Figure 3

PNRC stimulates the transcription of tRNA ^arg gene by Pol III. MCF7 breast cancer cells (A) were transiently transfected with 10 μg of reporter plasmid pArg-maxi which contains a Drosophila tRNA^arg gene and increasing amounts (0, 5, or 10 μg) of PNRC expression plasmid, pSG5-PNRC. Cells were cultured for 24 hours and the tRNA^arg transcripts in 1 μg of total RNA isolated from the transfected cells was determined by RNase protection assay using a ³²P-labeled antisense riboprobe. An example of an autoradiogram of tRNA^arg transcript (indicated by an arrow) and quantification of the levels of tRNA^arg mRNA from three independent experiments are shown. B, Western blot analysis of PNRC protein in MCF7/vector and MCF7/PNRC stably transfected cells. The procedures for transfection, G418 selection, and individual clone screening were described in Methods. An aliquot of cell lysate, from MCF7/vector or MCF7/PNRC cells, equal to 100 μg of protein was analysed by Western blot using PNRC antiserum (1:500 dilution) or anti-actin antibody (1:1000 dilution) as previously described [2]. The protein bands with molecular weights corresponding to those of PNRC or actin were indicated by arrows. C, MCF-7/vector or MCF7/PNRC stable expression cells were transiently transfected with 10 μg of reporter plasmid pArg-maxi. Twenty-four hours after transfection, the tRNA^arg mRNA levels in the transfected cells were analysed by RNase protection assay as described for A. An example of an autoradiogram of tRNA^arg transcript (indicated by an arrow) and quantification of the levels of tRNA^arg mRNA from three independent experiments are shown.