Estandarización de un protocolo sencillo para la extracción de ADN genómico de levaduras / Standardising a simple protocol for extracting yeast from genomic DNA

Se estandarizó un protocolo rápido, sencillo y de bajo costo para la extracción de ADN genómico de levaduras a partir de lisis de la pared celular mediante tratamiento enzimático y precipitación por alcoholes. El empleo de la enzima Beta-glucoronidasa en reemplazo de la enzima Zimolasa, permitió obtener ADN en alta concentración (124,9±30,2 ng/λl) y de buena calidad (A260/A280 nm =1,86±0,1), ideal para su uso en estudios de biología molecular. Además, se adicionó un paso de incubación del ADN obtenido a 100° C para inactivar ADNasas. La calidad del ADN obtenido fue evaluada por medio de la amplificación de la región ITS1-5.8S-ITS2, presentando bandas definidas y cuantificables (entre 380 y 880 pb) ideales para estudios de identificación molecular y filogenia.

A quick, simple and low-cost protocol for extracting genomic DNA from yeast by cell wall lysis involving enzymatic treatment and alcoholic precipitation was standardised. Higher DNA yields (124.9±30.2 ng/λl) were obtained by using beta-glucuronidase instead of zymolyase; these had very high quality (A260/A280 nm = 1.86±0.1) and would be suitable for use in molecular biology assays. Moreover, a DNAse inactivation step was also introduced by incubation at 100 °C to further ensure DNA stability. DNA quality was assayed by PCR amplification of the ITS1-5.8S-ITS2 region, revealing defined, quantifiable 380 to 880 bp bands. These results show that the protocol is ideal for molecular identification and phylogenetic studies.

Composición química y digestibilidad del mote / Chemical composition and digestibility of mote

Se analizaron granos de maíz (Zea mays amylacea, capia) blanco y amarillo descascarados por un tratamiento térmico con cal y cenizas (maíz para mote); y se estudiaron la composición química y digestibilidad in vitro de dichos granos cocidos en agua en ebullición (mote), obtenidos bajo condiciones estandarizadas en laboratorio, para comparalos con los adquiridos en los comercios. La composición de nutrientes fue similar en los granos descascarados, detacándose los blancos procesados con cenizas de laboratorio con un contenido de proteínas y de sodio de 9,95 g/100 y 80,33 mg/100g y en los tratados con cal 979,70 mg de calcio/100 g respectivamente. Los motes comerciales analizados resultaron semejantes químicamente con los de laboratorio. En los blancos con cenizas de éste último grupo, los valores de sodio, hierro y potasio (28,02; 7,95 y 75,64 mg/100 g) fueron mayores que los que recibieron tratamiento con cal (blanco y amarillo), los que presentaron un alto contenido de calcio de 464,97 y 430,79 mg/100g respectivamente. La digestibilidad fue superior en las muestras con cal, 79 por ciento en las comerciales y 80 por ciento en las estandarizadas

Thioglycollate-elicited murine macrophages are cytotoxic to Mycoplasma arginini-infected YAC-1 tumor cells

Macrophages are important components of natural immunity involved in inhibition of tumor growth and destruction of tumor cells. It is known that these cells can be activated for tumoricidal activity by lymphokines and bacterial products. We investigated whether YAC-1 tumor cells infected with Mycoplasma arginini stimulate nitric oxide (NO) release and macrophage cytotoxic activity. Thioglycollate-elicited macrophages from male BALB/c mice were co-cultured for 20 h with YAC-1 tumor cells infected or not with Mycoplasma arginini. The cytotoxic activity was evaluated by MTT assay and nitrite levels were determined with the Griess reagent. Thioglycollate-elicited macrophages co-cultured with noninfected YAC-1 cells showed low cytotoxic activity (34.7 ñ 8.6per cent) and low production of NO (4.7 ñ 3.1 µM NO2-). These macrophages co-cultured with mycoplasma-infected YAC-1 cells showed significantly higher cytotoxic activity (61.4 ñ 9.1 per cent; P=0.05) and higher NO production (48.5 ñ 13 µM NO2-; P=0.05). Addition of L-NAME (10 mM), an inhibitor of NO synthesis, to these co-cultures reduced the cytotoxic activity to 37.4 ñ 2per cent (P=0.05) and NO production to 3 ñ 4 µM NO2- (P=0.05). The present data show that Mycoplasma arginini is able to induce macrophage cytotoxic activity and that this activity is partially mediated by NO.

