《生物文献汇报》PPT课件.ppt

Functional genomics:the coming of age for Tetrahymena thermophila Aaron P.Turkewitz,Eduardo Orias and Geoffrey Kapler,Tetrahymena thermophila has been proven to be a valuable biological model for molecular and geneticstudies of eukaryotic cells.Some investigations on the ciliated protozoan have provided the insights into themechanism of ribozymes,self-splicing RNA,telomere structure and function,DNA sequence reorganizationand so on.Compartmentalization of the genome into two functionally distinct nuclei,the silent micronucleusand the transcriptionally active macronucleus,provides a powerful means for controlling the expression oftransgenes.,T.thermophila,and ciliated protozoa in general,have long captured the interests and imaginations of geneticists,because these unicellular organisms maintain two functionally distinct nuclei within the same cytoplasm,Fig.1.A pair of conjugating Tetrahymena,at the completion of meiosisbut before nuclear exchange.One of the meiotic products in each cellwill generate two gamete pronuclei that will be involved in reciprocalfertilization across the mating junction.Nuclei are colored green(withSytox);the cilia(blue)were labeled using an antibody that recognizes-tubulin(courtesy of Joe Frankel,University of Iowa,IA,USA);thecortex(magenta)is labeled using an antiserum that recognizes acortically-localized calcium-binding of Osamu Numata,Uniprotein(courtesy versity of Tsukuba,Japan).Photograph is courtesy ofEric Cole(St Olaf College,MN,USA).,The purpose of this article is to showcase the toolsthat are available for functional genomic analysis inTetrahymena,and to illustrate how differentapproaches can be harnessed to address a widerange of biological problems.At the heart of theseapproaches is a variety of methods for efficient DNAmediated cell transformation.An appreciation of thespectrum of possibilities requires a brief review of theunusual genetic organization of this organism.,Experimental investigation of the gene of interestOverexpression Antisense inhibition Germline knockout or gene replacement Heterokaryon analysis Somatic knockout or gene replacement(phenotypic assortment)Gene knock-in Inducible promoter,content,Versatility of DNA transformation methodsTetrahymena can be transformed using high-copy-numberautonomously replicating rDNA VECTORS,and gene targeting vectors.By varying the timing for DNA transformation,DNA molecules can be targeted to three different types of nuclei(Fig.3):the germline mic,the developing mac of conjugating cells,andthe 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 standpoint,different transformation methods can be combined to facilitatestructurefunction 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,For want of space,we have not discussed a numberof ongoing developments in Tetrahymena that willadvance the utility of this organism for experimentalbiologists further.These include extensive physicaland genetic mapping projects,and the generation and large-scale sequencing of expressed sequence tag(EST)libraries.T.thermophila is a highly developed unicell,belonging to a clade of ancient lineage whose cellular complexity rivals that of highly differentiated tissues.,It is precisely this complexity,the presence of features that are characteristic of metazoans but that are absent or less easily studied in other simple eukaryotic systems,that makes Tetrahymena so well suited for a widerange of fundamental problems including germlineor somatic differentiation,programmed DNArearrangements,distribution of mitotic chromosomes,histone modifications,microtubule diversity and tubulin modifications,basal body duplication,phagocytosis,function of rRNA,and regulated polypeptide secretion.These biological features and the functional genomics tools described in this article will greatly enhance the value of the genome sequencing project,currently underadvanced planning by the ciliate research community,Thank You!,Fig.3.Phenotypic assortment illustrated during two cell cycles.Thetop circle represents a heterozygous G1 macronucleus(mac)generatedeither after a cross or after DNA-mediated transformation followed byintegrative recombination.For clarity,the mac shows four allele copies(instead of 45):three are wild type and the fourth is mutant;forexample,a knockout allele where the gene is disrupted by insertion ofa neomycin-resistance cassette(red).The fused ovals represent macsundergoing division after DNA replication.Note that allele copies arepartitioned randomly at each mac division.If neither allele has selectiveadvantage,assortants pure for either allele are ultimately generated.In the presence of neomycin,vegetative descendants that acquire(bychance alone)a higher fraction of mac copies with the disrupted allelewill be selected for;if the disrupted gene is essential,both alleles will bemaintained by balanced selection.,rDNA vectors:Vectors based on the rDNA replicon.Because the rDNA chromosome is maintained at 9000 copies in wild-type cells,rDNA vectors will similarly be present at high copy number.,rDNA(rRNA-encoding DNA）,rDNA是大核形成过程中由单一拷贝的小核rDNA复制而成的，游离于大核染色体外的回文二聚体；同源性很高，没有外源基因的插入，是基因工程的良好材料；rDNA位于核内自主复制的DNA上，长度约为21kb；rDNA不同的遗传等位基因表现出不同的复制特点:当不同的rDNA等位基因进行比较时，序列的差异会显示出来，这些重复的保守序列主要位于5-非转录区（5-NTS）的复制原点，因此，这些序列被推测是复制的阳性调控因子。,过表达（Overexpression）：本来基因表达是受到各种内外信号的精确调控的，一旦这种调控机制的任何一个环节出现问题就会失控。基因的表达过程就可能进入失控状态，就有可能表现为表达过度，即该基因被过度的转录、翻译，最终基因表达产物超过正常水平。,反义抑制（Antisense inhibition）：主要相对于RNA来说，DNA分为正义链、反义链，相对转录出来的RNA也分为正义RNA、反义RNA。反义RNA是一种能与mRNA分子互补的RNA,可特异抑制某一mRNA的加工、翻译以及DNA的复制,特异阻断该基因的表达。,基因敲除是基因打靶技术的一种，类似于基因的同源重组。指外源DNA与受体细胞基因组中序列相同或相近的基因发生同源重组，从而代替受体细胞基因组中的相同/相似的基因序列，整合入受体细胞的基因组中。此法可产生精确的基因突变，也可正确纠正机体的基因突变。基因嵌入又称基因置换，它是利用内源基因序列两侧或外面的断裂点，用同源序列的目的基因整个置换内源基因。,基因敲入（gene knock in）是利用基因同源重组，将外源有功能基因（基因组原先不存在、或已失活的基因），转入细胞与基因组中的同源序列进行同源重组，插入到基因组中，在细胞内获得表达的技术。,诱导型启动子（inducible promoter）能被诱导表达的基因启动子。该启动子能被诱导物直接激活，或诱导物与启动子上的阻遏物结合，从而间接激活该启动子的转录。,