Nature of deletions formed in response to IS2 in a revertant of the gal3 insertion of E. coli

Summary

The gal3 mutation of E. coli, which arose by the insertion of IS2 in the OP region of the gal operon, reverts spontaneously by excision of the IS2 to produce inducible revertants or by mutational alterations of IS2 to produce constitutive revertants. However, gal3(λ) strains bearing chlD-pgl deletions produce constitutive revertants alone. We proposed that deletions formed in the presence of IS2 terminate specifically at its right end, so that revertants arising by excision of IS2 fuse the gal genes to other promoters. Therefore, the revertants are exclusively constitutive.

The above hypothesis was tested by electron microscopy of IS2-specific deletions. Spontaneous chlD-pgl deletions were isolated from gal ^c331 (a revertant of gal3 which retains IS2) and transferred to λgal genomes. Electron microscopy of DNA heteroduplexes from these phages confirmed that all of the deletions examined have one end-point fixed at the right end of IS2, whereas their other end-points are variable. In each case, the complete IS2 element was apparently retained. This specificity was also detectable in a revertant (gal ^c200) which retains only the right 1/5 portion of the IS2. The frequencies of these deletions were generally increased in constitutive revertants of gal3. Since a galO ^cmutant did not show a similar increase, it seems that this effect depends upon a base sequence provided by IS2. Moreover, the presence of prophage λ contributes to the specificity and, in some instances, the frequency of IS2-specific deletions.

A mechanism for the formation of the IS2-specific deletions has been proposed. A base sequence located at, or near, the right end of IS2 is recognized and nicked by a specific endonuclease. The nick is enlarged by unidirectional, exonucleolytic degradation to produce deletions extending outwards from the insertion. In constitutive revertants, the nicking site may be exposed to endonucleolytic attack more frequently.