生物文献汇报

1、 functional genomics: the functional genomics: the coming of age for tetrahymena coming of age for tetrahymena thermophilathermophila aaron p. turkewitz, eduardo orias and geoffrey kapler tetrahymena thermophila has been proven to be a valuable biological model for molecular and geneticstudies of eu

2、karyotic 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 micr

3、onucleusand 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 functionall

4、y distinct nuclei 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

5、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 a

6、cortically-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

7、 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 o

8、f this organism.experimental investigation of the gene of experimental investigation of the gene of interestinterestoverexpression antisense inhibition germline knockout or gene replacement heterokaryon analysis somatic knockout or gene replacement (phenotypic assortment) gene knock-in inducible pro

9、moterversatility of dna transformation methodsversatility 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 differen

10、t 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 transforma

11、tion 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 genefor want of space, we have not d

12、iscussed a for want of space, we have not discussed a numbernumberof ongoing developments in of ongoing developments in tetrahymena that tetrahymena that willwilladvance the utility of this organism for advance the utility of this organism for experimentalexperimentalbiologists further. these includ

13、e extensive biologists further. these include extensive physicalphysicaland genetic mapping projects, and the and genetic mapping projects, and the generation and large-scale sequencing of generation and large-scale sequencing of expressed sequence tag (est) libraries. expressed sequence tag (est) l

14、ibraries. t. t. thermophila is athermophila is a highly developed unicell, highly developed unicell, belonging to a clade of ancient lineage whose belonging to a clade of ancient lineage whose cellular complexity rivals that of highly cellular complexity rivals that of highly differentiated tissues.

15、differentiated tissues.it is precisely this complexity, the presence of it is precisely this complexity, the presence of features that are characteristic of metazoans features that are characteristic of metazoans but that are absent or less easily studied in but that are absent or less easily studie

16、d in other simple eukaryotic systems,other simple eukaryotic systems,that 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

17、dnaor somatic differentiation, programmed dnarearrangements, distribution of mitotic rearrangements, distribution of mitotic chromosomes, histone modifications, microtubule chromosomes, histone modifications, microtubule diversity and tubulin modifications, basal body diversity and tubulin modificat

18、ions, basal body duplication, phagocytosis, function of rrna, and duplication, phagocytosis, function of rrna, and regulated polypeptide secretion. these regulated polypeptide secretion. these biological features and the functional genomics biological features and the functional genomics tools descr

19、ibed in this article will greatly tools described in this article will greatly enhance the value of the genome sequencing enhance the value of the genome sequencing project, currently underproject, currently underadvanced planning by the ciliate research advanced planning by the ciliate research com

20、munitycommunityfig. 3. phenotypic assortment fig. 3. phenotypic assortment illustrated during two cell cycles. 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

21、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

22、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

23、disrupted allelewill be selected for; if the disrupted gene is essential, both alleles will bemaintained by balanced selection.rdna vectors: vectors based on rdna vectors: vectors based on the rdna replicon. the rdna replicon. because the rdna chromosome is maintained at 9000 copies in wild-type cel

24、ls,rdna vectors will similarly be present at high copy number. rdna(rrna-encoding rdna(rrna-encoding dnadna）rdnardna是大核形成过程中由单一拷贝的小核是大核形成过程中由单一拷贝的小核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；同源性很高，没有外源基因的插入，是基因工程的同源性很高，没有外源基因的插入，是基因工程的良好材料；良好材料； 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2

25、1kb21kb； 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（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：本来基因表达是受到各种内外信号的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。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。基因表达产物超过正常水平。反义抑制（反义抑制（antisense inhibition） ：主要相对于主要相对于rnarna来来说，说，dnadna分为正义链、反义链，相对分为正义链、反义链，相对转