Molecular cloning and characterization of winter flounder antifreeze cDNA

Double-stranded cDNA was synthesized from partially purified winter flounder antifreeze mRNA and inserted into the endonuclease Pst I site of plasmid pBR322 by the poly(dG).poly(dC) homopolymer extension technique. The recombinant plasmids wee used to transform Escherichia coli. Clones containing antifreeze cDNA inserts were identified by the hybridization-selection technique. One of the inserts, 380 nucleotides in length, was digested with endonucleases Sau3AI and HinfI, which cleaved the insert into three fragments. The nucleotide sequences of these fragments were determined. The cDNA contains the entire coding sequence for a possible antifreeze peptide, including the leader sequence. The predicted amino acid sequence is similar to but not identical to one of the known sequences of antifreeze peptide. Within the cDNA are three segments of repeating sequences. The basic repeating sequence of 11 amino acids is maintained in the amino acid sequence coded by the cDNA and in the antifreeze peptide.