家蚕mago nashi基因的克隆、序列分析及原核表达研究
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摘要
生殖质是在许多物种卵子发生中形成的一种特殊细胞质成分，含有这种成分的细胞才能发育为生殖细胞。对果蝇研究表明，mago nashi基因是生殖质组装和形成的一个关键基因，其编码的蛋白质能识别并结合某些生殖质决定基因的RNA，形成复合体，穿过核膜进入核质区域，指导生殖质的正确组装形成。从低等的真核生物酵母到高等的哺乳动物人，都存在mago nashi的同源基因并且相似性非常高。
为进一步阐明家蚕生殖发育的调控机制，在家蚕基因组、EST数据以及芯片数据分析的基础上，本论文对果蝇中已报道的生殖质决定相关基因在家蚕中的同源基因进行了鉴定，并对其表达模式进行了分析，然后对高度保守的mago nashi基因进行了克隆和原核表达，以期为以后深入了解该基因的功能研究奠定基础，获得结果如下：
1．果蝇生殖质决定相关基因在家蚕中的同源基因分析
从FlyBase下载果蝇生殖质形成相关基因18个(aubergine，spire，nanos，torso，tudor，vasa，pumilio，par-1，cappuccino，staufen，oskar，valois，germ-cell less，polar-granule component，mtlrRNA，mago nashi，tsunagi，exuperantia)，通过与家蚕基因组数据进行BLAST比对分析，发现除oskar，valois，germ-cell less，polar granule component，mtlrRNA 5个基因外，其余13个基因在家蚕中均有同源基因，并根据基因芯片数据分析了预测的家蚕生殖质形成相关基因的表达模式。发现在这些同源的生殖质形成相关基因中，有5个基因(Bmcapu，Bmnos，Bmvas，Bmaub，Bmexu)在雌雄发育后期有显著差异表达；有4个基因(Bmtud，Bmmago，Bmtsu，Bmstau)在雌雄发育过程中无差异表达；有3个基因(Bmnos，Bmvas，Bmaub)在生殖腺中特异表达；有6个基因(Bmcapu，Bmmago，Bmtsu，Bmexu，Bmstau，Bmtud)除在生殖腺中表达外，还在其它组织有高表达。
2．家蚕mago nashi基因的克隆及mRNA表达检测
根据预测序列设计引物，进行RT-PCR扩增和克隆测序验证，成功地克隆到家蚕mago nashi基因完整的编码区序列。分析发现该基因位于nscaf2655上，只有一个完整的拷贝，在基因组上有1611bp，共有两个外显子，分别为258bp和183bp，并且外显子、内含子边界符合典型的GT-AG结构。克隆的mago nashi基因完整的编码区序列为441 bp，编码146个氨基酸残基。RT-PCR检测了该基因在发育各时期各组织器官的表达情况，发现该基因在所检测的所有发育时期、组织器官均有表达，是一个非特异表达基因。
3．家蚕mago nashi基因的原核表达
将家蚕mago nashi基因的编码区序列克隆到原核表达载体pET28-a中，并将构建成的pET-mago nashi重组质粒转化表达宿主菌Escherichia coli BL21(DE3)细胞，IPTG诱导后进行SDS-PAGE检测分析。结果表明，同对照相比，重组质粒在23.5KD处有一条十分明显的新增蛋白带，与预测的蛋白质大小一致，说明该基因体外重组表达成功。进一步的分析发现该重组蛋白主要以包涵体的形式存在。
Germ plasm in many species formed during oogenesis ,it is a special cytoplasmic component. cells without the germ plasm can't develop into germ cells. Mago nashi ,a biological reproductive gene in Drosophila, plays an important role in assembling and formation of germ plasm. The protein encoded by Mago nashi gene can recognize and bind some RNA which decide the fate of germ cells .This RNP complex can guide germ plasm assembled correctly. From the lower eukaryotic like yeast to the higher mammals such as homo-sapiens,in which Mago nashi gene has the homology which has very high similarity with it. Mago nashi gene plays a conservative and important role in biological evolution.
