Abstract
The Bombyx mori doublesex (Bmdsx), a homologue of doublesex of Drosophila, is the bottom most gene of the sex determination cascade. Bmdsx plays a very crucial role in somatic sexual development. Its pre-mRNA sex-specifically splices to generate two splice variants; one encodes female-specific and the other encodes male-specific polypeptides which differ only at their C-termini. The open reading frame of Bmdsx consists of 5 exons, of which exons 3 and 4 are female-specific and are skipped in males. In the present study, we have identified a third splice form of the Bmdsx which is specific only to females and differs from the previously reported Bmdsxf isoform by the presence of 15 bp sequence. This new female splice form is generated as a result of alternative 5′ splice site selection in the third exon adding additional 15 bp sequence in exon 3 which results in alteration of the reading frame leading to incorporation of an early stop codon. Thus the protein encoded by this splice form is 20 aa shorter than the known BmDsxF. Initial results obtained from the study of dsx homologues in Saturniid silkmoths suggest that both the female-specific Dsx proteins are essential for female sexual differentiation. It remains to be seen whether female-specific multiple splice forms of dsx are characteristic feature of only silkmoths or widespread among lepidopterans. The findings that sex determination mechanism is unique in lepidopterans offer an opportunity to develop genetic sexing methods in beneficial as well as economically destructive lepidopteran pests.
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References
Abe H, Fujii T, Tanaka N, Yokoyama T, Kakehashi H, Ajimura M, Mita K, Banno Y, Yasukochi Y, Oshiki T, Nenoi M, Ishikawa T, Shimada T (2008) Identification of the female-determining region of the W chromosome in Bombyx mori. Genetica 133:269–282
Ajimura M, Ken S, Hiroaki A, Toshiki T, Toru S, Mita K (2006) Are the zinc-finger motif genes, z1 and z20, located in the W chromosome involved in the sex-determination of the domesticated silkworm, Bombyx mori? Abstracts from Seventh International Workshop on the Molecular Biology and Genetics of the Lepidoptera J Insect Sci [will be available online http://www.insectscience.org/]
Bachiller D, Sanchez L (1991) Production of X0 clones in XX females of Drosophila. Genet Res 57:23–28
Baker BS, Burtis K, Goralski T, Mattox W, Nagoshi R (1989) Molecular genetic aspects of sex determination in Drosophila melanogaster. Genome 31:638–645
Bell LR, Maine EM, Schedl P, Cline TW (1988) Sex-lethal, a Drosophila sex determination switch gene, exhibits sex-specific RNA splicing and sequence similarity to RNA binding proteins. Cell 55:1037–1046
Bell LR, Horabin JI, Schedl P, Cline TW (1991) Positive autoregulation of sex-lethal by alternative splicing maintains the female determined state in Drosophila. Cell 65:229–239
Belote JM, McKeown M, Boggs RT, Ohkawa R, Sosnowski BA (1989) Molecular genetics of transformer, a genetic switch controlling sexual differentiation in Drosophila. Dev Genet 10:143–154
Boggs RT, Gregor P, Idriss S, Belote JM, McKeown M (1987) Regulation of sexual differentiation in D. melanogaster via alternative splicing of RNA from the transformer gene. Cell 50:739–747
Bopp D, Bell LR, Cline TW, Schedl P (1991) Developmental distribution of female-specific Sex-lethal proteins in Drosophila melanogaster. Genes Dev 5:403–415
Bridges CB, Anderson EG (1925) Crossing over in the X Chromosomes of Triploid Females of DROSOPHILA MELANOGASTER. Genetics 10:418–441
Bull JJ (1983) Evolution of sex determining mechanisms. Benjamin Cummings Publishing, Menlo Park
