IDENTIFICATION OF GENES UP-REGULATED IN DEDIFFERENTIATING NICOTANIA-GLAUCA PITH TISSUE, USING AN IMPROVED METHOD FOR CONSTRUCTING A SUBTRACTIVE CDNA LIBRARY

Pith explants of Nicotiana glauca grown in vitro in synthetic medium s upplemented with 2,4 dichlorophenoxyacetic acid (2, 4 D), are induced to dedifferentiate. Treatment with actinomycin D within the first 4 - 8 h of culture (but not later) is lethal and the explants die, implyin g a requirement for de novo transcription. The genes expressed during the initial period of culture are presumably critical for subsequent c ell survival and proliferation, but so far their identity is unknown. We have constructed a subtractive cDNA library, enriched in sequences more abundant in dedifferentiating tissue than in pith. The subtractiv e library contains approximately seven major species, two of which, NG SUB7 and NGSUB8, are highly abundant. In Northern blots, these two hyb ridized to mRNA species whose abundance increased significantly but tr ansiently during the first 4 to 8 h of culture. The sequence of NGSUB7 showed no significant homology at a nucleotide or derived amino acid level with any previously reported sequence. NGSUB8 however, showed si gnificant homology over part of the derived amino acid sequence to sev eral yeast and bacterial proteins with DNA binding function. We propos e that the two recombinants represent transcripts from two novel genes edeA and edeB, which are expressed early in dedifferentiation.