RESUMEN
A method for identifying cDNA clones that hybridize to differentially expressed RNAs is described. Briefly, the RNA population in which the RNAs of interest are more abundant is used as a template for the synthesis of 35S-labeled cDNAs and another RNA population in which the RNAs of interest are less abundant is used as a template for the synthesis of 32P-labeled cDNAs. The labeled cDNAs are pooled and hybridized to plaque or colony lifts constructed from a cDNA library. Clones that hybridize to RNAs that are differentially expressed are identified using differential autoradiography/fluorography to discriminate between the 32P and 35S isotopes. We have used this method to identify cDNA clones that hybridize to mRNAs that are more abundant in the flowers of wild-type tomato than in the flowers of mutants that have low endogenous levels of gibberellins.
Asunto(s)
Sondas de ADN , Regulación de la Expresión Génica , Plantas/genética , ARN Mensajero/análisis , Autorradiografía/métodos , Células Clonales , ADN/genética , Genes , Técnicas Genéticas , Giberelinas/fisiología , Hibridación de Ácido Nucleico , Radioisótopos de Fósforo , Fotofluorografía/métodos , Poli A/genética , ARN Mensajero/genética , Radioisótopos de AzufreRESUMEN
A tomato (Lycopersicon esculentum) gene (GAST1) that encodes an RNA whose abundance increases > 20-fold in shoots of the GA-deficient gib1 mutant following spraying with GA3 has been characterized. An increase in GAST1 RNA levels is detectable 2 h after treatment and levels continue to increase for at least an additional 10 h. Between 12 and 24 h following treatment, the amount of GAST1 RNA begins to decline and at 48 h the level is nearly equivalent to that of water-treated control plants. Nuclear runoff analysis indicates that 8 h after treatment with GA3, transcription of the GAST1 gene has increased only threefold, suggesting that GA acts both transcriptionally and post-transcriptionally. ABA partially inhibits the GA-mediated increase in GAST1 RNA abundance while ethephon, kinetin, and 2,4-D have little effect. GAST1 RNA is detectable in untreated leaves, stems, petioles and flowers, but not in roots. The GAST1 gene encodes a 0.7 kb transcript. The sequence of the GAST1 cDNA and genomic clones indicates that the gene is interrupted by three introns and potentially encodes a 112 amino acid protein of unknown function.