Cloning and expression of chicken CLIP-170 and Restin isoforms

Abstract

We have cloned cDNA for the chicken homologues of human CLIP-170 and Restin and characterized expression of chicken CLIP-170 and Restin messages in a variety of chicken tissues. Chicken CLIP-170 and Restin, like the human homologues, differ only in a stretch of 35 amino acids present in Restin but missing from CLIP-170. This Restin-specific insert is perfectly conserved between the chicken and human sequences at both the protein and nucleotide level and contributes an additional five heptads to one of the heptad repeat regions in the central α-helical coiled-coil rod domain. Other highly conserved chicken and human CLIP-170/Restin regions confirm the importance of certain protein domains as crucial for protein function, including two CAP-Gly microtubule-binding motifs in the N-terminal globular head domain and two CCHC metal-binding motifs in the C-terminal globular tail domain. We have used Southern DNA blot analysis and PCR amplification of exon–intron junctions of chicken genomic DNA to confirm that CLIP-170 and Restin are isoforms encoded by the same gene. Semiquantitative RT-PCR analysis of CLIP-170 and Restin mRNA expression revealed expression of both isoforms in a variety of chicken tissues but in different ratios. In the tissues tested, except brain, the message for CLIP-170 was more abundant than that for Restin. Comparison of the levels of CLIP-170 and Restin messages in RNA from chicken and human intestinal epithelial cells revealed remarkably similar ratios in the two species. Our data suggest that expression of CLIP-170 and Restin is differentially regulated and that the two isoforms have distinct functions in a wide variety of cells.