Separation of specific segments of transforming DNA after treatment with endodeoxyribonuclease

Abstract

Hemophilus parainfluenzae endodeoxyribonuclease was used to degrade the DNA of H. influenzae and to follow the biological activity of 14 markers associated with this DNA. It was found that some H. influenzae markers were completely inactivated by endodeoxyribonuclease treatment, while others appeared to retain all or almost all of their original activity. The bulk of the H. influenzae DNA was reduced to double-stranded pieces of the order of 8 x 10(5) to 1 x 10(6) daltons. Velocity sedimentation of the DNA in sucrose gradients disclosed that markers that retained biological activity were present in DNA particles that were of the order of 1 x 10(6) daltons or larger, and indicated a close correlation between the size of the DNA fragment and the amount of biological activity retained. These data suggest that H. parainfluenzae endodeoxyribonuclease breaks DNA at specific sites. The nal(r) marker was shown to have twice as much biological activity after treatment with endodeoxyribonuclease when assayed at saturating DNA concentrations. In the linear portion of the DNA dose-response curve, the biological activity of this marker was reduced 3- to 10-fold compared to untreated DNA (in accord with the reduced size of its DNA). These data demonstrate a specific enrichment of the nal(r) marker by about 6- to 20-fold, and suggest a technique for the separation and purification of specific segments of DNA.