| We have developed a laboratory method combining a random mutagenesis method and the 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 α-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 α-spectrin. The α-spectrin DNA fragment and a DNA fragment containing the last 449 residues in β-spectrin were introduced into the yeast two-hybrid system for rapid screening of α- and β-spectrin interaction. Yeast colonies with interacting α- and β-peptides were blue, and those with non-interacting α- and β-peptides were white. Six single amino acid mutations (R27G, Y53N, 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 9 blue colonies. Thus this simple laboratory method allows us to study effects of mutation on interactions of α- and β-spectrin at the tetramerization site. |
