Rapid Amplification Of cDNA Ends PCR Research Articles

Swamp eel aldehyde reductase is involved in response to nitrosative stress via regulating NO/GSH levels.

Aldehyde reductase (ALR) plays key roles in the detoxification of toxic aldehyde. In this study, the authors cloned the swamp eel ALR gene using rapid amplification of cDNA ends-PCR (RACE-PCR). The recombinant protein (rALR) was expressed in Escherichia coli and purified using a Ni2+ -NTA chelating column. The rALR protein exhibited efficient reductive activity towards several aldehydes, ketones and S-nitrosoglutathione (GSNO). A spot assay suggested that the recombinant E. coli strain expressing rALR showed better resistance to formaldehyde, sodium nitrite and GSNO stress, suggesting that swamp eel ALR is crucial for redox homeostasis in vivo. Consequently, the authors investigated the effect of rALR on the oxidative parameters of the liver in swamp eels challenged with Aeromonas hydrophila. The hepatic glutathione (GSH) content significantly increased, and the hepatic NO content and levels of reactive oxygen species and reactive nitrogen species significantly decreased when rALR was administered. In addition, the mRNA expression of hepatic Alr, HO1 and Nrf2 was significantly upregulated, whereas the expression levels of NF-κB, IL-1β and NOS1 were significantly downregulated in the rALR-administered group. Collectively, these results suggest that ALR is involved in the response to nitrosative stress by regulating GSH/NO levels in the swamp eel.

A novel ferritin subunit gene from Asian green mussel, Perna viridis (Linnaeus, 1758)

Iron sequestration through ferritin forms a major part of innate immune response in molluscs and detailed understanding of ferritin gene and its functions can be directly applied in infection and disease management studies. Accordingly, identification and detailed molecular characterization of a ferritin subunit gene from a commercially significant marine mussel Perna viridis was targeted. Molecular screening using degenerate primers in total mantle RNA resulted in the amplification of a novel ferritin gene fragment having <87% identity to the reported ferritin gene sequences. Rapid amplification of cDNA ends-PCR was followed to generate complete cDNA sequence of P.viridis ferritin (PvFer). The complete cDNA was found to be 798 bp, containing an open reading frame of 522 bp, 5′ untranslated region (UTR) of 112 bp and 3′ UTR of 165 bp. The 5′ UTR and 3′ UTR were shown to contain an iron response element (IRE) and a polyadenylation signal (767AATAAA772) with poly (A) tail, respectively. Prediction of stem loop structure revealed that, PvFer-IRE can be folded into a typical secondary stem loop structure, having 5-CAGUGA-3’ loop, proximal stem of five paired bases followed by a bulged cysteine, and six nucleotide bottom stem, indicating that expression of PvFer is regulated by iron at the translational level. ORF was found to encode 175 amino acid protein with calculated molecular mass of 19.97 kDa and isoelectric point of 4.97. Examination for signal peptide and phylogenetic analysis confirmed that PvFer belonged to cytosolic ferritins of molluscs. Conserved domain analysis showed that PvFer contained both ferroxidase diiron center and ferrihydrite nucleation center, analogous to ferritin M subunit of bony fishes and amphibians. However, amino acid sequence and glycosylation site showed more homology to vertebrate ferritin H subunits. Predicted 3D models of PvFer resembled the typical spatial features of ferritin proteins. The study forms the first comprehensive identification of a ferritin subunit gene in a true/common mussel (Order: Mytilida). Further, the detailed molecular phylogeny conducted through the present study revealed certain thought provoking insights on ferritin genes of the phylum Mollusca.

Pro-Apoptotic Function Analysis of the Reaper Homologue IBM1 in Spodoptera frugiperda.

