Construction of interleukin-1 alpha mutants using unequal contamination of synthetic oligonucleotides
| Autor: | Kurt Poindexter, Richard B. Gayle, Rita Jerzy |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Genetics
Base Sequence Oligonucleotide Protein Conformation Mutant Molecular Sequence Data Nucleic acid sequence DNA Recombinant Mutagenesis (molecular biology technique) Biology Recombinant Proteins Protein structure Genetic Techniques Oligodeoxyribonucleotides Mutagenesis Humans Amino Acid Sequence Cloning Molecular Saturated mutagenesis Gene Peptide sequence Gene Library Interleukin-1 Plasmids |
| Zdroj: | Nucleic acids research. 19(8) |
| ISSN: | 0305-1048 |
| Popis: | Proteins without readily available three-dimensional structural data present a difficult problem in the exploration of structure/function relationships. Saturation mutagenesis using contaminated oligonucleotides can identify potentially interesting regions of such a protein. This technique, in which synthesized oligonucleotides contain low-level base substitutions, allows random mutations to be placed throughout a gene sequence. Using double-stranded cassettes, a region of the human interleukin-1 alpha gene has been altered using such mutagenic oligonucleotides. However, instead of contaminating both strands of the gene sequence at the same level, each strand of the insert was contaminated at a different level. Several recombinants were sequenced and the effects of the mutations on the activity of the proteins were examined. Contaminating the two oligonucleotides at different levels produced a significantly different distribution of nucleotide changes from that seen if both strands were contaminated at the same level. The observed distribution followed the average of the distributions for each of the two contamination levels. This resulted in roughly equal frequencies of 1 to 5 nucleotide changes per clone with very few clones containing the wild-type nucleotide sequence. This helped overcome the redundancy in the genetic code, resulting in a high frequency of amino acid changes, and allowed changes at every amino acid to be sampled in a small number of mutants. This procedure can allow a gene sequence to be screened rapidly by removing most wild-type sequences from analysis while making sure that there are many amino acid changes in the resultant mutants. |
| Databáze: | OpenAIRE |
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