Rat alpha-Crystallin A Chain with an Insertion of 22 Residues

Abstract

Rat lens α‐crystallin contains, besides the usual αA and αB subunits, an additional minor chain. This subunit was purified by ion‐exchange chromatography and its primary structure studied. It appeared to be an elongated αA‐like chain, having an insertion of 22 residues between position 63 and 64 of an otherwise normal αA2 chain. Therefore this subunit was called αAIns, i.e. an αA chain with an inserted sequence. This inserted region, which contains three methionyl, five basic and no acidic residues, apparently results in an adequately functioning αAIns chain. The αAIns chain may be the product of a gene which has originated, after duplication of the αA gene, by insertion in one of the copies of a stretch of 66 nucleotides of unknown origin, or alternatively be the result of unusual transcription or processing of precursor mRNA (pre‐mRNA), leaving an extra 66 nucleotides internally in the mRNA to be translated.

Domain structure and evolution in α‐crystallins and small heat‐shock proteins

“…of the spliced a-crystallin mRNA molecules [25,33]. This gives rise to an extra 22-23 residues of unknown function inserted into the protein sequence.…”

Exon shuffling mimicked in cell culture

“…It occurs as large globular complexes of up to 700 kDa, composed of two related types of 20-kDa subunits, ␣A-and ␣B-crystallin (4). In rodents and some other mammals, a minor ␣-crystallin subunit is present, resulting from alternative splicing of the ␣A-crystallin gene transcript (5)(6)(7). This process yields, in addition to the normal 173-residue ␣A-crystallin, the elongated ␣A ins -crystallin, with an insertion of 23 amino acid residues.…”

Two Structurally Closely Related Polypeptides Encoded by 14-S mRNA Isolated from Rat Lens

“…8 pl of a cell-free incubation, in which 1 4 4 mRNA was translated, wasmixed with 50pg of purified aAIns subunit [3]. The polypeptides in the mixture were precipitated with acetone to remove the salts and dissolved in electrophoresis buffer, suitable for polyacrylamide gel electrophoresis in Tris/glycine buffer pH 8.6, containing 6 M urea [12].…”

“…The cyanogen bromide (CB) fragments were lyophilized. Before tryptic digestion, 3 mg of the CB4 fragment of clAlns subunit [3] was added as an internal marker. Tryptic digestion was performed by incubation of the CB fragments with 1 % (w/w) trypsin (Worthington, treated with L-1 -tosylamido-2-phenylethyl chloromethyl ketone) in a 0.1 M ammonium bicarbonate buffer pH 8.9 for 1 h at 37 "C.…”

“…The latter polypeptide comigrates in dodecylsulphate/polyacrylamide gel electrophoresis and in isoelectrofocusing with a rat a-crystallin subunit (previously referred to as ax [2]), which is not found in calf cc-crystallin. We studied the primary structure of this subunit in more detail (as described in the following paper [3]); this led us to replace the designation ax by aAIns (referring to A chain with an inserted amino acid sequence). In the first part of this paper we present data about the immunological relationship between both translation products.…”