A new circadian class 2 gene, opcA, whose product is important for reductant production at night in Synechococcus elongatus PCC 7942

Gene expression in the cyanobacterium Synechococcus elongatus PCC 7942 is under the control of a circadian oscillator, such that peaks and troughs of expression recur with a periodicity of about 24 h in the absence of environmental cues. This can be monitored easily as light production from luciferase gene fusions to S. elongatus promoters. All promoters seem to exhibit circadian oscillation of expression, but the phasing of peak and trough times differs among different genes. The majority of genes are designated class 1, with expression peaks near dusk or subjective dusk (the time corresponding to dusk in the absence of a diurnal cycle). A minority, of which purF is an example, have expression peaks approximately 12 h out of phase with class 1 genes. A screen of Tn5 mutants for those in which purF phasing is altered revealed a mutant that carries an insertion in the opcA gene, previously identified as essential for glucose-6-phosphate dehydrogenase function. However, a different enzymatic reporter and in vitro luciferase assays revealed that the expression pattern of the purF promoter is not altered by opcA inactivation, but rather the reduced flavin mononucleotide substrate of luciferase is limiting at the time of the natural circadian peak. The results suggest that OpcA is involved in temporally separated reductant-generating pathways in S. elongatus and that it has a role outside of its function in activating glucose-6-phosphate dehydrogenase. The opcA gene, expected to be cotranscribed with fbp and zwf, was shown to have its own class 2 promoter, whereas the fbp promoter was determined to be in class 1. Thus, opcA expression is likely to be constitutive by virtue of the activity of two promoters in nearly opposite circadian phases.     

Functional dissection of an early Drosophila chorion gene promoter: expression throughout the follicular epithelium is under spatially composite regulation.

We have fused various DNA sequences located upstream of the Drosophila melanogaster s36 chorion gene TATA box to a heterologous basal promoter and reporter gene (hsp70/lacZ). The expression of these constructs, following P-element-mediated germline transformation, was examined in 144 independent lines by histological staining of dissected ovaries for beta-galactosidase activity. A short 84 bp segment of the proximal 5' flanking DNA was sufficient to confer a wild-type gene expression pattern, including temporal specificity for early choriogenic follicles. Surprisingly, initial expression was very localized at the anterior and posterior poles of the follicle. The downstream half of that DNA segment permitted expression at both poles, but especially at the anterior tip, while the upstream half only favored expression in the posterior pole; these results suggested the existence of multiple, spatially specific cis-regulatory elements. When the proximal 84 bp segment was placed 1.5 kb upstream of the basal promoter, beta-galactosidase activity was observed in an altered spatial pattern, indicating that the cis-regulatory element(s) that favor expression in the posterior half of the follicle are position independent, while the element(s) that favor expression elsewhere in the follicle are position sensitive. A distal regulatory segment containing redundant DNA element(s) specific for expression in the anterior pole was identified much further upstream of s36. Thus, the expression of this chorion gene throughout the follicular epithelium is actually composite, occurring in distinct spatial domains under the control of corresponding DNA elements.     

几种肿瘤细胞中ezrin基因增强子区转录调控特性的研究

该文采用Western blot技术检测人食管癌EC109细胞、鼻咽癌CNE2细胞和宫颈癌HeLa细胞Ezrin蛋白的表达：采用DNA片段定向克隆技术构建一系列携带ezrin基因增强子区-1541／-706序列的报告基因表达载体,将载体瞬时转染EC109、CNE2和HeLa细胞,检测荧光素酶活性;研究肿瘤细胞中ezrin基因增强子区的转录调控特性。实验结果显示,在被检测的三种肿瘤细胞中,Ezfin蛋白的表达水平没有明显不同。Ec109细胞中,当ezrin基因-1541／-706N段正向位于无启动子的报告基因上游时,表现出类似启动子的转录激活作用：当这一片段反向连接时转录激活作用几乎消失。当-1541／-706片段正向位于ezrin启动子或SV40启动子上游时,显著增强荧光素酶表达;然而,当这一片段反向位于启动子上游以及正向或反向位于启动子控制的报告基因下游时,转录增强作用消失。ezrin基因-1541／-706N段在CNE2和HeLa细胞中的转录调控作用,与其在EC109细胞中的转录调控作用部分相似,但不完全相同。结果表明,ezrin基因增强子区具有转录激活和转录增强双重作用,这种作用具有DNA序列位置和方向依赖性以及细胞特异性。     

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。     

Positive and negative DNA elements of the Drosophila grimshawi s18 chorion gene assayed in Drosophila melanogaster.

