FIG. 5.

TFIIIA with serine-16 replaced with glutamic acid fails to reactivate transcription of oocyte-type 5S rRNA genes in early embryos. Embryos were injected when the first cleavage furrow began to appear with capped mRNA encoding wild-type TFIIIA or the indicated mutant TFIIIA along with [α-³²P]UTP. (A) Embryos were collected at the beginning of gastrulation (stage 10), total RNA was isolated, and 3 embryo equivalents was analyzed by electrophoresis and autoradiography. Lanes: 1, embryos injected with [α-³²P]UTP only; 2 to 4, embryos injected with mRNA encoding wild-type TFIIIA or S16A or S16E mutant TFIIIA, respectively. (B) Autoradiographs of replicates of this experiment were scanned with a laser densitometer. The amount of 5S rRNA in each lane relative to the indicated band of tRNA, which serves as an internal control, is presented. Error bars indicate the standard deviation of the mean, and n is the number of independent measurements. C, control; WT, wild type. (C) The amount of ectopically expressed TFIIIA in injected embryos was determined with a Western blot assay. Whole-cell extract corresponding to one embryo was analyzed in each lane. Lanes: 1, purified TFIIIA; 2, embryos injected with [α-³²P]UTP only; 3 to 5, embryos injected with radiolabel and mRNA encoding wild-type TFIIIA or the S16A or S16E variant of TFIIIA, respectively.