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1、Model 5:THE FRUIT FLY,Drosophila melanogaster,we are approaching the 100th anniversary of the fruit fly as amodel organism for studies in genetics and developmental biology In 1908,Thomas Hunt Morgan and his research associates at Columbia University placed rotting fruit on the window ledge of their
2、 laboratory.,Among the menagerie of creatures that were captured,the fruit fly emerged as the animal of choice.Study the inheritance of quantitative traits,such as eye color.milk bottles,using inexpensive yeast and agarthe fruit fly adults produced large numbers of progeny innjust two weeks,Drosophi
3、la Has a Rapid Life Cycle,One of the key processes that occurs during larval development is the growth of the imaginal disksThe growth of the imaginal disks:arising from invaginations of epidermis in mid-stage embryos.imaginal disks differentiate into their appropriate adult structures during metamo
4、rphosis(or pupation,蛹化),There is a pair of disks for every set of appendagesfor example,a set of foreleg imaginal disks a set of antennae imaginal disks a set of mouthpart imaginal disks-Disks are composed of fewer than 100 cells in the embryo but ten thousands of cells in mature larvae.,Figure 21-1
5、6 Imaginal disks(器官芽)in Drosophila,The wing imaginal disk has become an important model system for the control of complex patterning processes by gradients of secreted signaling molecules.,The First Genome Maps Were Produced in DrosophilaIn 1910 the Morgan lab identified a spontaneous mutant male fl
6、y that had white eyes rather than the brilliant red seen for normal strains.This single fly lanched an incisive series of genetic studies-led two major discoveries:Genes are located on chromosome Each gene is composed of two allels,Two allels assort independently during meiosis(Mendels first law)Tha
7、t genes located on separate chromosomes segregate independently(Mendels second law)Whreras those linked on the same chromosome do not.,Mendels first law:Genes are located on chromosomes,and each gene is composed of two alleles that assort independently during meiosis.等位基因独立分离Mendels second law:Genes
8、 located on separate chromosomes segregate independently 不同染色体上的基因自由组合(形成合子时),Alfred H.Sturtevant(an undergradute at Columbia University,a member of the Morgan lab)developed a simple mathematical algorithm for mapping the distances between linked genes based on recombination frequenciesBy 1930s,exte
9、nsive genetic maps were produced that identified the relative position of numerous gene controlling a variety of physical characteristics of the adult,such as wing size and shape,eye color.,Useful features of the flies in experimental research:FecundityRapid life cycleFour chromosomes(two large auto
10、somes,a smaller X,and a very small fourth chromosome),Polytene chromosomesIn the salivary gland,extensive endoreplication without mitosis,Giant chromosomes composed of approximately 1000 copies of each chromatid-determine a physical map of the Drosophila genome,Genetic map,deficiency mappingfirst co
11、rrelate the occurrence of genes for certain traits with given physical segments of chromosomes,(phenotypes of flies white eyes were correlated with deletions in the chromosomes,Figure 21-17 Polytene chromosomes,Bridges used polytene chormosomes to determine a physical map of the Drosophila genome.id
12、entified 5000“bands”on the four chromosomes and established a correlation between the bands and the locations of genetic loci identified in the classical recombination maps.,For example:,Female fruit flies that are heterozygous for the recessive white mutation exhibit nomal red eyesFemale fruit flie
13、s that contain the white mutation and a small deletion in the other X chromosome,which removes polytene bands 3C2-3C3,exhibit white eyes.This type of analysis led to the conclusion:that the white gene is located between polytene bands 3C2-3C3 on the X chromosome,Balancer chromosomes contain inversio
14、ns,Figure 21-18,Such balancers fail to undergo recombination with the native chromosome.Thus,it is possible to maintain fruit flies that contain recessive,lethal mutations.For example:null allele of eve and a balancer,Embryos that are homozygous for this mutation die and fail to produce viable larva
15、e and adultsThe null mutation can be maintained in a population that is heterozygous for a“nomal”chromosome containing the null allele of eve and a balancer second chromosome which contain a nomal copy of the gene.Since the eve null allele is strictly recessive,these flies are completely viable.,Emb
16、ryos that contain two copies of the balancer chromosome die,because of the invresions produce recessive disruption in critical genes.In addition,embryos that contain two copies of“nomal”chromosome die,because they are homozygous for the eve null mutation,Genetic Mosaics Permit the Analysis of Lethal
17、 Genes in Adult Files,Mosaics are animals that contain small patches of mutant tissue in a generally“normal”genetic background.,For example:Small pathes engrailed/engrailed homozygous mutant tissue can be produced by inducing mitotic recombination in developing larvae using X-rays.When such pathes a
18、re created in posterior regions of the developing wings,then the resulting flies exhibit abnomal wings that have duplicted anterior structure in place of the nomal posterior structureThe analysis of genetic mosaics provided the first evidence that engrailed is required for subdividing segments of fl
19、ies into anterior and posterior compartments,Sexual identity in flies is determined by the number of X chromosomes.(two-female;one-male)Y is needed for production of spermRarely,one of the two X chromosomes is lost at the(only)first mitotic division.,Suppose that one of the X chromosomes contains th
20、e recessive white allele.Then one half of the fly,the male half,has white eyes.While the other female half,has red eyes.,Figure 21-19most spectacular genetic mosaics are gyandromorphs,Drosophila:several favorable attributes for molecular studies and whole-genome analysis Approximately 150 Mb-5%mouse
21、 and humam fewer 14000 protein coding genesNew experimental method-producingstable transgenic strains carrying recombinant DNA,The yeast FLP Recombinase Permits the Efficient Production of Genetic Mosaics,Earlier,genetic Mosaics are produced by mitotic recombination in somatic tissurs.X-rays were us
22、ed to induce recombination.it is inefficient-small pathes of mutant tissues More recently,The frequency of mitotic recombination was greatly enhanced by the use of the FLP recombinase from yeast.,FLP recognizes FRT and catalyzes DNA rearrangement.,FRT sequences were inserted near the centromere of e
23、ach of the four chromosomes using P-element transformation.Heterozygous flies contain a null allele in gene Z on one chromosome and a wild-type copy of that gene on the other.,Both chromosomes contain FRT sequencesThese flies are stable and viable as there is no endogenous FLP recombinase in Drosoph
24、ila.,However,it is possible to introduce the FLP recombinase in in transgenic strains that contain FLP protein coding sequence under the control of heat-inducible hsp70 promoter,FLP is synthesized upon heat shock.,FLP binds to the FRT motifs in the two homologs containing gene Z and catalyze mitotic
25、 recombination.,Figure 21-20 FLP-FRT,This method is quite efficient.In fact,short pulses are sufficient to produce enough FLP recombinase to produce large pathes of z-/z-tissue in different regions of an adult fly.,It Is Easy to Create Transgenic Fruit Flies that Carry Foreign DNA,P-elements are tra
26、nsposable DNA segments that can cause hybrid dysgenesis.,P-elements encode both a repressor of transposition and a transposase that promotes mobilization.The repressor is expressed in the developing P eggs.Thus M eggs lack the repressor that inhibits p-element mobilization.,Sometimes the P-elements
27、insert into genes that are essential for the development of progenitors of the gametes(配子),and,as a result,the adult flies derived from the these matings are sterile.,Figure 21-21 hybrid dysgenesis,The F1 progeny are often sterile,when mating females from the“M”strain with males from the“P”strain.,A
28、 full length P-element transposon is 3 kb in length.It contains inverted repeats at the termini that are essential for excision and insertion.,Recombinant DNA is inserted into defective P-elements that lack the internal genes encoding repressor and transposase.Transposase is injected along with the
29、recombination P-element vector.,P-elements can be used as vectors in the transformation of the fly embryos.,Figure 21-22,The recombinant P-elements insert into random positions in the pole cells.The amount of recombination p-element and transposase is calibrated so that,on average,a given pole cell
30、receives just a single integrated p-element.,The embryos are allowed to develop into adults and then mated with tester strains.The recombinant P-element contains a“marker”gene such as white+.,P-element transformation is routinely used to identify regulatory sequences.It can also be used to examine p
31、rotein coding genes in various genetic backgrounds.,Model 6:THE HOUSE MOUSE,The mouse is an excellent model for human development and disease,although,the life cycle(8-9 weeks)of the mouse is slow by the standard of the nematode worm and fruit fly.Special status due to its exalted position on the ev
32、olutionary tree.Mammal(mice,chimps,human),The predominance of the mouse model,The“knockout”technology continues to have an enormous impact on our understanding of the basic mechanisms underlying human development,behavior,and disease.,The mouse provides the link between the basic principles,discover
33、ed in simpler creatures like worms and flies,and human disease.The chromosome complement is similar between the mouse and human(autosomomes19,22 and X,Y sex chromosomes)Genome size and gene numbers 3*109 bp 30000,There is extensive synteny between mice and human:Extended regions of a given mouse chr
