生物文獻(xiàn)匯報課件

1、 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

2、 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

3、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 conj

4、ugating 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 us

5、ing 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

6、 (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

7、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 replaceme

8、nt Heterokaryon analysis Somatic knockout or gene replacement (phenotypic assortment) Gene knock-in Inducible promotercontentVersatility of DNA transformation methodsTetrahymena can be transformed using high-copy-numberautonomously replicating rDNA VECTORS, and gene targeting vectors. By varying the

9、 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 expl

10、oited 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

11、 development to study variant forms of the geneFor 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 an

12、d 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 characte

13、ristic 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 mod

14、ifications, 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 proj

15、ect, currently underadvanced planning by the ciliate research communityThank 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 r

16、ecombination. 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 replica

17、tion. 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

18、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是大核形成過程中由單一拷貝的小核rDNA復(fù)制而成的，游離于大核染色體外的回文二聚體；同源性很高，沒有外源基因的插入，是基因工程的良好材料； rDNA位于核內(nèi)自主復(fù)制的DNA上，長度約為21kb； rDNA不同的遺傳等位基因表現(xiàn)出不同的復(fù)制特點: 當(dāng)不同的rDNA等位基因進(jìn)行比較時，序列的差異會顯示出來，這些重復(fù)的保守序列主要位于5-非轉(zhuǎn)錄區(qū)（5-NTS）的復(fù)制原點，因此，這些序列被推測