Arthropod hemocyanins. Molecular cloning and sequencing of cDNAs encoding the tarantula hemocyanin subunits a and e

cDNA clones comprising the entire coding region of two out of the seven heterogeneous subunits of hemocyanin from the tarantula, Eurypelma californicum, were isolated from four cDNA libraries constructed from total RNA from the heart tissue of single spiders. Hybridization was first carried out using a tarantula hemocyanin subunit e partial cDNA, and several positive clones were isolated, including one containing a 2.2-kilobase full-length cDNA (lambda M1). The cDNA comprises an open reading frame for 623 amino acids, 34 nucleotides of the 5'noncoding region, and 286 nucleotides of the 3'-noncoding region. To select for other hemocyanin subunits, two 17-mer oligonucleotide mixtures, corresponding to the conserved regions in the copper A and copper B oxygen-binding site of chelicerate hemocyanins, were used as probes. Among the positive clones obtained, full-length cDNAs coding for subunit a were identified. The cDNA sequence determined from clone lambda K1 provides an open reading frame coding for 630 amino acids and includes the 5'- and 3'-noncoding regions. Northern blot analysis revealed single transcripts for subunits a and e, each 2.3 kilobases long. The cDNAs for subunits a and e were both found to lack any leader peptide sequence. This supports the idea that the mature protein accumulates in the cytoplasm and is released by cell rupture.