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Showing papers on "Complementary DNA published in 2010"


Journal ArticleDOI
TL;DR: This protocol can generate deep-sequencing libraries for 16 single-cell samples within 6 d and can capture up to 75% more genes expressed in early embryos compared with cDNA microarray techniques.
Abstract: We describe here a protocol for digital transcriptome analysis in a single mouse oocyte and blastomere using a deep-sequencing approach. In this method, individual cells are isolated and transferred into lysate buffer by mouth pipette, followed by reverse transcription carried out directly on the whole cell lysate. Free primers are removed by exonuclease I and a poly(A) tail is added to the 3' end of the first-strand cDNAs by terminal deoxynucleotidyl transferase. Single-cell cDNAs are then amplified by 20 + 9 cycles of PCR. The resulting 100-200 ng of amplified cDNAs are used to construct a sequencing library, which can be used for deep sequencing using the SOLiD system. Compared with cDNA microarray techniques, our assay can capture up to 75% more genes expressed in early embryos. This protocol can generate deep-sequencing libraries for 16 single-cell samples within 6 d.

517 citations


Journal ArticleDOI
TL;DR: A general model in which multiple APOBEC3 proteins function together to provide a constitutive barrier to foreign genetic elements, which can be fortified by transcriptional induction is suggested.
Abstract: The human APOBEC3 proteins are DNA cytidine deaminases that impede the replication of many different transposons and viruses. The genes that encode APOBEC3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G and APOBEC3H were generated through relatively recent recombination events. The resulting high degree of inter-relatedness has complicated the development of specific quantitative PCR assays for these genes despite considerable interest in understanding their expression profiles. Here, we describe a set of quantitative PCR assays that specifically measures the mRNA levels of each APOBEC3 gene. The specificity and sensitivity of each assay was validated using a full matrix of APOBEC3 cDNA templates. The assays were used to quantify the APOBEC3 repertoire in multiple human T-cell lines, bulk leukocytes and leukocyte subsets, and 20 different human tissues. The data demonstrate that multiple APOBEC3 genes are expressed constitutively in most types of cells and tissues, and that distinct APOBEC3 genes are induced upon T-cell activation and interferon treatment. These data help define the APOBEC3 repertoire relevant to HIV-1 restriction in T cells, and they suggest a general model in which multiple APOBEC3 proteins function together to provide a constitutive barrier to foreign genetic elements, which can be fortified by transcriptional induction.

350 citations


Journal ArticleDOI
TL;DR: A powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that is termed PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) is developed.
Abstract: RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1. A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample. We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs. Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

272 citations


Journal ArticleDOI
TL;DR: Deep RNA sequencing was used to simultaneously analyze vaccinia virus (VACV) and HeLa cell transcriptomes at progressive times following infection, revealing two clusters of early mRNAs that were synthesized in the presence of inhibitors of protein as well as DNA synthesis, indicating that they do not correspond to separate immediate- and delayed-early classes as defined for other DNA viruses.
Abstract: Deep RNA sequencing was used to simultaneously analyze vaccinia virus (VACV) and HeLa cell transcriptomes at progressive times following infection. VACV, the prototypic member of the poxvirus family, replicates in the cytoplasm and contains a double-stranded DNA genome with ≈200 closely spaced open reading frames (ORFs). The acquisition of a total of nearly 500 million short cDNA sequences allowed construction of temporal strand-specific maps of the entire VACV transcriptome at single-base resolution and analysis of over 14,000 host mRNAs. Before viral DNA replication, transcripts from 118 VACV ORFs were detected; after replication, transcripts from 93 additional ORFs were characterized. The high resolution permitted determination of the precise boundaries of many mRNAs including read-through transcripts and location of mRNA start sites and adjacent promoters. Temporal analysis revealed two clusters of early mRNAs that were synthesized in the presence of inhibitors of protein as well as DNA synthesis, indicating that they do not correspond to separate immediate- and delayed-early classes as defined for other DNA viruses. The proportion of viral RNAs reached 25–55% of the total at 4 h. This rapid change, resulting in a relative decrease of the vast majority of host mRNAs, can contribute to the profound shutdown of host protein synthesis and blunting of antiviral responses. At 2 h, however, a minority of cellular mRNAs was increased. The overrepresented functional categories of the up-regulated RNAs were NF-κB cascade, apoptosis, signal transduction, and ligand-mediated signaling, which likely represent the host response to invasion.