To further clarify the Silkworm reproductive and developmental mechanisms, based on the analysis of the silkworm genome ,the large scale EST datas and microarray data ,this paper analyzed the homology and Spatio-temporal expression of the key gene which had been reported to determine the formation of germ plasm; The cloning and expression of Mago nashi gene have been done with a view to understand the function of the gene, the results obtained are as follows :
1 The homology of reproductive genes playing essential roles in Drosophila germ plasm formation in the silkworm:
Downloaded 18 reproductive genes playing essential roles in germ plasm formation in the Drosophila from FlyBase (aub, spir,nos, tor, tud,vas,pum,par-1,capu,stau,osk,valois,germ-cell less,polar- granule component,mtlrRNA, mago nashi, tsunagi,exuperantia). Used BLAST to compare them with silkworm genome data, excepting oskar, valois,germ-cell less,polar granule component, mtlrRNA, the remaining 13 genes in Silkworm have homologous gene. According to the gene array and the gene expression microarray data to analyz the expression patterns of the predicted genes which may have influence on the formation of germ cells. Five of these genes have significant differences in expression of paroecious development in later stage, the expression in femina is significantly stronger than maleness; in the paroecious development process four of these genes have no difference in expression; Three of the genes differentially expressed only in gonad; six genes expressed not only in gonad,but also in other tissues and organs.
2 The cloning and expression pattern of Bmmago gene in Bombyx mori
For the expression analysis of Bmmago gene, we cloned the coding sequence of the Bmmago gene by RT-PCR,then the clone sequenced. The Bmmago gene, a single copy locating on the genome, spans 1611bp and is comprised of 2 exons and 1 intron. The gene comprised an open reading frame of 441bp,and the encoded protein was 146 amino acids. The expression of Bmmago gene was found in all kinds of tissues,organs and all stages,thus the gene is a non-specific expression gene.
3 The expression of Bmmago gene in E.coli
In order to further research of the characteristics and function of the encoding protein, a prokaryote express vector, pET-Bmmago, containing the complete ORF of Bmmago, had been constructed. The recombinant plasmid was induced by IPTG ,then performed the SDS-PAGE, we found the recombinant protein expressed. Comparing with the reference, between the marker 31KD-20.1KD, recombinant protein shown a clearly new band. This molecular weight correspond the predicted protein size. Further analysis revealed that the recombinant protein in the form of inclusion bodies.
为进一步阐明家蚕生殖发育的调控机制，在家蚕基因组、EST数据以及芯片数据分析的基础上，本论文对果蝇中已报道的生殖质决定相关基因在家蚕中的同源基因进行了鉴定，并对其表达模式进行了分析，然后对高度保守的mago nashi基因进行了克隆和原核表达，以期为以后深入了解该基因的功能研究奠定基础，获得结果如下：
1．果蝇生殖质决定相关基因在家蚕中的同源基因分析
从FlyBase下载果蝇生殖质形成相关基因18个(aubergine，spire，nanos，torso，tudor，vasa，pumilio，par-1，cappuccino，staufen，oskar，valois，germ-cell less，polar-granule component，mtlrRNA，mago nashi，tsunagi，exuperantia)，通过与家蚕基因组数据进行BLAST比对分析，发现除oskar，valois，germ-cell less，polar granule component，mtlrRNA 5个基因外，其余13个基因在家蚕中均有同源基因，并根据基因芯片数据分析了预测的家蚕生殖质形成相关基因的表达模式。发现在这些同源的生殖质形成相关基因中，有5个基因(Bmcapu，Bmnos，Bmvas，Bmaub，Bmexu)在雌雄发育后期有显著差异表达；有4个基因(Bmtud，Bmmago，Bmtsu，Bmstau)在雌雄发育过程中无差异表达；有3个基因(Bmnos，Bmvas，Bmaub)在生殖腺中特异表达；有6个基因(Bmcapu，Bmmago，Bmtsu，Bmexu，Bmstau，Bmtud)除在生殖腺中表达外，还在其它组织有高表达。
2．家蚕mago nashi基因的克隆及mRNA表达检测
根据预测序列设计引物，进行RT-PCR扩增和克隆测序验证，成功地克隆到家蚕mago nashi基因完整的编码区序列。分析发现该基因位于nscaf2655上，只有一个完整的拷贝，在基因组上有1611bp，共有两个外显子，分别为258bp和183bp，并且外显子、内含子边界符合典型的GT-AG结构。克隆的mago nashi基因完整的编码区序列为441 bp，编码146个氨基酸残基。RT-PCR检测了该基因在发育各时期各组织器官的表达情况，发现该基因在所检测的所有发育时期、组织器官均有表达，是一个非特异表达基因。
3．家蚕mago nashi基因的原核表达
将家蚕mago nashi基因的编码区序列克隆到原核表达载体pET28-a中，并将构建成的pET-mago nashi重组质粒转化表达宿主菌Escherichia coli BL21(DE3)细胞，IPTG诱导后进行SDS-PAGE检测分析。结果表明，同对照相比，重组质粒在23.5KD处有一条十分明显的新增蛋白带，与预测的蛋白质大小一致，说明该基因体外重组表达成功。进一步的分析发现该重组蛋白主要以包涵体的形式存在。
Germ plasm in many species formed during oogenesis ,it is a special cytoplasmic component. cells without the germ plasm can't develop into germ cells. Mago nashi ,a biological reproductive gene in Drosophila, plays an important role in assembling and formation of germ plasm. The protein encoded by Mago nashi gene can recognize and bind some RNA which decide the fate of germ cells .This RNP complex can guide germ plasm assembled correctly. From the lower eukaryotic like yeast to the higher mammals such as homo-sapiens,in which Mago nashi gene has the homology which has very high similarity with it. Mago nashi gene plays a conservative and important role in biological evolution.
