Part 1

Course video 24 of 68

Before Benzer’s experiments, most genetic mapping required a screening of all the progeny of a cross to calculate the recombination frequencies. The power of the rII system of phage T4 resides in the facts that many independent mutants can be identified by scoring plaque morphology on the permissive host strain, and that only wild type recombinants will grow on the restrictive host strain. This, together with the very high recombination of T4 DNA, allows the detection of recombination between mutations affecting adjacent nucleotides. The cis-trans complementation test showed that the rII locus consists of two genes. Using more than 100 deletion mutants, which do not revert to wild type, Benzer first demonstrated that the topology of DNA is linear. Using these deletions, he was able to carve the rIIA and rIIB genes in 47 segments. A very simple and rapid recombination test allowed him to map thousands of point mutants, both independent and mutagen-induced, in individual segments. This map was perfectly congruent with maps laboriously constructed by classical recombination tests. The topography of the map was strikingly non-random, with sites that are 100 times more likely to mutate than others and are called hot spots. The spectrum of sites detected after mutagenesis was strikingly different. It is now known that most of the spontaneous rII mutants are frame-shift, i.e. addition or deletion of one or a few base pairs that disrupt the translation of the mRNA into protein. In contrast, most mutagens used in this work induce base substitutions that do not usually arrest translation. Now that the sequence of the rII locus is known, the saturation of the map is such that there are about one mutation every 8 nucleotides.

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