Assuntos
Retículo Endoplasmático/metabolismo , Proteínas Fúngicas/metabolismo , Isomerases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Clonagem Molecular , Escherichia coli , Proteínas Fúngicas/biossíntese , Genes Fúngicos , Isomerases/biossíntese , Cinética , Isomerases de Dissulfetos de Proteínas , Mapeamento por Restrição , Saccharomyces cerevisiae/genéticaRESUMO
We have found an influence of cellular ubiquitin levels over the secretion of human leucocyte elastase inhibitor (elafin) by Saccharomyces cerevisiae. Inactivation of the UBI4 polyubiquitin gene reduced elafin secretion 3 to 4-fold. Conversely ubiquitin overexpression, by galactose induction of an integrated UBI4 gene under GAL1 promoter control, enhanced elafin secretion 7-fold compared to cells wild-type for ubiquitin genes. This influence of ubiquitin levels is exerted at a post-transcriptional step in elafin gene expression, and may represent a chaperone-like action. Ubiquitin overexpression did not affect production of alpha-factor and of certain natural yeast extracellular enzymes even though appreciable free ubiquitin became associated with the yeast periplasm.
Assuntos
Biopolímeros/genética , Expressão Gênica , Leucócitos/metabolismo , Proteínas , Saccharomyces cerevisiae/genética , Inibidores de Serina Proteinase/metabolismo , Ubiquitinas/genética , Sequência de Aminoácidos , Biopolímeros/fisiologia , Galactose/farmacologia , Expressão Gênica/efeitos dos fármacos , Técnicas de Transferência de Genes , Humanos , Elastase de Leucócito , Dados de Sequência Molecular , Elastase Pancreática/antagonistas & inibidores , Poliubiquitina , Regiões Promotoras Genéticas , Precursores de Proteínas/química , Proteínas Secretadas Inibidoras de Proteinases , Proteínas Recombinantes , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/genética , Ubiquitinas/fisiologiaRESUMO
The renin-encoding genes have been cloned from high (Ren-1d, Ren-2d)- and low (Ren-1c)-renin-producing strains of mice (DBA/2J and C57BL/10). Each of the genes is approx. 9.6 kb in length and consists of nine exons and eight introns. The entire nucleotide sequence of the Ren-1d gene has been determined and the 5'-flanking regions of the three genes, Ren-1c, Ren-1d and Ren-2d, have been compared. The significance of several potential regulatory signals found in the DNA is discussed.
Assuntos
Genes Reguladores , Genes , Renina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Éxons , Íntrons , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Dados de Sequência Molecular , Splicing de RNA , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Software , Especificidade da EspécieRESUMO
Replacement of the solvent-exposed residues of the DNA recognition helix of the 434 repressor with the corresponding residues of the P22 repressor generates a hybrid protein, 434R[alpha 3(P22R)], which binds specifically to P22 operators. We show here that a new DNA-binding specificity is generated by combining 434 and 434R[alpha 3(P22R)] repressor monomers to form a heterodimer. The heterodimer specifically recognizes a chimeric P22/434 operator that lacks two-fold rotational symmetry.
Assuntos
Regiões Operadoras Genéticas , Proteínas Repressoras/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Desoxirribonuclease I/farmacologia , MutaçãoRESUMO
Cells of Escherichia coli containing a chemically synthesized human alpha 1 interferon (IFN-alpha 1) gene, under control of the lac promoter, make a product with biological properties indistinguishable from those of the natural IFN-alpha 1 [antiviral activity, acid stability, species crossreactivity, inactivation by antisera directed against leukocyte or Namalwa cell interferon, and stimulation of (2'-5')oligoadenylate synthetase activity]. Similar levels of IFN synthesis were obtained when the expression unit (lac promoter plus synthetic IFN-alpha 1 gene) was transplanted into the obligate methylotroph Methylophilus methylotrophus.
Assuntos
Genes Sintéticos , Interferons/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Bioensaio , DNA Recombinante , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/genética , Regulação da Expressão Gênica , Humanos , Concentração de Íons de Hidrogênio , Interferons/farmacologia , Methylococcaceae/genética , PlasmídeosRESUMO
The glutamate dehydrogenase gene of Escherichia coli has been cloned into broad host-range plasmids and can complement glutamate synthase mutants of Methylophilus methylotrophus. Assimilation of ammonia via glutamate dehydrogenase is more energy-efficient than via glutamate synthase, thus the recombinant organism converts more growth substrate, methanol, into cellular carbon.
Assuntos
Proteínas de Bactérias/biossíntese , Glutamato Desidrogenase/genética , Methylococcaceae/metabolismo , Trifosfato de Adenosina/metabolismo , Amônia/metabolismo , DNA Recombinante , Escherichia coli/enzimologia , Engenharia Genética/métodos , Glutamato Sintase/metabolismo , Metanol/metabolismo , Methylococcaceae/enzimologia , PlasmídeosRESUMO
Pseudomonas aeruginosa PA01 was found to utilise both the D- and L-isomers of alpha-alanine and also beta-alanine as sole sources of carbon and energy for growth. Enzymological studies of wild-type cultures and comparison with mutants deficient in growth upon one or more isomers of alanine led to the following conclusions: (i) utilisation of D-alanine involved its direct oxidation by an inducible, membrane-bound, cytochrome-linked dehydrogenase; (ii) utilisation of L-alanine required its conversion to the directly oxidisable D-form by a soluble racemase; (iii) utilisation of beta-alanine, like L-alanine, involves both the racemase and D-alanine dehydrogenase enzymes, but in addition must involve other enzymes the identity of which is still speculative; (iv) P. aeruginosa, like Escherichia coli, appears to take up D-alanine and L-alanine by means of two specific permeases.
