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CLONING AND RESTRICTION MAP OF THE RECA-LIKE GENE OF ERWINIA AMYLOVORA
Legitimate recombination, which occurs between two homologous fragments of DNA, requires the expression of a gene called recA in Escherichia coli. By analogy, the corresponding gene in E. amylovora is referred to as the "recA-like" gene.
In E. coli the RecA protein catalyses DNA strand exchange required for homologous recombination.
Additionally, after DNA damage this protein is involved in the induction of several genes that function in DNA repair.
Thus, RecA– mutants of E. coli cannot promote legitimate recombination and are very sensitive to treatments that damage DNA, such as ultraviolet radiation or chemicals like nitroquinoline-n-oxide (NQO).
To construct stable isogenic recA-like mutants of E. amylovora, we chose to delete a part of the recA-like gene in vitro. This approach required the cloning and the construction of a restriction map of the chromosomal region containing the recA-like gene of E. amylovora.
A genomic DNA library of E. amylovora strain Ea 322 was made in the RecA– strain of E. coli, HB101. Clones harboring the recA-like gene were identified by taking advantage of the pleitrophic nature of the RecA protein, which is essential for DNA repair.
The library was spread on appropriate semi-solid medium and then exposed to UV radiation.
To confirm that clones surviving such treatment harbored the recA-like gene of E. amylovora, plasmids were isolated from these clones and reintroduced into a recA– strain of E. coli; the resulting clones were subjected to further analysis.
Two plasmids were identified that confer RecA-like activity in E. coli. The smallest of these, pCPP901, contains a 12 kb insert of E. amylovora DNA in the plasmid vector pBR325.
Deletions in pCPP901 were used to map the recA-like gene.
This plasmid was partially digested with the restriction endonuclease