Investigations on the nucleic acids coding for the sea anemone toxins, Anthopleurins A and B

Abstract

Anthopleurin B CAP-B) is a 49 amino acid cardiotonic polypeptide produced by the giant green sea anemone, Anthopleura xanthogrammica. Along with AP-B, another polypeptide, anthopleurin A (AP-A), of the same length, and differing only in 7 amino acids, is produced by the animal in higher quantities. The object of this study was to investigate the two peptides at the nucleic acid level. Genomic and cDNA libraries of the animal were constructed in A. replacement vectors. The genomic library was constructed in the Bam HI site of EMBL3, while the cDNA library was constructed in the unique Eco RI site of λ gtl0. Synthetic nucleotides spanning the first 10 amino acids of AP-B and the complement of the codons for the last 10 amino acids of the AP-A peptides were constructed from back translation of the peptides' sequences. The primers were. used to initiate a polymerase chain reaction, using cDNA as the template. A 144-147 base pair PCR product was obtained. The PCR product was purified from the primers and used to probe the genomic DNA and to screen both the genomic and cDNA libraries. Positive clones were isolated from both libraries. A 1.8 kb Bam HI genomic fragment common to 5 of the 14 genomic clones examined, and a 2.8 kb EeoRI genomic fragment common to 3 clones, that hybridized both to the PCR product and the primers were subcloned into M13mp18. The PCR product was cloned into the phagemid vector, pBluescript KSII+. The sequence of a random clone showed some sequence homology with the ambiguous sequence predicted for AP-A and AP-B. The cDNA clones were cut with two enzymes flanking the Eco RI site of the cloning vector and a 480 base pair fragment was subcloned into M13mp18.