Showing papers on "Complementary DNA published in 2013"

The microglial sensome revealed by direct RNA sequencing

Abstract: Microglia, the principal neuroimmune sentinels of the brain, continuously sense changes in their environment and respond to invading pathogens, toxins and cellular debris. Microglia exhibit plasticity and can assume neurotoxic or neuroprotective priming states that determine their responses to danger. We used direct RNA sequencing, without amplification or cDNA synthesis, to determine the quantitative transcriptomes of microglia of healthy adult and aged mice. We validated our findings by fluorescent dual in-situ hybridization, unbiased proteomic analysis and quantitative PCR. We report here that microglia have a distinct transcriptomic signature and express a unique cluster of transcripts encoding proteins for sensing endogenous ligands and microbes that we term the “sensome”. With aging, sensome transcripts for endogenous ligand recognition are downregulated, whereas those involved in microbe recognition and host defense are upregulated. In addition, aging is associated with an overall increase in expression of microglial genes involved in neuroprotection.

RNA-sequencing from single nuclei

Abstract: It has recently been established that synthesis of double-stranded cDNA can be done from a single cell for use in DNA sequencing. Global gene expression can be quantified from the number of reads mapping to each gene, and mutations and mRNA splicing variants determined from the sequence reads. Here we demonstrate that this method of transcriptomic analysis can be done using the extremely low levels of mRNA in a single nucleus, isolated from a mouse neural progenitor cell line and from dissected hippocampal tissue. This method is characterized by excellent coverage and technical reproducibility. On average, more than 16,000 of the 24,057 mouse protein-coding genes were detected from single nuclei, and the amount of gene-expression variation was similar when measured between single nuclei and single cells. Several major advantages of the method exist: first, nuclei, compared with whole cells, have the advantage of being easily isolated from complex tissues and organs, such as those in the CNS. Second, the method can be widely applied to eukaryotic species, including those of different kingdoms. The method also provides insight into regulatory mechanisms specific to the nucleus. Finally, the method enables dissection of regulatory events at the single-cell level; pooling of 10 nuclei or 10 cells obscures some of the variability measured in transcript levels, implying that single nuclei and cells will be extremely useful in revealing the physiological state and interconnectedness of gene regulation in a manner that avoids the masking inherent to conventional transcriptomics using bulk cells or tissues.

Reverse genetics with a full-length infectious cDNA of the Middle East respiratory syndrome coronavirus

Abstract: Severe acute respiratory syndrome with high mortality rates (∼50%) is associated with a novel group 2c betacoronavirus designated Middle East respiratory syndrome coronavirus (MERS-CoV). We synthesized a panel of contiguous cDNAs that spanned the entire genome. Following contig assembly into genome-length cDNA, transfected full-length transcripts recovered several recombinant viruses (rMERS-CoV) that contained the expected marker mutations inserted into the component clones. Because the wild-type MERS-CoV contains a tissue culture-adapted T1015N mutation in the S glycoprotein, rMERS-CoV replicated ∼0.5 log less efficiently than wild-type virus. In addition, we ablated expression of the accessory protein ORF5 (rMERS•ORF5) and replaced it with tomato red fluorescent protein (rMERS-RFP) or deleted the entire ORF3, 4, and 5 accessory cluster (rMERS-ΔORF3–5). Recombinant rMERS-CoV, rMERS-CoV•ORF5, and MERS-CoV-RFP replicated to high titers, whereas MERS-ΔORF3–5 showed 1–1.5 logs reduced titer compared with rMERS-CoV. Northern blot analyses confirmed the associated molecular changes in the recombinant viruses, and sequence analysis demonstrated that RFP was expressed from the appropriate consensus sequence AACGAA. We further show dipeptidyl peptidase 4 expression, MERS-CoV replication, and RNA and protein synthesis in human airway epithelial cell cultures, primary lung fibroblasts, primary lung microvascular endothelial cells, and primary alveolar type II pneumocytes, demonstrating a much broader tissue tropism than severe acute respiratory syndrome coronavirus. The availability of a MERS-CoV molecular clone, as well as recombinant viruses expressing indicator proteins, will allow for high-throughput testing of therapeutic compounds and provide a genetic platform for studying gene function and the rational design of live virus vaccines.

Engineering a Replication-Competent, Propagation-Defective Middle East Respiratory Syndrome Coronavirus as a Vaccine Candidate

Abstract: Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus infecting humans that is associated with acute pneumonia, occasional renal failure, and a high mortality rate and is considered a threat to public health. The construction of a full-length infectious cDNA clone of the MERS-CoV genome in a bacterial artificial chromosome is reported here, providing a reverse genetics system to study the molecular biology of the virus and to develop attenuated viruses as vaccine candidates. Following transfection with the cDNA clone, infectious virus was rescued in both Vero A66 and Huh-7 cells. Recombinant MERS-CoVs (rMERS-CoVs) lacking the accessory genes 3, 4a, 4b, and 5 were successfully rescued from cDNA clones with these genes deleted. The mutant viruses presented growth kinetics similar to those of the wild-type virus, indicating that accessory genes were not essential for MERS-CoV replication in cell cultures. In contrast, an engineered mutant virus lacking the structural E protein (rMERS-CoV-ΔE) was not successfully rescued, since viral infectivity was lost at early passages. Interestingly, the rMERS-CoV-ΔE genome replicated after cDNA clone was transfected into cells. The infectious virus was rescued and propagated in cells expressing the E protein in trans , indicating that this virus was replication competent and propagation defective. Therefore, the rMERS-CoV-ΔE mutant virus is potentially a safe and promising vaccine candidate to prevent MERS-CoV infection. IMPORTANCE Since the emergence of MERS-CoV in the Arabian Peninsula during the summer of 2012, it has already spread to 10 different countries, infecting around 94 persons and showing a mortality rate higher than 50%. This article describes the development of the first reverse genetics system for MERS-CoV, based on the construction of an infectious cDNA clone inserted into a bacterial artificial chromosome. Using this system, a collection of rMERS-CoV deletion mutants has been generated. Interestingly, one of the mutants with the E gene deleted was a replication-competent, propagation-defective virus that could only be grown in the laboratory by providing E protein in trans , whereas it would only survive a single virus infection cycle in vivo . This virus constitutes a vaccine candidate that may represent a balance between safety and efficacy for the induction of mucosal immunity, which is needed to prevent MERS-CoV infection.

Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing.

Abstract: Mobile group II introns encode reverse transcriptases (RTs) that function in intron mobility (“retrohoming”) by a process that requires reverse transcription of a highly structured, 2–2.5-kb intron RNA with high processivity and fidelity. Although the latter properties are potentially useful for applications in cDNA synthesis and next-generation RNA sequencing (RNA-seq), group II intron RTs have been difficult to purify free of the intron RNA, and their utility as research tools has not been investigated systematically. Here, we developed general methods for the high-level expression and purification of group II intron-encoded RTs as fusion proteins with a rigidly linked, noncleavable solubility tag, and we applied them to group II intron RTs from bacterial thermophiles. We thus obtained thermostable group II intron RT fusion proteins that have higher processivity, fidelity, and thermostability than retroviral RTs, synthesize cDNAs at temperatures up to 81°C, and have significant advantages for qRT-PCR, capillary electrophoresis for RNA-structure mapping, and next-generation RNA sequencing. Further, we find that group II intron RTs differ from the retroviral enzymes in template switching with minimal base-pairing to the 3′ ends of new RNA templates, making it possible to efficiently and seamlessly link adaptors containing PCR-primer binding sites to cDNA ends without an RNA ligase step. This novel template-switching activity enables facile and less biased cloning of nonpolyadenylated RNAs, such as miRNAs or protein-bound RNA fragments. Our findings demonstrate novel biochemical activities and inherent advantages of group II intron RTs for research, biotechnological, and diagnostic methods, with potentially wide applications.

Fusion of the ZC3H7B and BCOR genes in endometrial stromal sarcomas carrying an X;22-translocation.

