RESUMEN
Viable prokaryotes have been detected in basal sediments beneath the few Northern Hemisphere glaciers that have been sampled for microbial communities. However, parallel studies have not previously been conducted in the Southern Hemisphere, and subglacial environments in general are a new and underexplored niche for microbes. Unfrozen subglacial sediments and overlying glacier ice samples collected aseptically from the Fox Glacier and Franz Josef Glacier in the Southern Alps of New Zealand now have been shown to harbor viable microbial populations. Total direct counts of 2-7 x 10(6) cells g(-1) dry weight sediment were observed, whereas culturable aerobic heterotrophs ranged from 6-9 x 10(5) colony-forming units g(-1) dry weight. Viable counts in the glacier ice typically were 3-4 orders of magnitude smaller than in sediment. Nitrate-reducing and ferric iron-reducing bacteria were detected in sediment samples from both glaciers, but were few or below detection limits in the ice samples. Nitrogen-fixing bacteria were detected only in the Fox Glacier sediment. Restriction fragment analysis of 16S rDNA amplified from 37 pure cultures of aerobic heterotrophs capable of growth at 4 degrees C yielded 23 distinct groups, of which 11 were identified as beta-Proteobacteria. 16S rDNA sequences from representatives of these 11 groups were analyzed phylogenetically and shown to cluster with bacteria such as Polaromonas vacuolata and Rhodoferax antarcticus, or with clones obtained from permanently cold environments. Chemical analysis of sediment and ice samples revealed a dilute environment for microbial life. Nevertheless, both the sediment samples and one ice sample demonstrated substantial aerobic mineralization of 14C-acetate at 8 degrees C, indicating that sufficient nutrients and viable psychrotolerant microbes were present to support metabolism. Unfrozen subglacial sediments may represent a significant global reservoir of biological activity with the potential to influence glacier meltwater chemistry.
Asunto(s)
Bacterias/genética , Sedimentos Geológicos/microbiología , Cubierta de Hielo/microbiología , Bacterias/metabolismo , Secuencia de Bases , Carbono/análisis , Radioisótopos de Carbono , Recuento de Colonia Microbiana , Cartilla de ADN , ADN Ribosómico/genética , Funciones de Verosimilitud , Modelos Genéticos , Datos de Secuencia Molecular , Nueva Zelanda , Filogenia , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADNRESUMEN
Most standard procedures for extracting DNA from forensic substrates involve manipulations that expose the sample to potential contamination and which reduce yields. Furthermore, most methods require centrifugation and/or solvent extraction steps that render them difficult to automate. We describe a simple closed-tube DNA extraction procedure using a proteinase from the thermophilic Bacillus species EA1 that produces good DNA yields from a wide range of forensic substrates. The reaction is controlled by a temperature shift regime programmed into a thermal cycler and so eliminates the need for solvent extraction or column purification. The new method is ideally suited to forensic samples where exposure to extraneous contaminating DNA must be avoided. In addition, The simplicity of the procedure makes it suitable for automation.
Asunto(s)
Dermatoglifia del ADN/métodos , Metaloendopeptidasas/química , Autoanálisis , Bacillus/enzimología , ADN/análisis , ADN/sangre , Cabello/química , Humanos , Mucosa Bucal/química , Saliva/química , Solventes , TemperaturaRESUMEN
Sequence data for 16S rDNA of the type strains of Acidovorax avenae subsp. avenae, A. avenae subsp. cattleyae, A. avenae subsp. citrulli, A. konjaci and Herbaspirillum rubrisubalbicans were compared with GenBank library accessions of Burkholderia spp., Comamonas sp., Ralstonia solanacearum and Variovorax sp. Maximum Parsimony analysis produced two clusters: 1. Acidovorax spp., Comamonas sp., and Variovorax sp. (all in the Comamonadaceae), and 2. Burkholderia spp., Ralstonia solanacearum, and Herbaspirillum rubrisubalbicans. Maximum Likelihood analysis produced only one cluster (of the Comamonadaceae). Using nutritional and laboratory tests, all Acidovorax spp., Burkholderia spp., and Herbaspirillum rubrisubalbicans were discriminated in distinct clusters at the species level, and could be identified by selected determinative tests. There were no phenotypic tests constituted as a circumscription of the genera and which permitted the allocation of strains to genera. Strain identification as species allowed allocation to genera only by inference. The nomenclatural implications of these data are discussed.
