CLONING AND EXPRESSION OF CHICKEN CLIP-170 AND RESTIN ISOFORMS

We have cloned cDNA for the chicken homologues of human CLIP-170 and R estin and characterized expression of chicken CLIP-170 and Restin mess ages in a variety of chicken tissues. Chicken CLIP-170 and Restin, lik e the human homologues, differ only in a stretch of 35 amino acids pre sent 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 h eptads to one of the heptad repeat regions in the central alpha-helica l coiled-coil rod domain. Other highly conserved chicken and human CLI P-I7O/Restin regions confirm the importance of certain protein domains as crucial for protein function, including two CAP-Gly microtubule-bi nding motifs in the N-terminal globular head domain and two CCHC metal -binding motifs in the C-terminal globular tail domain. We have used S outhern DNA blot analysis and PCR amplification of exon-intron junctio ns of chicken genomic DNA to confirm that CLIP-170 and Restin are isof orms encoded by the same gene. Semiquantitative RT-PCR analysis of CLI P-170 and Restin mRNA expression revealed expression of both isoforms in a variety of chicken tissues but in different ratios. In the tissue s tested, except brain, the message for CLIP-170 was more abundant tha n that for Restin. Comparison of the levels of CLIP-170 and Restin mes sages in RNA from chicken and human intestinal epithelial cells reveal ed remarkably similar ratios in the two species. Our data suggest that expression of CLIP-170 and Restin is differentially regulated and tha t the two isoforms have distinct functions in a wide variety of cells. (C) 1998 Elsevier Science B.V.