310 



( HAPTER 23 



MASTER 

 PLATE 



T L B 



Contains 



-r -r -r 



♦ TLB, 



REPLICAS 



^ o o o 



- + + 

 T L B, 



+ - + + + - 



TLB, TLB, 



B Pa C 



^ o o o 



Medium 

 Contains 



t _ -r -i 



| B Pa C 



B Pa C 



+ - + 

 B Pa C 



B Pa C 



figure 23—4. Use of replica-plating {shown diagrammatically) to detect spontaneous 

 mutations in E. coli. Replica I detects one mutant to T . replica 3 detects one mutant 

 to B, . and replica 2' detects one mutant to Pa + . 



the genetic material of two different cells, 

 it is not a typical sexual process, since trans- 

 formation does not depend upon contact 

 between donor and recipient cells.) 



2. The integration process leading to ge- 

 netic recombination requires the presence of 

 only a portion of the entire genome of the 

 donor cell. (Integration results in a small 

 segment of the penetrant donor DNA replac- 

 ing a small homologous segment of the host 

 genome.) 



At this point it does not seem unreason- 

 able to hypothesize that any homologous 

 DNA penetrating a bacterial cell can inte- 

 grate by the same mechanism involved in 

 transformation. Of course there may be 

 other means of introducing DNA into a 

 cell and, consequently, experiments are now 

 designed to test whether or not DNA passes 

 from one bacterium to another when two 

 are in contact. 



One such experiment 2 starts with a proto- 

 trophic strain (K12) of E. coli treated with 



-' Based upon work of J. Lederberg and E. L. 

 latum ( 1946). 



a mutagen (like X rays or ultraviolet light) 

 to obtain single auxotrophic mutants which 

 require different nutritional supplements in 

 order to grow. The mutagenic treatment is 

 repeated — first on the single and then on 

 the double mutant auxotrophs — eventually 

 obtaining two lines which differ from each 

 other by three nutritional mutants, all six 

 mutants having arisen independently. One 

 triple mutant strain is auxotrophic for threo- 

 nine (T~), leucine (L ). and thiamin 

 ( #, ) ; the other triple mutant is auxotrophic 

 for biotin (B~), phenylalanine (Pa), and 

 cystine (C~). The genotypes of these two 

 lines can be given, respectively, as 



and 



T-L-B 1 B + Pa+C+ 



T + L + B, + BPaC-. 



Of course, the given gene sequence may be 

 different in the linkage map. 



The pure lines are grown separately on 

 complete liquid culture medium, that is, one 

 which contains all nutrients required for 

 growth and reproduction. To form a bac- 



