Aa Protein Research Articles

A reduced dimensionality NMR pulse sequence and an efficient protocol for unambiguous assignment in intrinsically disordered proteins.

Resonance assignment in intrinsically disordered proteins poses a great challenge because of poor chemical shift dispersion in most of the nuclei that are commonly monitored. Reduced dimensionality (RD) experiments where more than one nuclei are co-evolved simultaneously along one of the time axes of a multi-dimensional NMR experiment help to resolve this problem partially, and one can conceive of different combinations of nuclei for co-evolution depending upon the magnetization transfer pathways and the desired information content in the spectrum. Here, we present a RD experiment, (4,3)D-hNCOCAnH, which uses a combination of CO and CA chemical shifts along one of the axes of the 3-dimensional spectrum, to improve spectral dispersion on one hand, and provide information on four backbone atoms of every residue-HN, N, CA and CO chemical shifts-from a single experiment, on the other. The experiment provides multiple unidirectional sequential (i → i - 1) amide (1)H correlations along different planes of the spectrum enabling easy assignment of most nuclei along the protein backbone. Occasional ambiguities that may arise due to degeneracy of amide proton chemical shifts are proposed to be resolved using the HNN experiment described previously (Panchal et al. in J Biomol NMR 20:135-147, 2001). Applications of the experiment and the assignment protocol have been demonstrated using intrinsically disordered α-synuclein (140 aa) protein.

The Haemophilus ducreyi LspA1 protein inhibits phagocytosis by using a new mechanism involving activation of C-terminal Src kinase.

ABSTRACTHaemophilus ducreyi causes chancroid, a sexually transmitted infection. A primary means by which this pathogen causes disease involves eluding phagocytosis; however, the molecular basis for this escape mechanism has been poorly understood. Here, we report that the LspA virulence factors of H. ducreyi inhibit phagocytosis by stimulating the catalytic activity of C-terminal Src kinase (Csk), which itself inhibits Src family protein tyrosine kinases (SFKs) that promote phagocytosis. Inhibitory activity could be localized to a 37-kDa domain (designated YL2) of the 456-kDa LspA1 protein. The YL2 domain impaired ingestion of IgG-opsonized targets and decreased levels of active SFKs when expressed in mammalian cells. YL2 contains tyrosine residues in two EPIYG motifs that are phosphorylated in mammalian cells. These tyrosine residues were essential for YL2-based inhibition of phagocytosis. Csk was identified as the predominant mammalian protein interacting with YL2, and a dominant-negative Csk rescued phagocytosis in the presence of YL2. Purified Csk phosphorylated the tyrosines in the YL2 EPIYG motifs. Phosphorylated YL2 increased Csk catalytic activity, resulting in positive feedback, such that YL2 can be phosphorylated by the same kinase that it activates. Finally, we found that the Helicobacter pylori CagA protein also inhibited phagocytosis in a Csk-dependent manner, raising the possibility that this may be a general mechanism among diverse bacteria. Harnessing Csk to subvert the Fcγ receptor (FcγR)-mediated phagocytic pathway represents a new bacterial mechanism for circumventing a crucial component of the innate immune response and may potentially affect other SFK-involved cellular pathways.

Mutational profiling of the variability of individual amino acid positions in the hepatitis B virus matrix domain

The hepatitis B virus (HBV) is formed by budding. A stretch of 22 amino acids (aa) (matrix domain, MD, R103 – S124) in the large envelope protein L is crucial for virion formation and probably establishes contact to the nucleocapsid. Here, we assess the impact of sequence variations at numerous individual aa positions within the MD on virion formation. We generated panels of L mutants covering all 19 possible aa for 11 positions and tested the capacity of these mutants to rescue virus production by an L-defective HBV genome. At four positions (L112, R113, P117, W122), any replacement of the wild type (WT) aa reduced virus assembly to undetectable levels. Virus production was strongly diminished by substitutions at five other positions (R103, T106, S115, H116, A119). Only two tested positions (D114, Q118) tolerated several substitutions. The restricted positions may represent promising targets for the development of novel antiviral strategies.