206 citations


Journal ArticleDOI
TL;DR: Most of the Arabidopsis LRR-RLK genes have been isolated and the sequence analysis showed a number of alternatively spliced variants.
Abstract: Transmembrane receptor kinases play critical roles in both animal and plant signaling pathways regulating growth, development, differentiation, cell death, and pathogenic defense responses. In Arabidopsis thaliana, there are at least 223 Leucine-rich repeat receptor-like kinases (LRR-RLKs), representing one of the largest protein families. Although functional roles for a handful of LRR-RLKs have been revealed, the functions of the majority of members in this protein family have not been elucidated. As a resource for the in-depth analysis of this important protein family, the complementary DNA sequences (cDNAs) of 194 LRR-RLKs were cloned into the GatewayR donor vector pDONR/ZeoR and analyzed by DNA sequencing. Among them, 157 clones showed sequences identical to the predictions in the Arabidopsis sequence resource, TAIR8. The other 37 cDNAs showed gene structures distinct from the predictions of TAIR8, which was mainly caused by alternative splicing of pre-mRNA. Most of the genes have been further cloned into GatewayR destination vectors with GFP or FLAG epitope tags and have been transformed into Arabidopsis for in planta functional analysis. All clones from this study have been submitted to the Arabidopsis Biological Resource Center (ABRC) at Ohio State University for full accessibility by the Arabidopsis research community. Most of the Arabidopsis LRR-RLK genes have been isolated and the sequence analysis showed a number of alternatively spliced variants. The generated resources, including cDNA entry clones, expression constructs and transgenic plants, will facilitate further functional analysis of the members of this important gene family.

171 citations


Journal ArticleDOI
18 Aug 2010-PLOS ONE
TL;DR: It is proposed that most reported examples of non-canonical splicing in metazoans arise through template switching by reverse transcriptase during cDNA preparation, providing a simple diagnostic for identifying many of these experimental artifacts.
Abstract: Background: Trans-splicing, the in vivo joining of two independently transcribed RNA molecules, is well characterized in lower eukaryotes, but was long thought absent from metazoans. However, recent bioinformatic analyses of EST sequences suggested widespread trans-splicing in mammals. These apparently spliced transcripts generally lacked canonical splice sites, leading us to question their authenticity. Particularly, the native ability of reverse transcriptase enzymes to template switch during transcription could produce apparently trans-spliced sequences. Principal Findings: Here we report an in vitro system for the analysis of template switching in reverse transcription. Using highly purified RNA substrates, we show the reproducible occurrence of apparent trans-splicing between two RNA molecules. Other reported non-canonical splicing events such as exon shuffling and sense-antisense fusions were also readily detected. The latter caused the production of apparent antisense non-coding RNAs, which are also reported to be abundant in humans. Conclusions: We propose that most reported examples of non-canonical splicing in metazoans arise through template switching by reverse transcriptase during cDNA preparation. We further show that the products of template switching can vary between reverse transcriptases, providing a simple diagnostic for identifying many of these experimental artifacts.

143 citations


Journal ArticleDOI
TL;DR: This study presented a series of candidate genes and pathways that may contribute to DM resistance in grapes, and illustrated that the Solexa-based tag-sequencing approach was a powerful tool for gene expression comparison between control and treated samples.
Abstract: Downy mildew (DM), caused by pathogen Plasmopara viticola (PV) is the single most damaging disease of grapes (Vitis L.) worldwide. However, the mechanisms of the disease development in grapes are poorly understood. A method for estimating gene expression levels using Solexa sequencing of Type I restriction-endonuclease-generated cDNA fragments was used for deep sequencing the transcriptomes resulting from PV infected leaves of Vitis amurensis Rupr. cv. Zuoshan-1. Our goal is to identify genes that are involved in resistance to grape DM disease. Approximately 8.5 million (M) 21-nt cDNA tags were sequenced in the cDNA library derived from PV pathogen-infected leaves, and about 7.5 M were sequenced from the cDNA library constructed from the control leaves. When annotated, a total of 15,249 putative genes were identified from the Solexa sequencing tags for the infection (INF) library and 14,549 for the control (CON) library. Comparative analysis between these two cDNA libraries showed about 0.9% of the unique tags increased by at least five-fold, and about 0.6% of the unique tags decreased more than five-fold in infected leaves, while 98.5% of the unique tags showed less than five-fold difference between the two samples. The expression levels of 12 differentially expressed genes were confirmed by Real-time RT-PCR and the trends observed agreed well with the Solexa expression profiles, although the degree of change was lower in amplitude. After pathway enrichment analysis, a set of significantly enriched pathways were identified for the differentially expressed genes (DEGs), which associated with ribosome structure, photosynthesis, amino acid and sugar metabolism. This study presented a series of candidate genes and pathways that may contribute to DM resistance in grapes, and illustrated that the Solexa-based tag-sequencing approach was a powerful tool for gene expression comparison between control and treated samples.

135 citations


Journal ArticleDOI
TL;DR: The cloning and expression analysis of the two HSP70s provide theoretical basis to further study the mechanism of anti-adverseness and expression characteristics under stress conditions of Wuchang bream.

129 citations


Journal ArticleDOI
12 Aug 2010-Blood
TL;DR: It is concluded that the CL20i4-EF1alpha-hgamma(c)OPT vector may be suitable for testing in a clinical trial based on these preclinical demonstrations of efficacy and safety.

120 citations


Journal ArticleDOI
TL;DR: The results suggest that the novel TaNAC8 protein functions as a transcriptional activator involved in wheat defense responses to abiotic and biotic stresses.

117 citations


Journal ArticleDOI
TL;DR: It is demonstrated that mechanisms utilized by A3F to prevent HIV-1 viral DNA integration were different from those of A3G, and that their target specificities and/or their affinities for dsDNA may contribute to their distinct mechanisms.
Abstract: APOBEC3F (A3F) and APBOBEC3G (A3G) both are host restriction factors that can potently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Their antiviral activities are at least partially mediated by cytidine deamination, which causes lethal mutations of the viral genome. We recently showed that A3G blocks viral plus-strand DNA transfer and inhibits provirus establishment in the host genome (J. L. Mbisa, R. Barr, J. A. Thomas, N. Vandegraaff, I. J. Dorweiler, E. S. Svarovskaia, W. L. Brown, L. M. Mansky, R. J. Gorelick, R. S. Harris, A. Engelman, and V. K. Pathak, J. Virol. 81:7099-7110, 2007). Here, we investigated whether A3F similarly interferes with HIV-1 provirus formation. We observed that both A3F and A3G inhibit viral DNA synthesis and integration, but A3F is more potent than A3G in preventing viral DNA integration. We further investigated the mechanisms by which A3F and A3G block viral DNA integration by analyzing their effects on viral cDNA processing using Southern blot analysis. A3G generates a 6-bp extension at the viral U5 end of the 3′ long terminal repeat (3′-LTR), which is a poor substrate for integration; in contrast, A3F inhibits viral DNA integration by reducing the 3′ processing of viral DNA at both the U5 and U3 ends. Furthermore, we demonstrated that a functional C-terminal catalytic domain is more critical for A3G than A3F function in blocking HIV-1 provirus formation. Finally, we showed that A3F has a greater binding affinity for a viral 3′-LTR double-stranded DNA (dsDNA) oligonucleotide template than A3G. Taking these results together, we demonstrated that mechanisms utilized by A3F to prevent HIV-1 viral DNA integration were different from those of A3G, and that their target specificities and/or their affinities for dsDNA may contribute to their distinct mechanisms.