As an important type of programmed cell death, apoptosis plays a critical role in lepidopteran insects in response to various internal and external stresses. It is controlled by a network of genes such as those encoding the inhibitor of apoptosis proteins. However, there are few studies on apoptosis-related genes in Spodoptera frugiperda. In this study, an orthologue to the Drosophila reaper gene, named Sf-IBM1, was identified from S. frugiperda, and a full-length sequence was obtained by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends PCR (RACE-PCR). The expression pattern of Sf-IBM1 was determined in different developmental stages and various tissues. Apoptotic stimuli including azadirachtin, camptothecin, and ultraviolet radiation (UV) induced the expression of Sf-IBM1 at both transcript and protein levels. Overexpression of Sf-IBM1 induced apoptosis in Sf9 cells, and the Sf-IBM1 protein was localized in mitochondria. The apoptosis induced by Sf-IBM1 could be blocked by the caspase universal inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK) and Sf-IAP1. Our results provide valuable information that should contribute to a better understanding of the molecular events that lead to apoptosis in lepidopterans.

Molecular characterization and expressional quantification of lgp2, a modulatory co-receptor of RLR-signalling pathway in the Indian major carp Labeo rohita following pathogenic challenges and PAMP stimulations.

Lgp2 (laboratory of genetics and physiology 2) is a cytosolic viral sensor of the RLR (retinoic acid-inducible gene 1 like receptor) family member without the caspase recruitment domain, having both inhibitory and stimulatory roles in RLR-signalling pathway. In India, Labeo rohita (rohu) is one of the leading and economically favoured freshwater fish species. Several immunological sentry proteins have been reported in this fish species, but no information is available on the RLR members. This study was aimed at cloning and characterization of full-length lgp2-cDNA (complementary DNA) in rohu and investigation of its expressional modulations following various pathogen-associated molecular pattern stimulations and bacterial infections. The full-length lgp2-cDNA sequence obtained through rapid amplification of cDNA ends-PCR consisted of 2299 nucleotides with an open reading frame of 2034 bp encoding 677 amino acids. In rohu-Lgp2, four conserved domains - a DEAD/DEAH box helicase domain, Pfam type-III restriction enzyme domain, helicase superfamily c-terminal domain and RIG-I C-terminal regulatory domain - have been detected. Within these domains, several important functional motifs, such as ATP-binding site, ATPase motif, RNA unwinding motif and RNA-binding sites, have also been identified. In healthy rohu, lgp2 gene was abundantly expressed in gill, liver, kidney, spleen and blood. In response to both in vitro and in vivo treatments using double-stranded RNA (poly I:C), lgp2 gene expression was significantly (P < 0.05) upregulated in all tested tissues and also in the LRG (Labeo rohita gill) cells. lgp2 gene expression significantly (P < 0.05) increased on stimulation of LRG cells using γ-d-glutamyl-meso-diaminopimelic acid and muramyl dipeptide. In vivo treatment using lipopolysaccharide and Aeromonas hydrophila-derived RNA resulted in both up- and down-regulation of lgp2 gene expression. Upon gram-positive and gram-negative bacterial infections, the expression of the lgp2 gene increased at different times in almost all the tested tissues. These integrated observations in rohu suggest that Lgp2 is an antiviral and antibacterial cytosolic receptor. SIGNIFICANCE STATEMENT: Lgp2, a cytosolic viral sensor of retinoic acid-inducible gene 1 like receptor family member, has been cloned in Labeo rohita. The complete sequence of rohu lgp2-complementary DNA consisted of 2299 nucleotides with an open reading frame of 2034 bp encoding 677 amino acids. It consisted of a DExDc, RES-III, HELICc, Pfam RIG-I_C-RD, ATP-binding site, ATPase motif, RNA unwinding motif and RNA-binding site. Upon bacterial infection, double-stranded RNA and various pathogen-associated molecular pattern stimulations, lgp2 gene expression significantly increased, indicating its role as an antiviral and antibacterial cytosolic receptor.

A corepressor participates in LexA-independent regulation of error-prone polymerases in Acinetobacter.

