弓形虫LDH2基因启动子的克隆及初步鉴定
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摘要
刚地弓形虫(Toxoplasma gondii,T.gondii)是一种机会性致病原虫,能够感染包括人在内的所有哺乳动物,引起人兽共患寄生虫病。在机体免疫功能受累时,慢性感染或隐性感染阶段的弓形虫缓殖子迅速活化,可引起致命的危险,是弓形虫病防治的重点所在。但是弓形虫缓殖子寄生在宿主细胞内,常规抗生素治疗效果不理想,至今仍未找到治疗弓形虫慢性感染的有效措施。
研究表明,无论是在有氧条件下分裂增殖的速殖子,还是无氧条件下处于相对静止的缓殖子,其能量几乎全部来源于糖类代谢。乳酸脱氢酶(Lactatedehydrogenase,LDH)是糖类无氧酵解中催化丙酮酸和乳酸互相转换的酶,和辅酶NAD~+的循环再利用密切相关,在无氧条件下弓形虫能量代谢中具有举足轻重的地位。研究发现弓形虫LDH的结构、理化特性、免疫学特性及酶促动力学等方面和人类LDH存在很大的差异。所以,LDH能够成为杀灭弓形虫药物的潜在作用靶点。同时对LDH转化的相关研究还有助于对弓形虫速殖子向缓殖子转换机制的理解。
LDH2是刚地弓形虫RH株表达的两种乳酸脱氢酶同工酶中的一种,其mRNA仅在弓形虫缓殖子中检测到,其表达产物也只出现在缓殖子中;而LDH1的mRNA在速殖子和缓殖子中均有发现,但其表达产物只出现在速殖子中。序列分析表明两者核苷酸序列有64%一致,在蛋白水平二者约有71%的同源性,但在理化、免疫学特性和酶学方面等却有很大的差异。由此可以推测,在刚地弓形虫RH株中LDH两种同工酶之间的转换是一个阶段性的转变过程,LDH1被LDH2替代可能发生在速殖子向缓殖子转化的过程中。其具体的调节机制是发生在转录阶段还是转录后阶段,及其中的确切机制尚有待于实验进一步阐明。
鉴于缓殖子阶段是弓形虫致病的重要阶段,且危害性大,而LDH是弓形虫缓殖子能量唯一来源——糖类无氧代谢的终末催化酶,因此,LDH的种类及活性与弓形虫缓殖子的生存和致病能力密切相关,对LDH2基因的研究显得尤为重要。为此,本课题进行了以下几方面的研究:
第一部分弓形虫LDH2基因5′-侧翼序列的克隆
研究目的:获取刚地弓形虫LDH2基因的5′-侧翼序列,为研究该基因的表达调控机制奠定基础。
研究方法和结果:根据公用数据库的序列信息和文献资料设计合成引物,运用Thermal Asymmetric Interlaced PCR(TAIL-PCR)扩增刚地弓形虫LDH2基因的5′-侧翼序列,并TA克隆到pMD18-T载体中进行测序,得到一大小为993bp的弓形虫LDH2基因的5′-侧翼序列DNA片段。
结论:成功克隆了刚地弓形虫LDH2基因的5′-侧翼序列并测序,为研究该基因的表达调控机制奠定了试验基础。
第二部分弓形虫LDH2基因5′-侧翼序列生物信息学分析
研究目的:对生物信息学在弓形虫LDH2基因转录调控研究中的应用进行初步的摸索与探讨。
研究方法和结果:我们从多角度对LDH2基因5′-侧翼序列及编码区域进行生物信息学分析,总结前人对LDH2基因已有的研究成果,预测基因转录起始位点以及启动子的所在区域。在综合分析各方面的结果后,得出相关结论,并以此为依据,指导后续实验的进行。取得如下结果:
1.MethPrimer对CpG岛的预测结果并不唯一。在LDH2基因全序列中,MethPrimer搜索到11个潜在的CpG岛位点,其中5个CpG岛位于起始密码子上游,表明LDH2基因编码序列的确与CpG岛相连。
2.Promoterscan预测LDH2基因的候选启动子在输入序列的第320bp～509bp处(LDH2基因的-540bp～-351bp),其得分是53.25,预设阈值为50.00。
3.McPromoter预测在输入序列的第100bp和第600bp附近的序列有较高的得分,分别在0.02和0.03,但相对于预设阈值而言得分不是很高。而第600bp处的候选启动子与NNPP和Promoterscan的预测结果均有部分区域相重合。
4.Promoter2.0分析结果为,在输入序列的第900bp处很有可能存在具有启动活性的序列,但是没有给出转录起始位点。
5.NNPP得出唯一的分析结果,在输入序列的第326bp～376bp之间存在有启动子活性的序列,候选启动子的得分为0.98,远大于预设阈值0.80。该工具得出的候选启动子区域与Promoterscan分析得出的结果有部分区域重合,而NNPP的优势在于其所提供的启动子预测区间较短。其余的工具均未预测到具有启动活性的序列或可能存在的转录起始位点。
结论:各种生物信息学分析工具对LDH2基因候选启动子区域的预测结果并不完全相同。综合所有的分析结果,我们成功地将启动子和转录起始位点定位于输入序列的第300bp～700bp区间(LDH2基因的-559bp～-160bp)。
第三部分弓形虫LDH2基因候选启动子序列活性的测定
研究目的:对生物信息学所预测的刚地弓形虫LDH2基因的候选启动子区域进行试验鉴定。
研究方法和结果:PCR扩增LDH2基因的候选启动子区域和全长LDH2基因5′-侧翼序列,克隆到不含启动子和增强子的pTL_3-Basic载体的萤火虫荧光素酶报告基因上游,以构建候选启动子报告基因表达载体,分别命名为pTL_3-Lp和pTL_3-993,用以检测候选启动子的相对启动活性。然后以PRL-Tg为内参照质粒,电转化刚地弓形虫RH株速殖子,利用不同pH值的培养基继续培养,分别得到弓形虫速殖子和缓殖子。收集弓形虫,通过双荧光素酶报告基因检测系统定量分析候选启动子在速殖子和缓殖子中的活性。荧光素酶检测结果为:pTL_3-Lp在弓形虫速殖子中和缓殖子中的荧光素酶相对表达活性分别为0.054和0.053,与阴性对照pTL_3-Basic(0.052和0.051)基本相同;阳性对照pTL_3-Control的荧光素酶相对表达活性分别为1.753和1.802;pTL_3-993在弓形虫速殖子中和缓殖子中的荧光素酶相对表达活性为0.052和2.572。
结论:全长LDH2基因5′-侧翼序列在弓形虫速殖子中没有活性,而在缓殖子中有较强的启动活性;所检测的候选启动子片段在弓形虫速殖子中和缓殖子中均未表现出任何启动子活性。表明所克隆的LDH2基因5′-侧翼序列在弓形虫缓殖子中确具有启动子活性,而候选启动子片段不具有启动子活性。
第四部分弓形虫LDH2基因核心启动子鉴定
研究目的:确定弓形虫LDH2基因的核心启动子及转录起始位点,对该基因转录调控进行初步研究,为深入探讨该基因的表达调控机制奠定基础。
研究方法和结果:由于根据生物信息学分析所得出的候选启动子区没有检测到启动活性,本部分利用传统方法构建一系列巢式缺失突变体,构建系列突变体的报告基因表达载体,继而检测各缺失突变体的转录激活活性。引物延伸实验确定LDH2基因的转录起始位点。取得如下结果:
1.所有系列缺失突变体PCR扩增产物经1.0%琼脂糖凝胶电泳,均为单一条带,无其它杂带,且所得片段大小与预期一致。
2.5′-侧翼序列系列截短突变体荧光素酶表达载体经单、双酶切及测序鉴定正确。
3.所有系列截短突变体在弓形虫速殖子中的均没有检测到活性。而包含-259bp～+134bp区域的截短突变体在弓形虫缓殖子中的荧光素酶活性约是阴性对照pTL_3-Basic的50倍之多,是阳性对照pTL_3-Control的1.5倍左右;而缺少此区域的截短突变体荧光素酶活性和阴性对照pTL_3-Basic基本相同。
4.弓形虫LDH2基因的转录起始位点位于起始密码子ATG上游134bp处。
结论:成功地将弓形虫LDH2基因的核心启动子定位于-259 bp～+1bp区间。
第五部分弓形虫LDH2基因顺式作用元件的初步分析
研究目的:对弓形虫LDH2基因核心启动子区的顺式作用元件进行了初步分析,为深入了解参与该基因转录调控的转录因子提供研究基础。
研究方法和结果:运用凝胶阻滞实验(EMSA)对弓形虫LDH2基因核心启动区是否存在顺式作用元件进行了初步分析。弓形虫速殖子、缓殖子核蛋白提取物与核心启动子区结合后,经5%聚丙烯酰胺凝胶电泳分离,较未结合核蛋白提取物的靶序列位置明显滞后。随着反应体系中核蛋白提取物量的增加,滞后距离亦逐渐增加。而弓形虫速殖子、缓殖子核蛋白提取物与靶序列结合后,在凝胶上的位置不同,靶序列滞后程度亦不尽相同。此种结合可被未标记的冷探针竞争,而不被poly(dI:dC)竞争抑制,表明靶序列与核蛋白提取物结合的特异性。以上结果表明弓形虫速殖子、缓殖子核蛋白提取物中存在某种或者某些蛋白质因子与靶序列直接或间接结合,且弓形虫速殖子、缓殖子核蛋白提取物与靶序列的结合能力不同或者是与靶序列结合的蛋白因子不同。
结论:弓形虫LDH2基因核心启动子区存在顺式作用元件,且弓形虫速殖子和缓殖子核蛋白提取物有蛋白质因子与之特异性结合。
Toxoplasmosis,caused by an intracellular protozoan parasite named Toxoplasma gondii(T.gondii) which has the ability to infect a variety of warm-blooded vertebrates including human beings,is widespread throughout the world.In healthy humans,infection is often asymptomatic in immuno-competent individuals and progresses from a rapidly replicating tachyzoite stage to a dormant bradyzoite stage in response to the immune system,which is the most important stage in toxoplasmosis.The bradyzoites can remain dormant within tissue cysts protected from the host immune response and drugs.In patients with immuno-deficiencies such as AIDS or other malignancies,bradyzoites that differentiate into tachyzoites after release from the cysts can give rise to a recurrent infection that can be fatal.At present,no effective treatment for chronic toxoplasmosis is available,because bradyzoites can remain dormant within tissue cysts protected from the host immune response and drugs.
