Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 99
Текст из файла (страница 99)
Figure 8.17 shows plaques from progeny of a mixed infection with E. coli phage T4 mutants. The r(rapid lysis) allele results in large plaques, and the h- (host range) allele results in clear plaques. The cross iswritten asFour plaque types can be seen in Figure 8.17. Two—the large turbid plaque and the small clear plaque—correspond to the phenotypes of the parental phages. The other two phenotypes—the large clear plaqueFigure 8.17A phage cross is performed by infecting host cells with both parental types of phage simultaneously.
This example showsthe progeny of a cross between T4 phages of genotypes r- h+ and r+ h- when both parental phages infect cells of E. coli.The arrowheads point to plaques formed from progeny phages of the indicated genotypes.[Courtesy of Leslie Smith and John W. Drake.]Page 332Figure 8.18Circular genetic map of phage T4. Each arc connects three or four markers that were mappedin the crosses indicated. The circular map results when all data are considered together.[After G. Streisinger, R. S. Edgar, and G.
H. Denhardt. 1964. Proc. Natl. Acad. Sci. USA51: 775.]and the small turbid plaque—are recombinants that correspond to the genotypes r- h- and r+ h+, respectively. Whenmany bacteria are infected, approximately equal numbers of reciprocal recombinant types are usually found amongthe progeny phage. In an experiment like that in Figure 8.17, in which each of the four genotypes yields a differentphenotype of plaque morphology, the number of each of the genotypes can be counted by examining each of theplaques that is formed. The recombination frequency, expressed as a percentage, is defined asRecombination frequencies can be used to estimate map distances, just as they are in eukaryotes.
Early mappingexperiments indicated that mutations in T4 mapped in three separate clusters. However, all three clusters showedlinkage to one another. In elegant experiments with three-point crosses, George Streisinger and colleaguesdemonstrated in 1964 that the genetic map for T4 phage is actually circular.In each cross, they mapped three or four genetic markers with respect to one another and proceeded systematicallythrough the entire T4 genome, eventually demonstrating all of the linkages shown in Figure 8.18. Many additionalgenes were identified and mapped later by other researchers (Figure 8.19), and the results were fully consistentwith the circular map.
In Figure 8.19, the regions indicated in the innermost circle are the three clusters of T4markers that had initially been identified and mapped. The outermost circle in Figure 8.19 presents a much largerset of markersPage 333Figure 8.19Circular genetic map of bacteriophage T4 with additional markers. The middle circle contains the markersmapped and used in the earliest T4 experiments. The units along the innermost circle are kilobases;0 kb is at a fixed point between the rIIA and rIIB cistrons. The outer circle contains many genes definedby so-called conditional mutations, which cause a mutant phenotype under one condition(for example, at a high temperature) but not under other conditions (for example, at a low temperature).Many of the genes show clustering according to their functions.[After W.
B. Wood and H. R. Revel. 1976. Bacteriol. Rev. 40:847.]Page 334Connection ArtooSeymour Benzer 1955Purdue University,West Lafayette, IndianaFine Structure of a Genetic Region in BacteriophageJust as every fan of "Star Wars" can identify the lovable robot Artoo-Detoo (a.k.a. R2-D2), everygeneticist can identify the gene rII. The rII gene in bacteriophage T4 was the first experimentalexample of genetic fine structure.
Benzer used the special property that rII mutants cannot grow on E.coli strain K but can grow on strain B to examine recombination between different nucleotides withinthe rII gene. He demonstrated that the rII gene was divisible by recombination. (It is now known that,in principle, recombination can take place between any adjacent nucleotides.) But if the gene can besubdivided by recombination, then what is a gene, anyway? If two different mutations can undergorecombination whether or not they are in the same gene, then how can one decide, experimentally,whether two different mutations are, or are not, alleles? Benzer realized that the key experimentaloperation in the definition of allelism was not recombination but rather the complementation test.
Thisis a rare paper with two great ideas in it: recombination within a gene, and the use of thecomplementation test to determine experimentally whether two different mutations are, or are not,alleles of the same gene.The phenomenon of genetic recombination provides a powerful tool for separating mutations anddiscerning their positions along a chromosome. When it comes to very close neighboring mutations, adifficulty arises, since the closer two mutations lie to one another, the smaller is the probability thatrecombination between them will occur. Therefore, failure to observe recombinant types ordinarilydoes not justify the conclusion that the two mutations are inseparable .
