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1.
Yeast ; 37(9-10): 467-473, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32401376

RESUMO

Candida famata is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B2 ) in response to iron limitation. Overproduced riboflavin accumulates in the cultural medium rather than in the cells suggesting existence of the special mechanisms involved in riboflavin excretion. The corresponding protein and gene have not been identified in yeasts. At the same time, the corresponding gene BCRP has been identified in mammal mammary glands. Several homologs of the mammal BCRP gene encoding putative riboflavin efflux protein (excretase) were identified in Debaryomyces hansenii. The closest homolog was expressed under the control of D. hansenii TEF1 promoter in the riboflavin overproducing strain of C. famata. Resulted transformants overexpressed the corresponding gene and produced 1.4- to 1.8-fold more riboflavin as compared with the parental strain. They also were characterized by overexpression of RIB1 and RIB6 genes of riboflavin synthesis and exhibited elevated specific activity of GTP-cyclohydrolase II. Membrane localization of the riboflavin excretase was confirmed by fluorescent microscopy.


Assuntos
Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Candida/genética , Proteínas Fúngicas/genética , Mamíferos/genética , Riboflavina/metabolismo , Animais , Candida/classificação , Clonagem Molecular , DNA Fúngico/genética , Riboflavina/biossíntese
2.
Gene ; 643: 26-34, 2018 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-29208413

RESUMO

Mycobacterium leprae has a reduced genome size due to the reductive evolution over a long period of time. Lipid metabolism plays an important role in the life cycle and pathogenesis of this bacterium. In comparison to 26 lip genes (Lip A-Z) of M. tuberculosis, M. leprae retained only three orthologs indicating their importance in its life cycle. ML0314c (LipU) is one of them. It is conserved throughout the mycobacterium species. Bioinformatics analysis showed the presence of an α/ß hydrolase fold and 'GXSXG' characteristic of the esterases/lipases. The gene was expressed in E. coli and purified to homogeneity. It showed preference towards short chain esters with pNP-acetate as the preferred substrate. The enzyme showed optimal activity at 45°C and pH8.0. ML0314c protein was stable between temperatures ranging from 20 to 60°C and pH5.0-8.0, i.e., relatively acidic and neutral conditions. The active site residues predicted bioinformatically were confirmed to be Ser168, Glu267, and His297 by site directed mutagenesis. E-serine, DEPC and Tetrahydrolipstatin (THL) completely inhibited the activity of ML0314c. The protein was localized in cell wall and extracellular medium. Several antigenic epitopes were predicted in ML0314c. Protein elicited strong humoral immune response in leprosy patients, whereas, a reduced immune response was observed in the relapsed cases. No humoral response was observed in treatment completed patients. Overexpression of ml0314c in the surrogate host M. smegmatis showed marked difference in the colony morphology and growth rate. In conclusion, ML0314c is a secretary carboxyl esterase that could modulate the immune response in leprosy patients.


Assuntos
Lipólise/genética , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Sequência de Aminoácidos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Hidrolases de Éster Carboxílico/metabolismo , Domínio Catalítico/genética , Clonagem Molecular/métodos , Escherichia coli/genética , Humanos , Concentração de Íons de Hidrogênio , Hanseníase/metabolismo , Hanseníase/microbiologia , Lipase/genética , Metabolismo dos Lipídeos/genética , Lipídeos , Mutagênese Sítio-Dirigida/métodos , Mycobacterium tuberculosis/genética , Especificidade por Substrato/genética , Fatores de Virulência
3.
Phytopathology ; 107(8): 963-976, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28398876