Asunto(s)
Burkholderia/clasificación , Burkholderia/genética , Proteobacteria/clasificación , Proteobacteria/genética , Secuencia de Bases , Burkholderia/patogenicidad , Cartilla de ADN/genética , ADN Bacteriano/genética , ADN Ribosómico/genética , Filogenia , Plantas/microbiología , Proteobacteria/patogenicidad , ARN Bacteriano/genética , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Two psychrophilic Clostridium strains, DB1AT and R26, were isolated from incidences of 'blown-pack' spoilage of vacuum-packed chilled lamb. Vacuum packs of meat inoculated with these strains developed gas bubbles and pack distension within 14 d storage at 2 degrees C. The two main gases responsible for pack distension were carbon dioxide and hydrogen. 1-Butanol, butyric and acetic acid and butyl esters were the major volatile compounds produced by the strains in the artificially inoculated packs. The unknown strains were Gram-positive motile rods producing elliptical subterminal spores during the late-stationary growth phase. At pH 7.0, they grew from -1.5 to 26 degrees C, and their optimum growth temperature was 20-22 degrees C. At 20 degrees C, the pH range for growth was 5.4-8.9 and the optimum pH for growth was 6.2-8.6. In peptone/yeast extract broth, the organisms grew little or not at all in the absence of fermentable carbohydrates. Both strains hydrolysed gelatin, aesculin and starch. The fermentation products formed in peptone yeast extract glucose starch broth were ethanol, acetate, butyrate, lactate, butanol, carbon dioxide and hydrogen. The G+C contents of the DNA of strains DB1AT and R26 were 29.4 and 28.3 mol%, respectively. Phylogenetic analyses indicated that the strains belong to cluster I of the genus Clostridium (sensu Collins et al. 1994). The new strains differed from the phylogenetically related clostridia in cellular fatty acid composition, soluble protein profiles and phenotypic properties. On the basis of rDNA analysis and phenotypic and phylogenetic characterization, the strains were assigned to a new species for which the name Clostridium gasigenes is proposed. Strain DB1AT (= DSM 12272T) is designated as the type strain.
Asunto(s)
Clostridium/clasificación , Frío , Manipulación de Alimentos , Embalaje de Alimentos , Carne/microbiología , Composición de Base , Butanoles/metabolismo , Ácido Butírico/metabolismo , Clostridium/aislamiento & purificación , Clostridium/fisiología , Clostridium/ultraestructura , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Fermentación , Gases , Genes de ARNr/genética , Datos de Secuencia Molecular , Fenotipo , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , VacioRESUMEN
A psychrotolerant Clostridium species was isolated from vacuum-packed, temperature-abused raw lamb. Colonies of this micro-organism on sheep-blood agar were circular with an entire margin, grey-white, translucent and beta-haemolytic. Cells were single, tapered, motile rods. Elliptical subterminal spores were produced in the late stationary growth phase. Spores did not cause swelling of the maternal cells. The micro-organism was obligately anaerobic. In peptone yeast extract glucose starch (PYGS) broth at pH 7.0, the micro-organism grew optimally between 25.5 and 30.0 degrees C. The temperature range for growth was 2.5-32.2 degrees C. At 26 degrees C, the micro-organism grew optimally at pH 6.8 to 7.0. The pH range for anaerobic growth was 4.7-9.1. The micro-organism was saccharoclastic, hydrolysed starch and degraded xylan. The fermentation products formed in PYGS broth were acetate, formate, lactate, ethanol, butyrate, butanol, hydrogen and carbon dioxide. The G + C content of the DNA was 38.4 mol%. Phylogenetic analyses indicated that the strain belongs to cluster XIVa of the genus Clostridium (sensu Collins et al. 1994). The new strain differed from phylogenetically related clostridia in terms of cellular fatty acid composition, soluble protein profiles and phenotypic properties. On the basis of phenotypic and genotypic characterization data, the strain was assigned to a new species, namely Clostridium algidixylanolyticum. The type strain is strain SPL73T (= DSM 12273T).
