Primers were designed from the highly conserved protein regions of
luxl, one of the two component regulatory genes in prokaryotic, and an about 180 bp DNA from
Streptomyces cattleya was amplified by PCR. Using this fragment as a probe, Southern hybridization was accomplished with chromosome DNA of S.
cattleya, leading to the cloning of the foreign DNA into pBluescript (SK-). DNA sequencing and frame analysis showed an intact ORF
consisting of 1800 bp; the G+C composition was 69.3%. Upstream from the ORF was a 249 bp AT rich region, downstream of the
ORF several complicated secondary structures were found. DNA-binding assay by gel retardation demonstrated the presence of
a protein that specifically bound to the 249 bp AT rich region. The region also showed anomalous electrophoretic mobility
at low temperature, like that of a bent DNA molecule, indicating that the region contains a
cis-acting element.
Keywords
Streptomyces cattleya
- regulatory gene - gel retardation - “Bent” DNA