RESUMO

Citrus leprosis (CL) is a viral disease endemic to the Western Hemisphere that produces local necrotic and chlorotic lesions on leaves, branches, and fruit and causes serious yield reduction in citrus orchards. Samples of sweet orange (Citrus × sinensis) trees showing CL symptoms were collected during a survey in noncommercial citrus areas in the southeast region of Brazil in 2013 to 2016. Transmission electron microscopy analyses of foliar lesions confirmed the presence of rod-like viral particles commonly associated with CL in the nucleus and cytoplasm of infected cells. However, every attempt to identify these particles by reverse-transcription polymerase chain reaction tests failed, even though all described primers for the detection of known CL-causing cileviruses and dichorhaviruses were used. Next-generation sequencing of total RNA extracts from three symptomatic samples revealed the genome of distinct, although highly related (>92% nucleotide sequence identity), viruses whose genetic organization is similar to that of dichorhaviruses. The genome sequence of these viruses showed <62% nucleotide sequence identity with those of orchid fleck virus and coffee ringspot virus. Globally, the deduced amino acid sequences of the open reading frames they encode share 32.7 to 63.8% identity with the proteins of the dichorhavirids. Mites collected from both the naturally infected citrus trees and those used for the transmission of one of the characterized isolates to Arabidopsis plants were anatomically recognized as Brevipalpus phoenicis sensu stricto. Molecular and biological features indicate that the identified viruses belong to a new species of CL-associated dichorhavirus, which we propose to call Citrus leprosis N dichorhavirus. Our results, while emphasizing the increasing diversity of viruses causing CL disease, lead to a reevaluation of the nomenclature of those viruses assigned to the genus Dichorhavirus. In this regard, a comprehensive discussion is presented.


Assuntos
Citrus/virologia , Doenças das Plantas/virologia , Vírus de Plantas/genética , Vírus de Plantas/isolamento & purificação , Animais , Clonagem Molecular , Efeito Citopatogênico Viral , Genoma Viral , Ácaros/classificação , Ácaros/ultraestrutura , Ácaros/virologia , Filogenia , Folhas de Planta/ultraestrutura , Folhas de Planta/virologia , RNA Viral/genética
4.
Biosci Biotechnol Biochem ; 81(7): 1343-1347, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28417702

RESUMO

Quinolones are important antimicrobials for treatment of leprosy, a chronic infectious disease caused by Mycobacterium leprae. Although it is well known that mutations in DNA gyrase are responsible for quinolone resistance, the effect of those mutations on the enzymatic activity is yet to be studied in depth. Hence, we conducted in vitro assays to observe supercoiling reactions of wild type and mutated M. leprae DNA gyrases. DNA gyrase with amino acid substitution Ala91Val possessed the highest activity among the mutants. DNA gyrase with Gly89Cys showed the lowest level of activity despite being found in clinical strains, but it supercoiled DNA like the wild type does if applied at a sufficient concentration. In addition, patterns of time-dependent conversion from relaxed circular DNA into supercoiled DNA by DNA gyrases with clinically unreported Asp95Gly and Asp95Asn were observed to be distinct from those by the other DNA gyrases.


Assuntos
Substituição de Aminoácidos , Antibacterianos/farmacologia , DNA Girase/química , Mutação , Mycobacterium leprae/efeitos dos fármacos , Quinolonas/farmacologia , Clonagem Molecular , DNA Girase/genética , DNA Girase/metabolismo , DNA Bacteriano/química , DNA Bacteriano/metabolismo , DNA Circular/química , DNA Circular/metabolismo , DNA Super-Helicoidal/química , DNA Super-Helicoidal/metabolismo , Farmacorresistência Bacteriana , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Cinética , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade
5.
Dev Comp Immunol ; 71: 18-27, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28126556

RESUMO

Application of yeast is increasing to improve welfare and promotes growth in aquaculture. The halotolerant yeast Debaryomyces hansenii is normally a non-pathogenic yeast with probiotic properties and potential source of antioxidant enzymes as superoxide dismutase. Here, first, we characterized the sequence features of MnSOD and icCu/ZnSOD from Pacific red snapper, and second, we evaluated the potential antioxidant immune responses of the marine yeast Debaryomyces hansenii strain CBS004 in leukocytes which were then subjected to Vibrio parahaemolyticus infection. In silico analysis revealed that LpMnSOD consisted of 1186 bp, with an ORF of 678 bp encoding a 225 amino acid protein and LpicCu/ZnSOD consisted of 1090 bp in length with an ORF of 465 bp encoding a 154 amino acid protein. Multiple alignment analyzes revealed many conserved regions and active sites among its orthologs. In vitro assays using head-kidney and spleen leukocytes immunostimulated with D. hansenii and zymosan in response to V. parahaemolyticus infection reveled that D. hansenii strain CBS004 significantly increased transcriptions of MnSOD and icCu/ZnSOD genes. Flow cytometry assay showed that D. hansenii was able to inhibit apoptosis caused by V. parahaemolyticus in the Pacific red snapper leukocytes and enhanced the phagocytic capacity in head-kidney leukocytes. Immunological assays reveled an increased in superoxide dismutase and peroxidase activities, as well as, in nitric oxide production and reactive oxygen species production (respiratory burst) in fish stimulated with D. hansenii. Finally, our results. These results strongly support the idea that marine yeast Debaryomyces hansenii strain CBS004 can stimulate the antioxidant immune mechanism in head-kidney and spleen leukocytes.


