病原物诱导型植物启动子的研究方法

植物启动子是控制基因转录的DNA序列之一,按其表达方法可分为组成型(或非特异性)表达和诱导型(或特异性)表达启动子两种类型.当受到病原物侵染时,植物通常通过诱导型启动子中的W-box、RAV1 AAT和WRKY等元件/位点、调控下游基因的表达,作出相应的应答反应.近年来,通过计算机预测与试验测定方法的有效结合,发掘和鉴定出了一些病原物诱导型启动子,并进行了调控功能的解析.此外,在通过转基因方法改良植物抗病性的途径中,利用病原物诱导型启动子可以实现对抗病功能相关基因表达的精准控制,避免由组成型启动子持续驱动基因表达、对植株本身带来的负效应.结合本室对水稻启动子OsBTF3-p的研究结果,重点对近年来国内外有关病原物诱导型启动子及其调控元件/位点的研究方法和应用进展作一综述.     

泛素载体蛋白Rad6在基因表达调控及DNA损伤修复中的作用

泛素化修饰是蛋白质的一种重要的翻译后水平修饰,而且有着多种不同的生物学功能,对蛋白质的结构与功能、基因表达调控以及蛋白质-蛋白质/其它分子相互作用等多个方面有着重要的调控作用。Rad6即是酵母中的一种重要的泛素载体蛋白。Rad6通过泛素化修饰多种靶蛋白在DNA的损伤修复中发挥着重要作用。文章重点讨论了Rad6在DNA损伤修复方面的功能以及在正常情况下对染色质结构和基因表达调控的影响。     

SnRNA对基因表达的调控

基因表达的调控机制研究是生物学中十分活跃的领域,基因重组技术对基因的分离和结构分析的报道不胜枚举,其目的在于阐明基因表达的调控机理;在原核生物中,已发现蛋白质作为基因表达调节因子,因此,大部分研究者注重于蛋白质分子在基因表达中的调控作用,对于DNA分子不重视,只是把它看作是基因表达过程中的过渡阶段如mRNA、tRNA、rRNA。近年来发现RNA具有酶功能、参予mRNA成熟过程中的拼接、DNA复制、染色质结构及基因表达的调控,种类繁多,已引起了生物学者的关注。     

基因表达调控中的核因子作用

利用病毒和动物系统对基因表达调控进行了广泛和深入的研究,发现了顺式作用调节序列,鉴定了序列专一的DNA结合蛋白,DNA与蛋白质相互识别、结合及蛋白质与蛋白质相互作用中起作用的蛋白质结构域,并且对调节蛋白基因的克隆和序列进行了分析．基因表达调控领域又由于植物基因调控机制取得的发展而得到了补充,文章着重介绍植物基因中的DNA与蛋白质间的作用;植物调节蛋白基因的分离;这一领域的今后研究方向及展望．     

植物诱导型启动子及相关顺式作用元件研究进展

启动子是基因表达调控的重要元件,而启动子能正确调控基因的表达需要核心启动子以及上下游的顺式作用元件协同作用。诱导型启动子的应用减少了外源基因表达时产生的大量异源蛋白等代谢产物的积累和植物能量的浪费。综合近几年关于诱导型启动子的研究,从其启动子的结构及功能、分类、应用及相关顺式作用元件等方面进行概述,提出了在研究过程中存在的问题及展望。     

研究核酸-蛋白质系统的-种凝胶电泳方法

核酸蛋白质相互作用是生命科学研究的中心课题之一,凝胶电泳是研究核酸和蛋白质的常用技术。本文试图介绍一种研究核酸-蛋白质系统的凝胶电泳方法,以及该法在研究基因表达、调控和鉴定、分离DNA结合蛋白中的应用。     

康氏木霉中调控纤维素酶形成的两个调控因子的克隆及功能性分析

锌指蛋白ACEI和Xyrl是里氏木霉中调控纤维素酶和木聚糖酶形成的两个调控因子,它们能竞争性结合木聚糖酶基因xynl的启动子.为进一步研究ACEI和Xyr1调控纤维素酶基因表达的机制,本研究利用PCR技术扩增康氏木霉ACEI和Xyr1 DNA结合区的基因序列,并使其在大肠杆菌中得到表达.凝胶迁移率移动试验表明ACEI和Xyrl的DNA结合区均可与纤维素酶基因cbh1启动子的287 bp序列特异性结合.提示ACEI与Xyr1不仅能竞争性结合xynl启动子,也能竞争性结合cbhl启动子.     

MeCP2在Rett综合征中的调控机制

Rett综合征(Rett syndrome, RTT)是一种X连锁的神经发育障碍性遗传病, 是导致女性严重智力障碍的主要原因之一。编码甲基化CpG结合蛋白2(Methyl-CpG-binding protein 2, MeCP2)基因突变是RTT主要的遗传病理学改变, MeCP2作为转录抑制因子调控基因表达。在RTT发病机制中, 由于缺乏MeCP2与甲基化DNA的正确结合, 阻碍了它对下游靶基因表达的正常调控, 最终导致脑功能障碍。目前, 对MeCP2在脑发育过程中的作用以及如何导致RTT的发生, 其机制尚不清楚。文章从MECP2基因和MeCP2蛋白两个方面, 对基因结构、蛋白质功能以及在分子水平上的调控机制进行了综述, 以期为RTT的发病机制研究提供新思路。     

细菌RNA结合蛋白Hfq的DNA压缩和复制作用研究进展

RNA结合蛋白(RNA-Binding Protein)Hfq是一种重要的细菌转录后调节因子,之前对Hfq的研究大多集中在该蛋白对小分子非编码RNA (Small Non-Coding RNA,sRNA)和mRNA的作用上。Hfq最典型的功能是促进sRNA与其靶标mRNA碱基配对,在转录后介导对RNA的稳定性和翻译的调控。此外,Hfq也能与多种蛋白质直接或间接相互作用。然而,近年来的研究表明,除了RNA和蛋白质,Hfq还可以与DNA相互作用,在DNA压缩(DNA Compaction)和DNA复制(DNA Replication)等多种DNA代谢过程中发挥直接或间接的调控作用。额外的靶标和功能的鉴定将进一步夯实Hfq作为细菌中多种代谢途径核心调控因子的重要地位,也表明该蛋白的功能并不局限于其在RNA和蛋白质代谢中的作用。本文总结了Hfq在DNA代谢调控中的近几年最新研究进展,并展望了其前景。     

转移核糖核酸(tRNA)与癌症

转移核糖核酸(transfer RNA, tRNA)在蛋白质生物合成过程中起关键作用,是将遗传信息翻译成蛋白质一级结构的接头分子。tRNA长久以来一直被认为是基因表达调控过程中的执行者而不具备调控功能,更不曾与癌症的发生联系起来。最新研究表明,某些tRNA在癌细胞中异常表达,与癌症的发生和发展有密切联系。tRNA来源的小分子非编码RNA (tRFs和tiRNAs)是一类新的基因表达调控分子,tRFs可以调控癌基因的表达或者与RNA结合蛋白相互作用来调控癌细胞增殖和细胞周期进程。tRNA的转录后修饰能够调控mRNA翻译过程,进而影响癌细胞的生长。随着测序技术的发展,tRNA在癌症发生和发展中的调控作用成为近年来的研究热点,现将从"tRNA分子调控癌症的发生和发展"、"tRNA来源的小分子非编码RNA与癌症"以及"tRNA修饰与癌症"三个方面综述tRNA分子在癌症发生和发展中的调控功能。     

Identification of a nuclear protein interacting with a novel site on rat androgen receptor promoter after transcription factor NFkB is displaced from adjacent site

Sequence-specific DNA-protein interactions mediate the regulation of rat androgen receptor (rAR) gene expression. Previously, DNase I footprinting revealed that nuclear factor kappa B (NFkB) binds to region -574 to -554 on rAR promoter and represses its expression. In this study, we demonstrate that when NFkB protein is removed from its site by competitor DNA in DNase I footprinting reaction, a new DNase I protected region is formed overlapping adjacently (-594 to -561). This indicates that another nuclear protein (named here as FRN, factor repressed by NFkB) binds to rAR promoter only after NFkB protein is displaced. By competitive electrophoretic mobility shift assay and mutation analysis, we confirmed the formation of FRN-DNA complex. FRN interacts with a novel sequence on rAR promoter and may play a role in regulation of rAR gene expression in concert with NFkB.     

cis elements that control the expression of chick aggrecan

Aggrecan is a large chondroitin sulfate proteoglycan whose expression is both cell-specific and developmentally regulated. Cloning and sequencing of the 1.8-kilobase genomic 5'-flanking sequence of the chick aggrecan gene revealed the presence of potential tissue-specific control elements including a consensus sequence found in the cartilage-associated silencers, CSIIS1 and CSIIS2, that were first characterized in the type II collagen promoter sequences, as well as numerous other cis elements. Transient transfections of chick sternal chondrocytes and fibroblasts with reporter plasmids bearing progressively deleted portions of the chick aggrecan promoter and enhancer region demonstrated cell type-specific promoter activity and identified a 420-base pair region in the genomic 5-flanking region responsible for negative regulation of the aggrecan gene. In this report, three complementary methods, DNase I footprinting assays, transient transfections, and electrophoretic mobility shift assays (EMSA), provided an integral approach to better understand the regulation of the aggrecan gene. DNase I footprinting revealed that six regions of this genomic sequence bind to nuclear proteins in a tissue-specific manner. Transient transfection of reporter constructs bearing ablations of these protected sequences showed that four of the six protected sequences, which contain the sequence TCCTCC or TCCCCT, had repressor activities in transfected chick chondrocytes. Cross-competition EMSA using nuclear protein extracted from chondrocytes or fibroblasts explored the contributions of the different sequence elements in formation of DNA-protein complexes specific to cell type. This is the first parallel examination of the EMSA patterns for six functionally defined cis elements with highly similar sequences, using protein from primary cultured cells.     

