Skip to main content
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1993 Dec;13(12):7961–7970. doi: 10.1128/mcb.13.12.7961

Isolation of cDNAs encoding the Drosophila GAGA transcription factor.

W C Soeller 1, C E Oh 1, T B Kornberg 1
PMCID: PMC364868  PMID: 7504178

Abstract

To investigate the mechanisms involved in expression of the Drosophila melanogaster engrailed gene, we purified GAGA protein, one of several putative transcriptional activator proteins that binds to the proximal region of the engrailed promoter. Antibodies raised against GAGA protein were used to demonstrate that the protein is present in all nuclei of young embryos. We isolated cDNA clones encoding GAGA protein in which a putative 519-codon open reading frame contains general sequence motifs characteristic of other transcription factors. These include stretches of polyglutamine, a 60-amino-acid region with 18 (30%) lysine or arginine residues, and a single putative zinc finger motif. In addition, a 120-residue N-terminal region shares significant sequence homology with several other known Drosophila transcription factors, including those encoded by Broad Complex and tramtrack. Up to 35-fold GAGA protein-dependent stimulation of transcription in Schneider line 2 tissue culture cells was observed after transfection of GAGA protein-encoding sequences. The GAGA gene is present in one copy in the Drosophila genome, at cytological location 70EF, and it encodes RNAs which vary in size between 2.4 and 4.4 kb.

Full text

PDF
7963

Images in this article

Selected References

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

  1. Abarzúa P., Soeller W., Marians K. J. Mutational analysis of primosome assembly sites. I. Distinct classes of mutants in the pBR322 Escherichia coli factor Y DNA effector sequences. J Biol Chem. 1984 Nov 25;259(22):14286–14292. [PubMed] [Google Scholar]
  2. Bejsovec A., Martinez Arias A. Roles of wingless in patterning the larval epidermis of Drosophila. Development. 1991 Oct;113(2):471–485. doi: 10.1242/dev.113.2.471. [DOI] [PubMed] [Google Scholar]
  3. Benyajati C., Ewel A., McKeon J., Chovav M., Juan E. Characterization and purification of Adh distal promoter factor 2, Adf-2, a cell-specific and promoter-specific repressor in Drosophila. Nucleic Acids Res. 1992 Sep 11;20(17):4481–4489. doi: 10.1093/nar/20.17.4481. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Biggin M. D., Tjian R. Transcription factors that activate the Ultrabithorax promoter in developmentally staged extracts. Cell. 1988 Jun 3;53(5):699–711. doi: 10.1016/0092-8674(88)90088-8. [DOI] [PubMed] [Google Scholar]
  5. Brown J. L., Sonoda S., Ueda H., Scott M. P., Wu C. Repression of the Drosophila fushi tarazu (ftz) segmentation gene. EMBO J. 1991 Mar;10(3):665–674. doi: 10.1002/j.1460-2075.1991.tb07995.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Brown J. L., Wu C. Repression of Drosophila pair-rule segmentation genes by ectopic expression of tramtrack. Development. 1993 Jan;117(1):45–58. doi: 10.1242/dev.117.1.45. [DOI] [PubMed] [Google Scholar]
  7. Busturia A., Morata G. Ectopic expression of homeotic genes caused by the elimination of the Polycomb gene in Drosophila imaginal epidermis. Development. 1988 Dec;104(4):713–720. doi: 10.1242/dev.104.4.713. [DOI] [PubMed] [Google Scholar]
  8. Chi H. C., Juminaga D., Wang S. Y., Hui C. F. Structure of the Drosophila gene for the laminin B2 chain. DNA Cell Biol. 1991 Jul-Aug;10(6):451–466. doi: 10.1089/dna.1991.10.451. [DOI] [PubMed] [Google Scholar]
  9. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  10. Croston G. E., Kerrigan L. A., Lira L. M., Marshak D. R., Kadonaga J. T. Sequence-specific antirepression of histone H1-mediated inhibition of basal RNA polymerase II transcription. Science. 1991 Feb 8;251(4994):643–649. doi: 10.1126/science.1899487. [DOI] [PubMed] [Google Scholar]
  11. DiBello P. R., Withers D. A., Bayer C. A., Fristrom J. W., Guild G. M. The Drosophila Broad-Complex encodes a family of related proteins containing zinc fingers. Genetics. 1991 Oct;129(2):385–397. doi: 10.1093/genetics/129.2.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. DiNardo S., Kuner J. M., Theis J., O'Farrell P. H. Development of embryonic pattern in D. melanogaster as revealed by accumulation of the nuclear engrailed protein. Cell. 1985 Nov;43(1):59–69. doi: 10.1016/0092-8674(85)90012-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. DiNardo S., Sher E., Heemskerk-Jongens J., Kassis J. A., O'Farrell P. H. Two-tiered regulation of spatially patterned engrailed gene expression during Drosophila embryogenesis. Nature. 1988 Apr 14;332(6165):604–609. doi: 10.1038/332604a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dorn R., Szidonya J., Korge G., Sehnert M., Taubert H., Archoukieh E., Tschiersch B., Morawietz H., Wustmann G., Hoffmann G. P transposon-induced dominant enhancer mutations of position-effect variegation in Drosophila melanogaster. Genetics. 1993 Feb;133(2):279–290. doi: 10.1093/genetics/133.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. England B. P., Heberlein U., Tjian R. Purified Drosophila transcription factor, Adh distal factor-1 (Adf-1), binds to sites in several Drosophila promoters and activates transcription. J Biol Chem. 1990 Mar 25;265(9):5086–5094. [PubMed] [Google Scholar]
  16. Fjose A., McGinnis W. J., Gehring W. J. Isolation of a homoeo box-containing gene from the engrailed region of Drosophila and the spatial distribution of its transcripts. Nature. 1985 Jan 24;313(6000):284–289. doi: 10.1038/313284a0. [DOI] [PubMed] [Google Scholar]
  17. Gilmour D. S., Thomas G. H., Elgin S. C. Drosophila nuclear proteins bind to regions of alternating C and T residues in gene promoters. Science. 1989 Sep 29;245(4925):1487–1490. doi: 10.1126/science.2781290. [DOI] [PubMed] [Google Scholar]
  18. Harrison S. D., Travers A. A. The tramtrack gene encodes a Drosophila finger protein that interacts with the ftz transcriptional regulatory region and shows a novel embryonic expression pattern. EMBO J. 1990 Jan;9(1):207–216. doi: 10.1002/j.1460-2075.1990.tb08097.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hoey T., Levine M. Divergent homeo box proteins recognize similar DNA sequences in Drosophila. Nature. 1988 Apr 28;332(6167):858–861. doi: 10.1038/332858a0. [DOI] [PubMed] [Google Scholar]
  20. Jackson S. P., Tjian R. O-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation. Cell. 1988 Oct 7;55(1):125–133. doi: 10.1016/0092-8674(88)90015-3. [DOI] [PubMed] [Google Scholar]
  21. Kadonaga J. T., Tjian R. Affinity purification of sequence-specific DNA binding proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5889–5893. doi: 10.1073/pnas.83.16.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Karr T. L., Weir M. P., Ali Z., Kornberg T. Patterns of engrailed protein in early Drosophila embryos. Development. 1989 Mar;105(3):605–612. doi: 10.1242/dev.105.3.605. [DOI] [PubMed] [Google Scholar]
