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Showing papers on "Complementary DNA published in 2009"


Journal ArticleDOI
06 Aug 2009-PLOS ONE
TL;DR: A collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers based on the Gateway technology are described.
Abstract: The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we determined that both isoforms of the AT-Rich Interacting Domain 4B (ARID4B) protein could induce G1 arrest when overexpressed. As new technologies emerge, the vectors in this collection can be easily modified and adapted without the need for extensive recloning.

852 citations


Journal ArticleDOI
TL;DR: It is demonstrated that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes and helps to identify new genes and enables studying promoter-associated and antisense transcription.
Abstract: High-throughput complementary DNA sequencing (RNA-Seq) is a powerful tool for whole-transcriptome analysis, supplying information about a transcript's expression level and structure. However, it is difficult to determine the polarity of transcripts, and therefore identify which strand is transcribed. Here, we present a simple cDNA sequencing protocol that preserves information about a transcript's direction. Using Saccharomyces cerevisiae and mouse brain transcriptomes as models, we demonstrate that knowing the transcript's orientation allows more accurate determination of the structure and expression of genes. It also helps to identify new genes and enables studying promoter-associated and antisense transcription. The transcriptional landscapes we obtained are available online.

760 citations


Journal ArticleDOI
TL;DR: Given that RNA helicase encoded by MDA-5 is a critical molecule involved in the innate immune defense against viruses, viral infection may play an important role in the pathogenesis of C-ADM and rapidly progressive ILD.
Abstract: Objective To identify the autoantigen recognized by the autoantibody that is associated with clinically amyopathic dermatomyositis (C-ADM) and rapidly progressive interstitial lung disease (ILD). Methods An anti–CADM-140 antibody–positive prototype serum sample was used to screen a HeLa cell–derived complementary DNA (cDNA) library. Selected cDNA clones were further evaluated by immunoprecipitation of their in vitro–transcribed and in vitro–translated products using anti–CADM-140 antibody–positive and anti-CADM-140 antibody–negative sera. The lysates of COS-7 cells transfected with the putative antigen were similarly tested. An enzyme-linked immunosorbent assay (ELISA) to detect the anti–CADM-140 antibody was established using a recombinant CADM-140 antigen, and its specificity and sensitivity for C-ADM and rapidly progressive ILD were assessed in 294 patients with various connective tissue diseases. Results By cDNA library screening and immunoprecipitation of in vitro–transcribed and in vitro–translated products, we obtained a cDNA clone encoding melanoma differentiation–associated gene 5 (MDA-5). The anti–CADM-140 antibodies in patients' sera specifically reacted with MDA-5 protein expressed in cells transfected with full-length MDA-5 cDNA, confirming the identity of MDA-5 as the CADM-140 autoantigen. The ELISA, using recombinant MDA-5 protein as the antigen, showed an analytical sensitivity of 85% and analytical specificity of 100%, in comparison with the “gold standard” immunoprecipitation assay, and was useful for identifying patients with C-ADM and/or rapidly progressive ILD. Conclusion Given that RNA helicase encoded by MDA-5 is a critical molecule involved in the innate immune defense against viruses, viral infection may play an important role in the pathogenesis of C-ADM and rapidly progressive ILD. Moreover, our ELISA using recombinant MDA-5 protein makes detection of the anti–CADM-140 antibody routinely available.

485 citations


Journal ArticleDOI
TL;DR: The results suggest that RNA profiling might provide indirect support to antibodies' specificity, since whenever a evident correlation between the RNA and protein profiles exists, this can sustain that the antibodies used in the immunoassay recognized their cognate antigens.
Abstract: The Central Dogma of biology holds, in famously simplified terms, that DNA makes RNA makes proteins, but there is considerable uncertainty regarding the general, genome-wide correlation between levels of RNA and corresponding proteins. Therefore, to assess degrees of this correlation we compared the RNA profiles (determined using both cDNA- and oligo-based microarrays) and protein profiles (determined immunohistochemically in tissue microarrays) of 1066 gene products in 23 human cell lines. A high mean correlation coefficient (0.52) was obtained from the pairwise comparison of RNA levels determined by the two platforms. Significant correlations, with correlation coefficients exceeding 0.445, between protein and RNA levels were also obtained for a third of the specific gene products. However, the correlation coefficients between levels of RNA and protein products of specific genes varied widely, and the mean correlations between the protein and corresponding RNA levels determined using the cDNA- and oligo-based microarrays were 0.25 and 0.20, respectively. Significant correlations were found in one third of the examined RNA species and corresponding proteins. These results suggest that RNA profiling might provide indirect support to antibodies' specificity, since whenever a evident correlation between the RNA and protein profiles exists, this can sustain that the antibodies used in the immunoassay recognized their cognate antigens.