To further clarify the Silkworm reproductive and developmental mechanisms, based on the analysis of the silkworm genome ,the large scale EST datas and microarray data ,this paper analyzed the homology and Spatio-temporal expression of the key gene which had been reported to determine the formation of germ plasm; The cloning and expression of Mago nashi gene have been done with a view to understand the function of the gene, the results obtained are as follows :
1 The homology of reproductive genes playing essential roles in Drosophila germ plasm formation in the silkworm:
Downloaded 18 reproductive genes playing essential roles in germ plasm formation in the Drosophila from FlyBase (aub, spir,nos, tor, tud,vas,pum,par-1,capu,stau,osk,valois,germ-cell less,polar- granule component,mtlrRNA, mago nashi, tsunagi,exuperantia). Used BLAST to compare them with silkworm genome data, excepting oskar, valois,germ-cell less,polar granule component, mtlrRNA, the remaining 13 genes in Silkworm have homologous gene. According to the gene array and the gene expression microarray data to analyz the expression patterns of the predicted genes which may have influence on the formation of germ cells. Five of these genes have significant differences in expression of paroecious development in later stage, the expression in femina is significantly stronger than maleness; in the paroecious development process four of these genes have no difference in expression; Three of the genes differentially expressed only in gonad; six genes expressed not only in gonad,but also in other tissues and organs.
2 The cloning and expression pattern of Bmmago gene in Bombyx mori
For the expression analysis of Bmmago gene, we cloned the coding sequence of the Bmmago gene by RT-PCR,then the clone sequenced. The Bmmago gene, a single copy locating on the genome, spans 1611bp and is comprised of 2 exons and 1 intron. The gene comprised an open reading frame of 441bp,and the encoded protein was 146 amino acids. The expression of Bmmago gene was found in all kinds of tissues,organs and all stages,thus the gene is a non-specific expression gene.
3 The expression of Bmmago gene in E.coli
In order to further research of the characteristics and function of the encoding protein, a prokaryote express vector, pET-Bmmago, containing the complete ORF of Bmmago, had been constructed. The recombinant plasmid was induced by IPTG ,then performed the SDS-PAGE, we found the recombinant protein expressed. Comparing with the reference, between the marker 31KD-20.1KD, recombinant protein shown a clearly new band. This molecular weight correspond the predicted protein size. Further analysis revealed that the recombinant protein in the form of inclusion bodies.
引文
[1] Hamaguehi SA.light and electron microscopic study on the migration of primordial germ cell in teleost, Oryzias latipes.Cell Tissue Res, 1982,227:139-151
[2] Hamaguchi S.Changes in the morphology of the germinal dense bodies in primordial germ cells of the teleost Oryzia latipes.Cell Tissue Res, 1985,240:240-569
[3] 任素莲，王德秀，王如才。太平洋牡蛎卵母细胞发育及卵黄发生的超微结构。中国水产科学，1999，6(4)：4-6
[4] 尤永隆，林丹军，苏敏。黑脊倒刺卵子发生中生殖质的产生。动物学报，2004，50：231-239
[5] 桂建芳，易梅生。发育生物学。北京，科学出版社，2002
[6] T.A. Jongens, B. Hay, L.Y. Jan and Y.N. Jan.The germ cell-less gene product: a posteriorly localized component necessary for germ cell development in Drosophila. Cell 1992,70:569-584
[7] Kobayashi S, Yamada M, Asaoka M, Kitamura T.Essential role of the posterior morphogen nanos for germline development in Drosophila. Nature, 1996,380:708 -711
[8] Nakamura A, Amikura R, Mukai M, Kobayashi S, Lasko PF. Requirement for a noncoding RNA in Drosophila polar granules for germ cell establishment.Science,1996 274(5295): 2075--2079.
[9] Bounoure L. Recherches sur la lignee germinale chez la Grenouille rousse aux premiers stades du developpment. Ann.Sci. Natl, 10e Ser.1934, 17:67-248 (in French)
[10] Zhou Y and King M. L. RNA transport to the vegetal cortex of Xenopus oocytes. Dev. Bio1.1996,179:173-183.
