NOVEL COSMID VECTOR PDEL-BAC - CLONING OF LARGE GENOMIC DNA FRAGMENTS, GENERATION OF DELETIONS, AND SEQUENCING OF RESULTING DELETION MUTANTS

A vector was constructed for cloning long genome sequences, generating deletions, and selection and sequencing of the resulting deletion mut ants. It was used to clone genomic DNA fragments up to 185 kbp long, a nd the DNA was maintained in E. coli. The stability of DNA is due to t he fact that the vector constructed (pDEL-BAC) is related to the F fac tor, whose copy number is closely controlled. Deletions were generated at both ends of DNA owing to intramolecular transposition of Tn1000 t aking place in E. coil cells expressing the transposase of this mobile element. The vector obtained contains tetracycline resistance and suc rose sensitivity genes required for selecting deletion mutants. It als o bears kanamycin resistance and streptomycin sensitivity genes necess ary for selecting counterclockwise deletion mutants. For bidirectional sequencing of the mutants, the vector contains hybridization sites fo r universal primers SP6 and T7.