Genetic Dissection of T4 Lysis

t is the holin gene for coliphage T4, encoding a 218-amino-acid (aa) protein essential for the inner membrane hole formation that initiates lysis and terminates the phage infection cycle. T is predicted to be an integral membrane protein that adopts an N(in)-C(out) topology with a single transmembrane domain (TMD). This holin topology is different from those of the well-studied holins S105 (3 TMDs; N(out)-C(in)) of the coliphage lambda and S68 (2 TMDs; N(in)-C(in)) of the lambdoid phage 21. Here, we used random mutagenesis to construct a library of lysis-defective alleles of t to discern residues and domains important for holin function and for the inhibition of lysis by the T4 antiholin, RI. The results show that mutations in all 3 topological domains (N-terminal cytoplasmic, TMD, and C-terminal periplasmic) can abrogate holin function. Additionally, several lysis-defective alleles in the C-terminal domain are no longer competent in binding RI. Taken together, these results shed light on the roles of the previously uncharacterized N-terminal and C-terminal domains in lysis and its real-time regulation.

A new in situ hybridization and immunohistochemistry with a novel antibody to detect small T-antigen expressions of Merkel cell polyomavirus (MCPyV)

BackgroundApproximately 80% of Merkel cell carcinomas (MCCs) harbor Merkel cell polyomavirus (MCPyV) which monoclonally integrates into the genome and has prognostic significance. The presence or absence of MCPyV is usually diagnosed using CM2B4 immunohistochemistry (IHC) for MCPyV-large T antigen (LT) protein. However, this method poses a risk of misdiagnosis.MethodsIn this study, we determined MCPyV infection in MCCs using real-time PCR for MCPyV-LT DNA and prepared 16 cases of MCPyV-DNA-positive and -negative groups. Diagnostic sensitivity and specificity of conventional PCR for MCPyV-small T antigen (MCPyV-ST), IHC using a newly developed polyclonal antibody (ST-1) for MCPyV-ST protein (MCPyV-ST) (aa: 164–177), and in situ hybridization (ISH) as well as real-time PCR for MCPyV-ST mRNA were compared against CM2B4-IHC for sensitivity (0.94, 15/16) and specificity (0.94, 15/16).ResultsThe followings are the respective sensitivity and specificity results from examinations for MCPyV-ST gene: conventional PCR for the MCPyV-ST (0.94, 1.0), ST-1-IHC (0.69, 1.0), real-time PCR for ST mRNA (1.0, no data), ST mRNA ISH (0.94, 1.0). Each of the MCPyV-pseudonegative (1/16) and -pseudopositive (1/16) diagnoses evaluated using CM2B4-IHC were accurately corrected by examinations for MCPyV-ST or its expression as well as real-time PCR for MCPyV-LT. Sensitivity of CM2B4-IHC (0.94) was superior to that of ST-1-IHC (0.69) but equal to that of ST mRNA-ISH (0.94). Specificities of ST-1-IHC (1.0) and ST mRNA-ISH (1.0) were superior to that of CM2B4-IHC (0.94).ConclusionsTherefore, combined application of ST mRNA-ISH and ST-IHC as well as CM2B4-IHC is recommended and will contribute to the diagnostic accuracy for MCPyV infection in MCCs.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/9966295741144834

