Abstract
The synthesis and secretion of α-amylase (EC 3.2.1.1) from Xenopus 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 the Xenopus 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
References
Bush, D.S., Cornejo, M.-J., Huang, C.-N., Jones, R.L. (1986) Ca2+-stimulated secretion of α-amylase during development in barley aleurone protoplasts. Plant Physiol 82, 566–576
Chandler, P.M., Zwar, J.A., Jacobsen, J.V., Higgins, T.J.V., Inglis, A.S. (1984) The effect of gibberellic acid and abscisic acid on α-amylase mRNA levels in barley aleurone layers. Studies using an α-amylase cDNA clone. Plant Mol. Biol. 3, 407–418
Colman, A. (1984) Translation of eukaryotic messenger RNA in Xenopus oocytes and expression of exogenous DNA in Xenopus oocytes. In: Transcription and translation, a practical approach, pp. 49–69, Ames, B.D., Higgins, S.J., eds. IRL Press, Washington
Deikman, J., Jones, R.L. (1985) Control of α-amylase mRNA accumulation by gibberellic acid and calcium in barley aleurone layers. Plant Physiol. 78, 192–198
Fincher, G.B. (1989) Molecular and cellular biology associated with endosperm mobilization in germinating cereal grains. Annu. Rev. Plant Physiol. Plant Mol. Biol. 40, 305–346
Gatenby, A.A., Boccara, M., Baulcombe, D.C., Rothstein, S.J. (1986) Expression of a wheat α-amylase gene in Escherichia coli: recognition of the translational initiation site and the signal peptide. Gene 45, 11–18
Gurdon, J.B., Wickens, M.P. (1983) The use of Xenopus oocytes for the expression of cloned genes. Methods Enzymol. 101, 370–386
Gurdon, J.B., Woodland, H.R., Lingrel, J.B. (1974) The translation of mammalian globin mRNA injected into fertilized eggs of Xenopus laevis. Dev. Biol. 39, 125–133
Heupke, H.-J., Robinson, D.G. (1985) Intracellular transport of α-amylase in barley aleurone cells: evidence for the participation of the Golgi apparatus. Eur. J. Cell Biol. 39, 265–272
Huttly, A.K., Baulcombe, D.C. (1989) A wheat α-Amy2 promoter is regulated by gibberellin in transformed oat aleurone protoplasts. EMBO J. 8, 1907–1913
Jacobsen, J.V., Bush, D.S., Sticher, L., Jones, R.L. (1988) Evidence for precursor forms of the low isoelectric point α-amylase isozymes secreted by barley aleurone cells. Plant Physiol. 88, 1168–1174
Jones, R.L., Jacobsen, J.V. (1982) The role of the endoplasmic reticulum in the synthesis and transport of α-amylase in barley aleurone layers. Planta 156, 421–432
Jones, R.L., Bush, D.S., Sticher, L., Simon, P., Jacobsen, J.V. (1987) Intracellular transport and secretion of barley aleurone α-amylase. In: Plant membranes: structure, function, biogenesis, pp. 325–340, Sze, H., Leaver, C., eds. Liss, New York
Krieg, P., Strachan, R., Wallis, E., Tabe, L., Colman, A. (1984) Efficient expression of cloned complementary DNAs for secretory proteins after injection into Xenopus oocytes. J. Mol. Biol. 180, 615–643
Kursheed, B., Rogers, J.C. (1988) Barley α-amylase genes: quantitative comparison of steady-state mRNA levels from individual members of the two different families expressed in aleurone cells. J. Biol. Chem. 263, 18953–18960
Laemmli, U. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685
MacGregor, E.A., MacGregor, A.W. (1987) Studies of cerebral α-amylase using cloned DNA. CRC Crit. Rev. Biotechnol. 5, 129–142
Melroy, D., Jones, R.L. (1986) The effect of monensin on intracellular transport and secretion of α-amylase isoenzymes in barley aleurone. Planta 167, 252–259
Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K., Green, M.R. (1984) Efficient in vitro synthesis of biologically active RNA and DNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 121, 7035–7056
Mertz, J.E., Gurdon, J.B. (1977) Purified DNAs are transcribed after microinjection into Xenopus oocytes. Proc. Natl. Acad. Sci. USA 74, 1502–1506
O'Keefe, E., Bennett, V. (1980) Use of immunoglobulin-loaded protein A-bearing Staphylococci as a primary solid phase immun-oabsorbent in radioimmunoassay. J. Biol. Chem. 255, 561–568
Opresko, L.K., Karpf, R.A. (1987) Specific proteolysis regulates fusion between endocytic compartments in Xenopus oocytes. Cell 51, 557–568
Poreman, R.C., Judah, J.D. (1987) The processing and secretion of rat serum albumin by oocytes from Xenopus laevis. FEBS Lett. 219, 75–78
Rogers, J.C. (1985) Two barley α-amylase genes are regulated differently in aleurone cells. J. Biol. Chem. 260, 3731–3738
Rogers, J.C., Milliman, C. (1983) Isolation and sequence analysis of a barley α-amylase cDNA clone. J. Biol. Chem. 258, 2370–2375
Rothstein, S.J., Lazarus, C.M., Smith, W.E., Baulcombe, D.C., Gatenby, A.A. (1984) Secretion of a wheat α-amylase expressed in yeast. Nature 308, 662–665
Rothstein, S.J., Lahners, K.N., Lazarus, C.M., Baulcombe, D.C., Gatenby, A.A. (1987) Synthesis and secretion of wheat α-amylase in Saccharomyces cerevisiae. Gene 55, 353–356
Simon, P., Jones, R.L. (1988) Synthesis and secretion of catalytically active barley α-amylase isoforms by Xenopus oocytes injected with barley mRNAs. Eur. J. Cell Biol. 47, 213–221
Weselake, R.J., MacGregor, A.W., Hill, R.D. (1983) An endogenous α-amylase inhibitor in barley kernels. Plant Physiol. 72, 809–812
Wickens, M.P., Gurdon, J.B. (1983) Post-transcriptional processing of simian virus 40 late transcripts in injected frog oocytes. J. Mol. Biol. 163, 1–26
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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 in Xenopus laevis oocytes. Planta 180, 333–340 (1990). https://doi.org/10.1007/BF00198784
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DOI: https://doi.org/10.1007/BF00198784