470 citations


Journal ArticleDOI
TL;DR: An exhaustive real-time reverse transcriptase-polymerase chain reaction (PCR) quantification method was used to determine 15 of the catalytically active human UDP-glucuronosyltransferase (UGT) isoforms to provide valuable information about the medical efficacy or pharmacokinetic characteristics of a wide variety of UGT-metabolized drugs.
Abstract: An exhaustive real-time reverse transcriptase-polymerase chain reaction (PCR) quantification method was used to determine 15 of the catalytically active human UDP-glucuronosyltransferase (UGT) isoforms (1A1, 1A3, 1A4, 1A5, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B10, 2B11, 2B15, and 2B17). The specific primers for respective human UGTs were developed for differential determination. The cDNA derived from the 1A7 isoform was detected in the esophagus, the 1A8 and 1A10 isoforms were detected in the small intestine, and all other isoforms were detected in at least the liver by PCR. In all cases, single bands of the expected size on the agarose gel were confirmed to correspond with the predicted UGT isoform sequences. Each calibration curve showed linearity between the PCR crossing point and the calibrator copy number. The correlation coefficients were greater than 0.9957 with high reproducibility. This exhaustive measurement method was applied to UGT expression in 23 human tissue types. UGT was mostly expressed in the alimentary system and liver. We were surprised to find that extremely high expression in the liver was found for UGT2B4 and UGT2B15, which had, respectively, 8.98 and 4.38 times greater expression than UGT2B7 in the liver. In addition, even though expressed at low levels, several UGT isoforms were expressed in steroidogenic tissues, such as the breast, prostate, heart, and adrenal. Therefore, this quantification method may provide valuable information about the medical efficacy or pharmacokinetic characteristics of a wide variety of UGT-metabolized drugs.

426 citations


Journal ArticleDOI
TL;DR: Many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.
Abstract: Microarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR) and denervated (DL) forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration. Considerable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa). Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST) contigs from the Ambystoma EST database more than doubled (3935 to 9411) the number of non-redundant human-A. mexicanum orthologous sequences. Many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.

301 citations


Journal ArticleDOI
TL;DR: Transient expression experiments in agroinfiltrated Nicotiana benthamiana and A. annua leaves showed that AaWRKY1 protein transactivated the ADSpro2 promoter activity by binding to the W-box of the promoter; disruption of theW-box abolished the activation.
Abstract: Amorpha-4,11-diene synthase (ADS) of Artemisia annua catalyzes the conversion of farnesyl diphosphate into amorpha-4,11-diene, the first committed step in the biosynthesis of the antimalarial drug artemisinin. The promoters of ADS contain two reverse-oriented TTGACC W-box cis-acting elements, which are the proposed binding sites of WRKY transcription factors. A full-length cDNA (AaWRKY1) was isolated from a cDNA library of the glandular secretory trichomes (GSTs) in which artemisinin is synthesized and sequestered. AaWRKY1 encodes a 311 amino acid protein containing a single WRKY domain. AaWRKY1 and ADS genes were highly expressed in GSTs and both were strongly induced by methyl jasmonate and chitosan. Transient expression analysis of the AaWRKY1-GFP (green fluorescent protein) reporter revealed that AaWRKY1 was targeted to nuclei. Biochemical analysis demonstrated that the AaWRKY1 protein was capable of binding to the W-box cis-acting elements of the ADS promoters, and it demonstrated transactivation activity in yeast. Co-expression of the effector construct 35S::AaWRKY1 with a reporter construct ADSpro1::GUS greatly activated expression of the GUS (beta-glucuronidase) gene in stably transformed tobacco. Furthermore, transient expression experiments in agroinfiltrated Nicotiana benthamiana and A. annua leaves showed that AaWRKY1 protein transactivated the ADSpro2 promoter activity by binding to the W-box of the promoter; disruption of the W-box abolished the activation. Transient expression of AaWRKY1 cDNA in A. annua leaves clearly activated the expression of the majority of artemisinin biosynthetic genes. These results strongly suggest the involvement of the AaWRKY1 transcription factor in the regulation of artemisinin biosynthesis, and indicate that ADS is a target gene of AaWRKY1 in A. annua.