Synaptic vesicle tethering and the CaV2.2 distal C-terminal

Evidence that synaptic vesicles (SVs) can be gated by a single voltage sensitive calcium channel (CaV2.2) predict a molecular linking mechanism or “tether” (Stanley, 1993). Recent studies have proposed that the SV binds to the distal C-terminal on the CaV2.2 calcium channel (Kaeser et al., 2011; Wong et al., 2013) while genetic analysis proposed a double tether mechanism via RIM: directly to the C terminus PDZ ligand domain or indirectly via a more proximal proline rich site (Kaeser et al., 2011). Using a novel in vitro SV pull down binding assay, we reported that SVs bind to a fusion protein comprising the C-terminal distal third (C3, aa 2137–2357; Wong et al., 2013). Here we limit the binding site further to the last 58 aa, beyond the proline rich site, by the absence of SV capture by a truncated C3 fusion protein (aa 2137–2299). To test PDZ-dependent binding we generated two C terminus-mutant C3 fusion proteins and a mimetic blocking peptide (H-WC, aa 2349–2357) and validated these by elimination of MINT-1 or RIM binding. Persistence of SV capture with all three fusion proteins or with the full length C3 protein but in the presence of blocking peptide, demonstrated that SVs can bind to the distal C-terminal via a PDZ-independent mechanism. These results were supported in situ by normal SV turnover in H-WC-loaded synaptosomes, as assayed by a novel peptide cryoloading method. Thus, SVs tether to the CaV2.2 C-terminal within a 49 aa region immediately prior to the terminus PDZ ligand domain. Long tethers that could reflect extended C termini were imaged by electron microscopy of synaptosome ghosts. To fully account for SV tethering we propose a model where SVs are initially captured, or “grabbed,” from the cytoplasm by a binding site on the distal region of the channel C-terminal and are then retracted to be “locked” close to the channel by a second attachment mechanism in preparation for single channel domain gating.

Isolation and characterisation of two cDNAs encoding transglutaminase from Atlantic cod (Gadus morhua)

Two cDNAs encoding transglutaminase (TG) were identified in a subtractive cDNA library prepared from the head kidney of poly I:C stimulated Atlantic cod (Gadus morhua). Full-length TG-1 and TG-2 cDNA were cloned from the head kidney by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). The deduced amino acid (aa) sequence for TG-1 was 695 aa with an estimated molecular mass of 78.3 kDa, while TG-2 was a 698 aa protein with an estimated molecular mass of 78.8 kDa. The two proteins were named TG-1 and TG-2 and both possess transglutaminase/protease-like homologous domains (TGc) and full conservation of amino acids cysteine, histidine, and aspartate residues that form the catalytic triad. Sequence analysis showed high similarity (93.1%) with Alaska pollock TG, and the TGs were grouped together with TGs from chum salmon, Japanese flounder, Nile tilapia, and red sea bream in addition to Alaska pollock in phylogenetic analysis. Interestingly, they showed different tissue distribution with highest constitutive expression in reproductive and immunological organs, indicating important roles in these organs. Furthermore, the up-regulation of TG-1 and TG-2 in head kidney after stimulating Atlantic cod with poly I:C suggested a role of TGs in immune response in Atlantic cod.

Characterization of the small heat shock protein Hsp27 gene in Chironomus riparius (Diptera) and its expression profile in response to temperature changes and xenobiotic exposures

Small heat shock proteins constitute the most diverse and least conserved group within the large family of heat shock proteins, which play a crucial role in cell response to environmental insults. Chironomus riparius larvae are widely used in environmental research for testing pollutant toxicity in sediments and freshwater environments. Different genes, such as Hsp70, Hsc70, Hsp90, and Hsp40, have been identified in this species as sensitive biomarkers for xenobiotics, but small Hsps genes remain largely unknown. In this study, the Hsp27 has been characterized in C. riparius and its transcriptional response evaluated under several environmental stimuli. The Hsp27 gene was mapped by FISH on polytene chromosomes at region I-C4 and was found to encode a 195 aa protein, which contains an α-crystallin domain bounded by three conserved regions. This protein shows homology with Drosophila melanogaster HSP27, Ceratitis capitata HSP27, and Sarcophaga crassipalpis HSP25. Real-time reverse transcriptase-polymerase chain reaction analysis showed that heat shock (35°C) and cadmium dramatically upregulate this gene. Moreover, exposures to triclosan and bisphenol A were able to significantly increase mRNA levels. However, neither nonylphenol nor tributyltin altered Hsp27 gene expression. The transcriptional activity of Hsp27 gene was modulated during cold stress. Interestingly, cold shock (4°C) significantly reduced Hsp27 transcripts, but this gene was significantly overexpressed during the recovery time at the normal growing temperature. These results show that the Hsp27 gene is sensitive to different environmental stimuli, including endocrine-disrupting pollutants, suggesting its potential as a suitable biomarker for ecotoxicological studies in aquatic systems.