Abstract: Endometrial stromal sarcomas (ESS) are genetically heterogeneous uterine tumors in which a JAZF1-SUZ12 chimeric gene resulting from the chromosomal translocation t(7;17)(p15;q21) as well as PHF1 rearrangements (in chromosomal band 6p21) with formation of JAZF1-PHF1, EPC1-PHF1, and MEAF6-PHF1 chimeras have been described. Here, we investigated two ESS characterized cytogenetically by the presence of a der(22)t(X;22)(p11;q13). Whole transcriptome sequencing one of the tumors identified a ZC3H7-BCOR chimeric transcript. Reverse transciptase-PCR with the ZC3H7B forward and BCOR reverse primer combinations confirmed the presence of a ZC3H7-BCOR chimeric transcript in both ESS carrying a der(22)t(X;22) but not in a control ESS with t(1;6) and the MEAF6-PHF1 fusion. Sequencing of the amplified cDNA fragments showed that in both cases ESS exon 10 of ZC3H7B (from 22q13; accession number NM_017590 version 4) was fused to exon 8 of BCOR (from Xp11; accession number NM_001123385 version 1). Reciprocal multiple BCOR-ZC3H7B cDNA fragments were amplified in only one case suggesting that ZC3H7B-BCOR, on the der(22)t(X;22), is the pathogenetically important fusion gene. The putative ZC3H7B-BCOR protein would contain the tetratricopeptide repeats and LD motif from ZC3H7B and the AF9 binding site (1093-1233aa), the 3 ankyrin repeats (1410-1509 aa), and the NSPC1 binding site of BCOR. Although the presence of these motifs suggests various functions of the chimeric protein, it is possible that its most important role may be in epigenetic regulation. Whether or not the (patho)genetic subsets JAZF1-SUZ12, PHF1 rearrangements, and ZC3H7B-BCOR correspond to any phenotypic, let alone clinically important, differences in ESS remain unknown.

Welander distal myopathy is caused by a mutation in the RNA-binding protein TIA1.

Abstract: Objective A study was undertaken to identify the molecular cause of Welander distal myopathy (WDM), a classic autosomal dominant distal myopathy. Methods The genetic linkage was confirmed and defined by microsatellite and single nucleotide polymorphism haplotyping. The whole linked genomic region was sequenced with targeted high-throughput and Sanger sequencing, and coding transcripts were sequenced on the cDNA level. WDM muscle biopsies were studied by Western blotting and immunofluorescence microscopy. Splicing of TIA1 and its target genes in muscle and myoblast cultures was analyzed by reverse transcriptase polymerase chain reaction. Mutant TIA1 was characterized by cell biological studies on HeLa cells, including quantification of stress granules by high content analysis and fluorescence recovery after photobleaching (FRAP) experiments. Results The linked haplotype at 2p13 was narrowed down to A (p.E384K) in the RNA-binding protein TIA1, a key component of stress granules. Immunofluorescence microscopy of WDM biopsies showed a focal increase of TIA1 in atrophic and vacuolated fibers. In HeLa cells, mutant TIA1 constructs caused a mild increase in stress granule abundance compared to wild type, and showed slower average fluorescence recovery in FRAP. Interpretation WDM is caused by mutated TIA1 through a dominant pathomechanism probably involving altered stress granule dynamics. Ann Neurol 2013;73:500–509

Target of tae-miR408, a chemocyanin-like protein gene (TaCLP1), plays positive roles in wheat response to high-salinity, heavy cupric stress and stripe rust

Abstract: microRNAs (miRNAs) are novel and significant regulators of gene expression at the post-transcriptional level, and they are essential for normal growth and development and adaptation to stress conditions. As miRNAs are a kind of RNAs that do not code proteins, they play roles by repressing gene translation or degrading the corresponding target mRNAs. Plantacyanin-like (basic blue) proteins have been predicted and verified as the target gene of miR408 in wheat and Arabidopsis, respectively. Besides some biochemical characteristics, their detailed biological function remains unknown. In this study, the target gene of a wheat miRNA (tae-miR408), designated TaCLP1, was identified using degradome sequencing and co-transformation technology in tobacco leaves. We isolated the full-length cDNA clone, and defined its product as a chemocyanin-like protein, a kind of plantacyanin. Transcript accumulation of TaCLP1 and tae-miR408 showed contrasting divergent expression patterns in wheat response to Puccinia striiformis f. sp. tritici (Pst) and high copper ion stress. Overexpression of TaCLP1 in yeast (Schizosaccharomyces pombe) significantly increased cell growth under high salinity and Cu2+ stresses. Silencing of individual cDNA clones in wheat challenged with Pst indicated that TaCLP1 positively regulates resistance to stripe rust. The results indicate that the target of tae-miR408, TaCLP1, play an important role in regulating resistance of host plants to abiotic stresses and stripe rust, and such interactions can be a valuable resource for investigating stress tolerance in wheat.

High-Throughput Microfluidic Single-Cell Digital Polymerase Chain Reaction

Abstract: Here we present an integrated microfluidic device for the high-throughput digital polymerase chain reaction (dPCR) analysis of single cells This device allows for the parallel processing of single cells and executes all steps of analysis, including cell capture, washing, lysis, reverse transcription, and dPCR analysis The cDNA from each single cell is distributed into a dedicated dPCR array consisting of 1020 chambers, each having a volume of 25 pL, using surface-tension-based sample partitioning The high density of this dPCR format (118,900 chambers/cm(2)) allows the analysis of 200 single cells per run, for a total of 204,000 PCR reactions using a device footprint of 10 cm(2) Experiments using RNA dilutions show this device achieves shot-noise-limited performance in quantifying single molecules, with a dynamic range of 10(4) We performed over 1200 single-cell measurements, demonstrating the use of this platform in the absolute quantification of both high- and low-abundance mRNA transcripts, as well as micro-RNAs that are not easily measured using alternative hybridization methods We further apply the specificity and sensitivity of single-cell dPCR to performing measurements of RNA editing events in single cells High-throughput dPCR provides a new tool in the arsenal of single-cell analysis methods, with a unique combination of speed, precision, sensitivity, and specificity We anticipate this approach will enable new studies where high-performance single-cell measurements are essential, including the analysis of transcriptional noise, allelic imbalance, and RNA processing

Suppression of HIV-1 infection by APOBEC3 proteins in primary human CD4(+) T cells is associated with inhibition of processive reverse transcription as well as excessive cytidine deamination.

Abstract: The Vif protein of human immunodeficiency virus type 1 (HIV-1) promotes viral replication by downregulation of the cell-encoded, antiviral APOBEC3 proteins. These proteins exert their suppressive effects through the inhibition of viral reverse transcription as well as the induction of cytidine deamination within nascent viral cDNA. Importantly, these two effects have not been characterized in detail in human CD4(+) T cells, leading to controversies over their possible contributions to viral inhibition in the natural cell targets of HIV-1 replication. Here we use wild-type and Vif-deficient viruses derived from the CD4(+) T cells of multiple donors to examine the consequences of APOBEC3 protein function at natural levels of expression. We demonstrate that APOBEC3 proteins impart a profound deficiency to reverse transcription from the initial stages of cDNA synthesis, as well as excessive cytidine deamination (hypermutation) of the DNAs that are synthesized. Experiments using viruses from transfected cells and a novel method for mapping the 3' termini of cDNAs indicate that the inhibition of reverse transcription is not limited to a few specific sites, arguing that APOBEC3 proteins impede enzymatic processivity. Detailed analyses of mutation spectra in viral cDNA strongly imply that one particular APOBEC3 protein, APOBEC3G, provides the bulk of the antiviral phenotype in CD4(+) T cells, with the effects of APOBEC3F and APOBEC3D being less significant. Taken together, we conclude that the dual mechanisms of action of APOBEC3 proteins combine to deliver more effective restriction of HIV-1 than either function would by itself.

Identification of the N-Terminal Domain of the Influenza Virus PA Responsible for the Suppression of Host Protein Synthesis

Abstract: Cellular protein synthesis is suppressed during influenza virus infection, allowing for preferential production of viral proteins. To explore the impact of polymerase subunits on protein synthesis, we coexpressed enhanced green fluorescent protein (eGFP) or luciferase together with each polymerase component or NS1 of A/California/04/2009 (Cal) and found that PA has a significant impact on the expression of eGFP and luciferase. Comparison of the suppressive activity on coexpressed proteins between various strains revealed that avian virus or avian-origin PAs have much stronger activity than human-origin PAs, such as the one from A/WSN/33 (WSN). Protein synthesis data suggested that reduced expression of coexpressed proteins is not due to PA's reported proteolytic activity. A recombinant WSN containing Cal PA showed enhanced host protein synthesis shutoff and induction of apoptosis. Further characterization of the PA fragment indicated that the N-terminal domain (PANt), which includes the endonuclease active site, is sufficient to suppress cotransfected gene expression. By characterizing various chimeric PANts, we found that multiple regions of PA, mainly the helix α4 and the flexible loop of amino acids 51 to 74, affect the activity. The suppressive effect of PANt cDNA was mainly due to PA-X, which was expressed by ribosomal frameshifting. In both Cal and WSN viruses, PA-X showed a stronger effect than the corresponding PANt, suggesting that the unique C-terminal sequences of PA-X also play a role in suppressing cotransfected gene expression. Our data indicate strain variations in PA gene products, which play a major role in suppression of host protein synthesis.

Evidence that a consensus element found in naturally intronless mRNAs promotes mRNA export

Abstract: We previously showed that mRNAs synthesized from three genes that naturally lack introns contain a portion of their coding sequence, known as a cytoplasmic accumulation region (CAR), which is essential for stable accumulation of the intronless mRNAs in the cytoplasm. The CAR in each mRNA is unexpectedly large, ranging in size from ∼160 to 285 nt. Here, we identified one or more copies of a 10-nt consensus sequence in each CAR. To determine whether this element (designated CAR-E) functions in cytoplasmic accumulation of intronless mRNA, we multimerized the most conserved CAR-E and inserted it upstream of β-globin cDNA, which is normally retained/degraded in the nucleus. Significantly, the tandem CAR-E, but not its antisense counterpart, rescued cytoplasmic accumulation of β-globin cDNA transcripts. Moreover, dinucleotide mutations in the CAR-E abolished this rescue. We show that the CAR-E, but not the mutant CAR-E, associates with components of the TREX mRNA export machinery, the Prp19 complex and U2AF2. Moreover, knockdown of these factors results in nuclear retention of the intronless mRNAs. Together, these data suggest that the CAR-E promotes export of intronless mRNA by sequence-dependent recruitment of the mRNA export machinery.

Binding of RNA by APOBEC3G controls deamination-independent restriction of retroviruses

Abstract: APOBEC3G (A3G) is a host-encoded protein that potently restricts the infectivity of a broad range of retroviruses. This can occur by mechanisms dependent on catalytic activity, resulting in the mutagenic deamination of nascent viral cDNA, and/or by other means that are independent of its catalytic activity. It is not yet known to what extent deamination-independent processes contribute to the overall restriction, how they exactly work or how they are regulated. Here, we show that alanine substitution of either tryptophan 94 (W94A) or 127 (W127A) in the non-catalytic N-terminal domain of A3G severely impedes RNA binding and alleviates deamination-independent restriction while still maintaining DNA mutator activity. Substitution of both tryptophans (W94A/W127A) produces a more severe phenotype in which RNA binding and RNA-dependent protein oligomerization are completely abrogated. We further demonstrate that RNA binding is specifically required for crippling late reverse transcript accumulation, preventing proviral DNA integration and, consequently, restricting viral particle release. We did not find that deaminase activity made a significant contribution to the restriction of any of these processes. In summary, this work reveals that there is a direct correlation between A3G’s capacity to bind RNA and its ability to inhibit retroviral infectivity in a deamination-independent manner.

Discovery of WRKY transcription factors through transcriptome analysis and characterization of a novel methyl jasmonate-inducible PqWRKY1 gene from Panax quinquefolius

Abstract: Transcription factors (TFs) are important regulating factors that can mediate many life processes. However, no TF genes have previously been reported in Panax quinquefolius (American ginseng), with the exception of a few expressed sequence tags. In this study, 753 unigenes (unique sequences) have been annotated in the plant transcription factor database PlnTFDB (version 3.0) by mining a 454 transcriptome dataset of P. quinquefolius. After classification of the unigenes, 45 unigenes were discovered to be annotated as WRKY transcripts in the public databases. Furthermore, PqWRKY1, one of the WRKY family TF genes that respond to methyl jasmonate, was isolated according to the sequences in the 454 transcriptome dataset. The cDNA of PqWRKY1 (P. quinquefolius WRKY1) encodes a putative protein of 358 amino acids, including a WRKY domain and a zinc finger motif. A subcellular localization assay demonstrated that the protein localizes to the nucleus and has strong transcriptional activation activity in transgenic yeast. In comparison to control lines, the PqWRKY1 transgenic Arabidopsis line exhibited insensitive phenotypes when exposed to high salt or mannitol. Correspondingly, in transgenic Arabidopsis lines, the expression levels of some genes involved in the anti-stress process were relatively higher than those in the control lines. Additionally, genes involved in triterpene biosynthesis were expressed onefold to fivefold higher in the transgenic line compared to the control line. These results suggest that the transcription factor PqWRKY1 is a positive regulator related to osmotic stress and triterpene ginsenoside biosynthesis in P. quinquefolius.

