Mapping with Generalized Transducing Phage

Mapping with Generalized Transducing Phage

As we have already discussed, coliphage P1 is imperfect in its encapsulation process, and instead of packaging P1 DNA with com-plete fidelity, a small fraction of the phage package a segment of their host’s DNA. Upon infection of sensitive cells with such a lysate, the majority of infected cells are injected with a phage DNA molecule. A small fraction of the cells however, are injected with a segment of the chromosome of the cells on which the phage lysate was prepared. This DNA segment cannot permanently survive in the cells since it lacks a DNA replication origin and furthermore, exonucleases would degrade it. It is free to engage in genetic recombination before it is degraded. This type of transfer of genetic markers via a phage is called generalized transduction.

The ability to transduce cells with a phage like P1 facilitates fine structure genetic mapping. Three factor crosses may be performed with phage P1 to determine the order of genetic markers regardless of the sex of the cells rather than being restricted to using donor and receptor cell lines for bacterial conjugation. Even more useful, however, are the consequences of the fact that the length of DNA which a P1 phage particle carries is only 1% the size of the bacterial chromosome. There-fore, the frequency with which two genetic markers are simultaneously carried in a single transducing phage particle is high if they are sepa-rated by much less than 1% of the chromosome. This frequency falls as the separation between two markers increases, and it becomes zero if they are so widely separated that a segment of DNA carrying both alleles is too large to be encapsulated. As a result, the frequency of cotransduc-tion of two genetic markers provides a good measurement of their separation.

Cotransduction frequency often is easy to measure. For example, other experiments could have indicated that the genes for synthesis of leucine and the genes permitting utilization of the sugar arabinose were closely spaced on the chromosome. P1 mapping can measure their separation more accurately. P1 could be grown on an Ara⁺ Leu⁺ strain and used to transduce an Ara^- Leu^- strain to Ara⁺ by spreading the P1 infected cells on agar containing minimal salts, arabinose as a carbon source, and leucine. Then, the Ara⁺ transductants could be scored by spot testing for the state of their leucine genes.