反义技术及其在G蛋白研究中的应用

反义技术至少包括反义寡核苷酸技术、反义RNA技术和核酶技术.在G蛋白研究中,反义技术在G蛋白受体及受体亚型研究、G蛋白转导信号特异性研究方面及对G蛋白耦联信号传导途径与其他信号传导途径之间“cross talk”认识方面的研究中,有着广泛的应用.     

反义寡核苷酸的化学修饰

反义寡核苷酸的功能在很大程度上取决于其稳定性、生物利用度及与靶基因结合或反应的特性．通过特定的化学修饰可以改变这些物理、化学特性,从而增加其抗病毒、抗肿瘤及对其他特定基因的表达抑制活性．     

反义技术应用在细菌代谢调控中的研究进展

随着基因工程技术的蓬勃发展和代谢调控研究的深入,反义技术作为一种温和调控的基因工程技术,开始向世人展示其无穷的魅力.与基因敲除等功能缺失性研究方法相比,反义技术具有投入少、周期短、操作简单等优点,受到广泛的关注,成为细菌代谢调控的有力工具.以下对反义RNA、反义寡核苷酸,核酶这几种反义技术在细菌代谢工程操作中的研究进展及存在的问题进行了概述.     

反义RNA的作用机理及其在植物基因工程领域的应用

本文综述了反义RNA在原核生物和真核生物中的作用机理,并系统地介绍了反义RNA技术在植物基因过程中的研究进展。     

反义RNA及其在植物学研究中的应用

反义ＲＮＡ最初发现于细菌中,它们是一些较短的、散布的转录产物[1],本身缺乏编码能力,但可以通过碱基配对的方式与靶ＲＮＡ的特定互补区域结合,从而阻抑基因的正常表达,因此,反义ＲＮＡ是高度特异性的基因表达抑制因子。1　自然界中存在的反义ＲＮＡ参与基因表达调控的反义ＲＮＡ是在原核生物中发现的。一些实验结果表明,在质粒的复制、转座子的转座作用及噬菌体的发育进程中,反义ＲＮＡ的调控起着至关重要的作用。ＣｏｌＥ1以及其他相关质粒的复制过程中,ＤＮＡ的延伸起始于一种特殊引物的形成。而这一引物所在的区段,亦可产生与引物本身5…     

Achieving efficient delivery of morpholino oligos in cultured cells

One of the many features that make morpholino oligos unique among the antisense structural types is an uncharged backbone. While this feature eliminates the nonspecific interactions of traditional S-oligos, it also renders the morpholino undeliverable via the traditional lipid-based delivery systems. This article describes a highly efficient method of delivering morpholino oligos into adherent and nonadherent cultured cells. In this system, a nonionic morpholino oligo is paired to a complementary DNA "carrier." The DNA is then bound electrostatically to a partially ionized, weakly-basic ethoxylated polyethylenimine (EPEI). This morpholino/DNA/EPEI complex is efficiently endocytosed, and when the pH drops within the endosome, the EPEI more fully ionizes, resulting in permeabilization of the endosomal membrane and release of the morpholino into the cytosol. This article describes optimization of delivery in HeLa cells and provides the basis for delivery in any cultured endocytic cell type. genesis 30:94--102, 2001.     

登革热病毒反义寡核苷酸的合成及抗病毒活性

根据碱基互补原理, 反义寡核苷酸可与特定病毒基因结合从而选择性地抑制该病毒的复制, 这是病毒性疾病治疗的新途径. 基于上述原理设计并合成了D2-04 RNA特异的6个5′末端脂肪链修饰的硫代反义寡核苷酸. 体外抗病毒活性评价显示互补于5′端起始密码、3′端重复序列和末端序列的3个反义寡核苷酸呈较强的抗病毒活性.     

反义核酸技术应用难点及研究进展

反义核酸技术已被广泛用于治疗药物、药物靶点确认、探知病理基因的表达。目前对其作用原理的研究集中于其被吸收入细胞的机制、在细胞内的分布、反义核酸序列的最佳长度和性质,并针对体内可能抑制反义核酸活性的影响因素,采取了各种相应的反义核酸优化技术,如对反义核酸的化学修饰、联结高效的转运载体、确定最佳的反义结合位点等,通过这些技术来提高其体内稳定性、跨细胞转运的效率,识别靶序列的特异性,以获得更多更好的反义药物投入实用。     