Assuntos
Debaryomyces/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/metabolismo , Leucócitos/imunologia , Perciformes/imunologia , Superóxido Dismutase/metabolismo , Vibrioses/imunologia , Vibrio parahaemolyticus/imunologia , Sequência de Aminoácidos , Animais , Apoptose , Clonagem Molecular , Doenças dos Peixes/microbiologia , Proteínas de Peixes/genética , Imunidade Inata , Estresse Oxidativo , Fagocitose , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/genética , Regulação para Cima , Vibrioses/microbiologia
6.
Nucleic Acids Res ; 45(1): 1-14, 2017 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-27899559

RESUMO

RNase H enzymes sense the presence of ribonucleotides in the genome and initiate their removal by incising the ribonucleotide-containing strand of an RNA:DNA hybrid. Mycobacterium smegmatis encodes four RNase H enzymes: RnhA, RnhB, RnhC and RnhD. Here, we interrogate the biochemical activity and nucleic acid substrate specificity of RnhA. We report that RnhA (like RnhC characterized previously) is an RNase H1-type magnesium-dependent endonuclease with stringent specificity for RNA:DNA hybrid duplexes. Whereas RnhA does not incise an embedded mono-ribonucleotide, it can efficiently cleave within tracts of four or more ribonucleotides in duplex DNA. We gained genetic insights to the division of labor among mycobacterial RNases H by deleting the rnhA, rnhB, rnhC and rnhD genes, individually and in various combinations. The salient conclusions are that: (i) RNase H1 activity is essential for mycobacterial growth and can be provided by either RnhC or RnhA; (ii) the RNase H2 enzymes RnhB and RnhD are dispensable for growth and (iii) RnhB and RnhA collaborate to protect M. smegmatis against oxidative damage in stationary phase. Our findings highlight RnhC, the sole RNase H1 in pathogenic mycobacteria, as a candidate drug discovery target for tuberculosis and leprosy.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Peróxido de Hidrogênio/farmacologia , Mycobacterium smegmatis/efeitos dos fármacos , Ribonuclease H/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crescimento & desenvolvimento , Mycobacterium smegmatis/metabolismo , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonuclease H/metabolismo , Ribonucleotídeos/genética , Ribonucleotídeos/metabolismo , Alinhamento de Sequência , Especificidade por Substrato
7.
Enzyme Microb Technol ; 82: 58-65, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26672449

RESUMO

The gram-negative bacterium, Gluconacetobacter hansenii, produces cellulose of exceptionally high crystallinity in comparison to the cellulose of higher plants. This bacterial cellulose is synthesized and extruded into the extracellular medium by the cellulose synthase complex (CSC). The catalytic component of this complex is encoded by the gene AcsAB. However, several other genes are known to encode proteins critical to cellulose synthesis and are likely components of the bacterial CSC. We have purified an active heterodimer AcsA-AcsB from G. hansenii ATCC23769 to homogeneity by two different methods. With the purified protein, we have determined how it is post-translationally processed, forming the active heterodimer AcsA-AcsB. Additionally, we have performed steady-state kinetic studies on the AcsA-AcsB complex. Finally through mutagenesis studies, we have explored the roles of the postulated CSC proteins AcsC, AcsD, and CcpAx.


Assuntos
Proteínas de Bactérias/química , Gluconacetobacter/enzimologia , Glucosiltransferases/química , Complexos Multienzimáticos/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Catálise , Domínio Catalítico , Celulose/biossíntese , Centrifugação , Clonagem Molecular , Dimerização , Genes Bacterianos , Gluconacetobacter/genética , Glucosiltransferases/genética , Glucosiltransferases/isolamento & purificação , Glucosiltransferases/metabolismo , Cinética , Dados de Sequência Molecular , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/isolamento & purificação , Complexos Multienzimáticos/metabolismo , Mutagênese Insercional , Subunidades Proteicas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
8.
Protein Sci ; 24(1): 1-10, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25303009

