Clones; Transformation; Strand Recombination in Vitro 



297 



chromosomal DNA contains the chemical 

 units of the genetic material; all other trans- 

 formation studies support this conclusion. 



We should now re-examine the assump- 

 tion, made earlier in this chapter, that trans- 

 formation involves mutation. Because the 

 first transformation studies seemed to in- 

 volve novel, rare changes in the genetic ma- 

 terial, these were called mutations (p. 149). 

 We now know that transformation involves 

 replacement of one segment of genetic ma- 

 terial by another, that only a shuffling of 

 already-existent genes occurs and not a new 

 type of genetic material. Moreover, genetic 

 transformation has been found not only in 

 Pneumococcus but in Hemophilus, Xantho- 

 monas, Salmonella, Bacillus, Neisseria, Esch- 

 erichia, and other organisms as well. In 

 Neisseria, DNA is regularly liberated (into 

 the slime layer) by the cells which undergo 

 self-digestion, or autolysis, in aging cultures; 

 such DNA is effective in transformation, as 

 is the DNA obtained from penicillin-sensi- 

 tive pneumococci disintegrated or lysed 

 after treatment with penicillin. Using dif- 

 ferent genetically-marked pneumococci, it is 

 found 4 that genetic transformation — due to 

 DNA liberated from one strain transforming 

 members of the other strain — occurs spon- 

 taneously in the living mouse host. 



Two lines of the human cell strain D98S 

 maintained in vitro are genetically differen- 

 tiated by the presence or absence of the 

 enzyme, inosinic acid pyrophosphorylase 

 (IMPPase). Since the spontaneous muta- 

 tion frequency from IMPPase-negative to 

 -positive is found to be less than one cell 

 in 10 7 , and since a culture medium can be 

 employed which completely prevents cells 

 of the negative line — but not those of the 

 positive line — from forming colonies, it is 

 possible to detect as few as one genetic trans- 

 formant per 10 7 cells. Treatment of the 

 IMPPase-negative line with DNA isolated 



4 By E. Ottolenghi and C. M. MacLeod (1963). 



from IMPPase-positive cells results in the 

 appearance of IMPPase-positive, genetically- 

 transformed cells at rates as high as 4 times 

 10 4 transformations per recipient cell (one 

 transformation per 2500 treated cells). 

 Such rates offer clear proof that genetic 

 transformation of human cell lines occurs 

 under experimental conditions." 



Not only is transformation widespread, 

 but a given type can occur with a frequency 

 as high as 25%. Such results demonstrate, 

 of course, that transformation is not rare. 

 Because genetic transformation is not rare 

 and does not produce novel genotypes, it 

 should not be considered a type of mutation. 

 Accordingly, just as with segregation, inde- 

 pendent segregation, crossing over, and fer- 

 tilization, it is probably best to consider ge- 

 netic transformation as another mechanism 

 for genetic recombination. 



As determined from bacterial studies, the 

 complete transformation process requires a 

 series of discrete stages, as follows: 



Cell competence. During certain periods 

 in cell division or in the growth of a bacterial 

 culture, transformation does not occur; in 

 other periods the cells are competent to 

 react. 



Binding the transforming DNA. When 

 bacteria are in a competent stage, the trans- 

 forming DNA, transiently bound to the cell 

 at first, can be removed by several methods 

 including exposure to DNase, before the 

 DNA is permanently bound. 



Penetration of transforming DNA. Per- 

 manently-bound DNA is considered to have 

 penetrated the recipient bacterium. It 

 should be noted that the success of trans- 

 formation is inversely related to the thick- 

 ness of a polysaccharide coat which probably 

 acts as some kind of barrier to binding or 

 penetration. When transforming DNA is 

 fragmented sonically, the newly formed DNA 



5 According to W. Szybalski. E. H. Szybalska, and 

 G. Ragni (1962); see E. H. Szybalska and W. 

 Szybalski (1962). 