Journal ArticleDOI
23 Dec 2010-PLOS ONE
TL;DR: By co-expressing the fission yeast NatB complex with the target protein in E.coli, this work reports a simple and widely applicable method for the expression and purification of functional N-terminally acetylated eukaryotic proteins.
Abstract: One major limitation in the expression of eukaryotic proteins in bacteria is an inability to post-translationally modify the expressed protein. Amino-terminal acetylation is one such modification that can be essential for protein function. By co-expressing the fission yeast NatB complex with the target protein in E.coli, we report a simple and widely applicable method for the expression and purification of functional N-terminally acetylated eukaryotic proteins.

Journal ArticleDOI
TL;DR: A draft genome of Caenorhabditis sp.
Abstract: Efficient sequencing of animal and plant genomes by next-generation technology should allow many neglected organisms of biological and medical importance to be better understood. As a test case, we have assembled a draft genome of Caenorhabditis sp. 3 PS1010 through a combination of direct sequencing and scaffolding with RNA-seq. We first sequenced genomic DNA and mixed-stage cDNA using paired 75-nt reads from an Illumina GAII. A set of 230 million genomic reads yielded an 80-Mb assembly, with a supercontig N50 of 5.0 kb, covering 90% of 429 kb from previously published genomic contigs. Mixed-stage poly(A)+ cDNA gave 47.3 million mappable 75-mers (including 5.1 million spliced reads), which separately assembled into 17.8 Mb of cDNA, with an N50 of 1.06 kb. By further scaffolding our genomic supercontigs with cDNA, we increased their N50 to 9.4 kb, nearly double the average gene size in C. elegans. We predicted 22,851 protein-coding genes, and detected expression in 78% of them. Multigenome alignment and data filtering identified 2672 DNA elements conserved between PS1010 and C. elegans that are likely to encode regulatory sequences or previously unknown ncRNAs. Genomic and cDNA sequencing followed by joint assembly is a rapid and useful strategy for biological analysis.

Journal ArticleDOI
04 Oct 2010-RNA
TL;DR: Methods to minimize the effects of 2'-O-methyl modification of small RNA 3'-termini using T4 RNA ligase 2 truncated, and other optimized reaction conditions are described, demonstrating their use by quantifying representation of miRNAs and piRNAs in cDNA pools prepared from biological samples.
Abstract: Small regulatory RNA repertoires in biological samples are heterogeneous mixtures that may include species arising from varied biosynthetic pathways and modification events. Small RNA profiling and discovery approaches ought to capture molecules in a way that is representative of expression level. It follows that the effects of RNA modifications on representation should be minimized. The collection of high-quality, representative data, therefore, will be highly dependent on bias-free sample manipulation in advance of quantification. We examined the impact of 2'-O-methylation of the 3'-terminal nucleotide of small RNA on key enzymatic reactions of standard front-end manipulation schemes. Here we report that this common modification negatively influences the representation of these small RNA species. Deficits occurred at multiple steps as determined by gel analysis of synthetic input RNA and by quantification and sequencing of derived cDNA pools. We describe methods to minimize the effects of 2'-O-methyl modification of small RNA 3'-termini using T4 RNA ligase 2 truncated, and other optimized reaction conditions, demonstrating their use by quantifying representation of miRNAs and piRNAs in cDNA pools prepared from biological samples.

Journal ArticleDOI
TL;DR: The identified cDNA encodes a novel protein of 483 amino acids that displays significant homology to nSMase2 and possesses the same catalytic core residues as members of the extended N-SMase family and will contribute to the understanding of pathways regulated by sphingolipid metabolites, particularly with reference to the mitochondria and associated organelles.

