RESUMEN
The soil nematode Caenorhabditis elegans is an excellent research model in cell biology, human disease and developmental studies. In this study, a novel cryopreservation technique based on a rapid cooling procedure, previously established for juveniles, was applied to adult-worms. Here we demonstrated that adults of C. elegans, a complex metazoan organism, survive to a rapid cooling and storage in liquid nitrogen (-196 °C) with a very high survival percentage (85%). The procedure relies on a Low CryoProtectant Technique (LCPT) and Ultra Rapid Cooling (URC). The high cooling rate is achieved through the reduction of sample volumes and the effectiveness of a nylon carrier. Our technique complies with the requirements for vitrification to occur. The main distinctive characters of this cryopreservation technique compared to other methods, e.g. Slow Freezing and Vitrification, are presented. Our results show that this cryopreservation method is valid for both unicellular and multicellular organisms; it is suitable for short or long time storage in liquid-nitrogen. This technique promises to be a unique and simpler method for cryostorage of cells, organisms and tissues.
Asunto(s)
Criopreservación/métodos , Vitrificación , Animales , Caenorhabditis elegans , Crioprotectores/farmacología , Congelación , HumanosRESUMEN
A 7-kb fragment of Streptomyces rochei A2 chromosomal DNA was cloned into pAT153 and shown to confer endoglucanase (EglS) activity on Escherichia coli cells. In E. coli clones, the EglS was secreted into the periplasm. Deletion analysis revealed that an 827-bp fragment was enough for the enzymatic activity. Sequence analysis showed that the 827-bp fragment codes for the catalytic domain of the enzyme. The complete sequence of the gene (eglS) is 1149-bp long. A signal peptide, a catalytic domain and a cellulose-binding domain were identified from the nucleotide sequence, and the EglS found to belong to the family H of cellulase catalytic domains. These conclusions were substantiated by determination of the N-terminal sequence of the purified protein and zymogram analysis, which revealed protein species with a molecular mass equal to that deduced from the nt sequence analysis.
Asunto(s)
Celulasa/genética , Genes Bacterianos/genética , Streptomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Celulasa/biosíntesis , Celulasa/química , Celulasa/metabolismo , Clonación Molecular , Escherichia coli/genética , Expresión Génica , Datos de Secuencia Molecular , Peso Molecular , ARN Bacteriano/análisis , ARN Mensajero/análisis , Alineación de Secuencia , Análisis de Secuencia de ADN , Eliminación de Secuencia/fisiología , Streptomyces/enzimologíaRESUMEN
The dihydrofolate reductase gene from Candida albicans has been cloned and partially characterized. A genomic bank from C. albicans strain 10127/5 was constructed in Escherichia coli and screened for trimethoprim resistance. A plasmid pMF1, carrying the resistance marker was isolated and characterized by restriction mapping and Southern blotting. Cells harbouring pMF1 were as sensitive as the parental cells to a wide spectrum of antibacterial agents, except for trimethoprim; the dihydrofolate reductase activity from these cells was trimethoprim resistant.
Asunto(s)
Candida albicans/genética , Clonación Molecular , Tetrahidrofolato Deshidrogenasa/genética , Farmacorresistencia Microbiana/genética , Plásmidos , Trimetoprim/farmacologíaAsunto(s)
Celulasa/metabolismo , Streptomyces/enzimología , Celulasa/biosíntesis , Celulasa/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Clonación Molecular/métodos , Estabilidad de Enzimas , Enzimas Inmovilizadas/metabolismo , Escherichia coli , Genes Bacterianos , Calor , Cinética , Peso Molecular , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Streptomyces/genética , TermodinámicaRESUMEN
Streptomyces rochei A2 endoglucanase (eglS) and beta-glucosidase (bgs1) genes were used as probes to survey their distribution among 16 Streptomyces strains isolated from different sources and characterized for their cellulolytic activities. The eglS probe hybridized to the genomic DNA of 12 strains with a restriction pattern different from that of S. rochei A2. The DNA from all strains, except one, hybridized with the bgs1 probe and one strain showed the same restriction pattern as seen in S. rochei A2. The sequence localized by the eglS probe in S. thermoviolaceus and the one localized by the bgs1 probe in strain EC1 were cloned and expressed in E. coli in plasmids pTAE and pCSF203, respectively. The restriction maps showed that the cloned genes were identical to eglS and bgs1. The restriction enzyme analysis and genomic DNA from all the strains identified nine different groups, each characterized by a distinctive pattern and in agreement with the results of the hybridization experiments.
Asunto(s)
ADN Bacteriano/análisis , Genoma Bacteriano , Streptomyces/genética , ADN Bacteriano/genética , Mapeo Restrictivo , Streptomyces/aislamiento & purificaciónRESUMEN
A new binary vector encoding for Candida albicans dihydrofolate reductase (DFR1) has been constructed and used as a dominant selectable marker for plant transformation. Transgenic tobacco plants with an increased resistance to methotrexate (Mtx) were obtained by co-transformation of tobacco leaf discs with Agrobacterium tumefaciens strains carrying two new binary vectors: pTI20 and pTI18. Co-transformants of Nicotiana tabacum were directly selected for and rooted on medium containing both kanamycin (kan) and Mtx. Leaf discs of transgenic plants were assayed for capacity of regeneration at different Mtx concentrations. Analysis of transcripts was performed on total RNA extracted from two Mtx-resistant plants. The transgenic plants increased resistance to Mtx can be explained by the exceptionally low capacity of Mtx to bind C. albicans dihydrofolate reductase, accountable by the presence of two amino acid residues strategically important in Mtx binding.