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. 2000 Sep;156(1):229–244. doi: 10.1093/genetics/156.1.229

Genetic analysis of the Drosophila 63F early puff. Characterization of mutations in E63-1 and maggie, a putative Tom22.

M Vaskova 1, A M Bentley 1, S Marshall 1, P Reid 1, C S Thummel 1, A J Andres 1
PMCID: PMC1461229  PMID: 10978288

Abstract

The 63F early puff in the larval salivary gland polytene chromosomes contains the divergently transcribed E63-1 and E63-2 ecdysone-inducible genes. E63-1 encodes a member of the EF-hand family of Ca(2+)-binding proteins, while E63-2 has no apparent open reading frame. To understand the functions of the E63 genes, we have determined the temporal and spatial patterns of E63-1 protein expression, as well as undertaken a genetic analysis of the 63F puff. We show that E63-1 is expressed in many embryonic and larval tissues, but the third-instar larval salivary gland is the only tissue where increases in protein levels correlate with increases in ecdysone titer. Furthermore, the subcellular distribution of E63-1 protein changes dynamically in the salivary glands at the onset of metamorphosis. E63-1 and E63-2 null mutations, however, have no effect on development or fertility. We have characterized 40 kb of the 63F region, defined as the interval between Ubi-p and E63-2, and have identified three lethal complementation groups that correspond to the dSc-2, ida, and mge genes. We show that mge mutations lead to first-instar larval lethality and that Mge protein is similar to the Tom22 mitochondrial import proteins of fungi, suggesting that it has a role in mitochondrial function.

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Selected References

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  1. Ahting U., Thun C., Hegerl R., Typke D., Nargang F. E., Neupert W., Nussberger S. The TOM core complex: the general protein import pore of the outer membrane of mitochondria. J Cell Biol. 1999 Nov 29;147(5):959–968. doi: 10.1083/jcb.147.5.959. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andres A. J., Cherbas P. Tissue-specific ecdysone responses: regulation of the Drosophila genes Eip28/29 and Eip40 during larval development. Development. 1992 Dec;116(4):865–876. doi: 10.1242/dev.116.4.865. [DOI] [PubMed] [Google Scholar]
  4. Andres A. J., Thummel C. S. Methods for quantitative analysis of transcription in larvae and prepupae. Methods Cell Biol. 1994;44:565–573. doi: 10.1016/s0091-679x(08)60932-2. [DOI] [PubMed] [Google Scholar]
  5. Andres A. J., Thummel C. S. The Drosophila 63F early puff contains E63-1, an ecdysone-inducible gene that encodes a novel Ca(2+)-binding protein. Development. 1995 Aug;121(8):2667–2679. doi: 10.1242/dev.121.8.2667. [DOI] [PubMed] [Google Scholar]
  6. Ashburner M., Chihara C., Meltzer P., Richards G. Temporal control of puffing activity in polytene chromosomes. Cold Spring Harb Symp Quant Biol. 1974;38:655–662. doi: 10.1101/sqb.1974.038.01.070. [DOI] [PubMed] [Google Scholar]
  7. Avery J., Jahn R., Edwardson J. M. Reconstitution of regulated exocytosis in cell-free systems: a critical appraisal. Annu Rev Physiol. 1999;61:777–807. doi: 10.1146/annurev.physiol.61.1.777. [DOI] [PubMed] [Google Scholar]
  8. BECKER H. J. [The puffs of salivary gland chromosomes of Drosophilia melanogaster. Part 1. Observations on the behavior of a typical puff in the normal strain and in two mutants, giant and lethal giant larvae]. Chromosoma. 1959;10:654–678. doi: 10.1007/BF00396591. [DOI] [PubMed] [Google Scholar]
  9. Burtis K. C., Thummel C. S., Jones C. W., Karim F. D., Hogness D. S. The Drosophila 74EF early puff contains E74, a complex ecdysone-inducible gene that encodes two ets-related proteins. Cell. 1990 Apr 6;61(1):85–99. doi: 10.1016/0092-8674(90)90217-3. [DOI] [PubMed] [Google Scholar]
  10. Chao A. T., Guild G. M. Molecular analysis of the ecdysterone-inducible 2B5 "early' puff in Drosophila melanogaster. EMBO J. 1986 Jan;5(1):143–150. doi: 10.1002/j.1460-2075.1986.tb04188.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chen T., Bunting M., Karim F. D., Thummel C. S. Isolation and characterization of five Drosophila genes that encode an ets-related DNA binding domain. Dev Biol. 1992 May;151(1):176–191. doi: 10.1016/0012-1606(92)90225-6. [DOI] [PubMed] [Google Scholar]
  12. Cheney R. E., O'Shea M. K., Heuser J. E., Coelho M. V., Wolenski J. S., Espreafico E. M., Forscher P., Larson R. E., Mooseker M. S. Brain myosin-V is a two-headed unconventional myosin with motor activity. Cell. 1993 Oct 8;75(1):13–23. doi: 10.1016/S0092-8674(05)80080-7. [DOI] [PubMed] [Google Scholar]
  13. Cooley L., Thompson D., Spradling A. C. Constructing deletions with defined endpoints in Drosophila. Proc Natl Acad Sci U S A. 1990 Apr;87(8):3170–3173. doi: 10.1073/pnas.87.8.3170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Court D. A., Nargang F. E., Steiner H., Hodges R. S., Neupert W., Lill R. Role of the intermembrane-space domain of the preprotein receptor Tom22 in protein import into mitochondria. Mol Cell Biol. 1996 Aug;16(8):4035–4042. doi: 10.1128/mcb.16.8.4035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Crossgrove K., Bayer C. A., Fristrom J. W., Guild G. M. The Drosophila Broad-Complex early gene directly regulates late gene transcription during the ecdysone-induced puffing cascade. Dev Biol. 1996 Dec 15;180(2):745–758. doi: 10.1006/dbio.1996.0343. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Fehon R. G., Johansen K., Rebay I., Artavanis-Tsakonas S. Complex cellular and subcellular regulation of notch expression during embryonic and imaginal development of Drosophila: implications for notch function. J Cell Biol. 1991 May;113(3):657–669. doi: 10.1083/jcb.113.3.657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fletcher J. C., Thummel C. S. The ecdysone-inducible Broad-complex and E74 early genes interact to regulate target gene transcription and Drosophila metamorphosis. Genetics. 1995 Nov;141(3):1025–1035. doi: 10.1093/genetics/141.3.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hamilton B. A., Palazzolo M. J., Chang J. H., VijayRaghavan K., Mayeda C. A., Whitney M. A., Meyerowitz E. M. Large scale screen for transposon insertions into cloned genes. Proc Natl Acad Sci U S A. 1991 Apr 1;88(7):2731–2735. doi: 10.1073/pnas.88.7.2731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hasson T., Mooseker M. S. Porcine myosin-VI: characterization of a new mammalian unconventional myosin. J Cell Biol. 1994 Oct;127(2):425–440. doi: 10.1083/jcb.127.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Henrich V. C., Rybczynski R., Gilbert L. I. Peptide hormones, steroid hormones, and puffs: mechanisms and models in insect development. Vitam Horm. 1999;55:73–125. doi: 10.1016/s0083-6729(08)60934-6. [DOI] [PubMed] [Google Scholar]
