The isolation of ecdysterone inducible genes by hybridization subtraction chromatography

Abstract

We have developed a procedure for selectively enriching a mRNA population for inducible sequences. Other than the induced mRNA species, the population of mRNA in control cells is approximately the same as the mRNA population in induced cells. Cytoplasmic mRNA from control cells is bound to oligo (dT)-cellulose and used as a template for reverse transcriptase, the oligo (dT) serving as a primer. After removing the template mRNAs, the cDNA-cellulose column is used to hybridize a population of mRNAs from induced cells. The non-hybridized poly A+ RNAs are greatly enriched in the inducible sequences. We have used this technique of hybridization subtraction chromatography to select a mRNA population enriched for the mRNAs inducible by ecdysterone in Schneider's Line 2 Drosophila cells. This population of RNAs was used to screen a recombinant library. Preliminary results indicate that approximately 10% of the RNA in the probe population represents ecdysterone inducible sequences. Methods are described for optimizing the cDNA synthesis reaction (we obtain greater than or equal to 30% efficiency) and hybridizing RNA to the cDNA-cellulose resin. This method can be used to select induced mRNAs regardless of the way in which the induction is brought about.