RESUMEN
In recent years a number of hyperthermophilic micro-organisms of Sulfolobales have been found to produce trehalose from starch and dextrins. In our laboratory genes encoding the trehalosyl dextrin forming enzyme (TDFE) and the trehalose forming enzyme (TFE) of S. solfataricus MT4 have been cloned and expressed in E. coli (Rb791). Here we report the construction of a new protein obtained by fusion of TFE and TDFE coding sequences which is able to produce trehalose from dextrins at high temperature by sequential enzymatic steps. We demonstrate that the bifunctional fusion enzyme is able to produce trehalose starting from malto-oligosaccharides at 75 degrees C. Furthermore we partially purified the recombinant fusion protein from bacterial cell free extracts and from insoluble fractions in which the fusion protein was also found as aggregate in inclusion bodies.
Asunto(s)
Proteínas Bacterianas/genética , Glucosiltransferasas/genética , Proteínas Recombinantes de Fusión/metabolismo , Sulfolobus/genética , Trehalosa/biosíntesis , Proteínas Bacterianas/química , Clonación Molecular , Dextrinas/metabolismo , Genes Bacterianos , Genes Sintéticos , Glucosiltransferasas/química , Microbiología Industrial , Pliegue de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Sulfolobus/enzimología , TemperaturaRESUMEN
Molecular evolutionary analyses of mammalian ribonucleases have shown that gene duplication events giving rise to three paralogous genes occurred in ruminant ancestors. One of these genes encodes a ribonuclease identified in bovine brain. A peculiar feature of this enzyme and orthologous sequences in other ruminants are C-terminal extensions consisting of 17-27 amino acid residues. Evidence was obtained by Western blot analysis for the presence of brain-type ribonucleases in brain tissue not only of ox, but also of sheep, roe deer and chevrotain (Tragulus javanicus), a member of the earliest diverged taxon of the ruminants. The C-terminal extension of brain-type ribonuclease from giraffe deviates much in sequence from orthologues in other ruminants, due to a change of reading frame. However, the gene encodes a functional enzyme, which could be expressed in heterologous systems. The messenger RNA of bovine brain ribonuclease is not only expressed at a high level in brain tissue but also in lactating mammary gland. The enzyme was isolated and identified from this latter tissue, but was not present in bovine milk, although pancreatic ribonucleases A and B could be isolated from both sources. This suggests different ways of secretion of the two enzyme types, possibly related to structural differences. The sequence of the brain-type RNase from chevrotain suggests that the C-terminal extensions of ruminant brain-type ribonucleases originate from deletions in the ancestral DNA (including a region with stop codons), followed by insertion of a 5-8-fold repeated hexanucleotide sequence, coding for a proline-rich polypeptide.
Asunto(s)
Encéfalo/enzimología , Ribonucleasas/metabolismo , Rumiantes/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Ciervos , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Evolución Molecular , Femenino , Duplicación de Gen , Vectores Genéticos , Masculino , Glándulas Mamarias Animales/enzimología , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Ribonucleasas/genética , Ribonucleasas/aislamiento & purificación , Homología de Secuencia , OvinosRESUMEN
The nuclear protein CBF1 has been shown to function as an intermediate to target transcription factors,such as the activated Notch receptor,to specific DNA sites. In this paper,we show that CBF1 from cell lines of different origin is able to bind to the[kappa]B site of the IL-6 promoter. By transfection analyses performed in HeLa cells,we demonstrate that overexpressed CBF1 acts as a negative regulator of IL-6 gene transcription and is unable to elicit Notch-dependent activation of this gene. Analyses of protein-DNA interactions indicate that the topology of the complex formed by CBF1 and the target DNA is subtly affected by sequencessurrounding the recognition site. Furthermore,we show that CBF1 induces DNA bending. This finding suggests that CBF1 may influence IL-6 gene transcription by determining a specific conformation of the promoter region.
