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Clonado Y Expresión De Ubicuitina En Neurospora Crassa Taccioli, Guillermo 1989

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Clonado Y Expresión De Ubicuitina En Neurospora Crassa Taccioli, Guillermo 1989 Tesis de Posgrado Clonado y expresión de ubicuitina en Neurospora crassa Taccioli, Guillermo 1989 Tesis presentada para obtener el grado de Doctor en Ciencias Químicas de la Universidad de Buenos Aires Este documento forma parte de la colección de tesis doctorales y de maestría de la Biblioteca Central Dr. Luis Federico Leloir, disponible en digital.bl.fcen.uba.ar. Su utilización debe ser acompañada por la cita bibliográfica con reconocimiento de la fuente. This document is part of the doctoral theses collection of the Central Library Dr. Luis Federico Leloir, available in digital.bl.fcen.uba.ar. It should be used accompanied by the corresponding citation acknowledging the source. Cita tipo APA: Taccioli, Guillermo. (1989). Clonado y expresión de ubicuitina en Neurospora crassa. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. http://digital.bl.fcen.uba.ar/Download/Tesis/Tesis_2233_Taccioli.pdf Cita tipo Chicago: Taccioli, Guillermo. "Clonado y expresión de ubicuitina en Neurospora crassa". Tesis de Doctor. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. 1989. http://digital.bl.fcen.uba.ar/Download/Tesis/Tesis_2233_Taccioli.pdf Dirección: Biblioteca Central Dr. Luis F. Leloir, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Contacto: [email protected] Intendente Güiraldes 2160 - C1428EGA - Tel. (++54 +11) 4789-9293 UNIVERSIDAD DE BUENOS AIRES Facultad de Ciencias Exactas y Naturales CLONADO Y EXPRESION DE UBICUITINA EN NEUROSPORA CRASSA Guillermo Taccioli Tesis para optar al título de Doctor en Ciencias Químicas Director: Dr. Norberto Daniel Judewicz 1989 e? o? 33 Instituto de Investigaciones en "OZ Ingeniería Genética-y Biología Molecular (INGEBI) CONICET AGRADECIMIENTOS Al Dr. Héctor N. Torres por su constante apoyo y su 1nva­ luable aporte en la discusión de los proyectos de trabajo. A la Dra. Mirtha M. Flawiá por contar siempre con su in­ valorable ayuda en la solución de los problemas y su incesante empuje. Al Dr. Norberto D. Judewicz por la corrección del trabajo de tesis y por aceptar siempre la discusión de las ideas. A mis compañeros del inolvidable "Lab. 211", Gabriel 0. Aisemberg; Ricardo M. Attar; Daniel L. Altchuler; Erich Grotewold y Raúl Andino por la colaboración que me brindaron. Al Dr. Alberto R. Kornblihtt, por toda su ayuda y las in­ valorables sugerencias aportadas. A los Dres. Alejando A. Paladini (h); Mariano Levin; María T. Téllez-Iñón; Alejandro Mentaberry y Gerardo Glikin por el apo­ yo brindado en esta etapa. Al Dr. John Newbold por su colaboración en el proyecto de síntesis de oligodeoxinucleótidos. A los Dres. Richard Benarous; Jean Yves Picard y Louise Reubel por la ayuda brindada en la construcción de la genoteca de expresión. A mis compañeros, Graciela M. Bianchini; Gabriela Levitus; Patricia Levy-Yeyati; Betina Orman; Jorge Muschietti; Leonardo Erijman; Eduardo Cafferata y Ariel Wilner que además de compartir gratos momentoscolaboraron activamente en este trabajo. A la barra KILOMétrica de ingebianos que colaboró en hacer más placentero el trabajo de todos los días integrada por, Javier Cáceres; Viviana Bernath; Facundo Batista; Alejandra Mandel; Va­ lentina Carricarte; Ana Pastini; Enrique Mesri; Mercedes Goin; Andres Muro; Alejandro Schijman; Haría Laura Gómez; Laura Mora­ tinos; OmarCoeo; Fernando Bravo; Mónica Torruela; Horacio Marti­ netto; Lucio Castilla; Sonia Lafont; Claudio Eieenechloe; Claudio Alonso; Rosana Celnik; Nora Pafioe; Pablo Rabinovich y Claudia Ochat. A Norberto Malarini por eu contagioso (e incurable) buen humor y el esmerado trabajo gráfico. A Ursula Borrás por eu alegría y eficiente trabajo como secretaria. A Adriana Urmanpor su inagotable paciencia y profesiona­ lismo en la traducción, transcripción y replicación de esta te­ E316. A María J. Alvarez, Norberto Contreras, Gabriel Paissán, Mariano Rodríguez, Marcelo, Gustavo Peace y Leonor Acevedo por el excelente trabajo técnico. INDICE Página .IRLIEISUMJÍZN . .1 ABI-REVIA'I [JIQAS . .4 I N'1'13013UCJCII ON . .7 HBLQHLTIHA. ..8 H1S“ORIA . ..3 (“J ENZIMAS INVOLUCRADAS EN LA ACTIVACION Y CÚNJUGAClDN DE UBICUITINA . ..11 CONJUGADOS DE UBlCUITINA A HISTONAS . ..16 HIDROLASAS CARBOXITERMINAL DE UBICUITINA . ..20 PROTEASA DEPENDIENTE DE UBIQUITINA/ATP . ..23 coment...)(¿J PBDTEASAS DEPENDIENTES DE ATP EN UACTERIAS . .31 (¿J HHHHH ÑÜ'JCWHBGJ PAPEL DE LA CONEORMACIÜN DE LA UBICUITINA EN LA ESPECIFICIDAD DE LA DEGRADACION . .34 PAPEL DE tARN EN LA DEGRADACION DE SUSTRATOS . ..36 LA REGLA DEL N-TERHINAL . .38 (JJOO H .10. ALTERACION DE LA ESTRUCTURA DE UBLCUITINA Y SUS CONSECUENCIAS . ..45 SELECTIVIDAD DE UNION DE LA ENZIMA E3 . ..50 .12. POSIBLES SENALES PARA PROTEDLISIS . ‘ . ..54 .12. La hipótesis "PES”” . ‘ . ..55 O (‘J Degradación lieosomal . .J . ..60 .13. RESPUESTA AL ESTRES TERMICO . m..62 LOCALIZACION DE LA UBICUITINA EN LA SUPERFICIE n-dl-¡l-¡l-¡I-‘H .14. comunmente CELULAR Y EN EL CITOESQUELETO . ‘ . ..70 Indice GENETICA Y REGULACION DE LOS GENES DE UBICUITINA....76 Levadurae (Saccharomyees cerevísiae) . ..76 Plantas superiores . ..84 .15‘ Drosophila melanogaster . ..86 .15. Chenorfiabditis elegans . ..86 .15. Te trahymena pyr'ifoz'mís . .88 .15. Dictyostelium discoideum . ..88 .15. ÏTypanosomatidae . ..90 15. Mamíferos . ..91 .15 (OCDÑÜU'IACÚI‘JH Pollo . ..95 üJüJüJGJCIJCJüJü-‘IQJÜJGJ HHHHHHHHHHH .16. EVOLUCION . ..97 MMÏLWA (,Edfid, . .99 [‘J[‘J Características generales . .99 ¿o I‘J Organización del genoma . ..101 ü.) QLQLQHEXIHLDA . ... ..105 IRIESÜL'TADOS Y TJISCUSION . ... ..106 ANALISIS DE LA GENOTECA DE ADN GENOMICO DE ALflguxuüaí . .._ . ..107 Identificación de clones positivos . ..107 [‘J Subelonado y mapeo de restricción . ..117 CARACTERIZACION DEL GEN DE POLIUBICUITINA . ..123 Secuenciación . ..123 ¡buhnhhrh (“JH Identificación de los extremosdel transcripto.....123 “J(“J(“J(“JHH ¡b uu. La.) Análisis de la organización del gen.. ... ..131 (“J yt. Análisis del Southezw genómico . ..147 _ ii _ Indice ANALISIS DE LA EXPRESION DEL GEN DE POLIUBICUITINA . ‘ . ‘ . ..150 O Metodologia . ‘ . ‘ . .