279 citations


Journal ArticleDOI
TL;DR: A large portion of the transcriptome of Zea mays is presented, including ESTs representing 484,032 c DNA clones from 53 libraries and 36,565 fully sequenced cDNA clones, out of which 31,552 clones are non-redundant, suggesting that an identifiable class of genes in plants is associated with the Poaceae divergence.
Abstract: We present a large portion of the transcriptome of Zea mays, including ESTs representing 484,032 cDNA clones from 53 libraries and 36,565 fully sequenced cDNA clones, out of which 31,552 clones are non-redundant. These and other previously sequenced transcripts have been aligned with available genome sequences and have provided new insights into the characteristics of gene structures and promoters within this major crop species. We found that although the average number of introns per gene is about the same in corn and Arabidopsis, corn genes have more alternatively spliced isoforms. Examination of the nucleotide composition of coding regions reveals that corn genes, as well as genes of other Poaceae (Grass family), can be divided into two classes according to the GC content at the third position in the amino acid encoding codons. Many of the transcripts that have lower GC content at the third position have dicot homologs but the high GC content transcripts tend to be more specific to the grasses. The high GC content class is also enriched with intronless genes. Together this suggests that an identifiable class of genes in plants is associated with the Poaceae divergence. Furthermore, because many of these genes appear to be derived from ancestral genes that do not contain introns, this evolutionary divergence may be the result of horizontal gene transfer from species not only with different codon usage but possibly that did not have introns, perhaps outside of the plant kingdom. By comparing the cDNAs described herein with the non-redundant set of corn mRNAs in GenBank, we estimate that there are about 50,000 different protein coding genes in Zea. All of the sequence data from this study have been submitted to DDBJ/GenBank/EMBL under accession numbers EU940701-EU977132 (FLI cDNA) and FK944382-FL482108 (EST).

255 citations


Journal ArticleDOI
TL;DR: This study constitutes the first report of the silencing of a gene expressed specifically in the AG, which caused a transient adverse effect on male phenotypical gender differences and spermatogenesis in Macrobrachium rosenbergii.
Abstract: Androgenic glands (AGs) of the freshwater prawn Macrobrachium rosenbergii were subjected to endocrine manipulation, causing them to hypertrophy. Transcripts from these glands were used in the construction of an AG cDNA subtractive library. Screening of the library revealed an AG-specific gene, termed the M. rosenbergii insulin-like AG (Mr-IAG) gene. The cDNA of this gene was then cloned and fully sequenced. The cysteine backbone of the predicted mature Mr-IAG peptide (B and A chains) showed high similarity to that of other crustacean AG-specific insulin-like peptides. In vivo silencing of the gene, by injecting the prawns with Mr-IAG double-stranded RNA, temporarily prevented the regeneration of male secondary sexual characteristics, accompanied by a lag in molt and a reduction in growth parameters, which are typically higher in males of the species. In terms of reproductive parameters, silencing of Mr-IAG led to the arrest of testicular spermatogenesis and of spermatophore development in the terminal amp...