[11] Santos AC, Lehmann R. Germ cell specification and migration in Drosophila and beyond. Curr Biol 2004,14:78-89
[12] Glotzer, J.B., Saffrich, R., Glotzer, M. and Ephrussi, A.. Curt. Biol. 1997,7: 326-337
[13] Ephrussi, A, Lehmann, R.Induction of germ cell formation by oskar Nature ,1992,358(6385):387-392
[14] Erdelyi, M., Michon, A.M., Guichet, A., Glotzer, J.B., Ephrussi, A. Requirement for Drosophila cytoplasmic tropomyosin in oskar mRNA localization. Nature, 1995, 377(6549): 524-527
[15] Lipshitz HD, Smibert CA. Mechanism of RNA localizational regulation. Curr Opin Genet Dev, 2000,10(5):476—488
[16] P. J. Webster, L. Liang, C. A. Berg, P. Lasko, and P. M. Macdonald Translational repressor bruno plays multiple roles in development and is widely conserved.Genes & Dev., October 1, 1997;11(19): 2510 - 2521
[17] Gavis, E.R. and Lehmann, R.Translational regulation of nanos by RNA. localization. Nature, 1994,369:315-318
[18] Breitwieser W, Markussen FH, Horstmann H, Ephrussi A. Oskar protein interaction with Vasa represents an essential step in polar granule assembly. Genes Dev 1996,10(21):79-88
[19] Johnstone O, Lasko E Interaction with eIF5B is essential for Vasa function during development. Development 2004,131(41):67-78
[20] Amikura R, Hanyu K, Kashikawa M, Kobayashi S. Tudor protein is essential for the localization of mitochondrial RNAs in polar granules of Drosophila embryos. Mech Dev 2001, 107:97-104
[21] Thomson T, Lasko P. Drosophila tudor is essential for polar granule assembly and pole cell specification, but not for posterior patteming. Genesis 2004,40:164-70
[22] Lantz, V.A., Clemens, S.E., Miller, K.G. The actin cytoskeleton is required for maintenance of posterior pole plasm components in the Drosophila embryo. Mech. Dev. 1999, 85:111-122
[23] Robertson SE, DockendorffTC, Leatherman JL, Faulkner DL, Jongens TA.germ cell-less is required only during the establishment of the germ cell lineage of Drosophila and has activities which are dependent and independent of its localization to the nuclear envelope .Dev Biol, 1999,215(2):288-97
[24] Martinho, R.G., Kunwar, ES., Casanova, J., Lehmann, R.. A Noncoding RNA Is Required for the Repression of RNApolⅡ-Dependent Transcription in Primordial Germ Cells. Curr. Bioi.2004,14(2):159-165
[25] Schaner CE, Deshpande G, Schedl PD, Kelly WG, A conserved chromatin architecture marks and maintains the restricted germ cell lineage in worms and flies. Dev Cell. 2003 Nov,5(5):747-57
[26] Asaoka, M., Sano, H. and Kobayashi, S). Maternal Nanos regulates zygotic gene expression in germline progenitors of Drosophila melanogaster. Mech. Dev 1998,78:153-158
[27] Leatherman JL, Levin L, Boero J, Jongens TA.Germ cell-less acts to repress transcription duringthe establishment of the Drosophila germ cell lineage. Curt. Biol. 2002,12: 1681-1685
[28] Van Doren,M., Williamson,A.L., and Lehmann,R.Regulation of zygotic gene expression in Drosophila primordial germ cells. Curr. Biol. 1998,8:243-246
[29] Gonzales-Reyes, A., Elliot, H. and St Johnston, D. Polarity of both major body axes in Drosophila by gurken-torpedo signalling. Nature 1995,375:654-658
[30] Driever, W. Maternal control of anterior development in the Drosophila embryo. Bate, Martinez Arias, 1993,301--324.
[31] Ephrussi, A., Lehrnann, R. Induction of germ cell formation by oskar. Nature 1992,358(6385):387-392
[32] Ferrandon, D., et al. Staufen protein associates with the 3'UTR ofbicoid mRNA to form particles that move in a microtubule-dependent manner. Cell 1994,79:1221-1232
[33] Newmark and Boswell. The mago nashi locus encodes an essential product required for germ plasm assembly in Drosophila.Development 1994,120(5): 1303—1313
[34]Boswell, R., Prout, M., and Steichen, J. Mutations in a newly identified Drosophila melanogaster gene, mago nashi, disrupt germ cell formation and result in the formation of mirror-image symmetrical double abdomen embryos. Development, 1991,113:373-384
[35]Newmark, P. A., Mohr, S. E., Gong, L., and Boswell, R. E. mago nashi mediates the posterior follicle cell-to-oocyte signal to organize axis formation in Drosophila. Development, 1997, 124:3197-3207.
