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Title

Identification of the origin and direction of replication of the broad-host-range plasmid pLS1

AuthorsPuyet, A.; Solar, Gloria del ; Espinosa, Manuel
Issue Date1987
PublisherOxford University Press
CitationNucl. Acids Res. (1988) 16 (1): 115-133.
AbstractThe replication origin of the fully sequenced broad-hostrange streptococcal plasmid pLSl has been determined by the use of an in vitro replication system prepared from Escherichia colii, a host in which the plasmid can be established.Replicative intermediates were isolated from reaction mixtures that contained dideoxythymidine triphosphate, thus limiting the average extent of in vitro synthesis. Analysis of HinfI-cleaved intermediates demonstrated that the origin of replication is included within a 443-bp fragment. Replication proceeds unidirectionally in the same direction as transcription of plasmid mRNAs. Isolation of deletion derivatives allowed us to define the replication origin of pLS1 within a region of 284 bp.Replication of pLSI occurs through single-stranded intermediates by a rolling circle mechanism. Cleavage of supercoiled plasmid DNAs with endonuclease S1 followed by restriction mapping,allowed the positioning of three major specific S1 sites in regions of high potential to form secondary structures. One of these inverted repeats is located in the region where the origin of replication of pLS1 has been defined.
Description19 p.-7 fig.
Publisher version (URL)http://dx.doi.org/10.1093/nar/16.1.115
URIhttp://hdl.handle.net/10261/112706
DOI10.1093/nar/16.1.115
ISSN0305-1048
E-ISSN1362-4962
Appears in Collections:(CIB) Artículos
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