Skip to main content
NCBI 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
. 1992 Jul 1;89(13):6152–6156. doi: 10.1073/pnas.89.13.6152

Enhancer of splitD, a dominant mutation of Drosophila, and its use in the study of functional domains of a helix-loop-helix protein.

K Tietze 1, N Oellers 1, E Knust 1
PMCID: PMC402140  PMID: 1631102

Abstract

Helix-loop-helix proteins play important roles in developmental processes, such as myogenesis, neurogenesis, and sex determination. The gene Enhancer of split [E(spl)] of Drosophila, a member of a gene complex that is involved in early neurogenesis, encodes a protein with a basic domain and a helix-loop-helix motif. We took advantage of a dominant mutation of this gene, E(spl)D, to define in vivo structural features of this protein for proper function. The mutation renders the otherwise recessive eye phenotype of spl dominant. By germ-line transformation of different in vitro mutagenized versions of the E(spl) gene, we could demonstrate that the basic domain of this helix-loop-helix protein is functional and necessary for expression of the dominant phenotype. These results are supported by in vitro DNA-binding assays, which showed that the basic domain is in fact necessary for DNA binding, despite the presence of a proline residue. Furthermore, we could show that the dominant enhancement of spl is caused by truncation of the E(SPL)D protein in combination with deletion of a putative regulatory element.

