Section 3 — Molecular and Microbial Genetics 



with donor DNA. To the medium KC1 salt was 

 added to the final concentration of 0.075, 

 0.15, 0.2 and 0.3 m. The number of transform- 

 ants rose 6, 60, 120 and 20 times respectively 

 in comparison to the control without the added 

 salt. The optimal concentrations of 0.15 or 

 0.2 m KC1 were later used in the transformation 

 reactions. It was found that the salt showed its 

 effect only in the time of evolution of competency 

 when the rising tendency of competency in the 

 recipient culture was noticed and not during the 

 falling one. 



When small inocula of the recipient culture 

 were used and sometimes two waves of compe- 

 tency were obtained, the presence of KC1 salt 

 was still more effective in the rising period of 

 the second wave of competency. While in the 

 first rising period the difference between the 

 control without the added salt and the medium 

 with the salt was 2 to 5 times higher, the yield of 

 streptomycin resistant transformants in the 

 second rising period was even 10 to 15 times 

 higher. 



This effect of salts with monovalent cations 

 is not to be confused with the effect of divalent 

 cations (Fox and Hotchkiss). 



If we used in the transformation medium 

 different bovine serum albumin preparations 

 (BSA) in which various amounts of residual con- 

 tents of deoxyribonuclease could be determined, 

 we also found different answers to the presence 

 of KC1 salt added to the medium. Higher 

 amounts of DNAse in BSA (or in the cultiva- 

 tion medium) require the presence of KC1 salt 

 for a good yield of streptomycin resistance 

 transformants (1-10 per cent) while with a very 

 low content of deoxyribonuclease the presence 

 of KC1 salt is inhibitory. 



Series of our results may indicate that the 

 presence of small amounts of DNAse in the 

 transformation medium may play its active 

 role in the transformation reaction when properly 

 counterbalanced. 



3.32. Genetic Transformation Among Living Pneu- 

 mococci in the Mouse. Elena Ottolenghi and 

 Colin M. Macleod (New York, U.S.A.). 



Experiments were performed to determine 

 whether deoxyribonucleate-mediated transfor- 

 mation could take place spontaneously among 

 genetically distinguishable pneumococci growing 

 side by side in the same living host, the mouse. 

 It was found that when living, unencapsulated, 

 nonpathogenic, streptomycin-resistant pneumo- 

 cocci and living, streptomycin-sensitive pneumo- 

 cocci which synthesized only a small amount 



of type III polysaccharide capsule were injected 

 sequentially into the peritoneal cavity of mice, 

 recombinants could be recovered from about 

 30 per cent (26/83) of the mice. The recombinants 

 were streptomycin-resistant, synthesized type III 

 polysaccharide in almost the full amount, and 

 were more pathogenic for mice than either pa- 

 rental type. The recombination was most likely a 

 result of deoxyribonucleate-mediated transfor- 

 mation since it could be completely prevented 

 by exogenous deoxyribonuclease. It may be, 

 therefore, that transformation can serve as a 

 mechanism of recombination in nature for some 

 microorganisms such as pneumococci. 



This data will be submitted for publication in 

 the Proceedings of the National Academy of 

 Sciences of the United States in 1963. 



3.33. Linkage of Genetic Factors affecting Similar 

 Function and Derived from Different Bacterial 

 Species. Arnold W. Ravin and Joscelyn de 

 Sa (Rochester, U.S.A.). 



Certain strains of streptococcus and pneumo- 

 coccus are known to be capable of transforming 

 each other by means of their respective deoxy- 

 ribonucleic acids (DNAs). In pneumococcus, 

 moreover, various spontaneous mutations that 

 confer different levels of resistance to strepto- 

 mycin (str-r mutations) have been shown to be 

 genetically and physically linked. In DNA- 

 mediated transformations of pneumococcus, 

 for example, str-r mutations conferring high 

 levels of resistance generally replace (i.e. are 

 allelic with) str-r mutations conferring low 

 levels of resistance. In order to determine 

 whether str-r mutations arising spontaneously 

 in streptococcus and pneumococcus were also 

 genetically linked, therefore, a streptococcal 

 mutant resistant to a low streptomycin concen- 

 tration was exposed to DNA from a pneumococ- 

 cal mutant capable of resisting a high antibiotic 

 level. Conversely, pneumococcal mutants capa- 

 ble of resisting only low concentrations of the 

 antibiotic were exposed to DNA from a strep- 

 tococcal mutant capable of resisting high strep- 

 tomycin concentrations. Transformants pro- 

 duced in such interspecific reactions were isolated, 

 measured for level of streptomycin-resistance, 

 and DNA prepared from them for test on strep- 

 tomycin-sensitive strains. The latter test was 

 undertaken to determine whether the genome 

 of the transformed bacterium continued to 

 harbor its original str-r mutation and, if so, 

 whether the original mutation was linked to the 



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