Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1986 Nov;6(11):3973–3983. doi: 10.1128/mcb.6.11.3973

Developmental regulation of Dictyostelium discoideum actin gene fusions carried on low-copy and high-copy transformation vectors.

D A Knecht, S M Cohen, W F Loomis, H F Lodish
PMCID: PMC367161  PMID: 3025622

Abstract

The Dictyostelium discoideum genome contains an estimated 17 to 20 actin genes. We report the identification of a new member of this multigene family, actin 15, and its complete nucleotide sequence and transcription initiation sites. We constructed transformation vectors carrying either the actin 15 promoter fused to the neomycin phosphotransferase gene from transposon Tn903 or the actin 6 promoter fused to the neomycin phosphotransferase gene from Tn5. Cells transformed with the actin 15 vector carried less than five copies of vector DNA, while cells transformed with the actin 6 vector carried more than 200 copies. In both cases, the vector appeared to be integrated into the chromosome as a tandem array. Gene fusion RNAs transcribed from the actin 15 and actin 6 vectors were regulated like endogenous actin genes during D. discoideum development. DNA sequences required for temporal and cell type-specific regulation of these genes were contained within 2.8 kilobases of 5' noncoding DNA for actin 15 and 0.7 kilobases of 5' noncoding DNA for actin 6.

