Bacteria: Recombination (/) 



345 



DNA which is not integrated is also not con- 

 served as chromosomal gene material. If 

 integration occurs by copy-choice then the 

 transforming segment is not conserved either; 

 whereas if integration occurs by crossing over 

 the chromosomal segment replaced is not 

 conserved. 



We have already mentioned that DNA 

 from different sources and DNA particles of 

 different sizes behave differently in various 

 parts of the sequence leading to transforma- 

 tion. Several studies have been made of 

 DNA in vitro which may shed light on the 

 transformation process in particular, and 

 upon genes in general. When DNA in vitro 

 is exposed to dilute concentrations of 

 DNAase, the results '* indicate that single 

 strands of the double helix are attacked first, 

 and only later, when both strands have been 

 attacked at reasonably proximal positions, is 

 the molecule severed. Once severed, the 

 smaller molecule penetrates poorly, you re- 

 call. However, even if only the single strand 

 has been attacked, transformation rate de- 

 cHnes. This effect is attributed to the failure 

 of penetrant molecules to transform, because 

 the transforming locus, or a locus necessary 

 for synapsis or integration, has been in- 

 activated. 



We have already mentioned (refer to pp. 

 315, 325) that heating chromosomal DNA 

 denatures it by causing strand separation. 

 Under certain conditions, the single chain 

 can fold so that a large number of comple- 

 mentary base pairs are formed between bases 

 at different levels of the single chain. After 

 pneumococcal DNA has been heated for 10 

 minutes at 100° C, all chains are single and 

 all H bonds are broken.^ If such a sample is 

 cooled quickly under appropriate conditions, 

 almost no double chains are formed, almost 



■* Of L. S. Lerman and L. J. Tolmach. 



^ The following account is based primarily on work 



reported by P. Doty, J. Marmur, J. Eigner, and 



G. Schildkraut (1960), and J. Marmur and D. Lane 



(1960). 



all chains being single, with half the molecular 

 weight of the original DNA. This is called 

 denatured DNA (cf. p. 315). On the other 

 hand, if such a sample is cooled slowly, 

 double strands are produced, which are 

 united by complementary base pairing over 

 most of their length. This is called renatured 

 DNA. Denatured and renatured DNA differ 

 in several properties. These include their 

 appearance under the electron microscope 

 (renatured DNA looks very much like native 

 DNA while denatured DNA is irregularly 

 coiled with clustered regions), their density 

 (renatured and native DNA have similar and 

 lighter densities than denatured DNA), and 

 their ultraviolet absorbency (renatured and 

 native DNA have similar but lower absorben- 

 cies than denatured DNA). 



Several factors affect renaturation. Rena- 

 turation is dependent upon the concentration 

 of DNA in the slowly cooling mixture. When 

 the concentration of single strands is high, so 

 is the amount of renaturation, while slow 

 cooling of single strands in low concentration 

 does not produce any substantial recombina- 

 tion of strands. A second factor, influencing 

 union between strands, is the effect of salt 

 concentration. The negatively charged phos- 

 phate groups of single strands tend to prevent 

 union with other strands. This can be over- 

 come by adding KCl to the solution; this 

 acts as a shield against the repulsion between 

 phosphates. Accordingly, within a certain 

 range, the more KCl that is present the 

 greater is the amount of renaturation ob- 

 tained by slow cooling of heated DNA. A 

 third factor upon which strand recombination 

 depends is the source of DNA. Assuming 

 that the molecular weight of native DNA is 

 approximately the same in all organisms, 

 then a mammalian nucleus would have about 

 a thousand times as many DNA molecules as 

 a bacterial nucleus. Assume also, as is hkely 

 to be true, that the DNA molecules within a 

 genome all differ in base sequence. Then, for 

 a given concentration of denatured DNA, 