Journal ArticleDOI
TL;DR: Lipid analysis of the mutant yeast cells revealed that OtDGAT2B showed broad substrate specificity accepting saturated as well as mono- and poly-unsaturated acyl-CoAs as substrates, and analysis of their amino acid sequences showed that they share limited identity with other DGAT2 from different plant species, such as Ricinus communis and Vernicia fordii.

Journal ArticleDOI
TL;DR: The results suggest that the Wuchang bream HIF-1alpha and -2alpha would be involved in different physiological functions under normoxia and hypoxia situations.
Abstract: Transcriptional responses to hypoxia are primarily mediated by hypoxia-inducible factor (HIF) alpha subunits, i.e. HIF-1alpha, -2alpha and -3alpha. In fish, the molecular constructions, expression characteristics and hypoxic regulation of HIF-alpha subunits are still not well known. In this study, we identified the HIF-1alpha and HIF-2alpha full-length cDNAs in a hypoxia-sensitive fish species Wuchang bream, Megalobrama amblycephala. The whole length of HIF-1alpha cDNA was 3,815bp, consisting of an open reading frame (ORF) encoding 774 amino acid (aa) residues. The HIF-2alpha cDNA totaled 3,121-bp including an 835-aa ORF. The Wuchang bream HIF-1alpha and HIF-2alpha subunits were structurally similar in the DNA-binding and dimerization domains, but differed in the transactivation domain. In adult fish, both HIF-1alpha and HIF-2alpha mRNAs were detected in different tissues under normoxic conditions. HIF-1alpha mRNA was highly expressed in the liver, gill and testis, whereas HIF-2alpha mRNA was abundantly expressed in most of the Wuchang bream tissues. Both HIF-1alpha and HIF-2alpha mRNAs were detected in all stages of embryogenesis and expressed in a ubiquitous pattern. In contrast to HIF-1alpha, the mRNA levels of HIF-2alpha fluctuated in different stages, with higher expression in the zygote, 8-, 28-, 48- and 52-hr post fertilization (hpf) embryos. During hypoxic treatment, the mRNA levels of HIF-2alpha were significantly (p<0.01) up-regulated to 910% in the liver and 320% in the kidney, whereas no significant changes of HIF-1alpha mRNA were observed in the corresponding tissues. These results suggest that the Wuchang bream HIF-1alpha and -2alpha would be involved in different physiological functions under normoxia and hypoxia situations.

Journal ArticleDOI
TL;DR: The results indicate that LvHHSP60 and LvHSP70 may play important roles in mediating the immune responses of L. vannamei to bacterial challenge, and that the Ca(2+) signalling transduction pathway may be involved in the initiation of the shrimp's immune responses in early stages of infection.

Journal ArticleDOI
TL;DR: The data indicate that MgC1q is a pattern recognition molecule able to recognize pathogens during innate immune responses in Myitilus galloprovincialis, and the high sequence variability suggests that somatic diversification of these nonself recognition molecules could have occurred.
Abstract: In this study, we present the characterization of a newly identified gene, MgC1q, revealed in suppression subtractive hybridization and cDNA libraries from immunostimulated mussels. Based on sequence homology, molecular architecture and domain similarity, this new C1q-domain-containing gene may be classified as a member of the C1q family and, therefore, part of the C1q-TNF superfamily. The expression of MgC1q was detected all along the mussel ontogeny, being detectable within 2h post-fertilization, with a notable increase after 1 month and continuing to increase until 3 months. Measurable transcript levels were also evident in all analyzed tissues of naive adult mussels, and the hemocytes showed the highest expression levels. Experimental infection of adult mussels with Gram positive or Gram negative bacteria significantly modulated the MgC1q expression, and confirmed it as an immune-related gene. Intra- and inter-individual sequence analyses revealed extraordinary diversity of MgC1q at both the DNA and cDNA levels. While further research is needed to define its function, our data indicate that MgC1q is a pattern recognition molecule able to recognize pathogens during innate immune responses in Myitilus galloprovincialis. The high sequence variability suggests that somatic diversification of these nonself recognition molecules could have occurred.

