Isolation and Sequencing of the cDNA Encoding a-actinin 3 in Rat Skeletal Muscle

Abstract

Alpha-actinins are a family of proteins that have been characterized as crosslinkers of filamentous actin in eukaryotes. Their structure is marked by an actin-binding region followed by four spectrin-like repeats which in turn is followed by two EF-hand regions that may or may not bind calcium depending on the isoform. Alpha-actinin 3 (AA3) is a calcium insensitive isoform that is found only in skeletal muscle. Isolation of the cDNA that potentially encodes this protein in rat skeletal muscle was carried out using the Uni-ZAP XR cDNA library and immunoscreenmg with an anti-sarcomenc-cxactimn antibody. A phagemid containing this cDNA was transfected into E. coli and isolated. Primers designed against the multiple cloning site allowed for partial sequencing of the 5' and 3' ends of the insert. Partial sequences facilitated the designing of new pnmers to eventually bridge the two ends of the cDNA, and to determine a contiguous sequence of the insert using Sequencher software. The cDNA sequence shows strong identity to the AA3s found in mouse and human. The inferred protein sequence also showed 99.32% identity to AA3 in mouse and 96.00% to that of human. Each of the regions contained within the protein individually show strong identity with A A3 in mouse and human implying similar function and structure.