[36]Micklem et al. The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila., Curr. Biol. 1997, 7(7): 468—478:
[37]Li, W., Boswell, R., and Wood, W.B.mag-1, a homolog of Drosophila mago nashi, regulates hermaphrodite germ-line sex determination in Caenorhabditis elegans. Dev. Biol.2000,218: 172-82.
[38]R.W., and Aplan, P.D.The mammalian homologue of mago nashi encodes a serum-inducible protein. Genomics,1998 47:319-22.
[39]Zhao, X.F., Nowak, N.J., Shows, T.B., and Aplan, P.D. MAGOH interacts with a novel RNA-binding protein. Genomics,2000,63:145-148.
[40]Franco Cotelli. Carla Lora Lamia, Sveva Doniselli, Ombretta Pozzoli, Claudio N. Gilardelli, Paolo Sordino. Identification and expression pattern of mago nashi during zebrafish development. Gene Expression Patterns 2004, 5: 265-272
[41]Sergio DJ Pena,Elida ML Rabelo, Adriano BS Hobaika, Paulo Marcos Z Coelho. Sequencing and Expression Analysis of a Schistosoma mansoni Gene Homologue to a Drosophila Gene Involved in Germ Plasm Assembly. Mem Inst Oswaldo Cruz, Rio de Janeiro, 1998,Vol. 93, Suppl. I: 207-209
[42] Stephanie E. Mohr, Simon T. Dillon, and Robert E. Boswell.The RNA-binding protein Tsunagi interacts with Mago Nashi to establish polarity and localize oskar mRNA during Drosophila oogenesis. Genes Dev 2001,15:2886-2899
[43]Le Hir,H., Gatfield,D., Izaurralde,E. and Moore,M.J.The exon-exon junction complex provides a binding platform for factors involved in mRNA export and NMD. EMBO J 2001, 20:4987-4997
[44]Kim,V.N., Kataoka,N. and Dreyfuss.GRole of the nonsensemediated decay factor hUpf3 in the splicing-dependent exon-exon junction complex. Science,2001,293:1832-1836.
[45]Lykke-Andersen,J., Shu,M.-D. and Steitz,J.A. Human Upf proteins target an mRNA for nonsense-mediated decay when bound downstream of a termination codon. Cell, 2000,103:1121-1131.
[46]Lykke-Andersen,J., Shu.M.D. and Steitz,J.A.Communication of the position of exon-exon junctions to the mRNA surveillance machinery by the protein RNPS1. Science,2001, 293:1836-1839.
[47]Mendell,J.T., ap Rhys,C.M.J. and Dietz,H.C Separable roles for rent 1/hUpf1 in altered splicing and decay of nonsense transcripts.Science,2002,298:419-422
[48]Kataoka, N., Diem, M.D., Kim, V.N., Yong, J., and Dreyfuss, GMagoh, a human homolog of Drosophila mago nashi protein, is a component of the splicing-dependent exon-exon junction complex. EMBO J.2001 20:6424-6433.
[49]Kataoka, N., Yong, J., Kim, V.N., Velazquez, R, Perkinson, R.A., Wang R, Dreyfuss, G Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm. Mol. Cell,2000, 6:673-682.
[50]Taizo Kawano, Naoyuki Kataoka, Gideon Dreyfuss, Hiroshi Sakamoto. Ce-Y14 and MAG-1, components of the exon-exon junction complex,are required for embryogenesis and germline sexual switchingin Caenorhabditis elegans. Mechanisms of Development,2004,121: 27-35
[51]Xia Q, Zhou Z, Lu C, et al. A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science, 2004, 306(5703):1937-1940
[52]Burge C, Karlin S. Prediction of complete gene structures in human genomic DNA. J Mol Biol, 1997,268: 78-94
[53] Burge C, Karlin S. Prediction of complete gene structures in human genomic DNA. J Mol Biol, 1997, 268: 78-94
[54] Salamov A A, Solovyev V V. Ab initio gene finding in Drosophila genomic DNA. Genome Res, 2000,10:516-522
[55] Wang J, Li S, Zhang Y, Zheng H, Xu Z, Ye J, Yu J, Wong G K. Vertebrate gene predictions and the problem of large genes. Nature Rev Genet, 2003,4: 741-749
[56] Schmidt et al., Genetic and molecular characterization of sting, a gene involved in crystal formation and meiotic drive in the male germ line of Drosophila melanogaster. Genetics, 1999,151(2): 749-760
[57]Harris, A.N., Macdonald, P.M.. aubergine encodes a Drosophila polar granule component required for pole cell formation and related to eIP2C. Development,2001, 128(14): 2823-2832.
[58] van Eeden, R, St. Johnston, D.. The polarisation of the anterior-posterior and dorsal-ventral axes during Drosophila oogenesis. Curr. Opin. Genet. Dev. 1999, 9(4): 396-404.
[59] Lasko and Ashburner, The product of the Drosophila gene vasa is very similar to eukaryotic initiation factor 4A. 1988, Nature 335: 611-617
[60] Wang, C.I., Lehmann, R.. Nanos is the localized posterior determinant in Drosophila. Cell ,1991,66(4): 637-647.
[61] Murata, Y, Wharton, R.P. Binding of pumilio to maternal hunchback mRNA is required for posterior patterning in Drosophila embryos. Cell,1995, 80(5): 747-756.
[62] Lehmann R, Nusslein-Volhard C. Abdominal segmentation, pole cell formation, and embryonic polarity require the localized ac-tivity of oskar, a maternal gene in Drosophila. Cell, 1986,47(1): 141-152.
[63] Lehmann R, Ephrussi A. Germ plasm formation and germ cell determination in Drosophila. Ciba Found Symp, 1994, 182: 282-296.
[64] Purugganan M D. The molecular evolution of development. Bio-essays, 1998, 20(9): 700-711.
[2] Hamaguchi S.Changes in the morphology of the germinal dense bodies in primordial germ cells of the teleost Oryzia latipes.Cell Tissue Res, 1985,240:240-569
[3] 任素莲，王德秀，王如才。太平洋牡蛎卵母细胞发育及卵黄发生的超微结构。中国水产科学，1999，6(4)：4-6
[4] 尤永隆，林丹军，苏敏。黑脊倒刺卵子发生中生殖质的产生。动物学报，2004，50：231-239
[5] 桂建芳，易梅生。发育生物学。北京，科学出版社，2002
[6] T.A. Jongens, B. Hay, L.Y. Jan and Y.N. Jan.The germ cell-less gene product: a posteriorly localized component necessary for germ cell development in Drosophila. Cell 1992,70:569-584
[7] Kobayashi S, Yamada M, Asaoka M, Kitamura T.Essential role of the posterior morphogen nanos for germline development in Drosophila. Nature, 1996,380:708 -711
[8] Nakamura A, Amikura R, Mukai M, Kobayashi S, Lasko PF. Requirement for a noncoding RNA in Drosophila polar granules for germ cell establishment.Science,1996 274(5295): 2075--2079.
[9] Bounoure L. Recherches sur la lignee germinale chez la Grenouille rousse aux premiers stades du developpment. Ann.Sci. Natl, 10e Ser.1934, 17:67-248 (in French)
[10] Zhou Y and King M. L. RNA transport to the vegetal cortex of Xenopus oocytes. Dev. Bio1.1996,179:173-183.
[11] Santos AC, Lehmann R. Germ cell specification and migration in Drosophila and beyond. Curr Biol 2004,14:78-89
[12] Glotzer, J.B., Saffrich, R., Glotzer, M. and Ephrussi, A.. Curt. Biol. 1997,7: 326-337
[13] Ephrussi, A, Lehmann, R.Induction of germ cell formation by oskar Nature ,1992,358(6385):387-392
[14] Erdelyi, M., Michon, A.M., Guichet, A., Glotzer, J.B., Ephrussi, A. Requirement for Drosophila cytoplasmic tropomyosin in oskar mRNA localization. Nature, 1995, 377(6549): 524-527
[15] Lipshitz HD, Smibert CA. Mechanism of RNA localizational regulation. Curr Opin Genet Dev, 2000,10(5):476—488
[16] P. J. Webster, L. Liang, C. A. Berg, P. Lasko, and P. M. Macdonald Translational repressor bruno plays multiple roles in development and is widely conserved.Genes & Dev., October 1, 1997;11(19): 2510 - 2521
[17] Gavis, E.R. and Lehmann, R.Translational regulation of nanos by RNA. localization. Nature, 1994,369:315-318
[18] Breitwieser W, Markussen FH, Horstmann H, Ephrussi A. Oskar protein interaction with Vasa represents an essential step in polar granule assembly. Genes Dev 1996,10(21):79-88
[19] Johnstone O, Lasko E Interaction with eIF5B is essential for Vasa function during development. Development 2004,131(41):67-78
