Abstract
The synthesis and secretion of α-amylase (EC 3.2.1.1) fromXenopus laevis oocytes injected with plasmids containing barley α-amylase complementary DNA (cDNA), genomic DNA, or synthetic α-amylase mRNA were studied. α-Amylase accumulated within the oocytes beginning 12 h after injection of DNA and in the medium 12 h later as a result of secretion. S1 mapping showed that the transcription of genomic DNA was initiated at the same site in oocytes as in barley aleurone, but that the transcription of cDNAs was less precise than that of genomic DNA. The α-amylase secreted by oocytes injected with either RNA or DNA had a molecular mass (Mr) of 44000 daltons (Da) and was indistinguishable from native barley α-amylase in size, isoelectric point, antigenicity and enzymatic activity. Isoelectric focussing showed that two enzymatically active isoforms of α-amylase were synthesized and secreted from oocytes injected with synthetic RNA or DNA. The results permit us to assign specific electrophoretic bands to specific cDNA clones. We conclude that theXenopus oocyte is a promising surrogate system for the study of transcription and translation of plant genes.
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Abbreviations
- bp:
-
nucleotide base pair(s)
- cDNA:
-
complementary DNA
- Da:
-
dalton
- GA3 :
-
gibberellic acid
- IEF:
-
isoelectric focussing
- kb:
-
kilobase(s)
- poly(A)+RNA:
-
polyadenylated RNA
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
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This research was supported by grants from the U.S. Department of Energy and the National Science Foundation to R.L. Jones. The kindness of Dr. John Gerhart in supplying Xenopus oocytes, the helpful comments of Dr. Richard Harland and Dr. Jill Deikman, and the assistance of Eleanor Crump in the preparation of this manuscript are gratefully acknowledged.
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Aoyagi, K., Sticher, L., Wu, M. et al. The expression of barley α-amylase genes inXenopus laevis oocytes. Planta 180, 333–340 (1990). https://doi.org/10.1007/BF01160388
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DOI: https://doi.org/10.1007/BF01160388