:U4 



( II \l' MR 23 



Using this marker and the auxotrophic mu- 

 tants P (proline-requiring) and M (meth- 

 ionine-requiring), we can make the cross * 



B M /' / I , h\ B \i I' I I', and 

 select for the prototrophs B M /' / 

 Since the medium employed is virus-free, 



the I , locus is used as an unselected marker. 



The number o\ prototrophs thus obtained 

 are subsequently tested tor sensitivity and 

 resistance to virus T,. and 86% are found 

 typicall) resistant. IV. and L49S typically 

 sensitive, I', (Figure 23-6). Note that 

 both these alternatives are recombinant rela- 

 tive to some of the markers for prototrophy. 



When the reverse cross is made, V { enter- 

 ing with the B M + P~T~ parent and V x " 

 with B M~P + T+. the percentages of proto- 

 trophs sensitive and resistant to T, are found 

 to be approximately reversed. In other 

 words, the parent that provides P f and T^ 

 to the prototroph also contributes the K, 

 locus which it contains 79-86% of the time. 

 The imbalance in the frequency of resistants 

 and sensitives among prototrophs (that is, 

 their ratio is not 50% :50% ). and its re- 

 versal when the K, markers are reversed in 

 the parent cells, provide clear evidence that 

 the Vi locus does not integrate and segre- 

 gate independently of the other markers 

 with which it enters the zygote and which, 

 subsequently, are present in the haploid 

 prototroph. As a consequence V ', must be 



1 See J. Lederberg (1947). 



linked to /' and / and segregated from these 

 loei (or fail to be integrated in the same seg- 

 ment with them) onl\ about 20% of the 

 time. 



The linkage relationships for auxotrophic 

 markers can also be determined, as follows: 

 the cross / / B x -B~ by T L B, B is 

 made on a complete medium, plated on a 

 complete medium, and replicated four times 

 on a complete medium without T, or L, or 

 Bi, or B. As a result, prototrophic recom- 

 binants grow on all four replicas and single 

 auxotrophs grow on three, whereas double 

 auxotrophs grow on two of the replicas. 

 Since prototrophs are found to be more 

 frequent than either T L~Bi + B + or 

 T L + B : + B • , T and L must be linked. A 

 further analysis of the results, for other 

 markers and other experiments, reveals that 

 all the genetic markers tested in E. coli are 

 linked to each other and on a map can be 

 arranged in a linear order according to their 

 recombination (segregation or integration) 

 distances. In all likelihood, this analysis 

 indicates, that E. coli has a single ''chromo- 

 some. " 



Genetic recombination by the sexual proc- 

 ess of conjugation is known to occur in bac- 

 teria like Pseudomonas, and — under special 

 conditions — in Serratia and Salmonella, as 

 well as in Escherichia. Intergeneric conju- 

 gation between Escherichia and Salmonella 

 has been observed to occur in a mammalian 

 host. 



SUMMARY AND CONCLUSIONS 



Bacterial clones are excellent experimental material for the study of the mutation 

 process and its rate. Techniques for detecting and isolating mutants are described. 

 Mutants occur spontaneously, independent of the factors to which they may he adaptive. 

 Genetic recombination occurs in Escherichia coli and other bacteria after the sexual 

 process of conjugation. E. coli normally has a haploid nuclear body, in which all 

 tested genes belong to a single linear linkage group. 



