An SNP-based PCR assay to differentiate between Listeria monocytogenes lineages derived from phylogenetic analysis of the sigB gene

The alternative sigma factor sigB gene is involved in the stress response regulation of Listeria monocytogenes, and contributes towards growth and survival in adverse conditions. This gene was examined to determine if it could be a useful indicator of lineage differentiation, similar to the established method based on ribotyping. The sigB sequence was resolved in four local L. monocytogenes strains and the phylogenetic relationship among these, and a further 21 sigB gene sequences from strains of different serotype and lineage including two Listeria innocua strains, obtained from the GenBank database were determined. The sigB nucleotide sequences of these 25 Listeria strains were then examined for single nucleotide polymorphic (SNP) sites that could differentiate between the three lineages. Based on nucleotide sequences L. monocytogenes lineage F serotype 1/2b and 4b clustered together, lineage II/serotype 1/2a and 1/2c strains clustered together, lineage III/serotypes 4a and 4c strains clustered together and L. innocua strains clustered together as an outgroup. SNPs differentiating the three lineages were identified. Individual allele-specific PCR reactions based on these polymorphisms were successful in grouping known and a further 37 local L. monocytogenes isolates into the three lineages. (C) 2003 Elsevier B.V. All fights reserved.

Evaluating plant breeding strategies by simulating gene action and dryland environment effects

Functional genomics is the systematic study of genome-wide effects of gene expression on organism growth and development with the ultimate aim of understanding how networks of genes influence traits. Here, we use a dynamic biophysical cropping systems model (APSIM-Sorg) to generate a state space of genotype performance based on 15 genes controlling four adaptive traits and then search this spice using a quantitative genetics model of a plant breeding program (QU-GENE) to simulate recurrent selection. Complex epistatic and gene X environment effects were generated for yield even though gene action at the trait level had been defined as simple additive effects. Given alternative breeding strategies that restricted either the cultivar maturity type or the drought environment type, the positive (+) alleles for 15 genes associated with the four adaptive traits were accumulated at different rates over cycles of selection. While early maturing genotypes were favored in the Severe-Terminal drought environment type, late genotypes were favored in the Mild-Terminal and Midseason drought environment types. In the Severe-Terminal environment, there was an interaction of the stay-green (SG) trait with other traits: Selection for + alleles of the SG genes was delayed until + alleles for genes associated with the transpiration efficiency and osmotic adjustment traits had been fixed. Given limitations in our current understanding of trait interaction and genetic control, the results are not conclusive. However, they demonstrate how the per se complexity of gene X gene X environment interactions will challenge the application of genomics and marker-assisted selection in crop improvement for dryland adaptation.

Gene flow in great bustard populations across the Strait of Gibraltar as elucidated from excremental PCR and mtDNA sequencing

Recent advances in molecular biology have made it possible to use the trace amounts of DNA in faeces to non-invasively sample endangered species for genetic studies. Here we use faeces as a source of DNA and mtDNA sequence data to elucidate the relationship among Spanish and Moroccan populations of great bustards. 834 bp of combined control region and cytochrome-b mtDNA fragments revealed four variable sites that defined seven closely related haplotypes in 54 individuals. Morocco was fixed for a single mtDNA haplotype that occurs at moderate frequency (28%) in Spain. We could not differentiate among the sampled Spanish populations of Caceres and Andalucia but these combined populations were differentiated from the Moroccan population. Estimates of gene flow (Nm = 0.82) are consistent with extensive observations on the southern Iberian peninsular indicating that few individuals fly across the Strait of Gibraltar. We demonstrate that both this sea barrier and mountain barriers in Spain limit dispersal among adjacent great bustard populations to a similar extent. The Moroccan population is of high ornithological significance as it holds the only population of great bustards in Africa. This population is critically small and genetic and observational data indicate that it is unlikely to be recolonised via immigration from Spain should it be extirpated. In light of the evidence presented here it deserves the maximum level of protection.

Identification and characterization of pptA: a gene involved in the phase-variable expression of phosphorylcholine on pili of Neisseria meningitidis

Pili of pathogenic Neisseria are major virulence factors associated with adhesion, cytotoxicity, twitching motility, autoaggregation, and DNA transformation. Pili are modified posttranslationally by the addition of phosphorylcholine. However, no genes involved in either the biosynthesis or the transfer of phosphorylcholine in Neisseria meningitidis have been identified. In this study, we identified five candidate open reading frames (ORFs) potentially involved in the biosynthesis or transfer of phosphorylcholine to pilin in N. meningitidis. Insertional mutants were constructed for each ORF in N. meningitidis strain C311#3 to determine their effect on phosphorylcholine expression. The effect of the mutant ORFs on the modification by phosphorylcholine was analyzed by Western analysis with phosphorylcholine-specific monoclonal antibody TEPC-15. Analysis of the mutants showed that ORF NMB0415, now defined as pptA (pilin phosphorylcholine transferase A), is involved in the addition of phosphorylcholine to pilin in N. meningitidis. Additionally, the phase variation (high frequency on-off switching of expression) of phosphorylcholine on pilin is due to changes in a homopolymeric guanosine tract in pptA.

