Sequence Of cDNA Clone Research Articles

Cost-Effective Sequencing of Full-Length cDNA Clones Powered by a De Novo-Reference Hybrid Assembly

BackgroundSequencing full-length cDNA clones is important to determine gene structures including alternative splice forms, and provides valuable resources for experimental analyses to reveal the biological functions of coded proteins. However, previous approaches for sequencing cDNA clones were expensive or time-consuming, and therefore, a fast and efficient sequencing approach was demanded.MethodologyWe developed a program, MuSICA 2, that assembles millions of short (36-nucleotide) reads collected from a single flow cell lane of Illumina Genome Analyzer to shotgun-sequence ∼800 human full-length cDNA clones. MuSICA 2 performs a hybrid assembly in which an external de novo assembler is run first and the result is then improved by reference alignment of shotgun reads. We compared the MuSICA 2 assembly with 200 pooled full-length cDNA clones finished independently by the conventional primer-walking using Sanger sequencers. The exon-intron structure of the coding sequence was correct for more than 95% of the clones with coding sequence annotation when we excluded cDNA clones insufficiently represented in the shotgun library due to PCR failure (42 out of 200 clones excluded), and the nucleotide-level accuracy of coding sequences of those correct clones was over 99.99%. We also applied MuSICA 2 to full-length cDNA clones from Toxoplasma gondii, to confirm that its ability was competent even for non-human species.ConclusionsThe entire sequencing and shotgun assembly takes less than 1 week and the consumables cost only ∼US$3 per clone, demonstrating a significant advantage over previous approaches.

Basal Host Resistance of Barley to Powdery Mildew: Connecting Quantitative Trait Loci and Candidate Genes

The basal resistance of barley to powdery mildew (Blumeria graminis f. sp. hordei) is a quantitatively inherited trait that is based on nonhypersensitive mechanisms of defense. A functional genomic approach indicates that many plant candidate genes are involved in the defense against formation of fungal haustoria. It is not known which of these candidate genes have allelic variation that contributes to the natural variation in powdery mildew resistance, because many of them may be highly conserved within the barley species and may act downstream of the basal resistance reaction. Twenty-two expressed sequence tag or cDNA clone sequences that are likely to play a role in the barley-Blumeria interaction based on transcriptional profiling, gene silencing, or overexpression data, as well as mlo, Ror1, and Ror2, were mapped and considered candidate genes for contribution to basal resistance. We mapped the quantitative trait loci (QTL) for powdery mildew resistance in six mapping populations of barley at seedling and adult plant stages and developed an improved high-density integrated genetic map containing 6,990 markers for comparing QTL and candidate gene positions over mapping populations. We mapped 12 QTL at seedling stage and 13 QTL at adult plant stage, of which four were in common between the two developmental stages. Six of the candidate genes showed coincidence in their map positions with the QTL identified for basal resistance to powdery mildew. This co-localization justifies giving priority to those six candidate genes to validate them as being responsible for the phenotypic effects of the QTL for basal resistance.

Actin-depolymerizing factor of second-generation merozoite in Eimeria tenella: clone, prokaryotic expression, and diclazuril-induced mRNA expression

Actin depolymerizing factor (ADF) is an essential actin-binding protein that plays a key role in the control of actin dynamics and actin-based motility processes in intracellular parasites. To determine the effects of diclazuril on ADF gene of second-generation merozoites (mz-ADF) mRNA expression in Eimeria tenella, mz-ADF gene was cloned by RT-PCR from extracted RNA in second-generation merozoite of E. tenella and successfully expressed by pET-28a vector in Escherichia coli BL21(DE3). Results showed that the full length of the cloned cDNA sequence of the mz-ADF gene is 476 bp including an ORF of 375 bp. The sequence has 100% homology with a published sequence of sporozoite stage E. tenella ADF mRNA (GenBank EF195234.1). The recombinant protein was induced to be expressed by 1 mM isopropyl beta-D: -1-thiogalactopyranoside in vitro. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed that 16.99 kDa fusion protein existed in solvable form. Compared with the infected/control group, mz-ADF mRNA expression level was downregulated by 63.86% in the infected/treatment group with the treatment of diclazuril. In conclusion, the data presented here indicate that mz-ADF gene participates in an important role in the invasion host of E. tenella. Downregulation of mz-ADF mRNA expression enrich the mechanism study of diclazuril on E. tenella.

