分子生物學1chapter 21

1、chapter 21model organismsfundamental problems are solved in the simplest and most accessible system.these organisms are called model organisms.some important model organismsescherichia coli and its phage (the phage and m13 phage)bakers yeast saccharomyces cerevisiaethe nematode caenorhabditis elegan

2、sthe fruit fly drosophila melanogasterthe house mouse mus musculusfeatures of model systemsnthe availability of powerful tools of traditional and molecular genetics.nthe study of each model system attracted a critical mass of investigators. (ideas, methods, tools and strains could be shared)how to c

3、hoose a model organism? it depends on what question is being asked. when studying fundamental issues of molecular biology, simpler unicellular organisms or viruses are convenient. for developmental questions, more complicated organisms should be used.e.coli,t4-ideal system for tackling fundamental a

4、spects of the nature of the gene and information transferyeast-premier system for elucidating fundamental aspects of the eukaryotic cell ,powerful mating system for genetic analysisnematode,fruit flywell developed genetic systemmouse-best model sytem for gaining insights into human biology and human

5、 disease bacteriophage (viruses)the simplest systemtheir genomes are replicated only after being injected into a host cell.the genomes can recombine during these infections.figure bacteriophage each phage attaches to a specific cell surface molecule (usually a protein) and so only cells bearing that

6、 “receptor” can be infected by a given phage.two basic types1. lytic phage: eg. t phage infect a bacterial cell dna replication coat proteins expression host cell lysed to release the new phagefigure 21-1the lytic growth cycle2. lysogeny (溶源途徑溶源途徑) eg. phage nthe phage genome integrated into the bac

7、terial genome and replicated passively as part of the host chromosome, coat protein genes not expressed. the phage is called a prophage.figure 21-2 the lysogenic cycle of a bacteriophagenthe lysogenic state can switch to lytic growth, called induction. excision of the prophage dna dna replicationcoa

8、t proteins expressionlytic growthgrowth and induction of lysogenassays of phage growth propagate phage: by growth on a suitable bacterial host in liquid culture. quantify phage: plaque (嗜菌斑嗜菌斑) assaypropagate phagefind a suitable host cell that supports the growth of the virus.the mixture of viruses

9、 and bacteria are filtered through a bacterial-proof filter.quantify phagephage are mixed with and adsorb to bacterial cells.dilute the mix.add dilutions to “soft agar” (contain many uninfected bacterial cells).poured onto a hard agar base.incubated to allow bacterial growth and phage infection.soft

10、 agarhard agara petri dish this circle-of-death produces a hole or plaque in a lawn of living cells. these plaques can be easily seen and counted so that the numbers of virus can be quantitated. as the viruses replicate and are released, they spread and infect the nearby cells.the single-step growth

11、 curvefigure 21-4latent period-the time lapse between infection and release of progeny.burst size-the number of phage releasedthe single-step growth curveit reveals the life cycle of a typical lytic phage.it reveals the length of time it takes a phage to undergo one round of lytic growth, and also t

12、he number of progeny phage produced per infected cell.1. phage were mixed with bacterial cells for 10 minutes. (long enough for adsorption but too short for further infection progress.)2. the mixture is diluted by 10,000. (only those cells that bound phage in the initial incubation will contribute t

13、o the infected population; progeny phage produced from those infections will not find host cells to infect.)3. incubate the dilution. at intervals, a sample can be removed from the mixture and the number of free phage counted using a plaque assay.phage crosses and complementation tests mixed infecti

14、on: a single cell is infected with two phage particles at once.mixed infection (co-infection)1. it allows one to perform phage crosses. if two different mutants of the same phage co-infect a cell, recombination can occur between the genomes. the frequency of this genetic exchange can be used to orde

15、r genes on the genome.2. it allows one to assign mutations to complementation groups. if two different mutant phage co-infect the same cell and as a result each provides the function that the other was lacking, the two mutations must be in different genes (complementation groups). if not, the two mu

16、tations are likely located in the same gene.transduction and recombinant dna during infection, a phage might pick up a piece of bacterial dna (mostly happens when a prophage excises form the bacterial chromosome). the resulting recombinant phage can transfer the bacterial dna from one host to anothe

17、r. eg. phage phage displayfeatures of bacteriana single chromosomena short generation timenconvenient to study geneticallyassays of bacteria growth bacteria can be grow in liquid or on solid (agar) medium.bacterial cells are large enough to scatter light, allowing the growth of a bacterial culture t

18、o be monitored in liquid culture by the increase in optical density (od).bacterial cells can grow exponentially when not over-crowded, called exponential phase.as the population increase to high numbers of cells, the growth rate slows, called stationary phase.figure 21-5 bacteria growth curvequantif

19、y bacteriandilute the culture.nplate the cells on solid medium in a petri dish.nsingle cells grow into colonies; count the colonies.nknowing how many colonies are on the plate and how much the culture was diluted makes it possible to calculate the concentration of cells in the original culture.bacte

20、ria exchange dna by: sexual conjugation (性結合性結合)phage-mediated transduction(轉導轉導)dna-mediated transformation(轉化轉化)we use genetic exchange to:map mutations.construct strains with multiple mutations.build partially diploid strains for distinguishing recessive from dominant mutations and for carrying o

21、ut cis-trans analyses.sexual conjugationplasmids: autonomously replicating dna elements in bacteria.some plasmids are capable of transferring themselves from one cell to another. eg. f-factor (fertility plasmid of e.coli)( (育性質粒育性質粒-f-f因子因子). ).f+ cell: cell harboring an f-factor. hfr strain: a stra

22、in harboring an integrated f-factor in its chromosome.f-lac : an f-factor containing the lactose operon.f- cell ( (沒有沒有f f因子因子) ) f-factors is a fertility plasmid that contains a small segment of chromosomal dna.f-factors can be used to create partially diploid strains.eg. f-lacnf-factor-mediated co

23、njugation is a replicative process. the products of conjugating are two f+ cells. sexual conjugation f+ cell+ f- cell-2 f+ cell nhfr (high frequency recombination) strain 高頻重組菌株高頻重組菌株 ,不同的不同的hfr f-plasmid整合在染色體整合在染色體的的 不同位置不同位置 容易發(fā)生容易發(fā)生f+ cell的染色體的染色體dna dna 通過接合向通過接合向f- cellf- cell轉移轉移transfer the

24、host chromosome into the recipient cell takes place linearly, starting with the region closest to the integrated f-plasmid.根據整合的位置和交配的時間的不同根據整合的位置和交配的時間的不同,復制和轉移不同大小和位置的宿復制和轉移不同大小和位置的宿主染色體主染色體the f-factor can undergo conjugation only with other e.coli strains. ( (有選擇有選擇) )some plasmids can transfer

25、dna to a wide variety of unrelated strains(even to yeast), called promiscuous conjugative plasmids. ( (沒選擇沒選擇) )nthey provide a convenient means for introducing dna into bacteria strains that cant undergo genetic exchange.phage-mediated transductionlgeneralized transduction( (一般轉導一般轉導): ): a fragmen

26、t of chromosomal dna is occasionally packaged into phage . when such a phage infects a cell, it introduces the segment of chromosomal dna to the new cell. recombine-permanent transfer of genetic information from one cell to another (100kb)figure 21-7 phage-mediated :generalized transductionlspeciali

27、zed transduction lysogenic phage :原原,取代取代 （特異特異phagephage）transfer bacterial dna to a new bacterial host cell. dna-mediated transformationlsome bacterial species can take up and incorporate linear, naked dna into their own chromosome by recombination.lthe cells must be in a specialized state known a

28、s “genetic competence”.bacterial plasmids can be used as cloning vectors plasmid: circular dna in bacteria that can replicate autonomously. plasmids can serve as vectors for bacterial dna as well as foreign dna.dna should be inserted without impairing the plasmid replication.transposons can be used

29、to generate insertional mutations and gene -operon fusions eg1. transposons that integrate into the chromosome with low-sequence specificity(high random) such as tn5 mu can be used to generate a library of insertional mutations on a genome-wide basis.transposon-generated insertional mutagenesisinser

30、tional mutations generated by transposons have two advantages over traditional mutations(chemical mutagenesis). 1.the insertion of a transposon into a gene is more likely to result in complete inactivation of the gene. 不是改變一個堿基不是改變一個堿基, ,活力還剩余活力還剩余2.having inactivated the gene, the inserted dna is e

31、asy to isolate and clone that gene.分析轉座子和被插入基因的測序分析轉座子和被插入基因的測序,失活基因的確定變得容易失活基因的確定變得容易 operon or transcriptional fusion modified transposon: such as tn5lac -harbor a reporter gene lacz(no promoter) insert into the chromosome(in the appropriate orientation)transcription of the reporter gene is brough

32、t under thr control of the disrupted target gene測量測量laczlacz水平水平 , ,就可知靶基因的表達情況就可知靶基因的表達情況eg2. gene -operon fusions created by transopsonspromoter-less laczreporter genegene fusion: a fusion in which the reporter is joined both transcriptionally and translationally to the target gene.studies on the

33、molecular biology of bacteria have been enhanced by : recombinant dna technologywhole-genome sequencingtranscriptional profiling use of genetic competence in combination with recombinant methods:-creating precise mutations and gene fusions-expanding the kinds and number of molecular genetic manipula

34、tionmicroarray (representing all of the genes in a bacterium):-possible to study gene expressionon genome-wide basisbiochemical analysis is especially powerful in simple cells -with well-developed tools of traditional and molecular geneticsbacteria: center for biochemical study of dna replication, information transfer, gene regulation 1.large quantities of bacterial cells can be grown in a defined and homogenous physiological state.2.it is easier to purify protein complexes harboring precisely engineered alterations or to overproduce and obtain individual proteins in la