GILLEN, C.M.*; HEILMAN, N.R.; WATSON, N.; SOMPLE, M.; GILLEN, K.M.; Kenyon College, Gambier, OH: Tissue distribution of the cation-chloride cotransporter, masBSC, in Manduca sexta .

We examined the tissue distribution of the Manduca sexta cation-chloride cotransporter, masBSC. MasBSC has strong sequence similarity to vertebrate Na-K-Cl cotransporters. We expressed a 159 amino acid peptide from the C-terminus of masBSC in E. coli as a glutathione-S-transferase fusion protein. The fusion protein was gel-purified and used as antigen for polyclonal antibody production. The resultant polyclonal antibody (M6) recognized proteins at approximately 220 kDa in M. sexta midgut, Malpighian tubule, salivary gland, and ventral nerve cord. M6 also recognized a protein at approximately 220 kDa in Sf9 cells expressing full-length masBSC, but not in mock transfected Sf9 cells or Sf9 cells transfected with the expression vector alone. We isolated RNA from M. sexta midgut, Malpighian tubule, hindgut, trachea, nerve cord, and fat bodies and performed RT-PCR using masBSC gene-specific primers. Bands of the predicted size were amplified in all tissues. PCR products from hindgut, midgut, Malpighian tubule, and nerve cord were gel-purified and sequenced; all sequences matched masBSC with 97-98% identity. These results suggest that masBSC is expressed widely in M. sexta tissues.