While within a human host,the opportunistic pathogen T.gondii relies heavily on glycolysis for its energy needs no matter whether in rapidly replicating tachyzoite stage or in dormant bradyzoite stage.Lactate dehydrogenase(LDH),the terminal enzyme in anaerobic glycolysis,is necessary for NAD~+ regeneration.This enzyme plays an indispensable role when glycolysis becomes the only pathway to provide energy under anaerobic conditions,whereas glycolysis is the only energy source in bradyzoites.LDH is considered as a promising target for developing drugs for chronic toxoplasmosis because its unique structural,physical,chemical and kinetic properties differ from those of human host cells.What's more,study of the differentiation of LDH helps a lot to understand the genetic regulatory signals that control differentiation between tachyzoites and bradyzoites.
There are two isozymes of LDH expressed in T.gondii.The mRNA of LDH2 is detected in the bradyzoite stage only.The transcript of LDH1,on the other hand, is found in both bradyzoites and tachyzoites.But the LDH1 gene product is only expressed in tachyzoites,whereas that of LDH2 is expressed in only bradyzoites. The genes encoding the isozymes show 64%nucleotide sequence identity,and their gene products share 71%amino acid sequence identity.It is speculated that LDH1 is replaced by LDH2 during development from tachyzoites to bradyzoites. But it is unknown that whether the differentiation is a consequence of post-transcriptional induction or it happens during the course of transcription.
Since LDH plays an indispensable role when glycolysis becomes the only pathway to provide energy under anaerobic conditions,for bradyzoites in chronic toxoplasmosis,the pathogenecity of T.gondii depends very much on the type and activity of LDH.However,the regulatory mechanism of LDH differentiation in tachyzoites and bradyzoites is unknown at present.In this study,we extensively studied the promoter of LDH2 gene in T.gondii bradyzoites.
PART ONE Amplification of 5'-flanking Sequence of LDH2 Gene in T.gondii Bradyzoites
Objective:The aim of this part is to clone the 5'-flanking sequence of LDH2 gene in T.gondii bradyzoites.
Methods and results:In order to study the promoter and regulation of LDH2 gene,we amplified 5'-flanking sequence of T.gondii LDH2 gene using Thermal Asymmetric Interlaced PCR(TAIL-PCR).Then inserted it into pMD18-T vector and sent it for sequencing.
Conclusion:We got the right 5'-flanking sequence of LDH2 gene in T.gondii bradyzoites after checking the already-known information about LDH2.
PART TWO Analysis of T.gondii LDH2 Gene 5'-Flanking Sequence Using Bio-Informatic Tools
Objective:To examine T.gondii LDH2 gene candidate promoter and transcriptional start site(TSS) by online computer-based bio-informatics tools.
Methods and results:
1.Methprimer obtains 11 CpG islands by screening in the whole LDH2 gene sequence.Among these islands,there are 5 islands within about 1Kb fragment upstream the start code ATG.This indicates that the code sequence of LDH2 gene connects with CpG islands.
2.Promoterscan result shows a candidate promoter located in the fragment from 320bp to 509bp of input sequence,-540bp to -351bp of LDH2 gene.It gives a score of 53.25,whereas the threshold is 50.00.
3.McPromoter finds two candidate promoters,one is around 100bp of the input sequence,and another is near 600bp.The scores of them are 0.02 and 0.03, much lower than the threshold.While the candidate near 600bp has fragment overlapped with the candidate promoters of both NNPP and Pomoterscan.
4.Promoter2.0 finds that there is a highly likely promoter fragment near 900bp of the input sequence.
5.NNPP gives only one candidate promoter,which is from 327bp to 376bp of the input sequence.The score of this candidate is 0.98,much more than the threshold 0.8.This fragment is partially overlapped with the result of Promoterscan.But the advantage of this software is that it can give shorter fragment than the other tools used in this study.
6.The other software used in this study did not find any fragments with promoter similar activity,or TSS.
Conclusion:Although the results obtained from each software varies from each other,we put them together,and still can speculate that the candidate promoter should locate at the fragment from 300bp to 700bp of the sequence inputted.
PART THREE Identification of the Activity of the Candidate Promoter of T.gondii LDH2 Gene
Objective:The aim of this section is to identify the activity of the candidate promoter which we got in the second section by bio-informatics tools using dual luciferase reporter assay system.
Methods and results:At present,the exact mechanism of the regulation of T.gondii LDH2 gene has not been clearly elucidated and it will help us to understand the differentiation between tachyzoites and bradyzoites.Firstly,we inserted the candidate promoter into the upstream of the Firefly luciferase of pTL_3-Basic by KpnⅠ/XhoⅠto generate pTL_3-Lp,after PCR amplifying the fragment between 300bp to 700bp of the input sequence using primers by PCR with KpnⅠand XhoⅠrestriction sites.In the same way,we generated pTL_3-993 the luciferase reporter construct of 5'-flanking sequence of T.gondii LDH2 gene.pRL-Tg vector which has Renilla luciferases drived byβ-tubulin gene promoter used as an internal control to normalize transfection efficiencies.The constructs,negative control pTL_3-Basic(without any promoter or enhancer) and positive control pTL_3-Control(withβ-tubulin gene promoter upstream the Firefly luciferase ORF) were objectively transfected into T.gondii RH strain tachyzoites by electrotransformation.Ninety-six hours after transfection,tachyzoites and bradyzoites cells were harvested and assayed for the luciferase activity.The pTL_3-Lp gives 0.054 and 0.053 in luciferase activity in both tachyzoites and bradyzoites,similar with the negative control pTL_3-Basic(0.052 and 0.051). Whereas pTL_3-Control gives 1.753 in tachyzoites and 1.802 in bradyzoites, pTL_3-993 gives 0.052 and 2.572.
Conclusion:The candidate promoter has no function on the transcription of the downstream Firefly luciferase gene.
PART FOUR Identification of the core promoter of T.gondii LDH2 gene
Objective:In this section,we went back to traditional method to study the functional promoter within a not-very-known fragment.
Methods and results:We did not get any functional elements in the third section based on computer searching.In order to search the core promoter of T. gondii LDH2 gene,we generated 9 luciferase reporter constructs by 5'-deletion mutagenesis,and identified the activity using dual luciferase reporter assay system. To search the TSS of LDH2 gene,primer extension was performed according to the lab protocols.The identification of the core promoter(which is the minimal fragment to control the transcription) is very important to study the regulatory mechanism of T.gondii LDH2 gene.
1.In tachyzoites,all the 9 luciferase reporter constructs did not give any activities.This proves that mRNA of LDH2 is not detected in the tradyzoite stage at some aspects.
2.The constructs with 393-fragment located at the upstream of the luciferase reporter gene gives 50 fold of the activity of the negative control pTL_3-Basic, about 1.5 folds of the positive control pTL_3-Control.The luciferase activities of the constructs without this fragment are similar to the negative control.
3.Primer extension experiment shows that T.gondii LDH2 gene TSS is just 134bp upstream of the start code ATG.
4.All these results show that the core promoter of T.gondii LDH2 gene is located at -259～1 of LDH2 gene.
5.LDH2 gene expression is tightly regulated at the transcriptional level.And LDH2 replaces LDH1 during the development from tachyzoites to bradyzoites.It is therefore a regulation in transcription initiation stage instead of the post-transcription stage.
Conclusion:we successfully located the core promoter of T.gondii LDH2 gene at the place of-259bp～+1bp of LDH2 gene.
PART FIVE Identification of the Functional Cis-element of T.gondii LDH2 gene by EMSA
Objective:In this section,we checked whether there are some protein factors in the nuclear extracts that can bind the core promoter fragment of T.gondii LDH2 gene.
Methods and results:Whether a promoter is founctional or not depends on whether there are some transcriptional factors that can bind the promoter or not.So it is important to study the binding sites and transcriptional factors that bind to these sites.The results show that it does have some proteins in tachyzoites and bradyzoites nuclear extracts which can bind to the core promoter,but we have not identified these proteins factors yet.What's more,the results indicate that the protein that binds the promoter in tachyzoites nuclear extracts,might be different form that in bradyzoites.This also explains that why LDH2 only expresses in bradyzoites.
