Your browser doesn't support javascript.
loading
Functional characterization of the origin of replication of pRN1 from Sulfolobus islandicus REN1H1.
Joshua, Chijioke J; Perez, Luis D; Keasling, Jay D.
Afiliación
  • Joshua CJ; Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, United States of America ; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America ; Joint BioEnergy Institute, Emeryville, California, United States of America.
  • Perez LD; Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, United States of America ; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America ; Joint BioEnergy Institute, Emeryville, California, United States of America.
  • Keasling JD; Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, California, United States of America ; Department of Chemical & Biomolecular Engineering, University of California, Berkeley, Berkeley, California, United States of America ; Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, United States of America ; Joint BioEnergy Institute, Emeryville, California, United States of America ; Department of Bioengineering, Unive
PLoS One ; 8(12): e84664, 2013.
Article en En | MEDLINE | ID: mdl-24376833
Plasmid pRN1 from Sulfolobus islandicus REN1H1 is believed to replicate by a rolling circle mechanism but its origin and mechanism of replication are not well understood. We sought to create minimal expression vectors based on pRN1 that would be useful for heterologous gene expression in S. acidocaldarius, and in the process improve our understanding of the mechanism of replication. We constructed and transformed shuttle vectors that harbored different contiguous stretches of DNA from pRN1 into S. acidocaldarius E4-39, a uracil auxotroph. A 232-bp region 3' of orf904 was found to be critical for pRN1 replication and is therefore proposed to be the putative origin of replication. This 232-bp region contains a 100-bp stem-loop structure believed to be the double-strand origin of replication. The loop of the 100-bp structure contains a GTG tri-nucleotide motif, a feature that was previously reported to be important for the primase activity of Orf904. This putative origin and the associated orf56 and orf904 were identified as the minimal replicon of pRN1 because transformants of plasmids lacking any of these three features were not recovered. Plasmids lacking orf904 and orf56 but harboring the putative origin were transformable when orf904 and orf56 were provided in-trans; a 75-bp region 5' of the orf904 start codon was found to be essential for this complementation. Detailed knowledge of the pRN1 origin of replication will broaden the application of the plasmid as a genetic tool for Sulfolobus species.
Asunto(s)
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Plásmidos / Sulfolobus / Origen de Réplica / Replicación del ADN Límite: Animals Idioma: En Revista: PLoS One Asunto de la revista: CIENCIA / MEDICINA Año: 2013 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Plásmidos / Sulfolobus / Origen de Réplica / Replicación del ADN Límite: Animals Idioma: En Revista: PLoS One Asunto de la revista: CIENCIA / MEDICINA Año: 2013 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos