Laboratory method to study mutational effects on human erythrocyte spectrin tetramerization

We have developed a laboratory method combining a random mutagenesis method and a yeast two-hybrid system to study effects of mutation on human erythrocyte spectrin tetramerization. A PCR-based procedure was used to generate random mutations in DNA fragments of the first 55 residues of -spectrin. Each of the DNA fragments from random mutagenesis was fused with a DNA fragment of native spectrin consisting of residues 56 to 368 to give a DNA fragment of the first 368 residues in -spectrin. The -spectrin DNA fragment and a DNA fragment containing the last 449 residues in -spectrin were introduced into the yeast two-hybrid system for rapid screening of - and -spectrin interaction. Yeast colonies with interacting - and -peptides were blue, and those with non-interacting - and -peptides were white. Six single amino acid mutations (R27G, Y35N, F38S, L49H, Y53N, and Y53C) and a double amino acid mutation (K16M, I24N) were identified from 8 white colonies, but no mutations were found in the DNA fragments of 14 blue colonies. Thus this simple laboratory method allows us to study effects of mutation on interactions of - and -spectrin at the tetramerization site.