《分子生物学》2-cha.ppt

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1、CHAPTER 21Model OrganismsFundamental problems are solved in the simplest and most accessible system.These organisms are called model organisms.Features of Model SystemsnThe availability of powerful tools of traditional and molecular genetics.nThe study of each model system attracted a critical mass

2、of investigators.(Ideas,methods,tools and strains could be shared)E.coli,T4-ideal system for tackling fundamental aspects of the nature of the gene and information transferYeast-premier system for elucidating fundamental aspects of the eukaryotic cell,powerful mating system for genetic analysisNemat

3、ode,fruit flywell developed genetic systemMouse-best model sytem for gaining insights into human biology and human disease Features of bacteriana single chromosomen(E.coli 4.64 Mb,4397 genes)na short generation time(30min)nconvenient to study geneticallyBacteria Exchange DNA by:Sexual Conjugation(性结

4、合性结合)Phage-Mediated Transduction(转导转导)DNA-Mediated Transformation(转化转化)Bacteria:center for biochemical study of DNA replication,information transfer,gene regulation Model 3:BAKERS YEASTModel 3:BAKERS YEASTSaccharomyces cerevisiaeSaccharomyces cerevisiaeFeatures of S.cerevisiaeHave small genomes(12.1

5、Mb,about 6000 genes)grown rapidly in the lab(90 min per cell division)have central characteristics of all eukaryotic cells:1.contain a discrete(独立的独立的)nucleus:multiple linear chromosomes packaged into chromatin;2.cytoplasm includes a full intracellular organelles and cytoskeletal structures.The Exis

6、tence of Haploid and Diploid Cells Facilitate Genetic Analysis of S.cerevisiae Conversion between the two states is mediated by mating(haploid to diploid)and sporulation(diploid to haploid).S.Cerevisiae exist in three forms:two haploid cell types:a,One diploid cell:a/product of mating.Application in

7、 the LabGenetic complementation:by mating two haploid strains,each contains one of the two mutations whose complementation is being tested.Testing the function of an individual gene:mutations can be made in haploid cells in which there is only a single copy of that gene.Generating Precise Mutations

8、in Yeast Is Easy When linear DNA with ends homologous to any given region of the genome is introduced into S.cerevisiae cells,very high rates of homologous recombination are observed resulting in the transformation.Gene inactivation by homologus recombinationFigure 21-11 Recombinational transformati

9、on in yeastThis property can be used to make precise changes within the genome,allowing very detailed questions to be elucidated.delete an entire gene or change a base pair(in coding region or promoter)The use of a yeast deletion cassette（缺失盒两端连上新基因（缺失盒两端连上新基因DNA片段作为末端）片段作为末端）S.Cerevisiae Has a Smal

10、l,Well-Characterized Genome S.Cerevisiae was the first eukaryotic organism to have its genome entirely sequenced.(1996)1.3X106 bp,approximately 6,000 genes.The availability of the complete genome sequence has allowed“genome-wide”approaches to studies of this organism.Categories of gene in the yeast

11、genomenSingle orphans-看起来像基因，看起来像基因，但目前还没有同源基因但目前还没有同源基因nOrphans families-有同源基因，但有同源基因，但这些同源基因的功能不知这些同源基因的功能不知Yeast 6000 genes,30%genetic techniques 30%homology analysis 40%?nMixed yeast population:each cell contain a single inactivated gene by Tyl moves into different position in different cells(10

12、11)in a culture medium containing galactose induce the retrotransposonn在不同的条件下生长，确定不能最快生长的细胞。在不同的条件下生长，确定不能最快生长的细胞。PCR 扩增失活基因的扩增失活基因的DNA(Tyl primer),不利条件时不利条件时,PCR产物的丰度随时间而下降产物的丰度随时间而下降Genetic footprinting of yeast chromosome V Mixed yeast population:each contain a single inactivated gene In a cultu

13、re medium containing galactose Tyl moves into different position in different cells(1011).每个基因都有失活的拷每个基因都有失活的拷贝贝n在不同的条件下生长在不同的条件下生长，确定不能最快，确定不能最快生长的细胞。生长的细胞。nPCR PCR 扩增失活基因的扩增失活基因的DNA,PCRDNA,PCR产物产物的丰度随时间而下降的丰度随时间而下降酵母第酵母第3号染色体号染色体2167个密码子的个密码子的ORF，具有典型的酵母密码子倾向，具有典型的酵母密码子倾向，是功能基因，不是假是功能基因，不是假ORF。这个基

14、因失活突变株表型与正常株一样。也。这个基因失活突变株表型与正常株一样。也许这个基因是非必须的，其功能不重要。后来发现只有在许这个基因是非必须的，其功能不重要。后来发现只有在glucose和和 HAc 低低pH环境下，突变株死亡，正常株存活。该基因编码的蛋白可把环境下，突变株死亡，正常株存活。该基因编码的蛋白可把HAc 泵出细胞，细胞免受损害。泵出细胞，细胞免受损害。n有时从表型检查中很难发现新基因的功能有时从表型检查中很难发现新基因的功能Genetic footprinting of yeast chromosome V 5 5 号染色体号染色体268268个基因：个基因：大多数基因失活会导致

