A genetic switch in vitro: DNA inversion by Gin protein of phage Mu.

AUTOR(ES)

Plasterk, R H

RESUMO

Inversion of the G segment in the DNA of Escherichia coli phage Mu depends on the Mu Gin protein and alters the host range of the phage. The frequency of the inversion reaction is low both in the lysogenic state and during lytic growth. A sensitive assay was developed to detect low levels of G inversion: the E. coli lac operon was inserted within the invertible G segment in such a way that the lac operon was expressed only by G(-) clones. As a result Gin-catalyzed inversion from G(+) to G(-) can be monitored as a lactose-negative to lactose-utilizing switch. Using a crude extract from a Gin-overproducing strain and this assay plasmid, we could detect a low level of G inversion in vitro (1% in 30 min). The reaction depends on Mg2+ and a supercoiled substrate. Under optimized reaction conditions over 15% of the plasmids had the G segment inverted after incubation with Gin in vitro. The inversion was then visualized by agarose gel analysis of plasmid DNA digested by restriction endonucleases. The Gin protein retains its catalytic properties upon partial purification. The mechanism of this genetic switch can now be studied in vitro.

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