新肌纤生成调节因子MR-1基因功能的初步探讨
摘要
肌纤生成调节因子MR1（myofibrillogenesis regulator 1）为中国协和医科大学医药生物技术研究所代谢工程室克隆发现的新基因。它的完整cDNA由416个核苷酸组成，编码142个氨基酸，它的EST序列最初是从人卵巢肿瘤组织中克隆得到的，后来发现它也存在于子宫内膜组织及甲状旁腺肿瘤组织。初步研究表明该蛋白是一种膜蛋白，这种膜蛋白与肌肉收缩调控以及细胞信息传导和凋亡过程相关。
我的论文工作是在此基础之上进行的，主要分为以下三部分。
本论文第一部分是应用PCR-SSCP技术对MR1基因进行研究，筛查MR1的基因突变。针对MR1基因的三个外显子分别设计了三对引物，得到MR1三个外显子的PCR产物进行PCR-SSCP分析，只有外显子3存在基因突变，虽然该突变并未造成蛋白氨基酸组成的变化，但这种突变是否影响其基因拼接并导致功能的变化尚待进一步研究。
本研究第二部分是应用酵母双杂交技术研究与MR-1蛋白相互作用的蛋白，克隆它们的编码基因；并将MR1基因分段蛋白与得到的阳性AD蛋白相互作用，研究MR1蛋白的功能。本研究采用美国科隆技术公司酵母双杂交系统3中的酵母菌株AH109和pGBKT7诱饵载体（BD）。首先构建MR1的诱饵质粒，用这个诱饵质粒筛选人骨骼肌cDNA文库并通过β-gal分析验证，最终获得了9个阳性AD质粒，序列分析比较结果表明阳性AD质粒中的cDNA片段分别编码八个蛋白。然后根据MR1的跨膜区域结构，
将MR1从跨膜位点处分成两段，构建诱饵质粒，再分别与这些筛选出来的阳性AD蛋白进行酵母双杂交，结果显示这些AD蛋白与MR1的相互作用区域不一样，可能提示MR1在参与肌肉收缩调控的过程中，与不同蛋白之间的作用机制不同。本研究为深入研究MR-1蛋白在细胞生物学尤其是参与肌肉收缩调控中的作用打下了基础。
本研究第三部分是通过建立携带hMR1转基因小鼠模型，来进一步研究MR1基因的功能。首先利用受精卵原核的显微注射技术，将MR1基因显微注射至CD-1?∕(ICR)BR小鼠受精卵中，并移植到假孕受体鼠的输卵管内，然后应用PCR、Western Blot杂交技术分析子代鼠中基因的整合及表达情况。最终得到6只首建转基因小鼠，稳定遗传三代，得到三个稳定的家系，通过Western Blot分析，发现hMR1在转基因小鼠和正常小鼠中的表达有着明显的区别。结果证明采用受精卵显微注射的方法成功地建立MR1转基因小鼠，并证实所使用的基因片段能够在小鼠体内表达并随小鼠的传代而遗传，但由于位置效应或是其它原因，转基因小鼠的目的蛋白表达差异很大，但都和正常小鼠不同。对于hMR1对转基因小鼠的功能影响还需要进一步的研究。
MR-1（myofibrillogenesis regulator 1）gene is a novel human gene cloned in the lab of Dept. of Pathway Engineering at the Institute of Medicinal Biotechnology. The complete cDNA of MR-1 is made up of 416 nucleotides, encoding 142 amino acid residues. Its EST sequence was cloned the first time from human ovarian tumor tissue and was found also presented in uterine endomembrane tissue and parathyroid tissue. Pilot studies indicate that MR-1 is related with the proteins involved in regulation of muscle contraction and cell signal transmission, and there is a transmembrane region in this protein.
The first part of this research is to detect the mutation of hMR-1 gene by using non-isotopic polymerase reaction-based single-strand conformation polymorphism（PCR－SSCP）. PCR- products of three exons were obtained by designing three pairs primers which were aimed to three exons of hMR-1 gene. PCR-SSCP analysis showed that the synonymous mutation was found only in exon3 of the hMR-1 gene. The study seems to indicate that the mutation of the hMR-1 was not necessarily related to cardiac muscle hypertrophy, heart failure, coronary heart disease and tumour.
The second part is to find new proteins which interact with hMR-1 protein by using Yeast two-hybrid system and to clone their encoding sequences, and to research the interaction between these positive AD proteins and two segments which were from hMR-1. The yeast strain AH109 and bait vector (BD) pGBKT7 of MATCHMAKER GAL4 Two-Hybrid system 3, developed by CLONTECH Company of USA, were used in this research. Firstly, the
full-length of hMR-1 gene was cloned into the GAL4 DNA-binding domain of the vector pGBKT7 to construct a bait plasmid. Then the bait plasmid was used to screen Human Skeleton Muscle cDNA Library which was constructed on the pACT2 vector, and the β-gal analysis was carried out to verify the interaction between proteins. As a result, 9 positive AD plasmids were obtained. The results of DNA sequencing and homologous comparison showed that the cDNA fragments in the positive AD plasmids encoding the proteins belong to eight types, among which three proteins were related to muscle contraction. Subsection Yeast two-hybrid indicate that the interactional locations between these AD proteins and hMR-1 were different. This study facilitated the further elucidation of MR-1 function in cell biology, especially its function involved in the regulation of muscle contraction.
The third part is to further study function of the hMR-1 by constructing hMR-1-transgenic mice model . By microinjection techniques on germ nucleus of zygotes hMR-1 gene were injected into zygotes of CD-1?∕(ICR)BR mice and transplanted into the oviducts of pseudopregnancy female mice. The integration and expression of exogenous genes in offspring were detected by PCR and Western Blot, respectively. There were 6 founders of all injected mice, which had steadily inherited to the third generation, and three steady ancestry were obtained. Western Blot showed that there were obvious differences between transgenic mice and negative mice in the expression of hMR-1. The function of MR-1 need be further studied.
我的论文工作是在此基础之上进行的，主要分为以下三部分。
本论文第一部分是应用PCR-SSCP技术对MR1基因进行研究，筛查MR1的基因突变。针对MR1基因的三个外显子分别设计了三对引物，得到MR1三个外显子的PCR产物进行PCR-SSCP分析，只有外显子3存在基因突变，虽然该突变并未造成蛋白氨基酸组成的变化，但这种突变是否影响其基因拼接并导致功能的变化尚待进一步研究。
本研究第二部分是应用酵母双杂交技术研究与MR-1蛋白相互作用的蛋白，克隆它们的编码基因；并将MR1基因分段蛋白与得到的阳性AD蛋白相互作用，研究MR1蛋白的功能。本研究采用美国科隆技术公司酵母双杂交系统3中的酵母菌株AH109和pGBKT7诱饵载体（BD）。首先构建MR1的诱饵质粒，用这个诱饵质粒筛选人骨骼肌cDNA文库并通过β-gal分析验证，最终获得了9个阳性AD质粒，序列分析比较结果表明阳性AD质粒中的cDNA片段分别编码八个蛋白。然后根据MR1的跨膜区域结构，
将MR1从跨膜位点处分成两段，构建诱饵质粒，再分别与这些筛选出来的阳性AD蛋白进行酵母双杂交，结果显示这些AD蛋白与MR1的相互作用区域不一样，可能提示MR1在参与肌肉收缩调控的过程中，与不同蛋白之间的作用机制不同。本研究为深入研究MR-1蛋白在细胞生物学尤其是参与肌肉收缩调控中的作用打下了基础。
本研究第三部分是通过建立携带hMR1转基因小鼠模型，来进一步研究MR1基因的功能。首先利用受精卵原核的显微注射技术，将MR1基因显微注射至CD-1?∕(ICR)BR小鼠受精卵中，并移植到假孕受体鼠的输卵管内，然后应用PCR、Western Blot杂交技术分析子代鼠中基因的整合及表达情况。最终得到6只首建转基因小鼠，稳定遗传三代，得到三个稳定的家系，通过Western Blot分析，发现hMR1在转基因小鼠和正常小鼠中的表达有着明显的区别。结果证明采用受精卵显微注射的方法成功地建立MR1转基因小鼠，并证实所使用的基因片段能够在小鼠体内表达并随小鼠的传代而遗传，但由于位置效应或是其它原因，转基因小鼠的目的蛋白表达差异很大，但都和正常小鼠不同。对于hMR1对转基因小鼠的功能影响还需要进一步的研究。
MR-1（myofibrillogenesis regulator 1）gene is a novel human gene cloned in the lab of Dept. of Pathway Engineering at the Institute of Medicinal Biotechnology. The complete cDNA of MR-1 is made up of 416 nucleotides, encoding 142 amino acid residues. Its EST sequence was cloned the first time from human ovarian tumor tissue and was found also presented in uterine endomembrane tissue and parathyroid tissue. Pilot studies indicate that MR-1 is related with the proteins involved in regulation of muscle contraction and cell signal transmission, and there is a transmembrane region in this protein.