反义技术在抗HBV感染中的应用

反义技术是近些年来随着现代分子生物学技术的发展而产生的新的生物医学治疗技术。它采用反义核酸分子抑制、封闭或破坏靶基因组的技术手段,包括反义寡核苷酸、核酶及RNA干扰等。反义分子通过与靶基因异性互补配对结合,阻断靶基因的复制、转录或翻译过程,从而发挥抗病毒作用。针对乙型肝炎病毒的反义技术也有了广泛而深入的研究。根据反义技术在分子、细胞以及动物水平上的研究表明：反义技术能够高效、特异地抑制HBV的复制与表达。     

Profilin functions in cytokinesis, nuclear positioning, and stomatogenesis in Tetrahymena thermophila

Expression of the actin-binding protein profilin was disrupted in the ciliate Tetrahymena thermophila by an antisense ribosome method. In cells with the antisense disruption no profilin protein was detected. Cultures of cells with the antisense disruption could be maintained, indicating that profilin was not essential for cytokinesis or vegetative growth. Disruption of the expression of profilin resulted in many cells that were large and abnormally shaped. Formation of multiple micronuclei, which divide mitotically, was observed in cells with a single macronucleus, indicating a defect in early cytokinesis. Some cells with the antisense disruption contained multiple macronuclei, which in Tetrahymena may indicate a function late in cytokinesis. The lack of profilin also affected cytokinesis in the cells that could divide. Normal-sized and normal-shaped cells with the antisense disruption took significantly longer to divide than control cell types. The profilin disruption revealed two new processes in which profilin functions. In cells lacking profilin, micronuclei were not positioned at their normal site on the surface of the macronucleus and phagocytosis was defective. The defect in phagocytosis appeared to be due to disruption of the formation of oral apparatuses (stomatogenesis) and a possible failure in the internalization of phagocytic vacuoles.     

Morpholino oligos: making sense of antisense?

Since morpholino oligos were first introduced as a means to inhibit gene function in embryos, in the Spring of 2000, they have been tested in a range of model organisms, including sea urchin, ascidian, zebrafish, frog, chick, and mouse. This review surveys the results of these studies and examines the successes and limitations of the approach for targeting maternal and zygotic gene function. The evidence so far suggests that, with careful controls, morpholinos provide a relatively simple and rapid method to study gene function.     

反义寡聚核苷酸：生理学研究中的新工具

反义寡聚核苷酸（antisenseoligonucleotide,AS-ON）通常是指与体内某RNA或DNA序列具有互补顺序,并能通过碱基配对与互补链杂交,从而影响其转录或翻译过程的核酸片段。AS-ONs技术的近来应用为生理学研究开辟了一条新路,对将来了解基因的功能提供了一种新手段。本文综述了AS-ONs的设计策略、作用机理、修饰和导入方式等基本问题,旨在对AS-ONs的应用提供参考。     

Subunit control of Rubisco biosynthesis – a relic of an endosymbiotic past?

Subunits of multiprotein complexes in the chloroplasts of eukaryotic cells are frequently the products of protein synthesis in the nucleus-cytoplasm and the organelle. The mechanisms that integrate gene expression in the two compartments are poorly understood. Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a model of nuclear-chloroplast interactions because it is a relatively simple example of a multimeric complex, being composed of nuclear DNA-encoded (RbcS) small subunits (SS) and chloroplast DNA-encoded ( rbcL) large subunits (LS). One means by which RbcS and rbcL expression are coordinated is by the adjustment of subunit stoichiometries in response to the abundance of unassembled subunits. This type of integration occurs by two principal mechanisms. When SS accumulation is limiting (as in antisense mutants of tobacco), LS levels are primarily adjusted to those of the SS at the level of rbcL mRNA translation initiation. On the other hand, when LS accumulation is limiting (as in some rbcL nonsense and missense mutants), SS levels are adjusted to those of the LS at the level of protein degradation. These two mechanisms may be ubiquitous and serve as either fine-tune or course controls during normal growth and development. Autogenous control is a central theme of prokaryotic gene regulation, and intergenomic regulation of RbcS and rbcL expression by subunit concentrations may be a relic of an endosymbiotic past.     

反义核酸在肿瘤研究中的应用

反义核酸研究已活跃于肿瘤研究及基因治疗领域,反义核酸通过碱基配对待异性地抑制基因表达,因此为研究肿瘤中癌基因和生长因子的功能及癌基因突变检测提供了更为有效的手段,并为肿瘤的基因治疗提供了可能途径.文章综述了反义核酸在基因治疗中所面临的问题及部分解决办法.     