Asunto(s)
Clostridium/clasificación , Frío , Embalaje de Alimentos , Carne/microbiología , Xilanos/metabolismo , Animales , Composición de Base , Clostridium/metabolismo , Clostridium/fisiología , ADN Bacteriano/química , ADN Bacteriano/genética , ADN Ribosómico/química , ADN Ribosómico/genética , Conservación de Alimentos , Concentración de Iones de Hidrógeno , Ratones , Datos de Secuencia Molecular , Fenotipo , Filogenia , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , Ovinos , Esporas Bacterianas/fisiología , VacioRESUMEN
Two genes, xynB and xynC, coding for xylanases were isolated from Thermotoga maritima FjSS3B.1 by a genomic-walking-PCR technique. Sequencing of the genes showed that they encode multidomain family 10 xylanases. Only XynB exhibited activity against xylan substrates. The temperature optimum (87 degrees C) and pH optimum (pH 6.5) of XynB are different from the previously reported xylanase, XynA (also a family 10 enzyme), from this organism. The catalytic domain expressed without other domains has a lower temperature optimum, is less thermostable, and has optimal activity at pH 6.5. Despite having a high level of sequence similarity to xynB, xynC appears to be nonfunctional since its encoded protein did not show significant activity on xylan substrates.
Asunto(s)
Bacterias/enzimología , Genes Bacterianos , Xilosidasas/genética , Xilosidasas/metabolismo , Bacterias/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Southern Blotting , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Temperatura , Microbiología del Agua , Xilano Endo-1,3-beta-XilosidasaRESUMEN
The gene for a highly thermostable neutral proteinase (Npr) was isolated from Bacillus sp. strain EA1 by the polymerase chain reaction using consensus primers based on the sequences of npr genes from related species. The gene was sequenced and shown to be closely related to a neutral proteinase gene from Bacillus caldolyticus strain YP-T; the mature form of the enzyme differing by only a single amino acid. Enzyme samples were prepared from both the native organisms and also from recombinant Escherichia coli expressing the two npr genes. The proteinase from strain EA1 was shown to be significantly more thermostable than that from B. caldolyticus and that this difference is the result of a single amino acid substitution which is situated proximal to a region of the enzyme known to be crucial to conferring thermal stability. The phylogenetic relationship of EA1 to other Bacilli is also described.
Asunto(s)
Bacillus/genética , Proteínas Bacterianas/genética , Genes Bacterianos , Metaloendopeptidasas/genética , Secuencia de Aminoácidos , Bacillus/clasificación , Bacillus/enzimología , Clonación Molecular , Estabilidad de Enzimas , Escherichia coli/genética , Calor , Concentración de Iones de Hidrógeno , Metaloendopeptidasas/biosíntesis , Datos de Secuencia Molecular , Filogenia , Proteínas Recombinantes/biosíntesis , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
A genomic library of the extremely thermophilic eubacterial strain Rt8B.4 was constructed in lambda ZapII and screened for the expression of xylanase activity. One recombinant bacteriophage showed xylanase, xylosidase and arabinosidase activity. Sequence analysis and homology comparisons showed that this plasmid derivative, pNZ2011, was composed of 6.7 kb thermophilic DNA and contained what appeared to be an operon-like structure involving genes associated with xylose metabolism. The xylanase gene, xynA was shown to code for a multi-domain protein. Xylanase activity was shown to be associated with the carboxy-terminal domain (domain 2) by deletion analysis and also by selective polymerase chain reaction (PCR) amplification and expression of the individual domains. Denaturing polyacrylamide gel analysis of the protein encoded by the PCR product showed three main overexpressed proteins to be present in cell extracts, presumably caused by proteolytic degradation in the Escherichia coli host. The xylanase activity from domain 2 is associated with a 36-kDa protein, which is stable at 70 degrees C for at least 12 h at pH 7. The small size of this active enzymatic domain and its temperature stability suggest that it may be of value in the enzyme-enhanced bleaching of kraft pulp.