The first part of this research is to detect the mutation of hMR-1 gene by using non-isotopic polymerase reaction-based single-strand conformation polymorphism（PCR－SSCP）. PCR- products of three exons were obtained by designing three pairs primers which were aimed to three exons of hMR-1 gene. PCR-SSCP analysis showed that the synonymous mutation was found only in exon3 of the hMR-1 gene. The study seems to indicate that the mutation of the hMR-1 was not necessarily related to cardiac muscle hypertrophy, heart failure, coronary heart disease and tumour.
The second part is to find new proteins which interact with hMR-1 protein by using Yeast two-hybrid system and to clone their encoding sequences, and to research the interaction between these positive AD proteins and two segments which were from hMR-1. The yeast strain AH109 and bait vector (BD) pGBKT7 of MATCHMAKER GAL4 Two-Hybrid system 3, developed by CLONTECH Company of USA, were used in this research. Firstly, the
full-length of hMR-1 gene was cloned into the GAL4 DNA-binding domain of the vector pGBKT7 to construct a bait plasmid. Then the bait plasmid was used to screen Human Skeleton Muscle cDNA Library which was constructed on the pACT2 vector, and the β-gal analysis was carried out to verify the interaction between proteins. As a result, 9 positive AD plasmids were obtained. The results of DNA sequencing and homologous comparison showed that the cDNA fragments in the positive AD plasmids encoding the proteins belong to eight types, among which three proteins were related to muscle contraction. Subsection Yeast two-hybrid indicate that the interactional locations between these AD proteins and hMR-1 were different. This study facilitated the further elucidation of MR-1 function in cell biology, especially its function involved in the regulation of muscle contraction.
The third part is to further study function of the hMR-1 by constructing hMR-1-transgenic mice model . By microinjection techniques on germ nucleus of zygotes hMR-1 gene were injected into zygotes of CD-1?∕(ICR)BR mice and transplanted into the oviducts of pseudopregnancy female mice. The integration and expression of exogenous genes in offspring were detected by PCR and Western Blot, respectively. There were 6 founders of all injected mice, which had steadily inherited to the third generation, and three steady ancestry were obtained. Western Blot showed that there were obvious differences between transgenic mice and negative mice in the expression of hMR-1. The function of MR-1 need be further studied.
引文
Orita M, Iwahana H, Kanazawa H. Detection of polymorphisms??? of human DNA by gel electrophoresis as single-strand conformational polymorphisms. Proc Natl Acad Sci, USA,1989,86:??? 2766~2770
Orita M, Suzuki Y, Sekiya T, et al. A rapid and sensitive detection of point mutations and genetic polymorphism using polymerase chain reaction.Genomics,1989, 5:874~879
Sugano K, Kyogoku A, Fukayama N, Ohkura H, Shimosato Y, Sekiya T, et al. Methods in laboratory investigation. Rapid and simple detection of c-Ki-ras2 gene codon 12 mutations by non-radio isotopic single-strand conformation polymorphism analysis. Lab Invest 1993;68:361–6
Yasuyuki Miyakura, Kokichi Sugano et al. Concurrent Mutations of K-ras Oncogene at Codons 12 and 22 in Colon Cancer, Japanese Journal of Clinical Oncology (2002) 32:219-221
EP?Yap，JO?McGee Non-isotopic SSCP and competitive PCR for DNA quantification:p53 in breast cancer cells.Nucleic Acids Res.?1992 January 11;?20 (1): 145
PCR-SSCP的发展现状http://www.clinet.com.cn/edu/resource/article/2001101001.htm
Fields S, Song O: A novel system to detect protein-protein interactions. Nature 1989, 340: 245-246.