. .. A high degree of resolutioncan best be achieved if there is available a selective feature for the detection of small proportions ofrecombinants. Such a feature is offered by the case of the rII mutants of T4 bacteriophage described inthis paper. The wildtype phage produces plaques on either of two bacterial hosts, Escherichia colistrain B or strain K, while a mutant of the rII group produces plaques only on B.
Therefore, if a cross ismade between two different rII mutants any wildtype recombinants which arise, even in proportions aslow as 10-8, can be detected by plating on strain K . . .. In this way, a series of eight rII mutants of T4have been crossed with each other. The results of these crosses are given in the figure below.The distances are only roughly additive; there is some systematic deviation in the sense that a longdistance tends to be smaller than the sum of its component shorter ones, [which is accounted for bymultiple recombination events] .
. .. Thus, while all rII mutants in this set fall into a small portion of thephage linkage map, it is possible to seriate [order] them unambiguously, and their positions within theregion are well scattered . . .. Test for allelism. The functional relatedness of two closely linkedmutations causing similar defects may be tested by constructing diploid heterozygotes containing thetwo mutations . .
.. The transform, containing one of the mutations in each chromosome, may or maynot produce the wild phenotype. If it does [complementation], it is concluded that the two mutations inquestion are located in separate functional units. [They are alleles of different genes.] . . . In order tocharacterize a unit of genetic "function," it is necessary to define what function is meant . . .. On thebasis of phenotype tests of trans configuration heterozygotes [complementation tests], the rII regioncan be subdivided into two functionally separable segments. Each segment may have the "function" ofspecifying the sequence of amino acids in a polypeptide chain.Page 335establishing the overall circularity of the genetic map beyond doubt.
The T4 genetic map in Figure 8.19 alsoindicates that genes in T4 show extensive clustering according to the function for which they are required. Forexample, there is a large cluster of genes for DNA replication in the upper right quadrant, and there is a cluster ofgenes for phage head components near the bottom.In view of the circular nature of the T4 genetic map, it came as quite a surprise to find that the DNA molecule in aT4 phage particle is a single linear molecule. This discovery was completely unexpected and at first seemedinconsistent with the genetic data. However, the discrepancy was resolved by the finding that the very ends of thephage T4 DNA are duplicated, or have terminal redundancy.
Because of the redundancy, each molecule is about2 percent longer than would be expected. As the DNA is replicating inside the cell, recombination between each ofthe duplicated ends of one T4 genome with homologous sequences in other T4 genomes results in products muchlonger than can be contained in a T4 head (Figure 8.20).
Such concatenated molecules are formed becauserecombination in the T4 genome is very frequent, averaging about 20 recombination events per chromosome.When the DNA is packaged, it is enzymatically cleaved into "headful" packages consisting of about 102 percent ofthe minimum length of the T4 genome; hence the duplication at the ends. Because of the headful packagingmechanism, each T4 DNA molecule is terminally redundant. Moreover, in a whole population of DNA moleculespresent in a phage sample, except for the short terminal redundancy, each of the molecules is also a circularpermutation of the others. (A molecule is a circular permutation of another if the other sequence can be created byjoining its ends and cleaving at an internal position.) Because each T4 molecule is a circular permutation, differentmolecules begin at different points in the DNA sequence, but theyFigure 8.20Terminal redundancy and circular permutation of the phage T4 linear DNA molecule. A long concatenate,or series of molecules linked together, is formed inside the cell by genetic recombination.
Eachsuccessive headful of DNA is slightly longer than unit size and contains a terminal redundancy.Except for the terminal redundancy, the molecules are also circular permutations of each other.Page 336always incorporate a little more than one complete genome. The properties of terminal redundancy and circularpermutation account for the circular genetic map.Fine Structure of the rII Gene in Bacteriophage T4In Section 4.7, we described experiments with Drosophila that showed that recombination within a gene could takeplace. A genotype heterozygous for two different mutant alleles of the same gene could, at low frequency, generaterecombinant chromosomes carrying either both mutations or neither mutation.
![](https://s.studizba.com/z.php?f=/templates/si/i/noavatar.png&w=100&h=100&t=1"){kind=avatar}
