Skip to main content
The EMBO Journal logoLink to The EMBO Journal
. 1993 Feb;12(2):527–535. doi: 10.1002/j.1460-2075.1993.tb05684.x

The Drosophila doublesex proteins share a novel zinc finger related DNA binding domain.

S E Erdman 1, K C Burtis 1
PMCID: PMC413235  PMID: 8440242

Abstract

The doublesex gene of Drosophila melanogaster is the final member of a well characterized hierarchy of genes that controls somatic sex determination and differentiation. The male-specific and female-specific doublesex polypeptides occupy a terminal position in the hierarchy, and thus regulate those genes responsible for the development of sexually dimorphic characteristics of the fly. To investigate the molecular mechanism by which these two related proteins interact with specific target genes, we have identified and characterized their DNA binding domains. Using gel mobility shift experiments with sequentially deleted polypeptides, site-directed mutagenesis and spectrophotometric assays, we have shown that the two doublesex proteins share a common and novel zinc finger-related DNA binding domain distinct from any reported class of zinc binding proteins. We have further shown that of 10 null dsx alleles, six encode proteins deficient in DNA binding activity, and that three of these alleles are the result of mutations that alter cysteine and histidine residues in the metal binding domain. Our results provide evidence that both the male-specific and female-specific doublesex proteins share and depend upon the same DNA binding domain for function in vivo, suggesting that both proteins bind to, but differentially regulate, a common set of genes in both sexes.