Cellular Mechanisms For Transferrin Receptor Endocytosis Vital For Terminal Erythroid Differentiation

Clathrin-mediated endocytosis (CME) is a key endocytic portal that regulates transferrin receptor (TfR) endocytosis. Although CME is among the most-studied cellular systems in cell biology, little is known regarding how this key system physiologically functions in vivo.The CALM gene (for Clathrin Assembly Lymphoid Myeloid, also known as PICALM), located on human chromosome 11q14, encodes a 652 aa protein containing multiple domains functioning in clathrin-coated vesicle formation. Since CALM is predominantly expressed in erythroblasts, and hematopoietic-specific Calmconditional knockout mice, which we developed, exhibit a severe iron-deficient anemia, we hypothesized that CALM functions to increase efficiency of TfR endocytosis in erythroblasts so that iron uptake is not rate-limiting. To test this hypothesis, we performed FACS-based endocytosis assay in primary erythroblasts, in vitro erythroid differentiation assay, live-cell imaging and CALM add-back rescue experiments.TfR endocytosis was severely attenuated in Calm-deficient basophilic- and polychromatophilic erythroblasts: efficiency of TfR endocytosis was less than 25% of that of control, as revealed by FACS-based endocytosis assay. Unexpectedly, TfR endocytosis was not active in orthochromatophilic erythroblasts regardless of genotype, while they express TfR at high levels.For live cell imaging, we established immortalized mouse embryonic fibroblasts (MEFs), in which the Calm gene can be deleted in an inducible manner (CalmF/F ERT2-Cre+ MEFs). The MEFs were then stably transduced with the retrovirus encoding an EGFP-tagged AP2-bound adaptin σ2. The dynamics of clathrin-coated vesicle formation was determined using fast live cell fluorescence spinning disk confocal microscopy. Clathrin-coat dynamics was barely affected in the absence of Calm under regular culture condition; however, it was completely stalled under high membrane tension (e.g. hypo-osmotic medium, jasplakinolide treatment). To determine functional domain(s) of CALM, a series of CALM mutants were generated, “added-back” to Calm-deficient MEFs and coat dynamics examined by live cell imaging. Mutant that does not bind to phosphatidylinositol 4,5-bisphosphate of the plasma membrane (PIP2-mutant CALM) failed to rescue TfR endocytosis in Calm-deficient MEFs, suggesting that CALM binding to PIP2, but not to v-SNAREs or EPS15, is essential for CALM-mediated TfR endocytosis.Splenic erythroblasts were significantly expanded in Calm conditional knockout mice. In contrast, Calm-deficient hematopoietic progenitors cannot give rise to immature erythroblasts in vitro, suggesting that Calm-deficiency is partially compensated in vivo via non-cell autonomous mechanisms (e.g. central macrophages). Iron supplement treatment could significantly rescue erythroid development of Calm-deficient progenitors in vitro, indicating that iron-deficiency is a primary cause of developmental defects seen in Calm-deficient erythroblasts. As expected, retrovirus-mediated expression of WT-Calm, but not PIP2-mutant, completely rescued erythroid development in Calm-deficient erythroblasts in vitro.Our data indicate that CALM plays a key role in clathrin-mediated endocytosis in a context-dependent (high membrane tension) and cell-type-specific (erythroblasts) manner. We propose that CALM is essential for transferrin uptake in erythroblasts by functioning as an erythroid-specific clathrin adaptor. Disclosures:No relevant conflicts of interest to declare.

