Caracterización <strong>de</strong>l gen pnl 2 que codifica <strong>la</strong> pectín liasa 2 <strong>de</strong> Colletotrichum lin<strong>de</strong>muthianumniger are enco<strong>de</strong>d by a family of divergedgenes. Eur J Biochem 208:83–90.Cal<strong>de</strong>rón-Cortes, N. 2004. Detección <strong>de</strong>dos genes <strong>de</strong> patogenicidad a partird e u n a g e n o t e c a d e l h o n g oColletotrichum lin<strong>de</strong>muthianum raza1472. Tesis <strong>de</strong> licenciatura. Facultad<strong>de</strong> Biología. U.M.S.N.H.Dyrlov B. J., Nielsen, H., von Heijne, G.and Brunak, S. 2004. Improved predictionof signal pepti<strong>de</strong>s SignalP3.0. J Mol Biol 340:783-795Gysler, C., Harmsen, J.A.M., Kester,H.C.M., Visser, J. and Heim, J. 1990.Iso<strong>la</strong>tion and structure of the pectinlyase D-encoding gene from Aspergillusniger. Gene 89:101–108.Kozak, M. 1986. Point mutations <strong>de</strong>finea sequence f<strong>la</strong>nking the AUG initiatorcodon that modu<strong>la</strong>tes trans<strong>la</strong>tionby eukaryotic ribosomes. Cell44:283–292.Kupfer, M. D., Drabenstot, D. S., Buchanan,L. K ., Hua Zhu, L. H., Dyer, W.D., Roe, A. B. and Murphy, W. J.2004. Introns and splicing elementsof five diverse fungi. Euk Cell3:1088–1100.Kusters-van, Someten, M.A., Flipphi, M.,<strong>de</strong> Graaff, L., van <strong>de</strong>n Broeck, H.,Kester H, Hinnen, A. and Visser, J.1992. Characterization of the Aspergillusniger pelB gene: structure andregu<strong>la</strong>tion of expression. Mol GenGenet 234:113–120.Lara-Márquez, A. 2004. Construcción <strong>de</strong>una genoteca <strong>de</strong> Colletotrichumlin<strong>de</strong>muthianum para ais<strong>la</strong>r el genpectín liasa (pnl2). Tesis <strong>de</strong> licenciatu r a . Fa c u l t a d d e B i o l o g í a .U.M.S.N.H.Lara-Márquez, A. 2007. Caracterizaciónbioquímico-molecu<strong>la</strong>r <strong>de</strong> <strong>la</strong> enzimap e c t í n l i a s a 2 ( pnl2) d eColletotrichum lin<strong>de</strong>muthianum. Tesis<strong>de</strong> maestría. Instituto <strong>de</strong> Investigaciónen Biología Experimental.<strong>Universidad</strong> <strong>de</strong> Guanajuato.Mayans, O., Scott, M., Connerton, I.,Gravesen, T., Benen, T., Visser, J.,Pickersgill, R. and Jenkins, J. 1997.Two crystal structures of pectin lyaseA from Aspergillus reveal a pH drivenconformational change and strikingdivergence in the substrate-bindingclefts of pectin and pectate lyases.Structure 5:677-689.Pickersgill, R. W. and Jenkins, J. A. 2003.The structures and active sites of pectinases:Advances in pectin and pectinaseresearch. Kluwer Acad. Pub.The Nether<strong>la</strong>nds. 267-275.Ranveer, S. J., Shivalika, S. and Reena, G.2005. Microbial pectinolytic enzymes:a review. Proc Biochem40:2931-2944.Vitali, J., Schick, B., Kester, H.C.M., Visser,J. and Jurnak, F. 1996. The threedimensionalstructure of Aspergillusniger pectin lyase B at 1.7-A resolution.P<strong>la</strong>nt Physiol 116:69-80.Von Heijne. 1983. Patterns of amino acidsnear signal-sequence cleavagesites. Eur J Blochem 133:17-21.No. Especial 2008 Ciencia Nico<strong>la</strong>ita [117]