Polimorfismos genéticos no gene XRCC3 e dano no DNA em trabalhadores expostos ocupacionalmente a formaldeído

A exposição a formaldeído (FA), classificado como cancerígeno pela International Agency for Cancer Research (IARC), está epidemiologicamente associada a cancro e a alterações nucleares detectáveis pelo ensaio dos micronúcleos por bloqueio da citocinese (CBMN). Este método permite determinar vários marcadores de genotoxicidade, nomeadamente micronúcleos – biomarcadores de quebra ou perda de cromossomas; pontes nucleoplásmicas – biomarcador de re-arranjo cromossómico, pouca reparação e fusão de telómeros e, protusões nucleares – biomarcador de DNA amplificado. O gene X-ray repair cross-complementing group 3 (XRCC3) está envolvido na reparação de ligações cruzadas na recombinação de homólogos e quebras na cadeia dupla de DNA. Foi reportado pelo menos um polimorfismo no gene, o Thr241Met que tem sido associado a um aumento do dnao no DNA em vários estudos.

Papel do polimorfismo Pro12 Ala no gene PPAR Gama 2 na obesidade e diabetes mellitus tipo 2 - uma meta análise

A obesidade e a diabetes mellitus tipo 2 (DM2) são considerados dois grandes problemas de saúde pública. A má alimentação e a falta de atividade física encontram-se entre os principais desencadeadores de um crescente número de indivíduos obesos, diabéticos e com sensibilidade à insulina diminuída. Este aumento tem motivado a comunidade científica a investigar cada vez mais para o elevado contributo da herança genética associada aos fatores sociais e nutricionais. O gene dos recetores ativados por proliferadores do peroxissoma gama 2 (PPARγ2) desempenha um papel importante no metabolismo lipídico. Uma vez que o PPARγ2 é maioritariamente expresso no tecido adiposo, uma redução moderada da sua atividade tem influência na sensibilidade à insulina, diabetes, e outros parâmetros metabólicos. Vários estudos sugerem que tanto fatores genéticos como fatores ambientais (tais como a dieta), poderão estar envolvidos na formação de padrões associados ao polimorfismo Pro12Ala com a composição corporal em diferentes populações humanas. Os diversos estudos genéticos envolvendo o estudo do polimorfismo Pro12Ala do PPARγ2 na suscetibilidade de possuir risco de diabetes e obesidade em várias populações têm proposto conclusões diversas. Em alguns parece haver mais associações do que outros e, às vezes, não demonstram sequer associação. Desta forma, o presente trabalho teve como objectivo contribuir para a elucidação do impacto do polimorfismo Pro12Ala do PPARγ2 na resistência à insulina associada à DM2 e na obesidade, mediante estudo sistematizado da literatura existente até à data, através de meta análise. Do total de uma pesquisa de 63 publicações, foram incluídos 32 artigos no presente estudo, sendo que destes 25 foram incluídos na síntese qualitativa e 11 incluídos na sintese quantitativa. No presente trabalho pode-se concluir que existe evidência estatística que suporta a hipótese de que o polimorfismo Pro12Ala do PPARγ2 pode ser considerado um fator protetor para a DM2 [p <0,05 e OR (odds ratio) 0,702, com IC (intervalos de confiança) com valores que nunca incluem o 1]. No entanto, e mediante os mesmos pressupostos, o mesmo polimorfismo pode ser considerado um fator de risco ao desenvolvimento de obesidade, pela evidência estatística [p <0,05 e OR de 1,196, com IC com valores que nunca incluem o 1].

Implementação da técnica Silver In Situ Hibridization para avaliação do status do gene EGFR em doentes com CPNPC

Avaliação do estado do gene Epidermal Growth Factor Receptor (EGFR), por Silver In Situ Hibridization (SISH), tem-se destacado como biomarcador preditivo na resposta à terapêutica. O principal objectivo foi optimizar a etapa de recuperação por calor da metodologia automatizada SISH Dual-Colour, em carcinomas pulmonares fixados em formol durante 24 e 72 horas. A optimização levou a um aumento da preservação do contorno nuclear e da intensidade e contraste dos sinais para os dois tempos de fixação, permitindo avaliar o estado do EGFR em 83,3% dos casos em estudo. A SISH Dual-Colour é uma alternativa para avaliar o estado do EGFR.