Conclusion:There are some Cis-elements existed in the core promoter region of LDH2 gene of T.gondii.Some proteins could specifically bind to these Cis-elementshas.What we found provides a new insight into the transcriptional regulation of T.gondii LDH2 gene.
研究表明,无论是在有氧条件下分裂增殖的速殖子,还是无氧条件下处于相对静止的缓殖子,其能量几乎全部来源于糖类代谢。乳酸脱氢酶(Lactatedehydrogenase,LDH)是糖类无氧酵解中催化丙酮酸和乳酸互相转换的酶,和辅酶NAD~+的循环再利用密切相关,在无氧条件下弓形虫能量代谢中具有举足轻重的地位。研究发现弓形虫LDH的结构、理化特性、免疫学特性及酶促动力学等方面和人类LDH存在很大的差异。所以,LDH能够成为杀灭弓形虫药物的潜在作用靶点。同时对LDH转化的相关研究还有助于对弓形虫速殖子向缓殖子转换机制的理解。
LDH2是刚地弓形虫RH株表达的两种乳酸脱氢酶同工酶中的一种,其mRNA仅在弓形虫缓殖子中检测到,其表达产物也只出现在缓殖子中;而LDH1的mRNA在速殖子和缓殖子中均有发现,但其表达产物只出现在速殖子中。序列分析表明两者核苷酸序列有64%一致,在蛋白水平二者约有71%的同源性,但在理化、免疫学特性和酶学方面等却有很大的差异。由此可以推测,在刚地弓形虫RH株中LDH两种同工酶之间的转换是一个阶段性的转变过程,LDH1被LDH2替代可能发生在速殖子向缓殖子转化的过程中。其具体的调节机制是发生在转录阶段还是转录后阶段,及其中的确切机制尚有待于实验进一步阐明。
鉴于缓殖子阶段是弓形虫致病的重要阶段,且危害性大,而LDH是弓形虫缓殖子能量唯一来源——糖类无氧代谢的终末催化酶,因此,LDH的种类及活性与弓形虫缓殖子的生存和致病能力密切相关,对LDH2基因的研究显得尤为重要。为此,本课题进行了以下几方面的研究:
第一部分弓形虫LDH2基因5′-侧翼序列的克隆
研究目的:获取刚地弓形虫LDH2基因的5′-侧翼序列,为研究该基因的表达调控机制奠定基础。
研究方法和结果:根据公用数据库的序列信息和文献资料设计合成引物,运用Thermal Asymmetric Interlaced PCR(TAIL-PCR)扩增刚地弓形虫LDH2基因的5′-侧翼序列,并TA克隆到pMD18-T载体中进行测序,得到一大小为993bp的弓形虫LDH2基因的5′-侧翼序列DNA片段。
结论:成功克隆了刚地弓形虫LDH2基因的5′-侧翼序列并测序,为研究该基因的表达调控机制奠定了试验基础。
第二部分弓形虫LDH2基因5′-侧翼序列生物信息学分析
研究目的:对生物信息学在弓形虫LDH2基因转录调控研究中的应用进行初步的摸索与探讨。
研究方法和结果:我们从多角度对LDH2基因5′-侧翼序列及编码区域进行生物信息学分析,总结前人对LDH2基因已有的研究成果,预测基因转录起始位点以及启动子的所在区域。在综合分析各方面的结果后,得出相关结论,并以此为依据,指导后续实验的进行。取得如下结果:
1.MethPrimer对CpG岛的预测结果并不唯一。在LDH2基因全序列中,MethPrimer搜索到11个潜在的CpG岛位点,其中5个CpG岛位于起始密码子上游,表明LDH2基因编码序列的确与CpG岛相连。
2.Promoterscan预测LDH2基因的候选启动子在输入序列的第320bp～509bp处(LDH2基因的-540bp～-351bp),其得分是53.25,预设阈值为50.00。
3.McPromoter预测在输入序列的第100bp和第600bp附近的序列有较高的得分,分别在0.02和0.03,但相对于预设阈值而言得分不是很高。而第600bp处的候选启动子与NNPP和Promoterscan的预测结果均有部分区域相重合。
4.Promoter2.0分析结果为,在输入序列的第900bp处很有可能存在具有启动活性的序列,但是没有给出转录起始位点。
5.NNPP得出唯一的分析结果,在输入序列的第326bp～376bp之间存在有启动子活性的序列,候选启动子的得分为0.98,远大于预设阈值0.80。该工具得出的候选启动子区域与Promoterscan分析得出的结果有部分区域重合,而NNPP的优势在于其所提供的启动子预测区间较短。其余的工具均未预测到具有启动活性的序列或可能存在的转录起始位点。
结论:各种生物信息学分析工具对LDH2基因候选启动子区域的预测结果并不完全相同。综合所有的分析结果,我们成功地将启动子和转录起始位点定位于输入序列的第300bp～700bp区间(LDH2基因的-559bp～-160bp)。
第三部分弓形虫LDH2基因候选启动子序列活性的测定
研究目的:对生物信息学所预测的刚地弓形虫LDH2基因的候选启动子区域进行试验鉴定。
研究方法和结果:PCR扩增LDH2基因的候选启动子区域和全长LDH2基因5′-侧翼序列,克隆到不含启动子和增强子的pTL_3-Basic载体的萤火虫荧光素酶报告基因上游,以构建候选启动子报告基因表达载体,分别命名为pTL_3-Lp和pTL_3-993,用以检测候选启动子的相对启动活性。然后以PRL-Tg为内参照质粒,电转化刚地弓形虫RH株速殖子,利用不同pH值的培养基继续培养,分别得到弓形虫速殖子和缓殖子。收集弓形虫,通过双荧光素酶报告基因检测系统定量分析候选启动子在速殖子和缓殖子中的活性。荧光素酶检测结果为:pTL_3-Lp在弓形虫速殖子中和缓殖子中的荧光素酶相对表达活性分别为0.054和0.053,与阴性对照pTL_3-Basic(0.052和0.051)基本相同;阳性对照pTL_3-Control的荧光素酶相对表达活性分别为1.753和1.802;pTL_3-993在弓形虫速殖子中和缓殖子中的荧光素酶相对表达活性为0.052和2.572。
结论:全长LDH2基因5′-侧翼序列在弓形虫速殖子中没有活性,而在缓殖子中有较强的启动活性;所检测的候选启动子片段在弓形虫速殖子中和缓殖子中均未表现出任何启动子活性。表明所克隆的LDH2基因5′-侧翼序列在弓形虫缓殖子中确具有启动子活性,而候选启动子片段不具有启动子活性。
第四部分弓形虫LDH2基因核心启动子鉴定
研究目的:确定弓形虫LDH2基因的核心启动子及转录起始位点,对该基因转录调控进行初步研究,为深入探讨该基因的表达调控机制奠定基础。
研究方法和结果:由于根据生物信息学分析所得出的候选启动子区没有检测到启动活性,本部分利用传统方法构建一系列巢式缺失突变体,构建系列突变体的报告基因表达载体,继而检测各缺失突变体的转录激活活性。引物延伸实验确定LDH2基因的转录起始位点。取得如下结果:
1.所有系列缺失突变体PCR扩增产物经1.0%琼脂糖凝胶电泳,均为单一条带,无其它杂带,且所得片段大小与预期一致。
2.5′-侧翼序列系列截短突变体荧光素酶表达载体经单、双酶切及测序鉴定正确。
3.所有系列截短突变体在弓形虫速殖子中的均没有检测到活性。而包含-259bp～+134bp区域的截短突变体在弓形虫缓殖子中的荧光素酶活性约是阴性对照pTL_3-Basic的50倍之多,是阳性对照pTL_3-Control的1.5倍左右;而缺少此区域的截短突变体荧光素酶活性和阴性对照pTL_3-Basic基本相同。
4.弓形虫LDH2基因的转录起始位点位于起始密码子ATG上游134bp处。
结论:成功地将弓形虫LDH2基因的核心启动子定位于-259 bp～+1bp区间。
第五部分弓形虫LDH2基因顺式作用元件的初步分析
研究目的:对弓形虫LDH2基因核心启动子区的顺式作用元件进行了初步分析,为深入了解参与该基因转录调控的转录因子提供研究基础。
研究方法和结果:运用凝胶阻滞实验(EMSA)对弓形虫LDH2基因核心启动区是否存在顺式作用元件进行了初步分析。弓形虫速殖子、缓殖子核蛋白提取物与核心启动子区结合后,经5%聚丙烯酰胺凝胶电泳分离,较未结合核蛋白提取物的靶序列位置明显滞后。随着反应体系中核蛋白提取物量的增加,滞后距离亦逐渐增加。而弓形虫速殖子、缓殖子核蛋白提取物与靶序列结合后,在凝胶上的位置不同,靶序列滞后程度亦不尽相同。此种结合可被未标记的冷探针竞争,而不被poly(dI:dC)竞争抑制,表明靶序列与核蛋白提取物结合的特异性。以上结果表明弓形虫速殖子、缓殖子核蛋白提取物中存在某种或者某些蛋白质因子与靶序列直接或间接结合,且弓形虫速殖子、缓殖子核蛋白提取物与靶序列的结合能力不同或者是与靶序列结合的蛋白因子不同。
结论:弓形虫LDH2基因核心启动子区存在顺式作用元件,且弓形虫速殖子和缓殖子核蛋白提取物有蛋白质因子与之特异性结合。
Toxoplasmosis,caused by an intracellular protozoan parasite named Toxoplasma gondii(T.gondii) which has the ability to infect a variety of warm-blooded vertebrates including human beings,is widespread throughout the world.In healthy humans,infection is often asymptomatic in immuno-competent individuals and progresses from a rapidly replicating tachyzoite stage to a dormant bradyzoite stage in response to the immune system,which is the most important stage in toxoplasmosis.The bradyzoites can remain dormant within tissue cysts protected from the host immune response and drugs.In patients with immuno-deficiencies such as AIDS or other malignancies,bradyzoites that differentiate into tachyzoites after release from the cysts can give rise to a recurrent infection that can be fatal.At present,no effective treatment for chronic toxoplasmosis is available,because bradyzoites can remain dormant within tissue cysts protected from the host immune response and drugs.