Full text

PDF
3973

Images in this article

3978Image
on p.3978
3979Image
on p.3979
3980Image
on p.3980
3980Image
on p.3980

Selected References

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

  1. Alton T. H., Brenner M. Comparison of proteins synthesized by anterior and posterior regions of Dictyostelium discoideum pseudoplasmodia. Dev Biol. 1979 Jul;71(1):1–7. doi: 10.1016/0012-1606(79)90077-0. [DOI] [PubMed] [Google Scholar]
  2. Alton T. H., Lodish H. F. Developmental changes in messenger RNAs and protein synthesis in Dictyostelium discoideum. Dev Biol. 1977 Oct 1;60(1):180–206. doi: 10.1016/0012-1606(77)90118-x. [DOI] [PubMed] [Google Scholar]
  3. Barklis E., Lodish H. F. In situ localization of actin mRNA in Dictyostelium discoideum aggregates. Exp Cell Res. 1985 Aug;159(2):479–486. doi: 10.1016/s0014-4827(85)80021-5. [DOI] [PubMed] [Google Scholar]
  4. Barklis E., Lodish H. F. Regulation of dictyostelium discoideum mRNAs specific for prespore or prestalk cells. Cell. 1983 Apr;32(4):1139–1148. doi: 10.1016/0092-8674(83)90297-0. [DOI] [PubMed] [Google Scholar]
  5. Barklis E., Pontius B., Barfield K., Lodish H. F. Structure of the promoter of the Dictyostelium discoideum prespore EB4 gene. Mol Cell Biol. 1985 Jun;5(6):1465–1472. doi: 10.1128/mcb.5.6.1465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blumberg D. D., Lodish H. F. Complexity of nuclear and polysomal RNAs in growing Dictyostelium discoideum cells. Dev Biol. 1980 Aug;78(2):268–284. doi: 10.1016/0012-1606(80)90336-x. [DOI] [PubMed] [Google Scholar]
  7. Cappello J., Zuker C., Lodish H. F. Repetitive Dictyostelium heat-shock promotor functions in Saccharomyces cerevisiae. Mol Cell Biol. 1984 Apr;4(4):591–598. doi: 10.1128/mcb.4.4.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Carter P., Bedouelle H., Winter G. Improved oligonucleotide site-directed mutagenesis using M13 vectors. Nucleic Acids Res. 1985 Jun 25;13(12):4431–4443. doi: 10.1093/nar/13.12.4431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  10. Chung S., Landfear S. M., Blumberg D. D., Cohen N. S., Lodish H. F. Synthesis and stability of developmentally regulated dictyostelium mRNAs are affected by cell--cell contact and cAMP. Cell. 1981 Jun;24(3):785–797. doi: 10.1016/0092-8674(81)90104-5. [DOI] [PubMed] [Google Scholar]
  11. Cohen S. M., Cappello J., Lodish H. F. Transcription of Dictyostelium discoideum transposable element DIRS-1. Mol Cell Biol. 1984 Nov;4(11):2332–2340. doi: 10.1128/mcb.4.11.2332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Coloma A., Lodish H. F. Synthesis of spore- and stalk-specific proteins during differentiation of Dictyostelium discoideum. Dev Biol. 1981 Jan 30;81(2):238–244. doi: 10.1016/0012-1606(81)90287-6. [DOI] [PubMed] [Google Scholar]
  13. Deininger P. L. Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal Biochem. 1983 Feb 15;129(1):216–223. doi: 10.1016/0003-2697(83)90072-6. [DOI] [PubMed] [Google Scholar]
  14. Devine K. M., Loomis W. F. Molecular characterization of anterior-like cells in Dictyostelium discoideum. Dev Biol. 1985 Feb;107(2):364–372. doi: 10.1016/0012-1606(85)90318-5. [DOI] [PubMed] [Google Scholar]
  15. Donahue T. F., Daves R. S., Lucchini G., Fink G. R. A short nucleotide sequence required for regulation of HIS4 by the general control system of yeast. Cell. 1983 Jan;32(1):89–98. doi: 10.1016/0092-8674(83)90499-3. [DOI] [PubMed] [Google Scholar]
  16. Firtel R. A., Cockburn A., Frankel G., Hershfield V. Structural organization of the genome of Dictyostelium discoideum: analysis by EcoR1 restriction endonuclease. J Mol Biol. 1976 Apr 25;102(4):831–852. doi: 10.1016/0022-2836(76)90294-1. [DOI] [PubMed] [Google Scholar]
  17. Firtel R. A., Timm R., Kimmel A. R., McKeown M. Unusual nucleotide sequences at the 5' end of actin genes in Dictyostelium discoideum. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6206–6210. doi: 10.1073/pnas.76.12.6206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fitzgerald M., Shenk T. The sequence 5'-AAUAAA-3'forms parts of the recognition site for polyadenylation of late SV40 mRNAs. Cell. 1981 Apr;24(1):251–260. doi: 10.1016/0092-8674(81)90521-3. [DOI] [PubMed] [Google Scholar]
  19. Fyrberg E. A., Mahaffey J. W., Bond B. J., Davidson N. Transcripts of the six Drosophila actin genes accumulate in a stage- and tissue-specific manner. Cell. 1983 May;33(1):115–123. doi: 10.1016/0092-8674(83)90340-9. [DOI] [PubMed] [Google Scholar]
  20. Hope I. A., Struhl K. GCN4 protein, synthesized in vitro, binds HIS3 regulatory sequences: implications for general control of amino acid biosynthetic genes in yeast. Cell. 1985 Nov;43(1):177–188. doi: 10.1016/0092-8674(85)90022-4. [DOI] [PubMed] [Google Scholar]
  21. Ito H., Fukuda Y., Murata K., Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. doi: 10.1128/jb.153.1.163-168.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jones K. A., Tjian R. Sp1 binds to promoter sequences and activates herpes simplex virus 'immediate-early' gene transcription in vitro. Nature. 1985 Sep 12;317(6033):179–182. doi: 10.1038/317179a0. [DOI] [PubMed] [Google Scholar]
  23. Kimmel A. R., Firtel R. A. Sequence organization in Dictyostelium: unique structure at the 5'-ends of protein coding genes. Nucleic Acids Res. 1983 Jan 25;11(2):541–552. doi: 10.1093/nar/11.2.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kindle K. L., Firtel R. A. Identification and analysis of Dictyostelium actin genes, a family of moderately repeated genes. Cell. 1978 Nov;15(3):763–778. doi: 10.1016/0092-8674(78)90262-3. [DOI] [PubMed] [Google Scholar]
  25. Loomis W. F., Gilpin M. E. Multigene families and vestigial sequences. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2143–2147. doi: 10.1073/pnas.83.7.2143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Margolskee J. P., Lodish H. F. The regulation of the synthesis of actin and two other proteins induced early in Dictyostelium discoideum development. Dev Biol. 1980 Jan;74(1):50–64. doi: 10.1016/0012-1606(80)90052-4. [DOI] [PubMed] [Google Scholar]
