Abstract
The light-inducible nuclear gene coding for the small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco), produces a precursor protein with an amino-terminal transit peptide which is transported into the plastids and cleaved by a specific proteinase. To test whether the promoter and transit peptide-coding sequences of the small subunit gene can be used to direct the light-inducible synthesis and transport of a foreign protein into chloroplasts, a chimaeric gene was constructed consisting of the promoter, first exon and intron as well as part of the second exon of the small subunit Rubisco gene fused to the amino-terminal end of the neomycin phosphotransferase II gene, (nptII) of Tn5. Tobacco tissue, as well as whole plants, into which this chimaeric gene was introduced, were resistant to kanamycin. The transcription of the chimaeric gene as well as the NPTII activity of the resulting fusion protein were shown to be light inducible. The fusion protein is processed and located within the chloroplasts of the transformed plants.
Keywords: chloroplast protein transport, gene fusions, precursor proteins, plant cell transformation
Full text
PDFImages in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bailey J. M., Davidson N. Methylmercury as a reversible denaturing agent for agarose gel electrophoresis. Anal Biochem. 1976 Jan;70(1):75–85. doi: 10.1016/s0003-2697(76)80049-8. [DOI] [PubMed] [Google Scholar]
- Beck E., Ludwig G., Auerswald E. A., Reiss B., Schaller H. Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5. Gene. 1982 Oct;19(3):327–336. doi: 10.1016/0378-1119(82)90023-3. [DOI] [PubMed] [Google Scholar]
- Berry-Lowe S. L., Mc Knight T. D., Shah D. M., Meagher R. B. The nucleotide sequence, expression, and evolution of one member of a multigene family encoding the small subunit of ribulose-1,5-bisphosphate carboxylase in soybean. J Mol Appl Genet. 1982;1(6):483–498. [PubMed] [Google Scholar]
- Bohnert H. J., Crouse E. J., Pouyet J., Mucke H., Löffelhardt W. The subcellular localization of DNA components from Cyanophora paradoxa, a flagellate containing endosymbiotic cyanelles. Eur J Biochem. 1982 Aug;126(2):381–388. doi: 10.1111/j.1432-1033.1982.tb06791.x. [DOI] [PubMed] [Google Scholar]
- Broglie R., Coruzzi G., Fraley R. T., Rogers S. G., Horsch R. B., Niedermeyer J. G., Fink C. L., Chua N. H. Light-regulated expression of a pea ribulose-1,5-bisphosphate carboxylase small subunit gene in transformed plant cells. Science. 1984 May 25;224(4651):838–843. doi: 10.1126/science.6719112. [DOI] [PubMed] [Google Scholar]
- Capaldi R. A., Vanderkooi G. The low polarity of many membrane proteins. Proc Natl Acad Sci U S A. 1972 Apr;69(4):930–932. doi: 10.1073/pnas.69.4.930. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Caplan A., Herrera-Estrella L., Inzé D., Van Haute E., Van Montagu M., Schell J., Zambryski P. Introduction of genetic material into plant cells. Science. 1983 Nov 18;222(4625):815–821. doi: 10.1126/science.222.4625.815. [DOI] [PubMed] [Google Scholar]
- Cashmore A. R. Structure and expression of a pea nuclear gene encoding a chlorophyll a/b-binding polypeptide. Proc Natl Acad Sci U S A. 1984 May;81(10):2960–2964. doi: 10.1073/pnas.81.10.2960. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
- Chua N. H., Schmidt G. W. Post-translational transport into intact chloroplasts of a precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6110–6114. doi: 10.1073/pnas.75.12.6110. [DOI] [PMC free article] [PubMed] [Google Scholar]
- De Block M., Herrera-Estrella L., Van Montagu M., Schell J., Zambryski P. Expression of foreign genes in regenerated plants and in their progeny. EMBO J. 1984 Aug;3(8):1681–1689. doi: 10.1002/j.1460-2075.1984.tb02032.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- De Greve H., Dhaese P., Seurinck J., Lemmers M., Van Montagu M., Schell J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene. J Mol Appl Genet. 1982;1(6):499–511. [PubMed] [Google Scholar]
- Dhaese P., De Greve H., Gielen J., Seurinck L., Van Montagu M., Schell J. Identification of sequences involved in the polyadenylation of higher plant nuclear transcripts using Agrobacterium T-DNA genes as models. EMBO J. 1983;2(3):419–426. doi: 10.1002/j.1460-2075.1983.tb01439.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dron M., Rahire M., Rochaix J. D., Mets L. First DNA sequence of a chloroplast mutation: a missense alteration in the ribulosebisphosphate carboxylase large subunit gene. Plasmid. 1983 May;9(3):321–324. doi: 10.1016/0147-619x(83)90009-4. [DOI] [PubMed] [Google Scholar]
