cDNA cloning of the Octopus dofleini hemocyanin: sequence of the carboxyl-terminal domain.
A cDNA library was constructed in pUC 19, using poly(A+) RNA purified from Octopus dofleini branchial gland, which is the site of hemocyanin biosynthesis in cephalopods. The library was screened with an oligonucleotide probe derived from a portion of the partially known sequence of the C-terminal domain of Paroctopus dofleini dofleini. The clone with the longest insert--called pHC1--was sequenced and used as a probe for Northern blotting. It hybridized to a 9.5-kb RNA species, which was also visible as a band after ethidium bromide staining. The cDNA insert (approximately 1200 bp) of pHC1 contained an open reading frame of 1071 bp coding for 357 amino acids. In this insert, a region coding for 42 amino acids from the N-terminal end of the C-terminal domain is missing. These were obtained by sequencing a cloned primer extension product. By comparing our sequence with Helix pomatia beta c-hemocyanin unit D, we found 42.9% identical and 11.5% similar residues. One putative copper binding site (site B) was identified by homology to Helix hemocyanin and arthropodan hemocyanin. The location of a second possible site was identified.
Lang
W H
WH
Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D050260:Carbohydrate Metabolism; D003001:Cloning, Molecular; D003300:Copper / Q000378:metabolism; D004247:DNA / Q000235:genetics*; D006372:Helix, Snails / Q000235:genetics; D006433:Hemocyanins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009693:Nucleic Acid Hybridization; D049831:Octopodiformes / Q000235:genetics*; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid
D003300:Copper; D004247:DNA; D006433:Hemocyanins