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Enzymatic Removal of Intervening Sequences in the Synthesis of Yeast tRNAs

Richard C. Ogden, Gayle Knapp, Craig L. Peebles, Hyan S. Kang, Jacques S. Beckmann, Peter F. Johnson, Shella A. Fuhrman, John N. Abelson


The phenomenon of noncolinearity between a gene and its mature product has been shown to be a general one in the eukaryotic world. This discovery raised the question of how the cell removes the intervening sequences in the biosynthesis of RNA. Some answers to this question are presented here. The discovery by Hopper et al. (1978) that yeast tRNA precursors accumulate in a mutant strain (ts136) has considerably facilitated the study of the RNA splicing reaction. This mutant, isolated by Hutchison et al. (1969), defines the rna1 gene of yeast. It is presumed to be defective in a step in RNA transport from nucleus to cytoplasm. At the nonpermissive temperature, the 35S rRNA precursor accumulates (Hopper et al. 1978), the appearance of mRNA in the cytoplasm is halted, poly(A)-containing RNA accumulates in the nucleus (Shiokawa and Pogo 1974), and a particular subset of tRNA precursors accumulates (Knapp et al. 1978).

The separation of those tRNA precursors that accumulate in ts 136 has been accomplished by two-dimensional polyacrylamide gel electrophoresis. A typical two-dimensional separation is shown in Figure 1. Originally the precursor-specific spots were identified by hybridization of the RNA to a set of Escherichia coli recombinant plasmid clones, each of which carries one or more yeast tRNA genes (Beckmann et al. 1977). Five of the RNAs (spots indicated in Fig. 1) hybridized to clones that have been identified as containing genes for tRNATyr, tRNAPhe, tRNA3Leu, tRNAUCGSer, and tRNATrp. These identifications have been subsequently confirmed by RNA sequence analysis. Four other...

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