Journal ArticleDOI
TL;DR: It is found that, in activated T lymphocytes, viral integrase, which mediates HIV-1 cDNA integration into the host cell genome, is phosphorylated by JNK on a highly conserved serine residue in its core domain.
Abstract: Long-standing evidence indicates that quiescent human peripheral blood T lymphocytes (PBLs) do not support efficient HIV infection In resting PBLs, reverse transcription of viral RNA takes longer than in activated cells, partially because formation of the late products of reverse transcription is decreased by RNA binding by apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G) In a subsequent step, integration of the viral complementary DNA that is eventually formed is markedly impaired Here we show that cellular c-Jun N-terminal kinase (JNK), an enzyme that is not expressed in resting CD4+ T cells, regulates permissiveness to HIV-1 infection, and we unravel a new, sequential post-translational pathway of protein modification that regulates viral DNA integration We found that, in activated T lymphocytes, viral integrase, which mediates HIV-1 cDNA integration into the host cell genome, is phosphorylated by JNK on a highly conserved serine residue in its core domain Phosphorylated integrase, in turn, becomes a substrate for the cellular peptidyl prolyl-isomerase enzyme Pin1, which catalyzes a conformational modification of integrase These concerted activities increase integrase stability and are required for efficient HIV-1 integration and infection Lack of these modifications restricts viral infection in nonactivated, primary CD4+ T lymphocytes

Patent
Linnarsson Sten1
23 Mar 2010
TL;DR: In this paper, the authors present methods and compositions for the analysis of gene expression in single cells or in a plurality of single cells, and also provide a cDNA library produced by the methods described herein.
Abstract: The present invention provides methods and compositions for the analysis of gene expression in single cells or in a plurality of single cells. The invention provides methods for preparing a cDNΛ library from individual cells by releasing mRNΛ from each single cell to provide a plurality of individual mRNA samples, synthesizing cDNA from the individual mRNA samples, tagging the individual cDNA, pooling the tagged cDNA samples and amplifying the pooled cDNA samples to generate a cDNA library. The invention also provides a cDNA library produced by the methods described herein. The invention farther provides methods for analyzing gene expression in a plurality of cells by preparing a cDNA library as described herein and sequencing the library.

Journal ArticleDOI
TL;DR: The nucleic acid chaperone properties of ORF1p are reviewed in the context of L1 retrotransposition, a process which requires the two element-encoded proteins to act in cis on the L1 RNA.
Abstract: Long interspersed element-1 (LINE-1, or L1) is a non-long terminal repeat (LTR) retrotransposon that has amplified to hundreds of thousands of copies in mammalian evolution A small number of the individual copies of L1 are active retrotransposons which are presently replicating in most species, including humans and mice L1 retrotransposition begins with transcription of an active element and ends with a newly inserted cDNA copy, a process which requires the two element-encoded proteins to act in cis on the L1 RNA The ORF1 protein (ORF1p) is a high-affinity, non-sequence-specific RNA binding protein with nucleic acid chaperone activity, whereas the ORF2 protein (ORF2p) supplies the enzymatic activities for cDNA synthesis This article reviews the nucleic acid chaperone properties of ORF1p in the context of L1 retrotransposition

Journal ArticleDOI
TL;DR: Using fluorescent real-time quantitative PCR, the transcriptional expression of PtHsp70 showed a clear time-dependent response after challenge by Vibrio alginolyticus, the main causative agent of emulsification disease causing large mortality in P. trituberculatus.