Cells determine cell density using a small protein bound to a unique tissue-specific phospholipid

Cell density is the critical parameter controlling tendon morphogenesis. Knowing its neighbors allows a cell to regulate correctly its proliferation and collagen production. A missing link to understanding this process is a molecular description of the sensing mechanism. Previously, this mechanism was shown in cell culture to rely on a diffusible factor (SNZR [sensor]) with an affinity for the cell layer. This led to purifying conditioned medium over 4 columns and analyzing the final column fractions for band intensity on SDS gels versus biological activity – a 16 kD band strongly correlated between assays. N-terminal sequencing – EPLAVVDL – identified a large gene (424 AA), extremely conserved between chicken and human. In this paper we probe whether this is the correct gene. Can the predicted large protein be cleaved to a smaller protein? EPLAVVDL occurs towards the C-terminus and cleavage would create a small 94 AA protein. This protein would run at ∼10 kD, so what modifications or cofactor binding accounts for its running at 16 kD on SDS gels? This protein has no prominent hydrophobic regions, so can it be secreted? To validate its role, the chicken cDNA for this gene was tagged with myc and his and transfected into a human osteosarcoma cell line (U2OS). U2OS cells expressed the gene but not passively: differentiating into structures resembling spongy bone and expressing alkaline phosphatase, an early bone marker. Intracellularly, two bands were observed by Western blotting: the full length protein and a smaller form (26 kD). Outside the cell, a small band (28 kD) was detected, although it was 40% larger than expected, as well as multiple larger bands. These larger forms could be converted to the predicted smaller protein (94 AA + tags) by changing salt concentrations and ultrafiltering – releasing a cofactor to the filtrate while leaving a protein factor in the retentate. Using specific degradative enzymes and mass spectrometry, the bone cofactor was identified as a lipid containing a ceramide phosphate, a single chained glycerol lipid and a linker. Tendon uses a different cofactor made up of two fatty acid chains linked directly to the phosphate yielding a molecule about half the size. Moreover, adding the tendon factor/cofactor to osteosarcoma cells causes them to stop growing, which is opposite to its role with tendon cells. Thus, the cofactor is cell type specific both in composition and in the triggered response. Further support of its proposed role came from frozen sections from 5 week old mice where an antibody to the factor stained strongly at the growing ends of the tendon as predicted. In conclusion, the molecule needed for cell density signaling is a small protein bound to a unique, tissue-specific phospholipid yielding a membrane associated but diffusible molecule. Signal transduction is postulated to occur by an increased ordering of the plasma membrane as the concentration of this protein/lipid increases with cell density.

Effect of different temperature–time combinations on lipid and protein oxidation of sous-vide cooked lamb loins

Forty-five lamb loins were subjected to sous-vide cooking at different combinations of temperature (60, 70 and 80°C) and time (6, 12 and 24h) to assess the effect on the oxidative stability of lipids and proteins. Heating induced both lipid and protein oxidation in lamb loins. Higher cooking temperature–time combinations increased conjugated dienes and decreased thiobarbituric reactive substances (TBARS) values and hexanal. Total protein carbonyls increased throughout time at all cooking temperatures considered, while α-aminoadipic (AAS) and γ-glutamic semialdehydes (GGS) increased when cooking at 60°C but not at 80°C. Links between the decrease in secondary compounds from lipid oxidation due to cooking at higher temperatures and for longer times with the increased levels of 3-methylbutanal and greater differences between total protein carbonyls and AAS plus GGS were hypothesised.

Molecular characterization of the vitellogenin receptor from the tick, Amblyomma hebraeum (Acari: Ixodidae)

We have identified the full-length cDNA encoding a vitellogenin receptor (VgR) from the African bont tick Amblyomma hebraeum Koch (1844). VgRs are members of the low-density lipoprotein receptor superfamily that promote the uptake of the yolk protein vitellogenin (Vg), from the haemolymph. The AhVgR (GenBank accession No. JX846592) is 5703 bp, and encodes an 1801 aa protein with a 196.5 kDa molecular mass following cleavage of a 22 aa signal peptide. Phylogenetic analysis indicates that AhVgR is highly similar to other tick VgRs. AhVgR is expressed in only the ovary of mated, engorged females, and is absent in all other female tissues and in both fed and unfed males. Unfed, adult females injected with a VgR-dsRNA probe to knock-down VgR expression experienced a significant delay in ovary development and started oviposition significantly later than controls. These results indicate that the expression of AhVgR is important for the uptake of Vg and subsequent maturation of the oocytes.

Molecular characterization of a glycosyl hydrolase family 10 xylanase from Aspergillus niger

A gene coding for an endo-β-1,4-xylanase (XlnA) (glycosyl hydrolase family 10) from Aspergillus niger DSM 1957 was cloned and sequenced. The cDNA sequence (984bp) and its putative endoxylanase (327 aa protein with a predicted molecular mass of 35.5kDa and pI 6.23) showed 91.3–99.5% and 96.3–99.1% identities with cDNA sequences and their corresponding endoxylanases from A. niger strains from GenBank, respectively. The cDNA was expressed in Pichia pastoris GS115 under the control of AOX1 promoter at a level of 46.4U/ml culture supernatant, after 144h of growth at 30°C in YP medium induced with 0.5% (v/v) of methanol. The molecular mass of the purified XlnA determined by SDS–PAGE was 35.5kDa with a specific activity of 808.5U/mg towards 1% (w/v) of birch wood xylan. Temperature and pH optimum were observed at 50°C and pH 7.0, respectively. The enzyme was stable over a temperature range of 25–40°C and at pH range of 4.5–8.5 and resistant to Tween 80 and acetone. The Km and Vmax value obtained for the purified xylanase were 25.5mg/ml and 5000μmol/min/mg protein with birch wood xylan as substrate, respectively. The xylanase was free of cellulase and mannanase activity but highly active towards birch wood xylan. The major products of the birch wood xylan hydrolysis were predicted as xylotriose, xylotetraose, and xylopentose. The biochemical characteristics suggested that the recombinant xylanase has a potential application, including use as a feed enzyme.

Expression of immune relevant genes in rainbow trout following exposure to live Anisakis simplex larvae

Basic immune response mechanisms in vertebrates against helminths are still poorly understood. Fish-nematode models may prove valuable for elucidation of this question. In this study we orally challenged rainbow trout (Oncorhynchus mykiss) with larvae of Anisakis simplex (Nematoda: Anisakidae) and subsequently investigated the expression of 18 immune relevant genes in spleen and liver 1, 4 and 8days post infection (d.p.i.). Gene expression data were analysed with regard to the infection status of the challenged rainbow trout at the time of necropsy; “worms rejected” (÷worms), “worms present” (+worms) and a combined group consisting of samples pooled from both previous groups (÷/+worms). No significant regulation of cytokine genes was recorded but fish which had rejected worms up-regulated the CD4 gene (6.1-fold change, 8d.p.i.) in liver. The gene encoding CD8 was significantly down-regulated 24h post challenge in livers in fish still carrying worms (2.7-fold change) but not in the worm-free group. The immunoglobulin gene IgM was significantly down-regulated (2.9-fold change, 8d.p.i.) in liver samples from the +worms group. Complement factor C3 and precerebellin genes were significantly up-regulated twofold in liver samples from infected fish 4d.p.i. Significant up-regulation of the acute-phase protein SAA was observed in all three groups and in both tissues. To our knowledge, this is the first study to describe the expression of immune genes in a fish host challenged with live nematode larvae.

83-Year-Old Man With Nausea, Constipation, and Peripheral Neuropathy

83-Year-Old Man With Nausea, Constipation, and Peripheral Neuropathy

Induction of CYP3A gene and enzyme activity expression in kidney cell of grass carp (Ctenopharyngodon idellus)

