The TATA-dependent and TATA-independent promoters of the Drosophila melanogaster actin 5C-encoding gene

The major cytoskeletal actin of Drosophila melanogaster, actin 5C, is encoded by a gene (act5C) that has two promoters which are differentially controlled and possess distinct sets of regulatory elements. The distal basal promoter has a TATA motif, but the proximal does not. The distal strong positive domain, centered at nucleotide -290, can be shifted and fused directly to the distal basal promoter without losing its activity. It can also activate heterologous basal promoters containing either TATAAAT or TATTTAA signal when directly fused to them, but cannot activate the basal proximal promoter, which is TATA-less. When the entire distal regulatory region, which includes a remote enhancer-like region, is fused to the proximal promoter, it does not increase the proximal promoter activity. Fusion of the distal strong negative domain to the proximal promoter does not inhibit activity. Thus, all the three major strong regulatory domains of the distal promoter are unable to act on the proximal promoter.     

大肠杆菌yigP基因转录调控序列的鉴定

【目的】调查yigP基因启动子的活性,并对该转录调控序列进行分析。【方法】以lacZ为报告基因,克隆启动子片段至启动子探针质粒中,通过检测β-半乳糖苷酶活性判断启动子活性,并通过克隆一系列逐步缩短的启动子片段来确定启动子所在区域。利用定点突变技术,对启动子的重要序列进行定点突变,调查其对启动子活性的影响。【结果】确定了yigP基因启动子的区域,鉴定了启动子的-10区和-35区,并发现了启动子上游存在一个负调控序列,对该序列进行了初步的研究显示其中部分序列是这种负调控作用的核心序列。【结论】对yigP基因的转录调控序列进行了鉴定,丰富了我们对基因转录调控的认识。     

A single regulatory region modulates both cis activation and trans activation of the herpes simplex virus VP5 promoter in transient-expression assays in vivo.

A detailed analysis of the expression of the bacterial chloramphenicol acetyltransferase gene controlled by the herpes simplex virus major capsid protein (VP5) promoter showed that this promoter can be functionally separated into an 80-base core region, which has the minimal information required to serve as a pol II promoter but which is not fully activated by viral superinfection or by cotransfections with plasmids bearing functional alpha (immediate-early) genes, and an approximately 100-base regulatory region upstream of the core, which allowed full induction of VP5 promoter-driven chloramphenicol acetyltransferase activity but which repressed the ability of the VP5 core promoter to be cis activated by the simian virus 40 enhancer. This was in distinct contrast to the situation with the alkaline exonuclease promoter (a model early promoter) and defined the regions of this promoter which can be used to study the interaction between viral promoters and putative regulatory proteins induced by viral infection.     

Promoter interactions in retrovirus vectors introduced into fibroblasts and embryonic stem cells.

The activity of the Moloney murine leukemia virus promoter is restricted in mouse embryonic stem cells. Gene expression with retrovirus vectors can be achieved in these cells if internal promoters are used. To address the possible influence of the viral enhancer sequences on expression from the internal promoter, we have constructed high-titer, self-inactivating retrovirus vectors which delete viral regulatory sequences upon integration in the host genome. We show that deleting most of the viral enhancer sequences has no significant effect on viral titer. This enhancer deletion leads to either an increase or a decrease in the amount of RNA transcribed from the internal promoter, but no consistent change can be found with any type of vector. The same changes in expression from the internal promoter observed in embryonic stem cells are also observed in 3T3 fibroblast cells, in which the viral promoter is active. These results indicate that viral regulatory elements influence expression from an internal promoter independently of expression from the virus promoter.     

Comparative genomics and regulatory evolution: conservation and function of the Chs and Apetala3 promoters

DNA sequence variations of chalcone synthase (Chs) and Apetala3 gene promoters from 22 cruciferous plant species were analyzed to identify putative conserved regulatory elements. Our comparative approach confirmed the existence of numerous conserved sequences which may act as regulatory elements in both investigated promoters. To confirm the correct identification of a well-conserved UV-light-responsive promoter region, a subset of Chs promoter fragments were tested in Arabidopsis thaliana protoplasts. All promoters displayed similar light responsivenesses, indicating the general functional relevance of the conserved regulatory element. In addition to known regulatory elements, other highly conserved regions were detected which are likely to be of functional importance. Phylogenetic trees based on DNA sequences from both promoters (gene trees) were compared with the hypothesized phylogenetic relationships (species trees) of these taxa. The data derived from both promoter sequences were congruent with the phylogenies obtained from coding regions of other nuclear genes and from chloroplast DNA sequences. This indicates that promoter sequence evolution generally is reflective of species phylogeny. Our study also demonstrates the great value of comparative genomics and phylogenetics as a basis for functional analysis of promoter action and gene regulation.     

不同调控元件驱动下菊欧氏杆菌纤维素酶celY基因在大肠杆菌中表达的研究

目的:以菊欧氏杆菌(Erwinia chrysanthemi)基因组DNA为模板,通过PCR方法找到了该菌的β-1,4-内切葡聚糖酶celY基因及其调控元件并克隆至pUC19载体。为提高纤维素酶celY基因在原核细胞中的分泌表达量,比较了由不同启动子和信号肽调控的celY基因在大肠杆菌中的表达水平。方法:分别构建了由脂蛋白启动子、T7启动子、果胶酶信号肽调控的多种表达载体与由纤维素酶celY基因自身的启动子和信号肽调控的表达载体相比较。结果:由脂蛋白启动子、T7启动子、果胶酶信号肽调控的表达载体都不同程度提高了celY基因的分泌表达量。结论:脂蛋白启动子、T7启动子、果胶酶信号肽都不失为构建强分泌表达载体的可选元件。     

A calcium ionophore-inducible cellular promoter is highly active and has enhancerlike properties.

We examined the regulatory/promoter sequence of a calcium ionophore-inducible gene isolated from the rat genome. Whereas the promoter of this ubiquitously expressed gene is active under noninduced conditions, after induction by calcium ionophore A23187 this promoter is 10- to 25-fold more active than the simian virus 40 early promoter, as measured by chloramphenicol acetyltransferase activities. Within this regulatory/promoter region, we have identified a DNA fragment with enhancer-like properties immediately 5' to the TATA sequence. This 291-nucleotide fragment acts in cis to enhance expression of the neomycin phosphotransferase (neo) gene driven by the herpes simplex virus thymidine kinase promoter in an orientation-independent manner. In addition, this fragment can confer A23187 inducibility to the neo gene and effectively compete for positive regulatory factors involved in A23187 induction. Sequence analysis of this promoter reveals homology with viral core enhancer sequences, and the apparent organization of direct repeat domains is similar to those observed in viral enhancers.     

Analysis of Arabidopsis PsbQ A Gene Expression in Transgenic Tobacco Reveals Differential Role of Its Promoter and Transcribed Region in Organ-specific and Light-mediated Regulation

Arabidopsis PsbQ, encoding a 16 kDa protein of the oxygen-evolving complex, is regulated by light and is expressed preferentially in leaf tissues. To analyze the components required for light-regulated and organ-specific expression of PsbQA, several promoter constructs were generated and expressed in tobacco. The 2.2 kb promoter could confer organ-specific expression of the reporter gene, whereas regulatory elements for light-dependent induction could not be located within this promoter and the transcribed region extending up to a second exon, represented by a genomic fragment encompassing the gene. The genomic fragment representing the transcribed region, however, could confer light regulation even on a constitutive promoter, as observed by steady-state mRNA analysis in T0 and T1 tobacco plants. The results obtained have led to the conclusion that regulatory elements for organ-specificity mainly reside in the promoter region whereas the transcribed region of the gene has an important role in light regulation.