Activity of native vs. synthetic promoters in Brucella

Brucellosis caused by Brucella species is reportedly the most common zoonotic infection worldwide. The bacterial pathogen is also classified by the Centers for Disease Control and Prevention as a category (B) pathogen that has the potential for development as a bioweapon. Although eight genomes of Brucella have been sequenced, little information is available regarding the regulation of gene expression and promoter activity in Brucella spp. We therefore constructed a set of broad-host-range vectors expressing the lacZ reporter gene from various promoters. Four groups of promoters (Brucella native, antibiotic resistant, bacteriophage and synthetic promoters) were tested in vivo and in vitro in Brucella suis. The highest level of heterologous gene expression was achieved with synthetic hybrid trc promoter carrying the adenine-rich upstream element. Furthermore, this demonstrates the usefulness of synthetic promoters for enhanced level of gene expression in Brucella spp.     

Cis-acting regulatory elements: from random screening to quantitative design

The cis-acting regulatory elements, e.g., promoters and ribosome binding sites (RBSs) with various desired properties, are building blocks widely used in synthetic biology for fine tuning gene expression. In the last decade, acquisition of a controllable regulatory element from a random library has been established and applied to control the protein expression and metabolic flux in different chassis cells. However, more rational strategies are still urgently needed to improve the efficiency and reduce the laborious screening and multifaceted characterizations. Building precise computational models that can predict the activity of regulatory elements and quantitatively design elements with desired strength have been demonstrated tremendous potentiality. Here, recent progress on construction of cis-acting regulatory element library and the quantitative predicting models for design of such elements are reviewed and discussed in detail.     

拟南芥中影响基因对胁迫响应模式的顺式元件的挖掘

启动子中关键元件的数量、碱基变化等影响着所调控基因的表达模式。本文基于TAIR数据库的基因组序列和表达谱数据,首先通过BLAST比对和表达模式相关性分析,在拟南芥基因组范围内获得一批启动子序列相似程度很高而下游基因表达模式相反的启动子对,进而借助于PLACE顺式元件库,运用PatSearch软件找到这些启动子对中所含顺式元件种类与数量,并通过Culster聚类,GeneDoc和ContigExpress软件分析,获得了15个能影响胁迫条件下基因表达模式的关键顺式元件,这一发现暗示了生物系统利用较少的调控元件构建稳健与复杂的转录调控系统的方式。     

Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene.

Osteoblasts are cells of mesodermal origin that play a pivotal role during bone growth and mineralization. The mechanisms governing osteoblast-specific gene expression are still unknown. To understand these mechanisms, we analyzed the cis-acting elements of mouse osteocalcin gene 2 (mOG2), the best-characterized osteoblast-specific gene, by DNA transfection experiments in osteoblastic and nonosteoblastic cell lines and by DNA-binding assays. 5' deletion analysis of an mOG2 promoter-luciferase chimeric gene showed that a region located between -147 and -34 contained most if not all of the regulatory elements required for osteoblast-specific expression. Three different binding sites, called A, B, and C, for factors present in nuclear extracts of osteoblasts were identified in this short promoter by DNase I footprint assays. In gel retardation assays, the A element, located between bp -64 and -47, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing primary osteoblasts. The B element, located between bp -110 and -83, bound a ubiquitously expressed factor. The C element, located between bp -146 and -132, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing and mineralizing primary osteoblasts. When cloned upstream of a minimum osteocalcin promoter or a heterologous promoter, multimers of the A element strongly increased the activities of these promoters in osteoblastic cell lines at two different stages of differentiation but in no other cell line; we named this element osteocalcin-specific element 1 (OSE1). Multimers of the C element increased the activities of these promoters predominantly in a differentiated osteoblastic cell line; we named this element OSE2. This study demonstrates that two distinct cis-acting elements are responsible for osteoblast expression of mOG2 and provides for the first time a functional characterization of osteoblast-specific cis-acting elements. We speculate that these two elements may be important at several stages of osteoblast differentiation.     

Vascular-specific expression of the bean GRP 1.8 gene is negatively regulated.