Cytochrome P450 3A(CYP3A) is the major cytochrome P450 responsible for the metabolism of en-dogenous and xenobiotic substrates.Unexpected drug–drug interactions in fish are generally associated with the induction of CYP3A activity,and can lead to the formation of drug residues,which threaten the safety of fishery products.We determined the in vitro effects of rifampicin(RIF) on CYP3A mRNA expression and enzyme activity in a freshwater teleost,the grass carp(Ctenopharyngodon idellus).Recently,a number of CYP3A genes have been isolated from several teleost species.Although CYP3A genes in fish contain multiple paralogs differing in gene expression pattern and tissue distribution,CYP3A enzymes exhibit similar catalytic properties because of their structural similarities.The CYP3A gene and its full-length cDNA of 1 849 bp were cloned from grass carp by RT-PCR.The CYP3A gene has an open reading frame(ORF) of 1 542 bp,a stop codon,a 3'-untranslated regions(3' UTR),and a polyA signal is present in the 3'UTR region.The ORF encodes a 513 amino acid(aa) protein,which has a signal peptide(29aa),two transmembrane helixes(23aa and 23aa),a heme binding domain(HBD,21aa),and six substrate recognition sites(SRS1-6).Multiple alignments showed that the CYP3A amino acid se-quence from grass carp is 60% to 92% similar to other vertebrate CYP3As.CYP3A gene expression after induc-tion by RIF in grass carp kidney cells(CIK) was assayed by quantitative real-time PCR.CYP3A-dependent erythromycin N-demethylase(ERND) activity was determined by spectrophotometry,and CYP3A activity was assessed by measuring the formation of formaldehyde.In the induced group,CYP3A mRNA expression reached a plateau at 8h,while the highest level of CYP3A activity occurred at 10h.The results indicated that CYP3A mRNA expression and enzyme activity were significantly higher than in the control group(P0.05).The highest level of CYP3A mRNA expression appeared 2 hours before the maximum of enzyme activity.In addition,the transcription of level CYP3A was apparently higher than the enzyme activity,implying that induction by RIF affected enzyme activity by transcriptional regulation of the CYP3A gene.

Molecular characterisation of two homoeologous elicitor-responsive lipin genes in cotton

The identification and molecular characterisation of two lipin-like gene copies (GhLIPN) in cotton, Gossypium hirsutum, an allotetraploid derived from two progenitor diploid Gossypium species, is described. Sequence analyses of the GhLIPN copies, designated GhLIPN-1 and -2, revealed that they contain 11 exons, separated by ten introns. They each have a 2,643bp open reading frame that encodes 880 aa proteins, and share a 97.7 and 95.5% sequence similarity at the translated nucleotide and amino acid level, respectively. The GhLIPN genes have a distinct domain architecture consisting of an archetypical N-terminal lipin domain, followed by a haloacid dehalogenase (HAD) domain towards the C-terminus. A Southern blot did not distinguish between the two gene copies, which suggests that they may be homoeologs rather than paralogs. GhLIPN-2 is more similar to a homoeologous sequence from G. raimondii, representing the ancestral D-genome, compared to GhLIPN-1 that matches G. herbaceum and that represents the A-genome. Our data indicate that GhLIPN-1 and GhLIPN-2 are homoeologs that derive from the A- and the D-diploid genomes, respectively. The promoter sequences of GhLIPN-1 and -2 differ by 56%, as a result of multiple indels. In silico analysis of the promoter regions revealed that both genes contain numerous putative defence-related and elicitor-responsive cis-elements that support a role for GhLIPN in defence responses. Relative quantification real-time PCR confirmed the up-regulation in response to a cell-wall-derived V. dahliae elicitor, which supported the association of GhLIPN with defence signalling. The results add a new dimension to the proposed roles of lipins in plants by suggesting that lipins may have a role in defence signalling.

