RESUMEN
We have isolated and characterized a novel cDNA, C1q-Related Factor (CRF), that is predicted to encode a 258 amino acid polypeptide with a hydrophobic signal sequence, a collagenous region, and a globular domain at the carboxy terminus that shares homology to the C1q signature domain. Human CRF transcript is expressed at highest levels in the brain, particularly in the brainstem. In situ hybridization to mouse brain sections demonstrated that CRF transcripts are most abundant in areas of the nervous system involved in motor function, such as the Purkinje cells of the cerebellum, the accessory olivary nucleus, the pons and the red nucleus. The mouse CRF homolog is highly similar to the human gene at both the nucleotide and protein level, suggesting an important conserved role for this protein.
Asunto(s)
Química Encefálica/fisiología , Activación de Complemento , Complemento C1q/aislamiento & purificación , Actividad Motora/fisiología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Humanos , Ratones , Datos de Secuencia Molecular , Biosíntesis de Proteínas , ARN/biosíntesis , Homología de Secuencia de AminoácidoRESUMEN
An insertion sequence, designated as IST3091, was located adjacent to the putative origin of replication region of plasmid pTFI91 of Thiobacillus ferrooxidans TFI-91. The DNA sequence of the transposase gene of IST3091 revealed similarity with that of IS30, IS1086, IS4351, and the integrase gene of SpV1-R8A2 B (a bacteriophage of Spiroplasma citri). The sequence of IST3091 is 1063 bp long with partially matched 30-bp terminal inverted repeats. Several restriction fragments of plasmid pTFI91 of T. ferrooxidans containing the IST3091 element were cloned into the vector pHSG398. The hybrid plasmids (pBTL) were transformed into Escherichia coli NK7379 containing a miniF plasmid, which was devoid of transposable elements. The transposition function of the IST3091 element was confirmed by mobilizing hybrid plasmids via conjugation from transformed E. coli NK7379 (donor) to E. coli M8820 (recipient). The presence of the transposed element in transconjugants was detected by polymerase chain reaction amplification.
Asunto(s)
Elementos Transponibles de ADN/genética , Thiobacillus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Conjugación Genética , Cartilla de ADN/genética , ADN Bacteriano/genética , Escherichia coli/genética , Genes Bacterianos , Genoma Bacteriano , Datos de Secuencia Molecular , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Secuencias Repetitivas de Ácidos Nucleicos , Origen de Réplica/genética , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido NucleicoRESUMEN
A promoter probe shuttle vector suitable for the isolation of promoter elements from coryneform bacteria was constructed. This vector carried the neomycin phosphotransferase (NPTII) gene from transposon Tn5 as a reporter gene, and was capable of replication in both Escherichia coli and Brevibacterium flavum. The vector was used in the construction of a B. flavum library of 899 independently isolated promoter clones. Promoters with a wide range of activities in B. flavum, including some very strong promoter elements, were isolated. Comparative analysis suggests that significant differences between B. flavum and E. coli may exist in the determinants of promoter strength.
Asunto(s)
Brevibacterium/genética , Escherichia coli/genética , Regiones Promotoras Genéticas , Secuencia de Bases , Biotecnología , ADN Recombinante/genética , Escherichia coli/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica , Genes Reporteros , Ingeniería Genética , Vectores Genéticos , Resistencia a la Kanamicina/genética , Datos de Secuencia Molecular , Transformación GenéticaRESUMEN
Plasmids found in six strains of Thiobacillus ferrooxidans were mapped and compared in an effort to detect the origin of replication. Four strains yielded an identical 9.8-kb plasmid, pTFI91. Restriction mapping and Southern blot hybridization analysis were used to confirm this finding. Dissimilar plasmids found in two other strains contained a conserved 2.2-kb SacI region common to pTFI91. DNA sequence analysis of this region showed structural features common to bacterial plasmid replicons. A comparison of the pTFI91 origin with those of T. ferrooxidans pTF-FC2 and other broad host range vectors did not show significant homologous DNA sequences. To verify the replication function, a chloramphenicol acetyl transferase marker gene was ligated at the unique sites of pTFI91, and the plasmid was transformed into Escherichia coli DH5 alpha cells but no transformants were identified. To test the replication of pTFI91 independent of DNA polymerase I in E. coli, different restriction fragments of pTFI91 were cloned into pHSG398 (Cmr, ColEI origin) and transformed into the polA1 mutant SF800, but chloramphenicol-resistant transformants were not detected. Electrotransformation of T. ferrooxidans TFI-70 and Pseudomonas putida ATCC 19151 also failed to yield transformants. The results suggested that the pTFI91 plasmid replicon does not function either in E. coli or in P. putida. Since pTFI91 contains the same origin of replication as other plasmids in several other T. ferrooxidans strains, this replicon may be commonly distributed in T. ferrooxidans.