Burtis KC, Baker BS (1989) Drosophila doublesex gene controls somatic sexual differentiation by producing alternatively spliced mRNAs encoding related sex-specific polypeptides. Cell 56:997–1010
Cavaliere D, Di Cara F, Polito LC, Digilio FA (2009) Cloning and functional characterization of the intersex homologous gene in the pest lepidopteron maruca vitrata. Int J Dev Biol 53 (7):1057–1062
Chase BA, Baker BS (1995) A genetic analysis of intersex, a gene regulating sexual differentiation in Drosophila melanogaster females. Genetics 139:1649–1661
Cline TW (1978) Two closely linked mutations in Drosophila melanogaster that are lethal to opposite sexes and interact with daughterless. Genetics 90:683–698
Cline TW (1983) The interaction between daughterless and sex-lethal in triploids: a lethal sex-transforming maternal effect linking sex determination and dosage compensation in Drosophila melanogaster. Dev Biol 95:260–274
Cline TW (1984) Autoregulatory functioning of a Drosophila gene product that establish es and maintains the sexually determined state. Genetics 107:231–277
Cline TW, Meyer BJ (1996) Vive la difference: males vs females in flies vs worms. Annu Rev Genet 30:637–702
Erickson JW, Quintero JJ (2007) Indirect effects of ploidy suggest X chromosome dose, not the X:A ratio, signals sex in Drosophila. PLoS Biol 5:e332
Gandhe AS, John SH, Nagaraju J (2007) Noduler, a novel immune up-regulated protein mediates nodulation response in insects. J Immunol 179:6943–6951
Garrett-Engele CM, Siegal ML, Manoli DS, Williams BC, Li H, Baker BS (2002) intersex, a gene required for female sexual development in Drosophila, is expressed in both sexes and functions together with doublesex to regulate terminal differentiation. Development 129:4661–4675
Hoshijima K, Inoue K, Higuchi I, Sakamoto H, Shimura Y (1991) Control of doublesex alternative splicing by transformer and transformer-2 in Drosophila. Science 252:833–836
Kanginakudru S, Royer C, Edupalli SV, Jalabert A, Mauchamp B, Prasad SV, Chavancy G, Couble P, Nagaraju J (2007) Targeting ie-1 gene by RNAi induces baculoviral resistance in lepidopteran cell lines and in transgenic silkworms. Insect Mol Biol 16:635–644
Keyes LN, Cline TW, Schedl P (1992) The primary sex determination signal of Drosophila acts at the level of transcription. Cell 68:933–943
MacDougall C, Harbison D, Bownes M (1995) The developmental consequences of alternate splicing in sex determination and differentiation in Drosophila. Dev Biol 172:353–376
Marec F, Neven LG, Robinson AS, Vreysen M, Goldsmith MR, Nagaraju J, Franz G (2005) Development of genetic sexing strains in Lepidoptera: from traditional to transgenic approaches. J Econ Entomol 98:248–259
Nagaraja GM, Mahesh G, Satish V, Madhu M, Muthulakshmi M, Nagaraju J (2005) Genetic mapping of Z chromosome and identification of W chromosome-specific markers in the silkworm, Bombyx mori. Heredity 95:148–157
Ohbayashi F, Suzuki MG, Mita K, Okano K, Shimada T (2001) A homologue of the Drosophila doublesex gene is transcribed into sex-specific mRNA isoforms in the silkworm, Bombyx mori. Comp Biochem Physiol B Biochem Mol Biol 128:145–158
Peloquin JJ, Thibault ST, Staten R, Miller TA (2000) Germ-line transformation of pink bollworm (Lepidoptera: gelechiidae) mediated by the piggyBac transposable element. Insect Mol Biol 9:323–333
Penalva LO, Sanchez L (2003) RNA binding protein sex-lethal (Sxl) and control of Drosophila sex determination and dosage compensation. Microbiol Mol Biol Rev 67:343–359
Saccone G, Pane A, Polito LC (2002) Sex determination in flies, fruitflies and butterflies. Genetica 116:15–23
Sanchez L (2008) Sex-determining mechanisms in insects. Int J Dev Biol 52:837–856
Sanchez L, Nothiger R (1983) Sex determination and dosage compensation in Drosophila melanogaster: production of male clones in XX females. EMBO J 2:485–491
Satish V, Shukla JN, Nagaraju J (2006) CCCH-type zinc finger genes: candidate regulators of sex determination pathway in the silkworm, Bombyx mori. Abstracts from Seventh International Workshop on the Molecular Biology and Genetics of the Lepidoptera J Insect Sci [will be available online http://www.insectscience.org/]
Schutt C, Nothiger R (2000) Structure, function and evolution of sex-determining systems in Dipteran insects. Development 127:667–677
Shukla JN, Nagaraju J (2010) Two female-specific DSX proteins are encoded by the sex-specific transcripts of dsx, and are required for female sexual differentiation in two wild silkmoth species, Antheraea assama and Antheraea mylitta (Lepidoptera, Saturniidae). Insect Biochem Mol Biol. doi:10.1016/j.ibmb.2010.06.008
Siegal ML, Baker BS (2005) Functional conservation and divergence of intersex, a gene required for female differentiation in Drosophila melanogaster. Dev Genes Evol 215:1–12
Slee R, Bownes M (1990) Sex determination in Drosophila melanogaster. Q Rev Biol 65:175–204
Steinmann-Zwicky M, Amrein H, Nothiger R (1990) Genetic control of sex determination in Drosophila. Adv Genet 27:189–237
Suzuki MG, Ohbayashi F, Mita K, Shimada T (2001) The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori. Insect Biochem Mol Biol 31:1201–1211
Suzuki MG, Funaguma S, Kanda T, Tamura T, Shimada T (2003) Analysis of the biological functions of a doublesex homologue in Bombyx mori. Dev Genes Evol 213:345–354
Suzuki MG, Funaguma S, Kanda T, Tamura T, Shimada T (2005) Role of the male BmDSX protein in the sexual differentiation of Bombyx mori. Evol Dev 7:58–68
Suzuki MG, Imanishi S, Dohmae N, Nishimura T, Shimada T, Matsumoto S (2008) Establishment of a novel in vivo sex-specific splicing assay system to identify a trans-acting factor that negatively regulates splicing of Bombyx mori dsx female exons. Mol Cell Biol 28:333–343
Tamura T, Thibert C, Royer C, Kanda T, Abraham E, Kamba M, Komoto N, Thomas JL, Mauchamp B, Chavancy G, Shirk P, Fraser M, Prudhomme JC, Couble P (2000) Germline transformation of the silkworm Bombyx mori L using a piggyBac transposon-derived vector. Nat Biotechnol 18:81–84
Uhlirova M, Foy BD, Beaty BJ, Olson KE, Riddiford LM, Jindra M (2003) Use of Sindbis virus-mediated RNA interference to demonstrate a conserved role of Broad-Complex in insect metamorphosis. Proc Natl Acad Sci U S A 100:15607–15612
Valcarcel J, Singh R, Zamore PD, Green MR (1993) The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA. Nature 362:171–175
Waterbury JA, Jackson LL, Schedl P (1999) Analysis of the doublesex female protein in Drosophila melanogaster: role on sexual differentiation and behavior and dependence on intersex. Genetics 152:1653–1667
West SA, Reece SE, Sheldon BC (2002) Sex ratios. Heredity 88:117–124
Zarkower D (2001) Establishing sexual dimorphism: conservation amidst diversity? Nat Rev Genet 2:175–185
Acknowledgments
J Nagaraju acknowledges Centre of Excellence grant from the Department of Biotechnology, Government of India. JN Shukla is recipient of the fellowship from Council of Scientific and Industrial Research (CSIR), India.
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Shukla, J.N., Jadhav, S. & Nagaraju, J. Novel female-specific splice form of dsx in the silkworm, Bombyx mori . Genetica 139, 23–31 (2011). https://doi.org/10.1007/s10709-010-9479-3
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DOI: https://doi.org/10.1007/s10709-010-9479-3