:m 



CHAPTER 22 



than in several directions (to two or more 



S types i. 



To determine the chemical nature of the 

 mutagen involved, the transforming capacity 

 oi different tractions of the heat-killed S 

 bacteria is tested. Fractions containing 

 either the polysaccharide coat, protein, or 

 RNA are completely inactive; only the frac- 

 tion containing DNA has the ability to trans- 

 form. The purest DNA extracts retain the 

 full transforming ability, even though they 

 contain less than .02% protein or are treated 

 with protein-denaturing agents or proteolytic 

 enzymes. Chemical analyses and other tests 

 (serological, electrophorctic, ultracentrifugal, 

 and spectroscopic) also indicate that the ac- 

 tive DNA is not detectably contaminated by 

 either protein, unbound lipid, or polysac- 

 charide. RNase has no effect on the trans- 

 forming capacity of a purified DNA fraction, 

 but the transforming factor is completely de- 

 stroyed by DNase, showing that transforma- 

 tion requires DNA in highly polymerized 

 form. 



As revealed from its X-ray diffraction 

 pattern, transforming DNA has the double- 

 stranded configuration of chromosomal 

 DNA. Since pure DNA can be used to 

 transform, no contact need be made between 

 the cell acting as DNA donor and the one 

 acting as recipient. Moreover, genetic trans- 

 formation does not involve the mediation of 

 a virus. Therefore, beyond any reasonable 

 scientific doubt, DNA alone must be the 

 transforming agent. 



Transformation can occur in either direc- 

 tion (A^^A'), and, in bacteria, any chro- 

 mosomal gene can be transformed. Type A 

 cells can be transformed to an A' type which, 

 in turn, provides increased amounts of A'- 

 DNA capable of transforming other A cells 

 to A'. So, the DNA extracted from trans- 

 formed bacteria provides increased amounts 

 of the same transforming principle. 



One transforming principle (A') can 

 transform bacteria having any one of several 



alternative phenotypes (for example, A or 

 A"). If the A'-DNA, obtained from A-type 

 bacteria transformed to A', is then used to 

 transform bacteria of a third genotype. A", 

 only A' transformants are found — the only 

 transformations produced are those involv- 

 ing the genes of the immediate donor. This 

 result demonstrates that transformation pro- 

 duces a transmissible alteration based upon 

 the loss of host genetic material apparently 

 at the same time as the new genetic material 

 is acquired — not the simple addition of par- 

 ticular genetic material to the genotype. 

 Thus, the genetic change in transformation 

 is of a replacement type. 



The fate of transforming DNA can be 

 traced - by labeling its phosphate groups 

 with radioactive P 32 . At various times after 

 exposure to such labeled DNA, one portion 

 of the treated bacteria is killed and analyzed 

 for the presence of P :i - in its DNA, whereas 

 another portion is tested to determine trans- 

 formation frequency. Only after bacteria 

 have been exposed to the DNA extract for 

 a suitable period of time is the labeled DNA 

 found in the extract containing the host's 

 chromosomal DNA. Moreover, the fre- 

 quency with which the host cell is trans- 

 formed is directly proportional to the amount 

 of labeled DNA so incorporated. 



The preceding results demonstrate that the 

 transforming DNA actually enters the bac- 

 terium and replaces a segment of the host's 

 chromosomal DNA { (Pneumococcus con- 

 tains about 6 times 10 fi deoxyribotide pairs 

 per nuclear body), after which the newly- 

 introduced material replicates as a normal 

 part of the chromosome. Since it is DNA 

 which alone carries the genetic information 

 for transformation, genetic transformation 

 provides direct and a inclusive evidence that 

 DNA is genetic material. Accordingly, 



- Based upon work of L. S. Lerman and L. J. 

 Tolmach (1957). 



3 See H. Ephrussi-Taylor (1951) for specific evi- 

 dence on the latter observation. 



