Cloning the Recombinase

Cloning the Recombinase

Direct assay of the immune system recombinase is not easy, and unsuccessful attempts to detect the enzyme were made for years. An indirect approach was to seek the DNA that either codes for the recom-binase or that induces the synthesis of the recombinase. This was possible with the same trick described in the previous section. Recom-binational joining was used to invert a sequence and permit expression of a selectable marker. In this case, however, since the join was to occur within animal cells not normally expressing the recombinase, cells were transformed with vectors containing fragments of chromosomal DNA. Any vectors that expressed the immune system recombinase would flip the DNA sequence and express the selectable marker.

One might wonder why this experiment had a chance of working. Why should DNA transformed into cells be any more likely to express the recombinase than the recombinase gene resident on the chromo-some? One might picture that DNA in the chromosome was prevented from expressing by mechanisms similar to those that repress the yeast mating-type genes at the HML and HMR loci. Alternatively, newly arriving DNA might be able to express the recombinase gene until it is repressed just like zygotic induction of phage lambda or the P element transposition events that occur in Drosophila just after mating. None-theless, the assay did work, and it has been possible to detect and clone two clustered and divergently transcribed genes that code for the recom-binase or the recombinase activator gene RAG.