酵母基因上游序列中潜在的转录正调控位点分析

前期研究表明,高效转录酵母基因内含子在序列长度、寡核苷酸使用、以及位置分布等方面都有着区别于低转录内含子的特征 . 进一步观察发现：上游基因间区域的序列长度与基因转录频率也有与内含子序列相同的现象,转录频率高的上游基因间序列一般都比转录频率低的长 . 对高效转录和低效转录上游基因间序列的寡核苷酸使用频率进行统计比较分析,抽提出高转录基因上游区可能的转录正调控元件 . 与酵母的所有非编码序列比较,这些可能的正调控元件基本上也是过表达的 (over-represented) ,其中多数和实验所得的一些位点特征相吻合 . 这些元件富含 G 、 C ,这与内含子中可能的正调控元件在碱基组成上有一定的互补性 . 从这些特征看,高效转录基因上游的序列结构确实有利于基因的转录 .     

骨骼肌特异表达载体的构建与验证

目的:构建肌肉特异性表达线虫ω-3脂肪酸去饱和酶的肌肉特异性载体,证明其表达的有效性.方法:化学合成292bp的带有肌肉特异性转录因子结合位点的DNA序列,然后与线虫ω-3脂肪酸去饱和酶基因cDNA、去除CMV启动子的表达载体pcDNA3.1连接构成肌肉特异性表达载体pcDNA3.1 - SF,4w龄C57BL/6小鼠肌肉注射该重组载体,RT - PCR检测证明其表达有效性.结果:DNA测序结果证明合成的292 bp片段带有肌肉特异性表达元件;PCR鉴定显示启动子片段正确地插入到目的基因上游;RT - PCR鉴定显示肌肉内ω-3脂肪酸去饱和酶基因得到有效转录.结论:该试验为在转基因动物的肌肉中表达外源基因奠定了基础.     

肌肉特异性基因启动子的上游转录调控元件研究进展

真核生物启动子位于基因5’端上游转录起始位点附近,是包含核心启动子以及上游转录调控元件的一段DNA序列,这些转录调控元件控制着基因表达的强度和特异性。肌肉特异性启动子的上游调控元件种类、数量和排列顺序决定着基因在肌肉中的特异性表达。深入研究肌肉启动子的上游调控元件,可以进一步了解肌肉基因表达机制,从而为肌肉性状的改良、增殖分化的机理和疾病的基因治疗等研究提供重要依据。该文回顾了近年来肌肉特异性启动子研究领域中的新发现,包括肌肉特异性启动子转录调控元件的分子机制、建立人工合成肌肉启动子的方法及应用,并探讨该领域中急需解决的问题和发展前景。     

神经系统特异表达基因调控机理的研究进展

神经系统特异性基因正确的时空表达受细胞内外信号的调控,信号传导途径最终的靶位点是能结合特异转录因子的DNA序列.目前发现的决定神经系统基因特异性表达的顺式作用元件既有增强子,也有沉默子.它们可以特异性地增强基因在神经系统的表达,或特异性抑制基因在非神经系统的表达. 顺式元件要发挥这些作用,依赖于与其结合的反式因子,而这些反式因子又能与其他蛋白质或DNA序列发生互动, 通过协调作用,共同决定基因的时空表达顺序.     

植物非生物胁迫诱导启动子顺式元件及转录因子研究进展

顺式作用元件(cix-acting element)是与结构基因串联的特定DNA序列,是转录因子的结合位点,它们通过与转录因子结合调控基因转录的精确起始和转录效率,在植物基因表达调控过程中起着重要的作用.非生物胁迫诱导基因的表达受其上游启动子顺式作用元件及转录因子的调控,目前已发现了多种与非生物胁迫相关的顺势作用元件及转录因子,如DRE元件及DREB类转录因子、MYB元件及MYB类转录因子、GT-1元件及GT-1类转录因子等.顺式作用元件及转录因子的研究对研究植物非生物胁迫相关基因的表达调控具有重要意义,综述植物非生物胁迫诱导启动子功能元件及转录因子的研究进展.     