Full text

PDF
6152

Images in this article

6153Image
on p.6153
6153Image
on p.6153
6154Image
on p.6154
6155Image
on p.6155

Selected References

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

  1. Alonso M. C., Cabrera C. V. The achaete-scute gene complex of Drosophila melanogaster comprises four homologous genes. EMBO J. 1988 Aug;7(8):2585–2591. doi: 10.1002/j.1460-2075.1988.tb03108.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Artavanis-Tsakonas S., Simpson P. Choosing a cell fate: a view from the Notch locus. Trends Genet. 1991 Nov-Dec;7(11-12):403–408. doi: 10.1016/0168-9525(91)90264-q. [DOI] [PubMed] [Google Scholar]
  3. Benezra R., Davis R. L., Lockshon D., Turner D. L., Weintraub H. The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell. 1990 Apr 6;61(1):49–59. doi: 10.1016/0092-8674(90)90214-y. [DOI] [PubMed] [Google Scholar]
  4. Cai M., Davis R. W. Yeast centromere binding protein CBF1, of the helix-loop-helix protein family, is required for chromosome stability and methionine prototrophy. Cell. 1990 May 4;61(3):437–446. doi: 10.1016/0092-8674(90)90525-j. [DOI] [PubMed] [Google Scholar]
  5. Caudy M., Vässin H., Brand M., Tuma R., Jan L. Y., Jan Y. N. daughterless, a Drosophila gene essential for both neurogenesis and sex determination, has sequence similarities to myc and the achaete-scute complex. Cell. 1988 Dec 23;55(6):1061–1067. doi: 10.1016/0092-8674(88)90250-4. [DOI] [PubMed] [Google Scholar]
  6. Davis R. L., Cheng P. F., Lassar A. B., Weintraub H. The MyoD DNA binding domain contains a recognition code for muscle-specific gene activation. Cell. 1990 Mar 9;60(5):733–746. doi: 10.1016/0092-8674(90)90088-v. [DOI] [PubMed] [Google Scholar]
  7. Ellis H. M., Spann D. R., Posakony J. W. extramacrochaetae, a negative regulator of sensory organ development in Drosophila, defines a new class of helix-loop-helix proteins. Cell. 1990 Apr 6;61(1):27–38. doi: 10.1016/0092-8674(90)90212-w. [DOI] [PubMed] [Google Scholar]
  8. Garrell J., Campuzano S. The helix-loop-helix domain: a common motif for bristles, muscles and sex. Bioessays. 1991 Oct;13(10):493–498. doi: 10.1002/bies.950131002. [DOI] [PubMed] [Google Scholar]
  9. Garrell J., Modolell J. The Drosophila extramacrochaetae locus, an antagonist of proneural genes that, like these genes, encodes a helix-loop-helix protein. Cell. 1990 Apr 6;61(1):39–48. doi: 10.1016/0092-8674(90)90213-x. [DOI] [PubMed] [Google Scholar]
  10. González F., Romani S., Cubas P., Modolell J., Campuzano S. Molecular analysis of the asense gene, a member of the achaete-scute complex of Drosophila melanogaster, and its novel role in optic lobe development. EMBO J. 1989 Dec 1;8(12):3553–3562. doi: 10.1002/j.1460-2075.1989.tb08527.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gregor P. D., Sawadogo M., Roeder R. G. The adenovirus major late transcription factor USF is a member of the helix-loop-helix group of regulatory proteins and binds to DNA as a dimer. Genes Dev. 1990 Oct;4(10):1730–1740. doi: 10.1101/gad.4.10.1730. [DOI] [PubMed] [Google Scholar]
  12. Hu Y. F., Lüscher B., Admon A., Mermod N., Tjian R. Transcription factor AP-4 contains multiple dimerization domains that regulate dimer specificity. Genes Dev. 1990 Oct;4(10):1741–1752. doi: 10.1101/gad.4.10.1741. [DOI] [PubMed] [Google Scholar]
  13. Kerkhoff E., Bister K. Myc protein structure: localization of DNA-binding and protein dimerization domains. Oncogene. 1991 Jan;6(1):93–102. [PubMed] [Google Scholar]
  14. Klemenz R., Weber U., Gehring W. J. The white gene as a marker in a new P-element vector for gene transfer in Drosophila. Nucleic Acids Res. 1987 May 26;15(10):3947–3959. doi: 10.1093/nar/15.10.3947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Klämbt C., Knust E., Tietze K., Campos-Ortega J. A. Closely related transcripts encoded by the neurogenic gene complex enhancer of split of Drosophila melanogaster. EMBO J. 1989 Jan;8(1):203–210. doi: 10.1002/j.1460-2075.1989.tb03365.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Knust E., Bremer K. A., Vässin H., Ziemer A., Tepass U., Campos-Ortega J. A. The enhancer of split locus and neurogenesis in Drosophila melanogaster. Dev Biol. 1987 Jul;122(1):262–273. doi: 10.1016/0012-1606(87)90351-4. [DOI] [PubMed] [Google Scholar]
  17. Knust E., Tietze K., Campos-Ortega J. A. Molecular analysis of the neurogenic locus Enhancer of split of Drosophila melanogaster. EMBO J. 1987 Dec 20;6(13):4113–4123. doi: 10.1002/j.1460-2075.1987.tb02757.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Laski F. A., Rio D. C., Rubin G. M. Tissue specificity of Drosophila P element transposition is regulated at the level of mRNA splicing. Cell. 1986 Jan 17;44(1):7–19. doi: 10.1016/0092-8674(86)90480-0. [DOI] [PubMed] [Google Scholar]
