Binding of the C6-zinc cluster protein, AFLR, to the promoters of aflatoxin pathway biosynthesis genes in Aspergillus parasiticus

AFLR is a Zn₂Cys₆-type sequence-specific DNA-binding protein that is thought to be necessary for expression of most of the genes in the aflatoxin pathway gene cluster in Aspergillus parasiticus and A. flavus, and the sterigmatocystin gene cluster in A. nidulans. However, it was not known whether AFLR bound to the promoter regions of each of the genes in the cluster. Recently, A. nidulans AFLR was shown to bind to the motif 5′-TCGN₅CGA-3′. In the present study, we examined the binding of AFLR to promoter regions of 11 genes in the A. parasiticus cluster. Based on electrophoretic mobility shift assays, the genes nor1, pksA, adhA, norA, ver1, omtA, ordA, and, vbs, had at least one 5′-TCGN₅CGA-3′ binding site within 200 bp of the translation start site, and pksA and ver1 had an additional binding site further upstream. Although the promoter region of avnA lacked this motif, AFLR bound weakly to the sequence 5′-TCGCAGCCCGG-3′ at −110 bp. One region in the promoter of the divergently transcribed genes aflR/aflJ bound weakly to AFLR even though it contained a site with at most only 7 bp of the 5′-TCGN₅CGA-3′ motif. This partial site may be recognized by a monomeric form of AFLR. Based on a comparison of 16 possible sites, the preferred binding sequence was 5′-TCGSWNNSCGR-3′.     

纤枝短月藓BeLEA2基因的克隆及表达分析

为探究纤枝短月藓LEA2基因的结构和表达特征,该研究以纤枝短月藓为材料,首次利用PCR克隆技术得到纤枝短月藓BeLEA2基因序列,并对该基因进行分析。结果表明:(1)该基因序列中含有2个外显子和1个内含子,其开放阅读框(ORF)为456 bp,编码151个氨基酸,预测其相对分子质量为16515.96 Da。(2)将纤枝短月藓与其他植物LEA2基因氨基酸序列进行比对,构建系统进化树,结果显示纤枝短月藓与小立碗藓的亲缘关系最近。(3)利用HiTail-PCR技术克隆获得1072 bp的BeLEA2启动子序列,用PlantCARE在线工具对该启动子的顺式作用元件进行预测,结果表明该启动子除了含有核心启动子元件TATA-box和CAAT-box外,还含有ABRE、MYB、MYC、MYB结合位点(MBS)等其他顺式元件。(4)实时荧光定量PCR分析表明,BeLEA2基因在纤枝短月藓不同发育时期和不同组织中都有表达,且对脱水胁迫有响应。以上结果为进一步探究LEA2基因在苔藓植物中的功能及作用机制奠定了基础。     

肿瘤细胞中端粒酶催化亚基hTERT基因启动子结合因子的检测

为了研究端粒酶催化亚基ｈＴＥＲＴ基因在癌变细胞中组成型表达的调控机制,采用凝胶阻滞电泳（ＥＭＳＡ）实验方法检测人粒系白血病细胞ＨＬ－６０、人红系白血病细胞Ｋ５６２、人肺癌细胞Ａ５４９、人肝癌细胞ＨｅｐＧ２及正常人肺成纤维细胞２ＢＳ等体外传代的肿瘤和正常二倍体细胞核提取物中与ｈＴＥＲＴ启动子核心序列结合的核因子活性。结果在４种实验肿瘤细胞中均可检测出与ｈＴＥＲＴ基因启动子结合的核因子活性,而正常二倍     

Mechanisms of gene expression controlled by pressure in deep-sea microorganisms

A pressure-regulated operon has been cloned and sequenced from deep-sea barophilic Shewanella strains. To understand pressure-regulated mechanisms of gene expression, a regulatory element upstream of the pressure-regulated operon from Shewanella sp. strain DSS12 was studied. Regions A and B were classified by sequence analysis. A unique octamer motif, AAGGTAAG, was found to be repeated in tandem 13 times in region B. An electrophoretic mobility shift assay demonstrated that a σ⁵⁴-like factor recognizes region A and other unknown factors recognize region B. Different shift patterns of the protein–DNA complexes were observed when extracts of cells cultured at 0.1 MPa or 50 MPa were incubated with a DNA probe specific for region B. These results indicate that the deep-sea strain DSS12 expresses different DNA-binding factors under different pressure conditions. Received: January 22, 1998 / Accepted: February 16, 1998     

Induction of human aquaporin-1 gene by retinoic acid in human erythroleukemia HEL cells

