Genomic- and protein-based approaches for connectin (titin) identification in the ascidian Ciona intestinalis

Determining the complete primary structure of large proteins is difficult because of the large sequence size and low sequence homology among animals, as is the case with connectin (titin)-like proteins in invertebrate muscles. Conventionally, large proteins have been investigated using immuno-screenings and plaque hybridization screenings that require significant time and labor. Recently, however, the genomic sequences of various invertebrates have been determined, leading to changes in the strategies used to elucidate the complete primary structures of large proteins. In this paper, we describe our methods for determining the sequences of large proteins by elucidating the primary structure of connectin from the ascidian Ciona intestinalis as an example. We searched for genes that encode connectin-like proteins in the C. intestinalis genome using the BLAST search program. Subsequently, we identified some domains present in connectin and connectin-like proteins, such as immunoglobulin (Ig), fibronectin type 3 (Fn) and kinase domains in C. intestinalis using the SMART program and manual estimation. The existence of these domains and the unique sequences between each domain were confirmed using RT-PCR. We also examined the localization of mRNA using whole-mount in situ hybridization (WISH) and protein expression using SDS-PAGE. These analyses indicate that the domain structure and molecular weight of ascidian connectin are similar to those of vertebrate connectin and that ascidian connectin is also expressed in heart muscle, similarly to vertebrate connectin. The methods described in this study can be used to determine the primary structures of large proteins, such as novel connectin-like proteins in invertebrates.