《生物文献汇报》PPT课件.ppt

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1、 Functional genomics:the coming of Functional genomics:the coming of age for Tetrahymena thermophilaage 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 eukary

2、otic 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 micronucleusan

3、d 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 nuc

4、lei within the same cytoplasm Fig.1.A pair of conjugating Fig.1.A pair of conjugating Tetrahymena,at the completion of Tetrahymena,at the completion of meiosismeiosisbut before nuclear exchange.One of the meiotic products in each cellwill generate two gamete pronuclei that will be involved in recipr

5、ocalfertilization 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-bind

6、ing 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

7、 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

8、of the gene of interestExperimental 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 promoterVersatility of DNA transformation method

9、sVersatility 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 de

10、veloping 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 facilitatestructurefunc

11、tion 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 geneFor want of space,we have not discussed a numberFor want of space,we have not discussed

12、a numberof ongoing developments in of ongoing developments in Tetrahymena that willTetrahymena that willadvance the utility of this organism for experimentaladvance the utility of this organism for experimentalbiologists further.These include extensive physicalbiologists further.These include extens

13、ive physicaland genetic mapping projects,and the generation and and genetic mapping projects,and the generation and large-scale sequencing of expressed sequence tag large-scale sequencing of expressed sequence tag(EST)libraries.(EST)libraries.T.thermophila is aT.thermophila is a highly developed hig

14、hly developed unicell,belonging to a clade of ancient lineage whose unicell,belonging to a clade of ancient lineage whose cellular complexity rivals that of highly differentiated cellular complexity rivals that of highly differentiated tissues.tissues.It is precisely this complexity,the presence of

15、features It is precisely this complexity,the presence of features that are characteristic of metazoans but that are absent that are characteristic of metazoans but that are absent or less easily studied in other simple eukaryotic systems,or less easily studied in other simple eukaryotic systems,that

16、 makes that makes Tetrahymena so well suited for a wideTetrahymena so well suited for a widerange of fundamental problems including germlinerange of fundamental problems including germlineor somatic differentiation,programmed DNAor somatic differentiation,programmed DNArearrangements,distribution of

17、 mitotic chromosomes,rearrangements,distribution of mitotic chromosomes,histone modifications,microtubule diversity and tubulin histone modifications,microtubule diversity and tubulin modifications,basal body duplication,phagocytosis,modifications,basal body duplication,phagocytosis,function of rRNA

18、,and regulated polypeptide secretion.function of rRNA,and regulated polypeptide secretion.These biological features and the functional genomics These biological features and the functional genomics tools described in this article will greatly enhance the tools described in this article will greatly

19、enhance the value of the genome sequencing project,currently undervalue of the genome sequencing project,currently underadvanced planning by the ciliate research communityadvanced planning by the ciliate research communityThank You!Thank You!Fig.3.Phenotypic assortment illustrated during Fig.3.Pheno

20、typic assortment illustrated during two cell cycles.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 ar

21、e 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 all

22、ele 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 bema

23、intained by balanced selection.rDNA vectors:Vectors based on the rDNA vectors:Vectors based on the rDNA replicon.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 rDNA(rRNA-encoding

24、DNADNA）rDNArDNA是是大大核核形形成成过过程程中中由由单单一一拷拷贝贝的的小小核核rDNArDNA复复制而成的，游离于大核染色体外的回文二聚体；制而成的，游离于大核染色体外的回文二聚体；同同源源性性很很高高，没没有有外外源源基基因因的的插插入入，是是基基因因工工程程的的良良好材料；好材料；rDNArDNA位于核内自主复制的位于核内自主复制的DNADNA上，长度约为上，长度约为21kb21kb；rDNArDNA不同的遗传等位基因表现出不同的复制特点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（

25、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（过表达（Overexpression）：本来：本来基因表达是受到各种内外信号的精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，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，最终基因因被过度的转录、翻译，最终基因表达产物超过正常水平。表达产物超过正常水平。反义抑制（反义抑制（Antisense inhibition）：主要

26、相对于主要相对于RNARNA来说，来说，DNADNA分为正义分为正义链、反义链，相对转录出来的链、反义链，相对转录出来的RNARNA也分为正义也分为正义RNARNA、反义、反义RNARNA。反义。反义RNARNA是一种能与是一种能与mRNAmRNA分子互补的分子互补的RNA,RNA,可特异抑制某一可特异抑制某一mRNAmRNA的加工、的加工、翻译以及翻译以及DNADNA的复制的复制,特异阻断该基特异阻断该基因的表达。因的表达。基因敲除基因敲除是基因打靶技术的一种，类似于基是基因打靶技术的一种，类似于基因的同源重组。指外源因的同源重组。指外源DNADNA与受体细胞基因与受体细胞基因组中序列相同或

27、相近的基因发生同源重组，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从而代替受体细胞基因组中的相同/相似的相似的基因序列，整合入受体细胞的基因组中。此基因序列，整合入受体细胞的基因组中。此法可产生精确的基因突变，也可正确纠正机法可产生精确的基因突变，也可正确纠正机体的基因突变。体的基因突变。基因嵌入基因嵌入又称基因置换，它是利用内源基因又称基因置换，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。的基因整个置换内源基因。基因敲入基因敲入（gene knock ingene knock in）是利用）是利用基因同源重组，将外源有功能基因基因同源重组，将外源有功能基因（基因组原先不存在、或已失活的（基因组原先不存在、或已失活的基因），转入细胞与基因组中的同基因），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。组中，在细胞内获得表达的技术。诱导型启动子诱导型启动子（inducible promoter）能被诱导表达的基因启动子。该启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。间接激活该启动子的转录。