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Showing papers on "Complementary DNA published in 2019"


Journal ArticleDOI
TL;DR: It is shown that viral Reps evolved from Reps of bacterial and archaeal plasmids on multiple independent occasions, shedding light on the complex evolutionary history of a major class of viruses revealing its polyphyletic origins.
Abstract: Single-stranded (ss) DNA viruses are a major component of the earth virome. In particular, the circular, Rep-encoding ssDNA (CRESS-DNA) viruses show high diversity and abundance in various habitats. By combining sequence similarity network and phylogenetic analyses of the replication proteins (Rep) belonging to the HUH endonuclease superfamily, we show that the replication machinery of the CRESS-DNA viruses evolved, on three independent occasions, from the Reps of bacterial rolling circle-replicating plasmids. The CRESS-DNA viruses emerged via recombination between such plasmids and cDNA copies of capsid genes of eukaryotic positive-sense RNA viruses. Similarly, the rep genes of prokaryotic DNA viruses appear to have evolved from HUH endonuclease genes of various bacterial and archaeal plasmids. Our findings also suggest that eukaryotic polyomaviruses and papillomaviruses with dsDNA genomes have evolved via parvoviruses from CRESS-DNA viruses. Collectively, our results shed light on the complex evolutionary history of a major class of viruses revealing its polyphyletic origins.

104 citations


Journal ArticleDOI
TL;DR: Two pAgos from mesophilic bacteria are described that act as DNA-guided DNA nucleases at physiological temperatures that can perform programmable endonucleolytic cleavage of double-stranded DNA substrates.
Abstract: Argonaute (Ago) proteins are key players in RNA interference in eukaryotes, where they function as RNA-guided RNA endonucleases. Prokaryotic Argonautes (pAgos) are much more diverse than their eukaryotic counterparts but their cellular functions and mechanisms of action remain largely unknown. Some pAgos were shown to use small DNA guides for endonucleolytic cleavage of complementary DNA in vitro. However, previously studied pAgos from thermophilic prokaryotes function at elevated temperatures, which limits their potential use as a tool in genomic applications. Here, we describe two pAgos from mesophilic bacteria, Clostridium butyricum (CbAgo) and Limnothrix rosea (LrAgo), that act as DNA-guided DNA nucleases at physiological temperatures. In comparison with previously studied pAgos, CbAgo and LrAgo do not show strong preferences for the 5'-nucleotide in guide DNA and can use not only 5'-phosphorylated but also 5'-hydroxyl DNA guides. Both CbAgo and LrAgo can tolerate guide/target mismatches in the seed region, but are sensitive to mismatches in the 3'-guide region. Both pAgos can perform programmable endonucleolytic cleavage of double-stranded DNA substrates, showing enhanced activity at AT-rich regions and at elevated temperatures. The biochemical characterization of mesophilic pAgo proteins paves the way for their use for DNA manipulations both in vitro and in vivo.

75 citations


Posted ContentDOI
29 Jan 2019-bioRxiv
TL;DR: The functional and structural characterization of the pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo) is reported, providing an important step towards the development of pAgos for genetic engineering applications.
Abstract: Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity has been repurposed for programmable DNA cleavage in vitro. However, currently characterized DNA-cleaving pAgos require elevated temperatures (>65{degrees}C) for their activity, making them less suitable for in vivo applications at moderate temperatures. Here, using biochemistry, X-ray crystallography, and single-molecule fluorescence methods, we report the functional and structural characterization of the pAgo from the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo can be reprogrammed with siDNAs to cleave complementary DNA, but not RNA. CbAgo displays a preference for siDNAs that have a deoxyadenosine at the 5'-end and thymidines in the sub-seed segment (siDNA nucleotides 2-4). Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37{degrees}C). Taken together, this study provides an important step towards the development of pAgos for genetic engineering applications.

49 citations


Journal ArticleDOI
24 Jun 2019-PLOS ONE
TL;DR: A simplified workflow for amplification of IgG antibody variable regions from hybridoma RNA by a specialized RT-PCR followed by Sanger sequencing is described, which successfully sequenced the variable regions of five mouse monoclonal IgG antibodies and enabled the design of chimeric mouse/human antibody expression plasmids for recombinant antibody production in mammalian cell culture expression systems.
Abstract: The diversity of antibody variable regions makes cDNA sequencing challenging, and conventional monoclonal antibody cDNA amplification requires the use of degenerate primers. Here, we describe a simplified workflow for amplification of IgG antibody variable regions from hybridoma RNA by a specialized RT-PCR followed by Sanger sequencing. We perform three separate reactions for each hybridoma: one each for kappa, lambda, and heavy chain transcripts. We prime reverse transcription with a primer specific to the respective constant region and use a template-switch oligonucleotide, which creates a custom sequence at the 5' end of the antibody cDNA. This template-switching circumvents the issue of low sequence homology and the need for degenerate primers. Instead, subsequent PCR amplification of the antibody cDNA molecules requires only two primers: one primer specific for the template-switch oligonucleotide sequence and a nested primer to the respective constant region. We successfully sequenced the variable regions of five mouse monoclonal IgG antibodies using this method, which enabled us to design chimeric mouse/human antibody expression plasmids for recombinant antibody production in mammalian cell culture expression systems. All five recombinant antibodies bind their respective antigens with high affinity, confirming that the amino acid sequences determined by our method are correct and demonstrating the high success rate of our method. Furthermore, we also designed RT-PCR primers and amplified the variable regions from RNA of cells transfected with chimeric mouse/human antibody expression plasmids, showing that our approach is also applicable to IgG antibodies of human origin. Our monoclonal antibody sequencing method is highly accurate, user-friendly, and very cost-effective.