While within a human host,the opportunistic pathogen T.gondii relies heavily on glycolysis for its energy needs no matter whether in rapidly replicating tachyzoite stage or in dormant bradyzoite stage.Lactate dehydrogenase(LDH),the terminal enzyme in anaerobic glycolysis,is necessary for NAD~+ regeneration.This enzyme plays an indispensable role when glycolysis becomes the only pathway to provide energy under anaerobic conditions,whereas glycolysis is the only energy source in bradyzoites.LDH is considered as a promising target for developing drugs for chronic toxoplasmosis because its unique structural,physical,chemical and kinetic properties differ from those of human host cells.What's more,study of the differentiation of LDH helps a lot to understand the genetic regulatory signals that control differentiation between tachyzoites and bradyzoites.
There are two isozymes of LDH expressed in T.gondii.The mRNA of LDH2 is detected in the bradyzoite stage only.The transcript of LDH1,on the other hand, is found in both bradyzoites and tachyzoites.But the LDH1 gene product is only expressed in tachyzoites,whereas that of LDH2 is expressed in only bradyzoites. The genes encoding the isozymes show 64%nucleotide sequence identity,and their gene products share 71%amino acid sequence identity.It is speculated that LDH1 is replaced by LDH2 during development from tachyzoites to bradyzoites. But it is unknown that whether the differentiation is a consequence of post-transcriptional induction or it happens during the course of transcription.
Since LDH plays an indispensable role when glycolysis becomes the only pathway to provide energy under anaerobic conditions,for bradyzoites in chronic toxoplasmosis,the pathogenecity of T.gondii depends very much on the type and activity of LDH.However,the regulatory mechanism of LDH differentiation in tachyzoites and bradyzoites is unknown at present.In this study,we extensively studied the promoter of LDH2 gene in T.gondii bradyzoites.
PART ONE Amplification of 5'-flanking Sequence of LDH2 Gene in T.gondii Bradyzoites
Objective:The aim of this part is to clone the 5'-flanking sequence of LDH2 gene in T.gondii bradyzoites.
Methods and results:In order to study the promoter and regulation of LDH2 gene,we amplified 5'-flanking sequence of T.gondii LDH2 gene using Thermal Asymmetric Interlaced PCR(TAIL-PCR).Then inserted it into pMD18-T vector and sent it for sequencing.
Conclusion:We got the right 5'-flanking sequence of LDH2 gene in T.gondii bradyzoites after checking the already-known information about LDH2.
PART TWO Analysis of T.gondii LDH2 Gene 5'-Flanking Sequence Using Bio-Informatic Tools
Objective:To examine T.gondii LDH2 gene candidate promoter and transcriptional start site(TSS) by online computer-based bio-informatics tools.
Methods and results:
1.Methprimer obtains 11 CpG islands by screening in the whole LDH2 gene sequence.Among these islands,there are 5 islands within about 1Kb fragment upstream the start code ATG.This indicates that the code sequence of LDH2 gene connects with CpG islands.
2.Promoterscan result shows a candidate promoter located in the fragment from 320bp to 509bp of input sequence,-540bp to -351bp of LDH2 gene.It gives a score of 53.25,whereas the threshold is 50.00.
3.McPromoter finds two candidate promoters,one is around 100bp of the input sequence,and another is near 600bp.The scores of them are 0.02 and 0.03, much lower than the threshold.While the candidate near 600bp has fragment overlapped with the candidate promoters of both NNPP and Pomoterscan.
4.Promoter2.0 finds that there is a highly likely promoter fragment near 900bp of the input sequence.
5.NNPP gives only one candidate promoter,which is from 327bp to 376bp of the input sequence.The score of this candidate is 0.98,much more than the threshold 0.8.This fragment is partially overlapped with the result of Promoterscan.But the advantage of this software is that it can give shorter fragment than the other tools used in this study.
6.The other software used in this study did not find any fragments with promoter similar activity,or TSS.
Conclusion:Although the results obtained from each software varies from each other,we put them together,and still can speculate that the candidate promoter should locate at the fragment from 300bp to 700bp of the sequence inputted.
PART THREE Identification of the Activity of the Candidate Promoter of T.gondii LDH2 Gene
Objective:The aim of this section is to identify the activity of the candidate promoter which we got in the second section by bio-informatics tools using dual luciferase reporter assay system.
Methods and results:At present,the exact mechanism of the regulation of T.gondii LDH2 gene has not been clearly elucidated and it will help us to understand the differentiation between tachyzoites and bradyzoites.Firstly,we inserted the candidate promoter into the upstream of the Firefly luciferase of pTL_3-Basic by KpnⅠ/XhoⅠto generate pTL_3-Lp,after PCR amplifying the fragment between 300bp to 700bp of the input sequence using primers by PCR with KpnⅠand XhoⅠrestriction sites.In the same way,we generated pTL_3-993 the luciferase reporter construct of 5'-flanking sequence of T.gondii LDH2 gene.pRL-Tg vector which has Renilla luciferases drived byβ-tubulin gene promoter used as an internal control to normalize transfection efficiencies.The constructs,negative control pTL_3-Basic(without any promoter or enhancer) and positive control pTL_3-Control(withβ-tubulin gene promoter upstream the Firefly luciferase ORF) were objectively transfected into T.gondii RH strain tachyzoites by electrotransformation.Ninety-six hours after transfection,tachyzoites and bradyzoites cells were harvested and assayed for the luciferase activity.The pTL_3-Lp gives 0.054 and 0.053 in luciferase activity in both tachyzoites and bradyzoites,similar with the negative control pTL_3-Basic(0.052 and 0.051). Whereas pTL_3-Control gives 1.753 in tachyzoites and 1.802 in bradyzoites, pTL_3-993 gives 0.052 and 2.572.
Conclusion:The candidate promoter has no function on the transcription of the downstream Firefly luciferase gene.
PART FOUR Identification of the core promoter of T.gondii LDH2 gene
Objective:In this section,we went back to traditional method to study the functional promoter within a not-very-known fragment.
Methods and results:We did not get any functional elements in the third section based on computer searching.In order to search the core promoter of T. gondii LDH2 gene,we generated 9 luciferase reporter constructs by 5'-deletion mutagenesis,and identified the activity using dual luciferase reporter assay system. To search the TSS of LDH2 gene,primer extension was performed according to the lab protocols.The identification of the core promoter(which is the minimal fragment to control the transcription) is very important to study the regulatory mechanism of T.gondii LDH2 gene.
1.In tachyzoites,all the 9 luciferase reporter constructs did not give any activities.This proves that mRNA of LDH2 is not detected in the tradyzoite stage at some aspects.
2.The constructs with 393-fragment located at the upstream of the luciferase reporter gene gives 50 fold of the activity of the negative control pTL_3-Basic, about 1.5 folds of the positive control pTL_3-Control.The luciferase activities of the constructs without this fragment are similar to the negative control.
3.Primer extension experiment shows that T.gondii LDH2 gene TSS is just 134bp upstream of the start code ATG.
4.All these results show that the core promoter of T.gondii LDH2 gene is located at -259～1 of LDH2 gene.
5.LDH2 gene expression is tightly regulated at the transcriptional level.And LDH2 replaces LDH1 during the development from tachyzoites to bradyzoites.It is therefore a regulation in transcription initiation stage instead of the post-transcription stage.
Conclusion:we successfully located the core promoter of T.gondii LDH2 gene at the place of-259bp～+1bp of LDH2 gene.
PART FIVE Identification of the Functional Cis-element of T.gondii LDH2 gene by EMSA
Objective:In this section,we checked whether there are some protein factors in the nuclear extracts that can bind the core promoter fragment of T.gondii LDH2 gene.
Methods and results:Whether a promoter is founctional or not depends on whether there are some transcriptional factors that can bind the promoter or not.So it is important to study the binding sites and transcriptional factors that bind to these sites.The results show that it does have some proteins in tachyzoites and bradyzoites nuclear extracts which can bind to the core promoter,but we have not identified these proteins factors yet.What's more,the results indicate that the protein that binds the promoter in tachyzoites nuclear extracts,might be different form that in bradyzoites.This also explains that why LDH2 only expresses in bradyzoites.
Conclusion:There are some Cis-elements existed in the core promoter region of LDH2 gene of T.gondii.Some proteins could specifically bind to these Cis-elementshas.What we found provides a new insight into the transcriptional regulation of T.gondii LDH2 gene.