RESUMO

Protein production using recombinant DNA technology has a fundamental impact on our understanding of biology through providing proteins for structural and functional studies. Escherichia coli (E. coli) has been traditionally used as the default expression host to over-express and purify proteins from many different organisms. E. coli does, however, have known shortcomings for obtaining soluble, properly folded proteins suitable for downstream studies. These shortcomings are even more pronounced for the mycobacterial pathogen Mycobacterium tuberculosis, the bacterium that causes tuberculosis, with typically only one third of proteins expressed in E. coli produced as soluble proteins. Mycobacterium smegmatis (M. smegmatis) is a closely related and non-pathogenic species that has been successfully used as an expression host for production of proteins from various mycobacterial species. In this review, we describe the early attempts to produce mycobacterial proteins in alternative expression hosts and then focus on available expression systems in M. smegmatis. The advantages of using M. smegmatis as an expression host, its application in structural biology and some practical aspects of protein production are also discussed. M. smegmatis provides an effective expression platform for enhanced understanding of mycobacterial biology and pathogenesis and for developing novel and better therapeutics and diagnostics.


Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Mycobacterium smegmatis/genética , Proteínas Recombinantes/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Vetores Genéticos/genética , Modelos Moleculares , Mycobacterium/química , Mycobacterium/genética , Mycobacterium/metabolismo , Mycobacterium smegmatis/química , Mycobacterium smegmatis/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
9.
J Med Microbiol ; 62(Pt 7): 959-967, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23579398

RESUMO

The aim of this study is to examine the in vivo role of a small heat-shock protein (sHsp18) from Mycobacterium leprae in the survival of heterologous recombinant hosts carrying the gene encoding this protein under different environmental conditions that are normally encountered by M. leprae during its infection of the human host. Using an Escherichia coli system where shsp18 expression is controlled by its native promoter, we show that expression of shsp18 is induced under low oxygen tension, nutrient depletion and oxidative stress, all of which reflect the natural internal environment of the granulomas where the pathogen resides for long periods. We demonstrate the in vivo chaperone activity of sHsp18 through its ability to confer survival advantage to recombinant E. coli at heat-shock temperatures. Additional evidence for the protective role of sHsp18 was obtained when Mycobacterium smegmatis harbouring a copy of shsp18 was found to multiply better in human macrophages. Furthermore, the autokinase activity of sHsp18 protein demonstrated for what is believed to be the first time in this study implies that some of the functions of sHsp18 might be controlled by the phosphorylation state of this protein. Results from this study suggest that shsp18 might be one of the factors that facilitate the survival and persistence of M. leprae under stress and autophosphorylation of sHsp18 protein could be a mechanism used by this protein to sense changes in the external environment.


Assuntos
Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Proteínas de Choque Térmico Pequenas/metabolismo , Mycobacterium leprae/metabolismo , Mycobacterium smegmatis/metabolismo , Proteínas de Bactérias/genética , Linhagem Celular , Clonagem Molecular , Escherichia coli/genética , Genoma Bacteriano , Proteínas de Choque Térmico Pequenas/genética , Temperatura Alta , Humanos , Monócitos/microbiologia , Mycobacterium leprae/genética , Mycobacterium smegmatis/genética , Regiões Promotoras Genéticas , Estresse Fisiológico , Transcriptoma
10.
Appl Microbiol Biotechnol ; 97(4): 1613-23, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-22526783

RESUMO

Because of its natural ability to utilize both xylose and arabinose, the halotolerant and osmotolerant yeast Debaryomyces hansenii is considered as a potential microbial platform for exploiting lignocellulosic biomass. To gain better understanding of the xylose metabolism in D. hansenii, we have cloned and characterized a xylitol dehydrogenase gene (DhXDH). The cloned gene appeared to be essential for xylose metabolism in D. hansenii as the deletion of this gene abolished the growth of the cells on xylose. The expression of DhXDH was strongly upregulated in the presence of xylose. Recombinant DhXdhp was expressed and purified from Escherichia coli. DhXdhp was highly active against xylitol and sorbitol as substrate. Our results showed that DhXdhp was thermo-sensitive, and except this, its biochemical properties were quite comparable with XDH from other yeast species. Furthermore, to make this enzyme suitable for metabolic engineering of D. hansenii, we have improved its thermotolerance and modified cofactor requirement through modelling and mutagenesis approach.


