BmCycH基因及其在家蚕中表达特性的研究
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摘要
细胞周期依赖性蛋白激酶(Cdk7)是后生动物Cdk激酶(CAK)重要的催化、调节亚基,而Cdk的激活需要自身结合一个调节亚基,也就是细胞周期蛋白H(cyclin H)。在哺乳动物体内,cyclin H与Cdk7、MAT1形成的三元复合物具有CAK激酶的活性,它能磷酸化某些Cdk T环内保守的苏氨酸残基,继而行使Cdk的作用。目前,国内外对cyclin H的研究大多集中在脊椎动物上,而对无脊椎动物的研究较少。
通过RT-PCR的方法从家蚕蛹中扩增到家蚕cyclin H基因完整的ORF序列,GenBank No. AV406047,将其命名为家蚕细胞周期蛋白H(Bombyx mori Cyclin H,BmCycH)。该基因序列长为753 bp,编码250个氨基酸残基,预测分子量为28.34 kD。将扩增到的BmCycH基因克隆到重组表达载体pET-28a(+)相应的酶切位点上,构建重组蛋白表达质粒pET-28a(+)-BmCycH,经PCR、酶切鉴定以及序列分析证明重组正确。将该重组子转化大肠杆菌BL21(DE3),用终浓度为1 mmol/L的IPTG诱导工程菌表达重组蛋白,裂解菌体作SDS-PAGE分析,在32 kD左右的位置有一条特异性蛋白条带,与预期值相符。重组蛋白以包涵体形式存在,将超声裂解菌体后12000 rpm离心所得到的沉淀溶于N-十二烷基肌氨酸钠中,并以亲和层析的方法纯化目的蛋白His-tag BmCycH。以纯化蛋白为抗原免疫雄性新西兰兔,制备了该重组蛋白的多克隆抗体,ELISA检测该抗血清的效价可达1:12800以上。
利用荧光定量PCR技术,分析家蚕各个发育时期,以及五龄幼虫不同组织的靶mRNA的量。实验结果表明,BmCycH蛋白在家蚕各个时期都有表达,蛾、蛹、幼虫和卵表达量依次降低;BmCycH蛋白在家蚕五龄幼虫不同组织也均有表达,在头部表达量最高,丝腺表达量最低。提取家蚕各个时期以及五龄幼虫不同组织的蛋白,用于Western blotting实验。实验结果表明各个时期蛋白表达量与荧光定量PCR结果大致相同;而不同组织中蛋白表达量略有不同。这些研究表明BmCycH蛋白在家蚕发育分化过程中可能起到一定作用。利用免疫荧光化学法进行亚细胞定位,结果表明,该蛋白主要存在细胞核中。
The catalytic subunit of the metazoan Cdk-activating kinase(CAK) is cyclin-dependent kinase 7(Cdk7). Activation of Cdk7 requires its association with a regulatory subunit, cyclinH. In mammalian cells, Cdk7, cyclin H and MAT1, function as the CAK which phosphorylates the conserved threonine residue within the T loop of several Cdks. However, most researches of cyclin H focused on vertebrate, seldom were done on invertebrate.
The full open reading frame (ORF) of Bombyx mori cyclin H was obtained from silkworm pupa by RT-PCR and named BmCycH (Bombyx mori cyclin H), of which the accession number was AV406047. After cloned into pET-28a(+) and expressed in E.coli BL21(DE3), the BmCycH was fused with His-tag and purified on chelating columns. The molecular weight of this fusion protein was 32.0 kD, characterized by SDS-PAGE. This result proved that His-tag BmCycH was expressed in the right form. After being purified with Ni2+ affinity chromatography the recombinant protein was as antigen against which the antibody was raised by immune rabbit. The titer of the polyclonal antibody reaches 1:12800 measured by ELISA.
We also compared the quantity of BmCycH mRNA in different stages of the fifth instar larva and different tissues of silkworm. The expression level in the moth was highest, but in the egg was the lowest; and in the different tissues, the expression level in the head was the highest and the silk gland was the lowest. Western blot analysis revealed that the protein was expressed in all silkworm tissues and 4 developmental phases examined, with highest expression in the moth, pupa and larvae, lowest in egg. It may suggest that BmCycH plays a role in the differentiation and development of silkworm. Using fluorescent antibody for subcellular localization, we preliminarily localized the protein in the nucleus in BmN.