Gene expression profiles unique to chile ( Capsicum annuum L.) resistant to Phytophthora root rot

Transcriptome profiling in chile ( Capsicum annuum) roots was used to determine those genes with expression profiles correlated with a resistance phenotype to the pathogen, Phytophthora capsici. Two microarrays were generated; a 10K element array printed with cDNAs from a library of transcripts expressed in Criollo de Morelos-334 variety of C. annuum 6 h post-challenge with P. capsici and a 2K element array printed with sequenced cDNA clones selected based on their preliminary patterns of expression. This second array was enriched for clones that were differentially expressed in susceptible (New Mexico 6-4) vs. resistant (CM334) varieties of C. annuum. Gene expression profiles were revaluated at 0, 4 and 24 h post-inoculation. Control treatments included samples collected at 0, 4, and 24 h post-mock-inoculation. In addition to the parental CM334, a resistant backcross line (01-1688) was also used to identify gene expression patterns associated with the resistance phenotype. Based on a principal component analysis, CM334 samples showed the most significant transcription induction at 4 and 24 h post-inoculation, while the predominant variability in the susceptible line was in genes repressed at 24 h. A set of 168 genes with significant changes in expression following P. capsici challenge was identified; of these, 22 were uniquely expressed only in the resistant lines (CM334 or 01-1688). This set of genes represent candidates for further study as markers for recurrent selection programs and as candidate genes for the mechanism of disease resistance.

Functional characterization of the copper-transporting P-type ATPase gene of Penicillium janthinellum strain GXCR

Copper (Cu)-transporting P-type ATPase (CTPA) genes have been documented to play an important role in resistance to heavy metals. However, our understanding of roles of CTPA genes of the filamentous fungi was based only on sequence similarity prediction before. In a previous study, we isolated a Penicillum janthinellum strain GXCR of higher tolerance to Cu (200 mM). In this study, we cloned the partial cDNA of CTPA gene, named PcpA, from the strain GXCR. Sequence alignment indicated that the cloned cDNA sequence has the highest identity (94.4%) with a predictive CTPA gene of Aspergillus clavatus. The PcpA-encoded protein, termed PcpA, has classical functional domains of CTPAs, and shows differences from reported CTPAs in some specific sequence motifs and transmembrane regions. Expression of the PcpA was induced by extracellular Cu, cadmium (Cd), and silver (Ag). PcpA RNA interference (RNAi) mutants with a reduced level of PcpA mRNA were more sensitive to Cu, iron, Cd, and Ag than the wild-type (WT) strain GXCR. When grown in the presence of Cu, iron, and Cd, intracellular Cu and iron contents in the PcpA RNAi mutant were significantly (P<0.05) lower than those in the WT; However, intracellular Cd content in the mutant was significantly (P<0.05) higher than that in the WT. Taken together, it can be concluded that the PcpA functions in Cu uptake and homeostasis, iron uptake, and Cd export from the cytosol to the extracytosol.

Canine CXCL7 and its functional expression in dendritic cells undergoing maturation

Many cells, including leucocytes and stromal cells, express CXCL7, a member of the CXC chemokine family, also known as platelet basic protein. CXCL7 is a potent chemoattractant and activator of neutrophil function. Dendritic cells (DCs) play a pivotal role in antigen processing and presentation. Very little information is available on the ability of DCs to recruit neutrophils by producing chemokines. In this work, we have cloned canine CXCL7. Based on the predicted gene sequence and using the 3′RACE technique, the full-length gene was amplified from LPS-treated canine peripheral blood mononuclear cells. The cloned cDNA sequence consisted of 357 nucleotides and encoded a 118 amino acid protein, including a 38 amino acid signal peptide. The use of CXCL7-containing supernatants from CXCL7-transfected BALB/3T3 in the neutrophil migration assay confirmed that canine CXCL7 had chemoattractive activity for neutrophils. We then used canine monocyte-derived DCs to generate CXCL7 for the rest of the experiment. Expression of CXCL7 by DCs treated with LPS, IL-1β, IL-6, TGF-β, TNF-α, or IFN-γ was compared using real-time RT-PCR and Western blotting. When treated with IL-1β, IL-6, TNF-α, or TGF-β, canine DCs expressed significantly higher levels of CXCL7 mRNA and protein than when treated with IFN-γ or LPS. It is concluded that dog DCs express high levels of the neutrophil chemotactic factor CXCL7 when stimulated by proinflammatory cytokines, including IL-1β, IL-6, TNF-α, or TGF-β, and may play an important role in modulating inflammatory responses.