Ciencia Nico<strong>la</strong>ita, No. Especial, 2008: 118-126<strong>Universidad</strong> Michoacana <strong>de</strong> San Nicolás <strong>de</strong> HidalgoEstudio bacteriológico en granjas lecheras <strong>de</strong>traspatio <strong>de</strong>l municipio <strong>de</strong> Tarímbaro,MichoacánJesús Andrei Rosales-Castillo 1,2 , Ma. Soledad Vázquez-Garcidueñas 3 ,Hugo Álvarez-Hernán<strong>de</strong>z 2 , Alba Irene Vare<strong>la</strong>-Murillo 2 y Gerardo Vázquez-Marrufo 1,2 *1Centro Multidisciplinario <strong>de</strong> Estudios en Biotecnología, 2 Facultad <strong>de</strong> Medicina Veterinaria y Zootecnia; 3 División <strong>de</strong>Estudios <strong>de</strong> Postgrado, Facultad <strong>de</strong> Ciencias Médicas y Biológicas “Dr. Ignacio Chávez”. <strong>Universidad</strong> Michoacana <strong>de</strong> SanNicolás <strong>de</strong> Hidalgo. * Autor responsable para correspon<strong>de</strong>ncia: marrufo@umich.mx.ResumenLa producción <strong>de</strong> leche bovina a pequeña esca<strong>la</strong> constituye un riesgoepi<strong>de</strong>miológico, ya que <strong>la</strong>s heces <strong>de</strong>l ganado y el suelo son un factor <strong>de</strong>contaminación que pue<strong>de</strong> afectar <strong>la</strong> salud <strong>de</strong>l hato y <strong>de</strong>l consumidor. El objetivo <strong>de</strong>lpresente trabajo fue <strong>de</strong>terminar <strong>la</strong> presencia <strong>de</strong> enterobacterias en muestras <strong>de</strong> hecesy suelo <strong>de</strong>l sistema <strong>de</strong> producción <strong>de</strong> traspatio <strong>de</strong>l municipio <strong>de</strong> Tarímbaro,Michoacán y evaluar su re<strong>la</strong>ción con <strong>la</strong> contaminación <strong>de</strong> <strong>la</strong> leche. La lecheanalizada presentó <strong>de</strong> 86 a 4,100 UFC/mL. La amplificación mediante PCR <strong>de</strong>l gen16S <strong>de</strong> RNAr con iniciadores específicos para enterobacterias, generó una banda <strong>de</strong>544 pb, tanto en ais<strong>la</strong>dos bacterianos como en ADN extraído <strong>de</strong> heces y suelo. E<strong>la</strong>nálisis <strong>de</strong> <strong>la</strong>s secuencias obtenidas, a partir <strong>de</strong> los productos <strong>de</strong> amplificacióngenerados, muestra que <strong>la</strong>s principales enterobacterias presentes en leche, suelo yheces son Brenneria rubrifaciens, Escherichia coli, Pectobacterium carotovorum yPectobacterium wasabiae, lo que indica un intercambio <strong>de</strong> especies y posiblemente<strong>de</strong> clonas entre <strong>la</strong>s muestras analizadas.Pa<strong>la</strong>bras c<strong>la</strong>ve: Enterobacterias, granjas <strong>de</strong> traspatio, PCR, RNAr 16SAbstractSmall-scale bovine dairy farms represent an epi<strong>de</strong>miological risk because themanure generated and the soil that constitutes an important contamination source,affecting milk quality and potentially the health of cattle and humans. Therefore, theobjective of this work was to <strong>de</strong>termine the presence of enterobacteriaceae in cowfeces and soil samples from small-scale dairy farms located at Tarímbaro, Michoacán,and to evaluate their role as sources of milk contamination. The bacteriologica<strong>la</strong>nalysis of milk showed the presence of 86-4,100 CFU/mL. PCR assays, carried out toamplify the 16S gen of rRNA from bacterial iso<strong>la</strong>tes and from DNA obtained from soi<strong>la</strong>nd cow feces, produced a single band of 544 bp. The analysis of the DNAsequences obtained from the amplification products showed that theenterobacteriaceae present in milk, soil, and cow feces are Brenneria rubrifaciens,Ciencia Nico<strong>la</strong>ita No. Especial, 2008, Programa Institucional <strong>de</strong>Doctorado en Ciencias Biologicas
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