通过RT-PCR的方法从家蚕蛹中扩增到家蚕cyclin H基因完整的ORF序列,GenBank No. AV406047,将其命名为家蚕细胞周期蛋白H(Bombyx mori Cyclin H,BmCycH)。该基因序列长为753 bp,编码250个氨基酸残基,预测分子量为28.34 kD。将扩增到的BmCycH基因克隆到重组表达载体pET-28a(+)相应的酶切位点上,构建重组蛋白表达质粒pET-28a(+)-BmCycH,经PCR、酶切鉴定以及序列分析证明重组正确。将该重组子转化大肠杆菌BL21(DE3),用终浓度为1 mmol/L的IPTG诱导工程菌表达重组蛋白,裂解菌体作SDS-PAGE分析,在32 kD左右的位置有一条特异性蛋白条带,与预期值相符。重组蛋白以包涵体形式存在,将超声裂解菌体后12000 rpm离心所得到的沉淀溶于N-十二烷基肌氨酸钠中,并以亲和层析的方法纯化目的蛋白His-tag BmCycH。以纯化蛋白为抗原免疫雄性新西兰兔,制备了该重组蛋白的多克隆抗体,ELISA检测该抗血清的效价可达1:12800以上。
利用荧光定量PCR技术,分析家蚕各个发育时期,以及五龄幼虫不同组织的靶mRNA的量。实验结果表明,BmCycH蛋白在家蚕各个时期都有表达,蛾、蛹、幼虫和卵表达量依次降低;BmCycH蛋白在家蚕五龄幼虫不同组织也均有表达,在头部表达量最高,丝腺表达量最低。提取家蚕各个时期以及五龄幼虫不同组织的蛋白,用于Western blotting实验。实验结果表明各个时期蛋白表达量与荧光定量PCR结果大致相同;而不同组织中蛋白表达量略有不同。这些研究表明BmCycH蛋白在家蚕发育分化过程中可能起到一定作用。利用免疫荧光化学法进行亚细胞定位,结果表明,该蛋白主要存在细胞核中。
The catalytic subunit of the metazoan Cdk-activating kinase(CAK) is cyclin-dependent kinase 7(Cdk7). Activation of Cdk7 requires its association with a regulatory subunit, cyclinH. In mammalian cells, Cdk7, cyclin H and MAT1, function as the CAK which phosphorylates the conserved threonine residue within the T loop of several Cdks. However, most researches of cyclin H focused on vertebrate, seldom were done on invertebrate.
The full open reading frame (ORF) of Bombyx mori cyclin H was obtained from silkworm pupa by RT-PCR and named BmCycH (Bombyx mori cyclin H), of which the accession number was AV406047. After cloned into pET-28a(+) and expressed in E.coli BL21(DE3), the BmCycH was fused with His-tag and purified on chelating columns. The molecular weight of this fusion protein was 32.0 kD, characterized by SDS-PAGE. This result proved that His-tag BmCycH was expressed in the right form. After being purified with Ni2+ affinity chromatography the recombinant protein was as antigen against which the antibody was raised by immune rabbit. The titer of the polyclonal antibody reaches 1:12800 measured by ELISA.
We also compared the quantity of BmCycH mRNA in different stages of the fifth instar larva and different tissues of silkworm. The expression level in the moth was highest, but in the egg was the lowest; and in the different tissues, the expression level in the head was the highest and the silk gland was the lowest. Western blot analysis revealed that the protein was expressed in all silkworm tissues and 4 developmental phases examined, with highest expression in the moth, pupa and larvae, lowest in egg. It may suggest that BmCycH plays a role in the differentiation and development of silkworm. Using fluorescent antibody for subcellular localization, we preliminarily localized the protein in the nucleus in BmN.
引文
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[27] Kunlin Jin, Tetsuya Nagayama, Jun Chen, et al. Molecular Cloning of a Cell Cycle Regulation Gene Cyclin H from Ischemic Rat Brain: Expression in Neurons after Global Cerebral Ischemia[J]. Journal of Neurochemistry, 1999, 73: 1598-1608.
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[31] Eberhard Schneidera, Sabine Kartarius, Norbert Schusterb, et al. The cyclin H/cdk7/Mat1 kinase activity is regulated by CK2 phosphorylation of cyclin H[J]. Oncogene, 2002, 21(33): 5031-5037.