Avaliação da expressão do gene BDNF em doenças neurodegenerativas

O Factor Neurotrófico Derivado do Cérebro (BDNF) está associado a processos de crescimento, diferenciação e sobrevivência das células neuronais. A expressão diferencial do BDNF, particularmente no hipocampo, está relacionada com a manifestação clínica de algumas doenças do foro psiquiátrico e cognitivo como a doença de Huntington, Alzheimer, depressão e esquizofrenia. Este trabalho pretende dar conhecimento das técnicas utilizadas para avaliar a expressão do gene BDNF. As técnicas de ELISA, IHC e Western blot, por permitirem a avaliação precisa da expressão de BDNF, são úteis para uma melhor compreensão, diagnóstico e tratamento de algumas doenças neurodegenerativas.

Can power laws help us understand gene and proteome information?

Proteins are biochemical entities consisting of one or more blocks typically folded in a 3D pattern. Each block (a polypeptide) is a single linear sequence of amino acids that are biochemically bonded together. The amino acid sequence in a protein is defined by the sequence of a gene or several genes encoded in the DNA-based genetic code. This genetic code typically uses twenty amino acids, but in certain organisms the genetic code can also include two other amino acids. After linking the amino acids during protein synthesis, each amino acid becomes a residue in a protein, which is then chemically modified, ultimately changing and defining the protein function. In this study, the authors analyze the amino acid sequence using alignment-free methods, aiming to identify structural patterns in sets of proteins and in the proteome, without any other previous assumptions. The paper starts by analyzing amino acid sequence data by means of histograms using fixed length amino acid words (tuples). After creating the initial relative frequency histograms, they are transformed and processed in order to generate quantitative results for information extraction and graphical visualization. Selected samples from two reference datasets are used, and results reveal that the proposed method is able to generate relevant outputs in accordance with current scientific knowledge in domains like protein sequence/proteome analysis.

Polimorfismo I/D do gene da enzima conversora da angiotensina

Em Portugal, no ano de 2009, existiam 983 mil indivíduos com Diabetes mellitus (DM), dos quais 90% DM tipo 2 (DM2). Um dos principais factores de risco para o desenvolvimento da DM2 é a obesidade. A DM2 está, desta forma, associada a um estilo de vida sedentário, quer pela diminuição do metabolismo da glicose, quer pela sua associação à obesidade devido ao aumento dos níveis de glicose como consequência da sobrealimentação. Outros factores como a lipotoxicidade e o stress oxidativo também têm sido considerados como responsáveis pela disfunção das células-beta pancreáticas. O gene da enzima conversora da angiotensina (ECA) é altamente polimórfico, sendo associado como factor predisponente para diferentes patologias como a DM. O polimorfismo melhor descrito até à actualidade é o de Inserção/Delecção (I/D), consistindo na inserção de um fragmento Alu de 287bp no intrão 16 do gene DCP1. A actividade pró-inflamatória e pró-oxidativa desta enzima sobre o pâncreas, bem como a sua actuação nos processos de fibrose, podem em parte auxiliar na compreensão do processo que origina esta patologia. O polimorfismo I/D torna-se assim um óptimo candidato para a detecção de indivíduos susceptíveis.Este estudo tem como objectivo analisar a distribuição do polimorfismo I/D, bem como a sua possível relação com a incidência de DM e com os níveis de glicose.

Regulation of antioxidant enzymes gene expression in the yeast Saccharomyces cerevisiae during stationary phase

Gene expression of three antioxidant enzymes, Mn superoxide dismutase (MnSOD), Cu,Zn superoxide dismutase (Cu,ZnSOD), and glutathione reductase (GR) was investigated in stationary phase Saccharomyces cerevisiae during menadione-induced oxidative stress. Both GR and Cu,ZnSOD mRNA steady state levels increased, reaching a plateau at about 90 min exposure to menadione. GR mRNA induction was higher than that of Cu,ZnSOD (about 14-fold and 9-fold after 90 min, respectively). A different pattern of response was obtained for MnSOD mRNA, with a peak at about 15 min (about 8-fold higher) followed by a decrease to a plateau approximately 4-fold higher than the control value. However, these increased mRNA levels did not result in increased protein levels and activities of these enzymes. Furthermore, exposure to menadione decreased MnSOD activity to half its value, indicating that the enzyme is partially inactivated due to oxidative damage. Cu,ZnSOD protein levels were increased 2-fold, but MnSOD protein levels were unchanged after exposure to menadione in the presence of the proteolysis inhibitor phenylmethylsulfonyl fluoride. These results indicate that the rates of Cu,ZnSOD synthesis and proteolysis are increased, while the rates of MnSOD synthesis and proteolysis are unchanged by exposure to menadione. Also, the translational efficiency for both enzymes is probably decreased, since increases in protein levels when proteolysis is inhibited do not reflect the increases in mRNA levels. Our results indicate that oxidative stress modifies MnSOD, Cu,ZnSOD, and GR gene expression in a complex way, not only at the transcription level but also at the post-transcriptional, translational, and post-translational levels.

