Genetic transformation in Haemophilus influenzae: physical and biological properties of three DNA constructs carrying nov gene
Joshi, V.P.; Notani, N.K.
Indian Journal of Biochemistry and Biophysics 25(6): 523-527
1988
ISSN/ISBN: 0301-1208 PMID: 3267145 Document Number: 311704
Some of the main features of the genetic transformation with Haemophilus system are reviewed. Three aspects in particular, viz. recognition and uptake of DNA, vector systems and gene clonings and mechanism of transformation were considered. The role of uptake sites and recognition signals in Haemophilus and Escherichia coli DNA for their recognition and uptake by H. influenzae is described. DNA cloning in H. influenzae has been accomplished using either a novel principle of insert detection with plasmids RSF 0885 or pJ1-8 or by insertional inactivation with a shuttle vector pDM2. Mechanism of transformation appears to be as follows: Homologous DNA is first taken up in transformasomes, membraneous extensions sprouted in H. influenzae at the attainment of competence. DNA thereafter exits (is translocated) into the cell. Recombinational machinery or recombination probably has a role in homologous recognition and entry. In the present study, physical and biological properties of 3 novr DNA clones were examined. Clone pJ1-8N2, pJ1-8N19 and pJ1-8N20 respectively contain Eco RI fragments in the insert as follows: a, b (.rarw.) a, b, c (.fwdarw.) and a, b (.rarw.) and z (arrows indicate orientation relative to vector DNA). Almost, two-thirds of AmpR transformants are recombinant for nov allele with pJ1-8N19 DNA compared to only about 5% with pJ1-8N2 or pJ1-8N20. A role for fragment c or orientation of fragments a and b relative to vector DNA for efficient transformation is indicated.