人era基因的表达分布及其功能的相关研究
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摘要
era基因是1986年测定大肠杆菌基因组序列时，在编码RNaseⅢ的rnc基
因下游发现的一个开放读框，其编码的蛋白具有鸟苷酸结合活性，氨基酸序列与
人和酵母的Ras有明显相似性，故命名为E.coli Ras-like（era）基因。后来的研究表
明，era的同源序列存在于所有探讨过的细菌中，era编码的Era蛋白并不属于
Ras家族，其独特的C端使其成为一个新的G蛋白亚家族。
Era蛋白由两个结构域组成，N端是一个典型的鸟苷酸结合蛋白结构域，与
其它G蛋白有较高的同源性；C端为Era家族所特有，与其它蛋白家族无任何同
源性，其中含有一个RNA结合功能结构域—KH结构域。大肠杆菌era是属于
rnc操纵元的一个细菌生存必需的基因，并参与细胞周期的调控，Era可以特异
地与16S-rRNA结合。陈苏民教授在美国NIH工作期间，以大肠杆菌Era的C
端序列为靶序列，通过计算机搜索，发现从线虫、小鼠到人都有与细菌Era同源
的EST序列，以此为线索，成功地克隆了人和小鼠全长的era-cDNA。在此基础
上，本文对人Era的组织表达谱及其功能进行了相关研究。
在本研究工作中，首先对人era的表达分布规律进行探索。1、人era-mRNA
的组织分布规律：以人era编码区基因为探针，用CLONTECH公司的MTN和MTE
＄四旱医大学俗士攀位论文 第7 页
杂交膜进行杂交，发现人。。－mRNA的大小为2．ZKb，分布于所有检测的组织和
细胞系中，但表达水平有很大差异。表达最高的组织为心尖部、肝脏、胎肺和甲
状腺。2、人。。。的蛋白表达分布规律：用课题组制备并经过亲和纯化的兔抗人
Era多克隆抗体对几种胎儿组织进行免疫组织化学染色，结果人Era蛋白分布于
绝大多数所检测的组织，由强到弱依次为胰脏、肺、心脏、脾脏、胃、小肠、肝
脏和肾脏。
经免疫电镜检测大肠杆菌Era定位于胞浆膜的内侧面，其C端对细胞内定
位有着重要意义。为分析人ERA在细胞内的定位，构建了人Era与绿色荧光蛋白
融合的真核表达载体pSMEGFP－hEra和PEGFP－CI－hEra，即目的蛋白分别位于荧
光标签蛋白（EGFP）的N端和C端。瞬时转染NIH3T3细胞，带荧光的融合蛋白
主要位于靠近核膜的胞浆部分；进一步用纯化的兔抗人Era多克隆抗体对类风湿
关节炎滑膜细胞系RA，骨肉瘤细胞系MG63、胃癌细胞系7901等几种培养细胞进
行问接免疫荧光检测，发现均有强弱不等的荧光，且主要分布于胞浆中。表明人
Era具有不同于大肠杆茵Era的细胞内定位，其细胞内分布以胞浆为主，可能具
有与细菌Era不同的信号转导途径。
为研究人Era对细胞形态、细胞周期的影响，进行以下工作：1、对人Era
的氨基酸序列进行了计算机分析，在此基础上构建了人Era的定点突变体，其突
变的位置分别针对N端结构域的GTP结合位点和C端结构域的KH基序；2、
带HA标签的野生型和突变型人Era的真核表达载体，转染Nll－I3T3细胞，筛选
到稳定表达的细胞株。3、对稳定表达人Era细胞株的细胞形态和细胞周期进行
分析，发现野生型人Era对细胞生长有促进作用，而KH基序突变后对细胞生长
起抑制作用。流式细胞仪分析表明，转染野生型Era的细胞株表现为GZ期和S
期细胞增多，可能为DNA合成旺盛的结果；转染Era S36N细胞株各时相细胞所
占百分数变化不明显；转染 Era 31 ON的细胞株 S期细胞和 GZ期细胞增多异常
显著，结合细胞形态和生长曲线，可能存在细胞周期阻滞；即在细胞DNA复制后
期不能向M期转化。
进一步使用完全可调控诱导表达系统表达突变型人Era，以深入研究该蛋白
的功能。在构建稳定高表达蜕皮激素受体基因的细胞株3V3的基础上，转染突
变型人Era的表达载体进行压力筛选，对所得到的单克隆细胞株进行诱导与未诱
DopGI’－’’’ent ofBtochthe andMolecularmp FMMU2001
窜回旱医大学博士学位论文 章 吕 页
导条件下细胞周期的分析，结果表明未诱导加酒精的对照细胞可以引起细胞凋
亡，而Era诱导表达后可以显著抑制凋亡细胞的数量，Western表明Era S36N
可以诱导细胞仇 1－2表达，提示人 Era可能与细胞凋亡相关。
为进一步研究人Era的作用机理，对其结合蛋白的功能区进行鉴定，将人
。。a基因克隆入融合表达载体 PRSETB中，转化大肠杆菌 BL21DE3（pLysS），经
IPTG诱导，表达6His－hEra融合蛋白。光密度扫描结果表明其占菌体总蛋白50％
以上，表达产物分子量约为42KD，以包涵体形式存在。在SM i存在条件下，包
涵体溶解，利用6Hk与过渡态金属离子N广高亲合力结合的性质，用N广一叮A
亲和树脂一步法纯化，即获得纯度达96％以上的6His－hEra融合蛋白。将纯化的
蛋白进行包被，从噬菌体表面呈现随机十＝肽库中经过三轮淘筛（Biopanning），
并用ELISA检测，筛选到具有人Era结合活性的阳性重组噬菌体克隆，测序后发
现大多（7／9）具有 HxHSxxH的氨基酸序列，初步认为是人 Era的结合基序，为
进一步Era结合蛋白基因的克隆及其功能研究提供了依据。
Era (Escherihia coil ras-like) was originally named on the basis of limited similarity
to the Ras protein. Further analysis of Era sequence suggested that the similarity was
confined to the GTP-binding domain and that Era was not a number of Ras family, Era