217 citations


Journal ArticleDOI
TL;DR: Overexpression of GhZFP1 in transgenic tobacco enhanced tolerance to salt stress and resistance to Rhizoctonia solani appears to be involved as an important regulator in plant responses to abiotic and biotic stresses.
Abstract: * Zinc finger proteins are a superfamily involved in many aspects of plant growth and development. However, CCCH-type zinc finger proteins involved in plant stress tolerance are poorly understood. * A cDNA clone designated Gossypium hirsutum zinc finger protein 1 (GhZFP1), which encodes a novel CCCH-type zinc finger protein, was isolated from a salt-induced cotton (G. hirsutum) cDNA library using differential hybridization screening and further studied in transgenic tobacco Nicotiana tabacum cv. NC89. Using yeast two-hybrid screening (Y2H), proteins GZIRD21A (GhZFP1 interacting and responsive to dehydration protein 21A) and GZIPR5 (GhZFP1 interacting and pathogenesis-related protein 5), which interacted with GhZFP1, were isolated. * GhZFP1 contains two typical zinc finger motifs (Cx8Cx5Cx3H and Cx5Cx4Cx3H), a putative nuclear export sequence (NES) and a potential nuclear localization signal (NLS). Transient expression analysis using a GhZFP1::GFP fusion gene in onion epidermal cells indicated a nuclear localization for GhZFP1. RNA blot analysis showed that the GhZFP1 transcript was induced by salt (NaCl), drought and salicylic acid (SA). The regions in GhZFP1 that interact with GZIRD21A and GZIPR5 were identified using truncation mutations. * Overexpression of GhZFP1 in transgenic tobacco enhanced tolerance to salt stress and resistance to Rhizoctonia solani. Therefore, it appears that GhZFP1 might be involved as an important regulator in plant responses to abiotic and biotic stresses.

198 citations


Journal ArticleDOI
TL;DR: Using a large number of quantitative PCR (qPCR) assays, more than previous studies, it is found that neither technology is decisively better at measuring differential gene expression.
Abstract: High-throughput cDNA synthesis and sequencing of poly(A)-enriched RNA is rapidly emerging as a technology competing to replace microarrays as a quantitative platform for measuring gene expression. Consequently, we compared full length cDNA sequencing to 2-channel gene expression microarrays in the context of measuring differential gene expression. Because of its comparable cost to a gene expression microarray, our study focused on the data obtainable from a single lane of an Illumina 1 G sequencer. We compared sequencing data to a highly replicated microarray experiment profiling two divergent strains of S. cerevisiae. Using a large number of quantitative PCR (qPCR) assays, more than previous studies, we found that neither technology is decisively better at measuring differential gene expression. Further, we report sequencing results from a diploid hybrid of two strains of S. cerevisiae that indicate full length cDNA sequencing can discover heterozygosity and measure quantitative allele-specific expression simultaneously.

Journal ArticleDOI
TL;DR: The procedure for the preparation of whole transcriptome cDNA libraries depleted of ribosomal RNA from only 1 μg of total RNA is developed and validated by profiling human whole brain and universal human reference RNA using ultra-high-throughput sequencing.
Abstract: To deplete abundant rRNA from total RNA during cDNA library generation, hexamer primers that perfectly match rRNA are removed. These 'not so randomly primed' cDNA libraries can be generated from small amount of total RNA, preserve strand orientation and are equally enriched in polyadenylated and non-polyadenylated transcripts. We developed a procedure for the preparation of whole transcriptome cDNA libraries depleted of ribosomal RNA from only 1 μg of total RNA. The method relies on a collection of short, computationally selected oligonucleotides, called 'not-so-random' (NSR) primers, to obtain full-length, strand-specific representation of nonribosomal RNA transcripts. In this study we validated the technique by profiling human whole brain and universal human reference RNA using ultra-high-throughput sequencing.

Journal ArticleDOI
Xiaoying Lü1, Xiang Bao1, Yan Huang1, Yinghua Qu1, Huiqin Lu1, Zu Hong Lu1 
TL;DR: Gene ontology analysis showed that the L-929 cells which responded to Ni(II) covered a broad range of functional gene groups including cellular biological process, molecular function, and cellular component.

