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Genética y Biología Molecular de Neurospora

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Genética y Biología molecular de Neurospora crassa. Nallely Cano. Instituto de Fisiología Celular. UNAM.

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Genética y Biología Molecular de Neurospora

  1. 1. Genética y Biología Molecular en Neurosporacrassa<br />Nallely Cano Domínguez<br />Instituto de Fisiología Celular UNAM<br />Laboratorio 107 Oriente<br />
  2. 2. Neurosporacrassaen la naturaleza<br />
  3. 3. De Moniliasitophilaa Neurospora<br />Premio de la Sociedad Americana de Botánica 1956<br />Bernard Ogilvie Dodge<br />
  4. 4. “Cada uno de esos miles de tipos de genes tienen, en general, una única especificidad. Esto significa que una enzima usualmente tendrá su final específicamente en un grupo y sólo un gen”<br />GEORGE WELLS BEADLE<br />EDWARD L. TATUM<br />Premio Nobel en Medicina 1958 por su hipótesis de “un gen-una enzima”<br />
  5. 5. Experimentos de Beadle y Tatum<br />Encuentran tres mutantes distintas que necesitaban arginina (arg-1, arg-2 y arg-3) para crecer y probaron si eran capaces de crecer en medio con ornitina y/o citrulina<br />arg-1<br />arg-3<br />arg-2<br />Citrulina<br />Arginina<br />Ornitina<br />Precursor<br />“Las características de una célula (su fenotipo) a su vez están producidas por su metabolismo interno, que está controlado por las enzimas que intervienen en los distintos pasos metabólicos”.<br />La adición de arginina al medio restaura el crecimiento<br />
  6. 6. reconoce a Neurosporacrassacomo uno de los 12 modelos eucariontes para la investigación biomédica<br />The<br />¿Por qué Neurosporacrassaes un buen modelo genético?<br />
  7. 7. Su ciclo de vida asexual <br />✔<br /><ul><li>Sus estructuras diferenciadas del ciclo asexual pueden identificarse fácilmente</li></ul>Conidio<br /><ul><li>Crece rápido</li></ul>Micelio<br /><ul><li>Suficiente biomasa para los análisis </li></li></ul><li>Su ciclo de vida sexual<br />✔<br />Ascosporas<br />Peritecio<br />Ascas<br /><ul><li>Tiene dos tipos sexuales (matingtypeA y a) lo que permite hacer cruzas
  8. 8. Pueden reconocerse las distintas estructuras diferenciadas
  9. 9. Produce ascas grandes lo que facilita hacer estudios de meiosis</li></ul>Ascogonia<br />Protoperitecio<br />
  10. 10. Fácil mantenimiento en el laboratorio<br />✔<br />Medio Vogel y 1.5% de sacarosa<br />Medio con 2% de sorbosa<br />3 días en oscuridad y 2 días en luz a 30°C<br />2 días en oscuridad a 30°C<br />
  11. 11. Fácil mantenimiento en el laboratorio<br />✔<br />Medio sintético de cruza<br />“Racetubes”<br />
  12. 12. ✔<br />No es patógeno pero ha servido como modelo para el estudio de procesos que se han conservado en los hongos patógenos<br />Las cepas se pueden preservar durante mucho tiempo <br />✔<br />
  13. 13. Mutantes de N. crassadisponibles en FungalGenetics Stock Center (www.fgsc.net)<br />✔<br />Peritecios de la cepa mutante per-1<br />Mutante “Band”<br />Cepa “algodonosa” (fluffy)<br />
  14. 14. ✔<br />Mutantes de N. crassadisponibles en FungalGenetics Stock Center afectadas en la formación de las ascosporas<br />Ascosporas mutante peak<br />Ascas de la mutante Perforated<br />Ascosporas gigantes mutante Banana<br />Ascas de una cepa silvestre<br />Ascosporas de las mutante cys-3<br />
  15. 15. Su genoma se encuentra secuenciado<br />✔<br />Nature. 2003 Apr 24;422(6934):859-68<br />
  16. 16. Características del genoma de N. crassa<br />Nature. 2003 Apr 24;422(6934):859-68<br />
  17. 17. ✔<br />La secuencia del genoma de Neurosporacrassase encuentra disponible en la red en la página del BROAD INSTITUTE<br />
  18. 18. Busquemos un gen… nor-1<br />
  19. 19. En la página de BROAD INSTITUTE existen herramientas que nos permiten conocer algunas características de la secuencia del gen de interés <br />