Asunto(s)
Plásmidos/genética , Origen de Réplica , Replicón , Thiobacillus/genética , Secuencia de Bases , Southern Blotting , Cloranfenicol O-Acetiltransferasa/genética , Cloranfenicol O-Acetiltransferasa/metabolismo , ADN Polimerasa I/genética , ADN Polimerasa I/metabolismo , ADN Bacteriano/genética , Escherichia coli/genética , Datos de Secuencia Molecular , Pseudomonas putida/genética , Mapeo Restrictivo , Transformación BacterianaRESUMEN
Neurospora mitochondrial tyrosyl-tRNA synthetase (mt tyrRS), which is encoded by nuclear gene cyt-18, functions in splicing of group I introns in mitochondria. Here, we overproduced functional cyt-18 protein in Escherichia coli and purified it to near homogeneity. The purified protein has splicing and tyrRS activities similar to those of cyt-18 protein isolated from mitochondria and is by itself sufficient to splice the mitochondrial large rRNA intron in vitro. Structure-function relationships in the cyt-18 protein were analyzed by in vitro mutagenesis. We confirmed that a small amino-terminal domain not found in bacterial tyrRSs is required for splicing activity, but not tyrRS activity. Two linker insertion mutations, which disrupt the predicted ATP-binding site, completely inhibit tyrRS activity but leave substantial splicing activity. Finally, deletions or linker insertion mutations in the putative carboxy-terminal tRNA-binding domain inhibit both tyrRS and splicing activities, although some have differential effects on the two activities. Our results show that the normal catalytic activity of the cyt-18 protein is not required for splicing and are consistent with the hypothesis that the protein functions by binding to the precursor RNA and facilitating formation of the correct RNA structure. Regions required for splicing are distributed throughout the cyt-18 protein and overlap, but are not identical to, regions required for tyrRS activity. The finding that the putative carboxy-terminal tRNA-binding domain is required for both tyrRS and splicing activities suggests that the mechanism for binding the intron has similarities to the mechanism for binding tRNA(Tyr).
Asunto(s)
Intrones , Mitocondrias/enzimología , Neurospora crassa/genética , Empalme del ARN , ARN de Transferencia/metabolismo , Tirosina-ARNt Ligasa/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN de Hongos , Genes Fúngicos , Datos de Secuencia Molecular , Mutagénesis Insercional , Neurospora crassa/enzimología , ARN de Hongos/metabolismo , Mapeo Restrictivo , Tirosina-ARNt Ligasa/genéticaRESUMEN
Neurospora mitochondrial tyrosyl-tRNA synthetase (mt TyrRS), which is encoded by nuclear gene cyt-18, functions in splicing group I introns. Analysis of intragenic partial revertants of the cyt-18-2 mutant and in vitro mutants of the cyt-18 protein expressed in E. coli showed that splicing activity of the cyt-18 protein is dependent on a small N-terminal domain that has no homolog in bacterial or yeast mt TyrRSs. This N-terminal splicing domain apparently acts together with other regions of the protein to promote splicing. Our findings support the hypothesis that idiosyncratic sequences in aminoacyl-tRNA synthetase may function in processes other than aminoacylation. Furthermore, they suggest that splicing activity of the Neurospora mt TyrRs was acquired after the divergence of Neurospora and yeast, and they demonstrate one mechanism whereby splicing factors may evolve from cellular RNA binding proteins.
Asunto(s)
Aminoacil-ARNt Sintetasas/genética , Mitocondrias/fisiología , Neurospora crassa/genética , Neurospora/genética , Empalme del ARN , Tirosina-ARNt Ligasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Genes Fúngicos , Datos de Secuencia Molecular , Peso Molecular , Mutación , Fenotipo , Conformación Proteica , Análisis Espectral , Relación Estructura-Actividad , Tirosina/fisiologíaRESUMEN
Transposition of insertion sequence IS10 is regulated by an anti-sense RNA which inhibits transposase expression when IS10 is present in multiple copies per cell. The anti-sense RNA (RNA-OUT) consists of a stem domain topped by a flexibly paired loop; the 5' end of the target molecule, RNA-IN, is complementary to the top of the loop, and complementarity extends for 35 base-pairs down one side of RNA-OUT. We present here genetic evidence that anti-sense pairing, both in vitro and in vivo, initiates by interaction of the 5' end of RNA-IN and the loop domain of RNA-OUT; other features of the reaction are discussed. In the context of this model, we discuss features of this anti-sense system which are important for its biological effectiveness, and suggest that IS10 provides a convenient model for design of efficient artificial anti-sense RNA molecules.
Asunto(s)
Elementos Transponibles de ADN , Nucleotidiltransferasas/metabolismo , ARN Bacteriano/genética , ARN/genética , Escherichia coli/genética , Enlace de Hidrógeno , Técnicas In Vitro , Hibridación de Ácido Nucleico , ARN/ultraestructura , ARN sin Sentido , ARN Bacteriano/ultraestructura , Ribonucleasas/metabolismo , Relación Estructura-Actividad , TransposasasRESUMEN
The effect of phenothiazine and 11 of its derivatives on the mutagenicity of benzo[a]pyrene, as measured by the Ames test was investigated. Significant anti-mutagenic activity was detected for 10 phenothiazine derivatives, with the 2-chloro derivative being the most effective inhibitor tested and promazine the only phenothiazine drug tested which has no demonstrable inhibitory activity. It is considered that the anti-mutagenic activity and therefore potentially anticarcinogenic activity of these derivatives should be of interest to epidemiologists.