生物文献汇报.ppt
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1、 Functional genomics:the ing 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 provid
2、ed 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 mean
3、s 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 Tetrahymen
4、a,at the pletion 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 recogni
5、zes-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
6、 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 DNA mediated cell tran
7、sformation.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
8、 gene replacement(phenotypic assortment)Gene knock-in Inducible promoterVersatility 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 tar
9、geted 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,diff
10、erent transformation methods can be bined 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,w
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