Full text

PDF
534

Images in this article

Selected References

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

  1. Baker B. S., Hoff G., Kaufman T. C., Wolfner M. F., Hazelrigg T. The doublesex locus of Drosophila melanogaster and its flanking regions: a cytogenetic analysis. Genetics. 1991 Jan;127(1):125–138. doi: 10.1093/genetics/127.1.125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baker B. S., Ridge K. A. Sex and the single cell. I. On the action of major loci affecting sex determination in Drosophila melanogaster. Genetics. 1980 Feb;94(2):383–423. doi: 10.1093/genetics/94.2.383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baker B. S. Sex in flies: the splice of life. Nature. 1989 Aug 17;340(6234):521–524. doi: 10.1038/340521a0. [DOI] [PubMed] [Google Scholar]
  4. Belote J. M., Handler A. M., Wolfner M. F., Livak K. J., Baker B. S. Sex-specific regulation of yolk protein gene expression in Drosophila. Cell. 1985 Feb;40(2):339–348. doi: 10.1016/0092-8674(85)90148-5. [DOI] [PubMed] [Google Scholar]
  5. Berg J. M. Potential metal-binding domains in nucleic acid binding proteins. Science. 1986 Apr 25;232(4749):485–487. doi: 10.1126/science.2421409. [DOI] [PubMed] [Google Scholar]
  6. Blumberg H., Eisen A., Sledziewski A., Bader D., Young E. T. Two zinc fingers of a yeast regulatory protein shown by genetic evidence to be essential for its function. 1987 Jul 30-Aug 5Nature. 328(6129):443–445. doi: 10.1038/328443a0. [DOI] [PubMed] [Google Scholar]
  7. Bownes M., Nöthiger R. Sex determining genes and vitellogenin synthesis in Drosophila melanogaster. Mol Gen Genet. 1981;182(2):222–228. doi: 10.1007/BF00269661. [DOI] [PubMed] [Google Scholar]
  8. Burtis K. C., Baker B. S. Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex-specific polypeptides. Cell. 1989 Mar 24;56(6):997–1010. doi: 10.1016/0092-8674(89)90633-8. [DOI] [PubMed] [Google Scholar]
  9. Burtis K. C., Coschigano K. T., Baker B. S., Wensink P. C. The doublesex proteins of Drosophila melanogaster bind directly to a sex-specific yolk protein gene enhancer. EMBO J. 1991 Sep;10(9):2577–2582. doi: 10.1002/j.1460-2075.1991.tb07798.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  11. Clark J. M. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 1988 Oct 25;16(20):9677–9686. doi: 10.1093/nar/16.20.9677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Cline T. W. The affairs of daughterless and the promiscuity of developmental regulators. Cell. 1989 Oct 20;59(2):231–234. doi: 10.1016/0092-8674(89)90280-8. [DOI] [PubMed] [Google Scholar]
  13. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Frankel A. D., Kim P. S. Modular structure of transcription factors: implications for gene regulation. Cell. 1991 May 31;65(5):717–719. doi: 10.1016/0092-8674(91)90378-c. [DOI] [PubMed] [Google Scholar]
  15. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  16. HILDRETH P. E. DOUBLESEX, RECESSIVE GENE THAT TRANSFORMS BOTH MALES AND FEMALES OF DROSOPHILA INTO INTERSEXES. Genetics. 1965 Apr;51:659–678. doi: 10.1093/genetics/51.4.659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harrison S. C. A structural taxonomy of DNA-binding domains. Nature. 1991 Oct 24;353(6346):715–719. doi: 10.1038/353715a0. [DOI] [PubMed] [Google Scholar]
  18. Hunt J. B., Neece S. H., Ginsburg A. The use of 4-(2-pyridylazo)resorcinol in studies of zinc release from Escherichia coli aspartate transcarbamoylase. Anal Biochem. 1985 Apr;146(1):150–157. doi: 10.1016/0003-2697(85)90409-9. [DOI] [PubMed] [Google Scholar]
  19. Jones N. Transcriptional regulation by dimerization: two sides to an incestuous relationship. Cell. 1990 Apr 6;61(1):9–11. doi: 10.1016/0092-8674(90)90207-u. [DOI] [PubMed] [Google Scholar]
  20. Luisi B. DNA transcription. Zinc standard for economy. Nature. 1992 Apr 2;356(6368):379–380. doi: 10.1038/356379a0. [DOI] [PubMed] [Google Scholar]
  21. Nagoshi R. N., Baker B. S. Regulation of sex-specific RNA splicing at the Drosophila doublesex gene: cis-acting mutations in exon sequences alter sex-specific RNA splicing patterns. Genes Dev. 1990 Jan;4(1):89–97. doi: 10.1101/gad.4.1.89. [DOI] [PubMed] [Google Scholar]
  22. Nagoshi R. N., McKeown M., Burtis K. C., Belote J. M., Baker B. S. The control of alternative splicing at genes regulating sexual differentiation in D. melanogaster. Cell. 1988 Apr 22;53(2):229–236. doi: 10.1016/0092-8674(88)90384-4. [DOI] [PubMed] [Google Scholar]
  23. Ota T., Fukunaga A., Kawabe M., Oishi K. Interactions between sex-transformation mutants of Drosophila melanogaster. I. Hemolymph vitellogenins and gonad morphology. Genetics. 1981 Nov-Dec;99(3-4):429–441. doi: 10.1093/genetics/99.3-4.429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Postlethwait J. H., Bownes M., Jowett T. Sexual phenotype and vitellogenin synthesis in Drosophila melanogaster. Dev Biol. 1980 Oct;79(2):379–387. doi: 10.1016/0012-1606(80)90123-2. [DOI] [PubMed] [Google Scholar]
  25. Schena M., Freedman L. P., Yamamoto K. R. Mutations in the glucocorticoid receptor zinc finger region that distinguish interdigitated DNA binding and transcriptional enhancement activities. Genes Dev. 1989 Oct;3(10):1590–1601. doi: 10.1101/gad.3.10.1590. [DOI] [PubMed] [Google Scholar]
  26. Severne Y., Wieland S., Schaffner W., Rusconi S. Metal binding 'finger' structures in the glucocorticoid receptor defined by site-directed mutagenesis. EMBO J. 1988 Aug;7(8):2503–2508. doi: 10.1002/j.1460-2075.1988.tb03097.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Slee R., Bownes M. Sex determination in Drosophila melanogaster. Q Rev Biol. 1990 Jun;65(2):175–204. doi: 10.1086/416718. [DOI] [PubMed] [Google Scholar]
  28. Smith D. L., Johnson A. D. A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an alpha 2 dimer. Cell. 1992 Jan 10;68(1):133–142. doi: 10.1016/0092-8674(92)90212-u. [DOI] [PubMed] [Google Scholar]
  29. Steinmann-Zwicky M., Amrein H., Nöthiger R. Genetic control of sex determination in Drosophila. Adv Genet. 1990;27:189–237. doi: 10.1016/s0065-2660(08)60026-7. [DOI] [PubMed] [Google Scholar]
  30. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Taylor B. J., Truman J. W. Commitment of abdominal neuroblasts in Drosophila to a male or female fate is dependent on genes of the sex-determining hierarchy. Development. 1992 Mar;114(3):625–642. doi: 10.1242/dev.114.3.625. [DOI] [PubMed] [Google Scholar]
  32. Vallee B. L., Coleman J. E., Auld D. S. Zinc fingers, zinc clusters, and zinc twists in DNA-binding protein domains. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):999–1003. doi: 10.1073/pnas.88.3.999. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Webster L. C., Zhang K., Chance B., Ayene I., Culp J. S., Huang W. J., Wu F. Y., Ricciardi R. P. Conversion of the E1A Cys4 zinc finger to a nonfunctional His2,Cys2 zinc finger by a single point mutation. Proc Natl Acad Sci U S A. 1991 Nov 15;88(22):9989–9993. doi: 10.1073/pnas.88.22.9989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wolfner M. F. Sex-specific gene expression in somatic tissues of Drosophila melanogaster. Trends Genet. 1988 Dec;4(12):333–337. doi: 10.1016/0168-9525(88)90052-2. [DOI] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES