Plant Molecular Biology

, Volume 31, Issue 1, pp 1–12 | Cite as

Expression of the Volvox gene encoding nitrate reductase: Mutation-dependent activation of cryptic splice sites and intron-enhanced gene expression from a cDNA

  • Heribert Gruber
  • Stefan H. Kirzinger
  • Rüdiger Schmitt
Research Article


Use of the nitrate reductase encoding gene (nitA) as selection marker has facilitated the successful nuclear transformation of Volvox carteri. The Volvox nitA gene contains 10 introns. A stable nitA mutation in the Volvox recipient strain 153–81 resides in a G-to-A transition of the first nucleotide in the 5′ splice site of nitA intron 2. This mutation resulted in at least three non-functional splice variants, namely: (1) intron 2 was not spliced at all; (2) a cryptic 5′ splice site 60 nt upstream or (3) a cryptic 5′ splice site 16 nt downstream of the mutation were activated and used for splicing. When we used nitA cDNA (pVcNR13) for transformation of V. carteri 153–81, a low efficiency of about 5×10-5 transformants per reproductive cell was observed. Re-integration of either intron 1 (pVcNR15) or introns 9 and 10 (pVcNR16) in the transforming cDNA increased transformation rates to 5×10-4. In parallel, pVcNR15-transformed Volvox exhibited growth rates that were 100-fold increased over the pVcNR13-transformed alga. This intron-enhancement of nitA gene expression appears to be associated with post-transcriptional processing and ‘channelling’ of the message. These data suggest an important role of splicing for gene expression in V. carteri.

Key words

cryptic splice sites intron-enhancement gene expression nitA cDNA Volvox transformation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Adams CR, Stamer KA, Miller JK, McNally JG, Kirk MM, Kirk DL: Patterns of organellar and nuclear inheritance among progeny of two geographically isolated strains of Volvox carteri. Curr Genet 18: 141–153 (1990).CrossRefPubMedGoogle Scholar
  2. 2.
    Ahn AH, Kunkel LM: The structural and functional diversity of dystrophin. Nature Genet 3: 283–291 (1993).PubMedGoogle Scholar
  3. 3.
    Bornstein P, McKay J, Morishima JK, Devarayalu S, Gelinas RE: Regulatory elements in the first intron contribute to transcriptional control of the human alpha-1 (I) collagen gene. Proc Natl Acad Sci USA 84: 8869–8873 (1987).PubMedGoogle Scholar
  4. 4.
    Buttgereit D: Redundant enhancer elements guide beta 1 tubulin gene expression in apodemes during Drosophila embryogenesis. J Cell Sci 105: 721–727 (1993).PubMedGoogle Scholar
  5. 5.
    Cahoon EB, Shanklin J, Ohlrogge JB: Expression of a coriander desaturase results in petroselinic acid production in transgenic tobacco. Proc Natl Acad Sci USA 89: 11184–11188 (1992).PubMedGoogle Scholar
  6. 6.
    Callis J, Fromm M, Walbot V: Introns increase gene expression in cultured maize cells. Genes Devel 1: 1183–1200 (1987).PubMedGoogle Scholar
  7. 7.
    Cavalier-Smith T: Intron phylogeny: a new hypothesis. Trends Genet 7: 145–148 (1991).PubMedGoogle Scholar
  8. 8.
    Choi T, Huang M, Gorman C, Jaenisch R: A generic intron increases gene expression in transgenic mice. Mol Cell Biol 11: 3070–3074 (1991).PubMedGoogle Scholar
  9. 9.
    Daniel-Vedele F, Dorbe M-F, Caboche M, Rouzé P: Cloning and analysis of the tomato nitrate reductase-encoding gene: protein domain structure and amino acid homologies in higher plants. Gene 85: 371–380 (1989).CrossRefPubMedGoogle Scholar
  10. 10.
    Debuchy R, Purton S, Rochaix J-D: The argininosuccinate lyase gene of Chlamydomonas reinhardtii: an important tool for nuclear transformation and for correlating the genetic and molecular maps of the arg7 locus. EMBO J 8: 2803–2809 (1989).PubMedGoogle Scholar
  11. 11.
    Diener DR, Ang LH, Rosenbaum JL: Assembly of radial spoke proteins in Chlamydomonas: identification of the axoneme binding domain of radial spoke protein 3. J Cell Biol 123: 183–190 (1993).CrossRefPubMedGoogle Scholar
  12. 12.
    Dietmeier W, Fabry S, Schmitt R: DISEC-TRISEC: di-and trinucleotide sticky-end cloning of PCR-amplified DNA. Nucl Acids Res 21: 3603–3604 (1993).PubMedGoogle Scholar
  13. 13.
    Fernandez E, Schnell R, Ranum LWP, Hussey SC, Silflow CD, Lefebvre PA: Isolation and characterization of the nitrate reductase structural gene of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 86: 6449–6453 (1989).PubMedGoogle Scholar
  14. 14.
    Gallie DR, Young RE: The regulation of gene expression in transformed maize aleurone and endosperm protoplasts. Analysis of promoter activity, intron enhancement, and mRNA untranslated regions on expression. Plant Physiol 106: 929–939 (1994).CrossRefPubMedGoogle Scholar
  15. 15.
    Gilbert W: Genes-in-pieces revisited. Science 228: 823–824 (1985).PubMedGoogle Scholar
  16. 16.
    Goodenough UW: Green yeast. Cell 70: 533–538 (1992).CrossRefPubMedGoogle Scholar
  17. 17.
    Green MR: Biochemical mechanisms of constitutive and regulated pre-mRNA splicing. Annu Rev Cell Biol 7: 559–599 (1991).PubMedGoogle Scholar
  18. 18.
    Gruber H: Ph.D. dissertation, Universität Regensburg, Regensburg, Germany (1992).Google Scholar
  19. 19.
    Gruber H, Goetinck SD, Kirk DL, Schmitt R: The nitrate reductase-encoding gene of Volvox carteri: map location, sequence and induction kinetics. Gene 120: 75–83 (1992).CrossRefPubMedGoogle Scholar
  20. 20.
    Hasnain SE, Manavathu EK, Leung WC: DNA-mediated transformation of Chlamydomonas reinhardtii: use of aminoglycoside 3′-phosphotransferase as a selectable marker. Mol Cell Biol 5: 3647–3650 (1985).PubMedGoogle Scholar
  21. 21.
    Huang MT, Gorman CM: Intervening sequences increase efficiency of RNA 3′ processing and accumulation of cytoplasmic RNA. Nucl Acids Res 18: 937–947 (1990).PubMedGoogle Scholar
  22. 22.
    Huskey RJ, Semenkonich CF, Griffin BE, Cecil PO, Callahan AM, Chace KV, Kirk DL: Mutants of Volvox carteri affecting nitrogen assimilation. Mol Gen Genet 169: 157–161 (1979).CrossRefGoogle Scholar
  23. 23.
    Inoue K, Ohno M, Sakamoto H, Shimura Y: Effect of the cap structure on pre-mRNA splicing in Xenopus oocyte nuclei. Genes Devel 3: 1472–1479 (1989).PubMedGoogle Scholar
  24. 24.
    Jonsson JJ, Converse A, McIvor RS: An enhancer in the first intron of the human purine nucleoside phosphorylase gene. Gene 140: 187–193 (1992).CrossRefGoogle Scholar
  25. 25.
    Kindle KL, Schnell RA, Fernandez E, Lefebvre PA: Stable nuclear transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J Cell Biol 109: 2589–2601 (1989).CrossRefPubMedGoogle Scholar
