Abstract
Ribozymes are RNAs that can be designed to catalyze the specific cleavage or ligation of target RNAs. We have explored the possibility of using ribozymes in maize to downregulate the expression of the stearoyl-acyl carrier protein (Delta9) desaturase gene. Based on site accessibility and catalytic activity, several ribozyme constructs were designed and transformed into regenerable maize lines. One of these constructs, a multimer hammerhead ribozyme linked to a selectable marker gene, was shown to increase leaf stearate in two of 13 maize lines. There were concomitant decreases in Delta9 desaturase mRNA and protein. The plants with the altered stearate phenotype were shown to express ribozyme RNA. The ribozyme-mediated trait was heritable, as evidenced by stearate increases in the leaves of the R1 plants derived from a high-stearate line. The increase in stearate correlated with the presence of the ribozyme gene. A catalytically inactive version of this ribozyme did not produce any significant effect in transgenic maize. This is evidence that ribozymes can be used to modulate the expression of endogenous genes in maize.
Full Text
The Full Text of this article is available as a PDF (328.4 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Akagi H., Baba T., Shimada H., Fujimura T. Nucleotide sequence of a stearoyl-acyl carrier protein desaturase cDNA from developing seeds of rice. Plant Physiol. 1995 Jun;108(2):845–846. doi: 10.1104/pp.108.2.845. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bennett M. J., Cullimore J. V. Selective cleavage of closely-related mRNAs by synthetic ribozymes. Nucleic Acids Res. 1992 Feb 25;20(4):831–837. doi: 10.1093/nar/20.4.831. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Browse J., McCourt P. J., Somerville C. R. Fatty acid composition of leaf lipids determined after combined digestion and fatty acid methyl ester formation from fresh tissue. Anal Biochem. 1986 Jan;152(1):141–145. doi: 10.1016/0003-2697(86)90132-6. [DOI] [PubMed] [Google Scholar]
- Cameron F. H., Jennings P. A. Specific gene suppression by engineered ribozymes in monkey cells. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9139–9143. doi: 10.1073/pnas.86.23.9139. [DOI] [PMC free article] [PubMed] [Google Scholar]
- DeLeon D. V., Cox K. H., Angerer L. M., Angerer R. C. Most early-variant histone mRNA is contained in the pronucleus of sea urchin eggs. Dev Biol. 1983 Nov;100(1):197–206. doi: 10.1016/0012-1606(83)90211-7. [DOI] [PubMed] [Google Scholar]
- Douwe de Boer A., Weisbeek P. J. Chloroplast protein topogenesis: import, sorting and assembly. Biochim Biophys Acta. 1991 Nov 13;1071(3):221–253. doi: 10.1016/0304-4157(91)90015-o. [DOI] [PubMed] [Google Scholar]
- Fawcett T., Simon W. J., Swinhoe R., Shanklin J., Nishida I., Christie W. W., Slabas A. R. Expression of mRNA and steady-state levels of protein isoforms of enoyl-ACP reductase from Brassica napus. Plant Mol Biol. 1994 Oct;26(1):155–163. doi: 10.1007/BF00039528. [DOI] [PubMed] [Google Scholar]
- Forster A. C., Symons R. H. Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell. 1987 Apr 24;49(2):211–220. doi: 10.1016/0092-8674(87)90562-9. [DOI] [PubMed] [Google Scholar]
- Haseloff J., Gerlach W. L. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature. 1988 Aug 18;334(6183):585–591. doi: 10.1038/334585a0. [DOI] [PubMed] [Google Scholar]
- Herschlag D., Cech T. R. Catalysis of RNA cleavage by the Tetrahymena thermophila ribozyme. 1. Kinetic description of the reaction of an RNA substrate complementary to the active site. Biochemistry. 1990 Nov 6;29(44):10159–10171. doi: 10.1021/bi00496a003. [DOI] [PubMed] [Google Scholar]