38 citations


Journal ArticleDOI
TL;DR: To develop a versatile host for basidiomycete genes, gene expression using genomic DNA sequences in the robust ascomycete host Aspergillus oryzae, which is frequently used for the production of metabolites from filamentous fungi is examined.
Abstract: Basidiomycete fungi are an attractive resource for biologically active natural products for use in pharmaceutically relevant compounds. Recently, genome projects on mushroom fungi have provided a great deal of biosynthetic gene cluster information. However, functional analyses of the gene clusters for natural products were largely unexplored because of the difficulty of cDNA preparation and lack of gene manipulation tools for basidiomycete fungi. To develop a versatile host for basidiomycete genes, we examined gene expression using genomic DNA sequences in the robust ascomycete host Aspergillus oryzae, which is frequently used for the production of metabolites from filamentous fungi. Exhaustive expression of 30 terpene synthase genes from the basidiomycetes Clitopilus pseudo-pinsitus and Stereum hirsutum showed two splicing patterns, i.e., completely spliced cDNAs giving terpenes (15 cases) and mostly spliced cDNAs, indicating that A. oryzae correctly spliced most introns at the predicted positions and lengths. The mostly spliced cDNAs were expressed after PCR-based removal of introns, resulting in the successful production of terpenes (14 cases). During this study, we observed relatively frequent mispredictions in the automated program. Hence, the complementary use of A. oryzae expression and automated prediction will be a powerful tool for genome mining.IMPORTANCE The recent large influx of genome sequences from basidiomycetes, which are prolific producers of bioactive natural products, may provide opportunities to develop novel drug candidates. The development of a reliable expression system is essential for the genome mining of natural products because of the lack of a tractable host for heterologous expression of basidiomycete genes. For this purpose, we applied the ascomycete Aspergillus oryzae system for the direct expression of fungal natural product biosynthetic genes from genomic DNA. Using this system, 29 sesquiterpene synthase genes and diterpene biosynthetic genes for bioactive pleuromutilin were successfully expressed. Together with the use of computational tools for intron prediction, this Aspergillus oryzae system represents a practical method for the production of basidiomycete natural products.

37 citations


Journal ArticleDOI
TL;DR: A bisulfite-free and base-resolution sequencing method based on peroxotungstate oxidation is presented for the identification of hm5C sites in the transcriptome and m5C can also be detected in a procedure termed TET-Assisted WO-Seq (TAWO- Seq).

31 citations


Journal ArticleDOI
TL;DR: It is found that, while ssDNA and ssRNA are well tolerated, TET2 is most proficient at dsDNA oxidation and discriminates strongly against dsRNA, suggesting a broad range of plausible roles for TET-mediated 5mC oxidation in cells.
Abstract: Enzymes of the ten-eleven translocation (TET) family add diversity to the repertoire of nucleobase modifications by catalyzing the oxidation of 5-methylcytosine (5mC). TET enzymes were initially found to oxidize 5-methyl-2'-deoxycytidine in genomic DNA, yielding products that contribute to epigenetic regulation in mammalian cells, but have since been found to also oxidize 5-methylcytidine in RNA. Considering the different configurations of single-stranded (ss) and double-stranded (ds) DNA and RNA that coexist in a cell, defining the scope of TET's preferred activity and the mechanisms of substrate selectivity is critical to better understand the enzymes' biological functions. To this end, we have systematically examined the activity of human TET2 on DNA, RNA, and hybrid substrates in vitro. We found that, while ssDNA and ssRNA are well tolerated, TET2 is most proficient at dsDNA oxidation and discriminates strongly against dsRNA. Chimeric and hybrid substrates containing mixed DNA and RNA character helped reveal two main features by which the enzyme discriminates between substrates. First, the identity of the target nucleotide alone is the strongest reactivity determinant, with a preference for 5-methyldeoxycytidine, while both DNA or RNA are relatively tolerated on the rest of the target strand. Second, while a complementary strand is not required for activity, DNA is the preferred partner, and complementary RNA diminishes reactivity. Our biochemical analysis, complemented by molecular dynamics simulations, provides support for an active site optimally configured for dsDNA reactivity but permissive for various nucleic acid configurations, suggesting a broad range of plausible roles for TET-mediated 5mC oxidation in cells.

30 citations


Journal ArticleDOI
TL;DR: The mechanistic details of Cas12a-mediated cis- and trans-cleavage of DNA cleavage are reviewed, and how bacteriophage-derived anti-CRISPR proteins can inhibit Cas 12a activity are discussed.
Abstract: CRISPR-Cas12a (previously named Cpf1) is a prokaryotic deoxyribonuclease that can be programmed with an RNA guide to target complementary DNA sequences. Upon binding of the target DNA, Cas12a induces a nick in each of the target DNA strands, yielding a double-stranded DNA break. In addition to inducing cis-cleavage of the targeted DNA, target DNA binding induces trans-cleavage of non-target DNA. As such, Cas12a-RNA guide complexes can provide sequence-specific immunity against invading nucleic acids such as bacteriophages and plasmids. Akin to CRISPR-Cas9, Cas12a has been repurposed as a genetic tool for programmable genome editing and transcriptional control in both prokaryotic and eukaryotic cells. In addition, its trans-cleavage activity has been applied for high-sensitivity nucleic acid detection. Despite the demonstrated value of Cas12a for these applications, the exact molecular mechanisms of both cis- and trans-cleavage of DNA were not completely understood. Recent studies have revealed mechanistic details of Cas12a-mediates DNA cleavage: base pairing of the RNA guide and the target DNA induces major conformational changes in Cas12a. These conformational changes render Cas12a in a catalytically activated state in which it acts as deoxyribonuclease. This deoxyribonuclease activity mediates cis-cleavage of the displaced target DNA strand first, and the RNA guide-bound target DNA strand second. As Cas12a remains in the catalytically activated state after cis-cleavage, it subsequently demonstrates trans-cleavage of non-target DNA. Here, I review the mechanistic details of Cas12a-mediated cis- and trans-cleavage of DNA. In addition, I discuss how bacteriophage-derived anti-CRISPR proteins can inhibit Cas12a activity.

30 citations


Journal ArticleDOI
TL;DR: Although it has lower throughput than droplet-based methods, rhTCRseq is well-suited to analysis of small sorted populations, especially when analysis of 96 or 384 single cells is sufficient to identify predominant T-cell clones.
Abstract: RNase H-dependent PCR-enabled T-cell receptor sequencing (rhTCRseq) can be used to determine paired alpha/beta T-cell receptor (TCR) clonotypes in single cells or perform alpha and beta TCR repertoire analysis in bulk RNA samples. With the enhanced specificity of RNase H-dependent PCR (rhPCR), it achieves TCR-specific amplification and addition of dual-index barcodes in a single PCR step. For single cells, the protocol includes sorting of single cells into plates, generation of cDNA libraries, a TCR-specific amplification step, a second PCR on pooled sample to generate a sequencing library, and sequencing. In the bulk method, sorting and cDNA library steps are replaced with a reverse-transcriptase (RT) reaction that adds a unique molecular identifier (UMI) to each cDNA molecule to improve the accuracy of repertoire-frequency measurements. Compared to other methods for TCR sequencing, rhTCRseq has a streamlined workflow and the ability to analyze single cells in 384-well plates. Compared to TCR reconstruction from single-cell transcriptome sequencing data, it improves the success rate for obtaining paired alpha/beta information and ensures recovery of complete complementarity-determining region 3 (CDR3) sequences, a prerequisite for cloning/expression of discovered TCRs. Although it has lower throughput than droplet-based methods, rhTCRseq is well-suited to analysis of small sorted populations, especially when analysis of 96 or 384 single cells is sufficient to identify predominant T-cell clones. For single cells, sorting typically requires 2-4 h and can be performed days, or even months, before library construction and data processing, which takes ~4 d; the bulk RNA protocol takes ~3 d.

21 citations


Book ChapterDOI
TL;DR: The STRT-C1 method is described and the steps involved in capturing 96 cells across C1 microfluidics chip, cDNA synthesis, and preparing single-cell libraries for Illumina short-read sequencing are described.
Abstract: Single-cell RNA sequencing (scRNA-seq) has become an established approach to profile entire transcriptomes of individual cells from different cell types, tissues, species, and organisms. Single-cell tagged reverse transcription sequencing (STRT-seq) is one of the early single-cell methods which utilize 5' tag counting of transcripts. STRT-seq performed on microfluidics Fluidigm C1 platform (STRT-C1) is a flexible scRNA-seq approach that allows for accurate, sensitive and importantly molecular counting of transcripts at single-cell level. Herein, I describe the STRT-C1 method and the steps involved in capturing 96 cells across C1 microfluidics chip, cDNA synthesis, and preparing single-cell libraries for Illumina short-read sequencing.