Molecular characterization, chromosomal location, alternative splicing and polymorphism of porcine GFAT1 gene

Glutamine: fructose-6-phosphate amidotransferase (GFAT) is the rate-limiting enzyme of the hexosamine synthesis pathway, which plays important roles in insulin resistance and glucose toxicity. GFAT1 is one of the two isoenzymes of GFAT. In the present study, we cloned cDNA sequence of the porcine GFAT1 gene and identified a GFAT1 splice variant (designed GFAT1-L) that contains a 54 bp insertion within the coding region. Nested RT-PCR revealed that GFAT1 was ubiquitously expressed in all tested tissues, but GFAT1-L was only expressed in skeletal muscle and heart, not in liver, spleen, lung, kidney, small intestine, stomach and fat tissue, suggested that GFAT1-L was selectively expressed in striate muscle in pig. Using both the somatic cell hybrid panel and radiation hybrid panel, the GFAT1 gene was mapped to porcine chromosome 3q21-q27, in which several significant QTLs for carcass traits were found. Among the SNPs we found in porcine GFAT1 gene, only the g. 101A>G polymorphism which located in intron 8 was polymorphic in two pig populations we investigated in the study. Association analyses revealed that the g. 101A>G polymorphism has a significant effect on lean meat percentage (P < 0.05), corrected backfat thickness (P < 0.05) and backfat at the rump (P < 0.05).

Identification and expression analysis of WRKY transcription factor genes in canola (Brassica napus L.) in response to fungal pathogens and hormone treatments.

BackgroundMembers of plant WRKY transcription factor families are widely implicated in defense responses and various other physiological processes. For canola (Brassica napus L.), no WRKY genes have been described in detail. Because of the economic importance of this crop, and its evolutionary relationship to Arabidopsis thaliana, we sought to characterize a subset of canola WRKY genes in the context of pathogen and hormone responses.ResultsIn this study, we identified 46 WRKY genes from canola by mining the expressed sequence tag (EST) database and cloned cDNA sequences of 38 BnWRKYs. A phylogenetic tree was constructed using the conserved WRKY domain amino acid sequences, which demonstrated that BnWRKYs can be divided into three major groups. We further compared BnWRKYs to the 72 WRKY genes from Arabidopsis and 91 WRKY from rice, and we identified 46 presumptive orthologs of AtWRKY genes. We examined the subcellular localization of four BnWRKY proteins using green fluorescent protein (GFP) and we observed the fluorescent green signals in the nucleus only.The responses of 16 selected BnWRKY genes to two fungal pathogens, Sclerotinia sclerotiorum and Alternaria brassicae, were analyzed by quantitative real time-PCR (qRT-PCR). Transcript abundance of 13 BnWRKY genes changed significantly following pathogen challenge: transcripts of 10 WRKYs increased in abundance, two WRKY transcripts decreased after infection, and one decreased at 12 h post-infection but increased later on (72 h). We also observed that transcript abundance of 13/16 BnWRKY genes was responsive to one or more hormones, including abscisic acid (ABA), and cytokinin (6-benzylaminopurine, BAP) and the defense signaling molecules jasmonic acid (JA), salicylic acid (SA), and ethylene (ET). We compared these transcript expression patterns to those previously described for presumptive orthologs of these genes in Arabidopsis and rice, and observed both similarities and differences in expression patterns.ConclusionWe identified a set of 13 BnWRKY genes from among 16 BnWRKY genes assayed, that are responsive to both fungal pathogens and hormone treatments, suggesting shared signaling mechanisms for these responses. This study suggests that a large number of BnWRKY proteins are involved in the transcriptional regulation of defense-related genes in response to fungal pathogens and hormone stimuli.