[20] Amikura R, Hanyu K, Kashikawa M, Kobayashi S. Tudor protein is essential for the localization of mitochondrial RNAs in polar granules of Drosophila embryos. Mech Dev 2001, 107:97-104
[21] Thomson T, Lasko P. Drosophila tudor is essential for polar granule assembly and pole cell specification, but not for posterior patteming. Genesis 2004,40:164-70
[22] Lantz, V.A., Clemens, S.E., Miller, K.G. The actin cytoskeleton is required for maintenance of posterior pole plasm components in the Drosophila embryo. Mech. Dev. 1999, 85:111-122
[23] Robertson SE, DockendorffTC, Leatherman JL, Faulkner DL, Jongens TA.germ cell-less is required only during the establishment of the germ cell lineage of Drosophila and has activities which are dependent and independent of its localization to the nuclear envelope .Dev Biol, 1999,215(2):288-97
[24] Martinho, R.G., Kunwar, ES., Casanova, J., Lehmann, R.. A Noncoding RNA Is Required for the Repression of RNApolⅡ-Dependent Transcription in Primordial Germ Cells. Curr. Bioi.2004,14(2):159-165
[25] Schaner CE, Deshpande G, Schedl PD, Kelly WG, A conserved chromatin architecture marks and maintains the restricted germ cell lineage in worms and flies. Dev Cell. 2003 Nov,5(5):747-57
[26] Asaoka, M., Sano, H. and Kobayashi, S). Maternal Nanos regulates zygotic gene expression in germline progenitors of Drosophila melanogaster. Mech. Dev 1998,78:153-158
[27] Leatherman JL, Levin L, Boero J, Jongens TA.Germ cell-less acts to repress transcription duringthe establishment of the Drosophila germ cell lineage. Curt. Biol. 2002,12: 1681-1685
[28] Van Doren,M., Williamson,A.L., and Lehmann,R.Regulation of zygotic gene expression in Drosophila primordial germ cells. Curr. Biol. 1998,8:243-246
[29] Gonzales-Reyes, A., Elliot, H. and St Johnston, D. Polarity of both major body axes in Drosophila by gurken-torpedo signalling. Nature 1995,375:654-658
[30] Driever, W. Maternal control of anterior development in the Drosophila embryo. Bate, Martinez Arias, 1993,301--324.
[31] Ephrussi, A., Lehrnann, R. Induction of germ cell formation by oskar. Nature 1992,358(6385):387-392
[32] Ferrandon, D., et al. Staufen protein associates with the 3'UTR ofbicoid mRNA to form particles that move in a microtubule-dependent manner. Cell 1994,79:1221-1232
[33] Newmark and Boswell. The mago nashi locus encodes an essential product required for germ plasm assembly in Drosophila.Development 1994,120(5): 1303—1313
[34]Boswell, R., Prout, M., and Steichen, J. Mutations in a newly identified Drosophila melanogaster gene, mago nashi, disrupt germ cell formation and result in the formation of mirror-image symmetrical double abdomen embryos. Development, 1991,113:373-384
[35]Newmark, P. A., Mohr, S. E., Gong, L., and Boswell, R. E. mago nashi mediates the posterior follicle cell-to-oocyte signal to organize axis formation in Drosophila. Development, 1997, 124:3197-3207.
[36]Micklem et al. The mago nashi gene is required for the polarisation of the oocyte and the formation of perpendicular axes in Drosophila., Curr. Biol. 1997, 7(7): 468—478:
[37]Li, W., Boswell, R., and Wood, W.B.mag-1, a homolog of Drosophila mago nashi, regulates hermaphrodite germ-line sex determination in Caenorhabditis elegans. Dev. Biol.2000,218: 172-82.
[38]R.W., and Aplan, P.D.The mammalian homologue of mago nashi encodes a serum-inducible protein. Genomics,1998 47:319-22.
[39]Zhao, X.F., Nowak, N.J., Shows, T.B., and Aplan, P.D. MAGOH interacts with a novel RNA-binding protein. Genomics,2000,63:145-148.
