Meeting Abstract
P2.72 Friday, Jan. 4 Cloning and expression of multiple Troponin-C isoforms in lobster muscle CHAO, Erica*; KIM, Hyun-Woo; THOMPSON, Matthew D; MYKLES, Donald L; Colorado State University; Colorado State University; Colorado State University; Colorado State University echao@lamar.colostate.edu
Troponin-C (Tn-C) is a protein which regulates muscle contraction by binding calcium. In lobster, 3 isoforms of Tn-C (Tn-C1, Tn-C2a and Tn-C2b) have been identified and sequenced at the amino acid (aa) level. We used RT-PCR to clone 6 novel Tn-C sequences: a full-length cDNA sequence designated Tn-C3 (1.2 kb, 150 aa, ~16.8 kDa); an EST from the Marine Genomics Project constituting a full-length cDNA sequence (3.1 kb; 149 aa; ~17.3 kDa); and four partial cDNA sequences (Cr, Cu, DA1 and DA3, cloned from the crusher (Cr), cutter (Cu), and deep abdominal (DA) muscles respectively, all 249 bp and encoding a 97-aa protein of ~13 kDa). These 6 sequences share 46 � 98% similarity, and 45 � 98% similarity to the 3 published Tn-C aa sequences. Using RACE, we sequenced 7 unique 5�-UTRs and 9 unique 3�-UTRs for Tn-C, suggesting that the lobster may express 9 Tn-C isoforms produced by distinct genes. We also sequenced an EST from the Marine Genomics Project encoding a Tn-C-like protein (19 � 35% similarity to Tn-C; 4 � 49% similarity to other muscle Ca2+-binding proteins such as muscle-specific calpain, calpain-B, calpain-T, calmodulin, and sarcoplasmic Ca2+-binding protein) (2.4 kb; 154 aa; ~16.9 kDa). This sequence contains only one EF-hand motif (i.e. two Ca2+-binding sites; most Tn-C isoforms contain two EF-hand motifs, i.e. four Ca2+-binding sites). The expression patterns of these Tn-C isoforms and Tn-C-like protein are currently under investigation in each lobster muscle type. Further work will determine: the relationship between Tn-C isoform expression patterns and muscle functional properties; TnC as an indicator of fiber type switching; and whether fiber type switching and changes in TnC isoforms change in response to myostatin, which inhibits muscle growth prior to molting. Supported by NSF (IBN-0618203).
