IDENTIFICATION OF A THERMOPHILIC PLASMID ORIGIN AND ITS CLONING WITHIN A NEW THERMUS-E-COLI SHUTTLE VECTOR

A pUC19-based vector has been generated for selecting functional therm ophilic origins (oris) of Thermus ssp. Once combined with thermophilic DNA, the vector can be amplified in ampicillin resistant (Ap(R)) E. c oli, prior to transformation and kanamycin (Km) selection in Thermus t hermophilus. The Km(R) Thermus transformants replicate any newly-forme d shuttle vectors via introduced thermophilic oris. Using this ''ori-s electing'' vector, three novel thermophilic oris were cloned from rand omly digested Thermus cryptic plasmid DNA. These shuttle vectors are u seful for genetic analyses, as well as protein engineering within ther mophiles. The smallest ori-containing sequence of 4.2 kb has been subc loned, sequenced, and further refined to 2.3 kb. A significant ORF of 341 amino acids (aa), with a Thermus promoter and RES, is found within the thermophilic ori. Deleting part of this ORF abolishes the shuttle vector's ability to replicate in T. thermophilus. Therefore, we postu late that this ORF encodes a replication protein (Rep) necessary for t hermophilic plasmid replication. The thermophilic ori also contains tw o sequences which resemble DnaA boxes. (C) 1997 Elsevier Science B.V.