[40]Franco Cotelli. Carla Lora Lamia, Sveva Doniselli, Ombretta Pozzoli, Claudio N. Gilardelli, Paolo Sordino. Identification and expression pattern of mago nashi during zebrafish development. Gene Expression Patterns 2004, 5: 265-272
[41]Sergio DJ Pena,Elida ML Rabelo, Adriano BS Hobaika, Paulo Marcos Z Coelho. Sequencing and Expression Analysis of a Schistosoma mansoni Gene Homologue to a Drosophila Gene Involved in Germ Plasm Assembly. Mem Inst Oswaldo Cruz, Rio de Janeiro, 1998,Vol. 93, Suppl. I: 207-209
[42] Stephanie E. Mohr, Simon T. Dillon, and Robert E. Boswell.The RNA-binding protein Tsunagi interacts with Mago Nashi to establish polarity and localize oskar mRNA during Drosophila oogenesis. Genes Dev 2001,15:2886-2899
[43]Le Hir,H., Gatfield,D., Izaurralde,E. and Moore,M.J.The exon-exon junction complex provides a binding platform for factors involved in mRNA export and NMD. EMBO J 2001, 20:4987-4997
[44]Kim,V.N., Kataoka,N. and Dreyfuss.GRole of the nonsensemediated decay factor hUpf3 in the splicing-dependent exon-exon junction complex. Science,2001,293:1832-1836.
[45]Lykke-Andersen,J., Shu,M.-D. and Steitz,J.A. Human Upf proteins target an mRNA for nonsense-mediated decay when bound downstream of a termination codon. Cell, 2000,103:1121-1131.
[46]Lykke-Andersen,J., Shu.M.D. and Steitz,J.A.Communication of the position of exon-exon junctions to the mRNA surveillance machinery by the protein RNPS1. Science,2001, 293:1836-1839.
[47]Mendell,J.T., ap Rhys,C.M.J. and Dietz,H.C Separable roles for rent 1/hUpf1 in altered splicing and decay of nonsense transcripts.Science,2002,298:419-422
[48]Kataoka, N., Diem, M.D., Kim, V.N., Yong, J., and Dreyfuss, GMagoh, a human homolog of Drosophila mago nashi protein, is a component of the splicing-dependent exon-exon junction complex. EMBO J.2001 20:6424-6433.
[49]Kataoka, N., Yong, J., Kim, V.N., Velazquez, R, Perkinson, R.A., Wang R, Dreyfuss, G Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm. Mol. Cell,2000, 6:673-682.
[50]Taizo Kawano, Naoyuki Kataoka, Gideon Dreyfuss, Hiroshi Sakamoto. Ce-Y14 and MAG-1, components of the exon-exon junction complex,are required for embryogenesis and germline sexual switchingin Caenorhabditis elegans. Mechanisms of Development,2004,121: 27-35
[51]Xia Q, Zhou Z, Lu C, et al. A draft sequence for the genome of the domesticated silkworm (Bombyx mori). Science, 2004, 306(5703):1937-1940
[52]Burge C, Karlin S. Prediction of complete gene structures in human genomic DNA. J Mol Biol, 1997,268: 78-94
[53] Burge C, Karlin S. Prediction of complete gene structures in human genomic DNA. J Mol Biol, 1997, 268: 78-94
[54] Salamov A A, Solovyev V V. Ab initio gene finding in Drosophila genomic DNA. Genome Res, 2000,10:516-522
[55] Wang J, Li S, Zhang Y, Zheng H, Xu Z, Ye J, Yu J, Wong G K. Vertebrate gene predictions and the problem of large genes. Nature Rev Genet, 2003,4: 741-749
[56] Schmidt et al., Genetic and molecular characterization of sting, a gene involved in crystal formation and meiotic drive in the male germ line of Drosophila melanogaster. Genetics, 1999,151(2): 749-760
[57]Harris, A.N., Macdonald, P.M.. aubergine encodes a Drosophila polar granule component required for pole cell formation and related to eIP2C. Development,2001, 128(14): 2823-2832.
[58] van Eeden, R, St. Johnston, D.. The polarisation of the anterior-posterior and dorsal-ventral axes during Drosophila oogenesis. Curr. Opin. Genet. Dev. 1999, 9(4): 396-404.
[59] Lasko and Ashburner, The product of the Drosophila gene vasa is very similar to eukaryotic initiation factor 4A. 1988, Nature 335: 611-617
[60] Wang, C.I., Lehmann, R.. Nanos is the localized posterior determinant in Drosophila. Cell ,1991,66(4): 637-647.
[61] Murata, Y, Wharton, R.P. Binding of pumilio to maternal hunchback mRNA is required for posterior patterning in Drosophila embryos. Cell,1995, 80(5): 747-756.
[62] Lehmann R, Nusslein-Volhard C. Abdominal segmentation, pole cell formation, and embryonic polarity require the localized ac-tivity of oskar, a maternal gene in Drosophila. Cell, 1986,47(1): 141-152.
[63] Lehmann R, Ephrussi A. Germ plasm formation and germ cell determination in Drosophila. Ciba Found Symp, 1994, 182: 282-296.
[64] Purugganan M D. The molecular evolution of development. Bio-essays, 1998, 20(9): 700-711.