引文
[1]Jackson MH, Hutchison WM, Baker JR, et al. Advances in Parasitology. Academic Press. London, 1989,55-86.
[2]Bhopale GM, Naik SR, Bhave GG, et al. Assessmentof enzyme linked immunosorbent assay based diagnostic kits(Toxokit-G and Toxokit-M) for the detection of IgG and IgM antibodiesto Toxoplasma gondii in human serum[J]. Comp Immun Microbial Infect Dis. 1997,1(20): 309-314.
[3]Lehmann T, Marcet PL, Graham DH, et al. Globalization and the population structure of Toxoplasma gondii [J]. Proceeding of the National Academy of Science of the Unite States of America.2006,103:11423-8.
[4]Luft BJ, Remington JS, Toxoplasmic encephalitis in AIDS[J]. Clin Infec Dis. 1992 ,3(15): 211-222.
[5]Zhang YW, Halonen SK, Ma YF, et al. Initial characterization of CST1, a Toxoplasma gondii cyst wall glycoprotein[J]. Infect Immun. 2001, 69: 501-507.
[6]Cleary, MD, Singh, U, Blader IJ,et al. Toxoplasma gondii asexual development:identification of developmentally regulated genes and distinct patterns of gene expression[J]. Eukaryot Cell. 2002 ,1:329-340
[7]Manger ID, Hehl A, Parmley S,et al. Expressed sequence tag analysis of the bradyzoite stage of Toxoplasma gondii: identification of developmentally regulated genes[J]. Infect Immun.1998,1 (66):32-1637.
[8]Yang S, Parmey SF. Toxoplasma gondii expresses two distinct lactate dehydrogenase homologous genes during its life cycle in intermediate hosts[J]. Gene. (Amst). 1997,184:1-12.
[9]Tomavo, S. Implementation and evaluation of a distance learning introductory course in medical informatics[J]. Int J Parasitol. 2001,10,1023-1031.
[10]Alexandra C, Florence D, Sandra L, et al. Developmentally regulated biosynthesis of carbohydrate and storage polysaccharide during differentiation and tissue cyst formation in Toxoplasma gondii[J]. Biochimie. 2003,85:353-361.
[11]David JP, Ferguson, Stephen FP, Stanislas T, et al. Evidence for nuclear localisation of two stage-specific isoenzymes of enolase in Toxoplasma gondii correlates with active parasite replication. International Journal for Parasitology[J]. 2002,32:1399-1410.
[12] Dzierszinski F, Mortuaire M, Dendouga N,et al. Differential expression of two plant-like enolases with distinct enzymatic and antigenic properties during stage conversion of the protozoan parasite Toxoplasma gondii[J].J Mol Biol. 2001,309:1017-1027.
[13] Ferguson DJ, Parmley SF, Tomavo S.Evidence for nuclear localisation of two stage-specific isoenzymes of enolase in Toxoplasma gondii correlates with active parasite replication[J]. Int J Parasitol. 2002,32(11):1399-410.
[14] Parmley SF, Weiss LM, Yang S. Cloning of a bradyzoite-specific gene of Toxoplasma gondii encoding a cytoplasmic antigen. Mol Biochem Parasitol [J]. 1995,73: 253-257.
[15] Kavanagh KL, Elling RA, Wilson DK. Structure of Toxoplasma gondii LDH1: active-site differences from human lactate dehydrogenases and the structural basis for efficient APAD+ use[J].Biochemistry. 2004,43(4):879-89.
[16]Dando C, Schroeder ER, Hunsaker LA, et al. The kinetic properties and sensitivities to inhibitors of lactate dehydrogenases (LDH1 and LDH2) from Toxoplasma gondii: comparisons with pLDH from Plasmodium falciparum[J].Mol Biochem Parasitol. 2001,118: 23-32
[17]Winter V J, Cameron A, Tranter R, et al. Crystal structure of Plasmodium berghei lactate dehydrogenase indicates the unique structural differences of these enzymes are shared across the Plasmodium genus[J].Mol Biochem Parasitol. 2003,131,1-10.
[18]Lakhdar-Ghazal F, Blonski C, Willson M,et al. Glycolysis and proteases as targets for the design of new anti-trypanosome drugs[J]. Curr Top Med Chem.2002,2:439-456.
[19]Louassini M, Foulquie M, Benitez R,et al. Citric-acidcycle key enzyme activities during in vitro growth and metacyclogenesisof Leishmania infantum promastigotes[J].J Parasitol. 1999, 85:595-602.
[20]Brady RL, Cameron A. Structure-based approaches to the development of novel anti-malarials[J]. Curr Drug Targets. 2004,5:137-149.
[21]Verlinde C L, Hannaert V, Blonski C,et al. Glycolysis as a target for the design of new antitrypanosome drugs[J]. Drug Resist Update. 2001,4:50-65.
[22]Pedersen AG,Baldi P, Chauvin Y, et al. The biology of eukaryoticpromoter prediction-a review [J]. Computers&chemistry. 1999,23:191-20.
[23] Zeng Y, Miao C, Tang L, Xu Y. Cloning and analysis of the promoter of Tibetan hulless barley β 21, 32glucanase gene II and construction of expression vector[J].Chin J Applenviron Biol. 2003,9 (2): 122-126.
[24]Zhang XL, Wang SH, Tang L. Cloning and function identifiction of rice spermpre dominant expressed gene RSSG58' s promoter[J]. Chin J Appl environ Biol. 2005,11(3):271-275.
[25] Boulikas T, Kong CF. A novel class of matrix attached regions (MARs) identified by random cloning and their implications in differential ion and carcinogenesis[J]. Int J Oncol. 1993,2:325-330.
[26]Strissel PL , Rafael Espinosa I, Rowley JD, etal. Scaffold attachment regions in centromere associated DNA[J]. Chromsoma. 1996,105:122-133.
[27]Janct Stein, Gary S, Stein et al. The tissue specific nulear matrix protein, NMP-2 ,is a member of the AML/CBF/PEBP/Runt Domain transcription factor family:Interactions with theosteo-calcin gene promoter[J].Biochemistry.1995,34:13125-13132.
[28]Kazama T, Toriyama KA. Pentatricopeptide repeat containing gene that promotes the processing of aberrant atp6 RNA of cytoplasmic malesterile rice [J]. FEBS Lett.2003,544:99-102.
[29]Liu YG, Norihiro M, Teruko O, et al. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junction by thermal asymmertric interlaced PCR[J]. The Plant J. 1995,8(3) :457 - 463.
[30]Liu YG , Robert FW. Thermal asymmetric interlaced PCR:automatable amplification and sequencing of insert end fragment from P1 and YAC clones for chromosome walking[J]. Genomics. 1995,25:674- 681.
[31]Liu YG,Huang N.Efficient amplification of insert end sequences from bacterial artificial chromosome clones by thermal asymmetric interlaced PCR[J].Plant Molecular Biology Reporter.1998,16(2):175-181.
[32]彭玮欣,刘国庆,马峙英.用于染色体步行的PCR技术[J].河北农业大学学报[J].2001,24(3):86-89.
[33]Terauchi R,Kahl G.Rapid isolation of promoter sequences by TAIL-PCR:the 5'-flanking regions of Pal and Pgi genes from Yams(Dioscorea)[J].Mol Gen Genet.2000,263:554-560.
[34]应革,何朝族.TAIL-PCR方法快速分离XCC致病相关基因序列[J].生物工程学报.2002,18(2):182-186.
[35]李一琨,王金发.高等植物启动子研究进展[J].植物学通报.1998,15:1-6.
[36]孙晓红,陈明杰,潘迎捷.启动子克隆概述[J].食用菌学报.2002,9(3):57-62.
[37]韩志勇,沈革志.基于PCR的染色体步行技术[J].高技术通讯.2000,10(11):102-105.
[38]Ueli G,Jean P,Vielle C.Maternal control of embrogenesis by MEDEA,a polycomb group gene in A rabi dopsis[J].Science.1998,280:446-450.
[39]Chin HG,Chin HG,Mi SC,et al.Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system[J].The Plant Journal.1999,9(5):615-623.
[40]方进,翟文学,王文明,等.转基因水稻T-DNA侧翼序列的扩增与分析[J].遗传学报.2001,28(4):345-351.
[41]Vincentz M,Leite A,Neshich G,et al.Gander ES1 ACGT and vicilin core sequences in a promoter domain required for seed specific expression of a 2S storage protein gene are recognized by the opaque regulatory protein[J].Plant Molecular Biology.1997.34:879-889.
[42]Siebert P D,Chenchik A,Kellogg DE,et al.An improved PCR method for walking in uncloned genomic DNA1.Nucleic Acids Research[J].1995,23:1087-1088.
[43]Hahn S A,Schutte M,Hooue A,et al.A candidate tumer2suppressor gene at human chromosome[J].Science.1996,271:350-353.
[44]Ochman H,Gerber AS.Genetic applications of an inversepolymerase chain reaction[J].Genetics.1988,120:621-623.
[45]宋达峰,韩凝,边红武,等.用改良TAIL-PCR技术分析转基因烟草cbfl基因插入区的侧翼序列[J].作物学报.2005,31(10):1377-1379.
[46]Antequera F,Bird A.Number of CpG islands and genes in human and mouse[J].Proc Natl Acad Sci U S A.1993,90(24):11995-9.
