Clones; Transformation; Strand Recombination in Vitro 



303 



ical properties, that is, functions genetically 

 in vivo. When transforming DNA is used 

 in an in vitro synthesis, the total transform- 

 ing capacity of the incubating mixture de- 

 creases with time as the synthesis continues. 

 It is very likely that the trace amounts of 

 DNases present in the polymerase prepara- 

 tion cause the loss of overall transforming 

 activity by interrupting the continuity of the 

 DNA strands, so that transforming capacity 

 is lost by DNase action faster than it is 

 gained by means of DNA synthesis. The 

 biological activity of newly-synthesized DNA 

 can be detected, however, by differentially 

 labeling the old and the new DNA and sep- 

 arately testing each for transforming capac- 

 ity. 



Consequently, 10 nonradioactive DNA con- 

 taining 5-bromo uracil is used as a primer- 

 template to synthesize radioactive DNA with 

 no 5-bromo uracil. After synthesis, density 

 gradient centrifugation of the DNA provides 

 a double-stranded fraction containing essen- 

 tially all newly-synthesized DNA (which is 

 radioactive and less dense than the DNA 

 with 5-bromo uracil). Since other explana- 

 tions seem to be ruled out, and this new 

 DNA is found capable of transforming a 

 variety of gene loci, it apparently is proved 

 that biologically-active genetic material, that 

 is, functionally-active genes, can be syn- 

 thesized in vitro. 



10 See R. M. Litman and W. Szybalski (1963). 



SUMMARY AND CONCLUSIONS 



Genetic recombination occurs in cells of bacteria and of other organisms by means 

 of genetic transformation. In bacteria, transformation involves a sequence of events in 

 which competent cells transiently and then permanently bind DNA. Once bound DNA 

 has penetrated, it apparently undergoes a synapsis-like process with a corresponding 

 segment of the bacterial genome. Transformation is completed when a small segment 

 of donor DNA becomes integrated and replaces a similar segment of the host genome. 



Transformation provides direct and conclusive evidence that chromosomal DNA 

 is genetic material. In bacteria, the smallest recombinational unit of the genetic ma- 

 terial is the smallest unit of DNA integrated or replaced in transformation. 



Strand separation and recombination /'/; vitro produce denatured and renatured DNA. 

 respectively. 



Strong evidence has been obtained that functionally-active genes, introduced into 

 bacteria via transformation, can be synthesized in vitro. 



REFERENCES 



Akinrimisi, E. O., Sander, C, and Ts'o, P. O. P., "Properties of Helical Polycytidvlic 

 Acid," Biochemistry, 2:340-344, 1963. 



Avery, O. T., MacLeod, C. M., and McCarty, M.. "Studies on the Chemical Nature of 

 the Substance Inducing Transformation of Pneumococcal Types," J. Exp. Med., 

 79:137-158, 1944. Reprinted in Papers on Bacterial Genetics, Adelberg. E. A. 

 (Ed.). Boston: Little, Brown, 1960, pp. 147-168, and in Classic Papers in Genetics, 

 Peters. J. A. (Ed.). Englewood Cliffs, N.J.: Prentice-Hall. 1959. pp. 173-192. 



