小鼠新基因克隆及功能预测
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摘要
该研究克隆了一个可能与小鼠核酸结合相关的新基因,并对其表达模式和预期编码蛋白质功能进行了预测分析。该研究采用启动子捕获技术获得了具有启动子活性的基因组片段,结合生物信息学方法预测得到该启动子活性片段下游基因。采用RT-PCR(reverse transcription PCR)技术扩增得到相应基因,其c DNA为1 504 bp,完整开放阅读框(open reading frame,ORF)为540 bp,编码179个氨基酸。同源性分析表明,该基因与褐鼠(Rattus norvegicus)核酸结合蛋白和黑猩猩(Pan troglodytes)RNA结合蛋白的基因序列分别有78%和40%的相似性。利用实时荧光定量PCR技术分析了不同组织和不同生理、病理状态下该基因的表达水平。结果表明,该基因在带瘤小鼠各组织中的表达水平均高于健康小鼠相应组织,提示该基因可能参与了肿瘤的发生、发展。
In this study, a new gene coding putative nucleic acid binding protein from mouse was cloned. The gene expression pattern and its predicted protein function had also been discussed. By utilizing promoter-trap technology, a DNA fragment with promoter activity was obtained from mouse genomic DNA. A putative gene located in the downstream of this DNA fragment was predicted by bioinformatics analysis. The predicted gene was 1 504 bp in length with an open reading frame of 540 bp. The predicted open reading frame was confirmed by reverse transcription PCR(RT-PCR). Homology analysis with other species showed that the sequence of this gene shares similarities with the nucleic acid binding protein from Rattus norvegicus and RNA binding protein from Pan troglodytes of 78% and 40%, respectively. The gene expression properties in diverse tissue types under different physiological status or pathological status were studied by quantitative Real-time PCR(q RT-PCR). As in all the tissue types tested from mice bearing tumor, the expression levels of this gene are much higher than that in healthy mice. These results indicate that this new gene is possibly involved in tumorigenesis.
In this study, a new gene coding putative nucleic acid binding protein from mouse was cloned. The gene expression pattern and its predicted protein function had also been discussed. By utilizing promoter-trap technology, a DNA fragment with promoter activity was obtained from mouse genomic DNA. A putative gene located in the downstream of this DNA fragment was predicted by bioinformatics analysis. The predicted gene was 1 504 bp in length with an open reading frame of 540 bp. The predicted open reading frame was confirmed by reverse transcription PCR(RT-PCR). Homology analysis with other species showed that the sequence of this gene shares similarities with the nucleic acid binding protein from Rattus norvegicus and RNA binding protein from Pan troglodytes of 78% and 40%, respectively. The gene expression properties in diverse tissue types under different physiological status or pathological status were studied by quantitative Real-time PCR(q RT-PCR). As in all the tissue types tested from mice bearing tumor, the expression levels of this gene are much higher than that in healthy mice. These results indicate that this new gene is possibly involved in tumorigenesis.
引文
1 Blazewicz J,Glover F,Kasprzak M.DNA Sequencing—Tabu and Scatter search combined.INFORMS 2004;16(3):232-40.
2 Consortium MGS,Waterston RH,Lindbladtoh K,Birney E,Rogers J,Abril JF,et al.Initial sequencing and comparative analysis of the mouse genome.Nature 2002;420(6915):520-62.
3 Lander ES,Linton LM,Birren B,Nusbaum C,Zody MC,Baldwin J,et al.Initial sequencing and analysis of the human genome.Nature 2001;409(6822):860-920.
4 Riley DE,Jeon JS,Krieger JN.Simple repeat evolution includes dramatic primary sequence changes that conserve folding potential.Biochem Biophys Res Commun 2007;355(3):619-25.
5 Huang JF,Guo YJ,Zhao CX,Yuan SX,Wang Y,Tang GN,et al.Hepatitis B virus X protein(HBx)-related long noncoding RNA(lnc RNA)down-regulated expression by HBx(Dreh)inhibits hepatocellular carcinoma metastasis by targeting the intermediate filament protein vimentin.Hepatology 2013;57(5):1882-92.
6 Fernández-Navarro F,Hervás-Martínez C,Ruiz R,Riquelme JC.Evolutionary Generalized Radial Basis Function neural networks for improving prediction accuracy in gene classification using feature selection.Applied Soft Computing 2012;12(6):1787-800.
