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Genetic Mapping of Lambda Bacteriophage |
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Genetic Mapping of Lambda Bacteriophage
Some of the earliest mapping experiments with γ phages were published by A. Kaiser in 1955. Starting with a wild-type strains, which can be designated as + ++++ he obtained five mutant strain by UV irradiation. Each mutant strain produced a variant type of plaque morphology: the s (small) strain created a small plaque, mi (minute), a minute plaque, c (clear), a completely clear plaque, co, (cocarde), a clear plaque except for a ring of colonies in the centre, and co2, where the central ring is denser than for co1. The three last gene mutations can be immediately recognized as interfering with a proper lysogenic response; whereas wild type γ will frequently lysogenize cells on bacterial lawn and thus will create a turbid plaque containing viable lysogenes, the clear and cocarde strains lyse virtually every cell they meet.
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Before mutant strains are used for genetic mapping purposes they are scrutinized to make sure that markers they carry are suitable. Suitability is usually defined by following five criteria-1. The mutant phenotype should be clearly distinguishable from the wild type and from one another so that the progeny can be easily classified (e.g., s and mi plaques of γ phage). 2. Each mutant strain should have a reliable phenotype to prevent ambiguity in classification (e.g., for γ bacteriophage a mutation is chosen whose expression is not highly dependant on the age of the bacteria in the lawn).3. Each mutant strain should ideally reproduce itself as effectively as the wild type (i.e., for bacteriophages the burst size-meaning the number of progeny emerging from a bacterium infected by a single particle- following a lytic cycle of growth should be comparable for both the wild and mutant strains.
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4. The marker used for mapping experiments should has low frequency of reversion, (e.g., cells are infected by phages carrying the mi mutation no more than one phage in 104 or 105 has a wild phenotype arising from a reversion, the marker is suitable for mapping). 5. The mutant should ideally differ from both the wild type and other strains being examined by a single mutation. In plaque-morphology mutant strains of phage this contigency can be investigated with one-factor crosses.
The genetic mapping of phage is done by the help of following kinds of crosses:
A. One-factor crosses-When only one phenotypic trait is followed during anyone cross, then it is called one-factor cross. To perform a one factor cross two types of phages, one wild and one mutant, are allowed to infect a suspension of E. coli. For example, a cross can be made between a wild type γ strain with a small γ strain (i.e., + x s).
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The infection is adjusted so that a dense suspension of E. coli is presented with an even denser suspension of the two types of phages, and on the average, about 10 phages (five of each type) infect each cell at the same time. In other words the multiplicity of infection is adjusted to have a value of approximately. 10, 109 phages and 108 bacteria are present. Phage multiplication, is then allowed to proceed until lysis occurs; the progeny phages in the lysate are diluted to appropriate concentrations for plating on to bacterial lawns; and the morphologies of the plaques that form on the lawns are individually scored. Kaiser (1955) found that among 4390 plaques examined after a cross of + x s, 2050 had the s phenotype and 2340 had the +phenotype. No- other plaque morphologies were detected on any of the plates. Similarly, for +xco1+ x co2 + x mi, and + x only the two parental types were found among the progeny and they were present in roughly equal numbers.
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Thus, one-factor crosses indicate that each of the five mutant strains differs by only a single factor from the wild type: no additional kinds of plaque phenotype emerge when a high multiplicity of infection to undergo genetic exchange.
B. Two-factor crosses-The hypothetical cross which is illustrated in figure, is infact, a two-factor cross. In it a double mutant parent, ab, was crossed with a wild-type parent, + +. When both mutant genes are carried by one parent and both their wild-type alleles are carried by the other parent, the cross is said to be in coupling-the two markers are coupled in the same chromosome. Thus in genetic shorthand the cross is written + + x ab, and recombination between a and b loci will produce single mutant progeny, designated +b and a+. The alternative in a two factor cross is that the markers be in repulsion-each parent carries only one of the two markers being followed in the cross (+ b x a+).
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Kaiser (1955) made two factor crosses in repulsion for different mutant strains of γ phage. For example, in a cross, +mi x c+, he obtained either wild type (++) or double recombinant or mutant (c mi) progeny. He isolated these double mutant phages and crossed them with wild type phages to yield data for crosses in coupling.
C. Three-point test crosses-In a three-factor cross the parental phages are distinguished by three traits. Laboratory stocks carrying three mutations can be obtained by crossing double mutant strains with single mutant strains and isolated recombinants. Three factor crosses are written for example, as s co1 mi x + + + or s + mi x +co1+.
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