from different organisms is highly conserved and has been found in nearly every bacterial
genome sequenced to date. Their unique C terminal make them form a new subfamily of
GTPase.
EAC0I1 Era is an essential membrane-associated GTPase. and has a regulatory role
in cell cycle by coupling bacterial cell growth rate with cytokinesis. Cell division is
signaled when a threshold of Era GTPase activity is reached. Artificially reducing the
expression of ERA result in cell cycle arrest at a predivitional two-cell stage. The arrest
lasts until Era activity accumulates to the threshold level.
There are two domains in Era: The N-terminal GTP-binding domain is closely
related to Ras p21, whereas the C-terminal domain is unique. Sequence study have
shown that the C-terminal region of Era contains an RNA binding KH domain, consistent
with the findings that Era bind RNA in vitro and in vivo. Recently, during professor CHEN
Su-min worked in NIH, he and his college searched the dbEST database with the
C-terminal of E.coli Era and found there are Era homologues in eukaryotes, such as
Caenorhabditis elegans, mouse and human. Then they identified mouse and human
Department of Biochemistry tzndMolecidar Biology, FMMU 2001
era-cDNA successfully.
The aim of this study is to identify the expressive and contributive rule of human Era
and the function of this protein on cell morphology and cell cycle. Here we report the
analysis of the expression pattern and function of human Era.
In the study, the expression pattern of human Era has been first analyzed.
The coding region of human era-cDNA was labelled and used to probe human
multiple-tissue Northen blot and human multiple-tissue Expression dot (CLONTECH) to
detect the expression level of human era mRNA. The result demonstrated that human
era mRNA is expressed in all tissues and cell lines analyzed but with different expression
level, The probe identified an mRNA product of approximately 2.2kb.
To analyze the expression level of human Era protein, the rabbit anti human Era
polyclonal antibody prepared and purified in our former study has been used as the first
antibody. The expression level of human Era in several fetal tissues was then identified
by immunohistochemistry, This assay showed that human Era is expressed in almost all
tissues detected. The order of positive signal strength is Pancreas >Lung>Heart>Spleen
>Stomach >Small intestine> Liver>Kidney.
