Expressed sequence tag analysis of genes expressed in the bay scallop, Argopecten irradians.

The bay scallop (Argopecten irradians) is a marine bivalve found along the eastern United States. As in other pectinids, the bay scallop has a single, large adductor muscle which acts to open and close the shell with great force. This muscle is a prominent feature observed when the shell is removed, and is a favorite of both scientists and consumers, as it offers an excellent model for understanding muscle physiology and provides a healthy, highprotein food source. Consumer demand has caused the increase in aquaculture of bay scallops for direct marketing or to seed local waters. The objective of the present study was to better understand the factors that contribute to the growth and development of the scallop adductor muscle. Because little is known about these processes in scallops, we have started by isolating and identifying genes from adductor muscle tissue. The results from this work could help facilitate future research. One example is in aquaculture, where these genes could be used as markers for a selective breeding programs. The use of such techniques could result in increasing the yield of adductor muscle or altering the size and density of the muscle fibers. To identify the factors or genes involved in scallop muscle structure and function, a cDNA library was constructed from bay scallop adductor muscle, and expressed sequence tags (ESTs) were sequenced. ESTs are small pieces of DNA sequence (usually 100 to 800 nucleotides long) generated by sequencing randomly selected cDNA clones from a library. In this paper, the sequences of genes expressed in the bay scallop adductor muscle are reported. To construct the cDNA library, adductor muscle tissue was dissected from four adult bay scallops obtained from Woods Hole, Massachusetts. Total RNA was extracted with Tri-Reagent (Molecular Research Center Inc.), and mRNA was isolated using the Poly-A-Tract mRNA Isolation System (Promega). The cDNA library was constructed using the Zap Express cDNA/Gigapack cloning kit (Stratagene), starting with 5 g of mRNA as previously described (1). To obtain ESTs, the library was mass excised to pBK-CMV phagemids and plated. From these plates, 454 randomly chosen cDNA clones were picked and sequenced from the 5 region by using the dideoxy chain termination method, with Big Dye Terminator (Applied Biosystems) and a vector-specific primer. The reactions were precipitated and resuspended in Hi-Di Formamide with EDTA (Applied Biosystems) and run on an ABI Prism 3730 automated sequencer (Applied Biosystems). Gene identification analysis was performed using Phred (base-calling) and Cross_ match (vector removal) software (http://www.phrap.org/); then EST sequences were compared with those in the NCBI database (nr) using the Blast-X and Blast-N programs (http://www.ncbi. nlm.nih.gov/BLAST) (2). Of the 454 cDNA clones, 20 yielded no sequence and an additional 9 produced sequences of less than 150 bp; these were not used for sequence analysis. The average length of the remaining 425 sequences was 792 bp. Based on top Blast hits, 137 distinct sequences were observed and 90 of these genes were putatively identified; 47 lacked similarity with known genes and could not be identified. Of the latter 47 distinct sequences, 38 were most similar to unidentified products (e.g., hypothetical protein, unnamed protein product) and 9 produced no hits in the database. Only 16 of the distinct sequences had previously been sequenced from the genus Argopecten and were either myosins, ribosomal proteins, or mitochondrial sequence. The mitochondrial sequence appeared 54 times (13%) in the ESTs and does not necessarily code for a protein; the presence of a poly-A region in the sequence leads to its isolation along with mRNA. Of the genes identified based on sequence similarity, 33% are involved in cell structure (Fig. 1). The two genes most prevalent in the cDNA library were actin and myosin because they are the