RESUMEN
A branching enzyme (EC 2.4.1.18) gene was isolated from an extremely thermophilic bacterium, Rhodothermus obamensis. The predicted protein encodes a polypeptide of 621 amino acids with a predicted molecular mass of 72 kDa. The deduced amino acid sequence shares 42-50% similarity to known bacterial branching enzyme sequences. Similar to the Bacillus branching enzymes, the predicted protein has a shorter N-terminal amino acid extension than that of the Escherichia coli branching enzyme. The deduced amino acid sequence does not appear to contain a signal sequence, suggesting that it is an intracellular enzyme. The R. obamensis branching enzyme was successfully expressed both in E. coli and a filamentous fungus, Aspergillus oryzae. The enzyme showed optimum catalytic activity at pH 6.0-6.5 and 65 degrees C. The enzyme was stable after 30 min at 80 degrees C and retained 50% of activity at 80 degrees C after 16 h. Branching activity of the enzyme was higher toward amylose than toward amylopectin. This is the first thermostable branching enzyme isolated from an extreme thermophile.
Asunto(s)
Enzima Ramificadora de 1,4-alfa-Glucano/metabolismo , Bacterias/enzimología , Enzima Ramificadora de 1,4-alfa-Glucano/química , Enzima Ramificadora de 1,4-alfa-Glucano/genética , Secuencia de Aminoácidos , Aspergillus oryzae/enzimología , Aspergillus oryzae/genética , Bacterias/genética , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/genética , Estabilidad de Enzimas , Escherichia coli/enzimología , Escherichia coli/genética , Genes Bacterianos , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Peso Molecular , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoAsunto(s)
Cisteína Endopeptidasas/metabolismo , Diabetes Mellitus Tipo 1/genética , Complejos Multienzimáticos/metabolismo , Proteínas/genética , Animales , Cisteína Endopeptidasas/genética , Diabetes Mellitus Tipo 1/metabolismo , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Ratones , Ratones Endogámicos NOD , Complejos Multienzimáticos/genética , Complejo de la Endopetidasa Proteasomal , Proteínas/metabolismoRESUMEN
Cenococcum geophilum is an ecologically important mycorrhizal fungus with a global distribution and a wide host range. It has been difficult to study since it forms only sterile mycelia and, occasionally, sclerotial bodies. Because of its lack of morphological variability, its taxonomy and phylogenetic origins have until recently remained unclear. To better understand the genetic variation and environmental adaptability of C. geophilum, a molecular phylogeny was constructed based on the nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) of 69 isolates from various hosts and habitats. The results suggest DNA sequence conservation in the ITS regions. Considering its broad geographic and host range, this ITS conservation was unexpected. Our data imply that the ITS2 region is under evolutionary pressure to maintain the RNA secondary structure (similar to the pressure on the CgSSU introns) involved in the post-transcriptional processing of rRNA. Also, C. geophilum has very short ITS regions, thus limiting the number of mutable sites. This limited ITS variability suggests a recent radiation of C. geophilum, having been geographically distributed by a variety of efficient processes. C. geophilum appears to be a single taxonomic entity, possibly a single species. Therefore, it is an extremely adaptable, as well as ecologically valuable, taxon.
Asunto(s)
ADN Ribosómico/genética , Variación Genética , Hongos Mitospóricos/genética , Filogenia , Secuencia de Bases , Evolución Molecular , Genética de Población , Intrones , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN de Hongos/química , Mapeo Restrictivo , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
Circadian clocks function to govern a wide range of rhythmic activities in organisms. An integral part of rhythmicity is the daily control of target genes by the clock. Here we describe the sequence and analysis of a novel clock-controlled gene, ccg-7, showing similarity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic enzyme widely used as a constitutive control in a variety of systems. That ccg-7 encodes GAPDH was confirmed by demonstrating that in vitro synthesized CCG-7 possesses GAPDH activity. Rhythms in both ccg-7 mRNA accumulation and CCG-7 (GAPDH) activity are observed in a clock wild-type strain where the peak in GAPDH activity lags several hours behind the peak in ccg-7 mRNA accumulation in the late night. Together with our previous observation that ccg-7 mRNA is not developmentally regulated, we show that ccg-7 is not induced by environmental stresses such as glucose or nitrogen deprivation (which also trigger development), heat shock, or osmotic stress. Thus, the finding that GAPDH is clock-regulated points to a specific role for the circadian clock in controlling aspects of general metabolism and provides evidence for circadian regulation of a gene found in most living organisms.
Asunto(s)
Ritmo Circadiano , Regulación Enzimológica de la Expresión Génica/fisiología , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Gliceraldehído-3-Fosfato Deshidrogenasas/metabolismo , Humanos , Datos de Secuencia Molecular , Neurospora crassa/enzimología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
An endogenous circadian biological clock controls the temporal aspects of life in most organisms, including rhythmic control of genes involved in clock output pathways. In the fungus Neurospora crassa, one pathway known to be under control of the clock is asexual spore (conidia) development. To understand more fully the processes that are regulated by the N. crassa circadian clock, systematic screens were carried out for genes that oscillate at the transcriptional level. Time-of-day-specific cDNA libraries were generated and used in differential screens to identify six new clock-controlled genes (ccgs). Transcripts specific for each of the ccgs preferentially accumulate during the late night to early morning, although they vary with respect to steady-state mRNA levels and amplitude of the rhythm. Sequencing of the ends of the new ccg cDNAs revealed that ccg-12 is identical to N. crassa cmt encoding copper metallothionein, providing the suggestion that not all clock-regulated genes in N. crassa are specifically involved in the development of conidia. This was supported by finding that half of the new ccgs, including cmt(ccg-12), are not transcriptionally induced by developmental or light signals. These data suggest a major role for the clock in the regulation of biological processes distinct from development.
Asunto(s)
Ritmo Circadiano/genética , Regulación Fúngica de la Expresión Génica , Neurospora crassa/fisiología , Mapeo Cromosómico , Cromosomas Fúngicos , ADN Complementario , Biblioteca de Genes , Ligamiento Genético , Genoma Fúngico , Neurospora crassa/genética , Plásmidos , Polimorfismo de Longitud del Fragmento de Restricción , ARN de Hongos/biosíntesis , ARN Mensajero/biosíntesis , Esporas Fúngicas , Transcripción GenéticaRESUMEN
A family of optional group-I introns was found near the 3' end of the nuclear small subunit rRNA genes in 61 out of 70 isolates of the deuteromycete mycorrhizal fungus Cenococcum geophilum. DNA sequence polymorphisms among the introns (termed CgSSU introns) from ten of the isolates were studied. The sequences, ranging in size from 488 to 514 nucleotides, were from 93.2% to 99.6% similar to each other. Mutations were less common in predicted base-paired regions (33% of all mutations) than in free-standing regions (67%). The introns were self-spliced in vitro and were closest to subgroup IC1 according to sequence and predicted secondary structure. Group-I intron pairing regions P1 through P10, including core regions P, Q, R and S, were present in all ten CgSSU introns studied. No lengthy open reading frames were found in any of the introns, indicating that the introns do not encode a protein, and therefore may not be mobile. It is likely that a single intron entered a progenote of C. geophilum and changed as the species evolved.