Abstract
Two experimental approaches demonstrate that different types of RNA complementary to α-amylase mRNA are present in barley. S1 nuclease assays identify an RNA that is complementary to essentially the full length of both the type A and type B α-amylase mRNAs. Complementarity, however, is imperfect: the S1 nuclease-resistant products can only be identified if they are electrophoresed as RNA-DNA hybrids. This RNA is present in developing endosperm + aleurone tissue and in mature aleurone tissue cultured in the absence of hormonal treatment or in the presence of abscisic acid, but not in shoot or root tissue. In mature aleurone tissue treated with abscisic acid, its steady-state abundance is similar to that of α-amylase mRNA. Northern blot analysis indicated the presence of a second type of antisense RNA. Under conditions of moderate stringency, antisense-specific probes detect discrete hybridizing species of 1.6, 1.4, and 1.0 kilobases in mature aleurone and shoot tissues that do not represent spurious “hybridization” to rRNA, α-amylase mRNA, or the abundant, G+C-rich mRNA for a probable amylase/protease inhibitor. The different results are consistent with the fact that the hybridization assay can tolerate relatively short regions of complementarity separated by large, nonhomologous sequences, while the nuclease protection assay cannot.
Similar content being viewed by others
References
Berk AJ, Sharp PA: Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell 12: 721–732 (1977).
Burke JF, High-sensitivity S1 mapping with single-stranded [32P] DNA probes synthesized from bacteriophage M13mp templates. Gene 30: 63–68 (1984).
Brown AHD, Jacobsen JV: Genetic basis and natural variation of α-amylase isozymes in barley: Genet Res Camb 40: 315–324 (1982).
Chandler PM, Zwar JA, Jacobsen JV, Higgins TJV, Inglis AS: The effects 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 (1984).
Crouch ML, Sussex IM: Development and storage-protein synthesis in Brassica napus L. embryos in vivo and in vitro. Planta 153: 64–74 (1981).
Crowley TE, Nellen W, Gomer RG, Firtel RA: Phenocopy of discoidin I-minus mutants by antisense transformation in dictyostelium. Cell 43: 633–641 (1985).
Dretzen G, Bellard M, Sassone-Corsi P, Chambon P: A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem 112: 295–298 (1981).
Favaloro JM, Treisman RH, Kamen R: Transcription maps of polyoma virus-specific RNA: analysis by two-dimensional nuclease S1 mapping. Meth Enzymol 65: 718–749 (1980).
Finkelstein RR, Tenbarge KM, Shumway JE, Crouch ML: Role of ABA in maturation of rapessed embryos. Plant Physiol 78: 630–636 (1985).
Harland R, Weintraub H: Translation of mRNA injected into Xenopus oocytes is specifically inhibited by antisense RNA. J Cell Biol 101: 1094–1099 (1985).
Higgins TJV, Zwar JA, Jacobsen JV: Gibberellic acid enhances the level of translatable mRNA for α-amylase in barley aleurone layers. Nature 260: 166–169 (1976).
Higgins TJV, Jacobsen JV, Zwar JA: Gibberellic acid and abscisic acid modulate protein synthesis and mRNA levels in barley aleurone layers. Plant Mol Biol 1: 191–215 (1982).
Izant JG, Weintraub H: Inhibition of thymidine kinase gene expression by anti-sense RNA: a molecular approach to genetic analysis. Cell 36: 1007–1015 (1984).
Izant JG, Weintraub H: Constitutive and conditional suppression of exogenous and endogenous genes by anti-sense RNA. Science 229: 345–352 (1985).
Jackson PJ, Lark KG: Ribosomal RNA synthesis in soybean suspension cultures growing in different media. Plant Physiol 69: 234–239 (1982).
Jacobsen JV, Beach LR: Control of transcription of α-amylase and rRNA genes in barley aleurone protoplasts by gibberellin and abscisic acid. Nature 316: 275–277 (1986).
Jeppesen PGN: A method for separating DNA fragments by electrophoresis in polyacrylamide concentration gradient slab gels. Anal Biochem 58: 195–207 (1974).
Kim SK, Wold BJ: Stable reduction of thymidine kinase activity in cells expressing high levels of anti-sense RNA. Cell 42: 129–138 (1985).
Melton DA: Injected antisense RNAs specifically block messenger RNA translation in vivo. Proc Natl Acad Sci USA 82: 144–148 (1985).
Mozer TJ: Control of protein synthesis in barley aleurone layers by the plant hormones gibberellic acid and abscisic acid. Cell 20: 479–485 (1983).
Mundy J, Svedsen I, Hejgaard J: Barley α-amylase/subtilisin inhibitor. I. Isolation and characterization. Carlsberg Res Commun 48: 81–90 (1983).
Mundy J, Rogers JC: Selective expression of a probable amylase/protease inhibitor in barley aleurone cells: comparison to the barley amylase/subtilisin inhibitor. Planta 169: 51–63 (1986).
Muthukrishnan S, Chandra GR, Maxwell ES: Hormonal control of α-amylase gene expression in barley. Studies using a cloned cDNA probe. J Biol Chem 258: 2370–2375 (1983).
Rogers JC, Milliman C: Isolation and sequence analysis of a barley α-amylase cDNA clone. J Biol Chem 258: 8169–8174 (1983).
Rogers JC, Milliman C: Coordinate increase in major transcripts from the high pI α-amylase multigene family in barley aleurone cells stimulated with gibberellic acid. J Biol Chem 259: 12234–12240 (1984).
Rogers JC: Two barley α-amylase gene families are regulated differently in aleurone cells. J Biol Chem 260: 3731–3738 (1985).
Rosenberg UB, Preiss A, Seifert E, Jäckle H, Knipple DC: Production of phenocopies by Krüppel antisense RNA injection into Drosophila embryos. Nature 313: 703–706 (1985).
Varner JE, Ho DT: The role of hormones in the initiation of seedling growth. In: Papaconstantinou J (ed.) The Molecular Biology of Hormone Action, pp. 173–194. Academic Press, New York (1976).
Weselake RJ, MacGregor AW, Hill RD: An endogenous α-amylase inhibitor in barley kernels. Plant Physiol 72: 809–812 (1983).
Whittier RH, Dean DA, Rogers JC: Nucleotide sequence analysis of alpha-amylase and thiol protease genes that are hormonally regulated in barley aleurone cells. Nucleic Acids Res 15: 2515–2535 (1987).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Rogers, J.C. RNA complementary to α-amylase mRNA in barley. Plant Mol Biol 11, 125–138 (1988). https://doi.org/10.1007/BF00015665
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00015665