cpc-3, the Neurospora crassa Homologue of Yeast GCN2, Encodes a Polypeptide with Juxtaposed eIF2α Kinase and Histidyl-tRNA Synthetase-related Domains Required for General Amino Acid Control

Based on characteristic amino acid sequences of kinases that phosphorylate the α subunit of eukaryotic translation initiation factor 2 (eIF2α kinases), degenerate oligonucleotide primers were constructed and used to polymerase chain reaction-amplify from genomic DNA of Neurospora crassa a sequence encoding part of a putative protein kinase. With this sequence an open reading frame was identified encoding a predicted polypeptide with juxtaposed eIF2α kinase and histidyl-tRNA synthetase- related domains. The 1646 amino acid sequence of this gene, called cpc-3, showed 35% positional identity over almost the entire sequence with GCN2 of yeast, which stimulates translation of the transcriptional activator of amino acid biosynthetic genes encoded by GCN4. Strains disrupted for cpc-3 were unable to induce increased transcription and derepression of amino acid biosynthetic enzymes in amino acid-deprived cells. The cpc-3 mutation did not affect the ability to up-regulate mRNA levels of cpc-1, encoding the GCN4 homologue and transcriptional activator of amino acid biosynthetic genes in N. crassa, but the mutation abolished the dramatic increase of CPC1 protein level in response to amino acid deprivation. These findings suggest that cpc- 3 is the functional homologue of GCN2, being required for increased translation of cpc-1 mRNA in amino acid-starved cells

Keeping the eIF2 alpha kinase Gcn2 in check

The protein kinase Gcn2 is present in virtually all eukaryotes and is of increasing interest due to its involvement in a large array of crucial biological processes. Some of these are universally conserved from yeast to humans, such as coping with nutrient starvation and oxidative stress. in mammals, Gcn2 is important for e.g. long-term memory formation, feeding behaviour and immune system regulation. Gcn2 has been also implicated in diseases such as cancer and Alzheimer's disease. Studies on Gcn2 have been conducted most extensively in Saccharomyces cerevisiae, where the mechanism of its activation by amino acid starvation has been revealed in most detail. Uncharged tRNAs stimulate Gcn2 which subsequently phosphorylates its substrate, eIF2 alpha, leading to reduced global protein synthesis and simultaneously to increased translation of specific mRNAs, e.g. those coding for Gcn4 in yeast and ATF4 in mammals. Both proteins are transcription factors that regulate the expression of a myriad of genes, thereby enabling the cell to initiate a survival response to the initial activating cue. Given that Gcn2 participates in many diverse processes, Gcn2 itself must be tightly controlled. Indeed, Gcn2 is regulated by a vast network of proteins and RNAs, the list of which is still growing. Deciphering molecular mechanisms underlying Gcn2 regulation by effectors and inhibitors is fundamental for understanding how the cell keeps Gcn2 in check ensuring normal organismal function, and how Gcn2-associated diseases may develop or may be treated. This review provides a critical evaluation of the current knowledge on mechanisms controlling Gcn2 activation or activity. (C) 2014 Published by Elsevier B.V.Massey University Research FundAuckland Medical Research FoundationMaurice & Phyllis Paykel TrustNutricia Research FoundationFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Massey UniversityInstitute of Natural and Mathematical SciencesUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilMassey Univ, Inst Nat & Math Sci, Auckland 0745, Albany, New ZealandUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, São Paulo, BrazilAuckland Medical Research Foundation: 4109024Auckland Medical Research Foundation: 4113010Nutricia Research Foundation: 2010-35FAPESP: 2009/52047-5CNPq: 478903/2012-0CNPq: 309860/2011-3CNPq: 153660/2010-4CAPES: 1915-13-4Web of Scienc

A Rapid Extraction Method for mammalian cell cultures, suitable for quantitative immunoblotting analysis of proteins, including phosphorylated GCN2 and eIF2α

Many studies require the detection and relative quantitation of proteins from cell culture samples using immunoblotting. Limiting factors are the cost of protease inhibitors, the time required to break cells and generate samples, as well as the high risk of protein loss during cell breakage procedures. In addition, a common problem is the viscosity of lysed samples due to the released genomic DNA. As a consequence, the DNA needs to be broken down prior to denaturing polyacrylamide protein gel electrophoresis (SDS-PAGE), e.g. by passing the sample through a syringe gauge needle, sonication, or DNase treatment. In a quest to find a more cost-effective, fast, and yet robust procedure, we found that cell lysis, protein denaturation, and DNA fragmentation can be done in only two steps: harvesting followed by a simple non-laborious 2nd step. Similarly to many pre-existing cell breakage procedures, in our Rapid Protein Extraction (RPE) method, proteins liberated from cells are immediately exposed to a denaturing environment. However, advantages of our method are: • No breaking buffer is needed, instead proteins are liberated directly into the denaturing protein loading buffer used for SDS-PAGE. Consequently, our RPE method does not require any expensive inhibitors. • The RPE method does not involve post-lysis centrifugation steps; instead all cell material is dissolved during the 2nd step, the mixing-heat-treatment step which is new to this method. This prevents potential protein loss that may occur during centrifugation. In addition, this 2nd step simultaneously shears the genomic DNA, making an additional step for DNA fragmentation unnecessary. • The generated samples are suitable for high-quality quantitative immunoblotting. With our RPE method we successfully quantified the phosphorylated forms of protein kinase GCN2 and its substrate eIF2α. In fact, the western signals were stronger and with less background, as compared to samples generated with a pre-existing method. Method name: Rapid Protein Extraction Method (RPE method), Keywords: Mammalian cell culture, cell lysis, SDS-PAGE, quantitation, western blot, GCN2, eIF2 alph

