Bacteria: Recombination {II) 



353 



--++r ++ s __++s ++ — r 



BMPTV, xBMPTV, F^ BMPTV, xBMPTV, 



Prototrophs B M P T 



Prototrophs B M P T 



86% 



r 

 V, 



14% 

 s 

 V, 



79% 

 s 

 V, 



21% 



r 



V, 



FIGURE 38-3. Genetic recombinations, involving iinselected markers, obtained 

 in reversed crosses. 



mains, at least with respect to the genes 

 showing recombination, that the recombinant 

 may be diploid. 



It is possible, however, to add markers 

 that are not selected for or against when one 

 is selecting for recombinant prototrophs. 

 These are called unselected markers. E. coli 

 is available in two genetic forms, one, Ki% is 

 resistant to infection by the bacterial viruses 

 Ti and Te while the other, Ki% is sensitive to 

 infection by these viruses, which cause the 

 bacteria to lyse. Using the auxotrophic mu- 

 tants P~ (proline-requiring) and M^ (methio- 

 nine-requiring), it is possible to make the 

 cross - B'M-P+T+V/ X B+M^P-T-V,^ and 

 select for the prototrophs B+M+P^T^. 



A number of prototrophs were obtained. 

 These were then tested for sensitivity to virus 

 Ti. Because Vi is an unselected marker both 

 of its alternatives are testable. Eighty-six 

 per cent of the prototrophs were typically 

 resistant, Ff, and 14% typically sensitive, 

 Vi' (Figure 38-3). Note that both of these 

 alternatives are recombinant relative to some 

 of the markers for prototrophy, and that the 

 alternative present is typically expressed. 



A diploid Vi^'Vi^ would be either re- 

 sistant, or sensitive, or intermediate in expres- 

 sion. The fact that both typical sensitivity 

 and typical resistance to virus occurs proves 

 that some recombinants contain only one 

 representative of this locus. It is therefore 

 2 See J. Lederberg (1947). 



reasonable to extrapolate from this finding 

 and say that only one alternative is present 

 of any gene in a recombinant, which is the 

 same as saying that all the genes in a nuclear 

 body of E. coli are normally haploid. This 

 means that conjugation produces a tempo- 

 rary (partial or complete) diploid condition 

 which, following integration, results in hap- 

 loid recombinants, just as is presumably the 

 case in transformation. The haploid recom- 

 binants may also be called segregants. 



When the reverse cross is made, V{ entering 

 with the B^M+P-P- parent and Ki' with 

 B M'P^T^, the prototrophs show approxi- 

 mately the reversed percentages which are 

 sensitive and resistant to Ti. In other words, 

 the parent that provides P+T+ to the proto- 

 troph also contributes the Vi locus which it 

 contains about 80% of the time. The 

 fact that there is an imbalance in the fre- 

 quency of resistants and sensitives among 

 prototrophs (i.e., their ratio is not 50% : 

 50%), which is reversed when the Vi markers 

 are reversed in the parent cells, is clear evi- 

 dence that Vi does not segregate (or integrate) 

 independently of the markers with which it 

 entered the zygote and which were subse- 

 quently integrated to form the haploid proto- 

 troph. Accordingly, Vi must be linked to 

 P T, segregating from these loci (or failing to 

 be integrated in the same segment with them) 

 about 20% of the time. On a hnkage map, 

 therefore, Vi is about 20 recombination 



