Promoter regulation in Candida albicans and related species

Regulation of gene expression has been studied extensively in Saccharomyces cerevisiae and Schizosaccharomyces pombe . Some, but by far not all, of the findings are also applicable to Candida albicans , an important ascomycete fungal pathogen of humans. Areas of research in C. albicans include the influence of key signal transduction cascades on morphology, and the response to host-generated influences, such as host immune effector cells, blood, pH or elevated carbon dioxide. The resistance to antifungal agents and response to stress are also well researched. Conditional gene expression and reporter genes adapted to the codon usage of C. albicans are now widely used in C. albicans . Here we present a comprehensive overview of the current techniques used to investigate regulation mechanisms for promoters in C. albicans and other Candida species. In addition, we discuss reporter genes used for the study of gene expression.     

通用真核质粒表达载体的构建

利用真核基因表达调控的原理,以pSV2-dhfr为起始材料,构建了两个通用的真核质粒表达载体pMAML1-dhfr和pMAML2-dhfr,它们包含人巨细胞病毒立即早期启动子/增强子调控元件,由两个转录方向相同或相反的表达单元组成.以荧火虫荧光素酶基因为报道基因,β-半乳苷酶基因为内对照,借助COS-7短暂表达系统,研究了它们对荧光素酶表达的影响,并比较了它们与出发载体pSV2-dhfr的相对强弱.     

Retro-Tet:一种基于逆转录病毒的诱导性基因表达系统

 Retro Tet基因表达系统是一种新型的高效、稳定、无毒、具有严密开 关功能的可诱导性真核细胞基因表达系统 .该系统兼备了逆转录病毒基因表达系统和Tet off Tet on基因表达系统的优点 ,在稳定表达细胞系的筛选、基因的表达与调控及基因功能研究等方面得到了成功的应用 ,同时也为基因治疗提供了一种理想的基因载体系统     

Human placental alkaline phosphatase as a histochemical marker of gene expression in transgenic mice

The human placental alkaline phosphatase (PLAP) gene was analysed for its utility as a histochemically detectable reporter gene in transgenic mice. A reporter gene was made by linking the PLAP structural gene to an enhancerpromoter element from the human -actin gene. This gene was inserted into the mouse genome by transfection of embryonic stem cells, and by microinjection of fertilized eggs. Histochemical staining showed that the transgene was uniformly expressed in four of four stable ES cell lines, and in all ten tissues examined from adult animals from five lines of transgenic mice. Non-transgenic cells did not stain. These results suggest that the human PLAP gene will be of utility in studies requiring phenotypic marking of cells in tissues of mice.     

一株受二噁英类化学物质诱导表达的萤光素酶肝癌细胞系

 构建了一对二英类化学物质敏感的人肝癌细胞株 ,用于二英类化学物质生物筛选和快速半定量检测 .首先构建一在二英增强子调控下的萤光素酶报告基因质粒 ,该质粒转染入人肝癌细胞株HepG2 ,筛选稳定转染细胞株 .2 ,3,7,8 四氯代二苯并二英 (TCDD)诱导稳定转染细胞株萤光素酶表达 ,发光检测萤光素酶活性 .该细胞株用于TCDD检测 ,检测下限为 1 1pmol L ,线性范围为 1～ 10 0pmol L .该细胞系的建立可用于二英类化学物质的生物筛选和快速半定量检测     

一株受二噁英类化学物质诱导表达的萤光素酶肝癌细胞系

构建了一对二英类化学物质敏感的人肝癌细胞株 ,用于二英类化学物质生物筛选和快速半定量检测 .首先构建一在二英增强子调控下的萤光素酶报告基因质粒 ,该质粒转染入人肝癌细胞株HepG2 ,筛选稳定转染细胞株 .2 ,3,7,8 四氯代二苯并二英 (TCDD)诱导稳定转染细胞株萤光素酶表达 ,发光检测萤光素酶活性 .该细胞株用于TCDD检测 ,检测下限为 1 1pmol L ,线性范围为 1～ 10 0pmol L .该细胞系的建立可用于二英类化学物质的生物筛选和快速半定量检测     

微生物细胞工厂中多基因表达的控制策略

微生物代谢工程和合成生物学是当今微生物技术领域研究的热点,微生物的生长速度快、容易进行大规模培养;遗传背景清楚、遗传操作简便可靠等性质使其在与人类生活相关的多个领域中起到重要的作用。微生物细胞工厂是指人工设计的能够进行物质生产的微生物代谢体系。许多微生物细胞工厂的构建由于引入多个基因或整条代谢途径,而可能导致代谢失衡、部分代谢中间产物积累等问题,需要使用一定的调控策略加以控制。以下对涉及多个基因作用的微生物细胞工厂中所使用的调控策略,分为若干层次进行了总结和探讨,并对今后多基因控制策略的发展方向进行了预测与展望。     

