Skip to main content
Log in

Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library

  • Short Communication
  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

We report the rescue of an arginine-requiring mutant (arg7-8) of Chlamydomonas reinhardtii by complementation using total DNA from a genomic cosmid library. Using the glass-bead transformation method of Kindle [8] four putative transformants able to grow in the absence of exogenous arginine were obtained from 3×109 treated cells. Southern blot analysis reveals that at least three of the clones have acquired an additional copy of the gene (ARG7) encoding argininosuccinate lyase (ASL). The arginine-independent phenotype is stable in the absence of selective pressure and high levels of ASL activity are detected in all four clones. We conclude that these represent true transformants and that any stable nuclear mutant of Chlamydomonas could be rescued using this approach.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

References

  1. Bates P: Double cos site vectors: simplified cosmid cloning. Meth Enzymol 153: 82–94 (1987).

    PubMed  Google Scholar 

  2. Clarke L, Carbon J: Selection of specific clones from colony banks by suppression or complementation tests. Meth Enzymol 68: 396–408 (1979).

    PubMed  Google Scholar 

  3. 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).

    PubMed  Google Scholar 

  4. DiLella AG, Woo SLC: Cloning large segments of genomic DNA using cosmid vectors. Meth Enzymol 153: 199–212 (1987).

    PubMed  Google Scholar 

  5. Farrell K, Overton S: Characterization of argininosuccinate lyase (EC 4.3.2.1) from Chlamydomonas reinhardtii. Biochem J 242: 261–266 (1987).

    PubMed  Google Scholar 

  6. Harris EH: The Chlamydomonas Sourcebook: A Comprehensive Guide to Biology and Laboratory Use. Academic Press, San Diego, CA (1989).

    Google Scholar 

  7. Jiang XM, Himanshu N, Quigley NB, Reeves PR: A low copy number cosmid. Plasmid 18: 170–172 (1987).

    PubMed  Google Scholar 

  8. Kindle KL: High frequency nuclear transformation of Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 87: 1228–1232 (1990).

    PubMed  Google Scholar 

  9. Kindle KL, Schnell RA, Fernández E, Lefebvre PA: Stable transformation of Chlamydomonas using the Chlamydomonas gene for nitrate reductase. J Cell Biol 109: 2589–2601 (1989).

    Article  PubMed  Google Scholar 

  10. Kioussis D, Wilson E, Daniels C, Leveton C, Taverne J, Playfair JHL: Expression and rescuing of a cloned human tumour necrosis factor gene using an EBV-based shuttle cosmid vector. EMBO J 6: 355–361 (1987).

    PubMed  Google Scholar 

  11. Konvalinkova V, Matagne RF, Loppes R: Induction and analysis of revertants from various arg-7 mutants lacking argininosuccinate lyase in Chlamydomonas reinhardii. Mutat Res 24: 69–72 (1974).

    PubMed  Google Scholar 

  12. Kurnit DM: Escherichia coli recA deletion strains that are highly competent for transformation and in vivo phage packaging. Gene 82: 313–315 (1989).

    Article  PubMed  Google Scholar 

  13. Mayfield SP, Kindle KL: Stable nuclear transformation of Chlamydomonas reinhardtii using a C. reinhardtii gene as the selectable marker. Proc Natl Acad Sci USA 87: 2087–2091 (1990).

    PubMed  Google Scholar 

  14. Sambrook J, Frisch EF, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

    Google Scholar 

  15. Smart EJ, Selman BR: Complementation of a Chlamydomonas reinhardtii mutant defective in the nuclear gene encoding the chloroplast coupling factor (CF1) γ-subunit (atpC). J Bioenerg Biomemb 3: 275–284 (1993).

    Google Scholar 

  16. Tam LW, Lefebvre PA: Cloning of flagellar genes in Chlamydomonas reinhardtii by DNA insertional mutagenesis. Genetics 135: 375–384 (1993).

    PubMed  Google Scholar 

  17. Triagla T, Peterson MG, Kemp DJ: A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucl Acids Res 16: 8186–8186 (1988).

    PubMed  Google Scholar 

  18. Weeks DP, Beerman N, Griffith OM: A small-scale five-hour procedure for isolating multiple samples of CsCl-purified DNA: application to isolations from mammalian, insect, higher plant, algal, yeast and bacterial sources. Anal Biochem 152: 376–385 (1986).

    PubMed  Google Scholar 

  19. Zilsel J, Ma PH, Beatty JT: Derivation of a mathematical expression useful for the construction of complete genomic libraries. Gene 120: 89–92 (1992).

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Rights and permissions

Reprints and permissions

About this article

Cite this article

Purton, S., Rochaix, JD. Complementation of a Chlamydomonas reinhardtii mutant using a genomic cosmid library. Plant Mol Biol 24, 533–537 (1994). https://doi.org/10.1007/BF00024121

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00024121

Key words

Navigation