Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 94
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Notethe complexity of this structure compared with the simplerorganization of the bacterial cell shown in part A.[Part A, courtesy of A. Benichou-Ryter; part B, courtesy of JeremyPickett-Heaps.]Page 310Figure 8.2Electron micrographs of four different viruses: (A) poliovirus; (B) tobacco mosaic virus; (C) E. coli phage λ; (D)E.
coli phage M13. In each case, the length of the bar is 10-5 cm, or 1000 Å units.[Courtesy of Robley Williams.]Figure 8.3A schematic diagram of the life cycle of a typical bacteriophage.Page 311by a single phage have the same genotype as the parental phage. However, if two phage particles with differentgenotypes infect a single bacterial cell, new genotypes can arise by genetic recombination.
This process differssignificantly from genetic recombination in eukaryotes in two ways: (1) the number of participating DNAmolecules differs from one cell to the next, and (2) reciprocal recombinants are not always recovered in equalfrequencies from a single infected cell.
Some phages also possess systems that enable phage DNA to recombinewith bacterial DNA. Phage-phage and phage-bacterium recombination are the topic of the second part of thischapter. The best-understood bacterial and phage systems are those of E. coli, and we will concentrate on thesesystems.8.2—Bacterial MutantsBacteria can be grown in liquid medium or on the surface of a semisolid growth medium gelled with agar. Bacteriaused in genetic analysis are usually grown on agar.
A single bacterial cell placed on an agar medium will grow anddivide many times, forming a visible cluster of cells called a colony (Figure 8.4). The number of bacterial cellspresent in a liquid culture can be determined by spreading a known volume of the culture on a solid medium andcounting the number of colonies that form. Typical E. coli cultures contain up to 109 cells/ml. The appearance ofcolonies, or the ability or inability to form colonies on particular media can, in some cases, be used to identify thegenotypes of bacterial cells.As we have seen in earlier chapters, genetic analysis requires mutants; with bacteria, three types are particularlyuseful:• Antibiotic-resistant mutants These mutants are able to grow in the presence of an antibiotic, such as streptomycin(Str) or tetracycline (Tet).
For example, streptomycin-sensitive (Str-s) cells have the wildtype phenotype and fail toform colonies on medium that contains streptomycin, but streptomycin-resistant (Str-r) mutants can form colonieson such medium.• Nutritional mutants Wildtype bacteria can synthesize most of the complex nutrients they need from simpleFigure 8.4A petri dish with bacterial colonies that have formed on a solid medium. The heavy streaks of growthresult from colonies so densely packed that there is no space between them.Page 312molecules present in the growth medium. The wildtype cells are said to be prototrophs. The ability to grow insimple medium can be lost by mutations that disable the enzymes used in synthesizing the complex nutrients.Mutant cells are unable to synthesize an essential nutrient and cannot grow unless the required nutrient is suppliedin the medium. Such a mutant bacterium is said to be an auxotroph for the particular nutrient.
For example, amethionine auxotroph cannot grow on a minimal medium that contains only inorganic salts and a source of energyand carbon atoms (such as glucose), but the methionine auxotroph can grow if the minimal medium issupplemented with methionine.• Carbon-source mutants Such mutant cells cannot utilize particular substances as sources of carbon atoms or ofenergy. For example, Lac- mutants cannot utilize the sugar lactose for growth and are unable to form colonies onminimal medium that contains only lactose as the carbon source.A medium on which all wildtype cells form colonies is called a nonselective medium. Mutants and wildtype cellsmay or may not be distinguishable by growth on a nonselective medium.
If the medium allows growth of only onetype of cell (either wildtype or mutant), then it is said to be selective. For example, a medium containingstreptomycin is selective for the Str-r phenotype and selective against the Str-s phenotype; similarly, minimalmedium containing lactose as the sole carbon source is selective for Lac+ cells and against Lac- cells.In bacterial genetics, phenotype and genotype are designated in the following way. A phenotype is designated bythree letters, the first of which is capitalized, with a superscript + or - to denote presence or absence of thedesignated phenotype, and with s or r for sensitivity or resistance.