Molecular cloning and characterization of the lipopolysaccharide and β-1, 3-glucan binding protein in Chinese mitten crab (Eriocheir sinensis)

The lipopolysaccharide and beta-1, 3-glucan binding protein (LGBP) is a pattern recognition protein which is fundamental for the innate immune response of crustaceans. A partial cDNA produced by the random sequencing of cDNA clones from a haemocyte cDNA library of Eriocheir sinensis showed similarity to the LGBP gene of the Chinese shrimp (Fenneropenaeus chinensis). The full-length cDNA was subsequently cloned and sequenced by the technique of rapid amplification of cDNA ends (RACE). The E. sinensis LGBP gene (designated as Es-LGBP) was 1236 bases long and was capable of encoding a polypeptide of 362 amino acids showing significant similarity to homologous genes in shrimp. The crab LGBP deduced amino acid sequence carrying conserved features of this gene family including a potential recognition motif for beta-1, 3 linkages of polysaccharides and putative RGD (Arg-Gly-Asp)cell adhesion sites. Real-time quantitative reverse transcription-PCR (RT-PCR) analysis showed that LGBP gene expresses in haemocytes, hepatopancreas, muscles, gills, stomachs, and intestines with the highest level in haemocytes and the lowest in the stomach. The LGBP gene expression is up-regulated upon bacterial infection and the binding of lipopolysaccharide and beta-1, 3-glucan to LGBP could induce a series of immune reactions. The temporal expression of the LGBP gene after bacterial challenge was measured by real-time quantitative RT-PCR. The result demonstrated that the LGBP gene expression in crab was up-regulated at 1.5 h post-injection of bacteria followed by a step by step recovery at 12 and 24 h. Our data suggest that the crab LGBP is an inducible acute-phase protein that could be critical in crab immunity.

Expressed sequence tags of Aspergillus fumigatus: Extension of catalogue and their evaluation as putative drug targets and/or diagnostic markers

Aspergillus fumigatus a fungal pathogen is implicated in a spectrum of allergic and invasive disorders in humans. Validation of transcriptome of pathogen is essential for understanding its virulence mechanism and to identify new therapeutic targets/diagnostic markers. In order to rapidly identify genes of Aspergillus fumigatus we adopted sequencing of cDNA clones. Our earlier effort has lead to identification of 68 expressed sequence tags of Aspergillus fumigatus. Present study describes 52 more expressed sequence tags generated by sequencing 200 phage clones of a non-normalized cDNA library. One of the cDNA clones comprised of the complete coding region for tetratricopeptide repeat domain protein gene. Various homology search algorithms were employed to assign functions to expressed sequence tags coding for hypothetical proteins, and relevance of these expressed sequence tags or their protein products as drug targets/diagnostic markers was examined by searching for homologues in fungi and human.

Differential expression of α-l-arabinofuranosidase and α-l-arabinofuranosidase/β-d-xylosidase genes during peach growth and ripening

Arabinose is the major neutral sugar in peach (Prunus persica (L.) Batsch) cell walls and substantial changes in arabinose content take place not only during peach melting, when a rapid-softening-related depolymerizing activity may be expected, but also at the onset of peach ripening. A full-length cDNA clone sequence referred to as PpARF1 (GenBank accession no. DQ486870) was obtained and determined by bioinformatics' analysis to be a peach alpha-l-arabinofuranosidase homologue. The deduced PpARF1 translation product is 677 amino acids in length while the mature protein has a predicted molecular mass of 71.6 kD and a theoretical pI of 4.94. Semi-quantitative RT-PCR reactions were conducted to evaluate the expression of both PpARF1 and PpARF/XYL (GenBank accession no. AB264280), the latter encoding a putative bifunctional protein displaying both alpha-l-arabinofuranosidase and beta-d-xylosidase activities. In peach fruit, the PpARF1 gene expression was detected at every developmental stage with a maximum during S2 (lag phase of development) and a subsequent decrease towards S4 (maximal fruit size). In contrast, PpARF/XYL transcript levels were relatively high at the end of S1 (fruit set) and at S3-E (beginning of the cell expansion). Substantial increases in PpARF1 mRNA levels were found at the beginning and end of the climacteric rise and also in melting fruit. In contrast, PpARF/XYL transcripts reached a maximum when fruit firmness was 22-26 N, with a slight decline during the melting stage. PpARF/XYL and PpARF1 were expressed differently in three fruit tissue types as well as in other plant tissues. Ethylene is regarded as the main regulator of peach ripening and the accumulation of PpARF/XYL and PpARF1 transcripts is coincident with the autocatalytic ethylene production during ripening. On the hand, other factors may also play a role in PpARF1 and PpARF/XYL expression, since transcripts accumulate at different developmental times and organs even when ethylene biosynthesis is barely detectable.

