Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 96
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The incorporated regions replace homologous regions in the recipient chromosome. The result is thatsome F- cells become recombinants containing one or more genes from the Hfr donor cell. For example, in amating between Hfr leu+ and F- leu-, some F- leu+ cells arise. However, the genotype of the donor Hfr cell remainsunchanged.Genetic analysis requires that recombinant recipients be identified. Because the recombinants derive from recipientcells, a method is needed to eliminate the donor cells. The usual procedure is to employ an F- recipient containingan allele that can be selected.
Genes that confer antibiotic resistance are especially useful for this purpose. Forinstance, after a mating between Hfr leu+ str-s and F- leu- str-r cells, the Hfr Str-s cells can be selectively killed byplating the mating mixture on medium containing streptomycin. A selective medium that lacks leucine can then beused to distinguish between the nonrecombinant and the recombinant recipients. The F- leu- parent cannot grow inmedium that lacks leucine, but recombinant F- leu+ cells can grow because they possess a leu+ gene.
Onlyrecombinant recipients—that is, cells having the genotype leu+ str-r—form colonies on a selective mediumcontaining streptomycin and lacking leucine. The selected allele should be located at such a place in thechromosome that most mating cells will have broken apart before the selected gene is transferred, and the selectedallele must not be present in the Hfr cell. The selective agent can then be used to select the F- cells and eliminatethe Hfr donors.When a mating is done in this way, the transferred marker that is selected by the growth conditions (leu+ in thiscase) is called a selected marker, and the marker used to prevent growth of the donor (str-s in this case) is calledthe counterselected marker.
Selection and counterselection are necessary in bacterial matings becauserecombinants constitute only a small proportion of the entire population of cells (in spite of the name "highfrequency of recombination").Time-of-Entry MappingGenes can be mapped by Hfr × F- matings. However, the genetic map is quite different from all maps that we haveseen so far in that it is not a linkage map but a transfer-order map. It is obtained by deliberate interruption of DNAtransfer in the course of mating—for example, by violent agitation of the suspension of mating cells in a kitchenblender.
The time at which a particular gene is transferred can be determined by breaking the mating cells apart atvarious times and noting the earliest time at which breakage no longer prevents recombinants from appearing. Thisprocedure is called the interrupted-mating technique. When this is done with Hfr × F- matings, the number ofrecombinants of any particular allele increases with the time during which the cells are in contact. Thisphenomenon is illustrated in Table 8.1. The reason for the increase is that different Hfr × F- pairs initiateconjugation and chromosome transfer at slightly different times.A greater understanding of the transfer process can be obtained by observing thePage 320Table 8.1 Data showing the production ofLeu+ Str-r recombinants in a cross betweenHfr leu+ str-s and F- leu- str-r cells whenmating is interrupted at various timesMinutes aftermatingNumber of Leu+ Str-rrecombinants per 100Hfr cells003066915122415331842214324432743Note: Minutes after mating means minutesafter the Hfr and F- cell suspensions aremixed.
Extrapolation of the recombinationdata to a value of zero recombinants indicatesthat the earliest time of entry of the leu+marker is 4 minutes.results of a mating with several genetic markers. For example, consider the matingin which a- cells require nutrient A, b- cells require nutrient B, and so forth. At various times after mixing of thecells, samples are agitated violently and then plated on a series of media that contain streptomycin and differentcombinations of the five substances A through E (in each medium, one of the five is left out). Colonies that formon the medium lacking A are a+ str-r, those growing without B are b+ str-r, and so forth. All of these data can beplotted on a single graph to give a set of curves, as shown in Figure 8.10A.
Four features of this set of curves arenotable.1. The number of recombinants in each curve increases with length of time of mating.2. For each marker, there is a time (the time of entry) before which no recombinants are detected.3. Each curve has a linear region that can be extrapolated back to the time axis, defining the time of entry of eachgene a+, b+, . .
., e+.4. The number of recombinants of each type reaches a maximum, the value of which decreases with successivetimes of entry.The explanation for the time-of-entry phenomenon is the following: All donor cells do not start transferring DNAat the same time, so the number of recombinants increases with time. Transfer begins at a particular point in theHfr chromosome (the replication origin of F). Genes are transferred in linear order to the recipient, and the time ofentry of a gene is the time at which that gene first enters a recipient in the population.