Retinoids have been implicated in the control of cell proliferation, differentiation, and developmental processes. We report here that aquaporin-1 (AQP1) is specifically induced by retinoic acid (RA) in human erythroleukemia HEL cells. Both all-trans-RA (ATRA) and 9-cis-RA (9CRA) strongly induced the AQP1 mRNA and protein in a dose-dependent manner. AQP1 protein was mainly expressed in plasma membrane in cells induced by RAs. To identify the RA response element (RARE) in the human AQP1 promoter, the 5(')-flanking region of AQP1 promoter was isolated and transient transfection experiment in HEL cells was performed. Deletion analysis of the AQP1 promoter revealed that one putative DR5-like RARE with five spaces was located in the region from -2218 to -2202; AGGGCAgggacAGGTGA. Electrophoretic mobility shift assay (EMSA) experiment demonstrated that two slowly migrated complexes (C1 and C2) capable of binding the RARE sequence were present in nuclear extracts prepared from cells and the complex C1 was strongly increased in nuclear extracts by RA stimulation. The complexes C1 and C2 were significantly abolished by an excess unlabeled probe. These results indicate that RAs strongly stimulate the human AQP1 gene expression through the RARE and define a novel role in the regulation of erythropoiesis.     

Aspirin upregulates expression of urokinase type plasminogen activator receptor (uPAR) gene in human colon cancer cells through AP1

In this study, the effects of acetylsalicylic acid (aspirin) on the expression of uPAR and the mechanism by which it regulates expression of uPAR was examined in two different colon cancer cell lines HCT116 and GEO, respectively. The study shows that under physiological concentration, aspirin upregulates steady-state level expression of uPAR mRNA as well as expression of uPAR protein. Using a transient transfection assay, a region corresponding to -1 to -398 region of uPAR promoter has been identified which shows maximum responsiveness to aspirin treatment and found that this region is sufficient for the aspirin-induced up-regulation of uPAR. A stable integration of a single copy of this region coupled to luciferase reporter gene into the HCT116 genome also behaved similarly. Using gel mobility shift assays, it is found that the distal AP1 region between -171 and -186 is responsible for the aspirin-induced up-regulation of uPAR. Mutation of this region reduced up-regulation. Supershift assays identify that the bound proteins at this region are c-Jun and Fra-1. Real-time PCR analysis showed more than 4-fold increase in the binding of c-Jun and a 1.6-fold increase in the binding of Fra-1 in this region and this up-regulation corresponds to an increased binding of acetylated histone H4 in this region. Since an increase in the expression of uPAR corresponds to an increase in the migration of the cell, a migration assay was performed and result showed a 3-fold increased migration of HCT116 cells through the vitronectin-coated layer. Thus, an AP1 mediated pathway for aspirin induced up-regulation of uPAR has been identified.     

蛇足石杉COBRA基因家族的分子生物信息学及表达分析

为探明蛇足石杉COBRA基因家族成员分子生物信息学特征及组织表达规律,该文基于蛇足石杉的全长转录组数据,通过生物信息学技术对该家族成员(HsCOBRAs)的理化性质、结构域、保守基序、顺式作用元件、基因表达量等进行分析。结果表明:(1)在蛇足石杉全长转录组中共筛选出24个HsCOBRAs家族成员,其中酸性蛋白9个,稳定蛋白11个,疏水性蛋白5个,具有跨膜结构的蛋白7个,具有信号肽的蛋白3个。(2)亚细胞定位在细胞壁、叶绿体、细胞核、细胞膜上。(3)结构分析发现HsCOBRAs有7种结构域和6种保守基序,部分成员具有高度保守的CCVS结构。(4)HsCOBRAs具有CAAT-box、TATA-box等45种顺式作用元件。(5)HsCOBRA2在叶、孢子、茎、芽胞中的表达量均最高。该研究结果可为HsCOBRAs的进一步研究及生物学功能验证等提供理论依据。     

Molecular cloning and expression of retinoic-acid synthesizing enzyme raldh2 from Takifugu rubripes

Retinaldehyde dehydrogenases (raldhs) synthesize retinoic acid (RA), which is required for pattern formation and organogenesis during embryogenesis. To elucidate the common role of RA on vertebrate embryos, we first sought to clone a homologous gene to human raldh2 from fugu, Takifugu rubripes. We cloned a 1837 bp cDNA that encodes fugu raldh. The deduced amino acid sequence of the fugu raldh comprises 502 amino acids. The fugu Raldh showed highest sequence identity to zebrafish, Danio rerio, Raldh2 (79.9%). The fugu Raldh also showed high sequence identity to other vertebrate Raldh2: Xenopus laevis (77.2%), human (77.4%), mouse (74.3%) and chick (73.9%). Comparative genomic analysis showed that the gene arrangement around fugu raldh agreed with that of human raldh2. Fugu raldh mRNA was expressed through embryogenesis similarly to raldh2 in other vertebrates. These results and phylogenetic analyses suggest that pufferfish raldh is a fugu orthologue of other species' raldh2.