NOVOTNEY, D.A.*; DING, X.; LAHEY, B.C.; LOVETT, D.L.; BORST, D.W.; Illinois State University, Normal; Illinois State University, Normal; Illinois State University, Normal; The College of New Jersey, Ewing; Illinois State University, Normal: The site of yolk protein synthesis in the green crab

Most crustacean eggs contain large amounts of yolk protein. However, in most species the site of its synthesis is poorly understood. Therefore, we cloned a fragment of the yolk protein cDNA. Mature oocytes were removed from the green crab, Carcinus maenas , and homogenized. Two abundant yolk proteins (YP74 = 74kDa and YP106 = 106kDa) were isolated from the homogenate by gel filtration chromatography and SDS-PAGE, digested with trypsin, and the resulting peptide fragments sequenced using LC-MS. The amino acid sequences of the YP74 fragments shared sequence similarities to crayfish and shrimp yolk proteins. Therefore, we designed degenerate primers based on the amino acid sequences of these fragments and used them to amplify a 545-bp cDNA fragment of YP74. The deduced amino acid sequence of this fragment had a close relationship (~60% identity and ~75% similarity) to yolk proteins found in other crustaceans. We designed specific primers based on the sequence of the cDNA fragment and used them to determine whether the corresponding mRNA was present in ovarian and hepatopancreas tissue. The primers for YP74 amplified a band of the expected size (442-bp) when used with cDNA from ovarian tissue at different stages of the reproduction cycle. No bands were observed with cDNA from the hepatopancreas of these animals. These results suggest that the ovary is the sole site of YP74 synthesis in the green crab. However, other yolk proteins may be produced in other, extra-ovarian tissues. Supported by NSF grant 0240903 to DWB. DN is a Beckman Foundation Undergraduate Scholar.