Microinjection of tRNA into amphibian oocytes.
The microinjection technique affords us the possibility to introduce purified components into living cells and to answer the question of what effects the change introduced has on cellular metabolism. This technique can therefore be used to test the hypothesis that transfer RNA plays a regulatory role in cellular protein synthesis. Prior to these experiments it is important, however, to test whether transfer RNA microinjected into amphibian oocytes is stable and functional inside this cells. These two questions are answered affirmatively in this report. The stability of tRNA was tested by following the content of TCA precipitable counts inside the oocytes at different times after microinjection of radioactive yeast and E. coli tRNA and by polyacrilamide gel electrophoresis of the material recovered from the cell. The results clearly indicate that tRNAs are resistant to the action of occyte ribonucleases that degrade other RNAs such as 5S RNA. The functionality of the injected tRNA was tested by assaying the intracellular aminoacylation of microinjected yeast tRNA. The aminoacylation of bulk yeast (3H) tRNA introduced into Xenopus laevis oocytes was tested by the capacity of the material recovered 5 hours after injection into the cell to form a ternary complex with wheat protein synthesis elongation factor 1 and GTP. The complex only forms with aminoacyl-tRNA and not with unacylated tRNA. This method showed that at least 80% of the tRNA introduced into the cell was aminoacylated in vivo. A direct assay for internal aminoacylation made use of microinjection of pure tRNAPhe and subsequent determination by phenol extraction of (14C)Phe-tRNA content of oocytes that had been incubated for 2 hours in a medium containing (14C)phenylalanine. The results obtained showed that the oocytes could internally aminoacylate 200-500 times more tRNAPhe that the cell normally contains. Appropiate controls demonstrated that the aminoacylation was aminoacid and tRNA specific and that periodate oxidized tRNAPhe could not be in vivo aminoacylated but tRNAPhe deprived of its Y base could accept the aminoacid. A brief study demonstrated that bulk yeast tRNA and tRNAPhe without its Y base did not inhibit endogenous protein synthesis but a similar amount of tRNAPhe caused 50% inhibition and periodate-oxidized tRNAPhe a 95% inhibition.
D000663:Amphibians; D000818:Animals; D005260:Female; D008845:Microinjections / Q000379:methods*; D009865:Oocytes / Q000032:analysis / Q000378:metabolism*; D010063:Ovum / Q000378:metabolism*; D014176:Protein Biosynthesis; D012343:RNA, Transfer / Q000032:analysis / Q000378:metabolism*; D013268:Stimulation, Chemical; D015003:Yeasts / Q000378:metabolism*
D012343:RNA, Transfer