大二上遗传学ch 8 genetic analysis and mapping in bacteria and phages

1、Chapter 9The Genetics of Bacteria andPhagesso far. recombination & mapping in eukaryotes now. prokaryotes & viruses resolutionBoth bacteria and bacteriophages(Bacterial viruses) demonstrate mechanisms by which genetic recombination occurs, processes that can serve as the basis for genetic mapping三、细

2、菌和病毒在遗传研究中的优越性世代周期短易于管理和进行生物化学分析遗传物质比较简单是单倍体，便于研究基因的突变便于遗传操作可用作研究高等生物的简单模型。KEY CONCEPTSAs a result, both groups have been the subject of extensive analysis.3 ways to incorporate & recombine DNA in bacteria:1. conjugation (结合） plasmid-mediated transfer2. transformation （转化） absorb from environment3.

3、transduction （转导） bacteriophage-mediated transfer1. conjugation plasmid-mediated transfeBacteria also contain extrachromosomal DNA in the form of plasmids, which can house a fertility factor that plays a key rolein genetic recombination.fertility factor (F) permits bacterial cells to transfer DNA to

4、 other bacteria cells through conjugationF can be integrated or cytoplasmic when integrated, F can transfer hostchromosome markers through conjugation2. transformation absorb from environment DNA from the medium can enter a bacterial cell and integrate into the chromosome, thereby transforming the g

5、enotype. The different methods of gene transfer in bacteria generate partial diploids that permit the study of recombination and gene interaction.3. transduction bacteriophage- mediated transferbacteriophages can transfer DNA from one bacterial cell to another in two ways .generalized transduction i

6、s the transfer of randomly incorporated bacterial chromosome fragmentsspecialized transduction is the transfer of specific genes near phage integration sitesthese methods of gene transfer facilitate construction of detailed maps of bacterial genomes 9.1 Bacterial Mutation and GrowthBacterial Phenoty

7、pesNon-motile To do genetics, we needphenotypic variation.Morphology/resistance/ prototroph(autotroph)/ auxotroph and so on Prior to1943adaptation hypothesis, spontaneous mutations fluctuation test, Non-capsulated Rough colony Nutritional Sugar fermentation Drug resistant Virus resistant Temperature

8、 sensitive Pigmentless Cold sensitive波动性试验(fluctuation test) Mutation: Spontaneous mutation is the primary source of genetic variation in bacteria. Prototroph: is a bacterium that can synthesize all essential organic compound on minimal medium. Auxotroph: is a bacterium that loses, through mutation,

9、 the ability to synthesize one to more organic components, and that can not live on minimal medium.strainsprototrophs = wild typegrow on minimal mediumauxotrophs = mutantsdo not grow on minimal mediumnutrition carbon sourceresistant mutantsBacterial PhenotypesDeduce the genotypes of the following fo

10、ur E. colistrains:FIGURE 9-1 Typical bacterial population growth curve showing the initial lag phase, the subsequent log phase where exponential growth occurs, and the stationary phase that occurs when nutrients are exhaustedWe need only select the dish in which the number of colonies can be counted

11、 accurately. Because each colony arose from a single bacterium, the number of colonies multiplied by the dilution factor represents the number of bacteria in each milliliter of the initial inoculum used to start the serial dilutions. Cells grown in liquid medium can be quantified by transferring the

12、m to semisolid medium in a Petri dish10-310 -410 -5In Figure 9-2, the right most dish has 15 colonies. The dilution factor for a 10-5 dilution is 105. Therefore, the initial number of bacteria is 15 105 per milliliter9.2 Genetic Recombination in Bacteria: Conjugation Lederberg and Tatum 1946coli K12

13、. mutants Strain A required methionine and biotin in order to grow, while （需甲硫氨酸,生物素） Strain B required threonine, leucine, and thiamine (Figure 9-3). （需苏氨酸，亮氨酸和硫胺 Neither strain would grow on minimal medium. The two strains were first grown separately in supplemented media, and cells from both were

14、 mixed and grown together for several more generations and then plated on minimal medium. Any bacterial cells that grew on minimal medium were prototrophs. highly improbablespontaneous mutation at two or three In this experiment, prototrophs were recovered at a rate of 1/107 (10-7) cells plated. The

15、 controls for this experiment involved separate plating of cells from strains A and B on minimal medium. No prototrophs were recovered. Based on these observations, Lederberg and Tatum proposed that genetic exchange had occurred.FIGURE 94When strain A and B auxotrophs are grown in a common medium bu