  26. 26.
    Kirk DL, Kirk MM: Protein synthetic patterns during the asexual life cycle of Volvox carteri. Devel Biol 96: 493–506 (1983).Google Scholar
  27. 27.
    Luehrsen K, Taha S, Walbot V: Nuclear pre-mRNA processing in higher plants. Progr Nucl Acids Res 47: 149–193 (1993).Google Scholar
  28. 28.
    Luehrsen KR, Walbot V: Intron enhancement of gene expression and the splicing efficiency of introns in maize cells. Mol Gen Genet 225: 81–93 (1991).CrossRefPubMedGoogle Scholar
  29. 29.
    Luehrsen KR, Walbot V: Addition of A- and U-rich sequence increases the splicing efficiency of a deleted form of a maize intron. Plant Mol Biol 24: 449–463 (1994).CrossRefPubMedGoogle Scholar
  30. 30.
    Madhani HD, Guthrie C: Dynamic RNA-RNA interactions in the spliceosome. Annu Rev Genet 28: 1–26 (1994).CrossRefPubMedGoogle Scholar
  31. 31.
    Mages W, Salbaum JM, Harper JF, Schmitt R: Organization and structure of Volvox α-tubulin genes. Mol Gen Genet 213: 449–458 (1988).PubMedGoogle Scholar
  32. 32.
    McCullough AJ, Lou H, Schuler MA: Factors affecting authentic 5′ splice site selection in plant nuclei. Mol Cell Biol 13: 1323–1331 (1993).PubMedGoogle Scholar
  33. 33.
    Moore MJ, Query CC, Sharp PA: Splicing of precursors to mRNA by the spliceosome. In: Gesteland RF, Atkins JF (eds) The RNA World, pp. 303–358. Cold Spring Harbor Laboratory Press, Plainview, NY (1993).Google Scholar
  34. 34.
    Newman AJ, Lin R-J, Cheng L-C, Abelson J: Molecular consequences of specific intron mutations on yeast mRNA splicing in vivo and in vitro. Cell 42: 335–344 (1985).PubMedGoogle Scholar
  35. 35.
    Noh EW, Minocha SC: Expression of a human S-adenosylmethionine decarboxylase cDNA in transgenic tobacco and its effects on polyamine biosynthesis. Transgenic Res 3: 26–35 (1994).PubMedGoogle Scholar
  36. 36.
    Okkema PG, Harrison SW, Plunger V, Arayana A, Fire A: Sequence requirements for myosin gene expression and regulation in Caenorhabditis elegans. Genetics 135: 385–404 (1993).PubMedGoogle Scholar
  37. 37.
    Rochaix J-D, vanDillewijn J: Transformation of Chlamydomonas reinhardtii with yeast DNA. Nature 290: 70–72 (1982).Google Scholar
  38. 38.
    Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).Google Scholar
  39. 39.
    Schiedlmeier B, Schmitt R, Müller W, Kirk MM, Gruber H, Mages W, Kirk DL: Nuclear transformation of Volvox carteri. Proc Natl Acad Sci USA 91: 5080–5084 (1994).PubMedGoogle Scholar
  40. 40.
    Schmitt R, Fabry S, Kirk DL: In search of the molecular origins of cellular differentiation in Volvox and its relatives. Int Rev Cytol 139: 189–265 (1992).PubMedGoogle Scholar
  41. 41.
    Silflow CD, Chisholm RL, Conner TW, Ranum LP: The two α-tubulin genes of Chlamydomonas reinhardtii code for slightly different proteins. Mol Cell Biol 5: 2389–2398 (1985).PubMedGoogle Scholar
  42. 42.
    Solomonsen LB, Barber M: Assimilatory nitrate reductase: functional properties and regulation. Annu Rev Plant Physiol Mol Biol 41: 225–253 (1990).CrossRefGoogle Scholar
  43. 43.
    Spector DL: Higher order nuclear organization: three-dimensional distribution of small nuclear ribonucleoprotein particles. Proc Natl Acad Sci USA 87: 147–151 (1990).PubMedGoogle Scholar
  44. 44.
    Starr RC, Jaenicke L: Purification and characterization of the hormone initiating sexual morphogenesis in Volvox carteri f. nagariensis Iyengar. Proc Natl Acad Sci USA 71: 1050–1054 (1974).PubMedGoogle Scholar
  45. 45.
    Svaren J, Chalkley R: The structure and assembly of active chromatin. Trends Genet 6: 52–56 (1990).CrossRefPubMedGoogle Scholar
  46. 46.
    Talerico M, Berget SM: Effect of 5′ splice site mutations on the preceding intron. Mol Cell Biol 10: 6299–6305 (1990).PubMedGoogle Scholar
  47. 47.
    Tam L-W, Kirk DL: Identification of cell-type-specific genes of Volvox carteri and characterization of their expression during the asexual life cycle. Devel Biol 145: 51–66 (1991).Google Scholar
  48. 48.
    Vincent M, Caboche M: Constitutive expression of nitrate reductase allows normal growth and development of Nicotiana plumbaginifolia plants. EMBO J 10: 1027–1035 (1991).PubMedGoogle Scholar
  49. 49.
    Xuedong L, Mertz JE: Polyadenylation site selection cannot occur in vivo after excision of the 3′-terminal intron. Nucl Acids Res 21: 5256–5263 (1993).PubMedGoogle Scholar
  50. 50.
    Zimmerman L, Parr B, Lendahl U, Cunningham M, McKay R, Gavin B, Mann J, Vassileva G, McMahon A: Independent regulatory elements in the nestin gene direct transgene expression to neural stem cells or muscle precursors. Neuron 12: 11–24 (1994).CrossRefPubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Heribert Gruber
    • 1
  • Stefan H. Kirzinger
    • 1
  • Rüdiger Schmitt
    • 1
  1. 1.Lehrstuhl für GenetikUniversität RegensburgRegensburgGermany

Personalised recommendations