Use of Transducing Phage to Study Integration and Excision

Use of Transducing Phage to Study Integration and Excision

One use of transducing phage is to demonstrate that lambda normally integrates and excises at precisely the same point. Of course, lambda can be integrated and excised many times from the bacterial att region, and the region apparently suffers no harm. Nonetheless, how do we know, without sequencing, that bases are not inserted or deleted in the process? The integration of lambda into secondary att sites provided the proof that, as far as the sequence of the host chromosome is concerned, excision is the exact opposite of integration.

When lambda integrates into a secondary att site, the gene into which lambda has inserted is disrupted and therefore inactivated, but when the lambda is induced and excises from these sites, the majority of the surviving cells possess a perfectly normal gene. Few of the lambda improperly excise and produce transducing phage as described in the previous section. That is, except for the products of the rare improper excision events, no nucleotides are inserted or deleted at the pseudo att site. Therefore it is reasonable to infer that the integration and excision cycle at the normal att site similarly does not alter its sequence. For example, one site for secondary lambda integration is a gene coding for a protein required for proline synthesis. The insertion of lambda makes the cells Pro^-, but heat-pulse curing leaves the cells Pro⁺ (Fig. 18.7).

Figure 18.7 Integration oflambda phage into a pseudo att site in a gene inactivates that gene, but excision restores the original nucleotide sequence and the gene is reactivated.

A second use of transducing phage is in the study of the biochemistry of the integration and excision reactions themselves. These site-specific recombination events take place at the att regions, but the partners need not be confined to a phage and host chromosome. For example, the enzymatic equivalent of an excision reaction can be performed between λdgaland a λpbioto form a λ and a λdgal-bio. To detect these recombi-nation products, the input phage must be genetically marked. This can be done by using nonsense mutations located in the A and R genes, which are at opposite ends of the lambda DNA. The cross might therefore be between λdgalA^-R⁺ and λpbioA⁺R^-; the frequency of genera-tion of wild-type lambda, those able to form plaques on su^- cells, could

be measured. Crosses between all combinations of att regions can be performed by similar approaches. The main results of such studies examining the site-specific recombination events catalyzed by Int and Xis proteins are that all combinations of att regions will recombine, but at different rates, and that the Xis protein is required only for the excision type of reaction.