- Fraley R. T., Rogers S. G., Horsch R. B., Sanders P. R., Flick J. S., Adams S. P., Bittner M. L., Brand L. A., Fink C. L., Fry J. S. Expression of bacterial genes in plant cells. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4803–4807. doi: 10.1073/pnas.80.15.4803. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hamer D. H., Leder P. Splicing and the formation of stable RNA. Cell. 1979 Dec;18(4):1299–1302. doi: 10.1016/0092-8674(79)90240-x. [DOI] [PubMed] [Google Scholar]
- Herrera-Estrella L., Van den Broeck G., Maenhaut R., Van Montagu M., Schell J., Timko M., Cashmore A. Light-inducible and chloroplast-associated expression of a chimaeric gene introduced into Nicotiana tabacum using a Ti plasmid vector. Nature. 1984 Jul 12;310(5973):115–120. doi: 10.1038/310115a0. [DOI] [PubMed] [Google Scholar]
- Joos H., Inzé D., Caplan A., Sormann M., Van Montagu M., Schell J. Genetic analysis of T-DNA transcripts in nopaline crown galls. Cell. 1983 Apr;32(4):1057–1067. doi: 10.1016/0092-8674(83)90290-8. [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]
- Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ortiz W., Reardon E. M., Price C. A. Preparation of chloroplasts from euglena highly active in protein synthesis. Plant Physiol. 1980 Aug;66(2):291–294. doi: 10.1104/pp.66.2.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reiss B., Sprengel R., Schaller H. Protein fusions with the kanamycin resistance gene from transposon Tn5. EMBO J. 1984 Dec 20;3(13):3317–3322. doi: 10.1002/j.1460-2075.1984.tb02297.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Reiss B., Sprengel R., Will H., Schaller H. A new sensitive method for qualitative and quantitative assay of neomycin phosphotransferase in crude cell extracts. Gene. 1984 Oct;30(1-3):211–217. doi: 10.1016/0378-1119(84)90122-7. [DOI] [PubMed] [Google Scholar]
- Robinson C., Ellis R. J. Transport of proteins into chloroplasts. The precursor of small subunit of ribulose bisphosphate carboxylase is processed to the mature size in two steps. Eur J Biochem. 1984 Jul 16;142(2):343–346. doi: 10.1111/j.1432-1033.1984.tb08292.x. [DOI] [PubMed] [Google Scholar]
- Schmidt G. W., Bartlett S. G., Grossman A. R., Cashmore A. R., Chua N. H. Biosynthetic pathways of two polypeptide subunits of the light-harvesting chlorophyll a/b protein complex. J Cell Biol. 1981 Nov;91(2 Pt 1):468–478. doi: 10.1083/jcb.91.2.468. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schmidt G. W., Devillers-Thiery A., Desruisseaux H., Blobel G., Chua N. H. NH2-terminal amino acid sequences of precursor and mature forms of the ribulose-1,5-bisphosphate carboxylase small subunit from Chlamydomonas reinhardtii. J Cell Biol. 1979 Dec;83(3):615–622. doi: 10.1083/jcb.83.3.615. [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]
- Spreitzer R. J., Ogren W. L. Rapid recovery of chloroplast mutations affecting ribulosebisphosphate carboxylase/oxygenase in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A. 1983 Oct;80(20):6293–6297. doi: 10.1073/pnas.80.20.6293. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stiekema W. J., Wimpee C. F., Tobin E. M. Nucleotide sequence encoding the precursor of the small subunit of ribulose 1,5-bisphosphate carboxylase from Lemna gibba L.G-3. Nucleic Acids Res. 1983 Nov 25;11(22):8051–8061. doi: 10.1093/nar/11.22.8051. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thomas P. S. Hybridization of denatured RNA transferred or dotted nitrocellulose paper. Methods Enzymol. 1983;100:255–266. doi: 10.1016/0076-6879(83)00060-9. [DOI] [PubMed] [Google Scholar]
- Van Haute E., Joos H., Maes M., Warren G., Van Montagu M., Schell J. Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J. 1983;2(3):411–417. doi: 10.1002/j.1460-2075.1983.tb01438.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Willmitzer L., Dhaese P., Schreier P. H., Schmalenbach W., Van Montagu M., Schell J. Size, location and polarity of T-DNA-encoded transcripts in nopaline crown gall tumors; common transcripts in octopine and nopaline tumors. Cell. 1983 Apr;32(4):1045–1056. doi: 10.1016/0092-8674(83)90289-1. [DOI] [PubMed] [Google Scholar]
- Zambryski P., Joos H., Genetello C., Leemans J., Montagu M. V., Schell J. Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J. 1983;2(12):2143–2150. doi: 10.1002/j.1460-2075.1983.tb01715.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zurawski G., Perrot B., Bottomley W., Whitfeld P. R. The structure of the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase from spinach chloroplast DNA. Nucleic Acids Res. 1981 Jul 24;9(14):3251–3270. doi: 10.1093/nar/9.14.3251. [DOI] [PMC free article] [PubMed] [Google Scholar]