  20. 20. En la página de BROAD INSTITUTE existen herramientas que nos permiten conocer algunas características de la secuencia del gen de interés <br />
  21. 21. Se obtiene la secuencia génica y las regiones 5´y 3´que flanquean al gen nor-1<br />
  22. 22. Se pueden obtener mutantes nulas en un gen fácilmentePor la técnica del “Double Joint PCR”<br />✔<br />
  23. 23. Los métodos de transformación de Neurosporacrassa<br />Por electroporación<br />Por esferoplastos<br />Enzimas Líticas<br />
  24. 24. Existen herramientas disponibles para etiquetar proteínas (GFP, RFP, 6H, HA)<br />his-3<br />amp<br />pMF272<br />Promotor de ccg-1<br />his-3<br />gfp<br />nor-1<br />
  25. 25. Localización de Proteínas mediante fusiones con GFP<br />
  26. 26. Microscopía confocal<br />Microscopía de TIRF de polimerización de microtúbulos de una hifa de una cepa WT Uchida M. et al. 2008. FungalGeneticBiology. 2008 May;45(5):683-92<br />Microscopía confocal de una cepa con la construcción arg-4::gfp(Bowman)<br />
  27. 27. Microscopía confocal y epifluorescencia<br />Hifas con una construcción vma-1::rfp(se localiza en las vacuolas)<br />Bardiya, N. et al 2008. Genetics. 178 (1): 593-6<br />
  28. 28. Microscopia de BarridoConidia Anastomosis Tubes (CAT´s)<br />Roca, M. G. et al 2005. EukaryoticCell. P. 911-919 <br />
  29. 29. Ritmo circadiano<br />
  30. 30. Proceso de conidiación en N. crassa<br />1<br />ERO<br />6 – 15 min<br />40 min<br />Hifas en crecimiento<br />Adhesión de las hifas<br />2<br />ERO<br />ERO<br />2.5 - 12 h<br />5 – 30 min<br />1.5 h – 2.5 h<br />3<br />ERO<br />6.5 h - 8.5 h<br />8.5 - 15 h<br />ERO: Especies <br /> Reactivas <br />de Oxígeno<br />Conidiación<br />Micelio Aéreo<br />
  31. 31. Hansbergy Aguirre, 1990 <br />Estado diferenciado<br />Estado no diferenciado<br />O2 + nutrientes<br />O2 + nutrientes<br />[ERO]<br />diferenciación<br />Muerte Celular<br />O2 + nutrientes<br />germinación<br />[ERO]<br />“La diferenciación celular de los microorganismos se da como respuesta a un estado hiperoxidante”<br />Aguirre, J. et al. (2005), Trends in Microbiol. 13(3):111-8 <br />
  32. 32. PREDICCIONES<br />Mutaciones en los mecanismos antioxidantes<br />Mutaciones en los mecanismos pro-oxidantes<br />
  33. 33. Las NADPH oxidasas en células fagocíticas y sus ortólogos en hongos<br />O2<br /> O2.-<br />Flavocitocromo b558<br />gp91phox<br />NOX<br />NOX<br />p22 phox<br />p22 phox<br />gp91phox<br />p47phox<br />Rac-GTP<br />p40phox<br />Proteínas reguladoras<br />p67phox<br />BEM-1<br />p40phox<br />NADPH<br />NADP+<br />p47phox<br />GEF<br />p67phox<br />Rac-GTP<br />Rac-GDP<br />RhoGDI<br />NOR-1<br />Modificado de Lambeth, D. (2004). Nature review.4:181-1189<br />
  34. 34. NOX-1 isessentialfor sexual and asexual development<br />Δnox-1strains<br />NOX<br />p22phox<br />gp91phox<br /> WT <br />WT<br />∆nox-1<br />∆nox-1 (a) <br />WT<br />nox-1<br />p40phox<br />NOX-1 isessentialfor sexual frutingbodiesdevelopmentandalsorequiredfor normal developmentofaerialhyphaeandconidiation, as well as forvegetativegrowth.<br />p47phox<br />Cano-Dominguezet al. 2008. EukaryoticCell, 7(8):1352-61 <br />NOR-1<br />Rac-GTP<br />p67phox<br />
  35. 35. NOX-2 isrequieredfor sexual sporefunction<br />Δnox-2strains<br />NOX<br />p22phox<br />gp91phox<br />nox-2 x nox-2<br />WT x WT<br />p40phox<br />p47phox<br />TheascosporesfromΔnox-2 homozygouscrossesfailedtogerminate<br />NOR-1<br />Rac-GTP<br />p67phox<br />
  36. 36. NOR-1 isrequiredfor NOX-1 and NOX-2 activity<br />Δnor-1strains<br />NOX<br />p22phox<br />gp91phox<br />WT<br />nor-1<br />p47phox<br />p40phox<br />∆nox-2 X ∆nor-1<br />WT X WT<br />NOR-1<br />Rac-GTP<br />p67phox<br />
  37. 37. Agradecimientos<br />Dr. Jesús Aguirre <br />Biol. Olivia Sánchez<br />Laboratorio 107 Oriente<br />Dr. Wilhelm Hansberg<br />Dr. Pablo Rangel<br />Laboratorio 103 Oriente<br />
  38. 38. GRACIAS!!!<br />

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