Homeodomain transcription factor Hesx1/Rpx occupies Prop-1 activation sites in porcine follicle stimulating hormone (FSH) beta subunit promoter

Homeodomain repressor factor Hesx1/Rpx plays a crucial role in the formation of Rathke's pouch at the start of pituitary organogenesis and represses the Prop-1-dependent expression of Pit-1 gene, which promotes the differentiation of Pit-1-dependent hormone producing cells. Recently, we discovered a novel function of Prop-1 by which it activates the porcine follicle stimulating hormone beta subunit (FSHbeta) gene through Fd2 region (-852/-746). The present study aimed to determine whether Hesx1 exerts its role in the Prop-1-dependent activation of FSHbeta gene. Transient transfection assay for the porcine FSHbeta promoter -985/+10, electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis for Fd2 region were carried out. Transfection assay in GH3 cells demonstrated that expression of Hesx1 alone does not change the promoter activity but the coexpression with Prop-1 represses the Prop-1-dependent activation of FSHbeta promoter. Similar results were obtained for the mutant reporter vector deleting the region -745/-104 indicating that Fd2 region is a target site of Hesx1 as well as Prop-1. EMSA and DNase I footprinting analysis using recombinant Hesx1 and Prop-1 protein demonstrated that Hesx1 and Prop-1 certainly bind to the AT-rich regions in a different manner. These results suggest that Hesx1 blocks the advanced expression of FSHbeta gene in the early stage of pituitary development, and Prop-1 thereafter appears and activates this gene.     

The interplay of ubiquitous DNA-binding factors, availability of binding sites in the chromatin, and DNA methylation in the differential regulation of phosphoenolpyruvate carboxykinase gene expression.

We have identified DNA elements in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter which are bound 'in vivo' by proteins under conditions of basal level gene expression and have evaluated several hypothesis to account for the tissue specific expression of the gene. In vitro DNase I footprinting demonstrated that factors which bind to basal expression elements of the PEPCK promoter, the BSE/CRE and NFI/CCAAT sites, are also present in HTC and XC cells which do not express the PEPCK gene. 'In vivo' DNase I footprinting demonstrated that the BSE/CRE, NFI/CCAAT, and three additional sites are bound by protein in H4IIE cells which express the PEPCK gene but not in the HTC or XC cells. No evidence for a repressor protein or for phased nucleosome binding to the PEPCK promoter in HTC or XC cells could be detected. Genomic sequencing was used to determine if differential methylation of the PEPCK promoter could account for the lack of factor binding in HTC and XC nuclei. None of the 14 cytosine residues in CpG dinucleotides was methylated in H4IIE or rat liver DNA, all were methylated in rat sperm DNA, and 6 were methylated in HTC DNA; including the cytosine at position--90 within the BSE/CRE. Only one cytosine residue, at position--90, was methylated in XC DNA. Treatment of XC cells with 5-azacytidine resulted in loss of methylation at the--90 position yet this was insufficient to allow synthesis of a detectable amount of PEPCK mRNA.     

Interpolated markov chains for eukaryotic promoter recognition.

MOTIVATION: We describe a new content-based approach for the detection of promoter regions of eukaryotic protein encoding genes. Our system is based on three interpolated Markov chains (IMCs) of different order which are trained on coding, non-coding and promoter sequences. It was recently shown that the interpolation of Markov chains leads to stable parameters and improves on the results in microbial gene finding (Salzberg et al., Nucleic Acids Res., 26, 544-548, 1998). Here, we present new methods for an automated estimation of optimal interpolation parameters and show how the IMCs can be applied to detect promoters in contiguous DNA sequences. Our interpolation approach can also be employed to obtain a reliable scoring function for human coding DNA regions, and the trained models can easily be incorporated in the general framework for gene recognition systems. RESULTS: A 5-fold cross-validation evaluation of our IMC approach on a representative sequence set yielded a mean correlation coefficient of 0.84 (promoter versus coding sequences) and 0.53 (promoter versus non-coding sequences). Applied to the task of eukaryotic promoter region identification in genomic DNA sequences, our classifier identifies 50% of the promoter regions in the sequences used in the most recent review and comparison by Fickett and Hatzigeorgiou ( Genome Res., 7, 861-878, 1997), while having a false-positive rate of 1/849 bp.