7 马宝山,朱义胜.基于隐马尔科夫模型的基因预测算法.大连海事大学学报(Ma Baoshan,Zhu Yisheng.Gene-prediction algorithm based on hidden Markov model.Journal of Dalian Maritime University)2008;34(4):41-4.
8 Lee JY,Shim WS.Cloning of Xenopus laevis TRPV2 by gene prediction.Genomics&Informatis 2005;3(1):24-9.
9 刘庆坡,冯英,董辉.水稻线粒体丝氨酸羟甲基转移酶基因的电子克隆.生物信息学[Liu Qingpo,Feng Ying,Dong Hui.In silico cloning of m SHMT gene from rice(Oryza sativa L.).Bioinformatiocs]2005;3(1):5-9.
10 Khattak NA,Rauf S,Sehgal SA,Shahwar D,Ismail M.In silico gene prediction for autosomal recessive cone-rod dystrophy 8(CORD8).JBI 2013;14(2):104-17.
11 Tabata S,Kaneko T,Nakamura Y,Kotani H,Kato T,Asamizu E,et al.Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana.Nature 2000;402(6763):761-8.
12 Sasaki T,Burr B.International rice genome sequencing project:the effort to completely sequence the rice genome.Curr Opin Plant Biol 2000;3(2):138-42.
13 Bradley A.Mining the mouse genome.Nature 2002;420(6915):512-4.
14 Eichler EE,Clark RA,She X.An assessment of the sequence gaps:Unfinished business in a finished human genome.Nat Rev Genet 2004;5(5):345-54.
15 Venter JC,Adams MD,Myers EW,Li PW,Mural RJ,Sutton GG,et al.The sequence of the human genome.Science 2015;291(5507):1304-51.
16 李元元,张靖溥.基因捕获技术及其最新进展(英文).遗传学报(Li Yuanyuan,Zhang Jingpu.Gene trapping techniques current progress.Journal of Genetics and Genomics)2006;33(3):189-98.
17 Lindsey K,Wei W,Clarke MC,Mcardle HF,Rooke LM,Topping JF.Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants.Transgenic Res 1993;2(1):33.
18 Bellen HJ,Levis RW,Liao G,He Y,Carlson JW,Tsang G,et al.The BDGP gene disruption project.Genetics 2004;167(2):761-81.
19 Buendia F,Cano JC.Webgene:A generative and web-based learning architecture to teach operating systems in undergraduate courses.IEEE Xplore 2006;49(4):464-73.
20 张胜利,李东方,张改生,王军卫,牛娜.应用软件Fgenesh预测水稻基因.中国农业科学(Zhang Shengli,Li Dongfang,Zhang Gaisheng,Wang Junwei,Niu Na.Prediction of rice gene by fgenesh.Scientia Agricultura Sinica)2008;41(6):1567-74.
21 谢婷婷,王芳,赵卫东.GATA3与恶性肿瘤的相关性研究进展.山东医药(Xie Tingting,Wang Fang,Zhao Weidong.Research progress on the correlation between GATA3 and malignant tumor.Shangdong Medical Journal)2008;48(19):149-50.
22 涂军伟,朱以军,朱寿田,朱丹,陈慧,赵建平,等.大鼠肺纤维化转录因子T-bet/GATA3的表达及其与相关细胞因子的关系.浙江医学(Tu Junwei,Zhu Yijun,Zhu Shoutian,Zhu Dan,Chen Hui,Zhao Jianping,et al.Expression of transcription factors T—bet/GATA3 and its association with other cytokines related to pulmonary fibrosis in rat.Zhejiang Medcine)2010;32(11):1621-4.
23 郭宇立,倪逸声.从一级结构预测蛋白质稳定性:Curuprasad,Reedy和Pandit方法.生物化学与生物物理进展(Guo Yuli,Ni Yisheng.Prediction of protein stability from primary structures:Curuprasad,reedy and pandit methods.Prog Biochem Biophys)1993;20(2):95-9.
24 赵元茵,王元忠,曹念,周度金,李渝萍.核定位信号及其分析策略.中国生物化学与分子生物学报(Zhao Yuanyin,Wang Yuanzhong,Cao Nian,Zhou Dujin,Li Yuping.Categories and research strategies of nuclear localization signal.Chinese Journal of Biochemistry and Molecular Biology)2009;25(8):683-9.
