Molecular Cloning of Promoters Functional in Escherichia coli from Chloroplast DNA of a Liverwort, Marchantia polymorpha

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DNA fragments cloned from chloroplast DNA of a liverwort, Marchantia polymorpha, functioned in E. coli as transcriptional promoters using gene fusion to the E. coli lacZ. gene. A recombinant plasmid gave as high a level of β-galactosidase activity as when it was induced by IPTG (isopropyl-β-D-thiogalactopyranoside) in E. coli wild strain W3110. The inserted chloroplast DNA fragment was sequenced and mapped on the locus upstream from the 16S ribosomal RNA gene in the chloroplast genome. SI nuclease mappings using both chloroplast and E. coli RNA showed that the transcription starts at almost the same position downstream from the consensus Pribnow-box-like region. This clone also had a higher activity of β-galactosidase in E. coli than those containing promoters of the large subunit (LS) gene of ribulose bisphosphate carboxylase/oxygenase (RubisCO) and the β subunit gene of H+-ATPase. These results revealed that chloroplast DNA fragments having promoter function can -be cloned in E. coli, and the efficiency of promoter function can be estimated by this gene fusion technique.
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