Assuntos
Clonagem Molecular , D-Xilulose Redutase/química , D-Xilulose Redutase/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Saccharomycetales/enzimologia , Sequência de Aminoácidos , D-Xilulose Redutase/metabolismo , Estabilidade Enzimática , Proteínas Fúngicas/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Saccharomycetales/química , Saccharomycetales/genética , Alinhamento de Sequência , Xilose/metabolismo
11.
Genet Mol Res ; 11(2): 1146-57, 2012 Apr 27.
Artigo em Inglês | MEDLINE | ID: mdl-22614283

RESUMO

Lactic acid bacteria (LAB) are an attractive and safe alternative for the expression of heterologous proteins, as they are nonpathogenic and endotoxin-free organisms. Lactococcus lactis, the LAB model organism, has been extensively employed in the biotechnology field for large-scale production of heterologous proteins, and its use as a "cell factory" has been widely studied. We have been particularly interested in the use of L. lactis for production of heat shock proteins (HSPs), which reportedly play important roles in the initiation of innate and adaptive immune responses. However, this activity has been questioned, as LPS contamination appears to be responsible for most, if not all, immunostimulatory activity of HSPs. In order to study the effect of pure HSPs on the immune system, we constructed recombinant L. lactis strains able to produce and properly address the Mycobacterium leprae 65-kDa HSP (Hsp65) to the cytoplasm or to the extracellular medium, using a xylose-induced expression system. Approximately 7 mg/L recombinant Hsp65 was secreted. Degradation products related to lactococcal HtrA activity were not observed, and the Limulus amebocyte lysate assay demonstrated that the amount of LPS in the recombinant Hsp65 preparations was 10-100 times lower than the permitted levels established by the U.S. Food and Drug Administration. These new L. lactis strains will allow investigation of the effects of M. leprae Hsp65 without the interference of LPS; consequently, they have potential for a variety of biotechnological, medical and therapeutic applications.


Assuntos
Proteínas de Bactérias/genética , Chaperonina 60/genética , Lactococcus lactis/metabolismo , Mycobacterium leprae/metabolismo , Sequência de Bases , Clonagem Molecular , Primers do DNA , Lactococcus lactis/genética , Mycobacterium leprae/genética , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética
12.
Biochim Biophys Acta ; 1814(12): 1802-11, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22001565

RESUMO

Mycobacterium leprae is closely related to Mycobacterium tuberculosis, yet causes a very different illness. Detailed genomic comparison between these two species of mycobacteria reveals that the decaying M. leprae genome contains less than half of the M. tuberculosis functional genes. The reduction of genome size and accumulation of pseudogenes in the M. leprae genome is thought to result from multiple recombination events between related repetitive sequences, which provided the impetus to investigate the recombination-like activities of RecA protein. In this study, we have cloned, over-expressed and purified M. leprae RecA and compared its activities with that of M. tuberculosis RecA. Both proteins, despite being 91% identical at the amino acid level, exhibit strikingly different binding profiles for single-stranded DNA with varying GC contents, in the ability to catalyze the formation of D-loops and to promote DNA strand exchange. The kinetics and the extent of single-stranded DNA-dependent ATPase and coprotease activities were nearly equivalent between these two recombinases. However, the degree of inhibition exerted by a range of ATP:ADP ratios was greater on strand exchange promoted by M. leprae RecA compared to its M. tuberculosis counterpart. Taken together, our results provide insights into the mechanistic aspects of homologous recombination and coprotease activity promoted by M. lepare RecA, and further suggests that it differs from the M. tuberculosis counterpart. These results are consistent with an emerging concept of DNA-sequence influenced structural differences in RecA nucleoprotein filaments and how these differences reflect on the multiple activities associated with RecA protein.


Assuntos
Mycobacterium leprae/enzimologia , Mycobacterium tuberculosis/enzimologia , Recombinases Rec A/química , Recombinases Rec A/fisiologia , Homologia Estrutural de Proteína , Sequência de Aminoácidos , Composição de Bases , Sítios de Ligação/genética , Clonagem Molecular , DNA de Cadeia Simples/metabolismo , Dados de Sequência Molecular , Mycobacterium leprae/química , Mycobacterium leprae/genética , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Ligação Proteica , Estrutura Secundária de Proteína , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência , Especificidade da Espécie , Especificidade por Substrato
13.
PLoS Genet ; 7(5): e1002078, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21637794