[32] Valay, J.G., M.F.Dubois, O.Bensaude, et al. Ccl1, a cyclin associated with protein kinase Kin28, controls the phosphorylation of RNA polymeraseⅡlargest subunit and mRNA transcription[J]. C.R.Acad. Sci.III, 1996, 319(3): 183–189.
[33] Mary J. Cisomowski, Geoffrey M. Laff, Mark J. Solomon, et al. KIN28 Encodes a C-Terminal Domain Kinase That Controls mRNA Transcription in Saccharomyces cerevisiae but Lacks Cyclin-Dependent Kinase-Activating Kinase (CAK) Activity[J]. Molecular and Cellular Biology, 1995, 15(6): 2983-2992.
[34] Michael Christopher Keogh, Eun Jung Cho, Vladimir Podolny, et al. Kin28 Is Found within TFⅡH and a Kin28-Ccl1-Tfb3 Trimer Complex with Differential Sensitivities to T-Loop Phosphorylation[J]. Molecular and Cellular Biology, 2002, 22(5): 1288-1297.
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[2] Andersen G, Poterszman A, Egly JM, et al. The crystal s tructure of human cyclinH[J]. FEBS Letters, 1996, 397: 65-69.
[3] William O’Gorman, Benjamin Thomas, Kon Yew Kwek, et al. Analysis of U1 Small Nuclear RNA Interaction with Cyclin H[J]. J Biol Chem, 2005, 280(44): 36920–36925.
[4] Paul F.Lambert, Fatah Kashanchi, Michael F.Radonovich, et al. Phosphorylation of p53 Serine 15 Increases Interaction with CBP[J]. J Biol Chem, 1998, 273(49): 33048–33053.
[5] Zhao Xin, The function of cyclinH in the regulation of cell division cycle[J]. Chin J Gastroenterol Hepatol, 2007, 16(6): 615-620.
[6] Joanna Bloom, Frederick R. Cross Multiple levels of cyclin specificity in cell-cycle control[J]. Nature Review, 2007, 8: 149-160.
[7] G. Andersen, A. Poterszman, J.M. Egly, et al. The crystal structure of human cyclin H[J]. FEBS, 1996, 397: 65-69.
[8]翟中和,王喜忠,丁明孝主编.细胞生物学[M],高等教育出版社, 2000.
[9] Meijer, L. Cyclin-dependent kinases inhibitors as potential anticancer, antineurodegenerative, antiviral and antiparasitic agents[J]. Drug Resistance Updates, 2000, 3: 83-88.
[10] Evelyne Derelle, Genome analysis of the smallest free-living eukaryote Ostreococcus tauri unveils many unique features[J]. PNAS, 2006, 103(31): 11647-11652.
[11] Qing YunLiu, Zhi LiWu, Wen JianLv, et al, Developmental expression of in zebrafish:the essential role of CyclinH during early embryo development[J]. Cell Research, 2007, 17: 163-173.
[12] Jesper Q. Svejstrup, William J. Feaver, Roger D. Kornberg. Subunits of Yeast RNA PolymeraseⅡTranscription Factor TFIIH Encoded by the CCL1 Gene[J]. J Biol Chem, 1996, 271(2): 643–645.
[13] Fisher,R.P., Morgan,D.O. A novel cyclin associates with MO15/CDK7 to form the CDK-activating kinase[J]. Cell, 1994, 78(4): 713-24.
[14] Jeffrey,P.D., Russo,A.A., Polyak,K., et al. Mechanism of CDK activation revealed by the structure of a cyclin A-CDK2 complex[J]. Nature, 1995, 376: 313–320.
[15] Gregers Andersen, Didier Busso, Arnaud Poterszman, et al. The structure of cyclin H: common mode of kinase activation and speciffic features[J]. The EMBO Journal, 1997, 16(5): 958–967.
[16] Nina Korsisaari. Functional analysis of Cdk7-interacting proteins Mat1 and Hint in model organisms[D]. Helsinki: University of Helsinki, 2002.
[17] Jorg P.Adamczewski, Mireille Rossignol, Jean-Pierre Tassan1, et al. MAT1, cdk7 and cyclin H form a kinase complex which is UV light-sensitive upon association with TFⅡH[J]. The EMBO Journal, 1996, 15(8): 1877-1884.