25 王玉明,李亚.组织和发育特异性基因的远距离转录调控.生命的化学(Wang Yuming,Li Ya.Tissue and development of specific genes for long-term transcriptional regulation.Chemistry of Life)2007;27(2):151-3.
26 Thackray VG.Fox tales:Regulation of gonadotropin gene expression by forkhead transcription factors.Mol Cell Endocrinol2014;385(1/2):62-70.
27 Kind J,Pagie L,de Vries SS,Nahidiazar L,Dey SS,Bienko M,et al.Genome-wide maps of nuclear lamina interactions in single human cells.Cell 2011;163(1):134.
28 林元山.原核生物和真核生物的基因组及其基因表达调控的比较研究.怀化学院学报(Lin Yuanshan.A comparative study on regulation of genome and gene expression of prokaryotes and eukaryotes.Journal of Huaihua University)2002;21(5):39-44.
29 Gregory SG,Sekhon M,Schein J,Zhao S,Osoegawa K,Scott CE,et al.A physical map of the mouse genome.Nature 2002;418(6899):743-50.
30 Keren H,Lev-Maor G,Ast G.Alternative splicing and evolution:Diversification,exon definition and function.Nat Rev Genet2010;11(5):345-55.
31 于丹丹,汲广东.HMGB基因的结构功能与演化.鲁东大学学报(自然科学版)[Yu Dandan,Ji Guangdong.Structural function and evolution of HMGB gene.Journal of Ludong University(Natural Science Edition)]2016;32(1):38-43.
32 Lacasse EC,Lefebvre YA.Nuclear localization signals overlap DNA-or RNA-binding domains in nucleic acid-binding proteins.Nucleic Acids Res 1995;23(10):1647-56.
33 王锐,胡晓愚.DNA结合蛋白结构域的研究进展.化学进展(Wang Rui,Hu Xiaoyu.Progress in research on DNA-binding protein domains.Progress in Chemistry)1995;7(4):277.
34 杜光伟,周严,袁建刚,强伯勤.RRM RNA结合蛋白的结构与功能.生物化学与生物物理进展(Du Guangwei,Zhou Yan,Yuan Jiangang,Qiang Boqin.Structure and function of RRM RNA-binding protein.Biochemistry and Biophysical Advances)1999;26(4):305-07.
35 Strano S,Munarriz E,Rossi M,Castagnoli L,Shaul Y,Sacchi A,et al.Physical interaction with Yes-associated protein enhances p73 transcriptional activity.J Biol Chem 2001;276(18):15164-73.
36 Chi SW,Ayed A,Arrowsmith CH.Solution structure of a conserved C-terminal domain of p73 with structural homology to the SAM domain.EMBO J 1999;18(16):4438-45.
37 Papageorgis P,Ozturk S,Lambert A W,Neophytou CM,Tzatsos A,Wong CK,et al.Targeting IL13Ralpha2 activates STAT6-TP63 pathway to suppress breast cancer lung metastasis.Breast Cancer Res 2015;17(1):98.
38 Hu QY,Jin TB,Wang L,Zhang L,Geng T,Liang G,et al.Genetic variation in the TP63 gene is associated with lung cancer risk in the Han population.Tumor Biol 2014;35(3):1863-6.
39 Parsons JK,Saria EA,Nakayama M,Vessella RL,Sawyers CL,Isaacs WB,et al.Comprehensive mutational analysis and m RNA isoform quantification of TP63 in normal and neoplastic human prostate cells.Prostate 2009;69(5):559-69.
40 Fagerberg L,Hallstr?m BM,Oksvold P,Kampf C,Djureinovic D,Odeberg J,et al.Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibodybased proteomics.Mol Cell Proteomics 2014;13(2):397.
2 Consortium MGS,Waterston RH,Lindbladtoh K,Birney E,Rogers J,Abril JF,et al.Initial sequencing and comparative analysis of the mouse genome.Nature 2002;420(6915):520-62.
3 Lander ES,Linton LM,Birren B,Nusbaum C,Zody MC,Baldwin J,et al.Initial sequencing and analysis of the human genome.Nature 2001;409(6822):860-920.
4 Riley DE,Jeon JS,Krieger JN.Simple repeat evolution includes dramatic primary sequence changes that conserve folding potential.Biochem Biophys Res Commun 2007;355(3):619-25.