RESUMO

The discovery of expression quantitative trait loci ("eQTLs") can help to unravel genetic contributions to complex traits. We identified genetic determinants of human liver gene expression variation using two independent collections of primary tissue profiled with Agilent (n = 206) and Illumina (n = 60) expression arrays and Illumina SNP genotyping (550K), and we also incorporated data from a published study (n = 266). We found that ∼30% of SNP-expression correlations in one study failed to replicate in either of the others, even at thresholds yielding high reproducibility in simulations, and we quantified numerous factors affecting reproducibility. Our data suggest that drug exposure, clinical descriptors, and unknown factors associated with tissue ascertainment and analysis have substantial effects on gene expression and that controlling for hidden confounding variables significantly increases replication rate. Furthermore, we found that reproducible eQTL SNPs were heavily enriched near gene starts and ends, and subsequently resequenced the promoters and 3'UTRs for 14 genes and tested the identified haplotypes using luciferase assays. For three genes, significant haplotype-specific in vitro functional differences correlated directly with expression levels, suggesting that many bona fide eQTLs result from functional variants that can be mechanistically isolated in a high-throughput fashion. Finally, given our study design, we were able to discover and validate hundreds of liver eQTLs. Many of these relate directly to complex traits for which liver-specific analyses are likely to be relevant, and we identified dozens of potential connections with disease-associated loci. These included previously characterized eQTL contributors to diabetes, drug response, and lipid levels, and they suggest novel candidates such as a role for NOD2 expression in leprosy risk and C2orf43 in prostate cancer. In general, the work presented here will be valuable for future efforts to precisely identify and functionally characterize genetic contributions to a variety of complex traits.


Assuntos
Genoma Humano , Fígado/metabolismo , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Regiões 3' não Traduzidas , Grupo com Ancestrais do Continente Africano , Fatores Etários , Mapeamento Cromossômico , Clonagem Molecular , Grupo com Ancestrais do Continente Europeu , Feminino , Perfilação da Expressão Gênica , Vetores Genéticos , Estudo de Associação Genômica Ampla , Genótipo , Células Hep G2 , Hispano-Americanos , Humanos , Medições Luminescentes , Masculino , Análise de Componente Principal , Regiões Promotoras Genéticas , Reprodutibilidade dos Testes , Fatores Sexuais , Transfecção
14.
Biotechnol Lett ; 33(8): 1643-8, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21479627

RESUMO

Industrial strains of a polyploid, distiller's Saccharomyces cerevisiae that produces glucoamylase and α-amylase was used for the direct fermentation of raw starch to ethanol. Strains contained either Aspergillus awamori glucoamylase gene (GA1), Debaryomyces occidentalis glucoamylase gene (GAM1) or D. occidentalis α-amylase gene (AMY), singly or in combination, integrated into their chromosomes. The strain expressing both GA1 and AMY generated 10.3% (v/v) ethanol (80.9 g l(-1)) from 20% (w/v) raw corn starch after 6 days of fermentation, and decreased the raw starch content to 21% of the initial concentration.


Assuntos
Etanol/metabolismo , Glucana 1,4-alfa-Glucosidase/metabolismo , Saccharomyces cerevisiae/metabolismo , Amido/metabolismo , alfa-Amilases/metabolismo , Clonagem Molecular , Debaryomyces/enzimologia , Debaryomyces/genética , Etanol/análise , Fermentação , Proteínas Fúngicas/metabolismo , Glucana 1,4-alfa-Glucosidase/biossíntese , Glucana 1,4-alfa-Glucosidase/genética , Microbiologia Industrial/métodos , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Amido/análise , alfa-Amilases/biossíntese , alfa-Amilases/genética
15.
Appl Environ Microbiol ; 77(10): 3311-9, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21421781

RESUMO

Molecular transport is a key process in cellular metabolism. This step is often limiting when using a nonnative carbon source, as exemplified by xylose catabolism in Saccharomyces cerevisiae. As a step toward addressing this limitation, this study seeks to characterize monosaccharide transport preference and efficiency. A group of 26 known and putative monosaccharide transport proteins was expressed in a recombinant Saccharomyces cerevisiae host unable to transport several monosaccharides. A growth-based assay was used to detect transport capacity across six different carbon sources (glucose, xylose, galactose, fructose, mannose, and ribose). A mixed glucose-and-xylose cofermentation was performed to determine substrate preference. These experiments identified 10 transporter proteins that function as transporters of one or more of these sugars. Most of these proteins exhibited broad substrate ranges, and glucose was preferred in all cases. The broadest transporters confer the highest growth rates and strongly prefer glucose. This study reports the first molecular characterization of the annotated XUT genes of Scheffersomyces stipitis and open reading frames from the yeasts Yarrowia lipolytica and Debaryomyces hansenii. Finally, a phylogenetic analysis demonstrates that transporter function clusters into three distinct groups. One particular group comprised of D. hansenii XylHP and S. stipitis XUT1 and XUT3 demonstrated moderate transport efficiency and higher xylose preferences.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Hexoses/metabolismo , Ribose/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Clonagem Molecular , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento
16.
Hum Genet ; 127(3): 337-48, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20035344