\...‘150 Inhibición de la sintesis proteica por 'antibióticos . , . ..150 ¡“J Tratamiento térmico y otros estrés relacionados . , . ‘ . ‘ . ..161 Tratamiento con análogos de aminoácidos . ..167 Tratamiento con poliaminas . ..189 Otras condiciones estudiadas . ..174 Grupo: Expresión de conidiosporas . \ . ‘ . ‘ ..176 ANALISIS DE LAS GEHOTECASDE cADN DE üi_Qzafifiai....179 H Identificación de los clones positivos . ..179 Subclonado y mapeo de restricción . ‘ . ..185 CARACTERIZACION DEL GEN DE FUSION . T. ‘ . ‘ . ..138 Secuenciación . ‘ . ‘ . ‘ ‘ . ‘ . ..138 Análisis de la secuencia . ..183 Análisis del Southern genómico . ..193 ANALISIS DE LA EXPRESION DEL GEN DE FUSION . ..197 hbhhfibvhrbrbhbphnh QLWÜ'IÜICWñrPIhÜJCOCOü-‘I C3C3rJCIIJIJEBJICJDJIEEB . ..201 DIAKÜFIZIIJZIKIQIEES SI D113ÏP<313CJES . ..207 REACTIVOS . ..208 Reactivos químicos . ..208 Enzimas . ..210 Enzimas de restricción . ‘ . ‘ . ... i . ‘ . ‘ . ..210 NNNH NH Fosfatasa alcalina de intestino de ternero (CIP)...211 - iii — Indice ADN ligaea del fago T4 . .. ...¿1 .0. [‘J[‘J (DO) RNaea H. Digestión del tracto de poli A de mARN... AISLAMIENTO DE ADN . .. ...2]4 Aislamiento de ADNde plásmidos . .; . .. ...214 Purificación de ADNen gran escala . .. ...2]4 Purificación de ADNen pequeña escala . .. [‘JHHH Aislamiento de ADNde bacteriófagos . .. ...216 OIG‘JCDGDÜ'JG'J HHHHHH Purificación de ADNdel bacteriófago X en gran escala . .. ...216 (‘J[‘J Purificación de ADNdel bacteriófago X en pequeña escala . i . i . .. ...217 I‘J Purificación de ADNsimple cadena de fagoe filamentoeoe . i . .. ...218 Extracción y purificación de ADNde p¡*r'vo o es . ca......o..a.n.o.-sun.gcaoauoo Cuantificación de ADN. .. ...220 ¿a Purificación de ADNpor columnas (Minicolumn—D).... ¡“J EXTRACCION Y PURIFICACION DE ARN DE Mmmm . .. ...222 Purificación de ARN total . .. ...222 Obtención de ARN poly A+ . .. ...223 [‘J[‘JI‘J DJ[‘JH Cuantificaoión de ARN. .. ...224 (¿J CONSTRUCCION DE LA GENÜTECA DE ADN COPIA EN thll . .. ...225 Síntesis de ADN copia' . .. ...225 Método de la RNasa H . .. ...225 Método de la nucleasa Sl . ..= . .. ...2°7 GOGO) CJCJÜJCIJ NHHH Acondicionamiento de los cADNdc para eu ligado... .230 _iv_ Indice 5.3.3. Clonado en el vector thll . ..235 6.3.3.1. Ligado a los brazos del vector . ..235 6.3.3.2. Empaquetado in vitro . ..235 6.3.4. Titulación de los fagoe reccmbinantee . ..735 6.4. ANALISIS DE GEHOTECAS DE FAGOS . ..237 6.5. TRANSFORMACIONEN Etcherichia coli . ..239 6.5.1. Preparación de células competentes “01081 Y HM522 . \ . ..239 6.5.1.1 Alta eficiencia de transformación . i . ..239 6.5.1.2‘ Protocolo simplificado . ‘ . ..239 6.5.2. Transformación con pláemidoe . ..239 6.8. MARCACION DE ADN . ‘ . ‘ . ..241 6.6.1 Técnica del rellenado de extremo (fill in) . ..241 6.6.2. Tecnica del iniciador múltiple de secuencia al azar (random priming) . .L . ..241 6.6.3. Técnica de extensión del iniciador (primer extension) . ..243 6.6.4. Técnica de ADN copia . 244 6.7. MAPEO CON NUCLEASA 81 . ..
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