RECOMBINATION ANALYSIS IN MICROBIAL SYSTEMS 49 



recombination would be established; and the mutational sites could be 

 distinguished. If, however, recombination cannot occur in a space 

 smaller than three or four nucleotides, then the two mutations of Fig- 

 ure 3 would be scored as involving identical sites. Calculations suggest, 

 however, that recombination may be possible between sites only one 

 nucleotide apart ( Benzer, 1957; Chase and Doermann, 1958 ) . 



We can satisfy ourselves for the moment that the functional gene 

 contains multiple mutational sites by a simple calculation, taking an ex- 

 ample from a Mendelian organism, Aspergillus nidulans (Pontecorvo 

 and Roper, 1956 ) . In a locus controlling the synthesis of adenine, four 

 independent adenineless mutations were isolated. The most distant 

 mutational sites among these four recombine with a frequency of 

 1.8/1,000. The closest pair recombine with a frequency of 1.2/100,000. 

 If we suppose a linear relationship between recombination frequency 

 and map distance, then the gene locus can be considered to contain 

 a minimum of 1,800 recombination units. If we suppose further that the 

 recombination frequency of 1.2/100,000 is the smallest that can be 

 measured, we obtain from the ratio of these two values the minimum 

 number of points at which a change of sequence can occur and be 

 separated by recombination. This ratio is 150, and it is a minimum 

 estimate for two reasons. First, the locus may, in fact, be larger than 

 is indicated by the two most distant markers; second, the shortest dis- 

 tance within which recombination can occur may be smaller than that 

 indicated from the closest sites. 



A geneticist is not always in the agreeable position of knowing, for 

 each mutant of a given phenotype, what enzyme (or primary gene 

 product) is being aflFected. He has, however, a genetic test of the 

 identity of the functions aflFected by independent mutations: the 

 cis-trans test, developed by Lewis (1951) for Drosophila and subse- 

 quently extended to microbial systems, largely as a result of the work 

 of Benzer (1957) on phage T4. This test consists in comparing the 

 phenotypes obtained from two kinds of crosses involving two muta- 

 tional sites of apparently identical phenotypic eflFect. One cross forms a 

 diploid hybrid, or heterocaryon, in which each parental chromosome 

 carries one of the two mutated sites under examination. In the second 

 cross, both mutated sites are in the chromosome of one parent, while 

 the chromosome of the second parent is normal. In these two kinds of 

 hybrids, the same total amounts of mutated and normal cliromosome 

 are present. It is only the relative positions of mutated and normal 

 regions that diflFer. In the first cross, the two mutational sites are in the 

 trans position, while in the second they are in the cis position (see 

 Figure 4). If the two mutational sites are in diflFerent functional 

 genes linked together, the phenotype of the hybrid will be nor- 

 mal, whatever the arrangement of the mutational sites in tlie hy- 



