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CHAPTER 37 



there would be of the order of one thousand 

 times fewer complementary chains present 

 when the sample came from calf thymus than 

 there would be in a sample that was obtained 

 from Pneumococcus. It has been possible to 

 show that when denatured DNA is heated to 

 80° C, a large amount of double strands is 

 formed by the bacterial DNA but no detect- 

 able amount is produced by the calf thymus 

 DNA. Therefore, what is important in 

 renaturation is the concentration of comple- 

 mentary strands. 



Another physical-chemical change may oc- 

 cur when DNA is heated in vitro. As already 

 mentioned, native pneumococcal DNA has a 

 molecular weight of about 10 million. When 

 certain preparations of this native DNA are 

 heated, the single chains obtained may have 

 a molecular weight less than half this value. 

 This can be explained, as the consequence of 

 the presence of DNAase, as follows. Single 

 chains are enzymatically severed by DNAase 

 while still part of the double chain, as de- 

 scribed previously, but the whole complex 

 retains the double helix configuration. How- 

 ever, once the complementary chains are 

 separated by heat denaturation, the frag- 

 ments of each single chain fall apart. 



It is also possible to make hybrid molecules 

 by renaturing a mixture of N-14 and N-15 

 DNA from E. coli. These synthetic mole- 

 cules can be identified by the position they 

 assume in the ultracentrifuge tube (see dis- 

 cussion beginning p. 312). Such hybrid mole- 

 cules are also formed between single DNA 

 strands from diff'erent species, but only if the 

 species are closely related genetically (as 

 would be suggested if they showed inter- 

 specific transformation), and if they have 

 similar base compositions. 



Because of the occurrence of genetic re- 

 combination in bacteria via transformation, 

 it is possible to test the biological activity of 

 the various DNA's produced by strand sepa- 

 ration and recombination. It is found, using 

 Pneumococcus, that denatured DNA has very 



little transforming activity. (It is likely that 

 this activity is due to the small amount of 

 renaturation which could have taken place in 

 the denatured solution.) On the other hand, 

 the transforming ability of renatured DNA 

 may be as much as 50% of that shown by an 

 equivalent concentration of native DNA. 

 Just as an increased concentration of DNA 

 and a high ionic strength increase renatura- 

 tion, so do they also increase transforming 

 ability. 



The transforming ability of various chemi- 

 cally hybrid molecules has been studied also. 

 The pneumococci to be transformed were 

 streptomycin-sensitive, and transformants 

 were counted by the streptomycin-resistant 

 colonies growing after these bacteria were 

 plated on an agar medium containing strepto- 

 mycin. The material to be tested for trans- 

 forming ability was obtained as follows. A 

 constant concentration of DNA, from strepto- 

 mycin-resistant pneumococci, and a series of 

 increasing concentrations of DNA, from 

 streptomycin-sensitive pneumococci, were 

 heat denatured together and then renatured 

 together (by cooling slowly). The larger the 

 amount of streptomycin-sensitive DNA in 

 the mixture, the larger was the transformation 

 rate to streptomycin resistance. This increase 

 proves that the higher the concentration of 

 streptomycin-sensitive DNA, the greater the 

 number of renatured molecules capable of 

 transformation. 



It should also be noted that the addition, 

 to cooling, denatured, streptomycin-resistant 

 DNA, of homologous DNA (DNA from the 

 same species, as from sensitive Pneumococcus) 

 in the native (double-stranded) state, showed 

 no increase in transformation ability over the 

 rate obtained in its absence. Also, if, during 

 the slow cooling, denatured heterologous 

 DNA (DNA from radically different species, 

 as from Salmonella, Micrococcus, Strepto- 

 coccus, or calf thymus) is added, even in large 

 quantities, there was also no increase in 

 transforming activity over the control rate. 