21 citations


Journal ArticleDOI
TL;DR: The improved efficacy and reassuring safety profile support the potential application of the proposed AAV-mediated gene therapy in the liver to other liver diseases.
Abstract: Non-integrative AAV-mediated gene therapy in the liver is effective in adult patients, but faces limitations in pediatric settings due to episomal DNA loss during hepatocyte proliferation. Gene targeting is a promising approach by permanently modifying the genome. We previously rescued neonatal lethality in Crigler-Najjar mice by inserting a promoterless human uridine glucuronosyl transferase A1 (UGT1A1) cDNA in exon 14 of the albumin gene, without the use of nucleases. To increase recombination rate and therapeutic efficacy, here we used CRISPR/SaCas9. Neonatal mice were transduced with two AAVs: one expressing the SaCas9 and sgRNA, and one containing a promoterless cDNA flanked by albumin homology regions. Targeting efficiency increased ~26-fold with an eGFP reporter cDNA, reaching up to 24% of eGFP-positive hepatocytes. Next, we fully corrected the diseased phenotype of Crigler-Najjar mice by targeting the hUGT1A1 cDNA. Treated mice had normal plasma bilirubin up to 10 months after administration, hUGT1A1 protein levels were ~6-fold higher than in WT liver, with a 90-fold increase in recombination rate. Liver histology, inflammatory markers, and plasma albumin were normal in treated mice, with no off-targets in predicted sites. Thus, the improved efficacy and reassuring safety profile support the potential application of the proposed approach to other liver diseases.

Posted ContentDOI
25 Mar 2019-bioRxiv
TL;DR: In this paper, the authors used reverse genetic tools optimized for primary human CD4+ T cells, macrophages, and dendritic cells to demonstrate that disruption of the CA-CypA interaction rendered HIV-1 susceptible to restriction by human TRIM5α, with the block occurring before reverse transcription.
Abstract: The capsid (CA) protein lattice of HIV-1 and other retroviruses encases viral genomic RNA and regulates steps that are essential to retroviral invasion of target cells, including reverse transcription, nuclear trafficking, and integration of viral cDNA into host chromosomal DNA1. Cyclophilin A (CypA), the first cellular protein reported to bind HIV-1 CA2, has interacted with invading lentiviruses related to HIV-1 for millions of years3–7. Disruption of the CA-CypA interaction decreases HIV-1 infectivity in human cells8–12, but stimulates infectivity in non-human primate cells13–15. Genetic and biochemical data suggest that CypA interaction with CA protects HIV-1 from a restriction factor in human cells16–20. Discovery of the CA-specific restriction factor TRIM5α21, and of TRIM5-CypA fusion genes that were independently generated at least four times in phylogeny4,5,15,22-25, pointed to human TRIM5α as the CypA-sensitive restriction factor. However, significant HIV-1 restriction by human TRIM5α21, let alone inhibition of such activity by CypA26, has not been detected. Here, exploiting reverse genetic tools optimized for primary human CD4+ T cells, macrophages, and dendritic cells, we demonstrate that disruption of the CA-CypA interaction renders HIV-1 susceptible to restriction by human TRIM5α, with the block occurring before reverse transcription. Identical findings were obtained with single-cycle vectors or with replication-competent HIV-1, including sexually-transmitted clones from sub-Saharan Africa. Endogenous TRIM5α was observed to associate with virion cores as they entered the macrophage cytoplasm, but only when the CA-CypA interaction was disrupted. These experiments resolve the long-standing mystery of the role of CypA in HIV-1 replication by demonstrating that this ubiquitous cellular protein shields HIV-1 from previously inapparent, but potent inhibition, imposed by human TRIM5α. Hopefully this reinvigorates development of CypA-inhibitors for treatment of HIV-1 and other CypA-dependent pathogens27–30.

Journal ArticleDOI
TL;DR: The cloned two alternative splicing variants of the chitin synthase 1 gene (SfCHS1) from the white-backed planthopper, Sogatella furcifera, indicate that Sf CHS1 may be a potential target gene for RNAi-based S. fur cifera control.
Abstract: Chitin synthase is responsible for chitin synthesis in the cuticles and cuticular linings of other tissues in insects. We cloned two alternative splicing variants of the chitin synthase 1 gene (SfCHS1) from the white-backed planthopper, Sogatella furcifera. The full-length cDNA of the two variants (SfCHS1a and SfCHS1b) consists of 6408 bp, contains a 4719-bp open reading frame encoding 1572 amino acids, and has 5′ and 3′ non-coding regions of 283 and 1406 bp, respectively. The two splicing variants occur at the same position in the cDNA sequence between base pairs 4115 and 4291, and consist of 177 nucleotides that encode 59 amino acids but show 74.6% identity at the amino acid level. Analysis in different developmental stages showed that expression of SfCHS1 and SfCHS1a were highest just after molting, whereas SfCHS1b reached its highest expression level 2 days after molting. Further, SfCHS1 and SfCHS1a were mainly expressed in the integument, whereas SfCHS1b was predominately expressed in the gut and fat body. RNAi-based gene silencing inhibited transcript levels of the corresponding mRNAs in S. furcifera nymphs injected with double-stranded RNA of SfCHS1, SfCHS1a, and SfCHS1b, resulted in malformed phenotypes, and killed most of the treated nymphs. Our results indicate that SfCHS1 may be a potential target gene for RNAi-based S. furcifera control.