[18] Paula Rickert, Jeffry L Corden and Emma Lees. Cyclin C/CDK8 and cyclin H/CDK7/p36 are biochemically distinct CTD kinases[J]. Oncogene, 1999, 18: 1093-1102.
[19] Arik Dvir, Karla Pfeil Garrett, Christian Chalut, et al. A Role for ATP and TFⅡH in Activation of the RNA PolymeraseⅡPreinitiation Complex Prior to Transcription Initiation [J]. The Journal of Biological Chemistry, 1996, 27(13): 7245-7248.
[20]尹慕军,王杉,马向涛等.崔忘荣细胞周期素依赖性激酶7及细胞周期素H在人结直中肠癌组织中的表达[J].中华消化杂志, 2002, 22(3): 184-186.
[21] Wang Shan, Hang Bin, Qiao Xinmin. A study on the expression of cyclin H and CDK7 in human breast cancer[J]. Chin J Gen Surg, 2002, 17(19): 611-613.
[22] Narayan Iyer, Michael S.Reagan, Kou Juey Wu, et al. Interactions Involving the Human RNA PolymeraseⅡTranscription/Nucleotide Excision Repair Complex TFⅡH, the Nucleotide Excision Repair Protein XPG, and Cockayne Syndrome Group B (CSB) Protein[J]. Biochemistry, 1996, 35(7): 2157–2167.
[23] Sebastiaan Winkler, Wim Vermeulen, Frederic Coin, et al. Affinity Purification of Human DNA Repair/Transcription Factor TFⅡH Using Epitope-tagged Xeroderma Pigmentosum B Protein[J]. J Biol Chem, 1998, 273(2): 1092-1098.
[24] Valay, J.G., Simon, M. and Faye, G. The kin28 protein kinase is associated with a cyclin in Saccharomyces cerevisiae[J]. J Mol Biol, 1993, 234: 307-310.
[25] Ying Liu, Shinichiro Ando, Xianmin Xia, et al. p62, A TFⅡH Subunit, Directly Interacts with Thyroid Hormone Receptor and Enhances T3-Mediated Transcription[J]. Molecular Endocrinology, 2004, 19(4): 879–884.
[26] Aya Fukuda1, Jun Yamauchi, Shwu Yuan Wu, et al. Reconstitution of recombinant TFⅡH that can mediate activator-dependent transcription[J]. Genes to Cells, 2001, 6: 707–719.
[27] Kunlin Jin, Tetsuya Nagayama, Jun Chen, et al. Molecular Cloning of a Cell Cycle Regulation Gene Cyclin H from Ischemic Rat Brain: Expression in Neurons after Global Cerebral Ischemia[J]. Journal of Neurochemistry, 1999, 73: 1598-1608.
[28] Linda J.KO, Sheau Yann Shieh, Xinbin Chen, et al. p53 Is Phosphorylated by CDK7-Cyclin H in a p36MAT1-Dependent Manner[J]. Molecular and Cellular Biology, 1997, 17(12): 7220-7229.
[29] Liu ZhenJie, Liu XinGuang, Liang NianCi. The role of protein kinase CK2 in cell cycle control [J]. Chinese Pharmacological Bulletin, 2006, 22(11): 1281-1285.
[30] Michael Faust,Sabine Kartarius, Sandra L. et al. Cyclin H is a new binding partner for protein kinase CK2[J]. Biochemical and Biophysical Research Communications, 2002, 296: 13–19.
[31] Eberhard Schneidera, Sabine Kartarius, Norbert Schusterb, et al. The cyclin H/cdk7/Mat1 kinase activity is regulated by CK2 phosphorylation of cyclin H[J]. Oncogene, 2002, 21(33): 5031-5037.
[32] Valay, J.G., M.F.Dubois, O.Bensaude, et al. Ccl1, a cyclin associated with protein kinase Kin28, controls the phosphorylation of RNA polymeraseⅡlargest subunit and mRNA transcription[J]. C.R.Acad. Sci.III, 1996, 319(3): 183–189.
[33] Mary J. Cisomowski, Geoffrey M. Laff, Mark J. Solomon, et al. KIN28 Encodes a C-Terminal Domain Kinase That Controls mRNA Transcription in Saccharomyces cerevisiae but Lacks Cyclin-Dependent Kinase-Activating Kinase (CAK) Activity[J]. Molecular and Cellular Biology, 1995, 15(6): 2983-2992.
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