A genotype is designated by lowercase italicizedletters. Thus a cell unable to grow without a supplement of leucine (a leucine auxotroph) has a Leu- phenotype, andthis would usually result from a leu- mutation. Often the - superscript is omitted, but using it prevents ambiguity.8.3—Bacterial TransformationBacterial transformation is a process in which recipient cells acquire genes from free DNA molecules in thesurrounding medium. Transformation with purified DNA was the first experimental proof that DNA is the geneticmaterial (Chapter 1).
In these experiments, a rough-colony phenotype of Streptococcus pneumoniae was changedto a smooth-colony phenotype by exposure of the cells to DNA from a smooth-colony strain. In the laboratory,donor DNA is usually isolated from donor cells and then added to a suspension of recipient cells.
In naturalsettings, such as soil, free DNA can become available by spontaneous breakage (lysis) of donor cells.Transformation begins with uptake of a DNA fragment from the surrounding medium by a recipient cell andterminates with one strand of donor DNA replacing the homologous segment in the recipient DNA.
Most bacterialspecies are probably capable of the recombination step, but many species have only a very limited ability to take upfree DNA efficiently. Even in a species capable of transformation, DNA is able to penetrate only some of the cellsin a growing population. However, many bacterial species can be made competent to take up DNA, provided thatthe cells are subjected to an appropriate chemical treatment (for example, treatment with CaCl2).Transformation is a convenient technique for gene mapping in some species. When DNA is isolated from a donorbacterium (Figure 8.5), it is invariably broken into small fragments. In most species, with suitable recipient cellsand excess external DNA, transformation takes place at a frequency of about 1 transformed cell per 103 cells.
Iftwo genes, a and b, used as genetic markers, are so widely separated in the donor chromosome that they are alwayscontained in two different DNA fragments, then the probability of simultaneous trans-Page 313Figure 8.5Cotransformation of linked markers. Markers a and b are near enough to each other that they are often present on the samedonor fragment, as are markers b and c.
Markers a and c are not near enough to undergo cotransformation. The gene order musttherefore be a b c. The size of the transforming DNA, relative to that of the bacterial chromosome, is greatly exaggerated.formation (cotransformation) of an a- b- recipient into wildtype is the product of the probabilities oftransformation of each genetic marker, or roughly 10-3 × 10-3, which equals one a+ b+ transformant per 106 recipientcells. However, if the two genes are so near one another that they are often present in a single donor fragment, thenthe frequency of cotransformation is nearly the same as thePage 314frequency of single-gene transformation, or one wildtype transformant per 103 recipients.
The general principle isas follows:Cotransformation of two genes at a frequency substantially greater than the product of the single-genetransformations implies that the two genes are close together in the bacterial chromosome.Studies of the ability of various pairs of genes to be cotransformed also yield gene order. For example, if genes aand b can be cotransformed, and genes b and c can be cotransformed, but genes a and c cannot, the gene order mustbe a b c (Figure 8.5). Note that cotransformation frequencies are not equivalent to the recombination frequenciesused in mapping eukaryotes, because they are determined by the size distribution of donor fragments and thelikelihood of recombination between bacterial DNA molecules rather than by the occurrence of chiasmata insynapsed homologous chromosomes (Chapter 4).8.4—ConjugationConjugation is a process in which DNA is transferred from a bacterial donor cell to a recipient cell by cell-to-cellcontact. It has been observed in many bacterial species and is best understood in E.
coli, in which it was discoveredby Joshua Lederberg in 1951.When bacteria conjugate, DNA is transferred to a recipient cell from a donor cell under the control of a set of genesthat give the donor cell its transfer capability. These genes are often present in a nonchromosomal, circular DNAmolecule called a plasmid. Plasmid-mediated recombination in E. coli usually results from the presence of aplasmid called the F factor or the fertility factor.
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