Journal ArticleDOI
TL;DR: High levels of transgene expression, including high level of the corresponding protein, was observed in brain tissue and not in heart or liver tissues, indicating accumulation of the Aβ peptide in the brain may develop at the age of 1–2 years.
Abstract: In an effort to develop a porcine model of Alzheimer’s disease we used handmade cloning to produce seven transgenic Gottingen minipigs. The donor fibroblasts had been stably transfected with a plasmid cassette containing, as transgene, the cDNA of the neuronal variant of the human amyloid precursor protein gene with the Swedish mutation preceded by beta-globin sequences to induce splicing and a human PDGFbeta promoter fragment to drive transcription. Transgene insertion had occurred only at the GLIS3 locus where a single complete copy of the transgene was identified in intronic sequences in opposite direction. Similar and robust levels of the transgene transcript were detected in skin biopsies from all piglets and the sequence of full-length transcript was verified. Consistent with PDGFbeta promoter function, high levels of transgene expression, including high level of the corresponding protein, was observed in brain tissue and not in heart or liver tissues. A rough estimate predicts that accumulation of the Aβ peptide in the brain may develop at the age of 1–2 years.

Journal ArticleDOI
TL;DR: A quantitative PCR method featuring a reusable single-cell cDNA library immobilized on beads for measuring the expression of multiple genes in a single cell with an experimental error of 15.9% or less is developed.
Abstract: We developed a quantitative PCR method featuring a reusable single-cell cDNA library immobilized on beads for measuring the expression of multiple genes in a single cell. We used this method to analyze multiple cDNA targets (from several copies to several hundred thousand copies) with an experimental error of 15.9% or less. This method is sufficiently accurate to investigate the heterogeneity of single cells.

Journal ArticleDOI
TL;DR: The most distinct finding was a novel amino acid substitution mutation, D1794Y, located within the extracellular linker between IVS5 and IVS6, which is concurrent with the known V1023G mutation in Aa-para of the Per-R strain.

Journal ArticleDOI
TL;DR: Results suggested that FcLec3 might function in the recognition of bacterial and viral pathogens in shrimp.

Journal ArticleDOI
TL;DR: The suitability of this novel and versatile puromycin-linker DNA method for in vitro selections of disulfide-rich proteins and other potential applications is shown, with an enrichment efficiency of 20-fold per selection round.
Abstract: We report a robust display technology for the screening of disulfide-rich peptides, based on cDNA-protein fusions, by developing a novel and versatile puromycin-linker DNA. This linker comprises four major portions: a 'ligation site' for T4 RNA ligase, a 'biotin site' for solid-phase handling, a 'reverse transcription primer site' for the efficient and rapid conversion from an unstable mRNA-protein fusion (mRNA display) to a stable mRNA/cDNA-protein fusion (cDNA display) whose cDNA is covalently linked to its encoded protein and a 'restriction enzyme site' for the release of a complex from the solid support. This enables not only stabilizing mRNA-protein fusions but also promoting both protein folding and disulfide shuffling reactions. We evaluated the performance of cDNA display in different model systems and demonstrated an enrichment efficiency of 20-fold per selection round. Selection of a 32-residue random library against interleukin-6 receptor generated novel peptides containing multiple disulfide bonds with a unique linkage for its function. The peptides were found to bind with the target in the low nanomolar range. These results show the suitability of our method for in vitro selections of disulfide-rich proteins and other potential applications.

Journal ArticleDOI
TL;DR: It is reported that human RBCs contain typical eukaryotic RNA with 28S- and18S-rRNA standard bands and a number of genes responsible for transcription, translation, RNA-stabilisation as well as for apoptosis and anti-apoptosis have been identified for the first time in circulating human R BCs.
Abstract: Understanding of molecular mechanisms governing the enucleating phenomena of human erythrocytes is of major importance in both fundamental and applied studies. Total RNA (n=7) from human RBCs (purity of erythrocyte preparation >99,99%) was tested using 2100 Bioanalyzer (Agilent, USA), and transcribed to cDNA. Microarray analysis was performed with the Human Genome Focus GeneChip (Affymetrix, USA), containing 8500 transcripts corresponding to 8400 human genes. Here we report that human RBCs contain typical eukaryotic RNA with 28S- and18S-rRNA standard bands. Microarray studies revealed the presence of transcripts of 1019 different genes in erythrocytic RNA. Gene Ontology analysis recognized 859 genes involved in general biological processes: 529 genes for cellular metabolism, 228 genes for signal transduction, 104 genes for development, 107 genes for immune response, 62 genes for protein localization, 53 genes for programmed cell death, and 5 genes for autophagy. A number of genes responsible for transcription, translation, RNA-stabilisation as well as for apoptosis and anti-apoptosis have been identified for the first time in circulating human RBCs. The presented data shed new light on the genetic determination of erythropoiesis, apoptosis and may have implications on the pathophysiology and diagnosis of various diseases involving red blood cells.