To analyze the intercellular localization of human Era, we constructed two eukaryotic
expressive vectors of EGFP-hEra fusion protein named pSMEGFP-hEra and
pEGFP-C1-hEra, in which human Era localized in N or C terminal of EGFP separately.
These vectors was transfected into mouse fibroblast cell line NIH3T3 by way of lipofectin.
Fluorescence microscopy analysis revealed that human Era localized in cytoplasm
around cell nucleus mainly. Then the intercellular localization of human Era with natural
expression level was analyzed by indirect immunofluorescence assay. The results also
demonstrated that human Era localized mainly in cytoplasm in several cultured cell lines
such as osteosarcoma cell line MG63, stomach cancer cell line 7901 and rheumatoid
arthritis cell line RA. This study suggested human Era ma
因下游发现的一个开放读框，其编码的蛋白具有鸟苷酸结合活性，氨基酸序列与
人和酵母的Ras有明显相似性，故命名为E.coli Ras-like（era）基因。后来的研究表
明，era的同源序列存在于所有探讨过的细菌中，era编码的Era蛋白并不属于
Ras家族，其独特的C端使其成为一个新的G蛋白亚家族。
Era蛋白由两个结构域组成，N端是一个典型的鸟苷酸结合蛋白结构域，与
其它G蛋白有较高的同源性；C端为Era家族所特有，与其它蛋白家族无任何同
源性，其中含有一个RNA结合功能结构域—KH结构域。大肠杆菌era是属于
rnc操纵元的一个细菌生存必需的基因，并参与细胞周期的调控，Era可以特异
地与16S-rRNA结合。陈苏民教授在美国NIH工作期间，以大肠杆菌Era的C
端序列为靶序列，通过计算机搜索，发现从线虫、小鼠到人都有与细菌Era同源
的EST序列，以此为线索，成功地克隆了人和小鼠全长的era-cDNA。在此基础
上，本文对人Era的组织表达谱及其功能进行了相关研究。
在本研究工作中，首先对人era的表达分布规律进行探索。1、人era-mRNA
的组织分布规律：以人era编码区基因为探针，用CLONTECH公司的MTN和MTE
＄四旱医大学俗士攀位论文 第7 页
杂交膜进行杂交，发现人。。－mRNA的大小为2．ZKb，分布于所有检测的组织和
细胞系中，但表达水平有很大差异。表达最高的组织为心尖部、肝脏、胎肺和甲
状腺。2、人。。。的蛋白表达分布规律：用课题组制备并经过亲和纯化的兔抗人
Era多克隆抗体对几种胎儿组织进行免疫组织化学染色，结果人Era蛋白分布于
绝大多数所检测的组织，由强到弱依次为胰脏、肺、心脏、脾脏、胃、小肠、肝
脏和肾脏。
经免疫电镜检测大肠杆菌Era定位于胞浆膜的内侧面，其C端对细胞内定
位有着重要意义。为分析人ERA在细胞内的定位，构建了人Era与绿色荧光蛋白
融合的真核表达载体pSMEGFP－hEra和PEGFP－CI－hEra，即目的蛋白分别位于荧
光标签蛋白（EGFP）的N端和C端。瞬时转染NIH3T3细胞，带荧光的融合蛋白
主要位于靠近核膜的胞浆部分；进一步用纯化的兔抗人Era多克隆抗体对类风湿
关节炎滑膜细胞系RA，骨肉瘤细胞系MG63、胃癌细胞系7901等几种培养细胞进
行问接免疫荧光检测，发现均有强弱不等的荧光，且主要分布于胞浆中。表明人
Era具有不同于大肠杆茵Era的细胞内定位，其细胞内分布以胞浆为主，可能具
有与细菌Era不同的信号转导途径。
为研究人Era对细胞形态、细胞周期的影响，进行以下工作：1、对人Era
的氨基酸序列进行了计算机分析，在此基础上构建了人Era的定点突变体，其突
变的位置分别针对N端结构域的GTP结合位点和C端结构域的KH基序；2、
带HA标签的野生型和突变型人Era的真核表达载体，转染Nll－I3T3细胞，筛选
到稳定表达的细胞株。3、对稳定表达人Era细胞株的细胞形态和细胞周期进行
分析，发现野生型人Era对细胞生长有促进作用，而KH基序突变后对细胞生长
起抑制作用。流式细胞仪分析表明，转染野生型Era的细胞株表现为GZ期和S
期细胞增多，可能为DNA合成旺盛的结果；转染Era S36N细胞株各时相细胞所
占百分数变化不明显；转染 Era 31 ON的细胞株 S期细胞和 GZ期细胞增多异常
显著，结合细胞形态和生长曲线，可能存在细胞周期阻滞；即在细胞DNA复制后
期不能向M期转化。
进一步使用完全可调控诱导表达系统表达突变型人Era，以深入研究该蛋白
的功能。在构建稳定高表达蜕皮激素受体基因的细胞株3V3的基础上，转染突
变型人Era的表达载体进行压力筛选，对所得到的单克隆细胞株进行诱导与未诱
DopGI’－’’’ent ofBtochthe andMolecularmp FMMU2001
窜回旱医大学博士学位论文 章 吕 页
导条件下细胞周期的分析，结果表明未诱导加酒精的对照细胞可以引起细胞凋
亡，而Era诱导表达后可以显著抑制凋亡细胞的数量，Western表明Era S36N
可以诱导细胞仇 1－2表达，提示人 Era可能与细胞凋亡相关。
为进一步研究人Era的作用机理，对其结合蛋白的功能区进行鉴定，将人
