CONSTRUCTION OF AN ORI CASSETTE FOR ADAPTING SHUTTLE VECTORS FOR USE IN HAEMOPHILUS-INFLUENZAE

An ori (origin of DNA replication) cassette, pORC, containing the P15a ori and the kanamycin-resistance-encoding gene from Tn5, was construc ted. The cassette was used to convert an Escherichia coli promoter sel ection vector, which contains a promoterless chloramphenicol (Cm) acet yltransferase-encoding gene (cat) downstream from a multiple cloning s ite (MCS) [Brosius and Lupski, Methods Enzymol. 153 (1987) 54-68], to an E. coli-Haemophilus influenzae shuttle vector. The shuttle vector, pQL1, was shown to transform E. coli and H. influenzae efficiently. H. influenzae promoters were cloned into pQL1 by ligation of Sau3A-diges ted H. influenzae chromosomal fragments. Selection and semiquantitativ e analysis of promoter strength were performed on agar plates containi ng different concentrations of Cm. With the use of pQL1, H. influenzae gene regulation can now be studied in either H. influenzae or E. coli . In addition, elements of pORC can be used to convert other specializ ed E. coli vectors to E. coli-H. influenzae shuttle vectors.