technology and data used to construct transcripts, and the computational methods to predict the coding potential of a transcript. The databases of lncRNAs are also listed. Then the biological properties of lncRNAs are analyzed and discussed, including the length of Open Reading Frame (ORF), codon usage bias and codon substitution rate in their nucleotide sequences, and our study on their secondary structure is also discussed. Next, specific functions of lncRNAs are also described and analyzed. Finally, the key problems in the study of lncRNAs are brought forward and the progress in their future study is also indicated.

Cloning, expression of the major capsid protein gene from marine algae Emiliania huxleyi virus and the possible use in detection of virus infection

Here we described the cloning, bioinformatic characterization and expression in Escherichia coli of the major capsid protein (MCP) from marine unicellular algae Emiliania huxleyi virus EhV-99B1 isolate. The purified recombinant MCP was used to develop a polyclonal antibody for testing viral infection. The full length open-reading frame (ORF) of MCP encodes a protein of 496 amino acids with a calculated molecular mass of 55 kDa and Ip 6.34. Hydropathy analysis of MCP showed that there were 6 largely hydrophobic domains, which may be important for the interaction with the envelope protein. The conserved region of EhV strains MCP had high similarity in amino acid sequence and secondary structure which allow us to develop a specific biomarker for EhVs infection detection. The full length ORF was subcloned into expression vector pGEX-4T-3 for overexpression in E. coli as glutathione-Stransferase-L1 (GST-L1) fusion protein and the soluble recombinant protein was used to generate polyclonal antibodies in mice. The obtained antisera reacted in Western immunoblots with the same protein both in purified EhV-99B1 virions and infected host cells sample. These shows that the antiserum against recombinant EhV-MCP offers the potential to develop immunofluorescence techniques for the detection of EhVs infected cells.

Isolation and phylogenetic analysis of novel γ-gliadin genes in genus Dasypyrum

As the most ancient member of the wheat gluten family, the γ-gliadin genes are suitable for phylogenetic analysis among wheat and related species. Species in the grass genus Dasypyrum have been widely used for wheat cross breeding. However, the genomic relationships among Dasypyrum species have been little studied. We isolated 22 novel γ-gliadin gene sequences, among which 10 are putatively functional. The open reading frame lengths of these sequences range from 642 to 933 bp, and these putative proteins consist of five domains. Phylogenetic analyses showed that all Dasypyrum γ-gliadin gene sequences clustered in a large group; D. villosum and tetraploid D. breviaristatum γ-gliadin gene sequences clustered in a subgroup, while diploid D. breviaristatum γ-gliadin gene sequences clustered at the edge of the subgroup. All of the Dasypyrum γ-gliadin gene sequences were absent in three major T cell-stimulatory epitopes binding to HLA-DQ2/8 in celiac disease patients. Based on the phylogenetic analyses, we suggest that D. villosum and tetraploid D. breviaristatum evolved in parallel from a diploid ancestor D. breviaristatum.

Phylogenetic patterns of emergence of new genes support a model of frequent de novo evolution

BackgroundNew gene emergence is so far assumed to be mostly driven by duplication and divergence of existing genes. The possibility that entirely new genes could emerge out of the non-coding genomic background was long thought to be almost negligible. With the increasing availability of fully sequenced genomes across broad scales of phylogeny, it has become possible to systematically study the origin of new genes over time and thus revisit this question.ResultsWe have used phylostratigraphy to assess trends of gene evolution across successive phylogenetic phases, using mostly the well-annotated mouse genome as a reference. We find several significant general trends and confirm them for three other vertebrate genomes (humans, zebrafish and stickleback). Younger genes are shorter, both with respect to gene length, as well as to open reading frame length. They contain also fewer exons and have fewer recognizable domains. Average exon length, on the other hand, does not change much over time. Only the most recently evolved genes have longer exons and they are often associated with active promotor regions, i.e. are part of bidirectional promotors. We have also revisited the possibility that de novo evolution of genes could occur even within existing genes, by making use of an alternative reading frame (overprinting). We find several cases among the annotated Ensembl ORFs, where the new reading frame has emerged at a higher phylostratigraphic level than the original one. We discuss some of these overprinted genes, which include also the Hoxa9 gene where an alternative reading frame covering the homeobox has emerged within the lineage leading to rodents and primates (Euarchontoglires).ConclusionsWe suggest that the overall trends of gene emergence are more compatible with a de novo evolution model for orphan genes than a general duplication-divergence model. Hence de novo evolution of genes appears to have occurred continuously throughout evolutionary time and should therefore be considered as a general mechanism for the emergence of new gene functions.

