《分子生物学》1-cha.ppt

CHAPTER 21 Model Organisms Fundamental problems are solved in the simplest and most accessible system. These organisms are called model organisms. Some Important Model Organisms Escherichia coli and its phage (the phage and M13 phage) Bakers yeast Saccharomyces cerevisiae The nematode Caenorhabditis elegans The fruit fly Drosophila melanogaster The house mouse Mus musculus Features of Model Systems nThe 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 choose 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 aspects of the nature of the gene and information transfer Yeast-premier system for elucidating fundamental aspects of the eukaryotic cell ,powerful mating system for genetic analysis Nematode,fruit flywell developed genetic system Mouse-best model sytem for gaining insights into human biology and human disease Model 1: BACTERIOPHAGEModel 1: BACTERIOPHAGE Bacteriophage (Viruses) The simplest system Their 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 “receptor” can be infected by a given phage. Two Basic Types Lytic phage: eg. T phage infect a bacterial cell DNA replication coat proteins expression host cell lysed to release the new phage Figure 21-1 The lytic growth cycle 2. Lysogeny (溶源途径) eg. Phage nthe phage genome integrated into the bacterial 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 bacteriophage nThe lysogenic state can switch to lytic growth, called induction. Excision of the prophage DNA DNA replication Coat proteins expression Lytic growth Growth and induction of lysogen Assays of Phage Growth Propagate phage: by growth on a suitable bacterial host in liquid culture. Quantify phage: plaque (嗜菌斑) assay Propagate phage Find a suitable host cell that supports the growth of the virus. The mixture of viruses and bacteria are filtered through a bacterial-proof filter. Quantify phage Phage 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 agar Hard agar a 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 Curve Figure 21-4 Latent period-the time lapse between infection and release of progeny. Burst size-the number of phage released The Single-Step Growth Curve It 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 the 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 to 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 infection: 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 order 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 mutations 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 another. eg. Phage Phage display Model 2: BACTERIAModel 2: BACTERIA 有自己的复制有自己的复制, ,转录和翻译系统转录和翻译系统 Features of bacteria na single chromosome na short generation time nconvenient to study genetically Assays 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 to 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 curve Quantify bacteria nDilute 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. Bacteria Exchange DNA by: Sexual Conjugation (性结合) Phage-Mediated Transduction(转导) DNA-Mediated Transformation(转化) 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 out cis- trans analyses. Sexual Conjugation Plasmids: 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) (育性质粒-F因子). F+ cell: cell harboring an F-factor. Hfr strain: a strain harboring an integrated F-factor in its chromosome. F-lac : an F-factor containing the lactose operon. F- cell (没有F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-lac nF-factor-mediated conjugation 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 高频重组菌株 ,不同的Hfr F-plasmid整合在染色 体的 不同位置 容易发生F+ cell的染色体DNA 通过接合向F- cell转移 Transfer the host chromosome into the recipient cell takes place linearly, starting with the region closest to the integrated F-plasmid. 根据整合的位置和交配的时间的不同,复制和转移不同大小和位置的 宿主染色体 The F-factor can undergo conjugation only with other E.coli strains. (有选择) Some plasmids can transfer DNA to a wide variety of unrelated strains(even to yeast), called promiscuous conjugative plasmids. (没选择) nThey provide a convenient means for introducing DNA into bacteria strains that cant undergo genetic exchange. Phage-mediated transduction lGeneralized transduction(一般转导): A fragment 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 transduction lSpecialized transduction Lysogenic phage :原,取代 （特异phage） Transfer bacterial DNA to a new bacterial host cell. DNA-mediated transformation lSome 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 as “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 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 mutagenesis Insertional 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. 不是改变一个碱基,活力还剩余 2.Having inactivated the gene, the inserted DNA is easy to isolate and clone that gene. 分析转座子和被插入基因的测序,失活基因的确定变得容 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 brought under thr control of the disrupted target gene 测量lacZ水平 ,就可知靶基因的表达情况 eg2. Gene -operon fusions created by transopsons Promoter-less lacZ Reporter gene Gene fusion: a fusion in which the reporter is joined both transcriptionally and translationally to the target gene. Studies on the Molecular Biology of Bacteria Have Been Enhanced by : Recombinant DNA Technology Whole-Genome Sequencing Transcriptional Profiling Use of genetic competence in combination with recombinant methods: -creating precise mutations and gene fusions -expanding the kinds and number of molecular genetic manipulation Microarray (representing all of the genes in a bacterium): -possible to study gene expressionon genome-wide basis Biochemical Analysis Is Especially Powerful in Simple Cells -with Well-Developed Tools of Traditional and Molecular Genetics Bacteria: 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 large quantities. 3.It is much simpler to carry ou