Asunto(s)
Proteínas de Unión al ADN/genética , Interleucina-6/genética , Proteínas Nucleares/genética , Regiones Promotoras Genéticas/genética , Secuencia de Bases , Células HeLa , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas , Datos de Secuencia Molecular , Transcripción GenéticaRESUMEN
Molecular evolutionary analyses of mammalian ribonucleases have shown that gene duplication events giving three paralogous genes occurred in ruminant ancestors. The enzymes of the bovine species encoded by these genes, isolated from pancreas, brain and seminal vesicles, present similar enzymological properties but distinct structural features. In other ruminant species, genomic sequences orthologous to the bovine genes of pancreas and brain ribonucleases encode active enzymes. In mammalian species other than ruminant artiodactyls, only one gene encoding ribonuclease of the pancreatic type is generally present. In this work, we describe a differential pattern of transcriptional expression of the pancreas and brain ribonuclease genes in the ox species and report transcription of the human ribonuclease gene in brain as well as in pancreas and in mammary gland. We also report the molecular cloning of the gene encoding the bovine seminal ribonuclease in which the structural organization already described for the two paralogous genes is conserved. The seminal RNAase is exclusively expressed in seminal vesicles of Bos taurus, whereas in other ruminant species, the orthologous sequence is a pseudogene. Previous studies from a number of research groups demonstrated that, unlike other mammalian ribonucleases, the seminal enzyme is a covalent dimer, and its unique quaternary structure correlates with special biological activities. The major determinant of dimer formation, i.e. the presence of two adjacent cysteine residues, is absent in the pseudogenes. We advance the hypothesis that the differentiation of distinct expression patterns could represent an important evolutionary determinant for the genes encoding pancreas and brain ribonucleases in ruminants, whereas the differentiation of a quaternary structure endowed with new biological functions could be the main determinant for the evolutionary success of the seminal gene in the bovine species.
Asunto(s)
Regulación de la Expresión Génica/genética , Ribonucleasas/genética , Rumiantes/metabolismo , Transcripción Genética/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/enzimología , Bovinos , Clonación Molecular , Evolución Molecular , Masculino , Glándulas Mamarias Animales/enzimología , Datos de Secuencia Molecular , Páncreas/enzimología , Polimorfismo de Longitud del Fragmento de Restricción , Conformación Proteica , ARN Mensajero/metabolismo , Ribonucleasas/química , Vesículas Seminales/enzimología , Análisis de Secuencia de ADNRESUMEN
Phylogenetic analysis, based on the primary structures of mammalian pancreatic-type ribonucleases, indicated that gene duplication events, which occurred during the evolution of ancestral ruminants, gave rise to the three paralogous enzymes present in the bovine species. Herein we report data that demonstrate the existence of the orthologues of the bovine pancreatic, seminal, and cerebral ribonucleases coding sequences in the genomes of giraffe and sheep. The "seminal" sequence is a pseudogene in both species. We also report an analysis of the transcriptional expression of ribonuclease genes in sheep tissues. The data presented support a model for positive selection acting on the molecular evolution of ruminant ribonuclease genes.
Asunto(s)
Evolución Molecular , Ribonucleasas/genética , Rumiantes/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Datos de Secuencia Molecular , Alineación de Secuencia , Análisis de SecuenciaRESUMEN
Mammalian pancreatic ribonucleases form a family of homologous proteins that has been extensively investigated. The primary structures of these enzymes were used to derive phylogenetic trees. These analyses indicate that the presence of three strictly homologous enzymes in the bovine species (the pancreatic, seminal, and cerebral ribonucleases) is due to gene duplication events which occurred during the evolution of ancestral ruminants. In this paper we present evidence that confirms this finding and that suggests an overall structural conservation of the putative ribonuclease genes in ruminant species. We could also demonstrate that the sequences related to ox ribonuclease coding regions present in genomic DNA of the giraffe species are the orthologues of the bovine genes encoding the three ribonucleases mentioned above.
Asunto(s)
Bovinos/genética , ADN , Mamíferos/genética , Ribonucleasa Pancreática/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , Datos de Secuencia Molecular , Familia de Multigenes , Especificidad de la EspecieRESUMEN
In this paper we report the molecular cloning of the gene encoding the bovine brain ribonuclease. The nucleotide sequence determined in this work shows a high degree of identity to the homologous gene encoding the bovine pancreatic ribonuclease. Processing of the primary transcripts of these genes also follows a similar pathway, splicing of the unique intron in the 5' untranslated region occurs at corresponding positions. Expression of the bovine brain ribonuclease gene can be detected both at the transcriptional and translational levels in all the regions of the brain examined.