。。a基因克隆入融合表达载体 PRSETB中，转化大肠杆菌 BL21DE3（pLysS），经
IPTG诱导，表达6His－hEra融合蛋白。光密度扫描结果表明其占菌体总蛋白50％
以上，表达产物分子量约为42KD，以包涵体形式存在。在SM i存在条件下，包
涵体溶解，利用6Hk与过渡态金属离子N广高亲合力结合的性质，用N广一叮A
亲和树脂一步法纯化，即获得纯度达96％以上的6His－hEra融合蛋白。将纯化的
蛋白进行包被，从噬菌体表面呈现随机十＝肽库中经过三轮淘筛（Biopanning），
并用ELISA检测，筛选到具有人Era结合活性的阳性重组噬菌体克隆，测序后发
现大多（7／9）具有 HxHSxxH的氨基酸序列，初步认为是人 Era的结合基序，为
进一步Era结合蛋白基因的克隆及其功能研究提供了依据。
Era (Escherihia coil ras-like) was originally named on the basis of limited similarity
to the Ras protein. Further analysis of Era sequence suggested that the similarity was
confined to the GTP-binding domain and that Era was not a number of Ras family, Era
from different organisms is highly conserved and has been found in nearly every bacterial
genome sequenced to date. Their unique C terminal make them form a new subfamily of
GTPase.
EAC0I1 Era is an essential membrane-associated GTPase. and has a regulatory role
in cell cycle by coupling bacterial cell growth rate with cytokinesis. Cell division is
signaled when a threshold of Era GTPase activity is reached. Artificially reducing the
expression of ERA result in cell cycle arrest at a predivitional two-cell stage. The arrest
lasts until Era activity accumulates to the threshold level.
There are two domains in Era: The N-terminal GTP-binding domain is closely
related to Ras p21, whereas the C-terminal domain is unique. Sequence study have
shown that the C-terminal region of Era contains an RNA binding KH domain, consistent
with the findings that Era bind RNA in vitro and in vivo. Recently, during professor CHEN
Su-min worked in NIH, he and his college searched the dbEST database with the
C-terminal of E.coli Era and found there are Era homologues in eukaryotes, such as
Caenorhabditis elegans, mouse and human. Then they identified mouse and human
Department of Biochemistry tzndMolecidar Biology, FMMU 2001
era-cDNA successfully.
The aim of this study is to identify the expressive and contributive rule of human Era
and the function of this protein on cell morphology and cell cycle. Here we report the
analysis of the expression pattern and function of human Era.
In the study, the expression pattern of human Era has been first analyzed.
The coding region of human era-cDNA was labelled and used to probe human
multiple-tissue Northen blot and human multiple-tissue Expression dot (CLONTECH) to
detect the expression level of human era mRNA. The result demonstrated that human
era mRNA is expressed in all tissues and cell lines analyzed but with different expression
level, The probe identified an mRNA product of approximately 2.2kb.
To analyze the expression level of human Era protein, the rabbit anti human Era
polyclonal antibody prepared and purified in our former study has been used as the first
antibody. The expression level of human Era in several fetal tissues was then identified
by immunohistochemistry, This assay showed that human Era is expressed in almost all
tissues detected. The order of positive signal strength is Pancreas >Lung>Heart>Spleen
>Stomach >Small intestine> Liver>Kidney.
To analyze the intercellular localization of human Era, we constructed two eukaryotic
expressive vectors of EGFP-hEra fusion protein named pSMEGFP-hEra and
pEGFP-C1-hEra, in which human Era localized in N or C terminal of EGFP separately.
These vectors was transfected into mouse fibroblast cell line NIH3T3 by way of lipofectin.
Fluorescence microscopy analysis revealed that human Era localized in cytoplasm
around cell nucleus mainly. Then the intercellular localization of human Era with natural
expression level was analyzed by indirect immunofluorescence assay. The results also
demonstrated that human Era localized mainly in cytoplasm in several cultured cell lines
such as osteosarcoma cell line MG63, stomach cancer cell line 7901 and rheumatoid
arthritis cell line RA. This study suggested human Era ma
引文
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Gastroenterol. 1997;32(2) :282-287．
22． Weintraub HM. Antisense RNA and DNA. Sci Am 1990;262(1) :40-46
23． Fedor MJ. Structure and function of the hairpin ribozyme.J Mol Biol. 2000 ;297(2) :269-291．
24． Rossi JJ. Ribozyme therapy for HIV infection.Adv Drug Deliv Rev. 2000 ;44( 1) :71-78． .
25． Vaish NK, Kore AR, Eckstein F. Recent developments in the hammerhead ribozyme field-Nucleic Acids Res. 1998 ;26(23) :5237-5242．
26． Vaucheret H, Fagard M. Transcriptional gene silencing in plants: targets, inducers and regulators.Trends Genet. 2001;17(1) :29-35．
27． Meins F Jr. Related Articles RNA degradation and models for post-transcriptional gene-silencing.Plant Mol Biol. 2000 ;43(2-3) :261-273．
28． Bosher JM, Labouesse M. RNA interference: genetic wand and genetic watchdog.Nature Cell Biology 2000;2,E31-E36
29． Sharp, P. A. RNAi and double-strand RNA. Genes Dev.l999;13, 139-141 .
30． Bernstein E, Caudy AA, Hammond SM, Hannon GJ.Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001;409(6818) :363-366
31． Baulcombe D RNA silencing: Diced defence Nature 2001 ;409(6818) :295-296
32． Scott M. Hammond, Emily Bernstein, et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.Nature 2000;404, 293-296
33． Elbashir SM, Lendeckel W, Tuschl T.RNA interference is mediated by 21-and 22-nucleotide RNAs.Genes Dev. 2001; 15(2) : 188-200
34． Bosher JM, Dufourcq P, Sookhareea S, et al .RNA interference can target pre-mRNA:concequences for gene expression in the C. elegans operon. Genetics 1999;153,1245-1256
35． Maeda I I, Kohara Y, Yamamoto M, et al. Large-scale analysis of gene function-in Caenorhabditis elegans by high-throughput RNAi. Curr Biol 2001; 11(3) : 171-176
36． Wianny F, Zernicka-Goetz M. Specific interference with gene function by double-strand RNA in early mouse development. Nat Cell Biol 2000;2:70-75
37． Fassler R, Martin K, Forsberg E, et al. Knockout mice: How to make them and why. The immunological approach. Int Arch Allergy Immunol, 1995,106:323-324．
38． 王伯云 病理学技术,人民卫生出版社第一版2000年 1044-1047
39． Liu JL, Yakar S, LeRoith D. Conditional knockout of mouse insulin-like growth factor-1 gene using the Cre/loxP system. Proc Soc Exp Biol Med 2000;223(4) :344-351．
40． Jaenisch R, Mintz B. Simian virus 40 DNA sequences in DNA of healthy adult mice derived from preimplantation blastocytes injected with viral DNA. Proc Natl Acad Sci USA, 1974,71:1250-1254．
41． Kirk R, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell, 1987,51:503-512．
42． Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage PI. Proc Natl Acad Sci USA, 1988,85:5166-5170．
43． Gu H, Zou YR, Rajewskey K, et al. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-Mediated gene targerting. Cell, 1993,73:1155-1164．
44． Trends Genet 1997;13(2) :61-6 Production of transgenic mice with yeast artificial chromosomes.Peterson KR, Clegg CH, Li Q, Stamatoyannopoulos G.
