The Phenomenon of alpha-Complementation in E. coli Cloning Systems.

The E. coli enzyme beta-galactosidase is a homo-tetramer of the protein product of the lacZ gene. Certain mutations in the 5' region of lacZ prevent subunit association. Because monomers lack enzyme activity, the failure to assemble leads to a Lac- phenotype. In some such mutants, subunit assembly (and enzyme activity) are restored by the presence of a small (26 amino acids) amino-terminal fragment of the lacZ product (the so-called alpha polypeptide). Such lacZ mutants are said to be subject to "alpha-complementation."

The product of the allele lacZ D M15 lacks amino acids 11-41 of wild-type beta-galactosidase and is subject to a complementation.

Messing and co-workers took advantage of alpha-complementation in constructing the M13mp phage and pUC plasmid series of cloning vectors. These vectors carry a polycloning region embedded in the sequence for an alpha polypeptide gene fragment. The polycloning site does not alter the reading frame or destroy the ability of the fragment to alpha-complement.

Recombinant inserts at the polycloning site almost always eliminate alpha-complementation. Indicator (i.e., X-gal) media provide a convenient method to detect transformants with recombinant inserts in the polycloning region.

The virtue of this seemingly arcane situation is that it mitigates a typical problem in cloning strategies, namely that the stability of many vectors decreases with their size, thus limiting the amount of DNA that can be cloned. For example, the ability to maintain recombinant pUC plasmids decreases significantly as their size approaches 15 kb. Because the alpha polypeptide is small, the total size of the vector can be minimized, allowing it to carry a correspondingly larger insert. The bulk of the lacZ coding sequence is contained in the chromosome of the host. Compare the total size of the pUC vectors with the DNA necessary to code for wild-type beta-galactosidase (monomer = 1,173 amino acids).

Obviously, alpha complementation requires special host strains carrying an "alpha-complementable" lacZ mutation such as lacZ D M15. E. coli DH5a is such a strain.


Yanisch-Perron et al. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene , 33: 103-109.