Transcriptome Analysis and Identification of Genes Related to Immune Function in Skin of the Chinese Brown Frog

The Chinese brown frog (Rana chensinensis) is a special amphibian in northern China, as it has been used widely in traditional Chinese medicine. The skin of the Chinese brown frog is also a promising resource for producing diverse antimicrobial peptides. To obtain a more comprehensive view of the metabolism and effective pharmacological components of Chinese brown frog skin, we constructed a non-normalized cDNA library from the skin. By sequencing cDNA clones at the 5' end, we obtained 5,976 high-quality EST sequences, which clustered into 512 contigs and 1379 singletons (in all 1,891 clusters). After BLAST searches of the protein and nucleic acid databases in GenBank, we found 46.7% of clusters to have significant similarity to known sequences; 28% matched Xenopus tropicalis ESTs and 29.1% matched Xenopus laevis ESTs. Gene annotation results indicated that genes related to secretion and defensive function, such as ubiquitin, lectin, and proteinase inhibitors, are highly expressed in the skin. Whey acidic-domain proteins are also highly abundant in the skin. Furthermore, both a beta-defensin and a lysozyme are transcribed in the frog skin, providing antibacterial protection. Analyses of gene ontology and KEGG metabolic pathways indicated the physiological roles of Chinese brown frog skin.

Insights into corn genes derived from large-scale cDNA sequencing.

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).Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-008-9415-4) contains supplementary material, which is available to authorized users.

Detection of small RNAs in Pseudomonas aeruginosa by RNomics and structure-based bioinformatic tools

Inactivation of the Pseudomonas aeruginosa (PAO1) hfq gene, encoding the Sm-like Hfq protein, resulted in pleiotropic effects that included an attenuated virulence. As regulation by Hfq often involves the action of small regulatory RNAs (sRNAs), we have used a shotgun cloning approach (RNomics) and bioinformatic tools to identify sRNAs in strain PAO1. For cDNA library construction, total RNA was extracted from PAO1 cultures either grown to stationary phase or exposed to human serum. The cDNA libraries were generated from small-sized RNAs of PAO1 after co-immunoprecipitation with Hfq. Of 400 sequenced cDNA clones, 11 mapped to intergenic regions. Band-shift assays and Northern blot analyses performed with two selected sRNAs confirmed that Hfq binds to and affects the steady-state levels of these RNAs. A proteome study performed upon overproduction of one sRNA, PhrS, implicated it in riboregulation. PhrS contains an ORF, and evidence for its translation is presented. In addition, based on surveys with structure-based bioinformatic tools, we provide an electronic compilation of putative sRNA and non-coding RNA genes of PAO1 based on their evolutionarily conserved structure.

Alternative spliced variants in the pantetheinase family of genes expressed in human neutrophils

Pantetheinase (EC 3.5.1.92) is an enzyme that hydrolyzes pantetheine, an intermediate metabolite of coenzyme A, into pantothenic acid (vitamin B 5) and cysteamine, a potent antioxidant. The pantetheinase gene family consists of three independent genes, pantetheinase/vanin-1/VNN1, GPI-80/VNN2 and vanin-3/VNN3 that are each composed of seven exons. We herein report that human neutrophils express transcripts encoding at least nine splice variants of VNN3 and four splice variants of GPI-80/VNN2. Analysis of the DNA sequence of the human VNN3 gene demonstrated that the VNN3 locus in the human genome as well as the sequence of cDNA clones obtained in this study does not encode the complete VNN3 protein, as previously reported due to a frame shift caused by lack of one nucleotide. Moreover, the VNN3 locus indeed encodes smaller peptides compared to the proteins encoded by the mouse orthologous gene, vanin-3. The anti-GPI-80 monoclonal antibody 3H9 recognized amino acids 120–179 of the GPI-80/VNN2 protein as shown by the results of immunoblotting with recombinant GPI-80/VNN2 variant proteins. Immunoblotting with human neutrophil lysate suggests that the GPI-80/VNN2 variants exist in human neutrophils. The existence of splice variants in the pantetheinase gene family suggests the possibility of alternative roles in addition to canonical enzymatic activity in human neutrophils.