  19. Ludwig S. R., Wessler S. R. Maize R gene family: tissue-specific helix-loop-helix proteins. Cell. 1990 Sep 7;62(5):849–851. doi: 10.1016/0092-8674(90)90259-h. [DOI] [PubMed] [Google Scholar]
  20. Lüscher B., Eisenman R. N. New light on Myc and Myb. Part I. Myc. Genes Dev. 1990 Dec;4(12A):2025–2035. doi: 10.1101/gad.4.12a.2025. [DOI] [PubMed] [Google Scholar]
  21. Mellor J., Jiang W., Funk M., Rathjen J., Barnes C. A., Hinz T., Hegemann J. H., Philippsen P. CPF1, a yeast protein which functions in centromeres and promoters. EMBO J. 1990 Dec;9(12):4017–4026. doi: 10.1002/j.1460-2075.1990.tb07623.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Murre C., McCaw P. S., Baltimore D. A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell. 1989 Mar 10;56(5):777–783. doi: 10.1016/0092-8674(89)90682-x. [DOI] [PubMed] [Google Scholar]
  23. Murre C., McCaw P. S., Vaessin H., Caudy M., Jan L. Y., Jan Y. N., Cabrera C. V., Buskin J. N., Hauschka S. D., Lassar A. B. Interactions between heterologous helix-loop-helix proteins generate complexes that bind specifically to a common DNA sequence. Cell. 1989 Aug 11;58(3):537–544. doi: 10.1016/0092-8674(89)90434-0. [DOI] [PubMed] [Google Scholar]
  24. Olson E. N. MyoD family: a paradigm for development? Genes Dev. 1990 Sep;4(9):1454–1461. doi: 10.1101/gad.4.9.1454. [DOI] [PubMed] [Google Scholar]
  25. Preiss A., Hartley D. A., Artavanis-Tsakonas S. The molecular genetics of Enhancer of split, a gene required for embryonic neural development in Drosophila. EMBO J. 1988 Dec 1;7(12):3917–3927. doi: 10.1002/j.1460-2075.1988.tb03278.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Robertson H. M., Preston C. R., Phillis R. W., Johnson-Schlitz D. M., Benz W. K., Engels W. R. A stable genomic source of P element transposase in Drosophila melanogaster. Genetics. 1988 Mar;118(3):461–470. doi: 10.1093/genetics/118.3.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Rushlow C. A., Hogan A., Pinchin S. M., Howe K. M., Lardelli M., Ish-Horowicz D. The Drosophila hairy protein acts in both segmentation and bristle patterning and shows homology to N-myc. EMBO J. 1989 Oct;8(10):3095–3103. doi: 10.1002/j.1460-2075.1989.tb08461.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. 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]
  29. Sayers J. R., Schmidt W., Eckstein F. 5'-3' exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis. Nucleic Acids Res. 1988 Feb 11;16(3):791–802. doi: 10.1093/nar/16.3.791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Shepard S. B., Broverman S. A., Muskavitch M. A. A tripartite interaction among alleles of Notch, Delta, and Enhancer of split during imaginal development of Drosophila melanogaster. Genetics. 1989 Jun;122(2):429–438. doi: 10.1093/genetics/122.2.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  32. Van Doren M., Ellis H. M., Posakony J. W. The Drosophila extramacrochaetae protein antagonizes sequence-specific DNA binding by daughterless/achaete-scute protein complexes. Development. 1991 Sep;113(1):245–255. doi: 10.1242/dev.113.1.245. [DOI] [PubMed] [Google Scholar]
  33. Villares R., Cabrera C. V. The achaete-scute gene complex of D. melanogaster: conserved domains in a subset of genes required for neurogenesis and their homology to myc. Cell. 1987 Jul 31;50(3):415–424. doi: 10.1016/0092-8674(87)90495-8. [DOI] [PubMed] [Google Scholar]
  34. Vässin H., Vielmetter J., Campos-Ortega J. A. Genetic interactions in early neurogenesis of Drosophila melanogaster. J Neurogenet. 1985 Nov;2(5):291–308. doi: 10.3109/01677068509102325. [DOI] [PubMed] [Google Scholar]
  35. Weintraub H., Tapscott S. J., Davis R. L., Thayer M. J., Adam M. A., Lassar A. B., Miller A. D. Activation of muscle-specific genes in pigment, nerve, fat, liver, and fibroblast cell lines by forced expression of MyoD. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5434–5438. doi: 10.1073/pnas.86.14.5434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Welshons W. J. Analysis of a gene in drosophila. Science. 1965 Nov 26;150(3700):1122–1129. doi: 10.1126/science.150.3700.1122. [DOI] [PubMed] [Google Scholar]
  37. Yun R. H., Anderson A., Hermans J. Proline in alpha-helix: stability and conformation studied by dynamics simulation. Proteins. 1991;10(3):219–228. doi: 10.1002/prot.340100306. [DOI] [PubMed] [Google Scholar]
  38. Ziemer A., Tietze K., Knust E., Campos-Ortega J. A. Genetic analysis of enhancer of split, a locus involved in neurogenesis in Drosophila melanogaster. Genetics. 1988 May;119(1):63–74. doi: 10.1093/genetics/119.1.63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. de-la-Concha A., Dietrich U., Weigel D., Campos-Ortega J. A. Functional interactions of neurogenic genes of Drosophila melanogaster. Genetics. 1988 Mar;118(3):499–508. doi: 10.1093/genetics/118.3.499. [DOI] [PMC free article] [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