RESUMO
To achieve broader range of the defensin antimicrobial activity, based on the sd2 gene sequence, the modified gene, sd2mod, was constructed. Hybrid genes, sd2-licBM2, licBM2-sd2, licBM2-sd2mod, and sd2mod-licBM2, in which the wild-type and modified gene sequences were fused in frame with the reporter gene encoding thermostable lichenase, were constructed. Expression of the wild-type, modified, and hybrid genes was examined in the cells of pro- and eukaryotes. It was demonstrated that these genes were efficiently expressed in the cells of lower eukaryotes, the yeast. Inhibiting effect of the SD2 and SDmod proteins as the components of the hybrid proteins, SD2-LicBM2 and SD2mod-LicBM2, on the growth of the Fusarium culmorum hyphae was similar to that of the wild-type and modified proteins. It was shown that the presence of lichenase in the hybrid proteins facilitated selection and analysis of the hybrid proteins expression in transgenic organisms.
Assuntos
Defensinas/genética , Escherichia coli/genética , Expressão Gênica , Helianthus/genética , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Defensinas/farmacologia , Fusarium/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Dados de Sequência Molecular , Proteínas de Plantas/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologiaRESUMO
The original yeast vector system was proposed for study of regulation functions of eukaryotic noncoding sequences. This system consists of two reporter genes that arranges in opposition to one another. The promoter activity of LTR HERV-K of locus 22-19 in 7p22 human chromosome was studied. It was shown that the LTR initiates transcription of reporter gene in yeasts in forward and reverse orientation with respect to the reporter. It was displayed that the LTR 22-19 HERV-K possesses bidirectional promoter activity in the yeast vector system. Comparison of the promoter activity LTR 22-19 and strong GAL1 and TDH promoters in yeasts Saccharomyces cerevisiae shown that promoter activity of the LTR amount approximately 0.34% for promoter activity of the inducible GAL1 promoter and 0.26%--of the constitutive TDH promoter.
Assuntos
Vetores Genéticos , Saccharomyces cerevisiae/genética , Sequência de Bases , Cromossomos Humanos Par 7 , DNA , Genes Reporter , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Sequências Repetidas Terminais , Transcrição Gênica/genéticaRESUMO
Hybrid genes containing the reporter gene for thermostable lichenase and model genes recA, recA1, cry3a, cry3aM, and ssp1 were constructed. The expression of these genes was studied in prokaryotic and eukaryotic cells. The presence of lichenase in the hybrid proteins was shown to facilitate analysis of the hybrid protein expression in transgenic organisms. Owing to high relative activity and thermostability of lichenase, the activity of this enzyme can be measured by simple, rapid and sensitive qualitative and quantitative methods that do not require costly equipment and reagents. Using the zymograms method, molecular masses of the lichenase-containing hybrid proteins can be precisely estimated. This method is proposed instead of Western blotting using lichenase as a translational reporter. Our results showed that the use of thermostable lichenase as a translational reporter yields the data that are problematic to obtain using traditional methods of gene expression analysis, which is of importance for fundamental and applied research.
Assuntos
Genes Reporter , Glicosídeo Hidrolases/metabolismo , Biossíntese de Proteínas , Sequência de Bases , Western Blotting , DNA Bacteriano , Estabilidade Enzimática , Glicosídeo Hidrolases/genética , Dados de Sequência MolecularRESUMO
Analysis of clinical material obtained from the individuals (49 psoriasis patients and 357 individuals without this disease) representing three ethnic populations of Dagestan (Avars, Dargins, and Kumyks) was performed. Polymorphism of the blood group loci AB0, Rhesus (RH), Kell, P, and Lewis, as well as of the protein-encoding loci for haptoglobin (HP), group-specific component (GC), and the enzymes, including glycosylase (GL01), esterase D (ESD), 6-phosphate dehydrogenase (6PDG), and acid phosphatase (ACP), was studied. It was demonstrated that in the pooled sample of Avars and Kumyks the Lewis system phenotype Le(a-b-) and the RH homozygotes (ee/ee) were statistically significantly more frequent among the psoriasis patients (P = 0.0488 and P = 0.0166, respectively), than among healthy controls of the same ethnic groups. It was suggested that for the pooled sample of Avars and Kumyks, homozygosity for the recessive RH allele (ee/ee) in combination with the Le(a-b-) phenotype, representing homozygosity for recessive allele le, was the risk factor for the development of psoriasis.