Asunto(s)
Bacterias Anaerobias/enzimología , Bacterias Anaerobias/genética , Escherichia coli/genética , Genes Bacterianos , Xilosidasas/genética , Secuencia de Aminoácidos , Bacterias Anaerobias/clasificación , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , ADN Bacteriano/genética , Expresión Génica , Biblioteca Genómica , Datos de Secuencia Molecular , Estructura Molecular , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Xilano Endo-1,3-beta-Xilosidasa , Xilosidasas/químicaRESUMEN
A gene expressing xylanase activity was isolated from a genomic library of Thermotoga sp. strain FjSS3-B.1. The sequence of the gene shows that it encodes a single domain, family 10 xylanase. The recombinant enzyme has extremely high thermal stability, activity over a relatively broad pH range, and activity on Pinus radiata kraft pulp.
Asunto(s)
Genes Bacterianos , Bacterias Anaerobias Gramnegativas/enzimología , Bacterias Anaerobias Gramnegativas/genética , Xilosidasas/genética , Xilosidasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Biotecnología , ADN Bacteriano/genética , Estabilidad de Enzimas , Expresión Génica , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Papel , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura , Xilano Endo-1,3-beta-XilosidasaRESUMEN
The extreme thermophile Thermus sp. strain Rt41A produces an extracellular alkaline serine proteinase during growth. This enzyme is stable for more than 24 h at 70 degrees C and has a pH optimum of 8.0. The proteinase gene was identified using primers designed to amplify a region between two highly conserved amino acid motifs in subtilisin-like proteinases and the PCR product was used to identify a genomic fragment containing the gene. The amino acid sequence deduced from the Rt41A gene contained a region identical to that obtained by amino-terminal sequencing of purified Rt41A proteinase. Comparison of the entire derived peptide sequence with other subtilisin-like serine proteinases revealed significant homologies, especially with aqualysin I from Thermus aquaticus YT-1 and with exoprotease A from Vibrio alginolyticus. The Rt41A proteinase was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase as an aid for purification and to overcome difficulties experienced with other plasmid vectors which produced inactive protein. The enzyme is inactive as synthesized and activation was shown to be temperature-dependent, with shorter incubation times required at higher temperatures; removal of the hydrophobic signal peptide from the start of the gene reduced the time required for activation to less than a third of that required if the signal peptide was present.
Asunto(s)
Genes Bacterianos , Serina Endopeptidasas/genética , Thermus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Quelantes/química , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Estabilidad de Enzimas , Escherichia coli/metabolismo , Datos de Secuencia Molecular , Fluoruro de Fenilmetilsulfonilo/química , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/biosíntesis , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/química , Serina Endopeptidasas/aislamiento & purificación , Serina Endopeptidasas/metabolismo , Subtilisinas/genética , Temperatura , Factores de TiempoRESUMEN
The celA, manA, and celB genes from Caldocellulosiruptor saccharolyticus compose a cellulase-hemicellulase gene cluster and are arranged on a 12-kb C. saccharolyticus genomic fragment of the recombinant lambda bacteriophage NZP lambda 2. The beginning of a fourth open reading frame (celC) which was homologous to the C. saccharolyticus manA and celA genes was located at the 3' end of the 12-kb NZP lambda 2 genomic fragment. Genome-walking PCR was used to isolate DNA fragments downstream of the C. saccharolyticus celB gene, and the entire nucleotide sequence of celC was obtained. From the preliminary nucleotide sequence, celC appeared to encode yet another multidomain bifunctional enzyme (CelC) consisting of an N-terminal endo-1,4-beta-D-glucanase domain (75% similar to CelA domain 1), two central cellulose-binding domains, and a C-terminal endo-1,4-beta-D-mannanase domain (98% similar to ManA domain 1). However, upon completion of the celC sequencing, two -1 frameshifts were identified in the region encoding the putative CelC mannanase domain. The isolated CelC mannanase domain exhibited no beta-mannanase activity, which supported this observation. Recombinant PCR was used to correct the celC frameshifts by inserting the appropriate nucleotides into the gene. The repaired celC fragment containing the base insertions (manB) expressed strong beta-mannanase activity on soluble mannan substrates and showed significant activity on kraft pulp as judged by the release of reducing sugars.
