Isolation of DNA Fragments Bound by Transcriptional Factors, AdpA and ArpA, in the A-Factor Regulatory Cascade.

Abstract

DNA fragments recognized and bound by transcriptional factors, AdpA and ArpA, both of which are members in the A-factor regulatory cascade, were isolated. A library of fragmented chromosomal DNA from Streptomyces griseus was constructed either by HaeIII digestion or by sonication, followed by attachment of “catch” linkers at the ends. The library was incubated with a transcriptional activator, AdpA, to allow formation of protein-DNA complexes. The complexes were separated from free DNA by gel mobility shift and the DNA in the complexes was purified by extraction from gel pieces. The purified DNA was then amplified by PCR with the catch linker as primers. For enrichment of the DNA fragments actually bound by the transcriptional factor, the PCR product was subjected to the second and further cycles of the gel mobility shift-PCR procedure. The DNA fragments recovered after cycles of this procedure were cloned in an Escherichia coli plasmid for further characterization. The gel mobility shift-PCR procedure yielded 16 DNA sequences specifically bound by AdpA. This procedure was applied to the isolation of target sequences of another transcriptional factor, ArpA, and two DNA sequences were obtained.