16、t separated by a filter, no recombination occurs and no prototrophs are produced.The apparatus shown is a Davis U-tube.F+and F-Bacteria initial crosses of Lederberg and Tatum (Figure 9-3) can be designated as follows: STRAIN A F+DONORSTRAIN BF RECIPIENTConjugationPilus needed for cell contactDNA syn

17、thesis needed for transfer rolling circle replication begins at origin (ori)one strand nickedfertility geneConjugation The F factor is replicated during transferMGA2e Fig. 4-10Hayes, et. al., 1950stransfer one-way, not reciprocaldonor and recipient strains (+ and strains)donor: F+ recipient: F-matin

18、g of F+ x F-2 F+F is F plasmidbacterial gene transfer rareHfr Bacteria and Chromosome Mapping In 1950, Cavalli-Sforza treated an F+ strain of E. coli K12 with nitrogen mustard, a chemical known to induce mutations. From these treated cells, he recovered a genetically altered strain of donor bacteria

19、 that underwent recombination at a rate of 1/104 (or 104), 1000 times more frequently than the original F+ strains. In 1953, Hayes isolated another strain that demonstrated an elevated frequency. Both strains were designated Hfr, for high-frequency recombination. Because Hfr cells behave as donors,

20、they are a special class of F+ cells. F+ F- F+ (low rate of recombination)Hfr F F (higher rate of recombination) In the mid-1950s, experimentation by Ellie Wollman and Francois Jacob explained the difference between Hfr and F+Interrupted mating techniqueandmappingchromosomeSodium Azide8 no10azi ,no

21、other15 70% azi30% ton2025,It appeared that the chromosome of the Hfr bacterium was transferred linearly and that the gene order and distance between genes, as measured in minutes, could be predicted from such experiments (Figure 9-8). This information served as the basis for the first genetic map o

22、f the E. coli chromosome. Minutes in bacterial mapping are equivalent to map units in eukaryotes.mapping in E. coli by interrupted-mating distance measured in time (min)Wollman and Jacob then repeated the same type of experiment with other Hfr strains, obtaining similar results with one important di

23、fference. While genes were always transferred linearly with time, as in their original experiment, which genes entered first and which followed later seemed to vary from Hfr strain to Hfr strain Figure 9-9(a). The major difference between each strain was simply the point of origin (O) and the direct

24、ion in which entry proceeded from that point Figure 9-9(b).E coli gene map According to interrupted map technique,recombination genes and other gene mapping approaches,cyclemap of the bacteria E coli is created linkage map，thr（0m）， total 100m。52gene site are markedRecombination in F+ x F Matings: A

25、Reexamination The F State and MerozygotesIn 1959, during experiments with Hfr strains of E. coli, Edward Adelberg discovered that the F factor could lose its integrated status, causing the cell to revert to the F+ state (Figure 9-11, step 1). When this occurs, the F factor frequently carries several

26、 adjacent bacterial genes along with it (step 2). Adelberg labeled this condition F to distinguish it from F+ and Hfr. F, like Hfr, is thus another special case of F+. This conversion is described as one from Hfr to F.Ffactor and sex-ductionSexduction is process whereby a bacterium gains access to a

27、nd incorporates foreign DNA brought in by a modified F factor during conjugation.sex-duction F is different from Hfr strain in gene transfer。For example：（）Hfr：thr+leu+strsF-：thr-leu-strrresult： produce F-：thr+leu+strr recombinants（）F：thr+leu+strsF-：thr-leu-strr result：produce F：thr+leu+strr recombin

28、ants 产生F因子的Hfr菌株仍保持单倍体状态， 当F因子转入到受体细胞之后，由于引入了供体细胞的部分基因，从而构成了部分二倍体。如图6-13中Flac+可转移到F-lac- 后构成Flac+/F-lac-部分二倍体。这种利用F因子将供体细胞的基因导人受体形成部分二倍体的过程叫性导（sexduction或F- duction）。 性导在大肠杆菌的遗传学分析中十分有用。这种部分二倍体如果不发生重组，那么 F因子自主复制，可在细菌细胞中延续下去； 性导所形成的部分二倍体可用作不同突变型之间的互补测验，以确定这两个突变型是属于同一个基因或者是两个不同的基因； 观察由性导形成的杂合的部分二倍体中某一