[47]Larsen F,Gundersen G,Lopez R,et al.CpG islands as gene markers in the human genome[J].Genomics.1992,13(4):1095-107.
[48]Zhang MQ.Using CorePromoter to find human core promoters[J].Curr Protoc Bioinformatics.2005,30-32.
[49]Werner T.Finding and decrypting of promoters contributes to the elucidation of gene function[J].In Silico Biol.2002,2(3):249-55.
[50]Scherf M,Klingenhoff A,Werner T.Highly specific localization of promoter regions in large genomic sequences by PromoterInspector:a novel context analysis approach[J].J dlol Biol.2000,297(3):599-606.
[51]Gangal R,Sharma P.Human pol Ⅱ promoter prediction:time series descriptors and machine learning[J].Nucleic Acids Res.2005,33(4):1332-6.
[52]Anwar F,Murtuza Baker S,Jabid T,et al.Pol Ⅱ promoter prediction using characteristic 4-mer motifs:a machine learning approach[J].BMC Bioinformatics.2008,9(1):414.
[53]Knudsen S.Promoter2.0:for the recognition of PolⅡ promoter sequences[J].Bioinformatics.1999,15(5):356-61.
[54]Bird AP.CpG-rich islands and the function of DNA methylation[J].Nature.1986,321:209-213.
[55]Li M,Paik HI,Balch C,et al.Enriched transcription factor binding sites in hypermethylated gene promoters in drug resistant cancer cells[J].Bioinformatics.2008,24(16):1745-8.
[56]Rozenberg JM,Shlyakhtenko A,Glass K,et al.All and only CpG containing sequences are enriched in promoters abundantly bound by RNA polymerase Ⅱ in multiple tissues[J].BMC Genomics.2008 Feb 5;9:67.
[57]Oei SL, Babich VS, Kazakov VI, et al. Clusters of regulatory signals for RNA polymerase Ⅱ transcription associated with Alu family repeats and CpG islands in human promoters[J].Genomics. 2004 May;83(5):873-82.
[58]Thomas W. Target gene identification from expression array data by promoter analysis [J].Biomolecular Engineering. 2001,7:87-94.
[59]Cheng L, Ziegelhoffer PR, Yang NS. In vivo promoter activity and transgene expression in mammalian somatic tissues evaluated by using particle bombardment[J]. Proc Natl Acad Sci U S A. 1993,90(10):4455-9.
[60]Roeder RG The role of general initiation factors in transcription by RNA polymeraseⅡ [J].Trends Biochem Sci. 1996,21:327-335.
[61]Pedersen AG, Baldi P, Chauvin Y, et al. The biology of eukaryotic promoter prediction review[J] .Computers&chemistry. 1999,23:191-120.
[62]Huang RC, Smith MM. Nucleic acid hybridization and the nature of chromosomal protein bound RNA[J]. Results Probl Cell Differ. 1972;3:65-76.
[63]Coulombe B, Burton Z. DNA bending and wrapping around RNA Polymerase: a "revolutionary" model describing transcriptional mechanisms[J]. Microbiol Mol Biol Rev. 1999,63:457-478.
[64] Mu W, Burt DR. Transcriptional regulation of GABAA receptor gamma subunit gene[J]. Brain Res Mol Brain Res.l999,67(1):137-47.
[65]He X, Treacy MN, Simmons DM, et al. Expressiono falarge family of POU-domain regulatory genes in mammalian brain development[J] .Natuer.1989,340 (6228):35-41.
[66]Li L, Suzuki T, Mori N,et al. Identification of a Functional S ilencer Element Involved in Neuron-Specific Expression of the Synapsin Ⅰ[J]. Gene Proceedings of the National Academyof Science. 1993,90:1460-1468.
[67]Sanyal S, Narayanan R, Consoulas C. et al. Evidence for cell autonomous AP1 function in regulation of Drosophila motor-neuron plasticity [J]. BMC Neurosci.2003, 11;4(1): 20.
[68]Groth RD, Mermelstein PG Brain-derived neurotrophic factor activation of NFAT (nuclear factor of activated T-cells)-dependent transcription: a role for the transcription factor NFATc4 in neurotorphin-mediated gene expression[J].J Neuorsc.2003 3;23(22):8125-34.
[69] Soldati D, Boothroyd JC. A selector of transcription initiation in the protozoan parasite Toxoplasma gondii[J]. Mol Cell Biol. 1995,15(1):87-93.
[70]Mercier C, Garber GE, Lecordier L, et al.Common cis-acting elements critical for the expression of several genes of Toxoplasma gondii[J].Mol Microbiol.1996,21(2):421-8.
[71]Rountree MR, Bachman KE, Herman JG. et al. DNA methylation chromat in inheritance and cancer[J]. Oncogene. 2001,20(24):3156-3158.
[72]Siegfired Z, Cedar H. DNA methylation: a molecular lock[J].Curr Biol.1997,7 (5):305-307.
[73]Cross SH, Birda P. CpG islands and genes[J].Curr Opin Genet Dev. 1995,5 (3):309-314.
[74]Birda P. CpG-rich islands and the function of DNA methylation[J].Natuer.1986,32 1(6067): 209-13
[75]Jones PA, Takai D. The role of DNA methylation in mammaliane pigenetics[J].Science. 2001,293(5532): 1068-70.
[76]Kass S, Landsberger N, Wolfea P. DNA methylation directs a time-dependent repression of transcription initiation[J].Curr Biol. 1997,7(3): 157-165.
[77]Zhang Y,Niu Z, Cohena J et al. The internal chondrocyte-specific promoter of the chick type Ⅲ collagen gene is activated by AP1 and is repressed in fibroblasts by a complex containing an LBP1-related protein[J]. Nucleic Acids Res. 1999,27(20):4090-9.
[77]Das G, Hinkley CS, Herr W. Basal promoter elements as a selective determinant of transcriptional activator function [J]. Nature. 1995,374:657-660.
[78]Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase[J]. Science. 1990, 249 (4968) :5052-51011.
[79]Kim S, Shi H, Lee DK, et al. Specific SR protein dependent splicing substrates identified through genomic SELEX[J]. Nucleic Acids Res. 2003, 31(7): 1955-1961.
[80]Choo Y,Klug A.Toward a code for the interactions of zinc fingers with DNA:selection of randomized fingers disp layed on phage[J].Proc Natl Acad Sci USA.1994,91:11163-11167.
[81]Buly KM,Gentalen E,Lockhart D,et al.Quantifying DNA-protein interactions by double-stranded DNA arrays[J].Nat Biotechnol.1999,17:573-577.
[82]Deplancke B,Dupuy D,Vidal M,et al.Program in genefunction and expression[J].Genome Res.2004,14(10B):2093-2101.
[83]Garner M,Revzin A.A gel electrophoresismethod for quantifying the binding of proteins to specific DNA regions:application to components of the Escherichia coli lactose operon regulatory system[J].Nucleic Acids Res.1981,9:3047-3060.
[84]Rona IZ,Wang Y,Khandur JL,et al.Transcription factor binding study by capillary zoneelectrophoretic mobility shift assay[J].Electrophoresis.2003,24:96-100.
[85]Okuno Y,Huang G,Rosenbure F,et al.Potential auto-regulation of transcription factor PU1 by an up stream regulatory element[J].Mol Cell Biol.2005,25(7):2832-2845.
[86]Molnar G,O'Leary N,Pardee A,et al.Quantification of DNA-protein interaction by UV crosslinking[J].Nucleic Acids Res.1995,23:3318-3326.
[87]Chorley BN,Wang X,Campbell MR,et al.Discovery and verification of functional single nucleotide polymorphisms in regulatory genomic regions:current and developing technologies[J].Mutat Res.2008,659(1-2):147-57.
[88]Geser F,Seppi K,Stampfer-Kountchev M,K(o|¨)llensperger M,et al.The European Multiple System Atrophy-Study Group(EMSA-SG)[J].J Neural Transm.2005,112(12):1677-86.
[89]Veenstra TD.Electrospray ionization mass spectrometry:a promising new technique in the study of protein/DNA noncovalent complexes[J].Biochem Biophys Res Commun.1999,257(1):1-5.
[90]朱玉贤,李毅.现代分子生物.第2版.北京:高等教育出版社.2002:166-170.
[91]Kwon JA,Rho HM.Transcriptional repression of the Human P53 gene by hepatitis B viral core protein(HBc) in human liver cells[J].Bio Chem 2003;384:203-212.
[92]Rho HR, Kwon JA. Hepatitis B viral core protein activities the hepatitis B viralenhancer Ⅱ/pregenomic promoter through the nuclear factor B binding site[J]. Biochem Cell Biol. 2002;80: 445-463.
[93] Johnson JJ, Raney AK, Mclachlan A. Characterization of a functional hepatocyte nuclear factor 3 binding site in the hepatitis B virus nucleocapsid promoter[J].Virology 1995;208:147-158.
[94]Aouacheria A, Navratil V, Lopez-Perez R,et al. In silico whole-genome screening for cancer-related single-nucleotide polymorphisms located in human mRNA untranslated regions[J].BMC Genomics. 2007, 3:8-2.
[95]Garuti R, Croce MA, Piccinini L, Tiozzo R, Bertolini S, Calandra S. Functional analysis of the promoter of human sterol 27-hydroxylase gene in HepG2 cells[J].Gene.2002, 283:133-143.
