Amylase synthesis as a simple model system for translation and hybrid arrest in Xenopus oocytes.
A human alpha-amylase-encoding cDNA has been cloned in a transcription vector. When messenger RNA (mRNA) made in vitro from this construct was injected into Xenopus oocytes, amylase (AMY) activity was detected both in oocyte homogenates and in the incubation medium, indicating that the oocyte machinery correctly translated and processed the protein. Because AMY activity is easy to detect with a blue-starch assay, this expression system was used to determine the parameters of antisense oligodeoxyribonucleotide (oligo) inhibition of translation in the oocytes. Unique oligos complementary to the AMY mRNA sequence were effective in arresting translation, at approximately stoichiometric levels. Mixed oligos also inhibited translation, at levels that suggest that some mismatches may be tolerated in the formation of DNA-RNA hybrids. The AMY system provides a convenient probe of oocyte protein synthesis and processing machinery and can serve as a control substrate in investigations of other mRNAs.
Urnes
M S
MS
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City 84132.
Carroll
D
D
D000681:Amylases / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D016373:DNA, Antisense / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D009865:Oocytes / Q000378:metabolism*; D014176:Protein Biosynthesis; D014158:Transcription, Genetic; D014981:Xenopus
D016373:DNA, Antisense; D009838:Oligodeoxyribonucleotides; D000681:Amylases