- Hertel K. J., Pardi A., Uhlenbeck O. C., Koizumi M., Ohtsuka E., Uesugi S., Cedergren R., Eckstein F., Gerlach W. L., Hodgson R. Numbering system for the hammerhead. Nucleic Acids Res. 1992 Jun 25;20(12):3252–3252. doi: 10.1093/nar/20.12.3252. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Jarvis T. C., Alby L. J., Beaudry A. A., Wincott F. E., Beigelman L., McSwiggen J. A., Usman N., Stinchcomb D. T. Inhibition of vascular smooth muscle cell proliferation by ribozymes that cleave c-myb mRNA. RNA. 1996 May;2(5):419–428. [PMC free article] [PubMed] [Google Scholar]
- Knutzon D. S., Thompson G. A., Radke S. E., Johnson W. B., Knauf V. C., Kridl J. C. Modification of Brassica seed oil by antisense expression of a stearoyl-acyl carrier protein desaturase gene. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2624–2628. doi: 10.1073/pnas.89.7.2624. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koizumi M., Iwai S., Ohtsuka E. Construction of a series of several self-cleaving RNA duplexes using synthetic 21-mers. FEBS Lett. 1988 Feb 15;228(2):228–230. doi: 10.1016/0014-5793(88)80004-8. [DOI] [PubMed] [Google Scholar]
- Kruger K., Grabowski P. J., Zaug A. J., Sands J., Gottschling D. E., Cech T. R. Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell. 1982 Nov;31(1):147–157. doi: 10.1016/0092-8674(82)90414-7. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lindqvist Y., Huang W., Schneider G., Shanklin J. Crystal structure of delta9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins. EMBO J. 1996 Aug 15;15(16):4081–4092. [PMC free article] [PubMed] [Google Scholar]
- Matzke M. A., Matzke AJM. How and Why Do Plants Inactivate Homologous (Trans)genes? Plant Physiol. 1995 Mar;107(3):679–685. doi: 10.1104/pp.107.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McIntyre C. L., Bettenay H. M., Manners J. M. Strategies for the suppression of peroxidase gene expression in tobacco. II. In vivo suppression of peroxidase activity in transgenic tobacco using ribozyme and antisense constructs. Transgenic Res. 1996 Jul;5(4):263–270. doi: 10.1007/BF01972880. [DOI] [PubMed] [Google Scholar]
- Milligan J. F., Uhlenbeck O. C. Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol. 1989;180:51–62. doi: 10.1016/0076-6879(89)80091-6. [DOI] [PubMed] [Google Scholar]
- Napoli C., Lemieux C., Jorgensen R. Introduction of a Chimeric Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell. 1990 Apr;2(4):279–289. doi: 10.1105/tpc.2.4.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Perriman R., Graf L., Gerlach W. L. A ribozyme that enhances gene suppression in tobacco protoplasts. Antisense Res Dev. 1993 Fall;3(3):253–263. doi: 10.1089/ard.1993.3.253. [DOI] [PubMed] [Google Scholar]
- Pley H. W., Flaherty K. M., McKay D. B. Three-dimensional structure of a hammerhead ribozyme. Nature. 1994 Nov 3;372(6501):68–74. doi: 10.1038/372068a0. [DOI] [PubMed] [Google Scholar]
- Ruffner D. E., Stormo G. D., Uhlenbeck O. C. Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry. 1990 Nov 27;29(47):10695–10702. doi: 10.1021/bi00499a018. [DOI] [PubMed] [Google Scholar]
- Saghai-Maroof M. A., Soliman K. M., Jorgensen R. A., Allard R. W. Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci U S A. 1984 Dec;81(24):8014–8018. doi: 10.1073/pnas.81.24.8014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Scaringe S. A., Francklyn C., Usman N. Chemical synthesis of biologically active oligoribonucleotides using beta-cyanoethyl protected ribonucleoside phosphoramidites. Nucleic Acids Res. 1990 Sep 25;18(18):5433–5441. doi: 10.1093/nar/18.18.5433. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shanklin J., Somerville C. Stearoyl-acyl-carrier-protein desaturase from higher plants is structurally unrelated to the animal and fungal homologs. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2510–2514. doi: 10.1073/pnas.88.6.2510. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Slocombe S. P., Cummins I., Jarvis R. P., Murphy D. J. Nucleotide sequence and temporal regulation of a seed-specific Brassica napus cDNA encoding a stearoyl-acyl carrier protein (ACP) desaturase. Plant Mol Biol. 1992 Oct;20(1):151–155. doi: 10.1007/BF00029157. [DOI] [PubMed] [Google Scholar]
