Molecular and General Genetics MGG

, Volume 238, Issue 3, pp 339–349 | Cite as

Functional in vivo analyses of the 3′ flanking sequences of the Chlamydomonas chloroplast rbcL and psaB genes

  • Alan D. Blowers
  • Uwe Klein
  • George S. Ellmore
  • Lawrence Bogorad
Originals Articles

Abstract

Possible roles of untranslated sequences at the 3′ ends of chloroplast genes, which include inverted repeat elements, were investigated in Chlamydomonas reinhardtii in vivo. Chlamydomonas chloroplast rbcL or psaB 3′ flanking regions were coupled in various arrangements 3′ to a chimeric gene consisting of a Chlamydomonas chloroplast atpB promoter sequence fused 5′ to the Escherichia coli uidA (GUS) structural gene. These genes were introduced into the Chlamydomonas chloroplast genome at the same location by homologous recombination following microprojectile bombardment. Transformants harboring chimeric GUS genes fused to rbcL or psaB gene 3′ inverted repeat sequences in their normal forward orientations accumulated GUS transcripts of a single size, whereas GUS transcripts of heterogenous sizes accumulated in transformants harboring the same gene lacking an inverted repeat sequence at its 3′ end. Thus, the 3′ flanking regions of the rbcL and psaB genes can define the location of the 3′ terminus of a transcript in vivo. In chloroplast transformants harboring chimeric GUS genes fused to multiple inverted repeat sequences in their normal forward orientations, only GUS transcripts accumulated that were terminated by the first inverted repeat sequence. The latter data suggest that the 3′ ends of these RNAs are the products of either transcription termination or endonucleolytic cleavage. Analyses of GUS transcripts in transformants harboring GUS genes terminated by rbcL or psaB gene 3′ flanking regions in reversed orientations indicate that transcript 3′ end formation in vivo requires nucleotide sequences located outside the inverted repeat elements. Inasmuch as decay rates of GUS transcripts were found to be independent of the presence of a 3′ inverted repeat sequence, RNA stabilization does not appear to be a major in vivo function of these elements in the Chlamydomonas chloroplast transcripts studied.

Key words

GUS reporter gene RNA 3′ and formation RNA stability Inverted repeat RNA stem-loop structure 