吴乃虎 , 基因工程原理 , 科学出版社 , 第二版 1998:365.
Finley， R. L. and Brent， R. Proc. Natl. Acad. Sci. USA 1994; 91: 12980-12984.
Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions Engelender, S et al. Nat. Genet., 22 (1999) 110–114.
Michael Neystata, et al .Analysis of synphilin-1 and synuclein interactions by yeast two hybrid b-galactosidase liquid assay Neuroscience Letters 325 (2002) 119–123.
Geert De Jaeger, et al. Analysis of the interaction between single-chain variable fragments and their antigen in a reducing intracellular environment using the two-hybrid system. FEBS Letters 467 (2000) 316~320.
Subhash G.et al. Characterisation of inter- and intra-molecular interactions of the dengue virus RNA dependent RNA polymerase as potential drug targets. Farmaco 56 (2001) 33–36.
Shao-bing Hua, et al. Construction of a modular yeast two-hybrid cDNA library from human EST clones for the human genome protein linkage map. Gene 215 (1998) 143–152.
PalmiterRD and Brinster RL et al . Germ-line transformation of mice Ann Rev Genet,1986;20:465
Palmiter RD, Brinster RL, Hammer RE，et al. Dramatic growth of mice that develop from eggs microinjected with a metallothionein growth hormone fusion gene. Nature, 1982, 300: 611 ~ 615.
马先勇等 ，转基因技术在瘤基因结构和功能研究中的应用 ，国外医学 生理、病理科学与临床分册 1997,17(2).97~100.
JC Engert, S Servaes Activation of a muscle-specific enhancer by the Ski proto-oncogene Nucleic Acids Research, 1995;23:2988
Hogan B, Beddington R, Costantinii F, et al. Manipulating the Mouse Embryo: A Laboratory Manual. NewYork, Cold Spring Harbor Laboratory Press,1994,117~119.
Wang, T-H., Yang, W. K., Hoyt, P. R., Ch'ang, L-Y. and Savin, T. J. (1993) Effect of maternal aging on transgene heritability in transgenic founder mice derived from zygotes microinjected with retroviral long terminal repeat-containing recombinant deoxyribonucleic acid. Biology of
Reproduction 1993 48: 1104-1112
Wilmut I et al. Genetic manipulation of mammals and its application in reproductive-biology ,JReprodFert.1991;92:245
马先勇，姚开泰。 同源重组技术研究进展. 生物工程进展，1996，16：16～23.
郭长占等。转基因小鼠研究进展，中华微生物学和免疫学杂志2000年第20卷第3期 278~282.
Kirk R, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo derived stemcells.Cell,1987,51:503~512.
Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage Pl. Proc Natl Acad Sci USA,1988,85:5166～5170.
Gu H, Zou YR, Rajewdkey K. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre loxP-Mediated gene targetting.Cell,1993,73:1155~1164.
CarlT,DaridJ.RapidcycleDNAamplification:timeandtem-peratureoptimization.Biotechniques,1991,10(1)∶76-83.
Gordon, K., Lee, E., Vitale, J.A., Smith, A.E., Westphal, H. and Hennighausen, L. (1987). Production of human tissue plasminogen activator in transgenic mouse milk. BIO/TECHNOLOGY 5, 1183-1187.
Schwartz K, Mercadier JJ. Molecular and cellular biology of heart failure. Curr Opin Cardiol. 1996 May;11(3):227-36.
Murry CE, Kay MA, Bartosek T, et al. Muscle differentiation during repair of myocardial necrosis in rats via gene transfer with MyoD. J Clin Invest,1996,98:2209-2217.
Schneider JW, Gu W, Zhu L, et al. Reversal of terminal differentiation mediated by p107 in Rb-/-muscle cells. Science 1994 Jun 3;264(5164):1467-1471.