单纯疱疹Ⅰ型病毒即刻早期基因上游调控序列分析

感染人体的单纯疱疹病毒(HSV)在宿主细胞中,以级联反应的方式表达病毒蛋白.作为最早表达的立即早期基因,其上游都具有相似的调控序列.通过α4基因上游转录相关元件的依次递减,以CAT作为报告基因,评估了在病毒感染细胞早期阶段中,各DNA序列对立即早期基因转录所发挥的可能作用.实验数据表明,HSV立即早期基因在细胞中的表达受到多种元件的共同调控,这些成簇分布的调控元件的排列分布将为深入了解HSV的复制周期提供有益的线索.     

POLD1基因启动子中CDE/CHR元件的鉴定及功能分析

POLD1基因编码真核生物DNA聚合酶δ的催化亚基,其表达调控与细胞周期密切相关.为了探讨POLD1基因表达的调控机理,本研究首次在POLD1基因启动子中鉴定出CDE/CHR元件,随后通过分析元件序列定点突变对其转录活性的影响,以及与细胞周期相关的转录因子E2F和CDK及抑制因子p21WAF1/Cip1对其转录活性的调控作用,对此元件的功能进行了分析.结果显示,CDE/CHR元件序列突变后POLD1基因启动子转录活性明显升高,其转录活性不再受到E2F和p21WAF1/Cip1的调控,转录活性的细胞周期依赖性调控消失.与此同时本研究对直接参与CDE/CHR元件调控的蛋白进行了初步检测,结果显示,至少存在3种蛋白复合体能够与POLD1基因启动子中的CDE/CHR元件结合.由此证实,POLD1基因启动子中存在CDE/CHR元件,此元件与POLD1基因转录的细胞周期依赖性调控直接相关.     

香菇顺式调控元件的克隆及其序列分析*

应用顺式调控元件探测载体G221构建了一个香菇Lentinula edodes基因组文库。G221为大肠杆菌-酿酒酵母穿梭载体,含有一个由酵母Cyc1基因基本启动子控制的lacZ标记基因,能以转录增强活性筛选香菇DNA片段。用这个基因组文库转化酵母菌,获得了一批lacZ阳性转化子。对其中表达较强的阳性转化子进行质粒抽提和双酶切鉴定,筛选到50个香菇顺式调控元件DNA片段。对其中部分片段进行了测序,并对其中一个序列进行了序列分析,鉴别了该序列上的几个与转录相关的特征序列。该研究也探讨了利用酵母表达系统克隆香菇顺式调控元件的可行性。     

香菇顺式调控元件的克隆及其序列分析

应用顺式调控元件探测载体G221构建了一个香茹Lentinula edodes基因组文库。G221为大肠杆菌-酿酒酵母穿梭载体,含有一个由酵母Cycl基因基本启动子控制的lacZ标记基因,能以转录增强活性筛选香茹DNA片段。用这个基因组文库转化酵母菌,获得了一批lacZ阳性转化子。对其中表达较强的阳性转化子进行质粒抽提和双酶切鉴定,筛选到50个香茹顺式调控元件DNA片段。对其中部分片段进行了测序,并对其中一个序列进行了序列分析,鉴别了该序列上的几个与转录相关的特征序列。该研究也探讨了利用酵母表达系统克隆香茹顺式调控元件的可行性。     

沙冬青AmCIP基因结构及其内含子生物信息学分析

旨在分析沙冬青Am CIP基因DNA序列结构特性及功能。以沙冬青Am CIP基因的c DNA序列设计引物,采用PCR扩增其DNA序列,并利用生物信息学方法分析基因的结构。序列分析表明,沙冬青Am CIP基因DNA序列长1 548 bp,包含2个外显子和1个内含子,Gen Bank登录号KU744005。在该基因的ORF内存在一个78 bp的内含子序列,同时内含子序列中含有参与厌氧诱导的类增强子元件(GC-motif)和光响应元件(TCCC-motif),3'-UTR区域含有多个参与光响应、胁迫响应和激素响应等相关的顺式作用元件,而5'-UTR区域只含有赤霉素响应元件(GARE-motif),这些元件可能对Am CIP基因转录表达起调控作用。此外,该基因具有较高的A+T碱基含量(64.7%),Am CIP的内含子不具有GT-AG规则的保守序列,即内含子5'碱基是GT,3'碱基是TG,推测Am CIP的m RNA具有独特的剪切机制。     

