The word bacteriophage, which is a name for bacterial viruses, means “eater of bacteria.” These viruses parasitize and kill bacteria. Pioneering work on the genetics of bacteriophages in the middle of the twentieth century formed the foundation of more recent research on tumor-causing viruses and other kinds of animal and plant viruses. In this way, bacterial viruses have provided an important model system.

These viruses can be used in two different types of genetic analysis. First, two distinct phage genotypes can be crossed to measure recombination and hence map the viral genome. Mapping of the viral genome by this method is the topic of this section. Second, bacteriophages can be used as a way of bringing bacterial genes together for linkage and other genetic studies. We will study the use of phages in bacterial studies in Section 5.5. In addition, as we will see in Chapter 10, phages are used in DNA technology as carriers, or vectors, of foreign DNA. Before we can understand phage genetics, we must first examine the infection cycle of phages.

Most bacteria are susceptible to attack by bacteriophages. A phage consists of a nucleic acid “chromosome” (DNA or RNA) surrounded by a coat of protein molecules. Phage types are identified not by species names but by symbols—for example, phage T4, phage λ, and so forth. Figures 5-22 and 5-23 show the structure of phage T4. During infection, a phage attaches to a bacterium and injects its genetic material into the bacterial cytoplasm, as diagrammed in Figure 5-22. An electron micrograph of the process is shown in Figure 5-24. The phage genetic information then takes over the machinery of the bacterial cell by turning off the synthesis of bacterial components and redirecting the bacterial synthetic machinery to make phage components. Newly made phage heads are individually stuffed with replicates of the phage chromosome. Ultimately, many phage descendants are made and are released when the bacterial cell wall breaks open. This breaking-open process is called lysis. The population of phage progeny is called the phage lysate.

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How can we study inheritance in phages when they are so small that they are visible only under the electron microscope? In this case, we cannot produce a visible colony by plating, but we can produce a visible manifestation of a phage by taking advantage of several phage characters.

Let’s look at the consequences of a phage infecting a single bacterial cell. Figure 5-25 shows the sequence of events in the infectious cycle that leads to the release of progeny phages from the lysed cell. After lysis, the progeny phages infect neighboring bacteria. This cycle is repeated through progressive rounds of infection, and, as these cycles repeat, the number of lysed cells increases exponentially. Within 15 hours after one single phage particle infects a single bacterial cell, the effects are visible to the naked eye as a clear area, or plaque, in the opaque lawn of bacteria covering the surface of a plate of solid medium (Figure 5-26). Such plaques can be large or small, fuzzy or sharp, and so forth, depending on the phage genotype. Thus, plaque morphology is a phage character that can be analyzed at the genetic level. Another phage phenotype that we can analyze genetically is host range, because phages may differ in the spectra of bacterial strains that they can infect and lyse. For example, a specific strain of bacteria might be immune to phage 1 but susceptible to phage 2.

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Two phage genotypes can be crossed in much the same way that we cross organisms. A phage cross can be illustrated by a cross of T2 phages originally studied by Alfred Hershey. The genotypes of the two parental strains in Hershey’s cross were h⁻ r⁺ × h⁺ r⁻. The alleles correspond to the following phenotypes:

To make the cross, E. coli strain 1 is infected with both parental T2 phage genotypes. This kind of infection is called a mixed infection or a double infection (Figure 5-27). After an appropriate incubation period, the phage lysate (containing the progeny phages) is analyzed by spreading it onto a bacterial lawn composed of a mixture of E. coli strains 1 and 2. Four plaque types are then distinguishable (Figure 5-28). Large plaques indicate rapid lysis (r⁻), and small plaques indicate slow lysis (r⁺). Phage plaques with the allele h⁻ will infect both hosts, forming a clear plaque, whereas phage plaques with the allele h⁺ will infect only one host, forming a cloudy plaque. Thus, the four genotypes can be easily classified as parental (h⁻ r⁺ and h⁺ r⁻) and recombinant (h⁺ r⁺ and h⁻ r⁻), and a recombinant frequency can be calculated as follows:

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If we assume that the recombining phage chromosomes are linear, then single crossovers produce viable reciprocal products. However, phage crosses are subject to some analytical complications. First, several rounds of exchange can take place within the host: a recombinant produced shortly after infection may undergo further recombination in the same cell or in later infection cycles. Second, recombination can take place between genetically similar phages as well as between different types. Thus, if we let P₁ and P₂ refer to general parental genotypes, crosses of P₁ × P₁ and P₂ × P₂ take place in addition to P₁ × P₂. For both these reasons, recombinants from phage crosses are a consequence of a population of events rather than defined, single-step exchange events. Nevertheless, all other things being equal, the RF calculation does represent a valid index of map distance in phages.

Because astronomically large numbers of phages can be used in phage-recombination analyses, very rare crossover events can be detected. In the 1950s, Seymour Benzer made use of such rare crossover events to map the mutant sites within the rII gene of phage T4, a gene that controls lysis. For different rII mutant alleles arising spontaneously, the mutant site is usually at different positions within the gene. Therefore, when two different rII mutants are crossed, a few rare crossovers may take place between the mutant sites, producing wild-type recombinants, as shown here:

As distance between two mutant sites increases, such a crossover event is more likely. Thus, the frequency of rII⁺ recombinants is a measure of that distance within the gene. (The reciprocal product is a double mutant and indistinguishable from the parentals.)

Benzer used a clever approach to detect the very rare rII⁺ recombinants. He made use of the fact that rII mutants will not infect a strain of E. coli called K. Therefore, he made the rII × rII cross on another strain and then plated the phage lysate on a lawn of strain K. Only rII⁺ recombinants will form plaques on this lawn. This way of finding a rare genetic event (in this case, a recombinant) is a selective system: only the desired rare event can produce a certain visible outcome. In contrast, a screen is a system in which large numbers of individuals are visually scanned to seek the rare “needle in the haystack.”

This same approach can be used to map mutant sites within genes for any organism from which large numbers of cells can be obtained and for which wild-type and mutant phenotypes can be distinguished. However, this sort of intragenic mapping has been largely superseded by the advent of inexpensive chemical methods for DNA sequencing, which identify the positions of mutant sites directly.

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