基因非编码区与转录调控元件的识别研究

随着后基因组时代的到来,非编码区的研究已经成为科学家面临的挑战,对基因非编码区的一个主要研究方向就是对调控元件的研究。识别转录调控元件是理解基因转录机制和表达模式的关键。较全面地介绍了基因非编码区以及调控元件,包括功能和作用,常用识别算法,并对常用数据库进行介绍,提出可能的研究方法和发展方向。     

人Daintain 基因5'调控区序列的生物信息学分析

目的：探讨人Daintain 基因5''调控区的序列特征。方法：利用在线软件BLAST、Neural Network Promoter Prediction、Promoter 2.0、Promoter SCAN、EMBOSS、CpG Island Searcher 和TF SEARCH预测人Daintain 基因启动子区域、CpG 岛分布和转录因子结 合位点。结果：人Daintain 基因5''调控区存在1 个CAAT盒。Daintain 基因可能存在6 个启动子位点,CpG岛可能位于216 bp 区 间( 23 ~ 238 bp)。评分85 分以上时,该序列存在251 个可能的转录因子结合位点;评分90 分以上时,该序列存在70 个可能的转 录因子结合位点;评分95 分以上时,该序列存在16个可能的转录因子结合位点;评分100 分以上时,该序列存在7 个可能的转 录因子结合位点;这些结合的转录因子基本是与免疫细胞增殖或性别发生有关。结论：人Daintain 基因5''调控区的生物信息学研 究表明其转录受甲基化和多种转录因子的调控,为研究Daintain 基因启动子的功能提供理论基础。     

Sox2基因3'非翻译区保守元件对基因表达的调控作用

转录因子Sox2是脊椎动物早期发育中最早表达的神经系统特异性基因之一,同时在干细胞的维持中也起着关键作用。通过生物信息学分析,作者发现在脊椎动物Sox2 mRNA 3'非翻译区中存在4段非常保守的富含AU的区域。将这些片段按照不同的组合克隆到GFP和荧光素酶两种报告基因载体中,在非洲爪蟾胚胎和培养细胞中检测了这些片段对报告基因表达的影响。结果显示,Sox2的3'UTR可影响报告基因的表达水平,特别是其中的保守片段2可显著提高报告基因的表达水平,表明Sox2 3'非翻译区有可能参与Sox2表达的转录后调控。     

人类原钙粘蛋白基因簇调控元件的克隆及对其启动子活性的影响

人类原钙粘蛋白(Protocadherin,Pcdh)基因簇包含53个成串排列非常相似的基因,组成3个紧密相连的基因簇(α,β和γ)。原钙粘蛋白基因簇γ通过启动子选择性表达产生神经元细胞膜表面的分子多样性,但是,该多样性产生的分子机制还不清楚。调控元件HS7L和HS5-1aL作为候选的增强子可能具有调控Pcdhγ基因表达的作用。利用分子克隆的方法,将调控元件HS7L和HS5-1aL分别克隆至包含γa9、γa10、γb3、γb7和γc3启动子的荧光素酶报告基因的下游。通过荧光素酶报告基因试验检测其对该5种Pcdhγ启动子活性的影响,发现HS7L对5种启动子活性具有增强作用,HS5-1aL对γa10启动子活性具有增强作用。之后,通过基因沉默绝缘子CTCF,发现下调CTCF不仅降低γb1基因表达,而且能够显著降低γb1启动子报告基因活性。试验结果表明调控元件HS7L和HS5-1aL能够增强Pcdhγ启动子活性,推测可能通过CTCF介导的增强子-启动子相互作用调控Pcdhγ的细胞特异性基因表达。     

黑曲霉糖化酶基因上游调控区的克隆与分析

目的:通过基因工程方法改造糖化酶基因上游调控区,提高糖化酶产量提供研究。方法:以糖化酶生产菌黑曲霉为材料,通过PCR扩增获得糖化酶基因上游调控区片段PglaA。经测序比对发现该序列与GenBank中编号AM270061的序列相似性为100%。进一步的分析结果表明此序列含有2个保守的激活蛋白结合位点序列CCAAT和1个可能的葡萄糖阻遏位点CT-GGGG。结果:通过不同浓度葡萄糖对糖化酶合成的影响确定该菌株存在葡萄糖阻遏效应,当浓度到到3%以上时阻遏率高达70%以上,为葡萄糖阻遏位点预测结果提供了实验证据,并且经实验测得糖化酶活力随着接种量的增加而增加,当增加到5%时,相对酶活最高。     

油菜APETALA3基因5′调控序列的克隆及分析

利用单引物PCR方法获得了甘蓝型油菜APETALA3重复基因拷贝BnAP3-3和BnAP3-4的5' 调控序列.分析结果表明,两段调控区序列一致性为80.43%,大多数的保守功能元件如TATA盒和CAAT盒没有明显差异,都含有一些应对环境胁迫及诱导的元件,如MYB1AT、GATABOX和DOFCOREZM等.少数顺式作用元件存在较大差异,其中CGACGOSAMY3、CArG box、CARGCW8GAT及CAREOSREP1为BnAP3-3的5'调控序列所特有,而BnAP3-4的5'调控序列则含有GT1CONSENSUS.半定量RT-PCR显示,BnAP3-3表达量远高于BnAP3-4,可能与二者调控区某些功能元件序列差异有关.     