Journal ArticleDOI
TL;DR: The analysis of over 9000 expressed sequence tags from cDNA libraries obtained from various life cycle stages of Globodera pallida is consistent with the suggestion that the SPRYSEC proteins suppress host defences, as suggested previously, and that they achieve this through interaction with a range of host targets.
Abstract: In this article, we describe the analysis of over 9000 expressed sequence tags (ESTs) from cDNA libraries obtained from various life cycle stages of Globodera pallida. We have identified over 50 G. pallida effectors from this dataset using bioinformatics analysis, by screening clones in order to identify secreted proteins up-regulated after the onset of parasitism and using in situ hybridization to confirm the expression in pharyngeal gland cells. A substantial gene family encoding G. pallida SPRYSEC proteins has been identified. The expression of these genes is restricted to the dorsal pharyngeal gland cell. Different members of the SPRYSEC family of proteins from G. pallida show different subcellular localization patterns in plants, with some localized to the cytoplasm and others to the nucleus and nucleolus. Differences in subcellular localization may reflect diverse functional roles for each individual protein or, more likely, variety in the compartmentalization of plant proteins targeted by the nematode. Our data are therefore consistent with the suggestion that the SPRYSEC proteins suppress host defences, as suggested previously, and that they achieve this through interaction with a range of host targets.

Journal ArticleDOI
TL;DR: Two cDNA clones of rat alkaline phosphatase (AP) were isolated from a rat osteosarcoma λgt 11 cDNA library (ROS 17/2.8) utilizing a human bone‐liver‐kidney (BLK) type AP cDNA, containing overlapping DNA sequences corresponding to the 3' noncoding region of AP mRNA.
Abstract: Two cDNA clones of rat alkaline phosphatase (AP) were isolated from a rat osteosarcoma lambda gt 11 cDNA library (ROS 17/2.8) utilizing a human bone-liver-kidney (BLK) type AP cDNA. These clones contain overlapping DNA sequences of 597 and 520 bp, respectively, corresponding to the 3' noncoding region of AP mRNA. The sequence homology with the human BLK AP cDNA is 61%. In Northern blot analysis the rat cDNA hybridizes to a single band of 2.5 kb mRNA from ROS 17/2.8 and rat liver, under highly stringent conditions. Steady state levels of AP mRNAs measured in several rat osteosarcoma cell lines (ROS 17/2.8, ROS 2/3, ROS 25/1, UMR 106) correlate with the level of AP enzymatic activity in these cells. Dexamethasone, which stimulates AP enzymatic activity in ROS 17/2.8 cells, increases the relative abundance of AP mRNA in a dose-dependent manner. This probe can be used to study AP expression in rat tissues and cells.

Journal ArticleDOI
TL;DR: RNAi-mediated silencing of PmPPAE1 gene significantly decreased the total PO activity in shrimp and additionally increased the mortality of V. harveyi infected shrimp, the latter of which correlated with an increase in the number of viable bacteria in the hemolymph.
Abstract: The prophenoloxidase (proPO) activating system is an important innate immune response against microbial infections in invertebrates. The major enzyme, phenoloxidase (PO), is synthesized as an inactive precursor and its activation to an active enzyme is mediated by a cascade of clip domain serine proteinases (clip-SPs). In this study, a cDNA encoding a proPO activating enzyme (PPAE) from the black tiger shrimp, Penaeus monodon, designated as PmPPAE1, was cloned and characterized. The full-length cDNA contains an open reading frame (ORF) of 1392 bp encoding a predicted protein of 463 amino acids including an 18 amino acid signal peptide. The PmPPAE1 protein exhibits a characteristic sequence structure of clip-SPs consisting of the clip domain at the N-terminus and a SP domain at the C-terminus. Sequence analysis showed that PmPPAE1 exhibited the highest amino acid sequence similarity (70%) to a PPAE of the crayfish, Pacifastacus leniusculus. PmPPAE1 mRNA is abundantly expressed in hemocytes, and this is regulated after systemic Vibrio harveyi infection supporting that it is an immune-responsive gene. RNA interference-mediated suppression of PmPPAE1, performed by injection of double-stranded RNA (dsRNA) corresponding to the PmPPAE1 gene into shrimp, resulted in a significant reduction of PmPPAE1 but not other clip-SP and related gene transcript levels of P. monodon, suggesting gene-specific knockdown. RNAi-mediated silencing of PmPPAE1 gene significantly decreased the total PO activity (36.7%) in shrimp and additionally increased the mortality of V. harveyi infected shrimp, the latter of which correlated with an increase in the number of viable bacteria in the hemolymph. These results indicate that PmPPAE1 functions in the proPO system and is an important component in the shrimp immune system.