Sequence Analysis of the Genome of Piscine Orthoreovirus (PRV) Associated with Heart and Skeletal Muscle Inflammation (HSMI) in Atlantic Salmon (Salmo salar)

Piscine orthoreovirus (PRV) is associated with heart- and skeletal muscle inflammation (HSMI) of farmed Atlantic salmon (Salmo salar). We have performed detailed sequence analysis of the PRV genome with focus on putative encoded proteins, compared with prototype strains from mammalian (MRV T3D)- and avian orthoreoviruses (ARV-138), and aquareovirus (GCRV-873). Amino acid identities were low for most gene segments but detailed sequence analysis showed that many protein motifs or key amino acid residues known to be central to protein function are conserved for most PRV proteins. For M-class proteins this included a proline residue in μ2 which, for MRV, has been shown to play a key role in both the formation and structural organization of virus inclusion bodies, and affect interferon-β signaling and induction of myocarditis. Predicted structural similarities in the inner core-forming proteins λ1 and σ2 suggest a conserved core structure. In contrast, low amino acid identities in the predicted PRV surface proteins μ1, σ1 and σ3 suggested differences regarding cellular interactions between the reovirus genera. However, for σ1, amino acid residues central for MRV binding to sialic acids, and cleavage- and myristoylation sites in μ1 required for endosomal membrane penetration during infection are partially or wholly conserved in the homologous PRV proteins. In PRV σ3 the only conserved element found was a zinc finger motif. We provide evidence that the S1 segment encoding σ3 also encodes a 124 aa (p13) protein, which appears to be localized to intracellular Golgi-like structures. The S2 and L2 gene segments are also potentially polycistronic, predicted to encode a 71 aa- (p8) and a 98 aa (p11) protein, respectively. It is concluded that PRV has more properties in common with orthoreoviruses than with aquareoviruses.

Cloning and characterization of two allelic glyceraldehyde-3-phosphate dehydrogenase genes in Auricularia auricula-judae

Two allelic variants of the gpd gene, Gpd(a) and Gpd(b), were isolated based on a putative glyceraldehyde-3-phosphate dehydrogenase encoding sequence from the transcriptome of Auricularia auricula-judae strain Au916. The two alleles were found to have a 73 bp length discrepancy and 39 SNP variations. Both of the genomic DNA sequences of two alleles were interrupted by five introns, and encoded a same 340 aa protein. Intron positions analysis showed that the first intron was absent, but the last unique intron was gained in A. auricula-judae. Allele-specific expression analysis showed that the Gpd(a) and Gpd(b) were expressed with no significant difference in dikaryotic mycelia of A. auricula-judae. To the best of our knowledge, this is the first report about the detection of two allelic gpd genes in A. auricula-judae, as well as the application of allele-specific primers in gene expression analysis for this edible fungus.

Splice variants and promoter methylation status of the Bovine Vasa Homology (Bvh) gene may be involved in bull spermatogenesis

BackgroundVasa is a member of the DEAD-box protein family that plays an indispensable role in mammalian spermatogenesis, particularly during meiosis. Bovine vasa homology (Bvh) of Bos taurus has been reported, however, its function in bovine testicular tissue remains obscure. This study aimed to reveal the functions of Bvh and to determine whether Bvh is a candidate gene in the regulation of spermatogenesis in bovine, and to illustrate whether its transcription is regulated by alternative splicing and DNA methylation.ResultsHere we report the molecular characterization, alternative splicing pattern, expression and promoter methylation status of Bvh. The full-length coding region of Bvh was 2190 bp, which encodes a 729 amino acid (aa) protein containing nine consensus regions of the DEAD box protein family. Bvh is expressed only in the ovary and testis of adult cattle. Two splice variants were identified and termed Bvh-V4 (2112 bp and 703 aa) and Bvh-V45 (2040 bp and 679 aa). In male cattle, full-length Bvh (Bvh-FL), Bvh-V4 and Bvh-V45 are exclusively expressed in the testes in the ratio of 2.2:1.6:1, respectively. Real-time PCR revealed significantly reduced mRNA expression of Bvh-FL, Bvh-V4 and Bvh-V45 in testes of cattle-yak hybrids, with meiotic arrest compared with cattle and yaks with normal spermatogenesis (P < 0.01). The promoter methylation level of Bvh in the testes of cattle-yak hybrids was significantly greater than in cattle and yaks (P < 0.01).ConclusionIn the present study, Bvh was isolated and characterized. These data suggest that Bvh functions in bovine spermatogenesis, and that transcription of the gene in testes were regulated by alternative splice and promoter methylation.