individual loci; to be stably inherited, each marker has to recombine into the recipient chromosome by a double crossover spanning it. However, in the recom- binant frequency analysis, we have specifically selected trihybrids as a starting point, and now we have to consider the various possible combinations of the three donor alleles that can be inserted by double crossing over in the various intervals. We know that leu" must have entered and inserted because we selected it, but the leu" recombinants that we select may or may not have incorporated the other donor markers, depending on where the double crossover took place. Hence, the procedure is to first select leu" exconjugants and then isolate and test a large sam- ple of them to see which of the other markers were integrated. Let's look at an example. In the cross Hfr mer arg leu str x F- met arg leu- str, we would select leu recombinants and then examine them for the arg and met" alleles, called the unselected markers. Figure 5-17 depicts the types of double-crossover events expected. One crossover must be on the left side of the leu marker and the other must be on the right side. Let's assume that the leu exconjugants are of the following types and frequencies: FIGURE 5-17 The diagram shows how genes can be mapped by recombination in E col. In excorjugants, selection is made for merozygotes bearing the leu+ marker, which is donated late. The early markers (arg and met) may or may not be inserted, depending on the site where recombination between the Hir fragment and the F chromosome takes place. The frequencies of events diagrammed in parts a and b are used to abtain the relative sizes of the leu-arg and arg-met regions. Note that, in each case, only the DNA inserted into the F chromosome survives; the other fragment is lost. leu arg met 4% leu" arg mer OinthPod ANIMATED ART: Bactorial conjugation and mapping by recombination leu* arg mer 87% The generation of various recombinants by crossing over in different regions (a) Insertion of late marker only leu met Htr fragment met leu arg met chromosome (b) Insertion of late marker and one early marker met mét leu" arg" met (e) Insertion of all markers leu met leu arg met (d) Insertion of late and early markers, but not of marker in between met arg leu" arg" met met