Journal ArticleDOI
TL;DR: A protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit is detailed.
Abstract: Many important and complex laboratory procedures require an input of high quality, intact RNA. A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications. It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample. Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated. The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.

Journal ArticleDOI
TL;DR: The identification and characterization of a crustin (CrusSp) from the hemocyte of mud crab, Scylla paramamosain using an expressed sequence tag (EST) and rapid amplification cDNA end (RACE) approaches indicated the involvement of CrusSp in the innate immunity of S. paramamosains.
Abstract: Antimicrobial peptides (AMPs) are important components of the host innate immune response against microbial invasion. In the present study, we report the identification and characterization of a crustin (CrusSp) from the hemocyte of mud crab, Scylla paramamosain using an expressed sequence tag (EST) and rapid amplification cDNA end (RACE) approaches. Analysis of the nucleotide sequence revealed seven different variances of the CrusSp cDNA in mud crab. The open reading frame encodes a protein of 111 amino acids with 21 residues signal sequence. The predicted molecular mass of the mature protein (90 amino acids) is 10.27 kDa with an estimated pI of 8.54. Analysis of the protein domain features indicated typical conserved cysteine residues containing a single whey acidic protein (WAP) domain at the C-terminus. A neighbour-joining tree showed that S. paramamosain crustin is closely related to other crustin homologues, and displays the highest similarity to crustin antimicrobial peptide in shore crab Carcinus maenas. Four exons and three introns were identified within the 999 bp genomic DNA sequence of CrusSp. Tissue distribution analysis showed that CrusSp was highly expressed in hemocytes, gills, intestines and muscle but it was not expressed in hepatopancreas and eyestalks. To gain insight into the in vitro antimicrobial activities of CrusSp, the mature peptide coding region was cloned into E. coli for heterologous expression. The recombinant CrusSp could inhibit the growth of gram-positive bacteria but had no inhibition activity against gram-negative bacteria. These results indicated the involvement of CrusSp in the innate immunity of S. paramamosain.

Journal ArticleDOI
TL;DR: Analysis of expressed sequence tags from a bovine oocyte cDNA library identified a new member of the importin alpha family, KPNA7, which has a strong binding affinity for the nuclear protein nucleoplasmin 2 relative to that of other importin alphas.
Abstract: Nuclear proteins such as transcription and chromatin remodeling factors are required for initiation of transcription in early embryos before embryonic genome activation. The nuclear transport of these proteins is mediated by transport factors such as importins. Through analysis of expressed sequence tags from a bovine oocyte cDNA library, we identified a new member of the importin alpha family (named importin alpha8). The cloned cDNA for bovine importin alpha8 (KPNA7) is 1817 base pair in length, encoding a protein of 522 amino acids that contains a conserved importin beta-binding domain and seven armadillo motifs. The RT-PCR analysis revealed that KPNA7 mRNA is specifically expressed in ovaries and mature oocytes. Real-time PCR demonstrated that KPNA7 expression in germinal vesicle (GV) oocytes is 33 to 2396 times higher than that of other importin alpha genes and that KPNA7 mRNA is abundant in GV and metaphase II oocytes, as well as in early-stage embryos collected before embryonic genome activation, but is barely detectable in morula- and blastocyst-stage embryos. Similarly, expression of KPNA7 protein is very high in oocytes and early embryos but is low in blastocysts. A glutathione S-transferase pull-down assay revealed that KPNA7 has a strong binding affinity for the nuclear protein nucleoplasmin 2 relative to that of other importin alphas. RNA interference experiments demonstrated that knockdown of KPNA7 in early embryos results in a decreased proportion of embryos developing to the blastocyst stage. These results suggest that KPNA7 may have an important role in the transport of essential nuclear proteins required for early embryogenesis.

