1887

Abstract

Summary: We have developed a simple and accurate method to determine the amount of intact plasmid DNA taken up and retained by amoebae during various transformation protocols. We have used this method to compare the efficiency of three different methods for introducing foreign DNA into amoebae. Both a calcium phosphate and a spheroplast fusion procedure gave good uptake, but no intracellular plasmid DNA was detectable after calcium chloride treatment. The exogenous DNA was rapidly lost after transformation but was 20-fold more stable during starvation rather than growth conditions, suggesting a possible approach to improving transformation efficiency. No transient expression of neomycin phosphotransferase activity of any of the heterologous animal or plant promoters used could be detected using a sensitive gel assay procedure.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-131-12-3193
1985-12-01
2024-04-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/131/12/mic-131-12-3193.html?itemId=/content/journal/micro/10.1099/00221287-131-12-3193&mimeType=html&fmt=ahah

References

  1. Barclay S. L., Meller E. 1983; Efficient transformation of Dictyostelium discoideum amoebae.. Molecular and Cellular Biology 3:2117–2130
    [Google Scholar]
  2. Colbere-Garapin F., Horodniceanu F., Kourilsky P., Garapin A. -C. 1981; A new dominant hybrid selective marker for eukaryotic cells.. Journal of Molecular Biology 150:1–14
    [Google Scholar]
  3. Dimond R. L., Burns R. A., Jordan K. B. 1981; Secretion of lysosomal enzymes in the cellular slime mold Dictyostelium discoideum . Journal of Biological Chemistry 256:6565–6572
    [Google Scholar]
  4. Fregien N., Davidson N. 1985; Quantitative in situ gel electrophoretic assay for neomycin phosphotransferase activity in mammalian cell lysates.. Analytical Biochemistry (in the Press)
    [Google Scholar]
  5. Godfrey S. S., Sussman M. 1982; The genetics of development in Dictyostelium discoideum . Annual Review of Genetics 16:385–404
    [Google Scholar]
  6. Hirth K. -P., Edwards C. A., Firtel R. A. 1982; A DNA-mediated transformation system for Dictyostelium discoideum . Proceedings of the National Academy of Sciences of the United States of America 79:7356–7360
    [Google Scholar]
  7. Kimmel A. R., Firtel R. A. 1982; The organization of expression of the Dictyostelium genome.. In The Development of Dictyostelium discoideum pp 233–324 Edited by Loomis W. F. New York: Academic Press;
    [Google Scholar]
  8. Loomis W. F. (editor) 1982 The Development of Dictyostelium discoideum New York: Academic Press;
    [Google Scholar]
  9. Nellen W., Silan C., Firtel R. A. 1984; DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion.. Molecular and Cellular Biology 4:2890–2898
    [Google Scholar]
  10. Newell P. C. 1982; Genetics.. In The Development of Dictyostelium discoideum pp 35–70 Edited by Loomis W. F. New York: Academic Press;
    [Google Scholar]
  11. Ratner D. A., Ward T. E., Jacobson A. 1981; Evidence for the transformation of Dictyostelium discoideum with homologous DNA.. ICN-UCLA Symposia on Molecular and Cellular Biology 23:595–605
    [Google Scholar]
  12. Southern P., Berg P. 1982; Mammalian cell transformation with SV40 hybrid plasmid vectors.. In Eukaryotic Viral Vectors pp 41–45 Edited by Gluzman Y. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-131-12-3193
Loading
/content/journal/micro/10.1099/00221287-131-12-3193
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error