In French bean, the glycine-rich cell wall protein GRP 1.8 is specifically synthesized in the vascular tissue. To identify cis-acting sequences required for cell type-specific synthesis of GRP 1.8, expression patterns of fusion gene constructs were analyzed in transgenic tobacco. In these constructs, the uidA (beta-glucuronidase) gene was placed under control of 5' upstream deletions as well as internal deletions of the GRP 1.8 promoter. Four different cis-acting regulatory regions, SE1 and SE2 (stem elements), a negative regulatory element, and a root-specific element, were found to control the tissue-specific expression. Deletion of the negative regulatory element resulted in expression of the uidA gene in cell types other than vascular cells. The SE1 region was essential for expression in several cell types in the absence of further upstream regulatory sequences. Full-length promoters having insertions between the negative regulatory element and SE1 strongly expressed the gene in nonvascular cell types in stems and leaves. Thus, vascular-specific expression of the GRP 1.8 promoter is controlled by a complex set of positive and negative interactions between cis-acting regulatory regions. The disturbance of these interactions results in expression in additional cell types.     

水稻胚乳特异表达基因的挖掘及顺式元件分析

本研究旨在利用计算机方法对水稻胚乳特异性表达基因进行挖掘和功能分析,及特异性顺式调控元件的预测。我们将基因在不同组织中的表达信号谱看作多维空间内的向量,利用向量夹角余弦法计算其与理想状态下该基因在某一组织特异表达向量的相似度,以此来判断组织特异性表达基因。本文通过对水稻芯片数据的大规模分析,共挖掘出了127个在水稻胚乳中特异表达基因。并对其启动子进行顺式元件预测,发现两个与胚乳特异表达相关的顺式调控元件,其保守序列分别为CATGCATSCM和GATCGATCGR。与已知功能的顺式元件比较显示,前者为种子特异基因表达相关的RY repeat元件,而后者则与元件RNFG1相似,但其具体功能尚不明确。     

Analysis of stress-induced or salicylic acid-induced expression of the pathogenesis-related 1a protein gene in transgenic tobacco.

The cis-acting elements for regulating gene expression of the tobacco pathogenesis-related 1a protein gene were analyzed in transgenic plants. The 5'-flanking 2.4-kilobase fragment from the pathogenesis-related 1a protein gene was joined to the bacterial beta-glucuronidase gene and introduced into tobacco cells by Agrobacterium-mediated gene transfer. Promoter activity was monitored by quantitative and histochemical assay of beta-glucuronidase activity in leaves of regenerated transgenic plants. The level of beta-glucuronidase activity was clearly increased by treatment with salicylic acid, by cutting stress, and by local lesion formation caused by tobacco mosaic virus infection. Cytochemical studies of the induced beta-glucuronidase activity revealed tissue-specific and developmentally regulated expression of the pathogenesis-related 1a gene after stress or chemical treatment and after pathogen attack. To identify the cis-acting element more precisely, a series of 5'-deleted chimeric genes was constructed and transformed into tobacco plants. Transgenic plants with a 0.3-kilobase fragment of the 5'-flanking region of the pathogenesis-related 1a gene had the same qualitative response as those with the 2.4-kilobase fragment upon treatment with salicylic acid or infection with TMV. Thus, the 0.3-kilobase DNA sequence fragment was sufficient to allow the regulated expression of the pathogenesis-related 1a gene.     

Transcription of the chicken histone H5 gene is mediated by distinct tissue-specific elements within the promoter and the 3'' enhancer.

Molecular genetic analysis of a number of vertebrate erythroid cell-specific genes has identified at least two types of cis-acting regulatory sequences which control the complex developmental pattern of gene expression during erythroid cell maturation. Tissue-specific cellular enhancers have been identified 3' to three erythroid cell-specific genes, and additional regulatory elements have been identified in the promoters of many erythroid genes. We show that the histone H5 enhancer, like the adult beta-globin enhancer, is involved in mediating the developmental induction of histone H5 mRNA as erythroid cells mature. We also describe the preliminary characterization of a tissue-specific regulatory element within the 5' region of the H5 locus and describe investigations of the interaction between this element and the histone H5 enhancer in mediating histone H5 regulation.