45. Lamb BT, Gearhart JD. YAC transgenics and the study of genetics and human disease. Curr Opin Genet Dev 1995;5(3):342-348
46. Huxley C. Exploring gene function: use of yeast artificial chromosome transgenesis. Methods 1998 Feb;14(2):199-210
47. Peterson KR, Li QL, Clegg CH, et al. Use of yeast artificial chromosomes (YACs) in studies of mammalian development: production of beta-globin locus YAC mice carrying human globin developmental mutants. Proc Natl Acad Sci U S A 1995;92(12):5655-5659
48. Peterson KR, Navas PA, Stamatoyannopoulos G. beta-YAC transgenic mice for studying LCR function. Ann N Y Acad Sci 1998;850:28-37.
49. Wotten D, Ways DK, Parker PJ, et al. Activity of both Raf and Ras is necessary for activity of transcription of the human T cell receptor beta gene by protein kinase C Ras plays mutiple roles. J Biol Chem 1993,268(24)17975-17982
50. Hatzis P, Al-Madhoon AS, Jullig M, et al. The intercellular localization of deoxycytidine kinase. J Biol Chem 1998;273(46)30239-30243
51．张峰，任燕，陆长德。绿色荧光蛋白及其应用，生命科学1999；11(2)：61-65
52．司徒镇强，吴军正。细胞培养，世界图书出版公司，1996年第一版。175-178
53．范金水。通用型真核表达载体的构建，第四军医大学博士后研究工作报告，1998
54. Hartwell LH, Weinert TA. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989;246(4930):629-34.
55．桂建芳编著，RNA加工和细胞周期调控，科学出版社，第一版，1998年，103-108
56. Britton RA, Powell BS, Dasgupta S, et al. Cell cycle arrest in Era GTPase mutants: a potential growth rate-regulated checkpoint in Escherichia coli. Mol Microbiol. 1998;27(4):739-50
57．萨姆布鲁克等著，金冬雁、黎孟枫译。分子克隆实验指南，第二版，科学出版社，1992
2．陈润生。当前生物信息学的重要研究任务。《生物工程进展》1999年第19卷第4期
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18. M. Magdalena Gherardi, Juan C. Ramirez. IL-12 Delivery from Recombinant Vaccinia Virus Attenuates the Vector and Enhances the Cellular Immune Response Against HIV-1 Env in a Dose-Dependent Manner. J Immunol, 1999,162:6724-6733.,
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Gastroenterol. 1997;32(2) :282-287．
22． Weintraub HM. Antisense RNA and DNA. Sci Am 1990;262(1) :40-46
23． Fedor MJ. Structure and function of the hairpin ribozyme.J Mol Biol. 2000 ;297(2) :269-291．
24． Rossi JJ. Ribozyme therapy for HIV infection.Adv Drug Deliv Rev. 2000 ;44( 1) :71-78． .
25． Vaish NK, Kore AR, Eckstein F. Recent developments in the hammerhead ribozyme field-Nucleic Acids Res. 1998 ;26(23) :5237-5242．
26． Vaucheret H, Fagard M. Transcriptional gene silencing in plants: targets, inducers and regulators.Trends Genet. 2001;17(1) :29-35．
27． Meins F Jr. Related Articles RNA degradation and models for post-transcriptional gene-silencing.Plant Mol Biol. 2000 ;43(2-3) :261-273．
28． Bosher JM, Labouesse M. RNA interference: genetic wand and genetic watchdog.Nature Cell Biology 2000;2,E31-E36
29． Sharp, P. A. RNAi and double-strand RNA. Genes Dev.l999;13, 139-141 .
