An inducible promoter fused to the period gene in Drosophila conditionally rescues adult per-mutant arrhythmicity

The period (per) gene of Drosophila melanogaster is involved in the expression of circadian rhythms of locomotor activity in adult flies. Molecular studies of per (reviewed in ref. 2) have shown that the transcribed and translated products of this gene are present primarily at the embryonic, pupal and adult stages. Here we describe experiments with arrhythmic per mutants bearing an inducible form of this gene which indicate that strongly rhythmic adult behaviour can be obtained only if per expression is induced in the adult, independent of its history of expression earlier in development. Thus per-mutant locomotor-activity phenotypes seem not to result from abnormalities in the development of neural structures or in physiological processes that may be required at pre-adult stages for the expression of this circadian rhythm. Moreover, the action of per after light:dark cycle entrainment seems to be sufficient for activity rhythms to be exhibited in constant darkness; this suggests further that the per product is required only during the time that the rhythmic behaviour is being manifested. Our strategy used a heat-shock gene promotor fused to per coding sequences to obtain conditional gene expression. Heat-shock promoter-driven genes have previously been used to study the mode of action and tissue specificity of a variety of Drosophila genes; our experiments on circadian rhythms demonstrate the use of such gene constructions for the temporal manipulation of genes whose phenotypes, behavioural and otherwise, affect whole organisms.     

人IDE基因启动子生物信息学分析

对人IDE基因启动子进行生物信息学分析以获得人IDE基因启动子、CpG岛及转录因子结合位点特征.从UCSC基因组数据库成功获得人IDE基因5’调控区2 000 bp序列.Promoter 2.0、FPROM、NNPP预测人IDE基因分别有3个、2个、6个启动子.Relative profile score threshold选择80%、85%、90%、95%、100%时,JASPAR预测该序列存在5170、1771、454、87和5个可能的转录因子结合位点.Relative profile score threshold选择80%,搜索到6个潜在的TCF7L2转录因子结合位点.采用进化足迹法,LAGAN预测方法获得位于人和小鼠同源IDE基因启动子保守区域相同位置的转录因子结合位点为14个,包含转录因子SPI-1、cap、c-FOS、FREAC-3、c-ETS、Cdxa、HSF2等.发现一个CpG岛,位于1 303～1 705 bp 之间,大小为403 bp.人IDE基因启动子、CpG岛及转录因子结合位点的生物学信息学分析,为下一步基因表达调控实验奠定了基础.     

大肠杆菌可诱导启动子表达系统的构建

大肠杆菌作为现代基因工程最为常用的原核表达系统,其核心为基因启动元件的高效性和可控性．为了获得不依赖IPTG和温度诱导的新型启动子,克隆得到大肠杆菌中通过低pH诱导的启动子Padi,并采用点突变获得几个活性各异的启动子单元,启动子的转录效率最多提高18．8倍．结果表明：该受低pH条件诱导的启动子经点位突变后,提高转录活性的同时诱导的严谨性有一定下降;在调控蛋白和启动子的协同表达后,诱导的严谨性得到提高．     

人NOD8基因启动子绿色荧光蛋白表达载体的构建与鉴定

目的:构建人NOD8基因启动子的绿色荧光蛋白表达载体.方法:用特定的限制性内切酶位点,以人基因组DNA为模板,PCR扩增含有人NOD8基因启动子不同长度2段序列,并进行酶切以切除启动子的pEGFP-C2作框架结构,插入表达载体pEGFP-C2中,构建含有人NOD8基因启动子驱动的绿色荧光蛋白载体pEGFP-C2-NOD8(520 bp)、pEGFP-C2-NOD8(760 bp),用Vsp Ⅰ和Nhe Ⅰ双酶切和PCR鉴定重组质粒,再将重组质粒进行DNA序列分析.构建的重组质粒经脂质体(lipofectamine)TM2000介导转染HEK293、K562和HeLa细胞,转染48 h后在倒置荧光显微镜下观察.结果:pEGFP-C2-NOD8(520 bp)、pEGFP-C2-NOD8(760 bp)分别经酶切鉴定和序列测定证实目的基因已插入重组质粒;细胞转染结果表明,构建的2段重组质粒转染HEK293、K562及HeLa细胞均能表达绿色荧光,其中构建的pEGFP-C2-NOD8(760 bp)重组质粒绿色荧光表达强于pEGFP-C2-NOD8(520 bp).结论:成功构建2段不同长度的人NOD8基因启动子绿色荧光蛋白表达载体.     

