Isolation and characterization of Limulus C-reactive protein genes

Abstract

Three homologous genes coding for Limulus C-reactive protein (CRP) have been isolated and characterized from a lambda phage EMBL-3 library containing genomic DNA sequences from Limulus amebocytes. The genes have a typical promoter region with a CAAT (nucleotides 50-53) and a TATAA (nucleotides 77-81) box located, respectively, 178 and 149 base pairs 5' upstream from the initiation codon ATG. The polyadenylation site AATAAA is situated within 300 base pairs downstream from the stop codon TAG. Nucleotide sequence analysis reveals a 24-residue signal peptide preceding a coding region of 218 amino acids. Significant differences were found between the genes coding for human and Limulus CRPs. In the human CRP gene there is an intron separating the signal peptide and the coding region. In Limulus this intervening sequence is missing. The Drosophila heat shock consensus sequence CTnGAAnnTTnAG (Simon, J. A., Sutton, C. A., Lobell, R. B., Glaser, R. L., and Lis, J. T. (1985) Cell 40, 805-817), found in the genes of human (Woo, P., Korenberg, J. R., and Whitehead, A. S. (1985) J. Biol. Chem. 260, 13384-13388) and rabbit (Syin, C., Gotschlich, E. C., and Liu, T.-Y. (1986) J. Biol. Chem. 261, 5473-5479) CRP at the 5' end, is not found in the Limulus CRP genes. Whereas a single CRP gene was found in the human, multiple genes were found for the Limulus CRPs. All CRPs exhibit calcium-dependent phosphorylcholine ligand binding properties. The coding regions of the Limulus and human CRP genes share approximately 25% identity and two stretches of highly conserved regions, one of which falls in the region proposed as the phosphorylcholine binding site, while the other site is very similar to the consensus sequence required for calcium binding in calmodulin and related proteins. The nucleotide sequence analysis provides convincing evidence to support the evolutionary relatedness of the human and Limulus CRPs.