In the transformation of Diplococcus pneumonia, fragments of the transforming deoxyribonucleate (DNA) are inserted into the genome of recipient transformable bacteria. This entire process requires little or no net DNA synthesis., 2 Immediately following its fixation, the transforming DNA can be reisolated from the transformed bacterial population and can be demonstrated to be without biological activity. The newly introduced DNA recovers its activity with a half time of about 3 min and thereafter replicates in synchrony with the bulk DNA of the recipient bacteria." Lacks4 has examined the inactive transforming DNA extracted from transformed bacteria. Some of the material was degraded and the remainder was denatured. He concluded that the denatured DNA was singlestranded and proposed an integration mechanism on this basis.We will present evidence demonstrating that the transforming DNA extracted, prior to its replication, from transformed bacteria is a hybrid, which is apparently formed with the DNA of the recipient bacteria and which extends over a region of about one or two million daltons. Furthermore, the newly introduced DNA appears to be covalently linked to the DNA of the recipient bacteria.Experimental.-Transforming DNA carrying the density labels deuterium and nitrogen-15 was isolated from a streptomycin-resistant, p-nitrobenzoic acid-sensitive strain of pneumococcus,5 RF6S. The bacteria were allowed many generations of growth in a heavy-isotope-labeled medium containing p32 in the form of phosphate. The nutrients in the medium were in the form of sugar and amino acid extracts from algae that had been grown in a deuterium, nitrogen-15-substituted medium. The algal extracts were generously provided by H. Crespi.The bacteria were centrifuged, washed two times in 0.15 M NaCl, 0.01 M versene, pH 7.5, and then lysed with a mixture of sodium dodecyl sulphate and deoxycholate at a final concentration of 0.05% and 0.025%, respectively. The lysate was shaken with chloroform and isoamyl alcohol2 and either (a) treated with boiled ribonuclease, alcohol precipitated, redissolved in M/40 phosphate buffered saline, pH 7.5, and filtered through an HA Millipore filter before using, or (b) DNA reisolated from a preparative equilibrium density gradient centrifugation in CsCl.Equilibrium density gradient centrifugation was performed by adding DNA samples to a concentrated solution of CsCl (Trona) to give a final density of 1.70, a final volume of 1.5 ml, and a DNA concentration of less than 10 gg/ml. Samples were centrifuged at 36,000 rpm and 210C for 42 hr. The centrifuge tubes were punctured with a needle, and 26-32 fractions of two drops each were collected. The drops were diluted with 5 vol of M/40 phosphate buffered saline, pH 7.5, and aliquots were scanned for radioactivity and assayed for biological activity. Both the donor marker, streptomycin resistance, and the recipient marker, p-nitrobenzoic acid resistance, were assayed on the test strain RF6 which is sensitive to both drugs. Under these conditions, native "hea...

TRAINS which approach wild type in behavior can arise from mutant strains a result of either reversion (mutation of the gene altered by the original mutation), or suppression (mutation of some other gene). Recently it has become evident that reversion is an exceedingly complex phenomenon, whether analyzed at the level of genetic fine structure or in terms of modifications of specific proteins. Reversion may not only occur at the same site as the original mutational alteration but at other sites within the same gene (JINKS 1961;CRICK, BARNETT, BRENNER and WATTS-TOBIN 1961 ; YANOFSKY, HELINSKI and MALING 1961 ; HELINSKI and YANOFSKY 1963). The specific enzyme activities that are restored by reversion may be associated with proteins resembling the wild-type protein, or with proteins that are clearly different (MAAS and DAVIS