[2]Bhopale GM, Naik SR, Bhave GG, et al. Assessmentof enzyme linked immunosorbent assay based diagnostic kits(Toxokit-G and Toxokit-M) for the detection of IgG and IgM antibodiesto Toxoplasma gondii in human serum[J]. Comp Immun Microbial Infect Dis. 1997,1(20): 309-314.
[3]Lehmann T, Marcet PL, Graham DH, et al. Globalization and the population structure of Toxoplasma gondii [J]. Proceeding of the National Academy of Science of the Unite States of America.2006,103:11423-8.
[4]Luft BJ, Remington JS, Toxoplasmic encephalitis in AIDS[J]. Clin Infec Dis. 1992 ,3(15): 211-222.
[5]Zhang YW, Halonen SK, Ma YF, et al. Initial characterization of CST1, a Toxoplasma gondii cyst wall glycoprotein[J]. Infect Immun. 2001, 69: 501-507.
[6]Cleary, MD, Singh, U, Blader IJ,et al. Toxoplasma gondii asexual development:identification of developmentally regulated genes and distinct patterns of gene expression[J]. Eukaryot Cell. 2002 ,1:329-340
[7]Manger ID, Hehl A, Parmley S,et al. Expressed sequence tag analysis of the bradyzoite stage of Toxoplasma gondii: identification of developmentally regulated genes[J]. Infect Immun.1998,1 (66):32-1637.
[8]Yang S, Parmey SF. Toxoplasma gondii expresses two distinct lactate dehydrogenase homologous genes during its life cycle in intermediate hosts[J]. Gene. (Amst). 1997,184:1-12.
[9]Tomavo, S. Implementation and evaluation of a distance learning introductory course in medical informatics[J]. Int J Parasitol. 2001,10,1023-1031.
[10]Alexandra C, Florence D, Sandra L, et al. Developmentally regulated biosynthesis of carbohydrate and storage polysaccharide during differentiation and tissue cyst formation in Toxoplasma gondii[J]. Biochimie. 2003,85:353-361.
[11]David JP, Ferguson, Stephen FP, Stanislas T, et al. Evidence for nuclear localisation of two stage-specific isoenzymes of enolase in Toxoplasma gondii correlates with active parasite replication. International Journal for Parasitology[J]. 2002,32:1399-1410.
[12] Dzierszinski F, Mortuaire M, Dendouga N,et al. Differential expression of two plant-like enolases with distinct enzymatic and antigenic properties during stage conversion of the protozoan parasite Toxoplasma gondii[J].J Mol Biol. 2001,309:1017-1027.
[13] Ferguson DJ, Parmley SF, Tomavo S.Evidence for nuclear localisation of two stage-specific isoenzymes of enolase in Toxoplasma gondii correlates with active parasite replication[J]. Int J Parasitol. 2002,32(11):1399-410.
[14] Parmley SF, Weiss LM, Yang S. Cloning of a bradyzoite-specific gene of Toxoplasma gondii encoding a cytoplasmic antigen. Mol Biochem Parasitol [J]. 1995,73: 253-257.
[15] Kavanagh KL, Elling RA, Wilson DK. Structure of Toxoplasma gondii LDH1: active-site differences from human lactate dehydrogenases and the structural basis for efficient APAD+ use[J].Biochemistry. 2004,43(4):879-89.
[16]Dando C, Schroeder ER, Hunsaker LA, et al. The kinetic properties and sensitivities to inhibitors of lactate dehydrogenases (LDH1 and LDH2) from Toxoplasma gondii: comparisons with pLDH from Plasmodium falciparum[J].Mol Biochem Parasitol. 2001,118: 23-32
[17]Winter V J, Cameron A, Tranter R, et al. Crystal structure of Plasmodium berghei lactate dehydrogenase indicates the unique structural differences of these enzymes are shared across the Plasmodium genus[J].Mol Biochem Parasitol. 2003,131,1-10.
[18]Lakhdar-Ghazal F, Blonski C, Willson M,et al. Glycolysis and proteases as targets for the design of new anti-trypanosome drugs[J]. Curr Top Med Chem.2002,2:439-456.
[19]Louassini M, Foulquie M, Benitez R,et al. Citric-acidcycle key enzyme activities during in vitro growth and metacyclogenesisof Leishmania infantum promastigotes[J].J Parasitol. 1999, 85:595-602.
[20]Brady RL, Cameron A. Structure-based approaches to the development of novel anti-malarials[J]. Curr Drug Targets. 2004,5:137-149.
[21]Verlinde C L, Hannaert V, Blonski C,et al. Glycolysis as a target for the design of new antitrypanosome drugs[J]. Drug Resist Update. 2001,4:50-65.
[22]Pedersen AG,Baldi P, Chauvin Y, et al. The biology of eukaryoticpromoter prediction-a review [J]. Computers&chemistry. 1999,23:191-20.
[23] Zeng Y, Miao C, Tang L, Xu Y. Cloning and analysis of the promoter of Tibetan hulless barley β 21, 32glucanase gene II and construction of expression vector[J].Chin J Applenviron Biol. 2003,9 (2): 122-126.
[24]Zhang XL, Wang SH, Tang L. Cloning and function identifiction of rice spermpre dominant expressed gene RSSG58' s promoter[J]. Chin J Appl environ Biol. 2005,11(3):271-275.
[25] Boulikas T, Kong CF. A novel class of matrix attached regions (MARs) identified by random cloning and their implications in differential ion and carcinogenesis[J]. Int J Oncol. 1993,2:325-330.
[26]Strissel PL , Rafael Espinosa I, Rowley JD, etal. Scaffold attachment regions in centromere associated DNA[J]. Chromsoma. 1996,105:122-133.
[27]Janct Stein, Gary S, Stein et al. The tissue specific nulear matrix protein, NMP-2 ,is a member of the AML/CBF/PEBP/Runt Domain transcription factor family:Interactions with theosteo-calcin gene promoter[J].Biochemistry.1995,34:13125-13132.
[28]Kazama T, Toriyama KA. Pentatricopeptide repeat containing gene that promotes the processing of aberrant atp6 RNA of cytoplasmic malesterile rice [J]. FEBS Lett.2003,544:99-102.
[29]Liu YG, Norihiro M, Teruko O, et al. Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junction by thermal asymmertric interlaced PCR[J]. The Plant J. 1995,8(3) :457 - 463.
[30]Liu YG , Robert FW. Thermal asymmetric interlaced PCR:automatable amplification and sequencing of insert end fragment from P1 and YAC clones for chromosome walking[J]. Genomics. 1995,25:674- 681.
[31]Liu YG,Huang N.Efficient amplification of insert end sequences from bacterial artificial chromosome clones by thermal asymmetric interlaced PCR[J].Plant Molecular Biology Reporter.1998,16(2):175-181.
[32]彭玮欣,刘国庆,马峙英.用于染色体步行的PCR技术[J].河北农业大学学报[J].2001,24(3):86-89.
[33]Terauchi R,Kahl G.Rapid isolation of promoter sequences by TAIL-PCR:the 5'-flanking regions of Pal and Pgi genes from Yams(Dioscorea)[J].Mol Gen Genet.2000,263:554-560.
[34]应革,何朝族.TAIL-PCR方法快速分离XCC致病相关基因序列[J].生物工程学报.2002,18(2):182-186.
[35]李一琨,王金发.高等植物启动子研究进展[J].植物学通报.1998,15:1-6.
[36]孙晓红,陈明杰,潘迎捷.启动子克隆概述[J].食用菌学报.2002,9(3):57-62.
[37]韩志勇,沈革志.基于PCR的染色体步行技术[J].高技术通讯.2000,10(11):102-105.
[38]Ueli G,Jean P,Vielle C.Maternal control of embrogenesis by MEDEA,a polycomb group gene in A rabi dopsis[J].Science.1998,280:446-450.
[39]Chin HG,Chin HG,Mi SC,et al.Molecular analysis of rice plants harboring an Ac/Ds transposable element-mediated gene trapping system[J].The Plant Journal.1999,9(5):615-623.
[40]方进,翟文学,王文明,等.转基因水稻T-DNA侧翼序列的扩增与分析[J].遗传学报.2001,28(4):345-351.
[41]Vincentz M,Leite A,Neshich G,et al.Gander ES1 ACGT and vicilin core sequences in a promoter domain required for seed specific expression of a 2S storage protein gene are recognized by the opaque regulatory protein[J].Plant Molecular Biology.1997.34:879-889.
[42]Siebert P D,Chenchik A,Kellogg DE,et al.An improved PCR method for walking in uncloned genomic DNA1.Nucleic Acids Research[J].1995,23:1087-1088.
[43]Hahn S A,Schutte M,Hooue A,et al.A candidate tumer2suppressor gene at human chromosome[J].Science.1996,271:350-353.
[44]Ochman H,Gerber AS.Genetic applications of an inversepolymerase chain reaction[J].Genetics.1988,120:621-623.
[45]宋达峰,韩凝,边红武,等.用改良TAIL-PCR技术分析转基因烟草cbfl基因插入区的侧翼序列[J].作物学报.2005,31(10):1377-1379.
[46]Antequera F,Bird A.Number of CpG islands and genes in human and mouse[J].Proc Natl Acad Sci U S A.1993,90(24):11995-9.
