Locus assignment of alpha-globin structural mutations by hybrid-selected translation.
The two human alpha-globin genes, alpha 1 and alpha 2 located 3.4 kilobases apart on chromosome 16, encode identical alpha-globin proteins. A mutation in either gene could result in a structural hemoglobinopathy. It has only recently become possible to assign an alpha-chain mutant to one of these two loci by using recombinant DNA technology. While definitive, this approach has necessitated the cloning and sequencing of the specific gene in question. We present an alternative approach which results in rapid and definitive assignment of an alpha-globin mutation to its encoding genetic locus. This approach uses the technique of hybrid-selected translation. Reticulocyte RNA from individuals with alpha-globin mutations can be fractionated into beta-, alpha 9 (total)-, alpha 1-, and alpha 2-globin mRNA by selective hybridization of each mRNA species to its respective complementary DNA (cDNA) immobilized on nitrocellulose paper. Each mRNA purified in this way can be translated in vitro, and the mRNA species (and hence gene locus) encoding the globin mutant can then be directly identified by gel analysis of the radiolabeled translation products. This procedure can be used to identify globin mutants as alpha or beta and to localize alpha-globin mutants to the alpha 1 or alpha 2 gene. We have used this technique to localize the two alpha-globin mutants, alpha 125Pro (Hb Quong Sze) and alpha 47HIS (Hb Hasharon), to the alpha 2 locus. This approach could potentially be expanded to serve as an alternative to peptide analysis for the initial characterization of all globin structural mutants.
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D002874:Chromosome Mapping; D004720:Endonucleases / Q000502:physiology; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D005914:Globins / Q000235:genetics*; D006801:Humans; D006824:Hybridization, Genetic; D009154:Mutation; D014176:Protein Biosynthesis; D015719:Single-Strand Specific DNA and RNA Endonucleases
D005914:Globins; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases