Laboratory method to study mutational effects on human erythrocyte spectrin tetramerization
| Autor: | Senthil Ranganathan, N. Topouzian, N. Menhart, Leslie W.-M. Fung |
|---|---|
| Rok vydání: | 2001 |
| Předmět: |
Biology
medicine.disease_cause law.invention chemistry.chemical_compound law Two-Hybrid System Techniques medicine Methods Humans Protein Isoforms Spectrin A-DNA Polymerase chain reaction Mutation Binding Sites Mutagenesis Erythrocyte Membrane Hematology Molecular biology Yeast White (mutation) Biochemistry chemistry Dimerization DNA Protein Binding |
| Zdroj: | American journal of hematology. 67(4) |
| ISSN: | 0361-8609 |
| Popis: | 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 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, 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 α- and β-spectrin at the tetramerization site. Am. J. Hematol. 67:247–251, 2001. © 2001 Wiley-Liss, Inc. |
| Databáze: | OpenAIRE |
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