Persistent contamination of Salmonella, Campylobacter, Escherichia coli, and Staphylococcus aureus at a broiler farm in New Zealand

Intensive poultry production due to public demand raises the risk of contamination, creating potential foodborne hazards to consumers. The prevalence and microbial load of the pathogens Campylobacter, Salmonella, Staphylococcus aureus, and Escherichia coli was determined by standard methods at the farm level. After disinfection, swab samples collected from wall crevices, drinkers, and vents were heavily contaminated, as accumulated organic matter and dust likely protected the pathogens from the disinfectants used. The annex floor also showed high microbial concentrations, suggesting the introduction of pathogens from external environments, highlighting the importance of erecting hygiene barriers at the entrance of the main shed. Therefore, pathogen control measures and proper application of disinfectants are recommended as intervention strategies. Additionally, quantitative polymerase chain reaction (qPCR) was evaluated as a quantification tool. qPCR showed limitations with samples containing low microbial counts because of the low detection limit of the method. Thus, bacterial pre-enrichment of test samples may be necessary to improve the detection of pathogens by qPCR.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Interplay between GCN2 and GCN4 expression,

translation elongation factor 1 mutations and translational fidelity in yeas

Interplay Between GCN2 and GCN4 Expression, Translation Elongation Factor 1 Mutations and Translational Fidelity in Yeast

Genetic screens in Saccharomyces cerevisiae have identified the roles of ribosome components, tRNAs and translation factors in translational fidelity. These screens rely on the suppression of altered start codons, nonsense codons or frameshift mutations in genes involved in amino acid or nucleotide metabolism. Many of these genes are regulated by the General Amino Acid Control (GAAC) pathway. Upon amino acid starvation, the kinase GCN2 induces the GAAC cascade via increased translation of the transcriptional activator GCN4 controlled by upstream open reading frames (uORFs). Overexpression of the GCN2 or GCN4 genes enhances the sensitivity of translation fidelity assays that utilize genes regulated by GCN4, such as the suppression of a +1 insertion by S.cerevisiae translation elongation factor 1A (eEF1A) mutants. Paromomycin and the prion [PSI+], which reduce translational fidelity, do not increase GCN4 expression to induce the suppression phenotype and in fact reduce derepression. eEF1A mutations that reduce translation, however, reduce expression of GCN4 under non-starvation conditions. These eEF1A mutants also reduce HIS4 mRNA expression. Taken together, this system improves in vivo strategies for the analysis of translational fidelity and further provides new information on the interplay among translation fidelity, altered elongation and translational control via uORFs

Transcriptional defect of an inherited NKX2-5 haplotype comprising a SNP, a nonsynonymous and a synonymous mutation, associated with human congenital heart disease.

Germline mutations in cardiac-specific transcription factor genes have been associated with congenital heart disease (CHD) and the homeodomain transcription factor NKX2-5 is an important member of this group. Indeed, more than 40 heterozygous NKX2-5 germline mutations have been observed in individuals with CHD, and these are spread along the coding region, with many shown to impact protein function. In pursuit of understanding causes of CHD, we analyzed n = 49 cardiac biopsies from 28 patients and identified by direct sequencing two nonsynonymous NKX2-5 alterations affecting alanine 119, namely c.356C>A (p.A119E) and c.355G>T, (p.A119S), in patients with AVSD and HLHS, respectively. In functional assays, a significant reduction in transcriptional activities could be determined for the NKX2-5 variants. Importantly, in one family the mother, besides p.A119E, carried a synonymous mutant allele in the homeodomain (c.543G>A, p.Q181), and a synonymous dbSNP (c.63A>G, p.E21) in the transactivation domain of the protein, that were transmitted to the CHD daughter. The presence of these variants in-cis with the p.A119E mutation led to a further reduction in transcriptional activities. Such difference in activity may be in part related to reduced protein expression for the double variant c.356C>A and c.543G>A. We propose changes in mRNA stability and folding, due to a silent mutation and a dbSNP in the NKX2-5 coding region to contribute to the functional defect. Although the clinical significance of the NKX2-5 haplotype identified in the CHD patients remains to be ascertained, we provide evidence of an interaction of a dbSNP, with synonymous and nonsynonymous mutations to negatively impact NKX2-5 transcriptional activity