Assuntos
Antígenos de Grupos Sanguíneos/genética , Enzimas/genética , Polimorfismo Genético , Psoríase/genética , Etnicidade , Feminino , Marcadores Genéticos/genética , Homozigoto , Humanos , Masculino , Psoríase/etnologia , Federação RussaRESUMO
To obtain transgenic tobacco plants expressing recombinant analogs of spider dragline silk spidroin 1, artificial 1f5 and 1f9 coding for spidroin 1 analogs were 3'-fused in-frame with the reporter lichenase gene. The Tr2' weak constitutive promoter of Agrobacterium tumefaciens T-DNA and the strong constitutive promoter of the cauliflower mosaic virus 35S RNA gene were used as regulatory elements. The expression cassettes were used to transform agrobacteria and then introduced in tobacco leaf disks. On evidence of Southern hybridization, transgenic plants each carried a single copy of a hybrid gene, which corresponded in size to the constructed one. Zymography and Western blotting revealed full-length hybrid proteins in leaf extracts of transgenic plants. The results testified that plants can maintain and express synthetic genes for spider silks and, consequently, may be used as a convenient producer of recombinant silk analogs.
Assuntos
Fibroínas , Proteínas de Insetos/genética , Nicotiana/genética , Plantas Geneticamente Modificadas/genética , Engenharia de Proteínas/métodos , Proteínas/genética , Agrobacterium tumefaciens/genética , Animais , Caulimovirus/genética , Regulação da Expressão Gênica , Vetores Genéticos , Glicosídeo Hidrolases/genética , Proteínas de Insetos/química , Folhas de Planta/genética , Regiões Promotoras Genéticas , RNA Viral , Sequências Reguladoras de Ácido Ribonucleico , Seda , AranhasRESUMO
The bacterial gene of the thermostable endo-beta-1,4-glucanase (cellulase) was shown to retain its activity and substrate specificity when expressed in transgenic tobacco plants. The leader peptide of the carrot extensin was efficient in transferring the bacterial enzyme into the apoplast. The expression of the bacterial cellulase gene leads to changes in the plant tissue morphology. In the transgenic plant lines, regeneration of primary shoots from callus occurred at the three to five times higher cytokinin (6-BAP) concentration than in control plants. The transgenic plants that expressed the bacterial gene exhibited increased business and altered leaf shape. The transgenic plants developed can be used as models for studying the cellulases role and function in plants.
Assuntos
Celulase/genética , Nicotiana/genética , Plantas Geneticamente Modificadas , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Celulase/metabolismo , Daucus carota/genética , Estabilidade Enzimática , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Vetores Genéticos , Glicoproteínas/genética , Fenótipo , Folhas de Planta/anatomia & histologia , Proteínas de Plantas/genética , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/fisiologia , Transformação GenéticaRESUMO
Bifunctional reporter proteins were constructed to combine Clostridium thermocellum lichenase (LicBM2) with Aequorea victoria green fluorescent protein (GFP) or with Escherichia coli beta-glucuronidase (GUS). The major properties of the initial proteins were preserved in the hybrid ones: LicBM2 was active at 65 degrees C, GFP fluoresced, and GUS hydrolyzed its substrates. LicBM2 remained active after extension of its C of N end. Bifunctional reporter systems were shown to provide a convenient tool for studying the gene expression regulation in prokaryotic (E. coli) and eukaryotic (Saccharomyces cerevisiae, mammalian) cells, advantages of one reporter compensating for drawbacks of the other.
Assuntos
Células Eucarióticas/fisiologia , Genes Reporter , Células Procarióticas/fisiologia , Engenharia de Proteínas/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Animais , Clostridium/genética , Cnidários/genética , Escherichia coli/genética , Regulação da Expressão Gênica , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mamíferos , Células PC12 , Ratos , Saccharomyces cerevisiae/genéticaRESUMO
A new reporter system was developed to study the gene expression regulation in prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells. The system was based on the modified bacterial lichenase gene (licBM2), which was shown to meet the requirements for a reporter. The gene product was active and did not undergo modification in heterologous hosts. Simple and sensitive methods were used to detect and to quantitate the lichenase activity. Inducible licBM2 expression was demonstrated with E. coli and yeast cells, allowing the system to be employed in dynamic studies.
Assuntos
Clostridium/genética , Células Eucarióticas/fisiologia , Genes Reporter , Engenharia Genética/métodos , Glicosídeo Hidrolases/genética , Animais , Escherichia coli/genética , Regulação da Expressão Gênica , Glicosídeo Hidrolases/metabolismo , Células PC12 , Plasmídeos/genética , Ratos , Saccharomyces cerevisiae/genéticaRESUMO
The coding region of the licB gene from Clostridium thermocellum was truncated at the 3' end. The modified lichenase encoded by the construct (LicBM2) retained the most important properties of the enzyme - its high activity and thermostability. LicBM2 consists of the catalytic domain and part of the Pro-Thr-box. We demonstrated the application of the licBM2 gene as a reporter system for prokaryotic (Escherichia coli) and eukaryotic (Saccharomyces cerevisiae and mammalian) cells by expressing it either as a transcriptional fusion with selected promoters or as a translational fusion with the E. coli uidA gene. The assays available for LicB activity are sensitive, accurate and simple, and can be used for the analysis of various gene fusion systems or for screening of transformants.