Separation of a mating pairprevents further transfer and limits the number of recombinants seen at a particular time.The times of entry of the genes used in the mating just described can be placed on a map, as shown in Figure8.10B. The numbers on this map and the others are genetic distances between the markers, measured as minutesbetween their times of entry. Mating with another F- with genotype b- e- f- g- h- str-r could be used to locate thethree genes f, g, and h. Data for the second recipient would yield a map such as that shown in Figure 8.10C.Because genes b and e are common to both maps, the two maps can be combined to form a more comprehensivemap, as shown in Figure 8.10D.Studies with different Hfr strains (Figure 8.10E) also are informative.
It is usually found that different Hfr strainsare distinguishable by their origins and directions of transfer, which indicates that F can integrate at numerous sitesin the chromosome and in two different orientations. Combining the maps obtained with different Hfr strains yieldsa composite map that is circular, as illustrated in Figure 8.10F. The circularity of the map is a result of thecircularity of the E. coli chromosome in F- cells and the multiple points of integrationPage 321Figure 8.10Time-of-entry mapping. (A) Time-of-entry curves for one Hfr strain. (B) The linear map derived from the data inpart A.
(C) A linear map obtained with the same Hfr but with a different F- strain containing the alleles b- ef- g- h-. (D) A composite map formed from the maps in parts B and C. (E) A linear map from another Hfrstrain. (F) The circular map (gold) obtained by combining the two maps (green and blue) of parts D and E.Page 322Connection The Sex Life of BacteriaJoshua Lederberg and Edward L. Tatum 1946Yale University,New Haven, ConnecticutGene Recombination in Escherichia coliSince their discovery in the nineteenth century, bacteria were considered "things apart"—unlike otherorganisms in fundamental ways. Lederberg and Tatum's discovery of what at first appeared to be aconventional sexual cycle was a sensation, completely unexpected. It meant that bacteria could beconsidered "genetic organisms" along with yeast, Neurospora, Drosophila, and other genetic favorites.
Forthis and related discoveries, Lederberg and Tatum were awarded the 1958 Nobel Prize along with GeorgeW. Beadle. In this excerpt, you will note that the authors discuss bacterial recombination as requiring a cellfusion that would bring both parental genomes together. This interpretation shows that it is possible to makeexactly the right observation, and realize its significance, but not quite grasp what is really going on.
Theconclusion that bacterial recombination involved unidirectional transfer was reached much later, after thediscovery of Hfr strains and the development of the interrupted-mating technique.Analysis of mixed cultures of nutritional mutants has revealed the presence of new types which stronglysuggest the occurrence of a sexual process in the bacterium Escherichia coli. The mutants consist of strainswhich differ from their parent wildtype strain K-12, in lacking the ability to synthesize growth factors. As aresult of these deficiencies they will only grow in media supplemented with their specific nutritionalrequirements.
In these mutants single nutritional requirements are established as single mutational stepsunder the influence of x-rays or ultraviolet light. By successive treatments, strainsThese types can most reasonably be interpreted as instances of the assortment of genesin new combinationswith several requirements have been obtained.
In the recombination studies here reported, two triple mutantshave been used, one requiring threonine, leucine and thiamin, the other requiring biotin, phenylalanine andcystine. The strains were grown in mixed culture in complete medium. The cells were washed with sterilewater and inoculated heavily into synthetic agar medium, to which various supplements had been added toallow the growth of colonies of various nutritional types. This procedure readily allows the detection of verysmall numbers of cell types different from the parental forms. The only new types found in "pure" cultures ofthe individual mutants were occasional forms which had reverted for a single factor, giving strains whichrequired only two of the original three substances.
In mixed cultures, however, a variety of types has beenfound. These include wild-type strains with no growth-factor deficiencies and single mutant types requiringonly thiamin or phenylalanine . . .. These types can most reasonably be interpreted as instances of theassortment of genes in new combinations. In order that various genes may have the opportunity torecombine, a cell fusion would be required . . ..
The fusion presumably occurs only rarely, since in thecultures investigated only one cell in a million can be classified as a recombinant type . . .. These experimentsimply the occurrence of a sexual process in the bacterium Escherichia coli.Source: Nature 158:558of the F plasmid; if F could integrate at only one site and in one orientation, the map would be linear.A great many such mapping experiments have been carried out, and the data have been combined to provide anaccurate map of approximately 2000 genes throughout the E.
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