Hale AJ, Smith CA, Sutherland LC, et al. Apoptosis: molecular regulation of cell death. Eur J Biochem 1996 Feb 15;236(1):1-26.
Feig LA, Buchsbaum RJ. Cell signaling: life or death decisions of ras proteins. Curr Biol 2002 Apr 2;12(7):R259-61.
Yuan ZQ, Sun M, Feldman RI, et al. Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer. Oncogene 2000 May 4;19(19):2324-30.
Crackower MA, Oudit GY, Kozieradzki I, et al. Regulation of myocardial contractility and cell size by distinct PI3K-PTEN signaling pathways. Cell 2002 Sep 20;110(6):737-49.
Hein S, Kostin S, Heling A, et al. The role of the cytoskeleton in heart failure. Cardiovasc Res. 2000 Jan 14;45(2):273-8.Watkins SJ, Norbury CJ. Translation initiation and its deregulation during tumorigenesis. Br J Cancer 2002 Apr 8;86(7):1023-7.
Watkins SJ, Norbury CJ. Translation initiation and its deregulation during tumorigenesis. Br J Cancer 2002 Apr 8;86(7):1023-7
黄培堂等译。分子克隆实验指南。科学出版社，2002，第三版。
田小利，陈兰英，扈荣良。转基因动物原理、技术与应用。吉林科学技术出版社，1994，57-58.
Brinster RL, Allen JM, Behringer RR, Gelinas RE, Palmiter RD. Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci U S A. 1988, 85(3): 836-840.
Orita M, Suzuki Y, Sekiya T, et al. A rapid and sensitive detection of point mutations and genetic polymorphism using polymerase chain reaction.Genomics,1989, 5:874~879
Sugano K, Kyogoku A, Fukayama N, Ohkura H, Shimosato Y, Sekiya T, et al. Methods in laboratory investigation. Rapid and simple detection of c-Ki-ras2 gene codon 12 mutations by non-radio isotopic single-strand conformation polymorphism analysis. Lab Invest 1993;68:361–6
Yasuyuki Miyakura, Kokichi Sugano et al. Concurrent Mutations of K-ras Oncogene at Codons 12 and 22 in Colon Cancer, Japanese Journal of Clinical Oncology (2002) 32:219-221
EP?Yap，JO?McGee Non-isotopic SSCP and competitive PCR for DNA quantification:p53 in breast cancer cells.Nucleic Acids Res.?1992 January 11;?20 (1): 145
PCR-SSCP的发展现状http://www.clinet.com.cn/edu/resource/article/2001101001.htm
Fields S, Song O: A novel system to detect protein-protein interactions. Nature 1989, 340: 245-246.
吴乃虎 , 基因工程原理 , 科学出版社 , 第二版 1998:365.
Finley， R. L. and Brent， R. Proc. Natl. Acad. Sci. USA 1994; 91: 12980-12984.
Synphilin-1 associates with alpha-synuclein and promotes the formation of cytosolic inclusions Engelender, S et al. Nat. Genet., 22 (1999) 110–114.
Michael Neystata, et al .Analysis of synphilin-1 and synuclein interactions by yeast two hybrid b-galactosidase liquid assay Neuroscience Letters 325 (2002) 119–123.
Geert De Jaeger, et al. Analysis of the interaction between single-chain variable fragments and their antigen in a reducing intracellular environment using the two-hybrid system. FEBS Letters 467 (2000) 316~320.
Subhash G.et al. Characterisation of inter- and intra-molecular interactions of the dengue virus RNA dependent RNA polymerase as potential drug targets. Farmaco 56 (2001) 33–36.
Shao-bing Hua, et al. Construction of a modular yeast two-hybrid cDNA library from human EST clones for the human genome protein linkage map. Gene 215 (1998) 143–152.
PalmiterRD and Brinster RL et al . Germ-line transformation of mice Ann Rev Genet,1986;20:465
Palmiter RD, Brinster RL, Hammer RE，et al. Dramatic growth of mice that develop from eggs microinjected with a metallothionein growth hormone fusion gene. Nature, 1982, 300: 611 ~ 615.