Journal ArticleDOI
TL;DR: The zebrafish epg5 mutant is shown to be a valuable model for dissecting the contribution of EPG5 on the onset and progression of Vici syndrome as well as for the screening of autophagy-stimulating drugs.
Abstract: The EPG5 protein is a RAB7A effector involved in fusion specificity between autophagosomes and late endosomes or lysosomes during macroautophagy/autophagy. Mutations in the human EPG5 gene cause a rare and severe multisystem disorder called Vici syndrome. In this work, we show that zebrafish epg5-/- mutants from both heterozygous and incrossed homozygous matings are viable and can develop to the age of sexual maturity without conspicuous defects in external appearance. In agreement with the dysfunctional autophagy of Vici syndrome, western blot revealed higher levels of the Lc3-II autophagy marker in epg5-/- mutants with respect to wild type controls. Moreover, starvation elicited higher accumulation of Lc3-II in epg5-/- than in wild type larvae, together with a significant reduction of skeletal muscle birefringence. Accordingly, muscle ultrastructural analysis revealed accumulation of degradation-defective autolysosomes in starved epg5-/- mutants. By aging, epg5-/- mutants showed impaired motility and muscle thinning, together with accumulation of non-degradative autophagic vacuoles. Furthermore, epg5-/- adults displayed morphological alterations in gonads and heart. These findings point at the zebrafish epg5 mutant as a valuable model for EPG5-related disorders, thus providing a new tool for dissecting the contribution of EPG5 on the onset and progression of Vici syndrome as well as for the screening of autophagy-stimulating drugs. Abbreviations: ATG: autophagy related; cDNA: complementary DNA; DIG: digoxigenin; dpf: days post-fertilization; EGFP: enhanced green fluorescent protein; EPG: ectopic P granules; GFP: green fluorescent protein; hpf: hours post-fertilization; IL1B: interleukin 1 beta; Lc3-II: lipidated Lc3; mpf: months post-fertilization; mRNA: messenger RNA; NMD: nonsense-mediated mRNA decay; PCR: polymerase chain reaction; qPCR: real time-polymerase chain reaction; RAB7A/RAB7: RAB7a, member RAS oncogene family; RACE: rapid amplification of cDNA ends; RFP: red fluorescent protein; RT-PCR: reverse transcriptase-polymerase chain reaction; SEM: standard error of the mean; sgRNA: guide RNA; UTR: untranslated region; WMISH: whole mount in situ hybridization; WT: wild type.

Journal ArticleDOI
TL;DR: Evidence that Pif1 family helicases act on RNA-DNA hybrids and their potential roles in complementing RNase H for R-loop resolution are discussed and discussed.

Journal ArticleDOI
TL;DR: A DNA-templated copper nanoparticle probe has been developed for the determination of the human immunodeficiency virus oligonucleotide (HIV-DNA) and has large potential in biosensing because it may be extended to various other DNA targets.
Abstract: A DNA-templated copper nanoparticle (CuNP) probe has been developed for the determination of the human immunodeficiency virus oligonucleotide (HIV-DNA). The function of the probe relies on affinity binding-induced DNA hybridization associated with the use of double G-quadruplexes. Double-stranded DNA (dsDNA) with poly(AT-TA) bases was used as a template for synthesis of dsDNA-CuNPs. These have weak fluorescence. In the next step, two G-rich sequences that are linked to both sides of the ds-DNA are locked by HIV complementary DNA (cDNA). If HIV-DNA is introduced, it will hybridize with cDNA, thereby transforming the two G-rich sequences into G-quadruplexes. This enhances the fluorescence of the adjacent dsDNA-CuNPs. Fluorescence increases linearly in the 1 to 200 and 250–1000 nM HIV-DNA concentration range, and the detection limit is 13 pM. This enzyme-free fluorometric assay is time-saving, easily operated, and therefore has large potential in biosensing because it may be extended to various other DNA targets.

Journal ArticleDOI
30 Mar 2019-Gene
TL;DR: It is concluded that ABHD5 gene regulated by Evi1 and C/EBPα could be used as potential marker in marker assisted selection for the improvement of Qinchuan cattle breed for carcass quality traits.

