lambda Exonuclease-based subtractive hybridization approach to isolate differentially expressed genes from leaf cultures of Paulownia kawakamii

Genes that are preferentially expressed in a particular developmental pathw ay can be isolated by subtractive hybridization (SH). We developed a PCR-ba sed approach coupled with lambda exonuclease digestion that allows for gene rating single-stranded tester and driver nucleic acids suitable for SH star ting from cDNA libraries. An efficient subtraction strategy was developed t o overcome some of the problems in the previously described SH protocols, s uch as the need for large amounts of experimental tissue, RNase contaminati on during solution hybridization, and post-subtraction recovery of nucleic acids. We used this method to obtain cDNA corresponding to genes expressed during adventitious shoot regeneration from excised leaf cultures of the fa st-growing tree Paulownia kawakamii. Over 36 cDNA clones were isolated and 1 of the differentially expressed clones codes for a leucine zipper transcr iption factor. This clone showed about sixfold higher level of expression i n the shoot-forming tissues (tester) compared to that in the callus-forming tissues (driver) of Paulownia, suggesting that differentially expressed ge nes can be efficiently isolated using this simple lambda exonuclease-based subtractive hybridization method. (C) 2001 Academic Press.