Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 102
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(A) Formation of a gal transducingphage (λdgal). (B) Formation of a bio transducing phage (λdbio).genes are replaced by bacterial genes during formation of the gal+ -bearing or bio+-bearing λ molecules. Thephages are thus called λdgal (defective, gal+-transducing) or λdbio (defective, bio+ -transducing). They are unableby themselves to infect E. coli productively. Addition of a wildtype helper phage both allows production of a lysaterich in λdgal or λdbio transducing phage and formation of a double lysogen. Presumably, integration of thewildtype λ provides hybrid attachment sites POB' and BOP' that allow integration of λdbio or λdgal byhomologous recombination between identical hybrid attachment sites.8.8—Transposable ElementsDNA sequences that are present in a genome in multiple copies and that have the capability of occasionalmovement, or transposition, to new locations in thePage 347genome were described in Section 6.8.
These transposable elements are widespread in living organisms.Many transposable elements in bacteria have been extensively studied. The bacterial elements first discovered—called insertion sequences or IS elements—are small and do not contain any known host genes. Like manytransposable elements in eukaryotes, these bacterial elements possess inverted-repeat sequences at their termini,and most code for a transposase protein required for transposition and one or more additional proteins thatregulate the rate of transposition. The DNA organization of the insertion sequence IS50 is diagrammed in Figure8.30A.
Insertion sequences are instrumental in the origin of Hfr bacteria from F+ cells (Section 8.4), because the Fplasmid normally integrates through genetic exchange between insertion sequences present in F and homologouscopies present at various sites in the bacterial chromosome.Other transposable elements in bacteria contain one or more bacterial genes that can be shuttled between differentbacterial hosts by transposing into bacterial plasmids (such as the F plasmid), which are capable of conjugationaltransfer. These gene-containing elements are called transposons, and their length is typically several kilobases ofDNA, but a few are much longer. Some transposons have composite structures with the bacterial genes sandwiched between insertion sequences, as is the case with the Tn5 element illustrated in Figure 8.30B, whichterminates in two IS50 elements in inverted orientation.
Transposons are usually designated by the abbreviation Tnfollowed by an italicized number (for example, Tn5). When it is necessary to refer to genes carried in such anelement, the usual designations for the genes are used. For example, Tn5 (neo-r ble-r str-r) contains genes forresistance to three different antibiotics: neomycin, bleomycin, and streptomycin. Such genes provide markers,making it easy to detect transposition of the composite element, as is shown in Figure 8.31. An F' lac+ plasmid istransferred by conjugation into a bacterialFigure 8.30Transposable elements in bacteria.
(A) Insertion sequence IS50. The element is terminated by short, nearly perfectinverted-repeat sequences, the terminal nine base pairs of which are indicated. IS50 contains a region that codes for thetransposase and for a repressor of transposition. The coding regions are identical in the region of overlap, but the repressoris somewhat shorter because it begins at a different place. (B) Composite transposon Tn5. The central sequence contains genesfor resistance to neomycin, neo-r; bleomycin, ble-r, and streptomycin, str-r; it is flanked by two copies of IS50 in invertedorientation. The left-hand element (IS50L) contains mutations and is nonfunctional, so the transposase and repressor aremade by the right-hand element (IS50R).Page 348cell containing a transposable element that carries the neo-r (neomycin-resistance) gene.
The bacterial cell isallowed to grow, and in the course of multiplication, transposition of the transposon into the F' plasmidoccasionally takes place in a progeny cell. Transposition yields an F' plasmid containing both the lac+ and neo-rgenes. In a subsequent mating to a Neo-s Lac- cell, the lac+ and neo-r markers are transferred together and so aregenetically linked.In nature, sequential transposition of transposons containing different antibiotic-resistance genes into the sameplasmid results in the evolution of plasmids that confer resistance to multiple antibiotics. These multiple-resistanceplasmids are called R plasmids. The evolution of R plasmids is promoted by the use (and regrettable overuse) ofantibiotics, which selects for resistant cells because, in the presence of antibiotics, resistant cells have a growthadvantage over sensitive cells.
The presence of multiple antibiotics in the environment selects for multiple-drugresistance. Serious clinical complications result when plasmids resistant to multiple drugs are transferred tobacterial pathogens, or agents of disease. Infections with some pathogens containing R factors are extremelydifficult to treat because the pathogen is resistant to all known antibiotics.When transposition takes place, the transposable element can be inserted in any one of a large number of positions.The existence of multiple insertion sites can be shown when a wildtype lysogenic E. coli culture is infected with atemperate phageFigure 8.31An experiment demonstrating the transposition of transposon Tn5, whichcontains a neomycin-resistance gene, from the chromosome to an F'plasmid containing the bacterial gene for lactose utilization (F' lac).
Thebacterial chromosome is lac- and the cells are unable to grow on lactoseunless the F' lac is present. After the transposition, the F' lac plasmidalso contains Tn5, indicated by the linkage of the neo-r gene to the F'factor. Note that transposition to the F' plasmid does not eliminate the copyof the transposon in the chromosome.Page 349that is identical to the prophage (immunity prevents the phage from killing the cell) but carries a transposableelement with an antibiotic-resistance marker. Transposition events can be detected by the production of antibioticresistant bacteria that contain new mutations resulting from insertion of the transposon into a gene in the bacterialchromosome.
For example, a lac+ leu+ neo-s culture of E. coli infected with a neo-r transposon can yield both lacneo-r and leu- neo-r mutants. If many hundreds of Neo-r bacterial colonies are examined and tested for a variety ofnutritional requirements and for the ability to utilize different sugars as a carbon source, then colonies can usuallybe found bearing a mutation in almost any gene that is examined.
This observation indicates that potential insertionsites for transposons are scattered throughout the chromosomes of E. coli and other bacterial species.The end result of the transposition process is the insertion of a transposable element between two base pairs in arecipient DNA molecule. During the insertion process, most transposition events also create a duplication of a short(2–12 nucleotide pairs) sequence of host DNA, which after transposition flanks the insertion site (Section 6.8). Theinsertion of a transposable element does not require DNA sequence homology or the use of most of the enzymes ofhomologous recombination, because transposition is not inhibited even in cells in which the major enzyme systemsfor homologous recombination have been eliminated. Direct nucleotide sequence analysis of many transposableelements and their insertion sites confirms the absence of DNA sequence homology in the recipient with anysequence in the transposable element.Transposons in Genetic AnalysisTransposons can be employed in a variety of ways in bacterial genetic analysis.
Three features make themespecially useful for this purpose.1. Transposons can insert at a large number of potential target sites that are essentially random in their distributionthroughout the genome.2. Many transposons code for their own transposase and require only a small number of host genes for mobility.3. Transposons contain one or more genes for antibiotic resistance that serve as genetic markers for selection.Genetic analysis using transposons is particularly important in bacterial species that do not have readily exploitedsystems for genetic manipulation or large numbers of identified and mapped genes. Many of these species arebacterial pathogens, and transposons can be used to identify and manipulate the disease factors.The use of a neo-r transposable element to identify a particular disease gene in a pathogenic bacterial species isdiagrammed in Figure 8.32.
In this example, the transposon is introduced into the pathogenic bacterium by amutant bacteriophage that cannot replicate in the pathogenic host. A variety of other methods of introduction alsoare possible. After introduction of the transposon and selection on medium containing the antibiotic, the onlyresistant cells are those in which the transposon inserted into the bacterial chromosome, because the phage DNA inwhich it was introduced is incapable of replication. Any of a number of screening methods are then used to identifycells in which a particular disease gene became inactivated (nonfunctional) as a result of the transposon insertinginto the gene.
This method is known as transposon tagging, because the gene with the insertion is tagged(marked) with the antibiotic-resistance phenotype of the transposon.Once the disease gene has been tagged by transposon insertion, it can be used in many ways (for example, ingenetic mapping) because the phenotype resulting from the presence of the tagged gene is antibiotic resistance,which is easily identified and selected. The lower part of Figure 8.32 shows how the transposon tag is used totransfer the disease gene into E. coli. In the first step, DNA from the pathogenic strain that contains the tagged geneis purified, cut into fragments of suitable size, and inserted into a small plasmid capable of replication in E. coli.(Details of these genetic engineering methods are discussedPage 350Figure 8.32Transposon tagging making use of a neo-r transposonto mutate a disease gene by inserting intoit.
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