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Impact factor 2010: 31.1525-Year Impact Factor: 32.628 Issues per year: 26
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Demostrar que la replicación del DNA en mamíferos es bidireccional, con la formación de fragmentos de Okazaki en una de las cadenas (síntesis discontinua).
OBJETIVOS
Los fragmentos de Okazaki cambian de una cadena a otra dentro de un Origen de Replicación Bidireccional (OBR)
Identificar un OBR cerca del gen de DHFR.
Vaughn et al. (1990) described convincing evidence for the surprising conclusion that the DNA replication origin located downstream of the dihydrofolate reductase (DHFR) gene in Chinese hamster cells consists of a broad initiation zone extending over 26 kb. However, the data presented by Burhans et al. (1990) in this issue suggest the contradictory conclusion that replication forks must emanate bidirectionally from a site that is no larger than 450 nucleotides.
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FIGURE 1: PROTOCOLO EXPERIMENTAL
BrdU = Bromodeoxyuridine
BrdUTP: 5 - Bromo - 2’ - deoxyuridine 5’ - triphosphate
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FIGURE 2: MAPPING ORIGINS OF REPLICATION
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FIGURE 3: PULSE AND CHASE
Longitud en aprox. mamíferos: 25-300 nucleótidos.
High MW DNA
Broad peak of low MW
DNA (average 105 nt,
range 40-250 nt)
Heat denaturation
Alkaline denaturation
Características fragmentos de Okazaki:
Naturaleza transitoria (son rápidamente unidos a la cadena naciente).Mayoritariamente en la cadena retrasada.
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FIGURE 4: HIBRIDACION Y CLONADO EN COSMIDOS
Hibridación usando como sonda los fragmentos de Okazaki:
Buscando el origen de replicación…
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FIGURE 5: DOT BLOT
DOT BLOT
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FIGURE 6: CUANTIFICACION DEL RESULTADO DE DOT BLOT
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FIGURE 7: ACERCANDOSE AL ORIGEN DE REPLICACION, COMPARACION CON OTROS PAPERS