  22. Hurban P., Thummel C. S. Isolation and characterization of fifteen ecdysone-inducible Drosophila genes reveal unexpected complexities in ecdysone regulation. Mol Cell Biol. 1993 Nov;13(11):7101–7111. doi: 10.1128/mcb.13.11.7101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Iyengar B., Roote J., Campos A. R. The tamas gene, identified as a mutation that disrupts larval behavior in Drosophila melanogaster, codes for the mitochondrial DNA polymerase catalytic subunit (DNApol-gamma125). Genetics. 1999 Dec;153(4):1809–1824. doi: 10.1093/genetics/153.4.1809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kiebler M., Keil P., Schneider H., van der Klei I. J., Pfanner N., Neupert W. The mitochondrial receptor complex: a central role of MOM22 in mediating preprotein transfer from receptors to the general insertion pore. Cell. 1993 Aug 13;74(3):483–492. doi: 10.1016/0092-8674(93)80050-o. [DOI] [PubMed] [Google Scholar]
  25. Komiya T., Rospert S., Koehler C., Looser R., Schatz G., Mihara K. Interaction of mitochondrial targeting signals with acidic receptor domains along the protein import pathway: evidence for the 'acid chain' hypothesis. EMBO J. 1998 Jul 15;17(14):3886–3898. doi: 10.1093/emboj/17.14.3886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lang J. Molecular mechanisms and regulation of insulin exocytosis as a paradigm of endocrine secretion. Eur J Biochem. 1999 Jan;259(1-2):3–17. doi: 10.1046/j.1432-1327.1999.00043.x. [DOI] [PubMed] [Google Scholar]
  27. Lee H. S., Simon J. A., Lis J. T. Structure and expression of ubiquitin genes of Drosophila melanogaster. Mol Cell Biol. 1988 Nov;8(11):4727–4735. doi: 10.1128/mcb.8.11.4727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lithgow T., Junne T., Suda K., Gratzer S., Schatz G. The mitochondrial outer membrane protein Mas22p is essential for protein import and viability of yeast. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11973–11977. doi: 10.1073/pnas.91.25.11973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mahaffey J. W., Kaufman T. C. Distribution of the Sex combs reduced gene products in Drosophila melanogaster. Genetics. 1987 Sep;117(1):51–60. doi: 10.1093/genetics/117.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mangelsdorf D. J., Thummel C., Beato M., Herrlich P., Schütz G., Umesono K., Blumberg B., Kastner P., Mark M., Chambon P. The nuclear receptor superfamily: the second decade. Cell. 1995 Dec 15;83(6):835–839. doi: 10.1016/0092-8674(95)90199-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Moczko M., Bömer U., Kübrich M., Zufall N., Hönlinger A., Pfanner N. The intermembrane space domain of mitochondrial Tom22 functions as a trans binding site for preproteins with N-terminal targeting sequences. Mol Cell Biol. 1997 Nov;17(11):6574–6584. doi: 10.1128/mcb.17.11.6574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nakai M., Endo T. Identification of yeast MAS17 encoding the functional counterpart of the mitochondrial receptor complex protein MOM22 of Neurospora crassa. FEBS Lett. 1995 Jan 3;357(2):202–206. doi: 10.1016/0014-5793(94)01362-5. [DOI] [PubMed] [Google Scholar]
  33. Nargang F. E., Künkele K. P., Mayer A., Ritzel R. G., Neupert W., Lill R. 'Sheltered disruption' of Neurospora crassa MOM22, an essential component of the mitochondrial protein import complex. EMBO J. 1995 Mar 15;14(6):1099–1108. doi: 10.1002/j.1460-2075.1995.tb07093.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Neupert W. Protein import into mitochondria. Annu Rev Biochem. 1997;66:863–917. doi: 10.1146/annurev.biochem.66.1.863. [DOI] [PubMed] [Google Scholar]
  35. Pfanner N., Craig E. A., Hönlinger A. Mitochondrial preprotein translocase. Annu Rev Cell Dev Biol. 1997;13:25–51. doi: 10.1146/annurev.cellbio.13.1.25. [DOI] [PubMed] [Google Scholar]
  36. 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]
