Reduced Pol III Target Gene Expression in slj Larvae (A) Total tRNA levels are reduced relative to the amount of 5.8S rRNA (a gene transcribed by Pol I) in pooled 4-dpf and 5-dpf slj larvae compared with wild-type (WT) siblings, whereas levels of 5S rRNA, another Pol III target gene product, are normal. Identical RNA samples analyzed in (A and B). tRNA and 5S rRNA graphs represent an average of two measurements. Error bars indicate standard deviation. (B) Quantitative PCR data showing reduced levels of a tRNAleu gene in pooled 3-dpf and 5-dpf homozygous slj larvae compared with wild-type siblings relative to expression of hprt, a Pol II target gene. (C) Reduced levels of the Pol III target gene product 7SL RNA in pooled 4-dpf and 5-dpf slj larvae compared with wild-type siblings (as determined by quantitative real-time PCR). tRNAleu and 7SL expression are relative to the expression of the Pol II target gene hprt. (D) Northern analyses showing that the levels of mature tRNAleu and tRNAile transcripts are reduced 48% and 36% relative to U1 snRNA (a Pol II target gene) transcript levels in pooled 5-dpf slj larvae. This analysis also suggests a small reduction in slj 5S rRNA levels, although 5S rRNA levels relative to 5.8S RNA levels were normal when quantified by gel electrophoresis (Figure 5B). (E) Northern analyses showing reduced pre-tRNAile levels relative to U1 snRNA (unpublished data), in pooled 5-dpf slj versus sibling wild-type (WT) larvae. The pre-tRNAile transcript is the same size (89 bp) in slj and wild-type larvae. (M is a molecular weight marker.) (F) Quantitative real-time PCR data showing reduced pre-tRNAleu and pre-tRNAile expression relative to the Pol II target gene hprt in pooled 5-dpf slj versus pooled sibling wild-type larvae.
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