Oligonucleotide-based strategies to reduce gene expression

Research on embryonic development and differentiation provides a sensitive, but challenging opportunity to use a variety of techniques designed to modulate gene expression. Changes in the expression of a single gene can alter levels of other genes and provide information on developmentally regulated gene expression pathways. The morphological consequences of altered gene expression can link gene expression to developmental fate. Oligonucleotide-based approaches offer a variety of means to potentially disrupt normal gene expression. The basis for some of these approaches is presented in this review.     

嗜铬颗粒蛋白A基因的反义转基因小鼠模型的建立及该基因启动子的组织特异性

构建小鼠嗜铬颗粒蛋白A（Chromogranin,A,CGA)基因的反义DNA载体pGAS1C－lacZ。用电穿孔的方法将该载体转化大鼠肾上腺髓质细胞瘤细胞系PC－12,X－Gal染色后证明位于CGA基因启动子下游的报告基因lacX已经表达。用限制性内切酶除去载体的质粒骨架后,显微注射入供体昆明小鼠的受精卵中,随后将注射过DNA的受精卵移植入假母的输卵管中,完成正常的胚胎发育。用PCR的方法筛选假母产下的小鼠,得到CGA基因反义RNA基因首建鼠14只。将首建鼠分别与正常昆明鼠交配,产生后代。取首建鼠的肾上腺进行X－Gal染色,组织用于石蜡切片,根据各鼠肾上腺切片的蓝色深浅判定转入基因量的高低,筛选到两只表达量高的首建鼠,留下它们的后代。取转基因鼠的各种组织用于X－Gal染色,发现报告基因在肾上腺、胰腺的胰岛中有表达,而在肌肉、脂肪组织中无表达,说明CGA基因的启动子具有神经内分泌组织特异性。     

Combinatorial, selective and reversible control of gene expression using oligodeoxynucleotides in a cell-free protein synthesis system

Herein we describe the methods for selective and reversible regulation of gene expression using antisense oligodeoxynucleotides (ODNs) in a cell-free protein synthesis system programmed with multiple DNAs. Either a complete shut down or controlled level of gene expression was attained through the antisense ODN-mediated regulation of mRNA stability in the reaction mixture. In addition to the primary control of gene expression, we also demonstrate that the inhibition of protein synthesis can be reversed by using an anti-antisense ODN sequence that strips the antisense ODN off the target sequence of mRNA. As a result, sequential additions of the antisense and anti-antisense ODNs enabled the stop-and-go expression of protein molecules. Through the on-demand regulation of gene expression, presented results will provide a versatile platform for the analysis and understanding of the complicated networks of biological components.     

In Vitro Characterization of the Type I Toxin-Antitoxin System bsrE/SR5 from Bacillus subtilis

BsrE/SR5 is a new type I toxin/antitoxin system located on the prophage-like region P6 of the Bacillus subtilis chromosome. The bsrE gene encoding a 30-amino acid hydrophobic toxin and the antitoxin gene sr5 overlap at their 3′ ends by 112 bp. Overexpression of bsrE causes cell lysis on agar plates. Here, we present a detailed in vitro analysis of bsrE/SR5. The secondary structures of SR5, bsrE mRNA, and the SR5/bsrE RNA complex were determined. Apparent binding rate constants (k_app) of wild-type and mutated SR5 species with wild-type bsrE mRNA were calculated, and SR5 regions required for efficient inhibition of bsrE mRNA narrowed down. In vivo studies confirmed the in vitro data but indicated that a so far unknown RNA binding protein might exist in B. subtilis that can promote antitoxin/toxin RNA interaction. Using time course experiments, the binding pathway of SR5 and bsrE RNA was elucidated. A comparison with the previously well characterized type I TA system from the B. subtilis chromosome, bsrG/SR4, reveals similarities but also significant differences.     

Inhibition of gene expression with ribozymes

Summary 1. Ribozymes can be designed to cleavein trans, i.e. several substrate molecules can be turned over by one molecule of the catalytic RNA. Only small molecular weight ribozymes, or small ribozymes, are discussed in this review with particular emphasis on the hammerhead ribozyme as this has been most widely used for the inhibition of gene expression by cleavage of mRNAs.2. Cellular delivery of the ribozyme is of crucial importance for the success of inhibition of gene expression by this methodology. Two modes of delivery can be envisaged, endogenous and exogenous delivery. Of the former several variants exist, depending on the vector used. The latter is still in its infancy, even though chemical modification has rendered such ribozymes resistant against degradation by serum nucleases without impairment of catalytic efficiency.3. Various successful applications of ribozymes for the inhibition of gene expression are discussed, with particular emphasis on HIV1 and cancer targets. These examples demonstrate the promise of this methodology.