Chapter *37 



BACTERIA: RECOMBINATION 



(I. Transformation and Strand 

 Recombination In Vitro) 



I 



't has been known for some time 

 that Pneumococcus {Diplococcus 

 .pneumoniae) occurs in several 

 types which may be characterized by their 

 clonal phenotype. One type, S, produces a 

 smooth colony, and this trait is directly con- 

 nected with the fact that the bacterium pos- 

 sesses a capsule of polysaccharide material. 

 Another type of colony is rough, R, due to 

 bacteria which lack this polysaccharide cap- 

 sule. There are, moreover, several types of 

 S colonies, which can be distinguished from 

 one another because they differ antigenically, 

 and different antisera can be obtained which 

 specifically cause the clumping of each differ- 

 ent type of S. Antiserum can also be pro- 

 duced that will clump R cells, which can also 

 occur in several different antigenic types. 



When a large number of R cells is placed 

 in nutrient broth containing anti-R serum ^ 

 and growth continues, clumps of agglutinated 

 R cells settle to the bottom of the test tube, 

 leaving the supernatant fluid clear. If this 

 supernate is plated on nutrient agar, any 

 bacteria still present form typical R colonies, 

 so that any mutation which would cause R 

 to form S must occur so rarely as to be un- 

 detected using this particular technique. 



When the same experiment is performed, 

 with the exception that heat-killed (65° C for 

 30 minutes) S cells are also added to the nutri- 



^ The following account is based upon experiments of 

 F. Griffith, of M. H. Dawson and R. H. P. Sia, and of 

 O. T. Avery, C. M. MacLeod, and M. McCarty (1944). 

 340 



ent broth, plating of the supernate shows that 

 numerous clones of S type appear on the 

 agar.! This S phenotype is stable and is 

 clearly the result of a transmissible change. 

 We conclude, therefore, that the heat-killed 

 S cells are acting as a mutagen in the genetic 

 transformation of R to S cells. What is most 

 surprising is the fact that the type of S mutant 

 produced is always identical to that of the 

 heat-killed bacteria acting as mutagen. We 

 are apparently dealing with a unique situa- 

 tion in which the mutagen acts specifically to 

 produce mutations in only one predictable 

 direction (to one S type), rather than in 

 several directions (to two or more S types). 



In order to determine the chemical nature 

 of the mutagen involved, the transforming 

 capacity of different fractions of the heat- 

 killed S bacteria was tested. Fractions con- 

 taining only the polysaccharide coat, or pro- 

 tein, or RNA, were completely inactive. Only 

 the fraction containing DNA had transform- 

 ing capacity. The purest DNA extracts re- 

 tained the full transforming capacity, even 

 though they could have contained less than 

 .02% protein, and even after having been 

 treated with protein-denaturing agents or 

 proteolytic enzymes. Chemical analyses and 

 serological, electrophoretic, ultracentrifuge, 

 and spectroscopic tests also indicated that 

 this active DNA showed no detectable con- 

 tamination by protein, unbound lipid, or 

 polysaccharide. RNAase had no effect on 

 the transforming capacity of this purified 

 DNA fraction. On the other hand, the trans- 

 forming factor was completely destroyed by 

 DNAase. The last resuh shows that trans- 

 formation is not produced by exposure to 

 nucleotides or nucleosides of D-ribose type, 

 singly or in small groups. 



Transforming DNA has the double- 

 stranded configuration of chromosomal 

 DNA, as revealed from its X-ray diffraction 

 pattern. The fact that pure DNA can be 

 used to transform means that no contact 

 need be made between the cell acting as DNA 



