348 



CHAPTER 26 



about 300 arc different; that is. each is sepa- 

 rable from all the others b\ recombination 

 (Figure 26-7). Using overlapping deficien- 

 cies and point mutants, it is possible to ar- 

 range all the mutant loci of the A and B 

 subregions in a single linear sequence with 

 distances between mutants being approxi- 

 mately additive. Thus, even in its fine struc- 

 ture (within one "gene") the genetic recom- 

 bination map of bacteriophage is linear. 



The ability to select for r + reversions by 

 plating rll mutants on strain K12 permits the 

 detection of mutation frequencies as low as 

 one in 10\ This method also has approxi- 

 mately the same efficiency for detecting re- 

 combinants. Although numerous mutants 

 were crossed with each other, the smallest 

 reproducible frequency of recombination 

 found between two mutants (0.02%) was 

 relatively large, being at least one hundred 

 times greater than the lowest frequency de- 

 tectable. For recombination, therefore, 

 0.02% seems to be close to the lower limit, 

 a value which may be useful in estimating 

 how finitely divisible DNA is for purposes of 

 recombination. 



To calculate the smallest nucleotide dis- 

 tance between two markers of T4 which are 

 able to recombine with each other, we must 

 assume that: 



1. The probability of genetic recombina- 

 tion is constant per molecular distance on 

 the phage genetic map. 



2. The genetic markers studied are repre- 

 sentative of all other loci. 



3. The total length of the genetic map is 

 accurately estimated by the summation of a 

 number of small distances. 



Based on these assumptions, the total 

 genetic map of phage T4 is calculated to 

 be approximately 2500 recombination units 

 long; that is, it shows 2500% recombination 

 with respect to its total genetic content. 

 (Remember that a recombination map based 

 upon crossing over can be longer than one 



hundred recombination units — in this case, 

 crossover units.) The molecular weight of 

 the DNA of T4 is 130 to 160 times 10 ,; 

 which means 14 contains about 400,000 nu- 

 cleotides. These are arranged in the form 

 of a single double helix of 200,000 linearly- 

 arranged nucleotides. 



rr U c t . 2500% recombinants , 



The fraction A -^x ; —, — equals 



200,000 nucleotides 



0.0125% and expresses the percentage of 

 recombination per nucleotide pair. Assum- 

 ing that recombination cannot take place 

 within a nucleotide pair, a phage genome has 

 200,000 internucleotide points where ex- 

 changes can occur. Thus, we can say that 

 if two r mutants — different from wild-type in 

 single, adjacent nucleotides — are crossed, r+ 

 recombinants arc expected to occur among 

 0.0125 % ^ Qr 0006 25%, f their progeny 



(the undetected double mutant recombinant 

 also occurring with this frequency). 



Suppose that the lowest r+ recombinant 

 frequency observed, 0.02%, is actually the 

 minimal rate. This might mean that recom- 

 bination could occur in each internucleotide 

 position, but that the closest of the numerous 

 mutants tested still would be separated by 

 about three nucleotides ( .02 /.00625 ) . Sup- 

 posing the mutants actually affect adjacent 

 nucleotides, then only about every third 

 internucleotide point, on the average, would 

 be capable of undergoing recombination, and 

 an estimate of the average unit of recombi- 

 nation would be three nucleotides in length. 

 Because the observed value of 0.02% is a 

 maximum value for the least amount of re- 

 combination, and uncertainties exist concern- 

 ing the length of the genetic map and the 

 number of nucleotides in the phage genome, 

 the average nucleotide length between phage 

 recombination events is subject to consider- 

 able error. Since the DNA backbone does 

 not seem to have any structural feature 

 which occurs only every third nucleotide, it 

 is reasonable to accept as a working hypoth- 