Assuntos
Clostridium/enzimologia , Clostridium/genética , Genes Reporter , Glicosídeo Hidrolases/genética , Animais , Fusão Gênica Artificial , Sequência de Bases , DNA Bacteriano/genética , Estabilidade Enzimática , Escherichia coli/genética , Células Eucarióticas , Expressão Gênica , Genes Bacterianos , Vetores Genéticos , Células PC12 , Células Procarióticas , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/genética , TemperaturaRESUMO
The main properties (pH and temperature range, stability, substrate specificity) of the modified cellulase CelE (endo-beta-1,4-glucanase) from Clostridium thermocellum have been analyzed with the goal of its expression in plants. The modified enzyme is similar to plant cellulases. Deletions in the N-terminus of the enzyme do not affect its biochemical properties. Based on the present investigation, we conclude that the modified beta-1,4-glucanase CelEM1, when expressed in plants, will be a good model to study the role of cellulases in plants.
Assuntos
Carboximetilcelulose Sódica/metabolismo , Celulase/genética , Celulase/metabolismo , Clostridium/enzimologia , Glucanos/metabolismo , Deleção de Sequência/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Celulase/química , Clostridium/genética , Simulação por Computador , Ativação Enzimática , Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Modelos Genéticos , Mutação/genética , Plantas/enzimologia , Plantas/genéticaRESUMO
Major properties (pH and temperature optimum, stability) of lichenase (beta-1,3-1,4-glucanase) deletion variants from Clostridium thermocellum were comparatively studied. The deletion variant LicBM2 was used to create hybrid bifunctional proteins by fusion with sequences of the green fluorescent protein (GFP) from Aequorea victoria. The data show that in hybrid proteins both GFP and lichenase retain their major properties, namely, GFP remains a fluorescent protein and the lichenase retains activity and high thermostability. Based on the results of this investigation and results that have been obtained earlier, the use of the deletion variants of lichenase and the bifunctional hybrid proteins as reporter proteins is suggested.
Assuntos
Clostridium/enzimologia , Deleção de Genes , Glicosídeo Hidrolases/genética , Mutagênese , Proteínas Recombinantes de Fusão/metabolismo , Eletroforese em Gel de Poliacrilamida , Genes Reporter , Glicosídeo Hidrolases/química , Proteínas de Fluorescência Verde , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/metabolismo , Biologia Molecular/métodos , Plasmídeos/metabolismo , Desnaturação Proteica , Proteínas Recombinantes de Fusão/química , Temperatura , Fatores de Tempo , Raios UltravioletaRESUMO
Pseudomonas putida BS202 degrades naphthalene via a plasmid-encoded catabolic pathway. The nucleotide sequence of the nahC gene encoding one of this pathway enzymes, 1,2-dihydroxynaphthalene dioxygenase, has been determined. Analysis of nucleotide sequence of its flanking regions identified partially the nahF and putative nahQ genes. Comparison of these three genes with corresponding ones in the NAH7 plasmid and DOX operon showed a high degree of homology.
Assuntos
Dioxigenases , Naftalenos/metabolismo , Óperon , Oxigenases/genética , Plasmídeos , Pseudomonas putida/genética , Sequência de Aminoácidos , Sequência de Bases , Biodegradação Ambiental , Primers do DNA , Genes Bacterianos , Dados de Sequência Molecular , Pseudomonas putida/metabolismo , Mapeamento por Restrição , Homologia de Sequência de AminoácidosRESUMO
In order to take advantage of the high thermostability of its product, beta-1,3;1,4-glucanase (lichenase), we used a modified version of the licB gene from Clostridium thermocellum as a reporter gene for the analysis of gene expression in transformed plants. The coding region of the licB gene was truncated at both ends. The truncated enzyme retained its activity and thermostability. The modified gene (m-licB), with and without a plant leader peptide-encoding sequence, was expressed in tobacco plants under control of either the Agrobacterium octopine TR-DNA 2' gene promoter or the promoter of the gene for the small subunit of ribulose-1,5-bisphosphate carboxylase. Expression of licB can be measured quantitatively and accurately, the assay is sensitive and simple enough to be used for analysis of various gene fusion systems or for screening of transformants. The enzyme is very stable and remains active in tissue extracts even after storage for 1 year and survives many thawing-freezing cycles. The lichenase-encoding gene was expressed at high levels in transformed tobacco plants without any apparent detrimental effects on vegetative growth or flowering.