30． Bernstein E, Caudy AA, Hammond SM, Hannon GJ.Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001;409(6818) :363-366
31． Baulcombe D RNA silencing: Diced defence Nature 2001 ;409(6818) :295-296
32． Scott M. Hammond, Emily Bernstein, et al. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells.Nature 2000;404, 293-296
33． Elbashir SM, Lendeckel W, Tuschl T.RNA interference is mediated by 21-and 22-nucleotide RNAs.Genes Dev. 2001; 15(2) : 188-200
34． Bosher JM, Dufourcq P, Sookhareea S, et al .RNA interference can target pre-mRNA:concequences for gene expression in the C. elegans operon. Genetics 1999;153,1245-1256
35． Maeda I I, Kohara Y, Yamamoto M, et al. Large-scale analysis of gene function-in Caenorhabditis elegans by high-throughput RNAi. Curr Biol 2001; 11(3) : 171-176
36． Wianny F, Zernicka-Goetz M. Specific interference with gene function by double-strand RNA in early mouse development. Nat Cell Biol 2000;2:70-75
37． Fassler R, Martin K, Forsberg E, et al. Knockout mice: How to make them and why. The immunological approach. Int Arch Allergy Immunol, 1995,106:323-324．
38． 王伯云 病理学技术,人民卫生出版社第一版2000年 1044-1047
39． Liu JL, Yakar S, LeRoith D. Conditional knockout of mouse insulin-like growth factor-1 gene using the Cre/loxP system. Proc Soc Exp Biol Med 2000;223(4) :344-351．
40． Jaenisch R, Mintz B. Simian virus 40 DNA sequences in DNA of healthy adult mice derived from preimplantation blastocytes injected with viral DNA. Proc Natl Acad Sci USA, 1974,71:1250-1254．
41． Kirk R, Capecchi MR. Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell, 1987,51:503-512．
42． Sauer B, Henderson N. Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage PI. Proc Natl Acad Sci USA, 1988,85:5166-5170．
43． Gu H, Zou YR, Rajewskey K, et al. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-Mediated gene targerting. Cell, 1993,73:1155-1164．
44． Trends Genet 1997;13(2) :61-6 Production of transgenic mice with yeast artificial chromosomes.Peterson KR, Clegg CH, Li Q, Stamatoyannopoulos G.
45. Lamb BT, Gearhart JD. YAC transgenics and the study of genetics and human disease. Curr Opin Genet Dev 1995;5(3):342-348
46. Huxley C. Exploring gene function: use of yeast artificial chromosome transgenesis. Methods 1998 Feb;14(2):199-210
47. Peterson KR, Li QL, Clegg CH, et al. Use of yeast artificial chromosomes (YACs) in studies of mammalian development: production of beta-globin locus YAC mice carrying human globin developmental mutants. Proc Natl Acad Sci U S A 1995;92(12):5655-5659
48. Peterson KR, Navas PA, Stamatoyannopoulos G. beta-YAC transgenic mice for studying LCR function. Ann N Y Acad Sci 1998;850:28-37.
49. Wotten D, Ways DK, Parker PJ, et al. Activity of both Raf and Ras is necessary for activity of transcription of the human T cell receptor beta gene by protein kinase C Ras plays mutiple roles. J Biol Chem 1993,268(24)17975-17982
50. Hatzis P, Al-Madhoon AS, Jullig M, et al. The intercellular localization of deoxycytidine kinase. J Biol Chem 1998;273(46)30239-30243
51．张峰，任燕，陆长德。绿色荧光蛋白及其应用，生命科学1999；11(2)：61-65
52．司徒镇强，吴军正。细胞培养，世界图书出版公司，1996年第一版。175-178
53．范金水。通用型真核表达载体的构建，第四军医大学博士后研究工作报告，1998
54. Hartwell LH, Weinert TA. Checkpoints: controls that ensure the order of cell cycle events. Science. 1989;246(4930):629-34.
55．桂建芳编著，RNA加工和细胞周期调控，科学出版社，第一版，1998年，103-108
56. Britton RA, Powell BS, Dasgupta S, et al. Cell cycle arrest in Era GTPase mutants: a potential growth rate-regulated checkpoint in Escherichia coli. Mol Microbiol. 1998;27(4):739-50
57．萨姆布鲁克等著，金冬雁、黎孟枫译。分子克隆实验指南，第二版，科学出版社，1992