The DNA damage response of the multidrug-resistant pathogen Acinetobacter baumannii , which induces mutagenic UmuD′2C error-prone polymerases, differs from that of many bacteria. Acinetobacter species lack a LexA repressor, but induce gene transcription after DNA damage. One regulator, UmuDAb, binds to and represses the promoters of the multiple A. baumannii ATCC 17978 umuDC alleles and the divergently transcribed umuDAb and ddrR genes. ddrR is unique to the genus Acinetobacter and of unknown function. 5' RACE (rapid amplification of cDNA ends) PCR mapping of the umuDAb and ddrR transcriptional start sites revealed that their −35 promoter elements overlapped the UmuDAb binding site, suggesting that UmuDAb simultaneously repressed expression of both genes by blocking polymerase access. This coordinated control of ddrR and umuDAb suggested that ddrR might also regulate DNA damage-inducible gene transcription. RNA-sequencing experiments in 17 978 ddrR − cells showed that ddrR regulated approximately 25 % (n=39) of the mitomycin C-induced regulon, with umuDAb coregulating 17 of these ddrR-regulated genes. Eight genes (the umuDC polymerases, umuDAb and ddrR) were de-repressed in the absence of DNA damage, and nine genes were uninduced in the presence of DNA damage, in both ddrR and umuDAb mutant strains. These data suggest ddrR has multiple roles, both as a co-repressor and as a positive regulator of DNA damage-inducible gene transcription. Additionally, 57 genes were induced by mitomycin C in the ddrR mutant but not in wild-type cells. This regulon contained multiple genes for DNA replication, recombination and repair, transcriptional regulators, RND efflux, and transport. This study uncovered another regulator of the atypical DNA damage response of this genus, to help describe how this pathogen acquires drug resistance through its expression of the error-prone polymerases under DdrR and UmuDAb control.

Molecular cloning, characterization and tissue specificity of the expression in the TAC1 genes from Henan Huai goat (Capra hircus)

To further to understand the structure and function of the TAC1 gene, we cloned the full-length cDNAs of the TAC1 genes from goat by rapid amplification of cDNA ends-PCR and the qRT-PCR was used to analyze the TAC1 mRNA expression patterns of goat various tissues. The full-length cDNA of goat TAC1 was 1176 bp, with a 339 bp open reading frame encoding 112 amino acids. The amino acid sequence analysis revealed that goat TAC1 gene encoded a water-drain protein and its relative molecular weight and isoelectric point was 13,012.86 Da and 6.29 respectively. Alignment and phylogenetic analyses revealed that their amino acid sequences were highly similar to those of other vertebrates. TAC1 expression of the goat of the brain, cerebellum, medulla oblongata, heart, liver, spleen, lung, kidney, uterus, ovaries. These results serve as a foundation for further study on the Capra hircus TAC1 gene.

Transcriptional profile of gene clusters involved in the methylerythritol phosphate pathway in Bacillus subtilis 916

The methylerythritol phosphate pathway is responsible for the biosynthesis of terpenoids, the largest class of secondary metabolites. Although the structures and functions of the proteins involved in this pathway have been well studied in Bacillus subtilis, only a few studies have reported the transcriptional profile of the genes involved. Therefore, we analyzed methylerythritol phosphate pathway genes in the genome of B. subtilis 916, which has been developed as a biological control agent against some rice diseases in China. Our results showed that methylerythritol phosphate pathway genes were distributed throughout the genome of this strain. These genes were transcribed during both the exponential and stationary phases. We further confirmed the transcription units of dxs, dxr, ispD, ispF, ipK, ispG, ispH, idi, and ispA in B. subtilis 916 through reverse transcription-PCR analyses; the results showed that these nine genes were located in seven different operons. The transcript start sites of the seven different operons were determined by 5’-rapid amplification of cDNA ends-PCR. Thus, our study provides a molecular basis at the transcriptional level for investigating homoterpene synthesis in the methylerythritol phosphate pathway of B. subtilis 916.

