IDENTIFICATION AND CHARACTERIZATION OF GENES WITH UNSTABLE TRANSCRIPTS (GUTS) IN TOBACCO

Plants and other higher eukaryotes have the ability to recognize and target specific transcripts for rapid decay from among the majority of relatively stable mRNAs present within cells. However, little is known about the nature of unstable transcripts in plants, or the mechanisms that facilitate their rapid degradation. As a first step toward understanding how plants distinguish between unstable and stable transcripts, a novel differential screen was used to identify cDNAs for genes with unstable transcripts (GUTs), solely on the basis of the instability of their mRNAs. cDNA probes were prepared from tobacco cells that had been depleted of highly unstable mRNAs by treatment for 90 min with a transcriptional inhibitor, and from control, untreated cells. GUT clones were selected on the basis of weak hybridization to the former probe relative to the latter probe. Half-life measurements performed on the mRNAs hybridizing to eight GUT clones indicated that each was unstable, with a half-life on the order of about an hour or less. All eight of the cDNAs corresponded to new tobacco genes, and four showed sequence similarity with genes from other species, including the eukaryotic family of DNAJ homologs, a tomato wound-inducible protein, and histone H3. In addition to providing information about the types of transcripts that are inherently unstable in plants, the GUT clones should provide excellent tools for the identification of cis- and trans-acting determinants of mRNA instability.