Tubulin cofactor A gene silencing in mammalian cells induces changes in microtubule cytoskeleton, cell cycle arrest and cell death

Microtubules are polymers of alpha/beta-tubulin participating in essential cell functions. A multistep process involving distinct molecular chaperones and cofactors produces new tubulin heterodimers competent to polymerise. In vitro cofactor A (TBCA) interacts with beta-tubulin in a quasi-native state behaving as a molecular chaperone. We have used siRNA to silence TBCA expression in HeLa and MCF-7 mammalian cell lines. TBCA is essential for cell viability and its knockdown produces a decrease in the amount of soluble tubulin, modifications in microtubules and G1 cell cycle arrest. In MCF-7 cells, cell death was preceded by a change in cell shape resembling differentiation.

Q192R polymorphism of the paraoxonase-1 gene as a risk factor for obesity in Portuguese women

Introduction - Obesity became a major public health problem as a result of its increasing prevalence worldwide. Paraoxonase-1 (PON1) is an esterase able to protect membranes and lipoproteins from oxidative modifications. At the PON1 gene, several polymorphisms in the promoter and coding regions have been identified. The aims of this study were i) to assess PON1 L55M and Q192R polymorphisms as a risk factor for obesity in women; ii) to compare PON1 activity according to the expression of each allele in L55M and Q192R polymorphisms; iii) to compare PON1 activity between obese and normal-weight women. Materials and methods - We studied 75 healthy (35.9±8.2 years) and 81 obese women (34.3±8.2 years). Inclusion criteria for obese subjects were body mass index ≥30 kg/m2 and absence of inflammatory/neoplasic conditions or kidney/hepatic dysfunction. The two PON1 polymorphisms were assessed by real-time PCR with TaqMan probes. PON1 enzymatic activity was assessed by spectrophotometric methods, using paraoxon as a substrate. Results - No significant differences were found for PON1 activity between normal and obese women. Nevertheless, PON1 activity was greater (P<0.01) for the RR genotype (in Q192R polymorphism) and for the LL genotype (in L55M polymorphism). The frequency of allele R of Q192R polymorphism was significantly higher in obese women (P<0.05) and was associated with an increased risk of obesity (odds ratio=2.0 – 95% confidence interval (1.04; 3.87)). Conclusion - 55M and Q192R polymorphisms influence PON1 activity. The allele R of the Q192R polymorphism is associated with an increased risk for development of obesity among Portuguese Caucasian premenopausal women.

Relationship of PON1 192 and 55 gene polymorphisms to calcific valvular aortic stenosis

Introduction and Objectives - Paraoxonases may exert anti-atherogenic action by reducing lipid peroxidation. Previous studies examined associations between polymorphisms in the paraoxonase 1 (PON1) gene and development of coronary artery disease (CAD), with inconsistent results. Given the similarities in clinical and pathophysiological risk factors of CAD and calcific aortic valve stenosis (CAVS), we postulated a link between PON1 alleles and CAVS progression. Methods - We investigated the association between PON1 55 and 192 single nucleotide polymorphisms (SNPs), their enzyme activity, and CAVS progression assessed by aortic valve area and transvalvular peak velocity in 67 consecutive patients with moderate CAVS and 251 healthy controls. Results - PON1 paraoxonase activity was higher in CAVS patients (P<0.001). The PON1 genotype Q192R SNP (P=0.03) and variant allele (R192) (P=0.01) frequencies differed between CAVS patients and controls. Significant association existed between PON1 enzyme activity, phenotypic effects of PON1 192 genotype polymorphisms, and CAVS progression, but not between PON1 55 and high-density lipoprotein (P=0.44) or low-density lipoprotein cholesterol (P=0.12), between 192 genotype and high-density lipoprotein (P=0.24) or low-density lipoprotein cholesterol (P=0.52). Conclusion - The PON1 genotype Q192R SNP has an important effect on CAVS disease progression. This study helps outline a genotype-phenotype relationship for PON1 in this unique population.