In 1963, it was reported that deoxyribonucleic acid (DNA)-mediated transformations could take place spontaneously between genetically distinguishable pneumococci growing in a living host, the mouse. It was found in the present study that such transformations could be increased in frequency if infected animals were treated with a drug which kills only one of the two infecting strains. The frequency of in vivo transformations w4s also increased if the infection was prolonged. Interspecific transformation between pneumococci and streptococci without the addition of purified DNA was demonstrated both in vitro and in vivo. These results make it seem more likely that DNA-mediated transformations occur in nature.

A survey of pneumococci isolated from 19 healthy carriers and from 23 patients with pneumococcal disease showed that, for both groups, 25 to 30% of the isolates were competent for transformation by soluble deoxyribonucleic acid (DNA) in vitro. Untransformable type 3 and type 8 pneumococci, whose capsules had been hydrolyzed by the Bacillus palustris enzymes prior to exposure to DNA, remained untransformable. Thus, at least for these isolates, it was not the presence of capsule that prevented transformation. Type 9 pneumococci in a healthy human carrier were transformed by DNA released from living unencapsulated pneumococci sprayed onto the pharynx. The donor bacteria were resistant to 1,000 ,ug of streptomycin/ml. Two types of streptomycin-resistant bacteria were recovered from the carrier's pharynx: a type 9 pneumococcus and an alpha-hemolytic streptococcus. No streptomycin-resistant, gram-positive cocci were isolated from this individual prior to inoculation of the pharynx with the resistant organisms. It seems possible that transformations can occur in the natural environment of some gram-positive cocci.

The rate of clearance of pneumococci from the lungs of mice infected by aerosols was found to vary inversely with the virulence of the pneumococci. Treatment of of the mice with ethanol delayed the clearance and increased the rate of pneumococcal transformations in vivo. The transfer of genetic information from one Pneumococcus to another by transformation with freshly released deoxyribonucleic acid is known to occur both in vitro (1, 10, 16) and in vivo (4, 8, 18, 19). Conant and Sawyer (4) have recently reported that it will occasionally occur in mice infected by intrabronchial inoculation. The objective of the present study has been to design an experimental model in which pneumococcal transformation will occur consistently in the lungs. The design is based on the evidence that (i) pneumococci must multiply rapidly and accumulate in relatively large numbers for transformation to be efficient (4) and (ii) that ethanol intoxication will slow the pulmonary clearance of airborne bacteria (6, 12, 20, 23). MATERIALS AND METHODS Mice. Strain CD-1, male, pathogen-free albino mice weighing 21 to 25 g were obtained from Charles River Laboratories, Wilmington, Mass. The lungs of these mice are essentially devoid of bacteria. Antibiotics. Streptomycin sulfate (Sm; Eli Lilly & Co., Indianapolis, Ind.) was added to blood-agar medium to a final concentration of 500 jg/ml. Erythromycin gluceptate (ery; Eli Lilly & Co.) was added to a final concentration of 0.1 Ag/ml. Deoxyribonuclease. Pancreatic deoxyribonuclease (IX recrystallized, Worthington Biochemical Corp., Freehold, N.J.) was used in a final concentration of 10 Ag/ml together with a final concentration of 0.007% MgSO4-7H20. Ethanol and its determination in murine blood. Absolute ethanol, diluted to 10% in saline, was in-' This work was taken from a dissertation submitted in partial fulfillment of the requirements for the Ph.D. degree at the

Skin cells from albino mouse embryos have been shown to synthesize melanin after exposure to a nucleoprotein fraction of cells from pigmented mice. Although transformed clones were not found in any of 37 control experiments, at least 15 transformations took place in 10 of a series of 49 experiments using undegraded DNA. The active component in the transforming material appears to be DNA because DNase-treated preparations did not induce melanin synthesis.