[47]Larsen F,Gundersen G,Lopez R,et al.CpG islands as gene markers in the human genome[J].Genomics.1992,13(4):1095-107.
[48]Zhang MQ.Using CorePromoter to find human core promoters[J].Curr Protoc Bioinformatics.2005,30-32.
[49]Werner T.Finding and decrypting of promoters contributes to the elucidation of gene function[J].In Silico Biol.2002,2(3):249-55.
[50]Scherf M,Klingenhoff A,Werner T.Highly specific localization of promoter regions in large genomic sequences by PromoterInspector:a novel context analysis approach[J].J dlol Biol.2000,297(3):599-606.
[51]Gangal R,Sharma P.Human pol Ⅱ promoter prediction:time series descriptors and machine learning[J].Nucleic Acids Res.2005,33(4):1332-6.
[52]Anwar F,Murtuza Baker S,Jabid T,et al.Pol Ⅱ promoter prediction using characteristic 4-mer motifs:a machine learning approach[J].BMC Bioinformatics.2008,9(1):414.
[53]Knudsen S.Promoter2.0:for the recognition of PolⅡ promoter sequences[J].Bioinformatics.1999,15(5):356-61.
[54]Bird AP.CpG-rich islands and the function of DNA methylation[J].Nature.1986,321:209-213.
[55]Li M,Paik HI,Balch C,et al.Enriched transcription factor binding sites in hypermethylated gene promoters in drug resistant cancer cells[J].Bioinformatics.2008,24(16):1745-8.
[56]Rozenberg JM,Shlyakhtenko A,Glass K,et al.All and only CpG containing sequences are enriched in promoters abundantly bound by RNA polymerase Ⅱ in multiple tissues[J].BMC Genomics.2008 Feb 5;9:67.
[57]Oei SL, Babich VS, Kazakov VI, et al. Clusters of regulatory signals for RNA polymerase Ⅱ transcription associated with Alu family repeats and CpG islands in human promoters[J].Genomics. 2004 May;83(5):873-82.
[58]Thomas W. Target gene identification from expression array data by promoter analysis [J].Biomolecular Engineering. 2001,7:87-94.
[59]Cheng L, Ziegelhoffer PR, Yang NS. In vivo promoter activity and transgene expression in mammalian somatic tissues evaluated by using particle bombardment[J]. Proc Natl Acad Sci U S A. 1993,90(10):4455-9.
[60]Roeder RG The role of general initiation factors in transcription by RNA polymeraseⅡ [J].Trends Biochem Sci. 1996,21:327-335.
[61]Pedersen AG, Baldi P, Chauvin Y, et al. The biology of eukaryotic promoter prediction review[J] .Computers&chemistry. 1999,23:191-120.
[62]Huang RC, Smith MM. Nucleic acid hybridization and the nature of chromosomal protein bound RNA[J]. Results Probl Cell Differ. 1972;3:65-76.
[63]Coulombe B, Burton Z. DNA bending and wrapping around RNA Polymerase: a "revolutionary" model describing transcriptional mechanisms[J]. Microbiol Mol Biol Rev. 1999,63:457-478.
[64] Mu W, Burt DR. Transcriptional regulation of GABAA receptor gamma subunit gene[J]. Brain Res Mol Brain Res.l999,67(1):137-47.
[65]He X, Treacy MN, Simmons DM, et al. Expressiono falarge family of POU-domain regulatory genes in mammalian brain development[J] .Natuer.1989,340 (6228):35-41.
[66]Li L, Suzuki T, Mori N,et al. Identification of a Functional S ilencer Element Involved in Neuron-Specific Expression of the Synapsin Ⅰ[J]. Gene Proceedings of the National Academyof Science. 1993,90:1460-1468.
[67]Sanyal S, Narayanan R, Consoulas C. et al. Evidence for cell autonomous AP1 function in regulation of Drosophila motor-neuron plasticity [J]. BMC Neurosci.2003, 11;4(1): 20.
[68]Groth RD, Mermelstein PG Brain-derived neurotrophic factor activation of NFAT (nuclear factor of activated T-cells)-dependent transcription: a role for the transcription factor NFATc4 in neurotorphin-mediated gene expression[J].J Neuorsc.2003 3;23(22):8125-34.
[69] Soldati D, Boothroyd JC. A selector of transcription initiation in the protozoan parasite Toxoplasma gondii[J]. Mol Cell Biol. 1995,15(1):87-93.
[70]Mercier C, Garber GE, Lecordier L, et al.Common cis-acting elements critical for the expression of several genes of Toxoplasma gondii[J].Mol Microbiol.1996,21(2):421-8.
[71]Rountree MR, Bachman KE, Herman JG. et al. DNA methylation chromat in inheritance and cancer[J]. Oncogene. 2001,20(24):3156-3158.
[72]Siegfired Z, Cedar H. DNA methylation: a molecular lock[J].Curr Biol.1997,7 (5):305-307.
[73]Cross SH, Birda P. CpG islands and genes[J].Curr Opin Genet Dev. 1995,5 (3):309-314.
[74]Birda P. CpG-rich islands and the function of DNA methylation[J].Natuer.1986,32 1(6067): 209-13
[75]Jones PA, Takai D. The role of DNA methylation in mammaliane pigenetics[J].Science. 2001,293(5532): 1068-70.
[76]Kass S, Landsberger N, Wolfea P. DNA methylation directs a time-dependent repression of transcription initiation[J].Curr Biol. 1997,7(3): 157-165.
[77]Zhang Y,Niu Z, Cohena J et al. The internal chondrocyte-specific promoter of the chick type Ⅲ collagen gene is activated by AP1 and is repressed in fibroblasts by a complex containing an LBP1-related protein[J]. Nucleic Acids Res. 1999,27(20):4090-9.
[77]Das G, Hinkley CS, Herr W. Basal promoter elements as a selective determinant of transcriptional activator function [J]. Nature. 1995,374:657-660.
[78]Tuerk C, Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase[J]. Science. 1990, 249 (4968) :5052-51011.
[79]Kim S, Shi H, Lee DK, et al. Specific SR protein dependent splicing substrates identified through genomic SELEX[J]. Nucleic Acids Res. 2003, 31(7): 1955-1961.
[80]Choo Y,Klug A.Toward a code for the interactions of zinc fingers with DNA:selection of randomized fingers disp layed on phage[J].Proc Natl Acad Sci USA.1994,91:11163-11167.
[81]Buly KM,Gentalen E,Lockhart D,et al.Quantifying DNA-protein interactions by double-stranded DNA arrays[J].Nat Biotechnol.1999,17:573-577.
[82]Deplancke B,Dupuy D,Vidal M,et al.Program in genefunction and expression[J].Genome Res.2004,14(10B):2093-2101.
[83]Garner M,Revzin A.A gel electrophoresismethod for quantifying the binding of proteins to specific DNA regions:application to components of the Escherichia coli lactose operon regulatory system[J].Nucleic Acids Res.1981,9:3047-3060.
[84]Rona IZ,Wang Y,Khandur JL,et al.Transcription factor binding study by capillary zoneelectrophoretic mobility shift assay[J].Electrophoresis.2003,24:96-100.
[85]Okuno Y,Huang G,Rosenbure F,et al.Potential auto-regulation of transcription factor PU1 by an up stream regulatory element[J].Mol Cell Biol.2005,25(7):2832-2845.
[86]Molnar G,O'Leary N,Pardee A,et al.Quantification of DNA-protein interaction by UV crosslinking[J].Nucleic Acids Res.1995,23:3318-3326.
[87]Chorley BN,Wang X,Campbell MR,et al.Discovery and verification of functional single nucleotide polymorphisms in regulatory genomic regions:current and developing technologies[J].Mutat Res.2008,659(1-2):147-57.
[88]Geser F,Seppi K,Stampfer-Kountchev M,K(o|¨)llensperger M,et al.The European Multiple System Atrophy-Study Group(EMSA-SG)[J].J Neural Transm.2005,112(12):1677-86.
[89]Veenstra TD.Electrospray ionization mass spectrometry:a promising new technique in the study of protein/DNA noncovalent complexes[J].Biochem Biophys Res Commun.1999,257(1):1-5.
[90]朱玉贤,李毅.现代分子生物.第2版.北京:高等教育出版社.2002:166-170.
[91]Kwon JA,Rho HM.Transcriptional repression of the Human P53 gene by hepatitis B viral core protein(HBc) in human liver cells[J].Bio Chem 2003;384:203-212.
[92]Rho HR, Kwon JA. Hepatitis B viral core protein activities the hepatitis B viralenhancer Ⅱ/pregenomic promoter through the nuclear factor B binding site[J]. Biochem Cell Biol. 2002;80: 445-463.
[93] Johnson JJ, Raney AK, Mclachlan A. Characterization of a functional hepatocyte nuclear factor 3 binding site in the hepatitis B virus nucleocapsid promoter[J].Virology 1995;208:147-158.
[94]Aouacheria A, Navratil V, Lopez-Perez R,et al. In silico whole-genome screening for cancer-related single-nucleotide polymorphisms located in human mRNA untranslated regions[J].BMC Genomics. 2007, 3:8-2.
[95]Garuti R, Croce MA, Piccinini L, Tiozzo R, Bertolini S, Calandra S. Functional analysis of the promoter of human sterol 27-hydroxylase gene in HepG2 cells[J].Gene.2002, 283:133-143.