358 

 MINUTES RECOMBINANTS HAVING Hfr MARKERS 



None 



8 T 

 ^V2 T, L 



9 T, L, Az 

 n T, L, Az, T, 

 18 T, L, Az, T, , Lac 

 25 T, L, Az, T, , Lac, Gal 



CHAPTER 39 



FIGURE 39-2. Recombinants obtained 

 following artificial interruption of con- 

 jugation at various times after mixing F~ 

 and Hfr strains. The Hfr strain has 

 markers for T, L, Az, T\, Lac, Gal. 

 {After W. Hayes.) 



Particular strains of Hfr and F", both 

 marked with suitable genetic factors, are 

 grown separately and then mixed in the 

 proportion of 1 : 20, respectively, to assure 

 rapid contact of all Hfr with F~ cells. At 

 various intervals of time, up to 60 minutes 

 after mixing, samples are withdrawn and 

 subjected to a strong shearing force in a 

 Waring Blendor. This serves as a very 

 efficient means of separating bacteria in the 

 act of conjugation, the treatment affecting 

 neither the viability of the bacteria, nor their 

 ability to undergo the process of recombina- 

 tion, nor the expression of the various geno- 

 types under test. Once separated, the 

 bacteria are plated and scored for male 

 markers which have integrated. It is found 

 that minutes after mixing no recombinants 

 are obtained ; this is as expected, since at this 

 time no male markers have yet been trans- 

 ferred. After 50 minutes of conjugation 

 almost all the male markers that are going to 

 be transferred have done so. However, the 

 time at which different male markers enter 

 the female cell varies widely within this time 

 interval. For example, T and L markers do 

 not enter until after about nine minutes of 



conjugation, while the Gal marker (for galac- 

 tose) requires about 25 minutes of conjuga- 

 tion before it is transferred. T and L are 

 known to be close together and widely sepa- 

 rated from Gal in the recombination map of 

 Lfr referred to earlier (p. 354). Accordingly, 

 there is a definite relationship between time 

 of transfer from Hfr to F~ and the location of 

 the marker on the Hfr chromosome. 



If different portions of the Hfr chromo- 

 some were entering the F~ cell at random 

 times, the results mentioned would not be 

 obtained. We conclude, therefore, that the 

 Hfr chromosome is transferred in a prefer- 

 ential order, one particular end of the DNA 

 string usually entering the F~ cell first, since 

 the loci that transfer do so in a regular linear 

 procession (Figure 39-2). Other experiments 

 reveal that energy is required for the transfer 

 process and that the entrance rate is uniform 

 from the first part of the chromosome to be 

 transferred, O (representing the "origin"), up 

 to and including the locus of Lac (for 

 lactose). 



Whether or not they have received or lost 

 a segment of an artificially ruptured chromo- 

 some, both the female and male cells survive, 