Dermaseptin-PH: A Novel Peptide with Antimicrobial and Anticancer Activities from the Skin Secretion of the South American Orange-Legged Leaf Frog, Pithecopus (Phyllomedusa) hypochondrialis.

The dermaseptin peptides, mainly derived from the skin secretions of Hylidae frogs, belong to a superfamily of antimicrobial peptides and exhibit diverse antimicrobial and anticancer activities with low cytotoxicity. Here, we reported a novel dermaseptin peptide, from the South American orange-legged leaf frogs, Pithecopus (Phyllomedusa) hypochondrialis, processing the shortest peptide length, namely Dermaseptin-PH. The complementary DNA (cDNA) encoding biosynthetic precursor of Dermaseptin-PH was initially identified by the rapid amplification of cDNA ends PCR (RACE-PCR) technique from the skin secretion. The predicted primary structure was confirmed by a combination of reverse-phase high performance liquid chromatography (RP-HPLC) and MS/MS fragmentation from the skin secretion. Chemically-synthetic Dermaseptin-PH was investigated using a range of bioactivity assessment assays to evaluate the biological activities and cytotoxicity of Dermaseptin-PH. Dermaseptin-PH inhibited the growth of Gram-negative bacteria, Gram-positive bacteria, and pathogenic yeast Candida albicans. In addition, Dermaseptin-PH showed a broad-spectrum of anticancer activities against several cancer cell lines including MCF-7, H157, U251MG, MDA-MB-435S, and PC-3. The potent antimicrobial and anticancer activities of Dermaseptin-PH make it a promising candidate in the discovery of new drugs for clinical applications, and the relatively short sequence of Dermaseptin-PH can provide new insight for the research and structural modification of new peptide drugs.

Prevalence and genetic features of rabbit hepatitis E virus in Korea.

Hepatitis caused by hepatitis E virus (HEV) is a public health concern worldwide. HEV strains have been isolated from several animal species, some of which induce zoonosis. Recently, the isolation of HEV from rabbits was reported. Here, the partial capsid gene (320 bp) of HEV was detected in rabbit feces via reverse transcriptase-polymerase chain reaction (RT-PCR). Rabbit HEV was found in two of six rabbit farms and 17 of 264 rabbit fecal samples (6.4%). A phylogenetic analysis of the partial capsid gene classified the 17 HEV isolates into the putative rabbit HEV clade. A full genomic sequence, KOR-Rb-1, was obtained from one rabbit HEV isolate by 5' and 3' rapid amplification of cDNA ends-PCR and RT-PCR, and comprised 7275 bp excluding the 3' poly(A) tail. It shared 77.5-86.8%, 86.6%, and 80.2-84.3% nucleotide identities with rabbit HEV isolates from China, the US, and France, respectively. It also shared 72.3-73.0%, 71.4%, 76.7-78.3%, 72.8-73.3%, and 47.1-47.2% nucleotide identities with representative strains of HEV-1, HEV-2, HEV-3, HEV-4, and avian HEV, respectively. A full-genome phylogenetic analysis classified KOR-Rb-1 into the provisional rabbit HEV clade. This isolate could be used to study the pathogenesis and zoonotic potential of rabbit HEV.

Identification, Characterization and Expression Profiling of Stress-Related Genes in Easter Lily (Lilium formolongi)

Biotic and abiotic stresses are the major causes of crop loss in lily worldwide. In this study, we retrieved 12 defense-related expressed sequence tags (ESTs) from the NCBI database and cloned, characterized, and established seven of these genes as stress-induced genes in Lilium formolongi. Using rapid amplification of cDNA ends PCR (RACE-PCR), we successfully cloned seven full-length mRNA sequences from L. formolongi line Sinnapal lily. Based on the presence of highly conserved characteristic domains and phylogenetic analysis using reference protein sequences, we provided new nomenclature for the seven nucleotide and protein sequences and submitted them to GenBank. The real-time quantitative PCR (qPCR) relative expression analysis of these seven genes, including LfHsp70-1, LfHsp70-2, LfHsp70-3, LfHsp90, LfUb, LfCyt-b5, and LfRab, demonstrated that they were differentially expressed in all organs examined, possibly indicating functional redundancy. We also investigated the qPCR relative expression levels under two biotic and four abiotic stress conditions. All seven genes were induced by Botrytis cinerea treatment, and all genes except LfHsp70-3 and LfHsp90 were induced by Botrytis elliptica treatment; these genes might be associated with disease tolerance mechanisms in L. formolongi. In addition, LfHsp70-1, LfHsp70-2, LfHsp70-3, LfHsp90, LfUb, and LfCyt-b5 were induced by heat treatment, LfHsp70-1, LfHsp70-2, LfHsp70-3, LfHsp90, and LfCyt-b5 were induced by cold treatment, and LfHsp70-1, LfHsp70-2, LfHsp70-3, LfHsp90, LfCy-b5, and LfRab were induced by drought and salt stress, indicating their likely association with tolerance to these stress conditions. The stress-induced candidate genes identified in this study provide a basis for further functional analysis and the development of stress-resistant L. formolongi cultivars.

RETRACTION of: Molecular cloning and characterization of a salinity stress-induced gene encoding DEAD-box helicase from the halophyte Apocynum venetum.

The genes encoding DEAD-box helicases play a key role in various abiotic stresses, including temperature, light, oxygen, and salt stress. A salt-responsive gene, designated AvDH1, was isolated from the halophyte dogbane (Apocynum venetum) by using suppression subtractive hybridization and RACE (rapid amplification of cDNA ends) PCR. The deduced amino acid sequence has nine conserved helicase motifs of the DEAD-box protein family. The AvDH1 gene is present as a single copy in the dogbane genome. This gene is expressed in response to NaCl and not polyethlene glycol (PEG) nor abscisic acid, and its expression increases with time. The transcription of AvDH1 is also induced by low temperature (4 degrees C), but its accumulation first increases then decreases with time. The purified recombinant protein contains ATP-dependent DNA helicase activity, ATP-independent RNA helicase activity, and DNA- or RNA-dependent ATPase activity. The ATPase activity of AvDH1 is stimulated more by single-stranded DNA than by double-stranded DNA or RNA. These results suggested that AvDH1 belonging to the DEAD-box helicase family is induced by salinity, functions as a typical helicase to unwind DNA and RNA, and may play an important role in salinity tolerance.

Molecular cloning, characterization and tissue specificity of the expression of the ovine CSRP2 and CSRP3 genes from Small-tail Han sheep (Ovis aries)

Two genes, cysteine- and glycine-rich protein 2 (CSRP2) and cysteine- and glycine-rich protein 3 (CSRP3), play important roles in tissue-specific cell growth and development. However, few CSRP2 and CSRP3 genes have been functionally characterized in sheep. In this study, the full-length cDNAs of the CSRP2 and CSRP3 genes were cloned from Small-tail Han sheep by rapid amplification of cDNA ends-PCR. The GenBank accession numbers of the full-length CSRP2 and CSRP3 cDNA sequences are KJ743957 and KJ743958, respectively. The full-length cDNA of ovine CSRP2 was 917bp, with a 582-bp open reading frame encoding 193 amino acids. The complete ovine CSRP3 cDNA was 917bp, with a 585-bp open reading frame encoding 194 amino acids. Alignment and phylogenetic analyses revealed that their amino acid sequences are highly similar to those of other vertebrates, all of which contain two conserved LIM-only domains and a relatively conserved nuclear targeting sequence. To further validate the functions of the two genes, their mRNA expression patterns were evaluated in various Small-tail Han and Dorper sheep tissues using qRT-PCR analyses. CSRP2 was mainly detected in the aorta, whereas CSRP3 was highly concentrated in the heart and the muscle. CSRP3 was expressed to a higher level in the hearts of Small-tail Han sheep than in Dorper sheep (P<0.05). However, the opposite was found in the muscle (the longissimus dorsi and biceps femoris); CSRP3 was expressed to a higher level in Dorper sheep than in Small-tail Han sheep (P<0.05). We quantified the CRP3 protein (coded by the CSRP3 gene) levels in different tissues in Small-tail Han and Dorper sheep. We also detected a putative isoform of the CRP3 protein in sheep, which was significantly different in the heart tissue of the two breeds (P<0.05). The expression patterns of the two genes' mRNAs and CRP3 protein showed clear tissue specificity in both sheep breeds.

A half-type AvABCG1 transporter derived from Andropogon virginicus L. confers aluminum tolerance

Aluminum (Al) induced genes were isolated from a wild plant, Andropogon virginicus L. by a method involving arbitrarily primed PCR-based RNA finger printing. One of the clones, AP18-2-1, was induced in both roots and shoots by Al and diamide treatments, and slightly by Cu and Zn treatments. Full-length cDNA (2287bp) was obtained by RACE (rapid amplification of cDNA ends) PCR and DNA sequencing indicated that it encoded a half-type ABC transporter that was highly homologous to other ABCG family genes. The Al-induced transporter gene derived from A. virginicus L. was therefore designated AvABCG1. The AvABCG1 gene conferred tolerance to Al, Cu and diamide in Arabidopsis thaliana transformants. A fusion protein (AvABCG1::GFP) was localized in the cell membrane region. Immunohistological analysis of the native protein in A. virginicus L. indicated that it localized to both exodermis and endodermis in roots, and to the vascular bundle sheath and its extensions in both upper and lower epidermal cells in leaves. Fluorescent measurement showed that absorbed Al was detected in almost the same areas as the AvABCG1 protein in both root and leaf. We conclude that the AvABCG1 transporter may confer Al tolerance by accumulating absorbed Al ions in specific regions of the plant.

Proteome Analysis of Watery Saliva Secreted by Green Rice Leafhopper, Nephotettix cincticeps.

The green rice leafhopper, Nephotettix cincticeps, is a vascular bundle feeder that discharges watery and gelling saliva during the feeding process. To understand the potential functions of saliva for successful and safe feeding on host plants, we analyzed the complexity of proteinaceous components in the watery saliva of N. cincticeps. Salivary proteins were collected from a sucrose diet that adult leafhoppers had fed on through a membrane of stretched parafilm. Protein concentrates were separated using SDS-PAGE under reducing and non-reducing conditions. Six proteins were identified by a gas-phase protein sequencer and two proteins were identified using LC-MS/MS analysis with reference to expressed sequence tag (EST) databases of this species. Full -length cDNAs encoding these major proteins were obtained by rapid amplification of cDNA ends-PCR (RACE-PCR) and degenerate PCR. Furthermore, gel-free proteome analysis that was performed to cover the broad range of salivary proteins with reference to the latest RNA-sequencing data from the salivary gland of N. cincticeps, yielded 63 additional protein species. Out of 71 novel proteins identified from the watery saliva, about 60 % of those were enzymes or other functional proteins, including GH5 cellulase, transferrin, carbonic anhydrases, aminopeptidase, regucalcin, and apolipoprotein. The remaining proteins appeared to be unique and species- specific. This is the first study to identify and characterize the proteins in watery saliva of Auchenorrhyncha species, especially sheath-producing, vascular bundle-feeders.

Identification and Expression Analysis of Putative Chemosensory Receptor Genes in Microplitis mediator by Antennal Transcriptome Screening.

