In vitro Assay of Integration and Excision

In vitro Assay of Integration and Excision

Although ingenious genetic experiments can reveal much about biological systems, ultimately the details must be studied biochemically. There-fore it was of great interest to isolate the Int and Xis proteins. Such an isolation requires an assay for the proteins. The most reliable assay for Int protein activity is to seek a protein that carries out the entire integration reaction in vitro. As mentioned above, one likely require-ment for such a reaction to proceed in vitro is a high concentration of attB and attP regions. The easiest way to obtain these is to place bothon the same DNA molecule.

Can attB and attP be put on the same molecule, just as attL and attR could be put on λatt²? Nash constructed such phage by selecting the product of a rare recombination event in a region of nonhomology between a λdgal-bio containing attB and a λ (Fig. 18.14). Similar to the excision-type of reaction that occurs on λatt², on λattB-attP an integra-tion-type of reaction can occur. This removes the bio region and leaves the phage considerably more resistant to Mg⁺⁺ chelation or heat. Hence, the parental phage and derivatives that have undergone an integration reaction and have become smaller can easily be distinguished.

Initial tests with the λattB-attP phage showed that an in vitro integra-tion reaction catalyzed by an extract from cells would work. The experiment was performed by incubating the λattB-attP DNA with a cell extract prepared from heat-pulsed lambda lysogens. Then the DNA was extracted from the mix and used to transfect cells that had been made capable of taking up naked lambda DNA. The phage from the trans-fected spheroplasts were spread on a lawn of sensitive cells on a plate containing pyrophosphate. Under these conditions, only products from the integrative reaction would produce plaques. This provided an ex-tremely sensitive assay for the integration reaction. Later, as the assay conditions were improved, the integration reaction could be assayed merely by physical quantitation of the DNA products. The locations of restriction enzyme cleavage sites in the DNA are rearranged by the integration reaction. This creates new sizes of restriction fragments that can be detected by separating the resulting fragments by electrophoresis (Fig. 18.15).

The in vitro integration reaction required Mg⁺⁺, ATP, and spermidine. More careful examination showed that the use of supercoiled DNA eliminated the requirement for ATP and Mg⁺⁺. The assays permitted the purification and characterization of biologically active Int protein, and analogous experiments have been done with the λatt² for the purification of Xis protein.