  23. Kerrigan L. A., Croston G. E., Lira L. M., Kadonaga J. T. Sequence-specific transcriptional antirepression of the Drosophila Krüppel gene by the GAGA factor. J Biol Chem. 1991 Jan 5;266(1):574–582. [PubMed] [Google Scholar]
  24. Kornberg T., Sidén I., O'Farrell P., Simon M. The engrailed locus of Drosophila: in situ localization of transcripts reveals compartment-specific expression. Cell. 1985 Jan;40(1):45–53. doi: 10.1016/0092-8674(85)90307-1. [DOI] [PubMed] [Google Scholar]
  25. Krasnow M. A., Saffman E. E., Kornfeld K., Hogness D. S. Transcriptional activation and repression by Ultrabithorax proteins in cultured Drosophila cells. Cell. 1989 Jun 16;57(6):1031–1043. doi: 10.1016/0092-8674(89)90341-3. [DOI] [PubMed] [Google Scholar]
  26. Lee H., Kraus K. W., Wolfner M. F., Lis J. T. DNA sequence requirements for generating paused polymerase at the start of hsp70. Genes Dev. 1992 Feb;6(2):284–295. doi: 10.1101/gad.6.2.284. [DOI] [PubMed] [Google Scholar]
  27. Levis R., Bingham P. M., Rubin G. M. Physical map of the white locus of Drosophila melanogaster. Proc Natl Acad Sci U S A. 1982 Jan;79(2):564–568. doi: 10.1073/pnas.79.2.564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lu Q., Wallrath L. L., Granok H., Elgin S. C. (CT)n (GA)n repeats and heat shock elements have distinct roles in chromatin structure and transcriptional activation of the Drosophila hsp26 gene. Mol Cell Biol. 1993 May;13(5):2802–2814. doi: 10.1128/mcb.13.5.2802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Manoukian A. S., Krause H. M. Concentration-dependent activities of the even-skipped protein in Drosophila embryos. Genes Dev. 1992 Sep;6(9):1740–1751. doi: 10.1101/gad.6.9.1740. [DOI] [PubMed] [Google Scholar]
  30. Martizez Arias A., Baker N. E., Ingham P. W. Role of segment polarity genes in the definition and maintenance of cell states in the Drosophila embryo. Development. 1988 May;103(1):157–170. doi: 10.1242/dev.103.1.157. [DOI] [PubMed] [Google Scholar]
  31. Moazed D., O'Farrell P. H. Maintenance of the engrailed expression pattern by Polycomb group genes in Drosophila. Development. 1992 Nov;116(3):805–810. doi: 10.1242/dev.116.3.805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Mortin M. A., Zuerner R., Berger S., Hamilton B. J. Mutations in the second-largest subunit of Drosophila RNA polymerase II interact with Ubx. Genetics. 1992 Aug;131(4):895–903. doi: 10.1093/genetics/131.4.895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Poole S. J., Kauvar L. M., Drees B., Kornberg T. The engrailed locus of Drosophila: structural analysis of an embryonic transcript. Cell. 1985 Jan;40(1):37–43. doi: 10.1016/0092-8674(85)90306-x. [DOI] [PubMed] [Google Scholar]
  34. Read D., Levine M., Manley J. L. Ectopic expression of the Drosophila tramtrack gene results in multiple embryonic defects, including repression of even-skipped and fushi tarazu. Mech Dev. 1992 Sep;38(3):183–195. doi: 10.1016/0925-4773(92)90052-l. [DOI] [PubMed] [Google Scholar]
  35. Read D., Manley J. L. Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc finger proteins with distinct DNA binding specificities. EMBO J. 1992 Mar;11(3):1035–1044. doi: 10.1002/j.1460-2075.1992.tb05142.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Read D., Nishigaki T., Manley J. L. The Drosophila even-skipped promoter is transcribed in a stage-specific manner in vitro and contains multiple, overlapping factor-binding sites. Mol Cell Biol. 1990 Aug;10(8):4334–4344. doi: 10.1128/mcb.10.8.4334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rosenberg A. H., Lade B. N., Chui D. S., Lin S. W., Dunn J. J., Studier F. W. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene. 1987;56(1):125–135. doi: 10.1016/0378-1119(87)90165-x. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Soeller W. C., Poole S. J., Kornberg T. In vitro transcription of the Drosophila engrailed gene. Genes Dev. 1988 Jan;2(1):68–81. doi: 10.1101/gad.2.1.68. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Tabata T., Eaton S., Kornberg T. B. The Drosophila hedgehog gene is expressed specifically in posterior compartment cells and is a target of engrailed regulation. Genes Dev. 1992 Dec;6(12B):2635–2645. doi: 10.1101/gad.6.12b.2635. [DOI] [PubMed] [Google Scholar]
  42. Thummel C. S. The Drosophila E74 promoter contains essential sequences downstream from the start site of transcription. Genes Dev. 1989 Jun;3(6):782–792. doi: 10.1101/gad.3.6.782. [DOI] [PubMed] [Google Scholar]
  43. Tix S., Minden J. S., Technau G. M. Pre-existing neuronal pathways in the developing optic lobes of Drosophila. Development. 1989 Apr;105(4):739–746. doi: 10.1242/dev.105.4.739. [DOI] [PubMed] [Google Scholar]
  44. Topol J., Dearolf C. R., Prakash K., Parker C. S. Synthetic oligonucleotides recreate Drosophila fushi tarazu zebra-stripe expression. Genes Dev. 1991 May;5(5):855–867. doi: 10.1101/gad.5.5.855. [DOI] [PubMed] [Google Scholar]
  45. Wood W. I., Gitschier J., Lasky L. A., Lawn R. M. Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc Natl Acad Sci U S A. 1985 Mar;82(6):1585–1588. doi: 10.1073/pnas.82.6.1585. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES