Cloning of a portion of the chromosomal gene for human erythrocyte alpha-spectrin by using a synthetic gene fragment

Abstract

A region of minimal codon degeneracy was selected from the amino acid sequence of the amino-terminal alpha I domain of human erythrocyte spectrin to design a 90-base-pair DNA probe for the screening of a human genomic library. Five complementary oligonucleotides were assembled to form a full-length double-stranded DNA, which was then cloned in an M13 phage vector to generate hybridization probes. Under stringent conditions, a single hybridizing clone was isolated from a total human genomic library. Partial DNA sequence analysis established the 16.8-kilobase-pair isolate as erythrocyte alpha-spectrin by correlation to a known sequence of 131 amino acids. The spectrin 106 amino acid repeat segment is encoded by multiple exons separated by introns of various sizes. Of the 3074 base pairs of DNA sequenced thus far, 12.8% code for amino acids.