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 alpha-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 alpha-spectrin. The alpha-spectrin DNA fragment and a DNA fragment containing the last 449 residues in beta-spectrin were introduced into the yeast two-hybrid system for rapid screening of alpha- and beta-spectrin interaction. Yeast colonies with interacting alpha- and beta-peptides were blue, and those with non-interacting alpha- and beta-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 alpha- and beta-spectrin at the tetramerization site.
Ranganathan
S
S
Department of Chemistry, Loyola University of Chicago, Chicago, Illinois 60626, USA.
Menhart
N
N
Topouzian
N
N
Fung
L W
LW
D001665:Binding Sites; D019281:Dimerization; D004910:Erythrocyte Membrane / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D008722:Methods; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D013049:Spectrin / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D020798:Two-Hybrid System Techniques
D020033:Protein Isoforms; D013049:Spectrin