Pinstripe: a suite of programs for integrating transcriptomic and proteomic datasets identifies novel proteins and improves differentiation of protein-coding and non-coding genes

Comparing transcriptomic data with proteomic data to identify protein-coding sequences is a long-standing challenge in molecular biology, one that is exacerbated by the increasing size of high-throughput datasets. To address this challenge, and thereby to improve the quality of genome annotation and understanding of genome biology, we have developed an integrated suite of programs, called Pinstripe. We demonstrate its application, utility and discovery power using transcriptomic and proteomic data from publicly available datasets. To demonstrate the efficacy of Pinstripe for large-scale analysis, we applied Pinstripe's reverse peptide mapping pipeline to a transcript library including de novo assembled transcriptomes from the human Illumina Body Atlas (IBA2) and GENCODE v10 gene annotations, and the EBI Proteomics Identifications Database (PRIDE) peptide database. This analysis identified 736 canonical open reading frames (ORFs) supported by three or more PRIDE peptide fragments that are positioned outside any known coding DNA sequence (CDS). Because of the unfiltered nature of the PRIDE database and high probability of false discovery, we further refined this list using independent evidence for translation, including the presence of a Kozak sequence or functional domains, synonymous/non-synonymous substitution ratios and ORF length. Using this integrative approach, we observed evidence of translation from a previously unknown let7e primary transcript, the archetypical lncRNA H19, and a homolog of RD3. Reciprocally, by exclusion of transcripts with mapped peptides or significant ORFs (>80 codon), we identify 32 187 loci with RNAs longer than 2000 nt that are unlikely to encode proteins. Pinstripe (pinstripe.matticklab.com) is freely available as source code or a Mono binary. Pinstripe is written in C# and runs under the Mono framework on Linux or Mac OS X, and both under Mono and .Net under Windows. m.dinger@garvan.org.au or j.mattick@garvan.org.au Supplementary data are available at Bioinformatics online.

Predicting Statistical Properties of Open Reading Frames in Bacterial Genomes

An analytical model based on the statistical properties of Open Reading Frames (ORFs) of eubacterial genomes such as codon composition and sequence length of all reading frames was developed. This new model predicts the average length, maximum length as well as the length distribution of the ORFs of 70 species with GC contents varying between 21% and 74%. Furthermore, the number of annotated genes is predicted with high accordance. However, the ORF length distribution in the five alternative reading frames shows interesting deviations from the predicted distribution. In particular, long ORFs appear more often than expected statistically. The unexpected depletion of stop codons in these alternative open reading frames cannot completely be explained by a biased codon usage in the +1 frame. While it is unknown if the stop codon depletion has a biological function, it could be due to a protein coding capacity of alternative ORFs exerting a selection pressure which prevents the fixation of stop codon mutations. The comparison of the analytical model with bacterial genomes, therefore, leads to a hypothesis suggesting novel gene candidates which can now be investigated in subsequent wet lab experiments.

GC content dependency of open reading frame prediction via stop codon frequencies

A frequently used approach for detecting potential coding regions is to search for stop codons. In the standard genetic code 3 out of 64 trinucleotides are stop codons. Hence, in random or non-coding DNA one can expect every 21st trinucleotide to have the same sequence as a stop codon. In contrast, the open reading frames (ORFs) of most protein-coding genes are considerably longer. Thus, the stop codon frequency in coding sequences deviates from the background frequency of the corresponding trinucleotides. This has been utilized for gene prediction, in particular, in detecting protein-coding ORFs. Traditional methods based on stop codon frequency are based on the assumption that the GC content is about 50%. However, many genomes show significant deviations from that value. With the presented method we can describe the effects of GC content on the selection of appropriate length thresholds of potentially coding ORFs. Conversely, for a given length threshold, we can calculate the probability of observing it in a random sequence. Thus, we can derive the maximum GC content for which ORF length is practicable as a feature for gene prediction methods and the resulting false positive rates. A rough estimate for an upper limit is a GC content of 80%. This estimate can be made more precise by including further parameters and by taking into account start codons as well. We demonstrate the feasibility of this method by applying it to the genomes of the bacteria Rickettsia prowazekii, Escherichia coli and Caulobacter crescentus, exemplifying the effect of GC content variations according to our predictions. We have adapted the method for predicting coding ORFs by stop codon frequency to the case of GC contents different from 50%. Usually, several methods for gene finding need to be combined. Thus, our results concern a specific part within a package of methods. Interestingly, for genomes with low GC content such as that of R. prowazekii, the presented method provides remarkably good results even when applied alone.