Germ line transformation has been used to map the cis regulatory DNA elements responsible for the precise and evolutionarily stable developmental expression of the s18 chorion gene. Constructs containing chimeric combinations of Drosophila melanogaster and D. grimshawi DNA regions, as well as D. grimshawi sequences alone, can direct expression in the follicular epithelium, in an s18-specific temporal and spatial pattern. The results indicate that both positive and negative regulatory elements can function when transferred from D. grimshawi to D. melanogaster. The first ca. 100 bp of the 5'-flanking DNA region constitute a minimal, developmentally regulated promoter, expression of which is inhibited by the next 100-bp DNA segment and activated by positive elements located further upstream. Expression of the minimal promoter can also be enhanced by more distant chorion regulatory elements, provided the inhibitory DNA segment is absent.     

骨形成蛋白诱导型真核载体的构建及其表达

成骨分化相关基因骨钙素 (OC)等的启动子内均含有成骨特异性转录因子Cbfa1特异性作用元件 ,而骨形成蛋白 (bonemorphogeneticprotein ,BMP)的促成骨分化作用正是通过其首先引起Cbfa1的升高 ,而后Cbfa1激活这些基因的表达 ,最终出现成骨分化表型 .为解决BMP没有理想的活性测定方法的问题 ,在RT PCR结果证实BMP 2可促进NIH3T3和C2C12细胞Cbfa1表达后 ,构建了串联6个Cbfa1作用元件的小鼠OC部分启动子 (6OCP)控制萤光素酶 (luciferase)报告基因的真核表达质粒 ,以期来放大BMP诱导报告基因表达的作用效果 .即通过细胞转染、rhBMP 2刺激后检测萤光素酶活性变化 ,从而间接定量测定rhBMP 2的生物学活性 .结果表明 ,pcDNA3 6OCP Luc转染细胞后其报告基因的基础活性较pcDNA3 Luc大为降低 ;而且在一定剂量范围内 ,转染细胞的萤光素酶活性 (荧光值 )随rhBMP 2剂量增加而升高 ,并呈线性正相关 ,为建立BMP活性定量测定的方法打下基础     

A potent far-upstream enhancer in the mouse pro alpha 2(I) collagen gene regulates expression of reporter genes in transgenic mice

We have identified three DNase I-hypersensitive sites in chromatin between 15 and 17 kb upstream of the mouse pro alpha 2 (I) collagen gene. These sites were detected in cells that produce type I collagen but not in cells that do not express these genes. A construction containing the sequences from -17 kb to +54 bp of the mouse pro alpha 2 (I) collagen gene, cloned upstream of either the Escherichia coli beta- galactosidase or the firefly luciferase reporter gene, showed strong enhancer activity in transgenic mice when compared with the levels seen previously in animals harboring shorter promoter fragments. Especially high levels of expression of the reporter gene were seen in dermis, fascia, and the fibrous layers of many internal organs. High levels of expression could also be detected in some osteoblastic cells. When various fragments of the 5' flanking sequences were cloned upstream of the 350-bp proximal pro alpha 2(I) collagen promoter linked to the lacZ gene, the cis-acting elements responsible for enhancement were localized in the region between -13.5 and -19.5 kb, the same region that contains the three DNase I-hypersensitive sites. Moreover, the DNA segment from -13.5 to -19.5 kb was also able to drive the cell-specific expression of a 220-bp mouse pro alpha 1(I) collagen promoter, which is silent in transgenic mice. Hence, our data suggest that a far-upstream enhancer element plays a role in regulating high levels of expression of the mouse pro alpha 2(I) collagen gene.