RECOMBINATION ANALYSIS IN MICROBIAL SYSTEMS 47 



types indicated in Figure 2 as "selected recombinants." In order to ob- 

 serve these types by non-selective methods, one would have had to 

 characterize many millions of spores. As long as the spontaneous re- 

 version rates of markers are low, exceedingly rare recombination events 

 can be studied by such selective methods. Experiments have shown, in 

 microbial systems of all types including phage, that as a rule independ- 

 ent mutations affecting a single functional unit of the chromosome 

 almost always show recombination when crossed; they must, therefore, 

 have different locations on the chromosome. 



These results are readily interpreted in terms of the molecular 

 theory of the structure of genes. It is supposed that the primary gene 

 product is determined by a linear sequence of nucleotides whose length 

 is variously estimated as lying between 1,000 and 8,000 nucleotides 

 ( Pontecorvo and Roper, 1956 ) . This sequence comprises the functional 

 gene. An alteration of one or a few nucleotides at any one of many 

 points in the sequence would give rise to a mutant gene. However, a 

 genie alteration will be detected only if it leads to a quantitative or 

 quahtative modification of the gene product, or phenotype. Insofar as 

 an enzyme is concerned, an alteration in base sequence leading to a 

 change in composition of the protein will be detected only if a critical 

 aspect of protein structure is affected. In view of the structural restric- 

 tions imposed for an enzyme to be active, most alterations in critical 

 regions of an enzymatic protein would lead to total or partial inactiva- 

 tion or loss. Thus if one selects independent loss mutations affecting a 

 single gene product, very often these will be the result of changes of 

 sequence at different positions in the functional gene, even though the 

 phenotypes selected are identical. The fact that such alleles show re- 

 combination means that the recombination process may be initiated at 

 any one of many points along the functional gene sequence. We there- 

 fore distinguish the functional unit, which is a long sequence, from 

 mutational sites, which may be very small indeed and may be situated 

 at any one of many points in the functional gene sequence. We can dis- 

 tinguish also the recombination unit, which is much smaller than the 

 functional gene sequence. The smallest unit of recombination may 

 even be a single nucleotide ( Benzer, 1957 ) . Two mutations are said to 

 occupy the same site when no recombination is found to occur bet\veen 

 them and different sites when recombination does occur. The term 

 gene locus is reserved to specify the functional gene sequence. 



Limitations of fine-structure analysis 



Clearly there are as many possible positions at which sequence 

 can be altered as there are nucleotides in a sequence. However, not all 

 nucleotides of a functional sequence necessarily determine a critical as- 



