Several copies of insulator from MDG4 can determine the interaction between positively and negatively acting regulatory elements and promoter of the miniwhite gene of Drosophila melanogaster

Fine regulation of complex gene loci in higher eukaryotes is realized through the interaction of promoters with enhancers and repressors, which can be located long distance from the promoter regulated. A question arises, what mechanisms determine proper contacts between the regulatory elements over large distances in the genome. It is suggested that the important role in this process is played by a special class of regulatory elements, insulators, which block the interaction of enhancer and promoter, if they are positioned between them. Furthermore, enhancers do not directly inactivate the activities of enhancer and promoter. Nevertheless, an enhancer, isolated from one of the promoters by an insulator, can activate another, not isolated promoter. The best studied insulator of Drosophila melanogaster was found in the 5′ regulatory region of retrotransposon MDG4. It consists of 12 binding sites for the Su(Hw) protein, which is critical for the activity of this insulator. It was demonstrated that Su(Hw) insulator could protect the gene expression from the negative influence of heterochromatin and from repression, induced by the Polycomb group proteins (Pc proteins). In the present study, it was demonstrated that in transgenic lines, two or three copies of the Su(Hw) insulator could determine the interaction of the miniwhite enhancer and Pc dependant silencer with the miniwhite promoter. Thus, it was first demonstrated that insulators could participate in the regulation of the contacts between promoter and functionally opposite elements, responsible for either gene activation, or repression. Original Russian Text ? M.V. Kostyuchenko, E.E. Savitskaya, M.N. Krivega, P.G. Georgiev, 2008, published in Genetika, 2008, Vol. 44, No. 12, pp. 1693–1697.     

紫色红曲霉Mp-21 MpMcrA基因的克隆与生物信息学分析

McrA为最近在构巢曲霉(Aspergillus nidulans)中发现的全局调控因子，具有调控丝状真菌生长发育和次级代谢的作用，利用生物信息学分析方法找到并克隆紫色红曲霉（Monascus purpureus）中mcrA基因，将其命名为MpMcrA。分析MpMcrA蛋白质理化性质、亲疏水性、亚细胞定位、信号肽、跨膜区域及磷酸化位点、转录因子结合位点以及蛋白质二级结构。利用ProtParam、ProtScale、PSORTII、SignalP4.1等生物信息学软件对MpMcrA进行系统分析。 结果表明，MpMcrA基因长1 356 bp，其中含有3个外显子，2个内含子，编码410个氨基酸，与构巢曲霉序列比对蛋白相似性高达64％。预测结果显示，MpMcrA属于亲水蛋白，位于细胞核可能性大，不存在跨膜区域，不属于膜蛋白；不存在剪切位点，不属于分泌蛋白；基因含有54个潜在的磷酸化位点；可能存在5个转录因子结合位点；蛋白结构大部分为无规则卷曲，整体结构较松散。对MpMcrA基因进行了生物信息学分析，得到了基因特征和分析结果。初步确定MpMcrA基因为构巢曲霉同源mcrA基因，在红曲霉中未见有报道。     

Regulation of the phosphate regulon of Escherichia coli: Characterization of the promoter of the pstS gene

Summary The pstS gene belongs to the phosphate regulon whose expression is induced by phosphate starvation and regulated positively by the PhoB protein. The phosphate (pho) box is a consensus sequence shared by the regulatory regions of the genes in the pho regulon. We constructed two series of deletion mutations in a plasmid in vitro, with upstream and downstream deletions in the promoter region of pstS, which contains two pho boxes in tandem, and studied their promoter activity by connecting them with a promoterless gene for chloramphenicol acetyltransferase. Deletions extending into the upstream pho box but retaining the downstream pho box greatly reduced promoter activity, but the remaining activity was still regulated by phosphate levels in the medium and by the PhoB protein, indicating that each pho box is functional. No activity was observed in deletion mutants which lacked the remaining pho box or the-10 region. Therefore, the pstS promoter was defined to include the two pho boxes and the-10 region. The PhoB protein binding region in the pstS regulatory region was studied with the deletion plasmids by a gelmobility retardation assay. The results suggest the protein binds to each pho box on the pstS promoter. A phoB deletion mutant was constructed, and we demonstrated that expression of pstS was strictly dependent on the function of the PhoB protein.     

A multi-component upstream activation sequence of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter

Summary The majority of the activation potential of the Saccharomyces cerevisiae TDH3 gene promoter is contained within nucleotides –676 to –381 (relative to the translation initiation codon). An upstream activation sequence (UAS) in this region has been characterized by in vitro and in vivo assays and demonstrated to be composed of two small, adjacent DNA sequence elements. The essential determinant of this upstream UAS is a general regulatory factor 1 (GRF1) binding site at nucleotides –513 to –501. A synthetic DNA element comprising this sequence, or an analogue in which two of the degenerate nucleotides of the GRF1 site consensus sequence were altered, activated 5 deleted TDH3 and CYC1 promoters. The second DNA element of the UAS is a 7 by sequence which is conserved in the promoters of several yeast genes encoding glycolytic enzymes and occurs at positions –486 to –480 of the TDH3 promoter. This DNA sequence represents a novel promoter element: it contains no UAS activity itself, yet potentiates the activity of a GRF1 UAS. The potentiation of the GRFl UAS by this element occurs when placed upstream from the TATA box of either the TDH3 or CYC1 promoters. The characteristics of this element (termed GPE for GRF1 site potentiator element) indicate that it represents a binding site for a different yeast protein which increases the promoter activation mediated by the GRF1 protein. Site-specific deletion and promoter reconstruction experiments suggest that the entire activation potential of the –676 to –381 region of the TDH3 gene promoter may be accounted for by a combination of the GRF1 site and the GPE.     

甜荞花青素合成相关基因FeR2R3-MYB的克隆与表达分析

MYB 是一类常见的转录因子,广泛参与植物花青素生物合成的调控。为探究 MYB转录因子在甜荞花青素生物合成中的调控作用,该研究从红花甜荞和白花甜荞转录组学数据中筛选并克隆出一个和花青素生物合成相关的MYB基因,将其命名为 FeR2R3-MYB,GenBank 登录号为 MT151381.1,并对该序列进行生物信息学分析,以及利用 qRT-PCR 分析FeR2R3-MYB基因在白花甜荞和红花甜荞中的表达特征。结果表明:(1)FeR2R3-MYB基因全长 831 bp,编码 276 个氨基酸,蛋白的相对分子质量为 30.95 kD,理论等电点(pI)为 8.73,蛋白的不稳定指数为 69.64,属于不稳定蛋白,总疏水值为-0.679,整条肽链呈现亲水特性。(2)FeR2R3-MYB 具有典型的 R2R3-MYB 结构域,属于 R2R3-MYB 亚家族。(3)FeR2R3-MYB 与同属蓼科的苦荞和虎杖亲缘关系比较近。(4)FeR2R3-MYB 的启动子序列共含有 9 个光照响应元件、17 个转录因子结合位点、4 个非生物响应元件和 2 个激素响应元件。(5)亚细胞定位发现 FeR2R3-MYB 只在细胞核中表达。(6)FeR2R3-MYB 基因的表达量在叶片和花序中红花甜荞均高于白花甜荞,推测 FeR2R3-MYB 基因可以正向调节甜荞花青素生物合成。综上所述,该研究结果为进一步深化 FeR2R3-MYB 基因在甜荞花青素生物合成途径中的功能及表达调控方面的研究提供了基础。     

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。