马先勇等 ，转基因技术在瘤基因结构和功能研究中的应用 ，国外医学 生理、病理科学与临床分册 1997,17(2).97~100.
JC Engert, S Servaes Activation of a muscle-specific enhancer by the Ski proto-oncogene Nucleic Acids Research, 1995;23:2988
Hogan B, Beddington R, Costantinii F, et al. Manipulating the Mouse Embryo: A Laboratory Manual. NewYork, Cold Spring Harbor Laboratory Press,1994,117~119.
Wang, T-H., Yang, W. K., Hoyt, P. R., Ch'ang, L-Y. and Savin, T. J. (1993) Effect of maternal aging on transgene heritability in transgenic founder mice derived from zygotes microinjected with retroviral long terminal repeat-containing recombinant deoxyribonucleic acid. Biology of
Reproduction 1993 48: 1104-1112
Wilmut I et al. Genetic manipulation of mammals and its application in reproductive-biology ,JReprodFert.1991;92:245
马先勇，姚开泰。 同源重组技术研究进展. 生物工程进展，1996，16：16～23.
郭长占等。转基因小鼠研究进展，中华微生物学和免疫学杂志2000年第20卷第3期 278~282.
Kirk R, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo derived stemcells.Cell,1987,51:503~512.
Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage Pl. Proc Natl Acad Sci USA,1988,85:5166～5170.
Gu H, Zou YR, Rajewdkey K. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre loxP-Mediated gene targetting.Cell,1993,73:1155~1164.
CarlT,DaridJ.RapidcycleDNAamplification:timeandtem-peratureoptimization.Biotechniques,1991,10(1)∶76-83.
Gordon, K., Lee, E., Vitale, J.A., Smith, A.E., Westphal, H. and Hennighausen, L. (1987). Production of human tissue plasminogen activator in transgenic mouse milk. BIO/TECHNOLOGY 5, 1183-1187.
Schwartz K, Mercadier JJ. Molecular and cellular biology of heart failure. Curr Opin Cardiol. 1996 May;11(3):227-36.
Murry CE, Kay MA, Bartosek T, et al. Muscle differentiation during repair of myocardial necrosis in rats via gene transfer with MyoD. J Clin Invest,1996,98:2209-2217.
Schneider JW, Gu W, Zhu L, et al. Reversal of terminal differentiation mediated by p107 in Rb-/-muscle cells. Science 1994 Jun 3;264(5164):1467-1471.
Hale AJ, Smith CA, Sutherland LC, et al. Apoptosis: molecular regulation of cell death. Eur J Biochem 1996 Feb 15;236(1):1-26.
Feig LA, Buchsbaum RJ. Cell signaling: life or death decisions of ras proteins. Curr Biol 2002 Apr 2;12(7):R259-61.
Yuan ZQ, Sun M, Feldman RI, et al. Frequent activation of AKT2 and induction of apoptosis by inhibition of phosphoinositide-3-OH kinase/Akt pathway in human ovarian cancer. Oncogene 2000 May 4;19(19):2324-30.
Crackower MA, Oudit GY, Kozieradzki I, et al. Regulation of myocardial contractility and cell size by distinct PI3K-PTEN signaling pathways. Cell 2002 Sep 20;110(6):737-49.
Hein S, Kostin S, Heling A, et al. The role of the cytoskeleton in heart failure. Cardiovasc Res. 2000 Jan 14;45(2):273-8.Watkins SJ, Norbury CJ. Translation initiation and its deregulation during tumorigenesis. Br J Cancer 2002 Apr 8;86(7):1023-7.
Watkins SJ, Norbury CJ. Translation initiation and its deregulation during tumorigenesis. Br J Cancer 2002 Apr 8;86(7):1023-7
黄培堂等译。分子克隆实验指南。科学出版社，2002，第三版。
田小利，陈兰英，扈荣良。转基因动物原理、技术与应用。吉林科学技术出版社，1994，57-58.
Brinster RL, Allen JM, Behringer RR, Gelinas RE, Palmiter RD. Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci U S A. 1988, 85(3): 836-840.