FIG. 3.

Polysome profiles of wt and mutant selenoprotein P mRNAs. (A) Polysome profiles of mRNAs containing either SECIS 1 or SECIS 2 were assessed by sucrose gradient fractionation, followed by reverse transcription and PCR of gradient fractions, using primers specific for either SECIS element. The upper panel shows the gradient absorbance profile at 254 nm, with subunits, monosomes, and numbers of ribosomes indicated under each peak and the boundaries of collected fractions indicated across the bottom of the tracing. The middle panel shows the PCR products, and their quantitation by densitometry is shown in the lower panel. Arrows in the lower panel indicate the positions of the peaks of 80S monosomes and polysomes containing two, four, and eight ribosomes. (B) Polysome association for wt, UGU, and UGC no SECIS mRNAs. The upper panel shows the reverse transcription-PCR products of gradient fractions, and the lower panels show the quantitations of the products. Fractionation and quantitation were carried out as described above and in detail in Materials and Methods. Each experiment was carried out at least three times, with highly reproducible results.