Asunto(s)
Proteínas Bacterianas , Geobacillus stearothermophilus/genética , Manosidasas/genética , beta-Glucosidasa/genética , Secuencia de Bases , Mapeo Cromosómico , Activación Enzimática , Mutación del Sistema de Lectura , Geobacillus stearothermophilus/enzimología , Manosidasas/biosíntesis , Datos de Secuencia Molecular , Seudogenes/genética , Alineación de Secuencia , beta-Glucosidasa/biosíntesis , beta-ManosidasaRESUMEN
Caldocellum saccharolyticum is an extremely thermophilic anaerobic bacterium capable of growth on cellulose and hemicellulose as sole carbon sources. Cellulase and hemicellulase genes have been found clustered together on its genome. The gene for one of the cellulases (celA) was isolated on a lambda genomic library clone, sequenced and found to comprise a large open-reading frame of 5253 base pairs that could be translated into a peptide of 1751 amino acids. To date, it is the largest cellulase gene sequenced. The translated product is a multidomain structure composed of two catalytic domains and two cellulose-binding domains linked by proline-threonine-rich regions (PT linkers). The N-terminal domain of celA encodes for an endoglucanase activity on carboxymethylcellulose, consistent with its high homology to the sequences of several other endo-1, 4-beta-D-glucanases. The carboxyterminal domain shows sequence homology with a cellulase from Clostridium thermocellum (CelS), which is known to act synergistically with a second component to hydrolyze crystalline cellulose. In the absence of a Caldocellum homologue for this second protein, we can detect no activity from this domain.
Asunto(s)
Bacterias Anaerobias/genética , Celulasa/genética , Celulosa/metabolismo , Genes Bacterianos/genética , Complejos Multienzimáticos/genética , Secuencia de Aminoácidos , Bacterias Anaerobias/enzimología , Secuencia de Bases , Celulasa/metabolismo , Biblioteca Genómica , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismo , Conformación Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Especificidad por SustratoRESUMEN
The gene for a serine proteinase from a thermophilic Bacillus species was identified by PCR amplification, and the complete gene was cloned after identification and isolation of suitably sized restriction fragments from Southern blots by using the PCR product as a probe. Two additional, distinct PCR products, which were shown to have been derived from other serine proteinase genes present in the thermophilic Bacillus species, were also obtained. Sequence analysis showed an open reading frame of 1,206 bp, coding for a polypeptide of 401 amino acids. The polypeptide was determined to be an extracellular serine proteinase with a signal sequence and prosequence. The mature proteinase possessed homology to the subtilisin-like serine proteinases from a number of Bacillus species and had 61% homology to thermitase, a serine proteinase from Thermoactinomyces vulgaris. The gene was expressed in Escherichia coli in the expression vector pJLA602 and as a fusion with the alpha-peptide of the lacZ gene in the cloning vector pGEM5. A recombinant proteinase from the lacZ fusion plasmid was used to determine some characteristics of the enzyme, which showed a pH optimum of 8.5, a temperature optimum of 75 degrees C, and thermostabilities ranging from a half-life of 12.2 min at 90 degrees C to a half-life of 40.3 h at 75 degrees C. The enzyme was bound to a bacitracin column, and this method provided a simple, one-step method for producing the proteinase, purified to near homogeneity.
Asunto(s)
Bacillus/enzimología , Genes Bacterianos/genética , Serina Endopeptidasas/genética , Secuencia de Aminoácidos , Bacillus/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Escherichia coli/genética , Regulación Enzimológica de la Expresión Génica , Vectores Genéticos , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/biosíntesis , Serina Endopeptidasas/aislamiento & purificación , TemperaturaRESUMEN
The SSU rRNA gene of the extremely thermophilic bacterium strain AZ3 B.1, encodes an rRNA containing four large inserts. A secondary structure model has been constructed which predicts that the inserts form large stem loop structures with a common sequence motif at the base of the helices. To date, these structures have only been detected in related, thermophilic organisms.
