Random Mutagenesis by Oligonucleotides: A Probe for MHC Structure and Function

Abstract

The study of variants induced by mutagenesis has been extremely important in understanding many biological systems (Drake 1969). In the case of the mouse histocompatibility complex (MHC) H-2 mutants have been important in the definition of pleiotropic effects of single MHC genes. These mutants were laboriously isolated by screening for skin graft rejection (Melvold et al. 1982). The generation of such spontaneous mutants, however, is time consuming and difficult. Several laboratories have used in vitro selection techniques (by monoclonal antibodies) as an alternative, (Flores and Rajan 1977; Pious et al. 1982) others have used molecular genetics techniques (Shiroishi et al. 1985). These include using site directed mutagenesis and in vitro recombination. The in vitro recombinants have been crucial in establishing that the α1 and α2 domains of MHC class I molecules interact significantly to form the class I peptide binding structure recognized by T cells (Darsley et al. 1987). This was later confirmed using X-ray crystallography on the isolated HLA-A2 protein (Bjorkman et al. 1987a).