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References

  1. Adams CC, Stern DB (1990) Control of mRNA stability in chloroplasts by 3′ inverted repeats: effects of stem and loop mutations on degradation of psbA mRNA in vitro. Nucleic Acids Res 18:6003–6010Google Scholar
  2. Baker EJ, Schloss JA, Rosenbaum JL (1984) Rapid changes in tubulin RNA synthesis and stability induced by deflagellation in Chlamydomonas. J Cell Biol 99:2074–2081Google Scholar
  3. Bedbrook JR, Link G, Coen DM, Bogorad L, Rich A (1978) Maize plastid gene expressed during photoregulated development. Proc Natl Acad Sci USA 75:3060–3064Google Scholar
  4. Blowers AD, Bogorad L, Shark KB, Sanford JC (1989) Studies on Chlamydomonas chloroplast transformation: Foreign DNA can be stably maintained in the chromosome. Plant Cell 1:123–132Google Scholar
  5. Blowers AD, Ellmore GS, Klein U, Bogorad L (1990) Transcriptional analysis of endogenous and foreign genes in chloroplast transformants of Chlamydomonas. Plant Cell 2:1059–1070Google Scholar
  6. Boynton JE, Gillham NW, Harris EH, Hosler JP, Johnson AM, Jones AR, Randolph-Anderson BL; Robertson D, Klein TM, Shark KB, Sanford JC (1988) Chloroplast transformation in Chlamydomonas with high velocity microprojectiles. Science 240:1534–1538Google Scholar
  7. Brawerman G (1987) Determinants of messenger RNA stability. Cell 48:5–6Google Scholar
  8. Chen L-J, Orozco EM Jr (1988) Recognition of prokaryotic transcription terminators by spinach chloroplast RNA polymerase. Nucleic Acids Res 16:8411–8431Google Scholar
  9. Cheng S-WC, Lynch EC, Leason KR, Court DL, Shapiro BA, Friedman DI (1991) Functional importance of sequence in the stem-loop of a transcription terminator. Science 254:1205–1207Google Scholar
  10. Christopher DA, Kim M, Mullet JE (1992) A novel light-regulated promoter is conserved in cereal and dicot chloroplasts. Plant Cell 4:785–798Google Scholar
  11. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: Version 11. Plant Mol Biol Rep 1: 19–21Google Scholar
  12. Deng X-W, Gruissem W (1987) Control of plastid gene expression during development: The limited role of transcriptional regulation. Cell 49:379–387Google Scholar
  13. Dron M, Rahire M, Rochaix J-D (1982) Sequence of the chloroplast DNA region of Chlamydomonas reinhardii containing the large subunit of ribulose bisphosphate carboxylase and parts of its flanking genes. J Mol Biol 162:775–793Google Scholar
  14. Haley J, Bogorad L (1990) Alternative promoters are used for genes within maize chloroplast polycistronic transcription units. Plant Cell 2:323–333Google Scholar
  15. Herrin DL, Michaels AS, Paul A-L (1986) Regulation of genes encoding the large subunit of ribulose-1,5-bisphosphate carboxylase and the photosystem II polypeptides D-1 and D-2 during the cell cycle of Chlamydomonas reinhardtii. J Cell Biol 103:1837–1845Google Scholar
  16. Hsu-Ching C, Stern DB (1991a) Specific binding of chloroplast proteins in vitro to the 3′ untranslated region of spinach chloroplast petD mRNA. Mol Cell Biol 11:4380–4388Google Scholar
  17. Hsu-Ching C, Stern DB (1991b) Specific ribonuclease activities in spinach chloroplasts promote mRNA maturation and degradation. J Biol Chem 266:24205–24211Google Scholar
  18. Jefferson RA (1987) Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep 5: 387–405Google Scholar
  19. Jefferson RA, Burgess SM, Hirsh D (1986) β-Glucuronidase from Escherichia coli as a gene-fusion marker. Proc Natl Acad Sci USA 83:8447–8451Google Scholar
  20. Klein RR, Mullet JE (1990) Light-induced transcription of chloroplast genes. psbA transcription is differentially enhanced in illuminated barley. J Biol Chem 265:1895–1902Google Scholar
  21. Klein U, De Camp JD, Bogorad L (1992) Two types of chloroplast gene promoters in Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 89:3453–3457Google Scholar
  22. Krupinska K (1992) Transcriptional control of plastid gene expression during development of primary foliage leaves of barley grown under a daily light-dark regime. Planta 186:294–303Google Scholar
  23. Len S, White D, Michaels A (1990) Cell cycle-dependent transcriptional and post-transcriptional regulation of chloroplast gene expression in Chlamydomonas reinhardtii. Biochim Biophys Acta 1049:311–317Google Scholar
  24. Merchant S, Bogorad L (1986) Regulation by copper of the expression of plastocyanin and cytochrome c 552 in Chlamydomonas reinhardtii. Mol Cell Biol 6:462–469Google Scholar
  25. Mullet JE (1988) Chloroplast development and gene expression. Annu Rev Plant Physiol Plant Mol Biol 39:475–502Google Scholar
  26. Mullet JE, Klein RR (1987) Transcription and RNA stability are important determinants of higher plant chloroplast RNA levels. EMBO J 6:1571–1579Google Scholar
  27. Nickelsen J, Link G (1989) Interaction of a 3′ RNA region of the mustard trnK gene with chloroplast proteins. Nucleic Acids Res 17:9637–9648Google Scholar
  28. Nickelsen J, Link G (1991) RNA-protein interactions at transcript 3′ ends and evidence for trnK-psbA cotranscription in mustard chloroplasts. Mol Gen Genet 228:89–96Google Scholar
  29. Piechulla B, Gruissem W (1987) Diurnal mRNA fluctuations of nuclear and plastid genes in developing tomato fruits. EMBO J 6:3593–3599Google Scholar
  30. Platt T (1986) Transcription termination and the regulation of gene expression. Annu Rev Biochem 55:339–372Google Scholar
  31. Rodermel SR, Bogorad L (1985) Maize plastid photogenes: Mapping and photoregulation of transcript levels during lightinduced development. J Cell Biol 100:463–476Google Scholar
  32. Sager R, Granick S (1953) Nutritional studies with Chlamydomonas reinhardi. Ann NY Acad Sci 56:831–838Google Scholar
  33. Salvador ML, Klein U, Bogorad L (1993a) Light-regulated and endogenous fluctuations of chloroplast transcript levels in Chlamydomonas. Regulation by transcription and RNA degradation. Plant J, in pressGoogle Scholar
  34. Salvador ML, Klein U, Bogorad L (1993b) 5′ sequences are important positive and negative determinants of the longevity of Chlamydomonas chloroplast transcripts. Proc Natl Acad Sci USA, in pressGoogle Scholar
  35. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  36. Schrubar H, Wanner G, Westhoff P (1990) Transcriptional control of plastid gene expression in greening Sorghum seedlings. Planta 183:101–111Google Scholar
  37. Schuster G, Gruissem W (1991) Chloroplast mRNA 3′ end processing requires a nuclear-encoded RNA-binding protein. EMBO J 10:1493–1502Google Scholar
  38. Stern DB, Gruissem W (1987) Control of plastid gene expression: 3′ inverted repeats act as mRNA processing and stabilizing elements, but do not terminate transcription. Cell 51:1145–1157Google Scholar
  39. Stern DB, Jones H, Gruissem W (1989) Function of plastid mRNA 3′ inverted repeats. RNA stabilization and gene-specific protein binding. J Biol Chem 264:18742–18750Google Scholar
  40. Stern DB, Radwanski ER, Kindle KL (1991) A 3′ stem/loop structure of the Chlamydomonas chloroplast atpB gene regulates mRNA accumulation in vivo. Plant Cell 3:285–297Google Scholar
  41. Sueoka N (1960) Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardi. Proc Natl Acad Sci USA 46:83–91Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Alan D. Blowers
    • 1
  • Uwe Klein
    • 1
  • George S. Ellmore
    • 1
  • Lawrence Bogorad
    • 1
  1. 1.Department of Cellular and Developmental BiologyHarvard UniversityCambridgeUSA

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