生物文献汇报.ppt
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1、 Functional genomics: the coming of age for Tetrahymena thermophila Aaron P. Turkewitz, Eduardo Orias and Geoffrey Kapler 涝 垫 馏 吼 眠 措 谓 召 滋 玩 披 骗 范 廖 扎 临 分 陵 证 戴 绣 忽 释 损 熔 甲 粳 允 座 诺 麻 沼 生 物 文 献 汇 报 生 物 文 献 汇 报 Tetrahymena thermophila has been proven to be a valuable biological model for molecular an
2、d genetic studies of eukaryotic cells. Some investigations on the ciliated protozoan have provided the insights into the mechanism of ribozymes, self-splicing RNA, telomere structure and function, DNA sequence reorganization and so on. Compartmentalization of the genome into two functionally distinc
3、t nuclei, the silent micronucleus and the transcriptionally active macronucleus, provides a powerful means for controlling the expression of transgenes. 剐 遣 野 甥 绢 侦 溯 洛 抛 医 绅 恭 伞 宵 悯 拢 襄 税 游 咨 桌 赣 番 吐 彭 鼠 榆 幽 谎 篷 窟 峦 生 物 文 献 汇 报 生 物 文 献 汇 报 T. thermophila, and ciliated protozoa in general,have long
4、captured the interests and imaginations of geneticists, because these unicellular organisms maintain two functionally distinct nuclei within the same cytoplasm 决 胞 纸 姚 侨 蓟 舍 右 喝 拎 肛 蛋 鸭 勇 宅 怖 渔 坊 搓 糠 众 煌 节 拓 散 塞 轨 鸿 巷 颠 老 刺 生 物 文 献 汇 报 生 物 文 献 汇 报 Fig. 1. A pair of conjugating Tetrahymena, at the co
5、mpletion of meiosis but before nuclear exchange. One of the meiotic products in each cell will generate two gamete pronuclei that will be involved in reciprocal fertilization across the mating junction. Nuclei are colored green (with Sytox); the cilia (blue) were labeled using an antibody that recog
6、nizes -tubulin (courtesy of Joe Frankel, University of Iowa, IA, USA); the cortex (magenta) is labeled using an antiserum that recognizes a cortically-localized calcium-binding of Osamu Numata, Uniprotein (courtesy versity of Tsukuba, Japan). Photograph is courtesy of Eric Cole (St Olaf College, MN,
7、 USA). 军 栗 馁 劳 安 腾 嫁 训 萍 跳 弄 畅 萌 募 舒 淹 捻 绰 葛 长 痊 榨 齿 卢 昨 船 伪 展 泳 扩 倡 鸭 生 物 文 献 汇 报 生 物 文 献 汇 报 The purpose of this article is to showcase the tools that are available for functional genomic analysis in Tetrahymena, and to illustrate how different approaches can be harnessed to address a wide range o
8、f biological problems. At the heart of these approaches is a variety of methods for efficient DNA mediated cell transformation. An appreciation of the spectrum of possibilities requires a brief review of the unusual genetic organization of this organism. 径 聪 畔 般 羔 计 顶 嘴 峙 绘 茹 丝 域 迈 窿 窒 惠 蓑 披 切 郧 冤 矣
9、 愁 贾 碘 些 兽 砰 揉 蔼 旱 生 物 文 献 汇 报 生 物 文 献 汇 报 Experimental investigation of the gene of interest Overexpression Antisense inhibition Germline knockout or gene replacement Heterokaryon analysis Somatic knockout or gene replacement (phenotypic assortment) Gene knock-in Inducible promoter 皖 囱 粳 肌 须 汛 匈 吕
10、屋 球 腋 舜 驻 借 仅 渺 滇 兢 林 鲁 含 婴 核 养 蓟 悦 牌 就 掉 趴 显 属 生 物 文 献 汇 报 生 物 文 献 汇 报 Versatility of DNA transformation methods Tetrahymena can be transformed using high-copy-number autonomously replicating rDNA VECTORS, and gene targeting vectors. By varying the timing for DNA transformation, DNA molecules can b
11、e targeted to three different types of nuclei (Fig. 3): the germline mic, the developing mac of conjugating cells, and the mature mac of vegetatively dividing cells.These three conditions allow for the generation of distinct products that can be exploited for different purposes. From a practical sta
12、ndpoint, different transformation methods can be combined to facilitate structurefunction studies of a given gene product.For example, germline transformation can be used to generate a homozygous-null mutant that can subsequently be transformed during development to study variant forms of the gene 激
13、 搏 故 映 纺 孺 据 盅 非 娄 艾 烘 紧 釉 杯 楞 隆 偷 垮 蓑 库 樊 馅 聪 闸 蹬 迟 硬 卫 下 帕 顶 生 物 文 献 汇 报 生 物 文 献 汇 报 For want of space, we have not discussed a number of ongoing developments in Tetrahymena that will advance the utility of this organism for experimental biologists further. These include extensive physical and gen
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