Action of restriction endonucleases on transforming DNA of Haemophilus influenzae

Cleavage of DNA from H. influenzae with restriction endonucleases caused inactivation of transforming ability to an extent that depended on the genetic marker and the enzyme. The rate of inactivation, but not the final level of survival, depended on the concentration of enzyme in the restriction digest. In general, the greatest extent of inactivation of transforming activity was obtained with endonucleases that are known to produce the shortest fragments. The authors electrophoresed restriction digests of H. influenzae DNA in agarose gels and assayed transforming activity of DNA extracted from gel slices. In this way, they determined the lengths of restriction fragments that contain genetic markers of H. influenzae. For the marker that they studied most thoroughly (nov), the shortest restriction fragment that possessed detectable transforming activity was a 0.9-kilobase pair fragment produced by endonuclease R x PstI. The shortest marker-bearing restriction fragment that retained substantial transforming activity (50% of value for undigested DNA) was a 2.1-kilobase pair EcoRI fragment bearing the kan marker. Among marker-bearing restriction fragments 1 to 4 kilobase pairs in length, survival of transforming activity varied 10,000-fold. The authors relate these observations to the recent finding by Sisco and Smith that efficient entry of DNA into competent H. influenzae cells appears to require the presence of a recognition sequence that is scattered throughout the Haemophilus genome in many more copies than in unrelated genomes.