  37. Rodriguez-Cousiño N., Nargang F. E., Baardman R., Neupert W., Lill R., Court D. A. An import signal in the cytosolic domain of the Neurospora mitochondrial outer membrane protein TOM22. J Biol Chem. 1998 May 8;273(19):11527–11532. doi: 10.1074/jbc.273.19.11527. [DOI] [PubMed] [Google Scholar]
  38. Ryan M. T., Wagner R., Pfanner N. The transport machinery for the import of preproteins across the outer mitochondrial membrane. Int J Biochem Cell Biol. 2000 Jan;32(1):13–21. doi: 10.1016/s1357-2725(99)00114-4. [DOI] [PubMed] [Google Scholar]
  39. Sakurai T., Shiga T., Shirai T., Tanaka H., Grumet M. Biochemical characterization and immunolocalization of SC2 protein: SC2 protein is indistinguishable from the cell adhesion molecule axonin-1. Brain Res Dev Brain Res. 1994 Nov 18;83(1):99–108. doi: 10.1016/0165-3806(94)90183-x. [DOI] [PubMed] [Google Scholar]
  40. Schneider I. Cell lines derived from late embryonic stages of Drosophila melanogaster. J Embryol Exp Morphol. 1972 Apr;27(2):353–365. [PubMed] [Google Scholar]
  41. Segraves W. A., Hogness D. S. The E75 ecdysone-inducible gene responsible for the 75B early puff in Drosophila encodes two new members of the steroid receptor superfamily. Genes Dev. 1990 Feb;4(2):204–219. doi: 10.1101/gad.4.2.204. [DOI] [PubMed] [Google Scholar]
  42. Spradling A. C., Stern D., Beaton A., Rhem E. J., Laverty T., Mozden N., Misra S., Rubin G. M. The Berkeley Drosophila Genome Project gene disruption project: Single P-element insertions mutating 25% of vital Drosophila genes. Genetics. 1999 Sep;153(1):135–177. doi: 10.1093/genetics/153.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stöffler H. E., Bähler M. The ATPase activity of Myr3, a rat myosin I, is allosterically inhibited by its own tail domain and by Ca2+ binding to its light chain calmodulin. J Biol Chem. 1998 Jun 5;273(23):14605–14611. doi: 10.1074/jbc.273.23.14605. [DOI] [PubMed] [Google Scholar]
  44. Talbot W. S., Swyryd E. A., Hogness D. S. Drosophila tissues with different metamorphic responses to ecdysone express different ecdysone receptor isoforms. Cell. 1993 Jul 2;73(7):1323–1337. doi: 10.1016/0092-8674(93)90359-x. [DOI] [PubMed] [Google Scholar]
  45. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Thummel C. S., Boulet A. M., Lipshitz H. D. Vectors for Drosophila P-element-mediated transformation and tissue culture transfection. Gene. 1988 Dec 30;74(2):445–456. doi: 10.1016/0378-1119(88)90177-1. [DOI] [PubMed] [Google Scholar]
  47. Tower J., Karpen G. H., Craig N., Spradling A. C. Preferential transposition of Drosophila P elements to nearby chromosomal sites. Genetics. 1993 Feb;133(2):347–359. doi: 10.1093/genetics/133.2.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Truman J. W., Talbot W. S., Fahrbach S. E., Hogness D. S. Ecdysone receptor expression in the CNS correlates with stage-specific responses to ecdysteroids during Drosophila and Manduca development. Development. 1994 Jan;120(1):219–234. doi: 10.1242/dev.120.1.219. [DOI] [PubMed] [Google Scholar]
  49. Urness L. D., Thummel C. S. Molecular analysis of a steroid-induced regulatory hierarchy: the Drosophila E74A protein directly regulates L71-6 transcription. EMBO J. 1995 Dec 15;14(24):6239–6246. doi: 10.1002/j.1460-2075.1995.tb00314.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Whitaker J. E., Moore P. L., Haugland R. P., Haugland R. P. Dihydrotetramethylrosamine: a long wavelength, fluorogenic peroxidase substrate evaluated in vitro and in a model phagocyte. Biochem Biophys Res Commun. 1991 Mar 15;175(2):387–393. doi: 10.1016/0006-291x(91)91576-x. [DOI] [PubMed] [Google Scholar]
  51. Wilson I. A., Niman H. L., Houghten R. A., Cherenson A. R., Connolly M. L., Lerner R. A. The structure of an antigenic determinant in a protein. Cell. 1984 Jul;37(3):767–778. doi: 10.1016/0092-8674(84)90412-4. [DOI] [PubMed] [Google Scholar]
  52. Yeo S. L., Lloyd A., Kozak K., Dinh A., Dick T., Yang X., Sakonju S., Chia W. On the functional overlap between two Drosophila POU homeo domain genes and the cell fate specification of a CNS neural precursor. Genes Dev. 1995 May 15;9(10):1223–1236. doi: 10.1101/gad.9.10.1223. [DOI] [PubMed] [Google Scholar]
  53. van Wilpe S., Ryan M. T., Hill K., Maarse A. C., Meisinger C., Brix J., Dekker P. J., Moczko M., Wagner R., Meijer M. Tom22 is a multifunctional organizer of the mitochondrial preprotein translocase. Nature. 1999 Sep 30;401(6752):485–489. doi: 10.1038/46802. [DOI] [PubMed] [Google Scholar]

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