Host-seeking, ovipositional behavior and mating of insects are controlled mainly by odor perception through sensory organs such as antennae. Antennal chemoreception is extremely important for insect survival. Several antennal chemosensory receptors are involved in mediating the odor detection in insects, especially the odorant receptors (ORs) and ionotropic receptors (IRs), to ensure the specificity of the olfactory sensory neuron responses. In the present study, we identified the chemosensory receptor gene repertoire of the parasitoid wasp Microplitis mediator, a generalist endoparasitoid that infests more than 40 types of Lepidopterous larvae and is widely distributed in the Palaearctic region. By transcriptome sequencing of male and female antennae we identified 60 candidate odorant receptors, six candidate ionotropic receptors and two gustatory receptors in M. mediator. The full-length sequences of these putative chemosensory receptor genes were obtained by using the rapid amplification of cDNA ends PCR (RACE-PCR) method. We also conducted reverse transcription PCR (RT-PCR) combined with real-time quantitative PCR (qPCR) for investigating the expression profiles of these chemosensory receptor genes in olfactory and non-olfactory tissues. The tissue- and sex-biased expression patterns may provide insights into the roles of the chemosensory receptor in M. mediator. Our findings support possible future study of the chemosensory behavior of M. mediator at the molecular level.

Structural and functional characterization of Pseudomonas aeruginosa global regulator AmpR.

Pseudomonas aeruginosa is a dreaded pathogen in many clinical settings. Its inherent and acquired antibiotic resistance thwarts therapy. In particular, derepression of the AmpC β-lactamase is a common mechanism of β-lactam resistance among clinical isolates. The inducible expression of ampC is controlled by the global LysR-type transcriptional regulator (LTTR) AmpR. In the present study, we investigated the genetic and structural elements that are important for ampC induction. Specifically, the ampC (PampC) and ampR (PampR) promoters and the AmpR protein were characterized. The transcription start sites (TSSs) of the divergent transcripts were mapped using 5' rapid amplification of cDNA ends-PCR (RACE-PCR), and strong σ(54) and σ(70) consensus sequences were identified at PampR and PampC, respectively. Sigma factor RpoN was found to negatively regulate ampR expression, possibly through promoter blocking. Deletion mapping revealed that the minimal PampC extends 98 bp upstream of the TSS. Gel shifts using membrane fractions showed that AmpR binds to PampC in vitro whereas in vivo binding was demonstrated using chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR). Additionally, site-directed mutagenesis of the AmpR helix-turn-helix (HTH) motif identified residues critical for binding and function (Ser38 and Lys42) and critical for function but not binding (His39). Amino acids Gly102 and Asp135, previously implicated in the repression state of AmpR in the enterobacteria, were also shown to play a structural role in P. aeruginosa AmpR. Alkaline phosphatase fusion and shaving experiments suggest that AmpR is likely to be membrane associated. Lastly, an in vivo cross-linking study shows that AmpR dimerizes. In conclusion, a potential membrane-associated AmpR dimer regulates ampC expression by direct binding.

Phylogenetic and transcription analysis of chrysanthemum WRKY transcription factors.

WRKY transcription factors are known to function in a number of plant processes. Here we have characterized 15 WRKY family genes of the important ornamental species chrysanthemum (Chrysanthemum morifolium). A total of 15 distinct sequences were isolated; initially internal fragments were amplified based on transcriptomic sequence, and then the full length cDNAs were obtained using RACE (rapid amplification of cDNA ends) PCR. The transcription of these 15 genes in response to a variety of phytohormone treatments and both biotic and abiotic stresses was characterized. Some of the genes behaved as would be predicted based on their homology with Arabidopsis thaliana WRKY genes, but others showed divergent behavior.

Identification and analysis of a Marsupenaeus japonicus ferritin that is regulated at the transcriptional level by WSSV infection.