- Slocombe S. P., Piffanelli P., Fairbairn D., Bowra S., Hatzopoulos P., Tsiantis M., Murphy D. J. Temporal and tissue-specific regulation of a Brassica napus stearoyl-acyl carrier protein desaturase gene. Plant Physiol. 1994 Apr;104(4):1167–1176. doi: 10.1104/pp.104.4.1167. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
- Southern E. Gel electrophoresis of restriction fragments. Methods Enzymol. 1979;68:152–176. doi: 10.1016/0076-6879(79)68011-4. [DOI] [PubMed] [Google Scholar]
- Steinecke P., Herget T., Schreier P. H. Expression of a chimeric ribozyme gene results in endonucleolytic cleavage of target mRNA and a concomitant reduction of gene expression in vivo. EMBO J. 1992 Apr;11(4):1525–1530. doi: 10.1002/j.1460-2075.1992.tb05197.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thompson G. A., Scherer D. E., Foxall-Van Aken S., Kenny J. W., Young H. L., Shintani D. K., Kridl J. C., Knauf V. C. Primary structures of the precursor and mature forms of stearoyl-acyl carrier protein desaturase from safflower embryos and requirement of ferredoxin for enzyme activity. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2578–2582. doi: 10.1073/pnas.88.6.2578. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thompson J. D., Ayers D. F., Malmstrom T. A., McKenzie T. L., Ganousis L., Chowrira B. M., Couture L., Stinchcomb D. T. Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter. Nucleic Acids Res. 1995 Jun 25;23(12):2259–2268. doi: 10.1093/nar/23.12.2259. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Uhlenbeck O. C. A small catalytic oligoribonucleotide. Nature. 1987 Aug 13;328(6131):596–600. doi: 10.1038/328596a0. [DOI] [PubMed] [Google Scholar]
- Wegener D., Steinecke P., Herget T., Petereit I., Philipp C., Schreier P. H. Expression of a reporter gene is reduced by a ribozyme in transgenic plants. Mol Gen Genet. 1994 Nov 15;245(4):465–470. doi: 10.1007/BF00302259. [DOI] [PubMed] [Google Scholar]
- Werner M., Uhlenbeck O. C. The effect of base mismatches in the substrate recognition helices of hammerhead ribozymes on binding and catalysis. Nucleic Acids Res. 1995 Jun 25;23(12):2092–2096. doi: 10.1093/nar/23.12.2092. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wincott F., DiRenzo A., Shaffer C., Grimm S., Tracz D., Workman C., Sweedler D., Gonzalez C., Scaringe S., Usman N. Synthesis, deprotection, analysis and purification of RNA and ribozymes. Nucleic Acids Res. 1995 Jul 25;23(14):2677–2684. doi: 10.1093/nar/23.14.2677. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zaug A. J., Been M. D., Cech T. R. The Tetrahymena ribozyme acts like an RNA restriction endonuclease. Nature. 1986 Dec 4;324(6096):429–433. doi: 10.1038/324429a0. [DOI] [PubMed] [Google Scholar]
- Zuker M. On finding all suboptimal foldings of an RNA molecule. Science. 1989 Apr 7;244(4900):48–52. doi: 10.1126/science.2468181. [DOI] [PubMed] [Google Scholar]
- de Feyter R., Young M., Schroeder K., Dennis E. S., Gerlach W. A ribozyme gene and an antisense gene are equally effective in conferring resistance to tobacco mosaic virus on transgenic tobacco. Mol Gen Genet. 1996 Feb 25;250(3):329–338. doi: 10.1007/BF02174391. [DOI] [PubMed] [Google Scholar]
- van Hoof A., Green P. J. Premature nonsense codons decrease the stability of phytohemagglutinin mRNA in a position-dependent manner. Plant J. 1996 Sep;10(3):415–424. doi: 10.1046/j.1365-313x.1996.10030415.x. [DOI] [PubMed] [Google Scholar]
- van der Krol A. R., Mur L. A., Beld M., Mol J. N., Stuitje A. R. Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell. 1990 Apr;2(4):291–299. doi: 10.1105/tpc.2.4.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
