Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 101
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The genes in λ showextensive clustering by function. The left half of the map consists entirely of genes whose products (head and tailproteins) are required for assembly of the phage structure, and within this region, the head genes and the tail genesthemselves form subclusters. The right half of the λ genome also shows several gene clusters, which include genesfor DNA replication, recombination, and lysis. The genes are clustered not only by function but also according tothe time at which their products are synthesized. For example, the N gene acts early; genes O and P are active later;and genes Q, S, R, and the head-tail cluster are expressed last.
The transcription patterns for mRNA synthesis arethus very simple and efficient. There are only two rightward transcripts, and all late genes except for Q aretranscribed into the same mRNA.LysogenyThe lysogenic cycle comes about when the λ DNA molecule becomes integrated into the bacterial chromosomes,where it is passively replicated as part of the bacterialPage 341Figure 8.24Molecular and genetic maps of bacteriophage λ. The scale of the molecular map is length in kilobase pairs(kb); genetic distances are given in centimorgans (cM), equivalent in this case to percent recombination.Clusters of genes with related functions are indicated by the pastel colored boxes.
The positionsof the regulatory genes N and Q are indicated by vertical lines at about 35 and 45 kb, respectively. Promotersare indicated by the letter p. The direction of transcription and length of transcript are indicated byarrows. Many of the genes known through mutant phenotypes are identified.
Coding regionsof unidentified function are represented as unlabeled, light-colored rectangles; dark-coloredregions indicate sequences unlikely to code for proteins. The λ att (attachment site) is the site ofrecombination for integration of the λ prophage. The origin of replication (ori) is the region denotedO. The extents of possible substitutions of λ DNA with E. coli DNA in the specialized transducingphages λdgal and λdbio are indicated by arrows just above the gene map; such transducing phagesare discussed in the section on specialized transduction.chromosome; phage particles are not produced (Figure 8.25).
The inserted DNA is called a prophage, and thesurviving cell is called a lysogen. A strain lysogenic for λ has the symbol (λ) appended to its name. For example,the strain E. coli K12(λ) that made possible the fine-structure analysis of the rII region of phage T4 was a K12strain that had become lysogenic for λ.As noted, the cohesive ends of the λ DNA molecule are single-stranded, with 12 unpaired bases at either end. Theends are complementary, however.
Upon entering the cell, the complementary ends anneal to form a nicked circle,and ligation seals the nicks (Figure 8.26). Circularization, which takes place early in both the lytic and lysogeniccycles, is a necessary event in both cycles: for DNA replication in the lytic mode, for prophage integration in thelysogenic cycle. In about 75 percent of infected cells, the circular molecule replicates, and the lytic cycle ensues.However, in about 25 percent of infected cells, the circular λ molecule and the circular E.
coli DNA moleculeinteract and undergo a single, site-specific recombination event in which the phage DNA becomes incorporatedinto the bacterial chromosome. Because λ can exist either as an autonomous genetic element (in the lytic cycle) oras an integrated element in the chromosome (in lysogeny), λ, like the F factor, is classified as an episome.The positions of the site-specific recombination in the bacterial and phage DNA are called the bacterial and phageattachment sites, respectively.
Each attachment site consists of three segments. The centralPage 342Figure 8.25The general mode of lysogenization by integration of phage DNA into the bacterial chromosome.Some genes (those needed to establish lysogeny) are expressed shortly after infection and are thenturned off. The inserted brown DNA is the prophage.
For clarity, the phage DNA is drawn muchlarger than to scale; the size of phage λ DNA is actually about 1 percent of the size of the E. coli genome.Page 343Figure 8.26A diagram of a linear λ DNA molecule showing the cohesive ends(complementary single-stranded ends). Circularization by means ofbase pairing between the cohesive ends forms an open (nicked) circle,which is converted into a covalently closed (uninterrupted) circle bysealing (ligation) of the single-strand breaks. The length of the cohesiveends is 12 base pairs in a total molecule of approximately 50 kb.segment has the same nucleotide sequence in both attachment sites and is the region in which the recombinationactually takes place.
The phage attachment site is denoted POP' (P for phage), and the bacterial attachment site isdenoted BOB' (B for bacteria). A comparison of the genetic maps of the phage and the prophage indicates thatPOP' is located near the middle of the linear form of the phage DNA molecule (see Figure 8.24). A phage protein,integrase, catalyzes a site-specific recombination event; the integrase recognizes the phage and bacterialattachment sites and causes the physical exchange that results in integration of the λ DNA molecule into thebacterial DNA. The geometry of the exchange is shown in Figure 8.27.
As a result of the recombination event, thegenetic map of the prophage is not the same as the map of the phage; it is a circular permutation of it that arisesfrom the central location of λ att (the POP' site) and the circularization of the DNA.The correct model for prophage integration was first suggested by Allan Campbell in 1962. The model wasconfirmed by bacterial crosses of lysogens with nonlysogens, as well as by transduction by phage P1. Campbellfound that the order of genes in the integrated prophage waswhere m6 is a mutation affecting head formation and mi is one affecting lysis. In contrast, the gene order of genesin the free phage as determined by general recombination isPage 344Figure 8.27The geometry of integration and excision of phage λ.
The phage attachment site is POP'. The bacterialattachment site is BOB'. The prophage is flanked by two hybrid attachment sites denoted BOP' and POB'.The prophage map is thus a circular permutation of the map of the free phage. The prophage is inserted into the E.coli chromosome between the genes gal and bio, as indicated in Figure 8.28.
An additional finding that confirmedthe physical insertion of λ was that the integrated prophage increased the distance between gal and bio so that thesemarkers could no longer be cotransduced by phage P1. The distance between gal and bio in a λ lysogen is about 2minutes, compared to 1 minute in a nonlysogen.When a cell is lysogenized, the phage genes become part of the bacterial chromosome, so it might be expected thatthe phenotype of the bacterium would change. But most phage genes in a prophage are kept in an inactive state bya repressor protein, the product of one of the phage genes. The repressor protein is synthesized initially by theinfecting phage and then continually by the prophage. The gene that codes for the repressor is frequently the onlyprophage gene that is expressed in lysogens.
If a lysogen is infected with a phage of the same type as theprophage—for example, λ infecting a λ lysogen—then the repressor present within the cell from the prophageprevents expression of the genes of the infecting phage. This resistance to infection by a phage identical with theprophage, which is called immunity, is the usual criterion for determining whether a bacterial cell contains aparticular prophage.
For example, λ will not formPage 345Figure 8.28The map order of genes in phage λ as determined by phage recombination (lytic cycle) and in theprophage (prophage order). The genes have been selected arbitrarily to provide reference points.Bacterial DNA is labeled in red letters.plaques on bacteria that contain a λ prophage.A lysogenic cell can replicate nearly indefinitely without the release of phage progeny. However, the prophage cansometimes become activated to undergo a lytic cycle in which the usual number of phage progeny are produced.This phenomenon is called prophage induction, and it is initiated by damage to the bacterial DNA. The damagesometimes happens spontaneously but is more often caused by some environmental agent, such as chemicals orradiation.
The ability to be induced is advantageous for the phage because the phage DNA can escape from adamaged cell. The biochemical mechanism of induction is complex and will not be discussed, but the excision ofthe phage is straightforward.Excision is another site-specific recombination event that reverses the integration process. Excision requires thephage enzyme integrase plus an additional phage protein called excisionase.
Genetic evidence and studies ofphysical binding of purified excisionase, integrase, and λ DNA indicate that excisionase binds to integrase andthereby enables the latter to recognize the prophage attachment sites BOP' and POB'; once bound to these sites,integrase makes cuts in the O sequence and recreates the BOB' and POP' sites. This reverses the integrationreaction, causing excision of the prophage (Figure 8.27).Specialized Transducing PhageWhen a bacterium lysogenic for phage λ is subjected to DNA damage that leads to induction, the prophage isusually excised from the chromosome precisely. However, in about 1 cell per 106 to 107 cells, an excision error ismade (Figure 8.29), and a chance breakage in two nonhomologous sequences takes place—one break within theprophage and the other in the bacterial DNA. The free ends of the excised DNA are then joined to produce a DNAcircle capable of replication. The sites of breakage are not always located so as to produce a length of DNA thatcan fit in a λ phage head, and the DNA may be too large or too small.
Sometimes, however, a molecule forms thatcan replicate and be packaged. In λ lysogens, the prophage lies between the gal and bio genes, and because theaberrant cut in the host DNA can be either to the right or to the left of the prophage, particles can arise that carryeither the bio genes (cut at the right) or the gal genes (cut at the left). The resulting phage are called λbio and λgaltransducing particles. These are specialized transducing phages because they can transduce only certain bacterialgenes (gal or bio), in contrast with the P1-type generalized transducing particles, which can transduce any gene.Often the specialized transducing phages are defective, because essential λPage 346Figure 8.29Aberrant excision leading to the production of specialized λ transducing phages.
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