Genomic cis-regulatory architecture and trans-acting regulators of a single interneuron-specific gene battery in C. elegans

Gene batteries are sets of coregulated genes with common cis-regulatory elements that define the differentiated state of a cell. The nature of gene batteries for individual neuronal cellular subtypes and their linked cis-regulatory elements is poorly defined. Through molecular dissection of the highly modular cis-regulatory architecture of individual neuronally expressed genes, we have defined a conserved 16 bp cis-regulatory motif that drives gene expression in a single interneuron subtype, termed AIY, in the nematode Caenorhabditis elegans. This motif is bound and activated by the Paired- and LIM-type homeodomain proteins CEH-10 and TTX-3. Using genome-wide phylogenetic footprinting, we delineated the location, distribution, and evolution of AIY-specific cis-regulatory elements throughout the genome and thereby defined a large battery of AIY-expressed genes, all of which represent direct Paired/LIM homeodomain target genes. The identity of these homeodomain targets provides novel insights into the biology of the AIY interneuron.     

Multiple regulatory elements with spatially and temporally distinct activities control neurogenin1 expression in primary neurons of the zebrafish embryo

The basic Helix-Loop-Helix gene neurogenin1 (ngn1) is expressed in a complex pattern in the neural plate of zebrafish embryos, demarcating the sites of primary neurogenesis. We have dissected the ngn1 locus to identify cis-regulatory regions that control this expression. We have isolated two upstream elements that drive expression in precursors of Rohon-Beard sensory neurons and hindbrain interneurons and in clusters of neuronal precursors in the anterior neural plate, respectively. A third regulatory region mediates later expression. Thus, regulatory sequences with temporally and spatially distinct activities control ngn1 expression in primary neurons of the zebrafish embryo. These regions are highly similar to 5' sequences in the mouse and human ngn1 gene, suggesting that amniote embryos, despite lacking primary neurons, utilize related mechanism to control ngn1 expression.     

Regulatory elements from the related spec genes of Strongylocentrotus purpuratus yield different spatial patterns with a lacZ reporter gene.

The Spec1 and Spec2 genes of Strongylocentrotus purpuratus are closely associated with the differentiation of aboral ectoderm. To examine cis-regulatory elements involved in the spatial expression of the Spec genes, we fused the Escherichia coli lacZ gene containing a nuclear targeting signal to 5'flanking DNA plus 5' untranslated leader sequences from Spec1, Spec2a, and Spec2c. All three genes contain 700 bp of highly conserved DNA in their upstream regions, but in Spec1 and Spec2c large insertions interrupt the conserved regions. The Spec-lacZ reporter gene plasmids were microinjected into eggs of S. purpuratus, Lytechinus variegatus, and L. pictus, and beta-galactosidase activity was determined in situ by X-gal staining. The Spec2a-lacZ fusion gene, which contained 1516 bp of 5' flanking DNA and 18 bp of 5' untranslated leader sequence, was preferentially expressed in aboral ectoderm cells in all three species. The Spec1-lacZ fusion gene was expressed in a strikingly different fashion--preferentially in primary and secondary mesenchyme cells, occasionally in aboral ectoderm cells, and less often in oral ectoderm and endoderm cells. The staining pattern was the same in either homologous or heterologous embryos. The Spec2c-lacZ fusion gene, like Spec2a-lacZ, was preferentially expressed in aboral ectoderm, but staining of other cell types was frequently observed. To further delineate sequences required for correct spatial expression, we deleted 800 bp of 5' flanking DNA from the Spec2a-lacZ fusion gene, resulting in a delta Spec2a-lacZ fusion gene that contained only the conserved DNA region. This gene fusion showed preferential expression in aboral ectoderm cells. However, the cell type specificity was not as great as with the parental Spec2a-lacZ plasmid. These experiments implied that the conserved DNA region, associated with all Spec genes examined, was insufficient for complete aboral ectoderm specificity, and suggested that a spatial repressor element existed between -1516 and -697 bp in the 5' flanking DNA of Spec2a.