Monitoring gene expression: quantitative real-time rt-PCR.

Abstract: Two-step quantitative real-time RT-PCR (RT-qPCR), also known as real-time RT-PCR, kinetic RT-PCR, or quantitative fluorescent RT-PCR, has become the method of choice for gene expression analysis during the last few years. It is a fast and convenient PCR method that combines traditional RT-PCR with the phenomenon of fluorescence resonance energy transfer (FRET) using fluorogenic primers. The detection of changes in fluorescence intensity during the reaction enables the user to follow the PCR reaction in real time.RT-qPCR comprises several steps: (1) RNA is isolated from target tissue/cells; (2) mRNA is reverse-transcribed to cDNA; (3) modified gene-specific PCR primers are used to amplify a segment of the cDNA of interest, following the reaction in real time; and (4) the initial concentration of the selected transcript in a specific tissue or cell type is calculated from the exponential phase of the reaction. Relative quantification or absolute quantification compared to standards that are run in parallel can be performed.This chapter describes the entire procedure from isolation of total RNA from liver and fatty tissues/cells to the use of RT-qPCR to study gene expression in these tissues. We perform relative quantification of transcripts to calculate the fold-difference of a certain mRNA level between different samples. In addition, tips for choosing primers and performing analyses are provided to help the beginner in understanding the technique.

Establishment of a reverse genetics system for Schmallenberg virus, a newly emerged orthobunyavirus in Europe

Abstract: Schmallenberg virus (SBV) is a newly emerged orthobunyavirus that has caused widespread disease in cattle, sheep and goats in Europe. Like other orthobunyaviruses, SBV is characterized by a tripartite negative-sense RNA genome that encodes four structural and two non-structural proteins. This study showed that SBV has a wide in vitro host range, and that BHK-21 cells are a convenient host for both SBV propagation and assay by plaque titration. The SBV genome segments were cloned as cDNA and a three-plasmid rescue system was established to recover infectious virus. Recombinant virus behaved similarly in cell culture to authentic virus. The ORF for the non-structural NSs protein, encoded on the smallest genome segment, was disrupted by introduction of translation stop codons in the appropriate cDNA, and when this plasmid was used in reverse genetics, a recombinant virus that lacked NSs expression was recovered. This virus had reduced capacity to shut-off host-cell protein synthesis compared with the wild-type virus. In addition, the NSs-deleted virus induced interferon (IFN) in cells, indicating that, like other orthobunyaviruses, NSs functions as an IFN antagonist, most probably by globally inhibiting host-cell metabolism. The development of a robust reverse genetics system for SBV will facilitate investigation of its pathogenic mechanisms as well as the creation of attenuated strains that could be candidate vaccines.

Characterization of AFP1, an antifreeze protein from the psychrophilic yeast glaciozyma antarctica PI12

Abstract: The psychrophilic yeast Glaciozyma antarctica demonstrated high antifreeze activity in its culture filtrate. The culture filtrate exhibited both thermal hysteresis (TH) and ice recrystallization inhibition (RI) properties. The TH of 0.1 °C was comparable to that previously reported for bacteria and fungi. A genome sequence survey of the G. antarctica genome identified a novel antifreeze protein gene. The cDNA encoded a 177 amino acid protein with 30 % similarity to a fungal antifreeze protein from Typhula ishikariensis. The expression levels of AFP1 were quantified via real time-quantitative polymerase chain reaction (RT-qPCR), and the highest expression levels were detected within 6 h of growth at −12 °C. The cDNA of the antifreeze protein was cloned into an Escherichia coli expression system. Expression of recombinant Afp1 in E. coli resulted in the formation of inclusion bodies that were subsequently denatured by treatment with urea and allowed to refold in vitro. Activity assays of the recombinant Afp1 confirmed the antifreeze protein properties with a high TH value of 0.08 °C.