Journal ArticleDOI
TL;DR: Overexpression in yeast and Arabidopsis showed that TaPR-1-1 conferred tolerance to these stresses, concluding that screening cDNA yeast libraries following abiotic stress is an efficient way to identify stress-tolerance genes.
Abstract: Abiotic stress significantly impacts growth and yield of crop plants. It is imperative for crop improvement to discover and utilize stress-tolerant functional genes. In this study, genes responding to abiotic stresses, such as freezing, salt and osmotic stress, were screened from a cDNA yeast library that was constructed from the drought- and heat-tolerant wheat variety Hanxuan 10. After screening for surviving clones we isolated 7,249, 4,313 and 4,469 raw sequences, corresponding to 4,695, 2,641 and 2,771 genes following each treatment. Venn diagrams revealed 377 overlapping genes. GO analysis suggested that these genes were mainly involved in the metabolic and stress signal pathways. KEGG pathway enrichment analysis indicated that the isolated genes predominantly belonged to pathways concerning energy and metabolism. Overlapping gene TaPR-1-1 within the pathogenesis-related (PR) protein family was selected for detailed characterization. Although previous studies had shown that PR genes function during pathogen attack, our results demonstrated that TaPR-1-1 expression was also induced by freezing, salinity, and osmotic stresses. Overexpression in yeast and Arabidopsis showed that TaPR-1-1 conferred tolerance to these stresses. We concluded that screening cDNA yeast libraries following abiotic stress is an efficient way to identify stress-tolerance genes.

Journal ArticleDOI
TL;DR: Intriguingly, CCK assay suggested that CiSAMHD1 decreased cell viability and apoptosis, and TUNEL apoptosis assay and Hoechst 33258 staining assay indicated that apoptosis is induced by the overexpression of Ci SAMHD1.

Journal ArticleDOI
Hui Wei1, Chen Xu1, Ali Movahedi1, Weibo Sun1, Dawei Li1, Qiang Zhuge1 
TL;DR: PtHMGR overexpression significantly increased ABA, GA, carotene, and lycopene content, indicating that PtHMGR participates in the regulation of terpenoid compound synthesis.
Abstract: In the mevalonic acid (MVA) pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) is considered the first rate-limiting enzyme in isoprenoid biosynthesis. In this study, we cloned a full-length cDNA from Populus trichocarpa with an open reading frame of 1,734 bp. The deduced PtHMGR sequence contained two HMG-CoA motifs and two NADPH motifs, which exhibited homology with HMGR proteins from other species. Subsequently, truncated PtHMGR was expressed in Escherichia coli BL21 (DE3) cells, and enzyme activity analysis revealed that the truncated PtHMGR protein could catalyze the reaction of HMG-CoA and NADPH to form MVA. Relative expression analysis suggests that PtHMGR expression varies among tissues and that PtHMGR responds significantly to abscisic acid (ABA), NaCl, PEG6000, hydrogen peroxide (H2O2), and cold stresses. We used polymerase chain reaction (PCR) analysis to select transgenic Nanlin 895 poplars (Populus× euramericana cv.) and quantitative reverse-transcription PCR (qRT-PCR) to show that PtHMGR expression levels were 3- to 10-fold higher in transgenic lines than in wild-type (WT) poplars. qRT-PCR was also used to determine transcript levels of methylerythritol phosphate (MEP)-, MVA-, and downstream-related genes, indicating that overexpression of PtHMGR not only affects expression levels of MVA-related genes, but also those of MEP-related genes. We also measured the content of terpenoids including ABA, gibberellic acid (GA), carotenes, and lycopene. PtHMGR overexpression significantly increased ABA, GA, carotene, and lycopene content, indicating that PtHMGR participates in the regulation of terpenoid compound synthesis.

Journal ArticleDOI
TL;DR: The Trizol-based method is efficient as well as economical to use for quantification of circulating miRNAs and the storage of miRNA-derived cDNAs may be an alternative choice to avoid the stability effect.
Abstract: MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression at the post-transcriptional level. Since aberrant expression of miRNAs has been proposed as usage for blood-based biomarkers, hence reliable techniques for miRNA isolation as well as stability of miRNAs in various stored conditions needs to be explored. This present study aimed to investigate the efficacy of the Trizol-based isolation technique and the stability of miRNAs in stored serum and cDNA derivatives. Total RNA, including miRNAs, was isolated from human serum and a comparison of the efficiency of the Trizol®LS reagent isolation method against the miRNeasy®mini kit was conducted. Expression of RNU6, miR-145, and miR-20a was determined by quantitative real-time polymerase chain reaction (qRT-PCR). We showed that Trizol®LS isolation yielded significantly lower RNA concentrations than that of the miRNeasy®mini kit by approximately 35%. Purity of the isolated RNAs by both methods was similar. RNU6, miR-145, and miR-20a degraded at room temperature, but all genes were stable at 4oC, -20oC and -80oC for a 72-hrs period, in both serum and cDNA storage conditions. In the stored cDNA derivatives, we observed the stability of RNU6, miR-145, and miR-20a for 3 months at -20oC, and all genes also resisted 4 repeated freeze-thaw cycles at -20oC. In conclusion, the Trizol-based method is efficient as well as economical to use for quantification of circulating miRNAs. In addition, we proposed that the storage of miRNA-derived cDNAs may be an alternative choice to avoid the stability effect.

Journal ArticleDOI
Ling Duan1, Jingwen Yu1, Li Xu1, Ping Tian1, Xin Hu1, Xiaomei Song1, Pan Yu1 
TL;DR: It is demonstrated that CsMT4 might be obviously induced by the metal stress in cucumber, and improves tolerance to Cd ions but not Zn ions when heterologously expressed in E. coli, and suggested that the composition and arrangement of N-terminal Cys-residues in MT4 are associated with their binding capacity and preference for different metal ions.