34、omosome contain the same set genes(in the same order)as“homologous”regions of the corresponding human chromosomes.The mouse genome has been sequenced and assembled.,The mouse has virtually same complement of genes as those present in the human genome:each contains approximately 30000 genes and there
35、 is a one-to one correspondence for more than 85%of These genes.,Mouse Embryonic Development Depends on Stem Cells,The first obvious diversification of cell types is at the 16-cell stage,called the morula(桑椹胚).The cells in outer regions of the morula develop into the placenta(胎盘).Cells in internal r
36、egions generate the inner cell mass(ICM),The ICM cells can be cultured and induced to form any adult type upon addition of the appropriate growth factors.Human stem cells:offer the promise of providing a renwable source of tissues to replace defective cells in variety of degenerative diseases such a
37、s diabetes and Alzheimers,At the 64-cell stage(about 3-4 weeks after gertilization)the mouse embryo,called a blastocyst(胚泡),is ready for implantation.There are only 13 ICM cells form all the tissues of the adult mouse.The ICM is the prime source of embryonic stem cells.,Interactions between the blas
38、tocyst and uterine wall lead to the formation of the placenta.Shortly thereafter,a fetus emerges that contains a brain,a spinal cord,and internal organs(eg:heart and liver).,The first stage in gastrulation is the subdivision of the ICM into two cell lays:an inner hypoblast(下内胚层)and an outer epiblast
39、(上外胚层).Then the embryo enters gastrulation(原肠胚),and the ICM forms all three germ layers:endoderm(内胚层),mesoderm(中胚层),ectoderm(外胚层).,Then a groove called primitive streak(原条)forms along the length of the epiblast(上外胚层)and the cells that migrate into the groove form the internal mesoderm.The anterior e
40、nd of the primitive streak is the node.,Node;source of signaling moleculaes(including two secreted inhibitors of TGF-signaling,Chordin and Noggin)Double(both genes)mutant mouse embryos that lack head structure such as forebrain and nose.,It is Easy to Introduce Foreign DNA into the Mouse Embryo,Micr
41、oinjection methods have been developed for the efficient expression of recombinant DNA in transgenic strain of mice.1,Inject DNA into the egg pronucleus.2,place the embryos into the oviduct(输卵管)of a female mouse.3,the injected DNA integrates at random positions in the genome.,Figure 21-24,The effici
42、ency of integration is quite high and usually occurs during early steges of development,often in one-cell embryos.As result,the fusion gene insert into most or all of the cells in the embryo,including the ICM cells that form the somatic tissue and germline of the adult mouse Approximately 50%of the
43、transgenic mice that produced using this simple method of microinjection exhibit:germline transformation(生殖转化）:the offspring of transgenic mice also contain the foreign recombinant DNA.,Figure 21-25转基因小鼠的原位表达,A transgenic strain of mice was created that contains a portion of the Hoxb-2 regulatory re
44、gion attached to a lacZ report gene.There are two bands of staining detected in the hindbrain region of 10.5 day embryos.,Homologous Recombination Permits the Selective Ablation of Individual Genes,The single most powerful method of mouse transgenesis is the ability to disrupt,or“knock out,”single g
45、enetic loci.This permits the creation of mouse models for human disease.,For example,the p53 gene encodes a regulatory protein that activates the expression of genes required for DNA repair.when p53 fuction is lost,cancer cells become highly invasive due to rapid accumulation of DNA mutations.A stra
46、in of mice has been established that is completely normal except for the removal of the p53 gene.These mice are highly susceptible to cancer,die young.-drug discovery,Gene disruption experiments,They are done with embryonic stem(ES)cells.culturing mouse blastocyst so ICM cells proliferate without di
47、ferentiatingA recombinant DNA is created that contains a mutant form of the gene of interest.modified by deleting a small region near the beginning of the gene Modified form of the target gene is linked to a drug sistance gene,such as NEO.,The method is used to create a cell line lacking any given g
48、ene.,The process of the experiment,First,designing the recombination vector.It contains the modified target gene,the NEO gene(downstream of the target gene),the region of homology with the host cell chromosome resulting in the replacement of the target gene with the mutant gene and drug resistance g
49、ene(downstream of and flanking NEO)and a marker(TK,gene for thymidine kinase).,Second,transform the vector into ES cells.Third,select for NEO.Only the cells which undergo double recombination with the host cell chromosome can survive in the neomycin containing medium.,Fourth,select against TK.If ill
50、icit recombination occurs,TK gene will frequently be contained.In this case,the cells which undergo illicit recombination will die in the GANC(丙氧鸟苷负筛选)containing medium.同源重组TK 失活,非同源重组TK有活性,使丙氧鸟苷变成有毒化合物.,Fifth,harvest the homologous recombination ES cells and inject them into the ICM of normal blast
