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1、CHAPTER 21Model Organisms,Fundamental problems are solved in the simplest and most accessible system.These organisms are called model organisms.,Features of Model Systems,The availability of powerful tools of traditional and molecular genetics.The 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 analysisNem
3、atode,fruit flywell developed genetic systemMouse-best model sytem for gaining insights into human biology and human disease,Features of bacteria,a single chromosome(E.coli 4.64 Mb,4397 genes)a short generation time(30min)convenient to study genetically,Bacteria Exchange DNA by:,Sexual Conjugation(性
4、结合)Phage-Mediated Transduction(转导)DNA-Mediated Transformation(转化),Bacteria:center for biochemical study of DNA replication,information transfer,gene regulation,Model 3:BAKERS YEASTSaccharomyces cerevisiae,Features of S.cerevisiae,Have small genomes(12.1Mb,about 6000 genes)grown rapidly in the lab(90
5、 min per cell division)have central characteristics of all eukaryotic cells:1.contain a discrete(独立的)nucleus:multiple linear chromosomes packaged into chromatin;2.cytoplasm includes a full intracellular organelles and cytoskeletal structures.,The Existence of Haploid and Diploid Cells Facilitate Gen
6、etic 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 the Lab,Genetic complementation:by mating two
7、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 in Yeast Is Easy,When linear DNA with ends ho
8、mologous 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 recombination,Figure 21-11 Recombinational transformation in yeast,This property can be used to ma
9、ke 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DNA片段作为末端）,S.Cerevisiae Has a Small,Well-Characterized Genome,S.Cerevisiae was the first euk
10、aryotic 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 genome,Single orphans-看起来像基因，但目前还没有同源基因Orphans families-有
11、同源基因，但这些同源基因的功能不知,Yeast 6000 genes,30%genetic techniques 30%homology analysis 40%?Mixed yeast population:each cell contain a single inactivated gene by Tyl moves into different position in different cells(1011)in a culture medium containing galactose induce the retrotransposon在不同的条件下生长，确定不能最快生长的细胞。P
12、CR 扩增失活基因的DNA(Tyl primer),不利条件时,PCR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 culture medium containing galactose Tyl moves into different position in different cells(1011).每个基因都有失活的拷贝,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DNA
13、,PCR产物的丰度随时间而下降,酵母第3号染色体2167个密码子的ORF，具有典型的酵母密码子倾向，是功能基因，不是假ORF。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glucose和 HAc 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HAc 泵出细胞，细胞免受损害。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 号染色体268个基因：大多数基因失活会导致酵母细胞生长不良 51 个基因失活导致主要生长障碍（生长减慢25%以上）提 示:这些基因编码重要蛋白
14、质。有些基因功能已被确定 如氨基酸合成酶基因，DNA修复蛋白基因等14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 生长不良。如在富含醋酸的培养基 就涉及3个孤儿基因，高盐耐受，涉及2个孤儿基因。,酵母双杂交系统检测蛋白质相互作用,转录因子 调节真核生物的基因表达，它与靶基因上游的DNA结合，激活RNA聚合酶将基因转录为RNA。转录因子两部分由不同基因编码。酵母双杂交系统：1.缺少报道基因所需的转录因子的酵母株 2.DNA结合结构域基因与人的X蛋白基因相连 3.激活结构域基因与不同的DNA片段相连-许多克隆 4.将2和3共转化1，如果X蛋白与DNA片段表达的y蛋白有 相互作用，报道基
15、因开启（可看到绿色萤光，或蓝斑）,S.Cerevisiae Cells Change Shape as They Grow,S.Cerevisiae divides by budding.The bud grows until it reaches a size approximately equal to the size of the mother cell and is released from it.The emergence of a new bud is tightly connected to the initiation of DNA replication.,Start r
16、eplicating its genome,Chromosome segregation,Contribution to our understanding:eukaryotic transcription gene regulation,DNA replication,recombination,translation and spilicing,Model 4:THE NEMATODE WORM,Caenorhabditis elegans多细胞生物,Caenorhabditis elegans,Suitable characteristics:Rapid generation timeH
17、ermaphrodite(雌雄同体的)reproduction producing large numbers of“self-progeny”,Sexual reproduction so that genetic stocks could be constructedA small number of transparent cells so that development could be followed directly,over 300:morphological and behavioral mutantsover 100:complementation groups mapp
18、ing to 6 linkage groups30 years later:400 laboratories(worldwide)study C elegans,C.elegans Has a Very Raplid Life Cycle,C.elegans is cultured on petri dishes,fed a simple diet of bacteria and grow well at 25C.雌雄同体成虫4 天内产生300个自身后代或与罕见雄性交配产生1000多个后代,The lifecycle of the C.elegans,12 h,Eggs,Juvenile,Ad
19、ult,Death,12h,40h,15d,under stressful condition:low food,increased temperatures,high population dencityDauer larva(持续幼虫):resistant to environmental stresses living many months while waiting for environmental conditions to improve,Figure 21-14 b The body plan of the wrom,pharynx:咽oocyte:卵母细胞 intestin
20、e:肠 uterus:子宫vulva:阴孔 gonad:生殖腺anus:肛门,Prominent organ in the adult hermaphrodite is the gonad(生殖腺)which contains the proliferating and 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 o
21、utside through the vulva(22 epidermal cells),Mutations that disrupt the formation of the vulva do not interfere with production of embryos,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
22、 phenotype has allowed the isolation of hundreds of vulva-less mutants,so identifying many genes that function to control the generation specification and differentiation of the vulva cellsThe genes are components of a highly conserved receptor tyrosine kinase signaling pathway that controls cell pr
23、oliferation.,Mutations that inactivate this pathway eliminate vulva development.Mutations that activate this pathway cause overproliferation of the vulva precursor cells,resulting a multiple vulva phenotype.,The cell Death Pathway Was Discovered in C.elegans,The most notable achievement to date in C
24、.elegans research has been the elucidation of the molecular pathway that regulates apoptosis or cell deathAnalysis of cell lineages:cell death is under genetic control ced mutant cell corpses persited for many hoursAnalysis of the ced mutants showed:that the cell commits suicide.,Molecular identific
25、ation of the Ced genes:provided the means to identify proteins in mammals that carry out essentially the identical biochemical reactions to control cell death in all animals,RNAi Was Discovered in C.elegans,In 1998,RNAi was discovered in C.elegans,which is significant in two respects:1.RNAi appears
26、to be universal.2.Experimental investigation reveals the molecular mechanisms.,dsRNA-silenced homologous gene,Enzyme Dicer makes siRNAs,siRNAs direct a complex called RISC to repress gene in three ways,Translational inhibition,Motifying promoters,Digesting mRNA,tiny 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,
