Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Dec 1;88(23):10845–10849. doi: 10.1073/pnas.88.23.10845

Efficient transformation of cam2, a behavioral mutant of Paramecium tetraurelia, with the calmodulin gene.

J A Kanabrocki 1, Y Saimi 1, R R Preston 1, W J Haynes 1, C Kung 1
PMCID: PMC53028  PMID: 1961754

Abstract

An Ile-136----Thr substitution in calmodulin reduces the Ca(2+)-dependent K+ currents of cam2, a behavioral mutant of Paramecium tetraurelia, and renders it overly susceptible to BaCl2. DNA fragments carrying the wild-type CAM gene injected into cam2 macronuclei reverted these phenotypes in the clonal descendants of the recipients. Tetrahymena telomeric sequences, added in vitro to the fragment termini before injection, enhanced the efficiency and quality of transformation. Five times 10(4) copies of such fragments consistently restored the phenotypes to near normal; even 10(3) or fewer copies could still effect weak transformation. The restored phenotypes were stable for greater than 20 fissions in many clones and were lost after autogamy. We examined the fate of the injected fragments in the transformed clones and discuss the possible application of this efficient transformation in the cloning of other genes of P. tetraurelia.

Full text

PDF
10849

Images in this article

10848Image
on p.10848
10848Image
on p.10848
10848Image
on p.10848

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ascenzioni F., Lipps H. J. A linear shuttle vector for yeast and the hypotrichous ciliate Stylonychia. Gene. 1986;46(1):123–126. doi: 10.1016/0378-1119(86)90174-5. [DOI] [PubMed] [Google Scholar]
  2. Blackburn E. H. The molecular structure of centromeres and telomeres. Annu Rev Biochem. 1984;53:163–194. doi: 10.1146/annurev.bi.53.070184.001115. [DOI] [PubMed] [Google Scholar]
  3. Bourgain F. M., Katinka M. D. Telomeres inhibit end to end fusion and enhance maintenance of linear DNA molecules injected into the Paramecium primaurelia macronucleus. Nucleic Acids Res. 1991 Apr 11;19(7):1541–1547. doi: 10.1093/nar/19.7.1541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Burke D. T., Carle G. F., Olson M. V. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science. 1987 May 15;236(4803):806–812. doi: 10.1126/science.3033825. [DOI] [PubMed] [Google Scholar]
  5. Gilley D., Preer J. R., Jr, Aufderheide K. J., Polisky B. Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei. Mol Cell Biol. 1988 Nov;8(11):4765–4772. doi: 10.1128/mcb.8.11.4765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Godiska R., Aufderheide K. J., Gilley D., Hendrie P., Fitzwater T., Preer L. B., Polisky B., Preer J. R., Jr Transformation of Paramecium by microinjection of a cloned serotype gene. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7590–7594. doi: 10.1073/pnas.84.21.7590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Haga N., Saimi Y., Takahashi M., Kung C. Intra- and interspecific complementation of membrane-inexcitable mutants of Paramecium. J Cell Biol. 1983 Aug;97(2):378–382. doi: 10.1083/jcb.97.2.378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Hinrichsen R. D., Amberger E., Saimi Y., Burgess-Cassler A., Kung C. Genetic analysis of mutants with a reduced Ca2+-dependent K+ current in Paramecium tetraurelia. Genetics. 1985 Nov;111(3):433–445. doi: 10.1093/genetics/111.3.433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kink J. A., Maley M. E., Preston R. R., Ling K. Y., Wallen-Friedman M. A., Saimi Y., Kung C. Mutations in paramecium calmodulin indicate functional differences between the C-terminal and N-terminal lobes in vivo. Cell. 1990 Jul 13;62(1):165–174. doi: 10.1016/0092-8674(90)90250-i. [DOI] [PubMed] [Google Scholar]
  10. Lefort-Tran M., Aufderheide K., Pouphile M., Rossignol M., Beisson J. Control of exocytotic processes: cytological and physiological studies of trichocyst mutants in Paramecium tetraurelia. J Cell Biol. 1981 Feb;88(2):301–311. doi: 10.1083/jcb.88.2.301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Lukas T. J., Wallen-Friedman M., Kung C., Watterson D. M. In vivo mutations of calmodulin: a mutant Paramecium with altered ion current regulation has an isoleucine-to-threonine change at residue 136 and an altered methylation state at lysine residue 115. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7331–7335. doi: 10.1073/pnas.86.19.7331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Preston R. R., Saimi Y., Kung C. Evidence for two K+ currents activated upon hyperpolarization of Paramecium tetraurelia. J Membr Biol. 1990 Apr;115(1):41–50. doi: 10.1007/BF01869104. [DOI] [PubMed] [Google Scholar]
  13. Preston R. R., Wallen-Friedman M. A., Saimi Y., Kung C. Calmodulin defects cause the loss of Ca2(+)-dependent K+ currents in two pantophobiac mutants of Paramecium tetraurelia. J Membr Biol. 1990 Apr;115(1):51–60. doi: 10.1007/BF01869105. [DOI] [PubMed] [Google Scholar]
  14. Tondravi M. M., Yao M. C. Transformation of Tetrahymena thermophila by microinjection of ribosomal RNA genes. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4369–4373. doi: 10.1073/pnas.83.12.4369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Zakian V. A. Structure and function of telomeres. Annu Rev Genet. 1989;23:579–604. doi: 10.1146/annurev.ge.23.120189.003051. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES