Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 100
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These experiments gave the firstindication of intragenic recombination and the first evidence that genes have fine structure. Other studies of geneswere performed in the years that followed this experiment, but none could equal the finestructure mapping of therII gene in bacteriophage T4 carried out by Seymour Benzer.
Using novel genetic mapping techniques that reducedthe number of required crosses from more than half a million to several thousand, Benzer succeeded in mapping2400 independent mutations in the rII locus of phage T4.Wildtype T4 bacteriophage is able to multiply in E. coli strains B and K12(λ) and gives small ragged plaques.Mutations in the rII gene of T4 yield large round plaques on strain B but are completely unable to propagate instrain K12(λ). If E. coli cells of strain B are all infected with two different rII mutants, then recombination betweenthe mutants can be detected, even if the frequency is very low, by taking advantage of the inability of rII mutants togrow on K12(λ). Plating the progeny phages on K12(λ) selects for growth of the rII+ recombinant progeny,because only these recombinants can grow.
Furthermore, because very large numbers of progeny phage can beexamined (numbers of 1010 bacteriophages/ml are not unusual), even very low frequencies of recombination can bedetected. Typical results for three different rII mutations might yield a map like the following:where the numbers above the line designate rII alleles and the numbers below are map distances given as two timesthe percent of rII+, namelySome mutations failed to recombine with several mutations, each of which recombined with the others. These wereinterpreted to be deletion mutations, because they prevented recombination with two or more "point" mutationsknown to be at different sites in the gene.
Each deletion eliminated a part of the bacteriophage genome, including aregion of the rII gene. The use of deletions greatly simplified the ordering and mapping of thousands of mutations.Figure 8.21 depicts the array of deletion mutations used for mapping. Deletion mapping is based on the presence orabsence of recombinants; each cross yields a yes-or-no answer and so avoids many of the ambiguities of geneticmaps based on frequencies of recombination. In any cross between an unknown "point" mutation (for example, asimple nucleotide substitution) and one of the deletions, the presence of wildtype progeny means that the pointmutation is outside of the region missing in the deletion. (The reciprocal product of recombination, which carriesthe deletion plus the point mutation, is not detected in these experiments.) On the other hand, if the point mutationis present in the region missing in the deletion, then wildtype recombinant progeny cannot be produced.
Becauseeach cross clearly reveals whether a particular mutation is with in the region missing in the deletion, deletionmapping also substantially reduces the amount of work needed to map a large number of mutations.The series of crosses that would be made in order to map a particular rII mutation is presented in Figure 8.22, inwhich the large intervals A1 through A6 plus B are the same as those in Figure 8.21.
To illustrate the method,suppose that a particular mutation being examined is located in the region denoted A4. This mutation wouldPage 337Figure 8.21The array of deletion mutations used to divide the rII locus of bacteriophage T4 into 7 regions and 47 smallersubregions. The extent of each deletion is indicated by a horizontal bar. Any deletion endpoint used toestablish a boundary between regions or subregions is indicated with a vertical line.[After S. Benzer. 1961. Proc. Natl. Acad.
Sci. USA 47:403.]fail to yield wildtype recombinants in crosses with the large deletions r1272, r1241, rJ3, and rPT1, but it wouldyield wildtype recombinant progeny in crosses with the large deletions rPB242, rA105, and r638. Conversely, anymutation that yielded the same pattern of outcomes in crosses with the large deletions would be assigned to regionA4. Still finer resolution of the genetic map within region A4 is made possible by the set of deletions shown at thebottom of Figure 8.22, the endpoints of which define seven subregions (a through g) within A4. For example, amulation in region A4 that yields wildtype recombinants with the deletion rl368 but not with r221 would beassigned to the c subregion. At the finest level of resolution, mutationsPage 338Figure 8.22The location of rII in relation to other genetic markers in a linear version of the phage T4 genetic map.Major subdivisions of the rII region are defined by the left ends of seven deletion mutations.
All sevendeletions extend through the right end of the rII region. Genetic material deleted is indicated in red. Furthersubdivision of the A4 region is made possible by the additional deletions shown atthe bottom of the figure. For simplicity, only the deleted regions are shown.within a subregion are ordered by crosses among themselves. In phage T4, mutant sites that are very close can beseparated by recombination, because on the average, 1 percent recombination corresponds to a distance of about100 base pairs. Hence, any two mutations that fail to recombine can be assigned to the same site within the gene.The genetic map generated for a large number of independent rII mutations is given in Figure 8.23.The rII mutation and mapping studies were important because they gave experimental support to these conclusions:• Genetic exchange can take place within a gene and probably between any pair of adjacent nucleotides.Page 339Figure 8.23Genetic map of the rII locus of phage T4.
Each small square indicates a separate, independent occurrence of a mutation atthe indicated site. The arrangement of sites within each A or B segment is arbitrary.[After S. Benzer. 1961. Proc. Natl. Acad. Sci. USA 47:403.]• Mutations are not produced at equal frequencies at all sites within a gene. For example, the 2400 rII mutationswere located at only 304 sites.
One of these sites accounted for 474 mutations (Figure 8.23); a site that shows sucha high frequency of mutation is called a hotspot of mutation. At sites other than hotspots, mutations wererecovered only once or a few times.The rII analysis was also important because it helped to distinguish experimentally between three distinct meaningsof the word gene. Most commonly, the word gene refers to a unit of function. Physically, this corresponds to aprotein-coding segment of DNA. Benzer assigned the term cistron to this unit of function, and the term is stilloccasionally used.
The unit of function is normally defined experimentally by aPage 340complementation test (see Sections 2.2 and 4.8), and indeed, two units of function, the rIIA cistron and the rIIBcistron, were defined by complementation. The limits of rIIA and rIIB are shown in Figures 8.21 and 8.23. Thecomplementation between rIIA and rIIB is observed when two types of T4 phage, one with a mutation in rIIA andthe other with a mutation in rIIB, are used simultaneously to infect E. coli strain K12(λ). The multiply infectedcells produce normal numbers of phage progeny, most of which carry either the parental rIIA mutation or theparental rIIB mutation.
However, rare recombination between the mutant sites results in recombinant progenyphage that are rII+. In contrast, when K12(λ) is simultaneously infected with two phage having different rIIAmutations, or two phage having different rIIB mutations, no progeny phage are produced.Besides the meaning of function, clarified by use of the term cistron, the term gene has two other distinctmeanings: (1) the unit of genetic transmission that participates in recombination and (2) the unit of genetic changeor mutation. Physically, both the recombinational and the mutational units correspond to the individual nucleotidesin a gene.
Despite potential ambiguity, the term gene is still the most important word in genetics, and in most cases,the shade of meaning intended is clear from the context.8.7—Genetic Recombination in Temperate BacteriophagesTemperate bacteriophages have two alternative life cycles—a lytic cycle and a lysogenic cycle. The lytic cycle isdepicted in Figure 8.3.
A temperate phage, such as E. coli phage λ, when reproducing in its lytic cycle, undergoesgeneral recombination much as phage T4 does. A map of the phage λ genome is depicted in Figure 8.24; it is linearrather than circular. The DNA molecule in the λ phage particle is also linear. Unlike the DNA molecules in T4phage, however, every phage λ DNA molecule has identical ends. Indeed, the ends are single-stranded andcomplementary in sequence so that they can pair, forming a circular molecule.The single-stranded ends are called cohesive ends to indicate their ability to undergo base pairing.
The packagingof DNA in phage λ does not follow a headful mechanism, like T4. Rather, the λ packaging process recognizesspecific sequences that are cleaved to produce the cohesive ends.The complete DNA sequence of the genome of λ phage has been determined, and many of the genes and geneproducts and their functions have been identified.
The map of genes in Figure 8.24 shows where each gene islocated along the DNA molecule, scaled in kilobase pairs (kb), rather than in terms of its position in the geneticmap. However, a genetic map with coordinates in map units (centimorgans, or percent recombination) has beenplaced directly below the molecular scale. Comparing the scales reveals that the frequency of recombination is notuniform along the molecule. For example, the 5 map units between genes H and I span about 3.1 kb, whereas the 5map units between the genes int and cIII span about 4.8 kb.The λ map is also interesting for what it implies about the evolution of the phage genome.
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