5 Huang JF,Guo YJ,Zhao CX,Yuan SX,Wang Y,Tang GN,et al.Hepatitis B virus X protein(HBx)-related long noncoding RNA(lnc RNA)down-regulated expression by HBx(Dreh)inhibits hepatocellular carcinoma metastasis by targeting the intermediate filament protein vimentin.Hepatology 2013;57(5):1882-92.
6 Fernández-Navarro F,Hervás-Martínez C,Ruiz R,Riquelme JC.Evolutionary Generalized Radial Basis Function neural networks for improving prediction accuracy in gene classification using feature selection.Applied Soft Computing 2012;12(6):1787-800.
7 马宝山,朱义胜.基于隐马尔科夫模型的基因预测算法.大连海事大学学报(Ma Baoshan,Zhu Yisheng.Gene-prediction algorithm based on hidden Markov model.Journal of Dalian Maritime University)2008;34(4):41-4.
8 Lee JY,Shim WS.Cloning of Xenopus laevis TRPV2 by gene prediction.Genomics&Informatis 2005;3(1):24-9.
9 刘庆坡,冯英,董辉.水稻线粒体丝氨酸羟甲基转移酶基因的电子克隆.生物信息学[Liu Qingpo,Feng Ying,Dong Hui.In silico cloning of m SHMT gene from rice(Oryza sativa L.).Bioinformatiocs]2005;3(1):5-9.
10 Khattak NA,Rauf S,Sehgal SA,Shahwar D,Ismail M.In silico gene prediction for autosomal recessive cone-rod dystrophy 8(CORD8).JBI 2013;14(2):104-17.
11 Tabata S,Kaneko T,Nakamura Y,Kotani H,Kato T,Asamizu E,et al.Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana.Nature 2000;402(6763):761-8.
12 Sasaki T,Burr B.International rice genome sequencing project:the effort to completely sequence the rice genome.Curr Opin Plant Biol 2000;3(2):138-42.
13 Bradley A.Mining the mouse genome.Nature 2002;420(6915):512-4.
14 Eichler EE,Clark RA,She X.An assessment of the sequence gaps:Unfinished business in a finished human genome.Nat Rev Genet 2004;5(5):345-54.
15 Venter JC,Adams MD,Myers EW,Li PW,Mural RJ,Sutton GG,et al.The sequence of the human genome.Science 2015;291(5507):1304-51.
16 李元元,张靖溥.基因捕获技术及其最新进展(英文).遗传学报(Li Yuanyuan,Zhang Jingpu.Gene trapping techniques current progress.Journal of Genetics and Genomics)2006;33(3):189-98.
17 Lindsey K,Wei W,Clarke MC,Mcardle HF,Rooke LM,Topping JF.Tagging genomic sequences that direct transgene expression by activation of a promoter trap in plants.Transgenic Res 1993;2(1):33.
18 Bellen HJ,Levis RW,Liao G,He Y,Carlson JW,Tsang G,et al.The BDGP gene disruption project.Genetics 2004;167(2):761-81.
19 Buendia F,Cano JC.Webgene:A generative and web-based learning architecture to teach operating systems in undergraduate courses.IEEE Xplore 2006;49(4):464-73.
20 张胜利,李东方,张改生,王军卫,牛娜.应用软件Fgenesh预测水稻基因.中国农业科学(Zhang Shengli,Li Dongfang,Zhang Gaisheng,Wang Junwei,Niu Na.Prediction of rice gene by fgenesh.Scientia Agricultura Sinica)2008;41(6):1567-74.
21 谢婷婷,王芳,赵卫东.GATA3与恶性肿瘤的相关性研究进展.山东医药(Xie Tingting,Wang Fang,Zhao Weidong.Research progress on the correlation between GATA3 and malignant tumor.Shangdong Medical Journal)2008;48(19):149-50.
22 涂军伟,朱以军,朱寿田,朱丹,陈慧,赵建平,等.大鼠肺纤维化转录因子T-bet/GATA3的表达及其与相关细胞因子的关系.浙江医学(Tu Junwei,Zhu Yijun,Zhu Shoutian,Zhu Dan,Chen Hui,Zhao Jianping,et al.Expression of transcription factors T—bet/GATA3 and its association with other cytokines related to pulmonary fibrosis in rat.Zhejiang Medcine)2010;32(11):1621-4.