Journal ArticleDOI
TL;DR: Results demonstrate that the G, NV and L genes of VHSV are not, by themselves or in combination, major determinants of host-specific virulence in trout.
Abstract: Viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus belonging to the Novirhabdovirus genus, causes severe disease and mortality in many marine and freshwater fish species worldwide. VHSV isolates are classified into four genotypes and each group is endemic to specific geographic regions in the north Atlantic and Pacific Oceans. Most viruses in the European VHSV genotype Ia are highly virulent for rainbow trout (Oncorhynchus mykiss), whereas, VHSV genotype IVb viruses from the Great Lakes region in the United States, which caused high mortality in wild freshwater fish species, are avirulent for trout. This study describes molecular characterization and construction of an infectious clone of the virulent VHSV-Ia strain DK-3592B from Denmark, and application of the clone in reverse genetics to investigate the role of selected VHSV protein(s) in host-specific virulence in rainbow trout (referred to as trout-virulence). Overlapping cDNA fragments of the DK-3592B genome were cloned after RT-PCR amplification, and their DNA sequenced by the di-deoxy chain termination method. A full-length cDNA copy (pVHSVdk) of the DK-3592B strain genome was constructed by assembling six overlapping cDNA fragments by using natural or artificially created unique restriction sites in the overlapping regions of the clones. Using an existing clone of the trout-avirulent VHSV-IVb strain MI03 (pVHSVmi), eight chimeric VHSV clones were constructed in which the coding region(s) of the glycoprotein (G), non-virion protein (NV), G and NV, or G, NV and L (polymerase) genes together, were exchanged between the two clones. Ten recombinant VHSVs (rVHSVs) were generated, including two parental rVHSVs, by transfecting fish cells with ten individual full-length plasmid constructs along with supporting plasmids using the established protocol. Recovered rVHSVs were characterized for viability and growth in vitro and used to challenge groups of juvenile rainbow trout by intraperitoneal injection. Complete sequence of the VHSV DK-3592B genome was determined from the cloned cDNA and deposited in GenBank under the accession no. KC778774. The trout-virulent DK-3592B genome (genotype Ia) is 11,159 nt in length and differs from the trout-avirulent MI03 genome (pVHSVmi) by 13% at the nucleotide level. When the rVHSVs were assessed for the trout-virulence phenotype in vivo, the parental rVHSVdk and rVHSVmi were virulent and avirulent, respectively, as expected. Four chimeric rVHSVdk viruses with the substitutions of the G, NV, G and NV, or G, NV and L genes from the avirulent pVHSVmi constructs were still highly virulent (100% mortality), while the reciprocal four chimeric rVHSVmi viruses with genes from pVHSVdk remained avirulent (0–10% mortality). When chimeric rVHSVs, containing all the G, NV, and L gene substitutions, were tested in vivo, they did not exhibit any change in trout-virulence relative to the background clones. These results demonstrate that the G, NV and L genes of VHSV are not, by themselves or in combination, major determinants of host-specific virulence in trout.

Journal ArticleDOI
TL;DR: This study identifies RHA as a processivity factor of HIV-1 RT, and indicates that RHA in HIV- 1 virions is required for the efficient catalysis of (−)cDNA synthesis during viral infection before capsid core uncoating.

Journal ArticleDOI
20 Jul 2019-Gene
TL;DR: Pvfem-1 was found expressed in spermatogonia of both, subadult and adult shrimps indicating a function in male sexual differentiation and gametes generation and represents the first step for future functional analysis that is expected to contribute to clarifying the role of Pvfam-1 in sex differentiation and determination.

Journal ArticleDOI
TL;DR: Preliminary data is provided showing that IFI16 (but not cGAS) interacts with synthetic single-stranded HK2 oligos corresponding to the first product of reverse transcription, and it is shown that ssDNA detection by I FI16 has variability with respect to sequence features but is not dependent on strong secondary structures mimicking dsDNA.
Abstract: Human endogenous retroviruses (HERVs) are under genomic and epigenetic control but can be expressed in normal tissues, producing RNA transcripts some of which are translated. While it has not been demonstrated experimentally in modern humans, cDNA copies from HERV RNA (namely HERV-K HML-2 or HK2) were produced after the human-chimp split and until at least 250,000 years ago. We were interested in determining if such cDNA could be a ligand for pattern recognition receptors (PRRs) of the innate immune response. The AIM-2-like receptors for DNA, interferon-γ-inducible protein 16 (IFI16) and Cyclic GMP-AMP synthase (cGAS) were candidate PRRs. IFI16 can detect cDNA produced during HIV-1 replication, causing increased T cell death. While HIV-1 has emerged relatively recently as a human pathogen, the cDNA functionality of IFI16 could have been selected for during the course of human evolution. Here we present a novel hypothesis that the products of reverse transcription of HK2, which has been proliferating in the genome of human ancestors for 30 million years, could interact with IFI16. In support of our hypothesis, we provide preliminary data showing that IFI16 (but not cGAS) interacts with synthetic single-stranded HK2 oligos corresponding to the first product of reverse transcription. Further, we show that ssDNA detection by IFI16 has variability with respect to sequence features but is not dependent on strong secondary structures mimicking dsDNA. Among the HK2 oligos, IFI16 interacts more intensely with those derived from LTRs, suggesting these oligos have undetermined structural features that allow IFI16 to bind with greater affinity. Further, cells with stem cell features that naturally allow HK2 expression were found to express many components of the innate immune system including cGAS but not IFI16. Based on the presented preliminary data we further postulate another hypothesis: that the IFI16 functionality in human cells has been acting as "second-line" defense to control abnormal HK2 replication in somatic tissues. The absence of this protein in stem cells and a stem cell line could permit these cells to express HERVs which contribute to stem cell identity. Finally, we also comment on potential studies that could support or refute our hypothesis.