Molecular characterization of calcitonin gene-related peptide (CGRP) related peptides (CGRP, amylin, adrenomedullin and adrenomedullin-2/intermedin) in goldfish ( Carassius auratus): Cloning and distribution

To further characterize the structure and function of calcitonin gene-related peptide (CGRP) related peptides in fish, we have cloned cDNA sequences for CGRP, amylin, adrenomedullin (AM) and adrenomedullin-2/intermedin (IMD) in goldfish ( Carassius auratus) and examined their tissue distribution. CGRP, amylin, AM and IMD cDNAs were isolated by reverse transcription (RT) and rapid amplification of cDNA ends (RACE). The cloned sequences contain the complete four mature peptides, which present a high degree of identity with mature peptide sequences from other fish. Phylogenetic analyses show that goldfish AM and IMD form a sub-family within the CGRP-related peptides that is distinct from the CGRP/amylin sub-family. The distribution of goldfish CGRP-like peptides mRNA expression in different tissues and within the brain was studied by RT-PCR. CGRP, IMD and AM are detected throughout the brain, in pituitary and in most peripheral tissues examined. Amylin mRNA is mostly expressed in the brain, in particular posterior brain, optic tectum and hypothalamus, but is also present in pituitary, gonad, kidney and muscle. Our results suggest that goldfish CGRP, amylin, AM and IMD are conserved peptides that show the typical structure characteristics present in their mammalian counterparts. The widespread distributions of CGRP, AM and IMD suggest that these peptides could be involved in the regulation of many diverse physiological functions in fish. Amylin mRNA distribution suggests possible new roles for this peptide in teleosts, including the control of reproduction.

Molecular characterization of the porcine GBP1 and GBP2 genes

Experimental evidence indicated that interferon-inducible guanylate-binding proteins (GBPs) are similar with genes in the myxovirus (Mx) resistance protein subfamily of the large GTPases protein family and play important roles in the resistance to intracellular pathogens. As more diseases exert significant influence on pig industry, it is anticipated that more candidate disease-resistance genes could be found in future strategies aimed at improving genetic resistance to infectious diseases. In this study, we cloned cDNA sequences and analyzed the genomic structure of porcine GBP1 (poGBP1) and GBP2 (poGBP2). The two genes were mapped to SSC4q21-q23 and SSC4q24 by the SCHP panel respectively, further IMpRH panel analysis showed both genes were most closely linked to the marker SWR153. The deduced amino acid sequences of these two genes share the same three classical GTP-binding motifs at the amino terminus and are less conserved at the carboxyl termini except for a CaaX motif, compared with human and mouse counterparts. The reverse transcriptase-polymerase chain reaction (RT-PCR) revealed that poGBP1 and poGBP2 were both widely expressed in many tissues, and transient transfection indicated that poGBP1 and poGBP2 proteins were both located in cytoplasms within Pig Kidney Epithelial cells (PK15). Quantitative real-time PCR (Q-RT-PCR) analyses showed poGBP1 and poGBP2 had very similar expression patterns in PK15 cells at different time points after poly I:C stimulation, suggesting that ISRE (interferon-stimulated response element) plays a crucial role in the transcriptional regulation of these two genes. Four single nucleotide polymorphisms (SNPs) and three SNPs were detected in the cDNA sequences of poGBP1 and poGBP2, respectively. Association analyses revealed that the poGBP1 Eco81I and poGBP2 SspI polymorphisms both had significant associations ( p < 0.05) with red blood cell count (RBC), haemoglobin concentration (HGB) and hematocrit (HCT) of 17-day-old pigs.

