Clones; Transformation; Strand Recombination in Vitro 



299 



which bacteria are transformed with respect 

 to two markers present in the donor DNA. 

 In several cases (for example, penicillin- and 

 streptomycin-resistance), the frequency of 

 doubly-transformed bacteria is approxi- 

 mately equal to (actually somewhat less 

 than) the product of the frequencies for the 

 single transformations. Such results prob- 

 ably mean that the transforming DNA car- 

 ries the two loci either on separate particles 

 or in widely separated positions on the same 

 particle. On the other hand, the markers 

 for streptomycin-resistance and mannitol- 

 fermentation are transformed together with 

 a frequency (.1%) which is about 17 times 

 that expected from the product of the fre- 

 quencies of the single transformations 

 (.006%). This result implies that these 

 two genetic markers are located on the same 

 transforming particle; that is, they seem to 

 be reasonably close together in the same 

 bacterial chromosome. 



If two loci are closely linked, how can we 

 explain the occurrence of single and double 

 transformations for them? Because of frag- 

 mentation during extraction, a given pene- 

 trating DNA particle may not always have 

 the same composition relative to the two 

 markers; it may sometimes carry only one 

 and, at other times, may carry both of these 

 markers. The effect of reducing the particle 

 size of penetrating DNA upon the fre- 

 quencies of single and double transforma- 

 tions can be tested. When particle size is 

 reduced by DNase or sonic treatment, one 

 expects — according to the present hypothe- 

 sis — the particles sometimes to be broken 

 between the two markers, reducing the rela- 

 tive frequency of the double transformation 

 and increasing the relative frequencies of the 

 single transformations. When the particle 

 size is reduced, the overall rate of trans- 

 formation is lower, as expected. No change 

 is found, however, in the ratio of double to 

 single transformations, implying that the two 

 markers are so closely linked, they are rarely 



separated when particles are fragmented. 

 Accordingly, it seems that the penetrating 

 particles must usually carry both markers, 

 or neither, and the failure to obtain 100% 

 double-transformations from the former type 

 must be because only a small portion of a 

 penetrating, synapsing particle is integrated. 



Integration of a portion of a synapsed 

 particle can occur in two possible ways (Fig- 

 ure 22-5): One involves copy-choice (Fig- 

 ure 22-5B) in which a daughter chromo- 

 some is formed by the alternate use of the 

 host chromosome and the donor DNA as a 

 template. When completed, the daughter 

 chromosome is exactly like the original chro- 

 mosome except for the daughter segment 

 formed with transforming DNA as the tem- 

 plate. One expects the recombinant chro- 

 mosome produced by the copy-choice 

 method to contain all newly-synthesized 

 DNA. 



The second method involves breakage and 

 exchange of the kind that takes place in 

 chromosomal rearrangement or in crossing 

 over. In this case (Figure 22-5A), 

 "breaks" have to occur on each side of the 

 marker being integrated, so that a "double 

 crossover" (p. 134) is produced. Although 

 double crossovers within a short distance 

 are expected to be extremely rare between 

 two homologous chromosomes of higher 

 organisms, this kind of exchange can occur 

 under special circumstances and may be pos- 

 sible between the chemically less complex 

 chromosome of bacteria and the shorter, 

 synapsed segment of transforming DNA. 

 Linkage of transforming DNA to host mark- 

 ers does not require DNA synthesis in the 

 region involved, ,! although the integrated 

 segment — which must be at least 900 nu- 

 cleotide pairs long — appears to replicate in 

 synchrony with the host DNA. Experiments 

 with labeled DNA show that in transforma- 

 tion single-stranded donor DNA is inserted 



«See M. S. Fox (1962). 