Los cromosomas artificiales y sus aplicaciones / The artificial chromosomes and it applications

Los cromosomas artificiales son estructuras confeccionadas por ingeniería genética que facilitan el estudio del genoma y la comprensión del funcionamiento de los cromosomas; además, constituyen posibles vehículos de transferencia de genes en la terapia génica. Los primeros en desarrollarse fueron los cromosomas artificiales de levadura, los que se utilizan para la construcción de librerías de genes. Más recientemente se ha obtenido la primera generación de cromosomas artificiales humanos, los cuales han presentado una gran estabilidad, persistiendo en los cultivos celulares por varias generaciones

Análisis de la sección 8 del cromosoma x de drosophila melanogaster mediante el uso de clones yac de saccharomyces cerevisiae / Analysis of section 8 of x chromosome from drosophila melanogaster by use of yac clones in saccharomyces cerevisiae

En drosophila melanogaster, los genes estructurales que codifican para hexoquinasas A y B, se ubican estrechamente ligados en el cromosoma I (x). Estos loci corresponden a la región citológica 8D4.EI en el mapa citogenético de dicho cromosoma. Con el objetivo de determinar la ubicación exacta de los loci para hexoquinasas A y B se realizó un análisis molecular, mediante el uso de cromosomas artificiales de levadura, que contienen DNA de la sección 8 del cromosoma x. Para tal efecto, se aisló DNA cromosómico intacto de siete clones YAC de s. cerevisiae que cubren dicha región. El DNA fue resuelto mediante electroforesis de campo pulsado y analizado por hibridación con DNA del plásmido pB322 y con un fragmento de DNA de 0, 67 Kb de d. melanogaster, amplificado por PCR con partidores heterólogos específicos para hexoquinasas. Los resultados de estos experimentos permitieron concluir que, la sonda homóloga es capaz de hibridar con el YAC II, lo cual coincide con la cobertura de este clon en la región citológica esperada

Proceedings of the schistosome genome project

"The host-parasite relationship" is a vast and diverse research field which, despite huge human and financial input over many years, remains largely shrouded in mystery. Clearly, the adaptation of parasites to their different host species, and to the different environomental stresses that they represent, depends on interactions with, and responses to, various molecules of host and/or parasite origin. The schistosome genome project is a primary strategy to reach the goal; this systematic research project has successfully developed novel technologies for qualitative and quantitative characterization of schistosome genes and genome organization by extensive international collaboration between top quality laboratories. Schistosomes are a family of parasitic blood flukes (Phylum Platyhelminthes), which have seven pairs of autosomal chromosomes and one pair of sex chromosomes (ZZ for a male worm an ZW for a female), of a haploid genome size of 2.7X10 8 base pairs (Simpson et al. 1982). Schistosomes are ideal model organisms for the development of genome mapping strategies since they have a small genome size comparable to that of well-characterized model organisms such as Caenorhabditis elegans (100 Mb) and Drosophila (165Mb), and contain functional genes with a high level of homology to the host mammalian genes. Here we summarize the current progress in the schistosome genome project, the information of 3.047 transcribed genes (Expresses Sequence Tags; EST), complete sets of cDNA and genomic DNA libraries (including YAC and cosmid libraries) with a mapping technique to the well defined schistosome chromosomes. The schistosome genome project will further identify and charaterize the key molecules that are responsable for host-parasite adaptation, i.e., successful growth developement, maturation and reproduction of the parasite within its host in the near future.

Towards the physical map of the Trypanosoma cruzi nuclear genome: construction of YAC and BAC libraries of the reference clone T. cruzi CL-Brener

Strategies to construct the physical map of the Trypanosoma cruzi nuclear genome have to capitalize on three main advantage of the parasite genome, namely (a) its small size, (b) the fact that all chromosomes can be defined, and many of them can be isolated by pulse field gel electrophoresis, and (c) the fact that simple Southern blots of electrophoretic karyotypes can be used to map sequence tagged sites and expressed sequence tags to chromosomal bands. A major drawback to cope with is the complexity of T. cruzi genetics, that hinders the construction of a comprehensive genetic map. As a first step towards physical mapping, we report the construction and partial characterization of a T. cruzi CL-Brener genomic library in yeast artificial chromosomes (YACs) that consists of 2.770 individual YACs with a mean insert size of 365 kb encompassing around 10 genomic equivalents. Two libraries in bacterial artificial chromosomes (BACs) have been constructed, BACI and BACII. Both libraries represent about three genome equivalents. A third BAC library (BAC III) is being constructed. YACs and BACs are invaluable tools for physical mapping. More generally, they have to be considered as a common resource for research in Chagas disease.