A unique tetrameric structure of deer plasma haptoglobin - an evolutionary advantage in the Hp 2-2 phenotype with homogeneous structure

Similar to blood types, human plasma haptoglobin (Hp) is classified into three phenotypes: Hp 1-1, 2-1 and 2-2. They are genetically inherited from two alleles Hp 1 and Hp 2 (represented in bold), but only the Hp 1-1 phenotype is found in almost all animal species. The Hp 2-2 protein consists of complicated large polymers cross-linked by alpha2-beta subunits or (alpha2-beta)n (where n>or=3, up to 12 or more), and is associated with the risk of the development of diabetic, cardiovascular and inflammatory diseases. In the present study, we found that deer plasma Hp mimics human Hp 2, containing a tandem repeat over the alpha-chain based on our cloned cDNA sequence. Interestingly, the isolated deer Hp is homogeneous and tetrameric, i.e. (alpha-beta)4, although the locations of -SH groups (responsible for the formation of polymers) are exactly identical to that of human. Denaturation of deer Hp using 6 m urea under reducing conditions (143 mmbeta-mercaptoethanol), followed by renaturation, sustained the formation of (alpha-beta)4, suggesting that the Hp tetramers are not randomly assembled. Interestingly, an alpha-chain monoclonal antibody (W1), known to recognize both human and deer alpha-chains, only binds to intact human Hp polymers, but not to deer Hp tetramers. This implies that the epitope of the deer alpha-chain is no longer exposed on the surface when Hp tetramers are formed. We propose that steric hindrance plays a major role in determining the polymeric formation in human and deer polymers. Phylogenetic and immunochemical analyses revealed that the Hp 2 allele of deer might have arisen at least 25 million years ago. A mechanism involved in forming Hp tetramers is proposed and discussed, and the possibility is raised that the evolved tetrameric structure of deer Hp might confer a physiological advantage.

Gene profiling for studying the mechanism of aflatoxin biosynthesis in Aspergillus flavus and A. parasiticus

Aflatoxins are toxic and carcinogenic polyketide metabolites produced by certain fungal species, including Aspergillus flavus and A. parasiticus. Many internal and external factors, such as nutrition and environment affect aflatoxin biosynthesis; therefore, we analyzed the transcriptome of A. flavus using expressed sequence tags (ESTs) from a normalized cDNA expression library constructed from mycelia harvested under several conditions. A total of 7218 unique ESTs were identified from 26 110 sequenced cDNA clones. Functional classifications were assigned to these ESTs and genes, potentially involved in the aflatoxin contamination process, were identified. Based on this EST sequence information, a genomic DNA amplicon microarray was constructed at The Institute for Genomic Research (TIGR). To identify potential regulatory networks controlling aflatoxin contamination in food and feeds, gene expression profiles in aflatoxin-supportive media versus non-aflatoxin-supportive media were evaluated in A. flavus and A. parasiticus. Genes consistently expressed in several aflatoxin-supportive media are reported. †Yu, Ronning and Wilkinson contributed equally to this work.

Identification of the gene encoding a Dnak-type molecular chaperone as potentially down regulated in blue mussels (Mytilus edulis) following acute exposure to atrazine

Because of their widespread geographical distribution and high capacity of bioaccumulation, bivalve species, in particular blue mussels (Mytilus edulis), are considered as sentinel organisms in Ecotoxicology for monitoring the effects of xenobiotics in estuaries and coastal areas. The purpose of this study was to identify molecular biomarkers for atrazine exposure by using differential analysis of gene expression. For this, the digestive gland transcriptome of atrazine exposed mussels (1 μg l−1, 24 h) was compared to that of control animals. Candidate transcripts for differential expression were isolated by Subtractive PCR Suppressive Hybridization (SSH). A total of 543 potentially regulated clones was isolated. A part of these clones was sequenced and extensive similarity searches in genome data bases were carried out. Among the sequenced cDNA clones, gld15, gld122 and gld177 which corresponded to potentially down regulated mRNA, were likely to encode for a Mytilus edulis DnaK-type molecular chaperone of the Heat shock 70 protein family. As a matter of fact, the deduced peptide sequence of gld15 comprised 223 amino acids and possessed 87.4% of identity with the human Hsc70. Heat shock proteins and, in particular Hsc70 and Hsp70, are considered as molecular chaperones involved in various cellular processes including refolding of damaged proteins and apoptosis. The fact that atrazine potentially down regulates the expression of a cytoprotective gene was unexpected and further experiments are required to measure the kinetics and dose response effects of atrazine on DnaK gene expression. Differential gene expression analysis is one of most widely used approach of the postgenomics era in the biomedical field of research and it may prove to be a valuable tool for the identification of new and relevant biomarkers in Ecotoxicology.