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Nucleic Acids Research, 1993, Vol. 21, No. 24 5742-5747
© 1993

MOLECULAR BIOLOGY

Identification of genes up-regulated in dedifferentiating Nicotania glauca pith tissue, using an improved method for constructing a subtractive cDNA library

Edi Cecchini1,3, Peter J. Dominy1, Chiara Geri3,4, Kim Kaiser2, John Sentry2 and Joel J. Milner*

Plant Molecular Science Group, Glasgow University Glasgow G12 8QQ, UK 1Department of Botany, Glasgow University Glasgow G12 8QQ, UK 2Department of Genetics, Glasgow University Glasgow G12 8QQ, UK 3Dipartimento di Biologia Delle Piante Agrarie-Sezione di Genetica Via Matteotti 1/B 4Istituto di Mutagenesi e Differenziamento Via Svezia 10, 56124 Pisa, Italy

*To whom correspondence should be addressed

Received August 3, 1993. Revised August 27, 1993. Accepted August 27, 1993.

Pith explants of Nicotiana glauca grown in vitro in synthetic medium supplemented 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, implying a requirement for de novo transcription. The genes expressed during the initial period of culture are presumably critical for subsequent cell 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 subtractive library contains approximately seven major species, two of which, NGSUB7 and NGSUB8, are highly abundant. In Northern blots, these two hybridized to mRNA species whose abundance increased significantly but transiently 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 significant homology over part of the derived amino acid sequence to several yeast and bacterial proteins with DNA binding function. We propose that the two recombinants represent transcripts from two novel genes edeA and edeB, which are expressed early in dedifferentiation.


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