Expression of the E. coli uvrA gene is inducible

UvrA+-dependent excision repair is one of the most important systems in Escherichia coli for repairing UV-induced pyrimidine dimers and a variety of other forms of DNA damage. The uvrA protein acts in conjunction with the uvrB and uvrC gene products to introduce a nick at the of a DNA lesion and thus initiate the repair process. We have recently used the Mud(Ap, lac) operon fusion vector to identify a set of genes whose expression is induced by DNA damage. One Mud(Ap, lac) insertion mapped at the uvrA locus and made the cells sensitive to UV light. In this fusion strain, beta-galactosidase expression was induced by DNA-damaging agents in a recA+lexA+-dependent fashion. We were surprised by this result because uvrA+-dependent excision repair is observed both in cells in which protein synthesis has been inhibited and in recA- and lexA- cells, findings which have led to the conclusion that the uvrA gene product is constitutively expressed and not under the control of the complex recA+lexA+ regulatory circuitry (see below). We have investigated this possibility further and describe here the generation and characterization of a set of fusions of the lac genes to the promoter of the uvrA gene. We confirm that the uvrA gene product is induced by DNA damage in a recA+lexA+-dependent fashion.     

肝癌胰岛素样生长因子Ⅱ基因启动子P3甲基化的检测及意义

目的：建立检测肝癌胰岛素样生长因子Ⅱ(IGF-Ⅱ)基因启动子P3甲基化状态的方法。方法：培养三株肝癌细胞系(BEL7402、SMMC7721、HepG2)，选取正常肝组织3例；提取基因组DNA，亚硫酸氢钠修饰，巢式聚合酶链反应(nested PCR)加甲基化特异性PCR(MSP)检测IGF-Ⅱ基因启动子P3的甲基化状态，PCR产物经克隆、测序验证。结果：三株肝癌细胞系IGF-Ⅱ基因启动子P3去甲基化，正常肝组织IGF-Ⅱ基因启动子P3甲基化，目的片段无突变，甲基化修饰完全。结论：巢式PCR加甲基化特异性PCR可准确检测IGF-Ⅱ基因启动子P3的甲基化状态。     

肝癌胰岛素样生长因子II基因启动子P3甲基化的检测及意义

目的:建立检测肝癌胰岛素样生长因子II(IGF-II)基因启动子P3甲基化状态的方法.方法:培养三株肝癌细胞系(BEL7402、SMMC7721、HepG2),选取正常肝组织3例;提取基因组DNA,亚硫酸氢钠修饰,巢式聚合酶链反应(nested PCR)加甲基化特异性PCR(MSP)检测IGF-II基因启动子P3的甲基化状态,PCR产物经克隆、测序验证.结果:三株肝癌细胞系IGF-II基因启动子P3去甲基化,正常肝组织IGF-II基因启动子P3甲基化,目的片段无突变,甲基化修饰完全.结论:巢式PCR加甲基化特异性PCR可准确检测IGF-II基因启动子P3的甲基化状态.     

Deletion of the human T-cell receptor delta-gene by a site-specific recombination

The newly described T-cell receptor (TCR) delta locus is located inside the TCR alpha locus, between variable region (V)alpha and joining region (J)alpha. Although the delta and alpha TCR genes are physically linked on the same chromosome, they are sequentially expressed during T-cell development. This implies the existence of a highly efficient regulatory mechanism by which these two genes are independently rearranged. We have recently described a genetic element 'T early alpha' (TEA) in humans transcribed in foetal thymocytes, spliced alternatively to constant region (C)alpha, and located between the TCR-delta locus (5') and the group of J alpha segments (3'). Importantly, TEA flanks a common site of rearrangement in the thymus, and distinguishes cells using TCR-gamma/delta (TEA in germline configuration) from cells using TCR-alpha/beta (TEA deleted on both chromosomes). In order to understand this TEA-associated recombination we analysed genomic clones representing these thymic rearrangements. We show that the TEA-associated recombination deletes the delta locus before productive (V delta D delta J delta) rearrangement. The diversity (D)delta and J delta regions, which provide the major source of delta gene diversity, are eliminated as a consequence of delta gene deletion and cannot then be used in conjunction with an alpha-TCR. We propose that the TEA-associated deletion of TCR-delta precedes the formation of an alpha-TCR and could down-regulate TCR-delta formation in maturing thymus.     

The mapping of a cDNA from the human X-linked Duchenne muscular dystrophy gene to the mouse X chromosome

The recent discovery of sequences at the site of the Duchenne muscular dystrophy (DMD) gene in humans has opened up the possibility of a detailed molecular analysis of the genes in humans and in related mammalian species. Until relatively recently, there was no obvious mouse model of this genetic disease for the development of therapeutic strategies. The identification of a mouse X-linked mutant showing muscular dystrophy, mdx, has provided a candidate mouse genetic homologue to the DMD locus; the relatively mild pathological features of mdx suggest it may have more in common with mutations of the Becker muscular dystrophy type at the same human locus, however. But the close genetic linkage of mdx to G6PD and Hprt on the mouse X chromosome, coupled with its comparatively mild pathology, have suggested that the mdx mutation may instead correspond to Emery Dreifuss muscular dystrophy which itself is closely linked to DNA markers at Xq28-qter in the region of G6PD on the human X chromosome. Using an interspecific mouse domesticus/spretus cross, segregating for a variety of markers on the mouse X chromosome, we have positioned on the mouse X chromosome sequences homologous to a DMD cDNA clone. These sequences map provocatively close to the mdx mutation and unexpectedly distant from sparse fur, spf, the mouse homologue of OTC (ornithine transcarbamylase) which is closely linked to DMD on the human X chromosome.     