Long non-coding genes implicated in response to stripe rust pathogen stress in wheat (Triticum aestivum L.)

Abstract: The non-protein-coding genes have been reported as a critical control role in the regulation of gene expression in abiotic stress. We previously identified four expressed sequence tags numbered S18 (EL773024), S73 (EL773035), S106 (EL773041) and S108 (EL773042) from a SSH-cDNA library of bread wheat Shaanmai 139 infected with Puccinia striiformis f. sp. tritici (Pst). Here, we isolated four cDNA clones and referred them as TalncRNA18, TalncRNA73, TalncRNA106 and TalncRNA108 (GenBank: KC549675-KC549678). These cDNA separately consisted of 1,393, 667, 449 and 647 nucleotides but without any open reading frame. The alignment result showed that TalncRNA18 is a partial cDNA of E3 ubiquitin-protein ligase UPL1-like gene, TalncRNA73 is an antisense transcript of hypothetical protein, TalncRNA108 is a homolog to RRNA intron-encoded homing endonuclease, and lncRNA106 had no similarly sequence. Quantitative RT-PCR studies confirmed that these four lncRNAs were differentially expressed in three near isogenic lines. TalncRNA108 was significantly stepwise decreased at early stage of inoculation with Pst, while the others were upregulated, especially at 1 and 3 dpi (days post-inoculation). Using Chinese Spring nulli-tetrasomic lines and its ditelosomic lines, TalncRNA73 and TalncRNA108 were located to wheat chromosome 7A and the short arm of chromosome 4B, respectively, while TalncRNA18 and TalncRNA106 were located to chromosome 5B. Comparing the sequence of DNA and cDNA of four lncRNAs with polymerase chain reaction primers, the results showed that all of them have no introns. The kinetics analyses of lncRNAs expression as a result of pathogen challenge in immune resistant genotype indicated that they may play the roles of modulating or silencing the protein-coding gene into pathogen-defence response.

Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

Abstract: Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in soybean defence against soybean cyst nematode (Heterodera glycines Ichinohe, SCN). GmSAMT1 was identified as a candidate SCN defence-related gene in our previous analysis of soybean defence against SCN using GeneChip microarray experiments. The current study started with the isolation of the full-length cDNAs of GmSAMT1 from a SCN-resistant soybean line and from a SCN-susceptible soybean line. The two cDNAs encode proteins of identical sequences. The GmSAMT1 cDNA was expressed in Escherichia coli. Using in vitro enzyme assays, E. coli-expressed GmSAMT1 was confirmed to function as salicylic acid methyltransferase. The apparent Km value of GmSAMT1 for salicylic acid was approximately 46 μM. To determine the role of GmSAMT1 in soybean defence against SCN, transgenic hairy roots overexpressing GmSAMT1 were produced and tested for SCN resistance. Overexpression of GmSAMT1 in SCN-susceptible backgrounds significantly reduced the development of SCN, indicating that overexpression of GmSAMT1 in the transgenic hairy root system could confer resistance to SCN. Overexpression of GmSAMT1 in transgenic hairy roots was also found to affect the expression of selected genes involved in salicylic acid biosynthesis and salicylic acid signal transduction.

Ectopic expression of wheat TaCIPK14, encoding a calcineurin B‐like protein‐interacting protein kinase, confers salinity and cold tolerance in tobacco

Abstract: Calcineurin B-like protein-interacting protein kinases (CIPKs) are components of Ca(2+) signaling in responses to abiotic stresses. In this work, the full-length cDNA of a novel CIPK gene (TaCIPK14) was isolated from wheat and was found to have significant sequence similarity to OsCIPK14/15. Subcellular localization assay revealed the presence of TaCIPK14 throughout the cell. qRT-PCR analysis showed that TaCIPK14 was upregulated under cold conditions or when treated with salt, PEG or exogenous stresses related signaling molecules including ABA, ethylene and H2 O2 . Transgenic tobaccos overexpressing TaCIPK14 exhibited higher contents of chlorophyll and sugar, higher catalase activity, while decreased amounts of H2 O2 and malondialdehyde, and lesser ion leakage under cold and salt stresses. In addition, overexpression also increased seed germination rate, root elongation and decreased Na(+) content in the transgenic lines under salt stress. Higher expression of stress-related genes was observed in lines overexpressing TaCIPK14 compared to controls under stress conditions. In summary, these results suggested that TaCIPK14 is an abiotic stress-responsive gene in plants.

