Characterization of the primary structure of a size-variant antifreeze peptide and the organization and structure of antifreeze peptide genes from the Antarctic eel pout Rhigophila dearborni

• Main Author: Wang, Xin
• Other Authors: DeVries, Arthur L.
• Format: Text
• Language: English
• Published: 1993
• Subjects: Biology; Molecular; Animal Physiology; Chemistry; Biochemistry; Antarctic; The Antarctic; Antarc*
• Online Access: http://hdl.handle.net/2142/18947

The antarctic eel pout Rhigophila dearborni synthesizes three major antifreeze peptides (AFPs) which have been designated RD1, RD2 and RD3, and at least four minor ones. RD1 and RD2 both are 64 residues with a molecular weight (M.W.) of about 7000 Daltons. RD3 however appears to be twice as large with a M.W. of 14,000 Daltons. The primary structure of the size-variant RD3 was determined. The complete peptide sequence of RD3 was found to consist of two peptide sequences very similar to those of RD1 and RD2 joined head-to-tail by a 9-amino-acid connecting sequence that is not present in any characterized eel pout AFPs. In order to understand the genomic basis of the molecular heterogeneity and of the constant high level of AFPs present in this fish phage clones containing AFP genes were isolated from the R. dearborni genomic library. One of these clones was shown to contain two genes encoding RD2. The other clone (clone 1) was found to contain two AFP genes, one of which encodes RD2 as well. Based on restriction mapping analyses of the two genomic clones, the three RD2 genes appear to be arranged in direct tandem repeats of about 8 kbp. The other AFP gene in the clone 1 appears to be a partial "RD3" gene that contains six exons. The first exon and one from the other next four exons encode a 70-amino-acid sequence similar to that of the N-terminal half plus the 9-residue connecting sequence of RD3. The last exon encodes a 61-amino-acid sequence similar to that of the C-terminal half of RD3. The possibility exists that this "RD3" gene encodes "RD3" in the form of a polyprotein. Southern analysis of genomic DNA not only indicating the presence of multiple AFP genes but also the occurrence of AFP genes in regular 8 kbp intervals, which is in agreement with the results of restriction mapping and sequence analyses showing the presence of RD2 genes in 8 kbp tandem repeats. Northern analysis showed three distinctive bands of approximately 3 kb, 1 kb and 0.8 kb, confirming that the AFPs are encoded by a multigene family.