Journal ArticleDOI
TL;DR: Findings suggest that RfBGluc-1 may serve dual functions in cellulose digestion and immunity, and contributes to the development of next-generation termiticides and novel biocatalyst cocktails for use in biomass-to-bioethanol applications.

Journal ArticleDOI
TL;DR: This study contributes significantly to a better understanding of LEV receptor constitution in parasitic nematodes and highlights the putative role of aberrant mRNA encoding L-AChR subunits in LEV resistance.
Abstract: Objectives The molecular mechanisms of levamisole (LEV) activity and expression of resistance remain largely unknown in parasitic nematodes. In contrast, genetic screens for mutants that survive exposure to LEV in the free-living nematode Caenorhabditis elegans have led to the identification of five genes (unc-38, unc-63, unc-29, lev-1 and lev-8) that encode a LEV-sensitive acetylcholine receptor (L-AChR). Loss of these genes leads to LEV resistance. In this study, orthologues of these genes were identified in three species of trichostrongylid nematodes that have a major impact on small ruminants: Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis. Polymorphism associated with LEV resistance have been investigated by comparing transcripts of these subunits in LEV susceptible and LEV-resistant isolates of the three strongylid species. Basic methods Partial sequences were identified by PCR experiments and full-length cDNA sequences corresponding to AChR subunits in the three trichostrongylid species were obtained using 3'-rapid amplification of cDNA ends-PCR and 5' rapid amplification of cDNA ends anchored with the spliced leader sequence, SL1. Expression of L-AChR subunits was investigated in LEV-resistant and LEV-susceptible isolates of H. contortus, T. circumcincta and T. colubriformis using reverse transcription PCR. Main results We have identified a total of 20 full-length cDNA sequences corresponding to L-AChR subunits in three parasitic trichostrongylid species of which 14 correspond to novel sequences. Genes orthologous to unc-29, unc-63, unc-38 and lev-1 were found in each trichostrongylid species, whereas no gene corresponding to lev-8 has yet been identified. We have found 11 distinct paralogous sequences corresponding to the C. elegans unc-29 gene clustered in four groups revealing an unexpected diversity of unc-29-like genes. Complete coding sequences of the L-AChR subunits in two LEV-resistant and three susceptible isolates of H. contortus, T. circumcincta and T. colubriformis were essentially unchanged, but abbreviated transcripts of the unc-63 subunit were specifically expressed in resistant isolates of all three species. Conclusion The candidate gene strategy developed in this study revealed an unexpectedly high diversity of L-AChR subunits specific to the trichostrongylid parasites that are a principal target for the drug LEV. Abbreviated variants, predicted to produce nonfunctional unc-63, were associated with LEV resistance. This study contributes significantly to a better understanding of LEV receptor constitution in parasitic nematodes and highlights the putative role of aberrant mRNA encoding L-AChR subunits in LEV resistance.