RESUMO

The chromosomal region 10p13 has been linked to paucibacillary leprosy in two independent studies. The MRC1 gene, encoding the human mannose receptor (MR), is located in the 10p13 region and non-synonymous SNPs in exon 7 of the gene have been suggested as leprosy susceptibility factors. We determined that G396S is the only non-synonymous exon 7-encoded polymorphism in 396 unrelated Vietnamese subjects. This SNP was genotyped in 490 simplex and 90 multiplex leprosy families comprising 704 patients (47% paucibacillary; 53% multibacillary). We observed significant under-transmission of the serine allele of the G396S polymorphism with leprosy per se (P = 0.036) and multibacillary leprosy (P = 0.034). In a sample of 384 Brazilian leprosy cases (51% paucibacillary; 49% multibacillary) and 399 healthy controls, we observed significant association of the glycine allele of the G396S polymorphism with leprosy per se (P = 0.016) and multibacillary leprosy (P = 0.023). In addition, we observed a significant association of exon 7 encoded amino acid haplotypes with leprosy per se (P = 0.012) and multibacillary leprosy (P = 0.004). Next, we tested HEK293 cells over-expressing MR constructs (293-MR) with three exon 7 haplotypes of MRC1 for their ability to bind and internalize ovalbumin and zymosan, two classical MR ligands. No difference in uptake was measured between the variants. In addition, 293-MR failed to bind and internalize viable Mycobacterium leprae and BCG. We propose that the MR-M. leprae interaction is modulated by an accessory host molecule of unknown identity.


Assuntos
Éxons , Lectinas Tipo C/genética , Hanseníase/genética , Lectinas de Ligação a Manose/genética , Polimorfismo de Nucleotídeo Único , Receptores de Superfície Celular/genética , Estudos de Casos e Controles , Células Cultivadas , Clonagem Molecular , Predisposição Genética para Doença , Humanos , Lectinas Tipo C/metabolismo , Lectinas Tipo C/fisiologia , Desequilíbrio de Ligação , Lectinas de Ligação a Manose/metabolismo , Lectinas de Ligação a Manose/fisiologia , Proteínas Mutantes/genética , Mycobacterium bovis/metabolismo , Mycobacterium leprae/metabolismo , Polimorfismo de Nucleotídeo Único/fisiologia , Ligação Proteica , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/fisiologia , Transfecção
17.
Mem. Inst. Oswaldo Cruz ; 104(8): 1132-1138, Dec. 2009. tab, ilus
Artigo em Inglês | LILACS | ID: lil-538173

RESUMO

Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40°C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.


Assuntos
Humanos , Mycobacterium leprae/enzimologia , Serina Endopeptidases/biossíntese , Sequência de Bases , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Dados de Sequência Molecular , Mycobacterium leprae/genética , Espectroscopia de Infravermelho com Transformada de Fourier
18.
Int J Syst Evol Microbiol ; 59(Pt 5): 1242-51, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19406826

RESUMO

The delineation of species among strains assigned to Debaryomyces hansenii was examined using a gene genealogies-based approach in order to compare spliceosomal intron sequences found in four housekeeping genes (ACT1, TUB2, RPL31 and RPL33). This revealed four distinct groups of strains containing, respectively, D. hansenii var. hansenii CBS 767(T), D. hansenii var. fabryi CBS 789(T), Candida famata var. flareri CBS 1796(T) (the anamorph of D. hansenii var. fabryi CBS 789(T)) and Debaryomyces tyrocola CBS 766(T), whose species status was no longer accepted. The sequence divergence between these groups, reaching in some cases over 20 %, unambiguously isolated the groups as separate taxa, leading to a proposal for the reinstatement of the originally described species D. hansenii CBS 767(T) and D. tyrocola CBS 766(T). The variety D. hansenii var. fabryi was further subdivided into two taxa, Debaryomyces fabryi CBS 789(T) and Candida flareri CBS 1796(T) (previously C. famata var. flareri and Blastodendrion flareri). The comparison of intron sequences therefore exposed cryptic species whose phenotypic traits are not distinguishable from known species, but which have significantly diverged from the genetic point of view. Hence, we describe the new taxon Debaryomyces macquariensis sp. nov. CBS 5571(T) is related to, but clearly distinct from, the Debaryomyces species mentioned above. The approach used in this work has also revealed the existence of populations within the newly delineated species D. hansenii and genetic exchanges between these populations, indicating an unexpected genetic diversity within this part of the genus Debaryomyces.