鸡卵清蛋白基因第一内含子和3′-调控区对外源基因表达的调控作用

探明鸡卵清蛋白基因(ov)第一内含子和 3'-调控区对目的基因表达水平的影响,为鸡输卵管表达载体的构建提供科学依据.用牛生长激素基因(BGH)poly A 序列替换鸡输卵管表达载体中 ov 3'-调控区,用限制酶消化法逐步删减第一内含子序列,获得5个表达人组织激肽释放(hK1)cDNA 的鸡输卵管表达载体,分别命名为 pOV2K、pOV3K、pOV4K、pOV5K 和 pOV6K.经翅静脉给产蛋鸡注射聚乙烯亚胺包裹的相同拷贝数重组载体,通过蛋清中酶活性定量检测评价不同载体的表达水平.结果表明:受控于 3.0kb ov 5'-和3'-调控区的 pOV2K 表达的重组酶活性明显高于用 BGH poly A 替换 3'-调控区的pOV3K;无内含子的pOV6K表达水平显著低于舍不同长度第一内含子的pOV3K、pOV4K 和 pOV5K,重组酶表达水平随第一内舍子的缩短呈逐渐下降趋势.该试验结果表明 ov 第一内含子和 3'-调控区对目的基因在鸡榆卵管细胞中的表达具有正调节作用,应当包括在鸡输卵管表达载体中.     

Transposable Elements in the Evolution of Gene Regulatory Networks

Russian Journal of Genetics - Over the past decade, there has been an active study of the interactions between the population of transposable elements (TEs) and the rest of the genome. Many...     

The Main Regulatory Region in the Murine PSP Gene is a Parotid Gland Enhancer

The murine PSP gene is expressed at a high-level in the parotid glands. To extend the knowledge of parotid gland expression and develop tools for expression of heterologous proteins in this tissue, the regulation of the PSP gene was studied using transgenic mice. High-level parotid gland expression of the PSP gene was indicated to depend on a novel regulatory region situated between –8.0 and –6.5 kb. Together with previous results this indicates that the main regulatory elements in the PSP gene are situated between –8.0 to –3.1 kb. This region was shown to activate a heterologous SV40 early promoter in the parotid glands of transgenic mice, suggesting that the PSP gene is controlled by enhancer sequences. A novel Psp derived 9.7 kb parotid gland expression cassette, Lama IV, carrying all known regulatory regions in the PSP gene was expressed at high-levels in the parotid glands and should prove highly useful for expression of heterologous proteins in the saliva of transgenic mice.     

Phase-Specific Elements of the stringGene Regulatory Region in Drosophila melanogaster

The phases of the reporter gene expression controlled by different fragments of the string(stg) gene regulatory region were determined in Drosophilaneuroblasts by detection of -galactosidase activity and radioautography. In the D10 and D22 lines carrying the constructs pstg-E4.9 and pstg-E5.3, respectively, the reporter gene activity was detected in the G1 phase of the cell cycle. In the D12 and D20 lines (pstg-E6.4 and pstg-E2.6), no periodic expression was observed. The regulatory regions of the stgfrom lines D10 and D22 and that of Drosophilagene cyclin Dshared consensus aagaactttg, which was also expressed in the G1 phase. The phase-specific expression of the cell-cycle genes was compared in a model for the mitotic-wave cells of eye imaginal disk and neuroblasts of the nerve ganglia.     

人类连接蛋白基因Cx26的上游调控部位

人类连接蛋白２６（Ｃｏｎｎｅｘｉｎ ２６,Ｃｘ２６）已被看作乳腺癌上皮细胞中的抑瘤基因候选者．为了阐明此基因的调控机理,对其转译起始点上游的一个具有启动功能的１．６ ｋｂ 片段采用ｅｘｏ Ⅲ构建１０个单向删除重组体后进行了ＣＡＴ报告基因分析．结果表明,此１．６ ｋｂ 片段其启动子功能部位位于５′端２００ ｂｐ 范围内．其中含有－ＴＧＴ盒（位于１８２～１８７ ｂｐ）,一个ＴＴＡＡＡＡ 盒位于１５８～１６３ ｂｐ,这是人类Ｃｘ２６基因的又一启动区,这些发现无疑地对了解和阐明Ｃｘ２６基因在乳腺发育过程中及其病理生理作用的复杂调控具有特别重要的意义．     

Crystal Structure of the Src Family Kinase Hck SH3-SH2 Linker Regulatory Region Supports an SH3-dominant Activation Mechanism

Most mammalian cell types depend on multiple Src family kinases (SFKs) to regulate diverse signaling pathways. Strict control of SFK activity is essential for normal cellular function, and loss of kinase regulation contributes to several forms of cancer and other diseases. Previous x-ray crystal structures of the SFKs c-Src and Hck revealed that intramolecular association of their Src homology (SH) 3 domains and SH2 kinase linker regions has a key role in down-regulation of kinase activity. However, the amino acid sequence of the Hck linker represents a suboptimal ligand for the isolated SH3 domain, suggesting that it may form the polyproline type II helical conformation required for SH3 docking only in the context of the intact structure. To test this hypothesis directly, we determined the crystal structure of a truncated Hck protein consisting of the SH2 and SH3 domains plus the linker. Despite the absence of the kinase domain, the structures and relative orientations of the SH2 and SH3 domains in this shorter protein were very similar to those observed in near full-length, down-regulated Hck. However, the SH2 kinase linker adopted a modified topology and failed to engage the SH3 domain. This new structure supports the idea that these noncatalytic regions work together as a “conformational switch” that modulates kinase activity in a manner unique to the SH3 domain and linker topologies present in the intact Hck protein. Our results also provide fresh structural insight into the facile induction of Hck activity by HIV-1 Nef and other Hck SH3 domain binding proteins and implicate the existence of innate conformational states unique to individual Src family members that “fine-tune” their sensitivities to activation by SH3-based ligands.