Journal ArticleDOI
TL;DR: Chaperones are an important part of the cellular response to stress, and genes identified in this work may play important roles in resistance to thermal-stress.
Abstract: Daily and seasonal changes in temperature are challenges that fish within aquaculture settings cannot completely avoid, and are known to elicit complex organismal and cellular stress responses. We conducted a large-scale gene discovery and transcript expression study in order to better understand the genes that are potentially involved in the physiological and cellular aspects of stress caused by heat-shock. We used suppression subtractive hybridization (SSH) cDNA library construction and characterization to identify transcripts that were dysregulated by heat-shock in liver, skeletal muscle and head kidney of Atlantic cod. These tissues were selected due to their roles in metabolic regulation, locomotion and growth, and immune function, respectively. Fish were exposed for 3 hours to an 8°C elevation in temperature, and then allowed to recover for 24 hours at the original temperature (i.e. 10°C). Tissue samples obtained before heat-shock (BHS), at the cessation of heat-shock (CS), and 3, 12, and 24 hours after the cessation of heat-shock (ACS), were used for reciprocal SSH library construction and quantitative reverse transcription - polymerase chain reaction (QPCR) analysis of gene expression using samples from a group that was transferred but not heat-shocked (CT) as controls. We sequenced and characterized 4394 ESTs (1524 from liver, 1451 from head kidney and 1419 from skeletal muscle) from three "forward subtracted" libraries (enriched for genes up-regulated by heat-shock) and 1586 from the liver "reverse subtracted" library (enriched for genes down-regulated by heat-shock), for a total of 5980 ESTs. Several cDNAs encoding putative chaperones belonging to the heat-shock protein (HSP) family were found in these libraries, and "protein folding" was among the gene ontology (GO) terms with the highest proportion in the libraries. QPCR analysis of HSP90α and HSP70-1 (synonym: HSPA1A) mRNA expression showed significant up-regulation in all three tissues studied. These transcripts were more than 100-fold up-regulated in liver following heat-shock. We also identified HSP47, GRP78 and GRP94-like transcripts, which were significantly up-regulated in all 3 tissues studied. Toll-like receptor 22 (TLR22) transcript, found in the liver reverse SSH library, was shown by QPCR to be significantly down-regulated in the head kidney after heat-shock. Chaperones are an important part of the cellular response to stress, and genes identified in this work may play important roles in resistance to thermal-stress. Moreover, the transcript for one key immune response gene (TLR22) was down-regulated by heat-shock, and this down-regulation may be a component of heat-induced immunosuppression.

Journal ArticleDOI
TL;DR: Comparison of amino acid sequences showed that tiger shrimp GPx is more closely related to vertebrate GPx1, in accordance with those in Litopenaeus vannamei and Macrobrachium rosenbergii.
Abstract: Complementary (c)DNA encoding glutathione peroxidase (GPx) messenger (m)RNA of the tiger shrimp Penaeus monodon was obtained from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) method. The 1321-bp cDNA contained an open reading frame (ORF) of 564bp, a 69-bp 5'-untranslated region (UTR), and a 688-bp 3'-UTR containing a poly A tail and a conserved selenocysteine insertion sequence (SECIS) element. The molecular mass of the deduced amino acid (aa) sequence (188 aa) was 21.05kDa long with an estimated pI of 7.68. It contains a putative selenocysteine residue which is encoded by the unusual stop codon, (190)TGA(192), and forms the active site with residues Glu(75) and Trp(143). Comparison of amino acid sequences showed that tiger shrimp GPx is more closely related to vertebrate GPx1, in accordance with those in Litopenaeus vannamei and Macrobrachium rosenbergii. GPx cDNA was synthesised in lymphoid organ, gills, heart, haemocytes, the hepatopancreas, muscles, and intestines. After injected with either Photobacterium damsela or white spot syndrome virus (WSSV), the respiratory bursts of shrimp significantly increased in order to kill the pathogen, and induced increases in the activities of superoxide dismutase and GPx, and regulation in the expression of cloned GPx mRNA to protect cells against damage from oxidation. The GPx expression significantly increased at stage D(0/1), and then gradually decreased until stage C suggesting that the cloned GPx might play a role in the molt regulation of shrimp.

Journal ArticleDOI
TL;DR: The beta-defensin-like gene and its cloned isoforms (fBDI-1 to -5) were identified in an expressed sequence tag (EST) library from the early developmental stages of the olive flounder and confirmed that the fBDI isoforms cluster at the same locus and exhibit the conserved gene organisation reported for other fish defensin genes.

Journal ArticleDOI
TL;DR: Results showed that reduction in expression of the three APNs is functionally associated with the Cry1Ab resistance in D. saccharalis, and silencing of all three APN in vivo by RNAi resulted in a decrease inCry1Ab susceptibility.

Journal ArticleDOI
TL;DR: A new C-type lectin (Ec-CTL) gene was cloned from grouper, Epinephelus coioides by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) PCR and showed high similarity of 54% to the C- type lectin of killifish Fundulus heteroclitus.