Identification of a novel, recurrent SLC44A1-PRKCA fusion in papillary glioneuronal tumor.

Abstract: Mixed neuronal-glial tumors are rare and challenging to subclassify. One recently recognized variant, papillary glioneuronal tumor (PGNT), is characterized by prominent pseudopapillary structures and glioneuronal elements. We identified a novel translocation, t(9;17)(q31;q24), as the sole karyotypic anomaly in two PGNTs. A fluorescence in situ hybridization (FISH)-based positional cloning strategy revealed SLC44A1, a member of the choline transporter-like protein family, and PRKCA, a protein kinase C family member of serine/threonine-specific protein kinases, as the 9q31 and 17q24 breakpoint candidate genes, respectively. Reverse transcription-polymerase chain reaction (RT-PCR) analysis using a forward primer from SLC44A1 exon 5 and a reverse primer from PRKCA exon 10 confirmed the presence of a SLC44A1-PRKCA fusion product in both tumors. Sequencing of each chimeric transcript uncovered an identical fusion cDNA junction occurring between SLC44A1 exon 15 and PRKCA exon 9. A dual-color breakpoint-spanning probe set custom-designed for interphase cell recognition of the translocation event identified the fusion in a third PGNT. These results suggest that the t(9;17)(q31;q24) with the resultant novel fusion oncogene SLC44A1-PRKCA is the defining molecular feature of PGNT that may be responsible for its pathogenesis. The FISH and RT-PCR assays developed in this study can serve as valuable diagnostic adjuncts for this rare disease entity.

Association of RNAs with Bacillus subtilis Hfq

Abstract: The prevalence and characteristics of small regulatory RNAs (sRNAs) have not been well characterized for Bacillus subtilis, an important model system for Gram-positive bacteria. However, B. subtilis was recently found to synthesize many candidate sRNAs during stationary phase. In the current study, we performed deep sequencing on Hfq-associated RNAs and found that a small subset of sRNAs associates with Hfq, an enigmatic RNA-binding protein that stabilizes sRNAs in Gram-negatives, but whose role is largely unknown in Gram-positive bacteria. We also found that Hfq associated with antisense RNAs, antitoxin transcripts, and many mRNA leaders. Several new candidate sRNAs and mRNA leader regions were also discovered by this analysis. Additionally, mRNA fragments overlapping with start or stop codons associated with Hfq, while, in contrast, relatively few full-length mRNAs were recovered. Deletion of hfq reduced the intracellular abundance of several representative sRNAs, suggesting that B. subtilis Hfq-sRNA interactions may be functionally significant in vivo. In general, we anticipate this catalog of Hfq-associated RNAs to serve as a resource in the functional characterization of Hfq in B. subtilis.

Base Preferences in Non-Templated Nucleotide Incorporation by MMLV-Derived Reverse Transcriptases

Abstract: Reverse transcriptases derived from Moloney Murine Leukemia Virus (MMLV) have an intrinsic terminal transferase activity, which causes the addition of a few non-templated nucleotides at the 3´ end of cDNA, with a preference for cytosine. This mechanism can be exploited to make the reverse transcriptase switch template from the RNA molecule to a secondary oligonucleotide during first-strand cDNA synthesis, and thereby to introduce arbitrary barcode or adaptor sequences in the cDNA. Because the mechanism is relatively efficient and occurs in a single reaction, it has recently found use in several protocols for single-cell RNA sequencing. However, the base preference of the terminal transferase activity is not known in detail, which may lead to inefficiencies in template switching when starting from tiny amounts of mRNA. Here, we used fully degenerate oligos to determine the exact base preference at the template switching site up to a distance of ten nucleotides. We found a strong preference for guanosine at the first non-templated nucleotide, with a greatly reduced bias at progressively more distant positions. Based on this result, and a number of careful optimizations, we report conditions for efficient template switching for cDNA amplification from single cells.