携带红色荧光蛋白的RU486可诱导真核表达载体的构建及其表达

在基因治疗中, 实现目的基因的调控表达是非常重要的。然而, 传统基因载体的无调控地持续或不适当的表达会影响治疗效果, 甚至可能带来致命的副作用。在本研究中, 我们构建了一种带有DsRed红色荧光蛋白报告基因并可经RU486诱导的真核表达载体, 并在体外评估了其调控表达作用。利用分子生物学技术, 将DsRed基因和启动子, 以及RU486系统构建成单一的质粒载体PDC-RURED, 为减少RU486调控元件和基因表达元件之间的相互干扰, 在两者之间加入1.6 kb的绝缘子。经PCR检测和限制性酶切分析及序列测定均证实了载体的正确性。在转染HEK293细胞后, 运用荧光显微镜和流式细胞技术证实了该载体的调控能力。没有RU486时, 几乎没有红色荧光蛋白的表达, 而加入诱导剂RU486后, 最高可以实现红色荧光蛋白的40余倍的表达。实验结果表明构建的可经RU486诱导的新型真核表达载体可以实现对目的基因的表达时间和表达水平的调控, 为进一步的基因调控研究和和基因治疗提供了良好的工具。     

Isolation and characterization of cDNA clones for RNA species induced by substituted benzenesulfonamides in corn

A search of compounds capable of inducing specific gene expression in plants without affecting growth and development led to the examination of changes in the pattern of gene expression in corn after treatment with substituted benzenesulfonamide herbicide safeners. Following hydroponic treatment of corn with the safener N-(aminocarbonyl)-2-chlorobenzenesulfonamide (2-CBSU), the specific induction of new translatable mRNA species was observed. Replicate copies of a cDNA library made using RNA from 2-CBSU-treated corn roots were differentially screened with cDNA probes made from either the same mRNA fraction used for library construction or mRNA isolated from roots treated with 2-chlorobenzenesulfonamide (2-CBSA), an inactive analog of the safener. Colonies showing hybridization only with the probe made using mRNA from 2-CBSU-treated roots were further characterized to assess the specificity of the induction and decay of the corresponding induced RNA species. RNA blot analyses showed two clones, designated In2-1 and In2-2, contained plasmids that hybridized to RNAs that were induced from an undetectable background in corn roots within 30 minutes after treatment with 2-CBSU. Leaf and meristem tissues showed similar inductions of the In2-1 and In2-2 RNA species after a delay of several hours. In addition, both RNA species were induced in corn by foliar application of 2-CBSU. In contrast, neither RNA species was induced following stress treatments of plants. These results indicate a substituted benzenesulfonamide safener might be used with the promoters from the In2-1 and In2-2 genes to develop a new inducible gene expression system for plants.     

Construction, electroporatic transfer and expression of ZpβypGH and ZpβrtGH in zebrafish

Recombinant transformation vectors (ZPβypGH and ZpβrtGH) consisting of fish growth hormone cDNA, and a reporter geneβ-galactosidase driven by fish promoter (Zp) were constructed. Freshly fertilized eggs of zebrafish (Brachydanio rerio) were electroporated at optimum conditions (0.07 kV voltage; 25 μF capacitance; 8 ohm resistance and 2 pulses) in the presence of one of these transformation vectors (100 μg circular DNNml). In either cases 72% of the electroporated eggs successfully hatched, in comparison to the 85% hatchability of the control eggs. Genomic DNA extracted from fins of randomly chosenF ₀ individuals was screened (by Southern blot hybridization); the transgenes were retained in the host genome of all the randomly chosen adult transformants. Fin-positive presumptive founder parents were crossed with control counterparts and the DNA of randomly chosenF ₁ progenies was screened for germline transformation. Southern analysis of chosenF ₁ progenies revealed the persistence of ZPβypGH or ZpβrtGH in 53% of theF ₁ progenies. Southern analyses of chosenF ₁ progenies and the frequency (53% ofF ₁ ZpβrtGH and 53% ofF ₁ ZP{β}ypGH) of transmission revealed the degree of mosaicism inF ₀ transformants. Expression was confirmed from the 3–4 times elevated levels of activity of the reporter gene and 30–40% accelerated growth of transgenicF ₀ andF ₁ progenies. Construction of the transgenes was made at the Taiwan National Ocean University, Taiwan and all other work at the Madurai Kamaraj University, Madurai.     

Efficiency of expression of transfected genes depends on the cell cycle

Lipofection, a lipid-mediated DNA transfection procedure, was used to transfect synchronized L929 mouse fibroblast cells with a reporter plasmid containing the bacterial chloramphenicol acetyltransferase gene. The efficiency of gene expression was investigated on transfection of cells at different stages of the cell cycle. Our data show that expression of the reporter gene was minimal when transfection was performed in G0-phase and parallel experimental data disproved the possibility that the reduced expression observed was due to differential uptake at different times in the cell cycle. Investigation into the condensation state of the plasmid has shown that the low chloramphenicol acetyltransferase gene expression could be a direct consequence of the packaging of the plasmid into condensed chromatin when transfection occurs in G0-phase. The inactivation of the reporter gene is not reversed by growth of the cells in high serum or by treatment with Trichostatin A, a specific inhibitor of histone deacetylase, suggesting that the inactive chromatin formed in G0-phase cells lacks associated histone acetylase activity. In contrast, the high activity seen when cells in S-phase are transfected is enhanced even further by treatment with Trichostatin A.