302 



( II M'lI.K 22 



thej reveal what is responsible for holding 



the strands in a DNA double helix together 

 under in vivo or the usual in vitro conditions. 



Vnother physical-chemical change can 

 occur when DNA is heated in vitro. As 

 noted, native pneumococcal DNA has a 

 molecular weight of about six million. 

 When certain preparations of this native 

 DNA are heated, the single strands obtained 

 have a molecular weight o\' less than half 

 this value. This reduction in molecular 

 weight can be explained by the presence o\ 

 DNase as a contaminant. Even though 

 single strands in a double helix are enzy- 

 matically severed by DNase, the whole com- 

 plex can still retain the double-helix con- 

 figuration. Once these complementary 

 strands are separated by heat denaturation, 

 however, the fragments of each single strand 

 separate. 



As already mentioned, DNA from differ- 

 ent sources and DNA particles of different 

 sizes behave differently in various parts of 

 the sequence leading to transformation. 

 When DNA in vitro is exposed to dilute con- 

 centrations of DNase, the results 9 indicate 

 that single strands of the double helix are 

 attacked first, and only later — when both 

 strands have been attacked at reasonably 

 nearby positions — is the molecule severed. 

 This scission produces smaller DNA mole- 

 cules which, it should be recalled, penetrate 

 a host cell poorly. Even if only one strand 

 of the double helix has been attacked, how- 

 ever, transformation capacity declines. This 

 effect is attributed partly to the failure of 

 penetrant molecules to transform because 

 the transforming locus or because a locus 

 neeessary for synapsis or integration has 

 been inactivated. 



In Pneumococcus, denatured DNA has a 

 small amount of transforming ability. The 

 molecular basis for this is still undetermined. 

 On the other hand, the transforming ability 



1 Of L. S. Lerrrum and I.. J. Tolmach. 



of renaturcd DNA can be as much as 

 509? o[ that shown by an equivalent con- 

 centration o\' native DNA. An increased 

 concentration of DNA plus a high ionic 

 strength increase both renaturation and 

 transforming ability. 



Hybrid molecules can be made by rena- 

 turing a mixture of N-14 and N-15 DNA 

 from E. coli. ( Recall that these synthetic 

 molecules can be identified by the inter- 

 mediate position they assume in the ultra- 

 centrifuge tube.) Hybrid molecules can also 

 be formed between single DNA strands from 

 different speeies, but only if the species are 

 closely related genetically (as would be sug- 

 gested if they showed interspecific transfor- 

 mation) and, therefore, have similar base 

 sequences. Molecular hybrids are useful 

 for comparing base sequences in closely- 

 related organisms even when genetic recom- 

 bination between them cannot take place. 



Several additional observations should be 

 made: 



1 . Strand separation is accomplished by 

 heat in a matter of a few minutes or less. 

 One wonders if this kind of extensive strand 

 separation occurs in vivo. It has been sug- 

 gested that chain separation normally is pro- 

 duced enzymatically through the activity of 

 ravelase, or better, unravelase. 



2. The now-routine ability to separate and 

 combine single strands should lead to a better 

 understanding of transformation, in particu- 

 lar, the mechanism of integration. 



3. The smallest recombinational unit of 

 the genetic material in bacteria can he iden- 

 tified as the smallest unit of DNA capable 

 of being integrated or replaced in a host 

 genotype in a genetic transformation. 



Although the physical and chemical prop- 

 erties of the DNA product of an extensive 

 synthesis in vitro closely resemble those of 

 the natural DNA used as primer-template, 

 and although the synthesis is considered to 

 be a biological process, it has not been dem- 

 onstrated that the DNA product has biolog- 