Deletion of steroid 5 alpha-reductase 2 gene in male pseudohermaphroditism

The conversion of testosterone into dihydrotestosterone by steroid 5 alpha-reductase is a key reaction in androgen action, and is essential both for the formation of the male phenotype during embryogenesis and for androgen-mediated growth of tissues such as the prostate. Single gene defects that impair this conversion lead to pseudohermaphroditism in which 46X,Y males have male internal urogenital tracts, but female external genitalia. We have described the isolation of a human 5 alpha-reductase complementary DNA from prostate. Subsequent cloning and genetic studies showed that this gene (designated 5 alpha-reductase 1) was normal in patients with 5 alpha-reductase deficiency. We report here the isolation of a second 5 alpha-reductase cDNA by expression cloning and the polymerase chain reaction. The biochemical and pharmacological properties of this cDNA-encoded enzyme (designated 5 alpha-reductase 2) are consistent with it being the major isozyme in genital tissue. A deletion in this gene is present in two related individuals with male pseudohermaphroditism caused by 5 alpha-reductase deficiency. These results verify the existence of at least two 5 alpha-reductases in man and provide insight into a fundamental hormone-mediated event in male sexual differentiation.     

水稻Os05g0442400基因启动子分析以及由Os05g0442400基因启动子引导GUS报告基因在转基因水稻中的表达

采用Plant CARE和PLACE软件分析预测水稻Os05g0442400基因启动子序列中可能存在的顺式作用元件.结果显示,在起始密码子ATG上游1500bp区域内,除了启动子基本的核心作用元件外,还存在一些与植物抵御非生物胁迫过程有关的作用元件、脱落酸应答元件、光诱导启动子作用元件、病原菌诱发因子作用元件,以及根特异性结合位点.采用根癌农杆菌介导法成功将Os05g0442400 promoter::gus构建导入"中花11"水稻.由不同组织部位GUS染液检测表明:在水稻苗期,内源Os05g0442400基因可能主要在根部表达;随着水稻生殖期的延续,水稻内源Os05g0442400基因在颖壳中的表达区域由上向下面积增大,并在抽穗后达到最大表达区域.这些结果可能与Os05g0442400基因启动子上分别存在根特异性结合位点和光诱导启动子作用元件具有一定的联系.     

人RIP2基因启动子绿色荧光蛋白表达载体的构建与鉴定

目的:构建人RIP2基因启动子的绿色荧光蛋白表达载体。方法:根据特定限制性内切酶位点,以人基因组DNA为模板,PCR扩增含人RIP2基因启动子不同长度2段序列,构建含人RIP2基因启动子驱动的绿色荧光蛋白载体pEGFP-C2-RIP2(750 bp)wt、pEGFP-C2-RIP2(941 bp)wt,用VspⅠ和NheⅠ双酶切鉴定重组质粒,进行DNA序列分析,重组质粒经阳离子聚合物JetPeiTM介导转染HEK293细胞48 h后观察。结果:酶切鉴定和序列测定证实目的基因已插入重组质粒;细胞转染结果表明,重组质粒转染HEK293细胞均能表达绿色荧光,其中构建的pEGFP-C2-RIP2(750 bp)wt重组质粒绿色荧光表达强于pEGFP-C2-RIP2(941 bp)wt。结论:成功构建2段不同长度的人RIP2基因启动子绿色荧光蛋白表达载体。     

DNA sequence for a low-level promoter of the lac repressor gene and an 'up' promoter mutation.

The promoter for a weakly expressed constitutive gene, the lactose repressor gene (lacI), has been sequenced, along with an 'up' promoter mutation Iq. The 10-fold enhancement in I expression found in Iq is the result of a single base change at position -35. To facilitate the sequencing, the lacI gene was cloned in a small plasmid.     

中国明对虾卵黄蛋白原基因启动子克隆与分析

为了探明中国明对虾卵黄蛋白原基因启动子表达调控机制,利用DNA步移法克隆了中国明对虾卵黄蛋白原基因启动子及其上游调控序列,总长1 100bp.分析表明,在基因转录起始位点上游-30～-24bp处有1个TATA box,未发现有CAAT box和GC box.同时,在上游调控区还存在有多个可能影响启动子转录活性的顺式作用元件,如NF-κB,YY1和SP1等转录因子结合位点.这些结果为深入研究中国明对虾卵黄蛋白积累及卵子发生过程奠定了基础.