23 郭宇立,倪逸声.从一级结构预测蛋白质稳定性:Curuprasad,Reedy和Pandit方法.生物化学与生物物理进展(Guo Yuli,Ni Yisheng.Prediction of protein stability from primary structures:Curuprasad,reedy and pandit methods.Prog Biochem Biophys)1993;20(2):95-9.
24 赵元茵,王元忠,曹念,周度金,李渝萍.核定位信号及其分析策略.中国生物化学与分子生物学报(Zhao Yuanyin,Wang Yuanzhong,Cao Nian,Zhou Dujin,Li Yuping.Categories and research strategies of nuclear localization signal.Chinese Journal of Biochemistry and Molecular Biology)2009;25(8):683-9.
25 王玉明,李亚.组织和发育特异性基因的远距离转录调控.生命的化学(Wang Yuming,Li Ya.Tissue and development of specific genes for long-term transcriptional regulation.Chemistry of Life)2007;27(2):151-3.
26 Thackray VG.Fox tales:Regulation of gonadotropin gene expression by forkhead transcription factors.Mol Cell Endocrinol2014;385(1/2):62-70.
27 Kind J,Pagie L,de Vries SS,Nahidiazar L,Dey SS,Bienko M,et al.Genome-wide maps of nuclear lamina interactions in single human cells.Cell 2011;163(1):134.
28 林元山.原核生物和真核生物的基因组及其基因表达调控的比较研究.怀化学院学报(Lin Yuanshan.A comparative study on regulation of genome and gene expression of prokaryotes and eukaryotes.Journal of Huaihua University)2002;21(5):39-44.
29 Gregory SG,Sekhon M,Schein J,Zhao S,Osoegawa K,Scott CE,et al.A physical map of the mouse genome.Nature 2002;418(6899):743-50.
30 Keren H,Lev-Maor G,Ast G.Alternative splicing and evolution:Diversification,exon definition and function.Nat Rev Genet2010;11(5):345-55.
31 于丹丹,汲广东.HMGB基因的结构功能与演化.鲁东大学学报(自然科学版)[Yu Dandan,Ji Guangdong.Structural function and evolution of HMGB gene.Journal of Ludong University(Natural Science Edition)]2016;32(1):38-43.
32 Lacasse EC,Lefebvre YA.Nuclear localization signals overlap DNA-or RNA-binding domains in nucleic acid-binding proteins.Nucleic Acids Res 1995;23(10):1647-56.
33 王锐,胡晓愚.DNA结合蛋白结构域的研究进展.化学进展(Wang Rui,Hu Xiaoyu.Progress in research on DNA-binding protein domains.Progress in Chemistry)1995;7(4):277.
34 杜光伟,周严,袁建刚,强伯勤.RRM RNA结合蛋白的结构与功能.生物化学与生物物理进展(Du Guangwei,Zhou Yan,Yuan Jiangang,Qiang Boqin.Structure and function of RRM RNA-binding protein.Biochemistry and Biophysical Advances)1999;26(4):305-07.
35 Strano S,Munarriz E,Rossi M,Castagnoli L,Shaul Y,Sacchi A,et al.Physical interaction with Yes-associated protein enhances p73 transcriptional activity.J Biol Chem 2001;276(18):15164-73.
36 Chi SW,Ayed A,Arrowsmith CH.Solution structure of a conserved C-terminal domain of p73 with structural homology to the SAM domain.EMBO J 1999;18(16):4438-45.
37 Papageorgis P,Ozturk S,Lambert A W,Neophytou CM,Tzatsos A,Wong CK,et al.Targeting IL13Ralpha2 activates STAT6-TP63 pathway to suppress breast cancer lung metastasis.Breast Cancer Res 2015;17(1):98.
38 Hu QY,Jin TB,Wang L,Zhang L,Geng T,Liang G,et al.Genetic variation in the TP63 gene is associated with lung cancer risk in the Han population.Tumor Biol 2014;35(3):1863-6.
39 Parsons JK,Saria EA,Nakayama M,Vessella RL,Sawyers CL,Isaacs WB,et al.Comprehensive mutational analysis and m RNA isoform quantification of TP63 in normal and neoplastic human prostate cells.Prostate 2009;69(5):559-69.
40 Fagerberg L,Hallstr?m BM,Oksvold P,Kampf C,Djureinovic D,Odeberg J,et al.Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibodybased proteomics.Mol Cell Proteomics 2014;13(2):397.