Journal ArticleDOI
TL;DR: In pigs, the gene for glucosephosphate isomerase (GPI) is linked to the halothane (HAL) gene which is responsible for malignant hyperthermia (MH), and in situ hybridization with GPI8R assigned the GPI locus to bands p12-q22 of chromosome 6.
Abstract: Summary. In pigs, the gene for glucosephosphate isomerase (GPI) is linked to the halothane (HAL) gene which is responsible for malignant hyperthermia (MH). A single copy DNA probe, designated GPI8R, has been isolated from a pig genomic library using a porcine GPI cDNA probe. This probe detects, as was the case for the cDNA probe, a five allele polymorphism in Sac1 andPvuII digested pig DNA. Family studies show that this polymorphism is linked to the HAL locus and hence can be used in carrier detection. In situ hybridization with GPI8R assigned the GPI locus to bands p12-q22 of chromosome 6. We conclude that the HAL linkage group resides on chromosome 6.

Journal ArticleDOI
Aili Wang1, Zhiliang Gu1, B. Heid1, R.M. Akers1, Honglin Jiang1 
TL;DR: It is demonstrated that the bovine genome encodes functional GPR41 and GPR43 genes and suggested that GPR 41 and G PR43 may play a role in the regulatory effects of VFA in cattle.

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TL;DR: IL-2 expression was induced by the T cell mitogen PHA and by the mixed leucocyte reaction, where leucocytes from pairs of fish were cultured together for four days, and expression was also induced in vivo during bacterial infection.

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TL;DR: It is demonstrated that M5 exhibits tighter binding (lower Km) to template-primer, which likely protects against heat inactivation and generates higher cDNA yields and exhibits better RT–PCR performance compared to wild-type RT when used at high temperature to amplify RNA targets containing secondary structure.
Abstract: In an effort to increase the thermostability of Moloney Murine Leukemia Virus reverse transcriptase (MMLV RT), we screened random and site-saturation libraries for variants that show increased resistance to thermal inactivation. We discovered five mutations E69K, E302R, W313F, L435G and N454K that collectively increase the half-life of MMLV RT at 55 degrees C from less than 5 min to approximately 30 min in the presence of template-primer. In addition, these mutations alter the thermal profile by increasing specific activity of the pentuple mutant (M5) over a broad range of cDNA synthesis temperatures (25-70 degrees C). We further show that M5 generates higher cDNA yields and exhibits better RT-PCR performance compared to wild-type RT when used at high temperature to amplify RNA targets containing secondary structure. Finally, we demonstrate that M5 exhibits tighter binding (lower K(m)) to template-primer, which likely protects against heat inactivation.

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TL;DR: Analysis of the expression patterns of a new TCTP cDNA cloned from Fenneropenaeus chinensis suggests that Fc-TCTP could well be involved in the antiviral response in F. chinensis.
Abstract: The translationally controlled tumor protein (TCTP) is a multi-functioning protein that performs vital roles, particularly in various complicated life processes. In this study, a new TCTP cDNA was cloned from Fenneropenaeus chinensis and hence was designated as Fc-TCTP. Its length is 711 bp, and it is characterized by 507-bp open reading frame that encodes a deduced 168-amino acid protein, including a TCTP domain. Moreover, this study analyzed the expression patterns of this gene when it responds to infection specifically with Vibrio anguillarum and the white spot syndrome virus (WSSV). Based on the results, Fc-TCTP was present in all the analyzed tissues. Additionally, Fc-TCTP’s expression level decreased after having been infected by bacteria, but was upregulated in the hepatopancreas after having been exposed to WSSV. Likewise, the Fc-TCTP protein was upregulated during its exposure to the virus. These results suggest that Fc-TCTP could well be involved in the antiviral response in F. chinensis.