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TL;DR: The results showed that MuPR1 shares some conserved characteristics with its homologues from different plant families, and may have roles in mediating the rates of oxygen radical formation and detoxification in mulberry varieties.
Abstract: In the present study, the cDNA (designated MuPR1) of PR1 gene was obtained from mulberry. Our results showed that MuPR1 shares some conserved characteristics with its homologues from different plan...

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TL;DR: It was found that mRNA expressions of CaMyD88 and CaTRAF6 were generally up‐regulated after stimulation by polyI:C, flagellin, and Aeromonas hydrophila in spite of the down‐regulation appeared at some time points or tissues.

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31 Jan 2019-Cells
TL;DR: Dysregulations of LRRFIP1/GCF2 have been implicated in the causes of several experimental and clinico-pathological states and the responses to them, such as autoimmune diseases, excitotoxicity after stroke, thrombosis formation, inflammation and obesity, the wound healing process, and in cancers.
Abstract: Leucine Rich Repeat of Flightless-1 Interacting Protein 1/GC-binding factor 2 (LRRFIP1/GCF2) cDNA was cloned for a transcriptional repressor GCF2, which bound sequence-specifically to a GC-rich element of epidermal growth factor receptor (EGFR) gene and repressed its promotor. LRRFIP1/GCF2 was also cloned as a double stranded RNA (dsRNA)-binding protein to trans-activation responsive region (TAR) RNA of Human Immunodeficiency Virus-1 (HIV-1), termed as TAR RNA interacting protein (TRIP), and as a binding protein to the Leucine Rich Repeat (LRR) of Flightless-1(Fli-1), termed as Flightless-1 LRR associated protein 1 (FLAP1) and LRR domain of Flightless-1 interacting Protein 1 (LRRFIP1). Subsequent functional studies have revealed that LRRFIP1/GCF2 played multiple roles in the regulation of diverse biological systems and processes, such as in immune response to microorganisms and auto-immunity, remodeling of cytoskeletal system, signal transduction pathways, and transcriptional regulations of genes. Dysregulations of LRRFIP1/GCF2 have been implicated in the causes of several experimental and clinico-pathological states and the responses to them, such as autoimmune diseases, excitotoxicity after stroke, thrombosis formation, inflammation and obesity, the wound healing process, and in cancers. LRRFIP1/GCF2 is a bioregulator in multidisciplinary systems of the human body and its dysregulation can cause diverse human diseases.

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TL;DR: Overexpression of StGA2ox1 cDNA in transgenic potato plants exhibited an improved salt, drought, exogenous hormone, and low temperature stress tolerance in comparison to the non-transformed plant, which may be associated with the subsequent accumulation of proline osmoprotectant.
Abstract: It has been known that GA2ox is one kind of key enzyme gene in the gibberellin synthesis pathway, which plays important regulatory roles throughout plant whole growth and development. In this article, one of the GA2ox family genes, designated StGA2ox1, was isolated from potato (Solanum tuberosum L.). The full length of cDNA is 1005 bp, and the cDNA corresponds to a protein of 334 amino acids; this protein was classified in a group with NtGA2ox3 based on multiple sequence alignments and phylogenetic characterization. A plant expression vector pCAEZ1383-StGA2ox1 was established. qRT-PCR showed that the expression of RD28, DREB1, WRKY1, and SnRK2 genes in StGA2ox1 transgenic plant is higher than that in non-transformed control under dehydration, low temperature conditions, and abscisic acid treatments. Overexpression of StGA2ox1 cDNA in transgenic potato plants exhibited an improved salt, drought, exogenous hormone, and low temperature stress tolerance in comparison to the non-transformed plant. The enhanced stress tolerance may be associated with the subsequent accumulation of proline osmoprotectant in addition to a better control of chlorophyll, carotenoids, and water loss. These data suggest that the StGA2ox1 is involved in the regulation of plant growth and tolerance in potato by regulating the synthesis of gibberellin.