15、酵母细胞生长不良大多数基因失活会导致酵母细胞生长不良 51 51 个基因失活导致个基因失活导致主要主要生长障碍（生长减慢生长障碍（生长减慢25%25%以上）以上）提提 示示:这些基因编码重要蛋白质。有些基因功能已被确定这些基因编码重要蛋白质。有些基因功能已被确定 如氨基酸合成酶基因，如氨基酸合成酶基因，DNADNA修复蛋白基因等修复蛋白基因等1414个孤儿个孤儿 基因。有些孤儿基因失活只有在特殊情况下才会导致基因。有些孤儿基因失活只有在特殊情况下才会导致 生长不良。如在富含醋酸的培养基生长不良。如在富含醋酸的培养基 就涉及就涉及3 3个孤儿基因，个孤儿基因，高盐耐受，涉及高盐耐受，涉及2 2个

16、孤儿基因。个孤儿基因。酵母双杂交系统检测蛋白质相互作用酵母双杂交系统检测蛋白质相互作用n转录因子转录因子 调节真核生物的基因表达，它与靶基因上游的调节真核生物的基因表达，它与靶基因上游的DNADNA结合结合，n激活激活RNARNA聚合酶聚合酶将基因转录为将基因转录为RNARNA。n转录因子两部分由不同基因编码。转录因子两部分由不同基因编码。酵母双杂交系统：酵母双杂交系统：1.1.缺少报道基因所需的转录因子的酵母株缺少报道基因所需的转录因子的酵母株 2.DNA 2.DNA结合结构域基因与人的结合结构域基因与人的X X蛋白蛋白基因相连基因相连 3.3.激活结构域基因与不同的激活结构域基因与不同的D

17、NADNA片段相连片段相连-许多克隆许多克隆 4.4.将将2 2和和3 3共转化共转化1 1，如果，如果X X蛋白蛋白与与DNADNA片段表达的片段表达的y y蛋白蛋白有有 相互作用，报道基因开启（可看到绿色萤光，或蓝斑）相互作用，报道基因开启（可看到绿色萤光，或蓝斑）S.Cerevisiae Cells Change Shape as They GrowS.Cerevisiae divides by budding.The bud grows until it reaches a size approximately equal to the size of the mother cell

18、and is released from it.The emergence of a new bud is tightly connected to the initiation of DNA replication.Start replicating its genomeChromosome segregationContribution to our understanding:eukaryotic transcription gene regulation,DNA replication,recombination,translation and spilicingModel 4:THE

19、 NEMATODE WORM,Caenorhabditis elegans多细胞生物多细胞生物Caenorhabditis elegansSuitable characteristics:nRapid generation timenHermaphrodite(雌雄同体的雌雄同体的)reproduction producing large numbers of“self-progeny”nSexual reproduction so that genetic stocks could be constructednA small number of transparent cells so t

20、hat development could be followed directlyover 300:morphological and behavioral mutantsover 100:complementation groups mapping to 6 linkage groups30 years later:400 laboratories(worldwide)study C elegansC.elegans Has a Very Raplid Life CycleC.elegans is cultured on petri dishes,fed a simple diet of

21、bacteria and grow well at 25C.雌雄同体成虫雌雄同体成虫4 4 天内产生天内产生300300个自身后代个自身后代或与罕见雄性交配产生或与罕见雄性交配产生10001000多个后代多个后代The lifecycle of the C.elegans12 hEggsJuvenileAdultDeath12h40h15dunder stressful condition:low food,increased temperatures,high population dencityDauer larva(持续幼虫持续幼虫):):resistant to environment

22、al stresses living many months while waiting for environmental conditions to improveFigure 21-14 b The body plan of the wrompharynx:咽咽oocyte:卵母细胞卵母细胞 intestine:肠肠 uterus:子宫子宫vulva:阴孔阴孔 gonad:生殖腺生殖腺anus:肛门肛门Prominent organ in the adult hermaphrodite is the gonad(生殖腺)which contains the proliferating a

23、nd differentiating germ cells(生殖细胞sperm and oocytes精子 和卵子),fertilization chamber uterus for temporary storage of young embryosThe embryos pass from the uterus to the outside through the vulva(22 epidermal cells)Mutations that disrupt the formation of the vulva do not interfere with production of emb

24、ryos,but do preventthe eggs from being laid So form a“bag of worms”(the hatched worms devour their mother and become trapped inside her skin).This readily identified phenotype has allowed the isolation of hundreds of vulva-less mutants,so identifying many genes that function to control the generatio

25、n specification and differentiation of the vulva cellsThe genes are components of a highly conserved receptor tyrosine kinase signaling pathway that controls cell proliferation.nMutations that inactivate this pathway eliminate vulva development.nMutations that activate this pathway cause overprolife

26、ration of the vulva precursor cells,resulting a multiple vulva phenotype.The cell Death Pathway Was Discovered in C.elegansThe most notable achievement to date in C.elegans research has been the elucidation of the molecular pathway that regulates apoptosis or cell deathAnalysis of cell lineages:cell

27、 death is under genetic control ced mutant cell corpses persited for many hoursAnalysis of the ced mutants showed:that the cell commits suicide.Molecular identification of the Ced genes:provided the means to identify proteins in mammals that carry out essentially the identical biochemical reactions

28、to control cell death in all animalsRNAi Was Discovered in C.elegansIn 1998,RNAi was discovered in C.elegans,which is significant in two respects:1.RNAi appears to be universal.2.Experimental investigation reveals the molecular mechanisms.dsRNA-silenced homologous geneEnzyme Dicer makes siRNAssiRNAs

29、 direct a complex called RISC to repress gene in three waysTranslational inhibitionMotifying promotersDigesting mRNAtiny endogenous microRNA:1.regulate gene expression in plants,animals2.Genome rearragement in ciliates3.regulate chromatin structure in yeastThe first two microRNA were discovered in C.elegans Model 5:THE FRUIT FLYDrosophila melanogaster