Clones; Transformation: Strand Recombination in Vitro 



295 



tion, since it is not yet feasible to make 

 physiological and biochemical studies on 

 such a microscopic scale. One can. how- 

 ever, make use of the fact that, barring 

 mutation, clones are composed of genetically- 

 identical individuals. Genetically-different 

 clones can show phenotypic differences in 

 the size, shape, and color they produce on 

 agar. Genetically-different clones can also 

 respond differently to various dyes, drugs, 

 and viruses. Therefore, one can also estab- 

 lish the genotype of a single bacterium from 

 the phenotype of the clone it produces. 

 E. coli is easily cultured since it can grow 

 and reproduce on a simple, chemically-de- 

 fined food medium. Strains which grow on 

 such a basic, minimal medium are considered 

 to be prototrophic, or wild-type, capable of 

 synthesizing the numerous metabolic com- 

 ponents of the cell not supplied in the me- 

 dium. In this respect prototrophs of E. coli 

 or other bacteria are similar to the wild-type 

 of Neurospora which also grows on a min- 

 imal, chemically-defined medium. It is not 

 surprising, then, that in bacteria (and also 

 in Neurospora) the richest source of mu- 

 tants comes from the study of the biochem- 

 ical variations which occur in different 

 clones, particularly those involving changes 

 in nutritional requirements. For numerous 

 mutants to grow and reproduce — whether 

 they arise spontaneously or after treatment 

 with physical or chemical mutagens — one or 

 more of a variety of chemical substances 

 must be added to the basic medium. For 

 example, one strain of E. coli requires the 

 addition of the amino acid threonine to the 

 minimal medium; another strain requires the 

 amino acid methionine. Nutritionally de- 

 pendent strains whose growth depends on a 

 supplement to their basic food medium are 

 said to be auxotrophic. 



Transformation 



As characterized by clonal phenotypes. 

 Pneumococcus (Diplococcus pneumoniae) 



occurs in a number of uenctic types. One 

 type, S. produces a colony whose smooth 

 surface is directly related to the capsule of 

 polysaccharide material each bacterium pos- 

 sesses. Another type of colony, R, has a 

 rough surface because its bacteria lack this 

 polysaccharide capsule. Moreover, several 

 types of S colonies can be distinguished from 

 each other because they differ antigenically; 

 that is, different antisera can be obtained 

 which specifically cause the clumping of each 

 different type of S. R cells also occur in 

 several different antigenic types, and antisera 

 can also be produced which will clump them. 



In one experiment, a large number of R 

 cells is placed in a nutrient broth containing 

 corresponding anti-R serum. 1 When growth 

 continues, clumps of agglutinated R cells 

 settle to the bottom of the test tube, and the 

 initially cloudy supernatant fluid becomes 

 clear. If this supernate is plated on nutrient 

 agar, the bacteria still present form typical 

 R colonies; any mutation from R to S must 

 occur rarely, for it is not detected with this 

 particular technique. 



When the same experiment is performed 

 with heat-killed (65 : C for 30 minutes) S 

 cells also present in the nutrient broth, nu- 

 merous clones of S type appear on the agar 

 after plating the supernate. This S pheno- 

 type is stable and clearly the result of a 

 genetic change. Therefore, one is led to 

 assume 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 

 that the type of S mutant produced is always 

 identical to that of the heat-killed bacteria 

 presumably acting as mutagen. In this ap- 

 parently unique situation, the mutagen acts 

 specifically to produce mutations in only one 

 predictable direction (to one S type) rather 



1 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. 

 McCartv (1944). 