  27. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. McKeown M., Firtel R. A. Differential expression and 5' end mapping of actin genes in Dictyostelium. Cell. 1981 Jun;24(3):799–807. doi: 10.1016/0092-8674(81)90105-7. [DOI] [PubMed] [Google Scholar]
  29. McKeown M., Firtel R. A. Evidence for sub-families of actin genes in Dictyostelium as determined by comparisons of 3' end sequences. J Mol Biol. 1981 Oct 5;151(4):593–606. doi: 10.1016/0022-2836(81)90425-3. [DOI] [PubMed] [Google Scholar]
  30. McKeown M., Taylor W. C., Kindle K. L., Firtel R. A., Bender W., Davidson N. Multiple, heterogeneous actin genes in Dictyostelium. Cell. 1978 Nov;15(3):789–800. doi: 10.1016/0092-8674(78)90264-7. [DOI] [PubMed] [Google Scholar]
  31. McKnight S. L., Kingsbury R. C., Spence A., Smith M. The distal transcription signals of the herpesvirus tk gene share a common hexanucleotide control sequence. Cell. 1984 May;37(1):253–262. doi: 10.1016/0092-8674(84)90321-0. [DOI] [PubMed] [Google Scholar]
  32. Mehdy M. C., Ratner D., Firtel R. A. Induction and modulation of cell-type-specific gene expression in Dictyostelium. Cell. 1983 Mar;32(3):763–771. doi: 10.1016/0092-8674(83)90062-4. [DOI] [PubMed] [Google Scholar]
  33. Messing J., Crea R., Seeburg P. H. A system for shotgun DNA sequencing. Nucleic Acids Res. 1981 Jan 24;9(2):309–321. doi: 10.1093/nar/9.2.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Morrissey J. H., Devine K. M., Loomis W. F. The timing of cell-type-specific differentiation in Dictyostelium discoideum. Dev Biol. 1984 Jun;103(2):414–424. doi: 10.1016/0012-1606(84)90329-4. [DOI] [PubMed] [Google Scholar]
  35. Nellen W., Firtel R. A. High-copy-number transformants and co-transformation in Dictyostelium. Gene. 1985;39(2-3):155–163. doi: 10.1016/0378-1119(85)90309-9. [DOI] [PubMed] [Google Scholar]
  36. Nellen W., Silan C., Firtel R. A. DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion. Mol Cell Biol. 1984 Dec;4(12):2890–2898. doi: 10.1128/mcb.4.12.2890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ng R., Abelson J. Isolation and sequence of the gene for actin in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3912–3916. doi: 10.1073/pnas.77.7.3912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Oka A., Sugisaki H., Takanami M. Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol. 1981 Apr 5;147(2):217–226. doi: 10.1016/0022-2836(81)90438-1. [DOI] [PubMed] [Google Scholar]
  39. Pelham H. R. A regulatory upstream promoter element in the Drosophila hsp 70 heat-shock gene. Cell. 1982 Sep;30(2):517–528. doi: 10.1016/0092-8674(82)90249-5. [DOI] [PubMed] [Google Scholar]
  40. Pelham H. R., Bienz M. A synthetic heat-shock promoter element confers heat-inducibility on the herpes simplex virus thymidine kinase gene. EMBO J. 1982;1(11):1473–1477. doi: 10.1002/j.1460-2075.1982.tb01340.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Ratner D., Borth W. Comparison of differentiating Dictyostelium discoideum cell types separated by an improved method of density gradient centrifugation. Exp Cell Res. 1983 Jan;143(1):1–13. doi: 10.1016/0014-4827(83)90103-9. [DOI] [PubMed] [Google Scholar]
  42. Romans P., Firtel R. A. Organization of the Dictyostelium discoideum actin multigene family. Flanking sequences show subfamily homologies and unusual dyad symmetries. J Mol Biol. 1985 Jun 5;183(3):311–326. doi: 10.1016/0022-2836(85)90003-8. [DOI] [PubMed] [Google Scholar]
  43. Romans P., Firtel R. A. Organization of the actin multigene family of Dictyostelium discoideum and analysis of variability in the protein coding regions. J Mol Biol. 1985 Nov 20;186(2):321–335. doi: 10.1016/0022-2836(85)90108-1. [DOI] [PubMed] [Google Scholar]
  44. Romans P., Firtel R. A., Saxe C. L., 3rd Gene-specific expression of the actin multigene family of Dictyostelium discoideum. J Mol Biol. 1985 Nov 20;186(2):337–355. doi: 10.1016/0022-2836(85)90109-3. [DOI] [PubMed] [Google Scholar]
  45. Sanger F., Coulson A. R. The use of thin acrylamide gels for DNA sequencing. FEBS Lett. 1978 Mar 1;87(1):107–110. doi: 10.1016/0014-5793(78)80145-8. [DOI] [PubMed] [Google Scholar]
  46. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Southern P. J., Berg P. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J Mol Appl Genet. 1982;1(4):327–341. [PubMed] [Google Scholar]
  48. Staden R. Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing. Nucleic Acids Res. 1982 Aug 11;10(15):4731–4751. doi: 10.1093/nar/10.15.4731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Stinchcomb D. T., Thomas M., Kelly J., Selker E., Davis R. W. Eukaryotic DNA segments capable of autonomous replication in yeast. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4559–4563. doi: 10.1073/pnas.77.8.4559. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Struhl K., Stinchcomb D. T., Scherer S., Davis R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1035–1039. doi: 10.1073/pnas.76.3.1035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Tsang A. S., Mahbubani H., Williams J. G. Cell-type-specific actin mRNA populations in dictyostelium discoideum. Cell. 1982 Dec;31(2 Pt 1):375–382. doi: 10.1016/0092-8674(82)90131-3. [DOI] [PubMed] [Google Scholar]
  52. Tuchman J., Alton T., Lodish H. F. Preferential synthesis of actin during early development of the slime mold Dictyostelium discoideum. Dev Biol. 1974 Sep;40(1):116–128. doi: 10.1016/0012-1606(74)90113-4. [DOI] [PubMed] [Google Scholar]
  53. Vandekerckhove J., Weber K. Vegetative Dictyostelium cells containing 17 actin genes express a single major actin. Nature. 1980 Apr 3;284(5755):475–477. doi: 10.1038/284475a0. [DOI] [PubMed] [Google Scholar]
  54. Varnum B., Edwards K. B., Soll D. R. The developmental regulation of single-cell motility in Dictyostelium discoideum. Dev Biol. 1986 Jan;113(1):218–227. doi: 10.1016/0012-1606(86)90124-7. [DOI] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES