Cystine-rich type II antifreeze protein precursor is initiated from the third AUG codon of its mRNA

The primary translation product encoded by sea raven antifreeze protein mRNA was labeled during cell-free synthesis with [3H]leucine. N-terminal sequencing of the immunoselected translation product showed that the third AUG in the mRNA is used as the initiating methionine codon. The antifreeze protein precursor is therefore 163 amino acids long. Amino acid analysis and sequencing of the deblocked N-terminal peptide from the mature circulating form of the antifreeze indicated that glutamine at position 35 is the N-terminal residue. The most likely site of signal peptide cleavage is after alanine at position 17, suggesting that the sea raven antifreeze protein is produced as a preproprotein. Analysis of slot blots indicates that the gene for the antifreeze protein is present in 12-15 copies in the sea raven genome. A representative gene copy was sequenced. It is split into six exons spanning 2.2 kilobase pairs and, based on composite maps of genomic clones, is not accompanied by a second copy within at least 25 kilobase pairs of flanking DNA. The transcription start site was determined by primer extension. Ninety base pairs upstream from this point, beyond the CAAT and TATA boxes, is a putative cis-acting regulatory element in the form of a triplicated 21-base pair tandem repeat.