GC content dependency of open reading frame prediction

Motivations. A frequently used approach for detecting potential coding regions is to search for stop codons. In the standard genetic code 3 out of 64 trinucleotides are stop codons. Hence, in random or non-coding DNA one can expect every 21st trinucleotide to be equivalent to a stop codon. In contrast, the open reading frames (ORFs) of most protein coding genes are considerably longer. Thus, the stop codon frequency in coding sequences deviates from the background frequency of the corresponding trinucleotides. This has been utilized for gene prediction, in particular, in detecting ORFs. Traditional methods based on stop codon frequency are based on the assumption that the GC content is about 50 %. However, many genomes show significant deviations from that value. Methods and Results. With the presented method we can describe the effects of GC content on the selection of appropriate length thresholds of ORFs. Conversely, for a given length threshold, we can calculate the probability of observing it in a random sequence. Thus, we can derive the maximum GC content for which ORF length is practicable as a feature for gene prediction methods and the resulting false positive rates. A rough estimate for an upper limit is a GC content of 80 %. This estimate can be made more precise by including further parameters and by taking into account start codons as well. We demonstrate the feasibility of this method by applying it to the genomes of the bacteria Rickettsia prowazekii, Escherichia coli and Caulobacter crescentus, exemplifying the effect of GC content variations according to our predictions.

Characterization of Common Carp Transcriptome: Sequencing, De Novo Assembly, Annotation and Comparative Genomics

BackgroundCommon carp (Cyprinus carpio) is one of the most important aquaculture species of Cyprinidae with an annual global production of 3.4 million tons, accounting for nearly 14% of the freshwater aquaculture production in the world. Due to the economical and ecological importance of common carp, genomic data are eagerly needed for genetic improvement purpose. However, there is still no sufficient transcriptome data available. The objective of the project is to sequence transcriptome deeply and provide well-assembled transcriptome sequences to common carp research community.ResultTranscriptome sequencing of common carp was performed using Roche 454 platform. A total of 1,418,591 clean ESTs were collected and assembled into 36,811 cDNA contigs, with average length of 888 bp and N50 length of 1,002 bp. Annotation was performed and a total of 19,165 unique proteins were identified from assembled contigs. Gene ontology and KEGG analysis were performed and classified all contigs into functional categories for understanding gene functions and regulation pathways. Open Reading Frames (ORFs) were detected from 29,869 (81.1%) contigs with an average ORF length of 763 bp. From these contigs, 9,625 full-length cDNAs were identified with sequence length from 201 bp to 9,956 bp. Comparative analysis revealed that 27,693(75.2%) contigs have significant similarity to zebrafish Refseq proteins, and 24,371(66.2%), 24,501(66.5%) and 25,025(70.0%) to teraodon, medaka and three-spined stickleback refseq proteins. A total of 2,064 microsatellites were initially identified from 1,730 contigs, and 1,639 unique sequences had sufficient flanking sequences on both sides for primer design.ConclusionThe transcriptome of common carp had been deep sequenced, de novo assembled and characterized, providing the valuable resource for better understanding of common carp genome. The transcriptome data will facilitate future functional studies on common carp genome, and gradually apply in breeding programs of common carp, as well as closely related other Cyprinids.

Is There a Classical Nonsense-Mediated Decay Pathway in Trypanosomes?

In many eukaryotes, messenger RNAs with premature termination codons are destroyed by a process called “nonsense-mediated decay”, which requires the RNA helicase Upf1 and also, usually, an interacting factor, Upf2. Recognition of premature termination codons may rely on their distance from either a splice site or the polyadenylation site, and long 3′-untranslated regions can trigger mRNA decay. The protist Trypanosoma brucei relies heavily on mRNA degradation to determine mRNA levels, and 3′-untranslated regions play a major role in control of mRNA decay. We show here that trypanosomes have a homologue of Upf1, TbUPF1, which interacts with TbUPF2 and (in an RNA-dependent fashion) with poly(A) binding protein 1, PABP1. Introduction of a premature termination codon in either an endogenous gene or a reporter gene decreased mRNA abundance, as expected for nonsense-mediated decay, but a dependence of this effect on TbUPF1 could not be demonstrated, and depletion of TbUPF1 by over 95% had no effect on parasite growth or the mRNA transcriptome. Further investigations of the reporter mRNA revealed that increases in open reading frame length tended to increase mRNA abundance. In contrast, inhibition of translation, either using 5′-secondary structures or by lengthening the 5′-untranslated region, usually decreased reporter mRNA abundance. Meanwhile, changing the length of the 3′-untranslated region had no consistent effect on mRNA abundance. We suggest that in trypanosomes, translation per se may inhibit mRNA decay, and interactions with multiple RNA-binding proteins preclude degradation based on 3′-untranslated region length alone.