394 



CHAPTER 43 



the two mutants can act phenotypically in a 

 complementary manner, or show comple- 

 mentation, to produce the r+ phenotype (Fig- 

 ure 43-4). 



On the other hand, if muUiple infection 

 occurs with two different mutants both 

 located in subregion A (or B), they will not 

 be capable of phenotypic cooperation to 

 produce the r+ phenotype, since both phages 

 produce defective or no A (or B) product. 

 While the entire plate does not clear in about 

 a half hour in this case, one may observe the 

 occasional incidence of plaques in different 

 regions of the plate. Sometimes the fre- 

 quency of these plaques is no greater than 

 one would expect to find due to the rate of 

 spontaneous mutation of the two mutants. 

 This would indicate the nonoccurrence of 

 /•+ genetic recombinants, which failed to ap- 

 pear either because the two mutants are lo- 

 cated too close to each other on the genetic 

 map and/or because both have a common 

 nucleotide defect. Other times the frequency 

 of plaques is so clearly larger than that 

 expected from mutation that the excess can 

 be attributed to recombination between the 

 two A (or B) mutants which results in progeny 

 phages whose rll region is normal (and pro- 

 duce the r+ phenotype) or is doubly mutant 

 (and are undetected). 



Two mutants in subregion A, rl and r2, 

 may fail to show recombination with each 

 other. However, one of these, say rl, shows 

 recombination with mutant r3, while mutant 

 r2 does not. Such results can sometimes be 

 shown to be due to the fact that mutant r2 is 

 deficient not only in its own locus but for all 

 or part of rl and r3 also. Accordingly, the 

 mutant order must be 123 (or 321) in such 

 cases. Other mutants behave as point mu- 

 tants, giving no evidence of deficiency. Of 

 more than 1500 spontaneously occurring rll 

 mutants typed, about 300 were different, that 

 is, each was separable from all the others by 

 recombination (Figure 43-5). Using over- 

 lapping deficiencies and mutants showing no 



evidence of being deficiencies, it is possible 

 to arrange all the r loci in the A and B sub- 

 regions in a single linear sequence, the re- 

 combination rates between two mutants being 

 constant in different tests. Thus, even in its 

 fine structure, the genetic recombination map 

 of bacteriophage is linear. 



The great efficiency in detecting r+ mutants 

 by the plating of rll mutants on strain K has 

 already been impHed by the statement that 

 mutation rates as low as 1 in 10* can be 

 detected. This method also has approxi- 

 mately the same efficiency for detecting re- 

 combinations. The smallest reproducible 

 frequency of recombination, 0.02%, was 

 found between the mutants /•240 and r359. 

 Of numerous other mutants tested, all gave 

 either no recombination or a higher per- 

 centage of recombination. Note that the 

 methods used can detect recombination fre- 

 quencies that are a hundred or even more 

 times less frequent than 0.02%. Even so, 

 none were found, as if 0.02% is close to the 

 lower limit of recombination. To what use 

 may we put this observation? Since the 

 genetic material of phage T4 is DNA, a lower 

 limit for recombination frequency may give 

 us an idea to what degree DNA is finitely 

 divisible for purposes of recombination. Can 

 we translate genetic recombination distance 

 into DNA nucleotide distance? Or, in other 

 words, can we set any limits in terms of 

 nucleotides to the size of the recon in phage 

 T4? 



In order to attempt to estimate the nucleo- 

 tide scope of a recon we shall have to make 

 the following primary assumptions: (1) the 

 probability for genetic recombination is con- 

 stant per molecular distance throughout the 

 phage genetic map, and (2) phage DNA is 

 present as a single copy in the form of a 

 single double-stranded Watson-Crick hehx. 

 With reference to (1) it is further supposed 

 that the genetic markers studied are repre- 

 sentative of all loci present and that the total 

 length of the genetic map is accurately 