29、性状的表现，可以确定这一性状的等位基因中的显隐性关系； 不同的F因子带有不同的细菌DN段， 因此利用不同的F因子性导可以测定不同基因在一起性导的频率来作图。部分二部体中也会出现重组，即F因子所带的供体细菌染色体同受体细菌染色体之间的同源重组，如果发生单交换，就导致F整合形成Hfr品系，同时F因子上所携带的基因发生重组；如果双交换，则形成F品系，只是F因子的细菌基因和受体染色体上的等位基因之间发生互换。三种致育因子F, F，Hfr的关系是：(1). 有F因子的细菌为F+，没有F因子的为F-，具有致育因子（F, F或Hfr）的菌株就是雄性菌株(male strains) 。 (2). F因子可以整

30、合到细菌染色体上，形成Hfr染色体。不同的Hfr菌株F因子的整合位点不同。(3). F因子又可以从Hfr染色体上剪切下来，产生F因子。 如果剪切不准确而带有一段细菌染色体，则称为F因子。(4). F因子很容易转移到F-细胞中， F+ F-F+，但是供体染色体的转移频率则很低, 重组频率很低。(5). Hfr能以高频率把细菌染色体基因转移到F-细菌中， 却极少使F-变为F+（因为F因子位于Hfr染色体的最末 端）；(6). F因子的性质介于F+和Hfr之间，即可转移自身，又可以转移细菌基因。但频率较低。Bacterial GeneTransfer Conjugation Transformati

31、on Transductionconversion of one genotype to another by uptake of exogenous DNAtransformation principle demonstrated that DNA was responsible for inherited differences in polysaccharide character of S. pneumoniaeTransformationBackground knowledge:Griffiths, 1928 - vaccine for pneumoniaStreptococcus

32、pneumoniae smooth vs. rough strains transforming principleAvery, McCarty & MacLeod, 19448.5 Bacterial Transformation Transformation also provides a mechanism for recombining genetic information in some bacteria. In transformation, small pieces of extracellular DNA are taken up by a living bacterium,

33、 ultimately leading to a stable genetic change in the recipient cell. We are interested in transformation in this chapter because, in those bacterial species where it occurs, the process can be used to map bacterial genes, although in a more limited way than conjugation. 氯霉素（chloramphenical）和阻碍能量产生的

34、二 硝基苯（dinitrophenol）可抑制转化作用。显然 外源DNA只有在酶促旺盛的受体部位进人。 这种能接受外源DNA分子并被转化的细菌细胞称为 感受态细胞（competence cell），而促进转化作用的酶或蛋白质分子称为感受态因子（competence factor）。Transformation Genes can be transferred between bacteria as exogenous DNATransformation and Linked GenesFor DNA to be effective in transformation, it must incl

35、ude between 10,000 and 20,000 nucleotide pairs, about 1/200 of the E. coll chromosome. This size is sufficient to encode several genes. Genes adjacent or very close to one another on the bacterial chromosome can be carried on a single segment of this size. Because of this fact, a single event can re

36、sult in the cotransformation of several genes simultaneously. Genes that are close enough to each other to be contransformed are said to be linked. Transduction (Virus-Mediated Bacterial DNA transfer) Bacteriophages, or phages as they are commonly known, are viruses that have bacteria as their hosts

37、.Bacterial GeneTransfer Conjugation Transformation TransductionPhage T4: Structure and Life CycleBacteriophage T4 , one T-even phages. It exhibits an intricate structure, as shown in Figure 9-14. Its genetic material, DNA, is contained within an icosahedral (a polyhedron with 20 faces) protein coat,

38、 together making up the head of the virus. The DNA is sufficient in quantity to encode more than 150 average-size genes. The life cycle of phage T4 (Figure 9-15) is initiated when the virus binds by absorption to the bacterial host cell.The Plaque Assay Bacteriophages and other viruses play a critic

39、al role in our understanding of molecular genetics. Often, over 1010 viruses are produced per milliliter of culture medium. Many genetic studies rely on the ability to quantify the number of phages produced following infection under specific culture conditions. The technique used routinely is called

40、 the plaque assay.This assay is shown in Figure 9-16, where actual plaque morphology is also shown.TransductionBacteriophage or phage - bacterial viruses lytic or virulent phagelysogenic or temperate phageLysis Bacterial gene transfer mediated by bacteriophages (viruses) Phage life cycles: lyticMGA2

41、e Fig. 7-17MGA Fig. 7-18Lysogeny Phage life cycles: lysogenicMGA2e Fig. 7-23Prophage. Viruses that either lyse the cell or behave as a prophage are temperate. Those that only lyse the cell are referred to as virulent. A bacterium harboring a prophage is lysogenic; episome8.7 Transduction: Virus-Medi

42、ated Bacterial DNA Transfer In 1952, Norton Zinder and Joshua Lederberg were investigating possible recombination in the bacterium Salmonella typhimurium鼠伤寒沙门氏杆菌. Although they recovered prototrophs from mixed cultures of two different auxotrophic strains, subsequent investigations revealed that rec

43、ombination was occurring in a manner differentfrom that attributable to the presence of an F factor, as in E. coli. What they discovered was a process of bacterial recombination mediated by bacteriophages and now called transduction.The Lederberg-Zinder Experiment Lederberg and Zinder mixed the Salm

44、onella auxotrophic strains LA-22 and LA-2 together and, when the mixture was plated on minimal medium, they recovered prototrophic cells. LA-22 (phe trp met+ his+) ), LA-2 (phe+ trp+ met his). Prototrophs (phe+ trp+ met+ his+) were recovered at a rate of about 1/105 (or 105) cells.Since LA-2 cells a

45、ppeared to be the source of the new genetic information (phe+ and trp+), how that information crossed the filter from the LA-2 cells to the LA-22 cells, allowing recombination to occur, was a mystery.The unknown source was designated simply as a filterable agentTransductionNature of transductionGene

46、ralized TransductionGeneralized transduction lytic or lysogenic phage any suitably sized piece of DNA packagedSpecialised transductionAgain this represents the transfer ofbacterial DNA from one bacterial cell to another via phage particles.However instead of random packaging of bacterial DNA this fo

47、rm of gene exchange results from imprecise excision of an integrated phage(prophage) integrated in to the bacterial chromosome at a site known as the att site.Consequently only genes flanking the integration or att site are carried in the phage transducing particle, and accompany phage genes (by acc

48、ident).Lambda life cycleFollowing lambda infection there are 2 alternatives:1. LyticCircular ds l DNA is replicated many fold via the rolling circle mechanism, l gene products are made and phage particle are assembled before the cell islysed and the viral particles released.2. LysogenyThe l genome b

49、ecomes integrated in to the bacterial genome at a unique site, the att site, by a site-specific recombination event (non-homologous recombination). Behaves as a piece of chromosomal DNA, and is replicated like any other section of bacterial chrm DNA.l DNA has a genome size of 50 kb, with 48 genes. i

50、s a circular ds DNA molecule, and is replicated largely (but not totally) by rolling circle replication. Linear l DNAproduced by rolling circle mechanism and cleavage, form circular molecules via ss complementary DNA termini 12 nt in length, which are known as cos (cohesive) sites. DNA is contained

51、within a phage particle, and can only replicate by infecting cells, and exploiting host DNA replication proteins.Cos sitescoscos5aggtcgccgccc cosTransduction Specialized transductionattP attBl phage integrates by site specific recombination (RecA-independent)lPPatt siteBgalExcisionatt siteBacterial

52、chrmBbioIntegrationbioBPPBgal(l int + l xis IHF)(l int + IHF)Prophage/lysogenl bio or l gal transducing phage arise by imprecise excisiongalPBBPbiollPBPBTRANSDUCTION IN BACTERIAco-transductiondonor leu+ thr+ azir recipient leu thr azisTRANSDUCTION IN BACTERIAco-transductiondonor leu+ thr+ azir recip

53、ient leu thr azisthrleuazi 如作双因子转导（two-factor transduction）实验就是每次观察两个基因的转导，通过每两个基因 之间的共转导频率就可以确定这些基因在染色体上 的次序。若要分析3个基因则需做3次双因子转导实 验，才能确定这个基因的次序。假定这3次实验结果为： a基因和b基因共转导频率高； a和c基因的共转导频率也高； b和c基因的共转导频率很低，那么这3个基因的 次序就应为b、a、c。TRANSDUCTION IN BACTERIAspecialized transductionTRANSDUCTION IN BACTERIAtransduction:phage acquire host genes and transfer them to other bacterial cellsgeneralized transduction:transfers any host gene; and occurs when phage randomly package host DNAspecialized transduction:faulty separation of prophage (phage inco