Assuntos
Íntrons/genética , Técnicas de Tipagem Micológica , Saccharomycetales/classificação , Análise de Sequência de DNA/métodos , Animais , Clonagem Molecular , DNA Fúngico/análise , DNA Fúngico/genética , Proteínas Fúngicas/genética , Humanos , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase/métodos , Saccharomycetales/genética , Especificidade da Espécie
19.
Fish Shellfish Immunol ; 27(2): 181-91, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19376233

RESUMO

A selenium dependent glutathione peroxidase (Se-GPx) cDNA was cloned from haemocyte by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA (RACE). The 913 bp cDNA contained an open reading frame (ORF) of 558 bp encoded a deduced amino acid sequence of 186 amino acids. The prawn Se-GPx sequence contains a selenocysteine (Sec) residue which is encoded by the unusual stop codon, (115)TGA(117). According to the molecular modeling analysis, the active site Sec residue, located in the loop between beta3 and alpha2 in a pocket on the protein surface, and hydrogen bonded to Gln(73) and Trp(141). A GPx signature motif 2, (63)LAFPCNQF(70) and active site motif, (151)WNFEKF(156), two arginine (R) residues, R(89) and R(167) contribute to the electrostatic architecture that directs the glutathione donor substrate, and two putative N-glycosylation site, (75)NNT(77) and (107)NGS(109) were observed in the prawn Se-GPx sequence. In addition, the eukaryotic selenocysteine insertion sequence element is conserved in the 3'-UTR. Comparison of amino acid sequences showed that prawn Se-GPx is more closely related to vertebrate GPx 1. The prawn Se-GPx was synthesized in haemocyte, hepatopancreas, muscle, stomach, gill, intestine, eyestalk, heart, epidermis, lymph organ, ventral nerve cord, testis and ovary. The increase of respiratory burst in haemocyte was observed in pathogen, Debaryomyces hansenii-injected prawn in order to kill the pathogen, and the up-regulation in SOD and GPx acitivity, and prawn Se-GPx mRNA transcription were involved with the protection against damage from oxidation.


Assuntos
Glutationa Peroxidase/genética , Palaemonidae/enzimologia , Palaemonidae/genética , Selênio/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Debaryomyces/fisiologia , Regulação Enzimológica da Expressão Gênica , Glutationa Peroxidase/química , Glutationa Peroxidase/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Palaemonidae/imunologia , Palaemonidae/microbiologia , Filogenia , Estrutura Terciária de Proteína , Alinhamento de Sequência
20.
Mem Inst Oswaldo Cruz ; 104(8): 1132-8, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20140374

RESUMO

Members of the high temperature requirement A (HtrA) family of chaperone proteases have been shown to play a role in bacterial pathogenesis. In a recent report, we demonstrated that the gene ML0176, which codes for a predicted HtrA-like protease, a gene conserved in other species of mycobacteria, is transcribed by Mycobacterium leprae in human leprosy lesions. In the present study, the recombinant ML0176 protein was produced and its enzymatic properties investigated. M. lepraerecombinant ML0176 was able to hydrolyse a variety of synthetic and natural peptides. Similar to other HtrA proteins, this enzyme displayed maximum proteolytic activity at temperatures above 40 degrees C and was completely inactivated by aprotinin, a protease inhibitor with high selectivity for serine proteases. Finally, analysis of M. leprae ML0176 specificity suggested a broader cleavage preference than that of previously described HtrAs homologues. In summary, we have identified an HtrA-like protease in M. lepraethat may constitute a potential new target for the development of novel prophylactic and/or therapeutic strategies against mycobacterial infections.


Assuntos
Mycobacterium leprae/enzimologia , Serina Endopeptidases/biossíntese , Sequência de Bases , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Humanos , Dados de Sequência Molecular , Mycobacterium leprae/genética , Espectroscopia de Infravermelho com Transformada de Fourier
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