Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 97
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coli chromosome. Figure 8.11 is a map of thechromosome of E. coli containing a sample of the mapped genes. Both the DNA molecule and the genetic map arecircular. The entire chromosome requires 100 minutes to be transferred (it usually breaks first), so the total maplength is 100 minutes. In the outer circle, the arrows indicate the direction of transcription and the coding regionincluded in each transcript. The purple arrowheads show the origin and direction of transfer of a number of Hfrstrains. Transfer from HfrC, for example, goes counterclockwise starting with purE acrA lac.Page 323Figure 8.11Circular genetic map of E. coli. Map distances are given in minutes; the total map length is 100 minutes.
For some of theloci that encode functionally related gene products, the map order of the clustered genes is shown, along with the directionof transcriptionand length of transcript (black arrows). The purple arrowheadsshow the origin and direction of transfer of a numberof Hfr strains. For example, HfrH transfers thr very early, followed by leu and other genes in a clockwise direction.Page 324Figure 8.12A more dense genetic map showing genes in theregion between 84 and 86 minutes of the E. colichromosome. Arrows indicate the direction oftranscription and length of the messenger RNAfor each gene or gene cluster. Symbols to theleft of the light blue bar indicate untranscribedDNA sites and genes for which the direction oftranscription is not known.
For example,attTn7 is an insertion site for the transposonTn7.[From M. K. B. Berlyn, K. B. Low, andK. E. Rudd. 1996. In Escherichia coli and Salmonella:Cellular and Molecular Biology, 2nd ed. (F. C. Neidhardt,R. Curtiss III, J. L. Ingraham, E. C. C. Lin, K. B. Low,B. Magasanik, W. Reznikoff, M. Riley,M. Schaechter, and H. E.
Umbarger, eds.) Washington,DC: American Society for Microbiology.]Greater detail of part of the E. coli map for the 2 minutes between minutes 84 and 86 is shown in Figure 8.12.Genes and gene clusters transcribed as a unit are indicated by the arrows pointing in the direction of transcription,shown to the right of the blue line. To the left of the line are either regions that are not transcribed or genes inwhich the direction of transcription is unknown. The symbol oriC represents the origin of DNA replication of theE.
coli chromosomes.F' PlasmidsOccasionally, F is excised from Hfr DNA by an exchange between the same sequences used in the integrationevent. However, in some cases, the excision process is not a precise reversal of integration. Instead, breakage andreunion take place between nonhomologous sequences at the boundary of F and nearby chromosomal DNA (Figure8.13). Aberrant excision creates a plasmid containing a fragment of chromosomal DNA, which is called an F'plasmid ("F prime"). By the use of Hfr strains having different origins of transfer, F' plasmids with chromosomalsegments from many regions of the chromosome have been isolated.
These elements are extremely useful becausethey render any recipient cell diploid for the region of the chromosome carried by the plasmid. These diploidregions make possible dominance tests and gene-dosage tests (studies of the effects on gene expression ofincreasing the number of copies of a gene). Because only a part of the genome is diploid, cells containing an F'plasmid are partial diploids, also called merodiploids. Examples of genetic analysis using F' plasmids will beoffered in Chapter 11 in a discussion of the E. coli lac genes.Page 325Figure 8.13Formation of an F' lac plasmid by aberrant excision of F from an Hfr chromosome.Breakage and reunion are between nonhomologous regions.8.5—TransductionIn the process of transduction, a bacterial DNA fragment is transferred from one bacterial cell to another by aphage particle containing the bacterial DNA.
Such a particle is called a transducing phage. Two types oftransducing phages are known—generalized and specialized. A generalized transducing phage produces someparticles that contain only DNA obtained from the host bacterium, rather than phage DNA; the bacterial DNAfragment can be derived from any part of the bacterial chromosome.
A specialized transducing phage producesparticles that contain both phage and bacterial genes linked in a single phage DNA molecule, and the bacterialgenes are obtained from a particular region of the bacterial chromosome. In this section, we consider E. coli phageP1, a wellstudied generalized transducing phage. Specialized transducing particles will be discussed in Section 8.7.During infection by P1, the phage makes a nuclease that cuts the bacterial DNA into fragments. Single fragmentsof bacterial DNA comparable in size to P1 DNA are occasionally packaged into phage particles in place of P1DNA.
The positions of the nuclease cuts in the hostPage 326chromosome are random, so a transducing particle may contain a fragment derived from any region of the hostDNA. A large population of P1 phages will contain a few particles carrying any bacterial gene. On the average, anyparticular gene is present in roughly one transducing particle per 106 viable phages. When a transducing particleadsorbs to a bacterium, the bacterial DNA contained in the phage head is injected into the cell and becomesavailable for recombination with the homologous region of the host chromosome.
A typical P1 transducing particlecontains from 100 kb to 115 kb of bacterial DNA.Let us now examine the events that follow infection of a bacterium by a generalized transducing particle obtained,for example, by growth of P1 on wildtype E. coli containing a leu+ gene (Figure 8.14). If such a particle adsorbs toa bacterial cell of leu- genotype and injects the DNA that it contains into the cell, then the cell survives because thephage head contained only bacterial genes and no phage genes.
A recombination event exchanging the leu+ allelecarried by the phage for the leu- allele carried by the host converts the genotype of the host cell from leu- into leu+.In such an experiment, typically about 1 leu- cell in 106 becomes leu+. Such frequencies are easily detected onselective growth medium. For example, if the infected cell is placed on solid medium that lacks leucine, it is able tomultiply and a leu+ colony forms. A colony does not form unless recombination inserted the leu+ allele.The fragment of bacterial DNA contained in a transducing particle is large enough to include about 50 genes, sotransduction provides a valuable tool for genetic linkage studies of short regions of the bacterial genome.
Considera population of P1 prepared from a leu+ gal+ bio+ bacterium. This sample contains particles able to transfer any ofthese alleles to another cell; that is, a leu+ particle can transduce a leu- cell to leu+, or a gal+ particle can transduce agal- cell to gal+. Furthermore, if a leu- gal- culture is infected with phage, both leu+ gal- and leu- gal+ bacteria areproduced. However, leu+ gal+ colonies do not arise because the leu and gal genes are too far apart to be included inthe same DNA fragment (Figure 8.15A).The situation is quite different with a recipient cell with genotype gal- bio-, because the gal and bio genes are soclosely linked that both genes are sometimes present in a single DNA fragment carried in a transducing particle—namely, a gal+ -bio+ particle (Figure 8.15B).
However, not all gal+ transducing particles also include bio+, nor doall bio+ particles include gal+. The probability of both markers being in a single particle—and hence the probabilityof simultaneous transduction of both markers (cotransduction)—depends on how close to each other the genesare. The closer they are, the greater the frequency of cotransduction. Cotransduction of the gal+ -bio+ pair can bedetected by plating infected cells on the appropriate growth medium.
If bio+ transductants are selected by spreadingthe infected cells on a glucosecontaining medium that lacks biotin, then both gal+ bio+ and gal- bio+ colonies willgrow. If these colonies are tested for the gal marker, then 12 percent are found to be gal+ bio+ and the rest gal- bio+;similarly, if gal+ transductants are selected, then about 12 percent are found to be gal+ bio+. In other words, thefrequency of cotransduction of gal and bio is 12 percent, which means that 12 percent of all transducing particlesthat contain one gene also include the other.Studies of cotransduction can be used to map closely linked genetic markers by means of three-factor crossesanalogous to those described in Chapter 4.
That is, P1 is grown on wildtype bacteria and used to transduce cellsthat carry a mutation of each of three closely linked genes. Cotransductants containing various pairs of wildtypealleles are examined. The gene located in the middle can be identified because its wildtype allele is almost alwayscotransduced with the wildtype alleles of the genes that flank it. For example, in Figure 8.15B, a genetic markerlocated between gal+ and bio+ will almost always be present in gal+ bio+ transductants.How is the frequency of cotransduction between genes related to their map distance in minutes in the standard E.coli map? The theoretical relation depends on the size of a molecule in a transducing phage relative to the size ofthe entire chromosome.
For bacteriophage P1, the distance betweenPage 327Figure 8.14Transduction. Phage P1 infects a leu+ donor, yielding predominantly normal P1 phageswith an occasional one carrying bacterial DNA instead of phage DNA. If the phagepopulation infects a bacterial culture, then the normal phages produce progeny phages, whereas thetransducing particle yields a transductant. Note that the recombination step requires twocrossovers. For clarity, double-stranded phage DNA is drawn as a single line.Page 328Figure 8.15Demonstration of linkage of the gal and bio genes by cotransduction. (A) A P1 transducing particle carrying the leu+ allele canconvert a leu- gal- cell into a leu+ gal- genotype (but cannot produce a leu+ gal+ genotype).
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