Asunto(s)
Bacterias Anaerobias/genética , ARN Ribosómico/química , Secuencia de Bases , Datos de Secuencia Molecular , Estructura MolecularRESUMEN
The sequences of the 16S rRNA genes of 20 Thermus isolates were determined to a high fidelity by using automated DNA sequencing and fluorescent-dye-labelled primers. The strains tested included members of the three validly named Thermus species and representatives of major taxonomic clusters defined previously for this genus. The parsimony method was used to reconstruct the phylogeny of the strains from the aligned sequences, and a bootstrap analysis revealed a number of well-supported clades. Our results are not consistent with groupings inferred from numerical taxonomy data but support the conjecture that the genus Thermus contains more species than the three currently recognized species.
Asunto(s)
ADN Ribosómico/genética , Genes Bacterianos , ARN Ribosómico 16S/genética , Thermus/clasificación , Thermus/genética , Secuencia de Bases , Clonación Molecular , Secuencia de Consenso , Cartilla de ADN , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Homología de Secuencia de Ácido NucleicoRESUMEN
The complete sequence of a beta-mannanase gene from an anaerobic extreme thermophile was determined, and it shows that the expressed protein consists of two catalytic domains and two binding domains separated by spacer regions rich in proline and threonine residues. The amino-terminal catalytic domain has beta-mannanase activity, and the carboxy-terminal domain acts as an endoglucanase. Neither domain shows homology with any other cellulase or hemicellulase sequence at the nucleic acid or protein level.
Asunto(s)
Bacterias Anaerobias/enzimología , Bacterias Anaerobias/genética , Manosidasas/genética , Complejos Multienzimáticos/genética , Secuencia de Aminoácidos , Secuencia de Bases , Celulasa/genética , ADN Bacteriano/genética , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , beta-ManosidasaRESUMEN
"Caldocellum saccharolyticum" is an obligatory anaerobic thermophilic bacterium. A gene from this organism, designated celB, has been cloned in Escherichia coli as part of a bacteriophage lambda gene library. This gene produces a thermostable cellulase that shows both endoglucanase and exoglucanase activities on test substrates and is able to degrade crystalline cellulose to glucose. The sequence of celB has homology with both exo- and endoglucanases described by others. It appears to have a central domain without enzymatic activity which is joined to the enzymatic domains by runs of amino acids rich in proline and threonine (PT boxes). Deletion analysis shows that the exoglucanase activity is located in the amino-terminal domain of the enzyme and that endoglucanase activity is located in the carboxy-terminal domain. There are internal transcriptional and translational start sites within the gene. The intact gene has been cloned into a temperature-inducible expression vector, pJLA602, and overexpressed in E. coli. Polyacrylamide gel electrophoresis showed that celB produced a protein with a molecular weight of 118,000 to 120,000. A number of smaller proteins with activity against carboxymethyl cellulose and 4-methyl umbelliferyl-beta-D-cellobioside were also produced. These are believed to be the result of alternative translational start sites and/or proteolytic degradation products of the translated gene product.
Asunto(s)
Bacterias Anaerobias/enzimología , Proteínas Bacterianas/genética , Celulasa/genética , Genes Bacterianos , beta-Glucosidasa/genética , Secuencia de Aminoácidos , Bacterias Anaerobias/genética , Proteínas Bacterianas/aislamiento & purificación , Celulasa/aislamiento & purificación , Glucano 1,3-beta-Glucosidasa , Datos de Secuencia Molecular , Homología de Secuencia de Ácido Nucleico , Especificidad por Sustrato , beta-Glucosidasa/aislamiento & purificaciónRESUMEN
The WHI2 gene of Saccharomyces cerevisiae plays a key role in coordinating cell proliferation and nutrient availability. A 2.6 kb yeast DNA sequence has been cloned which fully suppresses the whi2 mutation. Integration of this sequence to demonstrate that the structural gene itself had been cloned proved difficult. Integration occurred only rarely and only at the LEU2 locus which was also present on the integrating plasmid. To circumvent these difficulties an adjacent sequence, present on the original isolate from the gene library, was subcloned onto the integrating vector YIp5, after which directed integration proved straightforward. The integrated sequence was closely linked to WHI2, confirming that the structural gene had been cloned. A chromosomal restriction map of the WHI2 region is presented; no gross changes were observed in the region as cells entered stationary phase.