Marsupenaeus japonicus is a shrimp species of great value in the Chinese aquaculture industry. Given the susceptibility to viral diseases, research efforts have focused on the molecular characteristics of the shrimp's immune mechanisms. Ferritin is well known for its iron storage function, but studies have also addressed its immune function in response to pathogens. In this study, an M. japonicus ferritin cDNA was identified by homology cloning and rapid amplification of cDNA ends-PCR. The full-length cDNA is 1244 bp long and contains an open reading frame (513 bp) that encodes a highly conserved protein of 170 amino acids. Quantitative real-time PCR detection of ferritin revealed high expression in eight tested tissues, with the highest levels in hemocytes-consistent with the iron storage capacity of ferritin. We infected M. japonicus with white spot syndrome virus and validated the model by viral copy analysis and histopathology, which demonstrated an increase in viral copies along with acute degeneration of tissues. Transcripts of ferritin increased by 3.1-fold, 2.1-fold, and 1.5-fold in the hepatopancreas, gill, and midgut at 24h post-injection, suggesting that ferritin played an important role in the immune response of M. japonicus.

RNA Targets and Specificity of Staufen, a Double-stranded RNA-binding Protein in Caenorhabditis elegans

The Staufen family consists of proteins that possess double-stranded RNA-binding domains (dsRBDs). Staufen proteins of Drosophila and mammals regulate mRNA localization, translation, and decay. We report analysis of Staufen in Caenorhabditis elegans, which we have designated STAU-1. We focus on its biochemical properties, mRNA targets, and possible role in RNAi. We show that STAU-1 is expressed as mRNA and protein at all stages of C. elegans development. The wild-type, full-length protein, purified from bacteria, binds duplex RNA with high affinity in vitro. Purified, mutant proteins lacking single dsRBDs still bind RNA efficiently, demonstrating that no single domain is required for binding to duplex RNA (although dsRBD2 could not be tested). STAU-1 mRNA targets were identified via immunoprecipitation with specific anti-STAU-1 antibodies, followed by microarray analysis (RIP-Chip). These studies define a set of 418 likely STAU-1 mRNA targets. Finally, we demonstrate that stau-1 mutants enhance exogenous RNAi and that stau-1;eri-1 double mutants exhibit sterility and synthetic germ line defects.

Identification of novel growth phase- and media-dependent small non-coding RNAs in Streptococcus pyogenes M49 using intergenic tiling arrays

BackgroundSmall non-coding RNAs (sRNAs) have attracted attention as a new class of gene regulators in both eukaryotes and bacteria. Genome-wide screening methods have been successfully applied in Gram-negative bacteria to identify sRNA regulators. Many sRNAs are well characterized, including their target mRNAs and mode of action. In comparison, little is known about sRNAs in Gram-positive pathogens. In this study, we identified novel sRNAs in the exclusively human pathogen Streptococcus pyogenes M49 (Group A Streptococcus, GAS M49), employing a whole genome intergenic tiling array approach. GAS is an important pathogen that causes diseases ranging from mild superficial infections of the skin and mucous membranes of the naso-pharynx, to severe toxic and invasive diseases.ResultsWe identified 55 putative sRNAs in GAS M49 that were expressed during growth. Of these, 42 were novel. Some of the newly-identified sRNAs belonged to one of the common non-coding RNA families described in the Rfam database. Comparison of the results of our screen with the outcome of two recently published bioinformatics tools showed a low level of overlap between putative sRNA genes. Previously, 40 potential sRNAs have been reported to be expressed in a GAS M1T1 serotype, as detected by a whole genome intergenic tiling array approach. Our screen detected 12 putative sRNA genes that were expressed in both strains. Twenty sRNA candidates appeared to be regulated in a medium-dependent fashion, while eight sRNA genes were regulated throughout growth in chemically defined medium. Expression of candidate genes was verified by reverse transcriptase-qPCR. For a subset of sRNAs, the transcriptional start was determined by 5′ rapid amplification of cDNA ends-PCR (RACE-PCR) analysis.ConclusionsIn accord with the results of previous studies, we found little overlap between different screening methods, which underlines the fact that a comprehensive analysis of sRNAs expressed by a given organism requires the complementary use of different methods and the investigation of several environmental conditions. Despite a high conservation of sRNA genes within streptococci, the expression of sRNAs appears to be strain specific.