Lamprey fibrinogen .gamma. chain: cloning, cDNA sequencing, and general characterization

Abstract

A cDNA library from lamprey liver was constructed in pBR322 and screened with a synthetic mixed oligonucleotide probe, the sequence of which was based on a partial amino acid sequence of the lamprey fibrinogen gamma chain determined by conventional procedures. Among the positive clones was one containing a 600-base insert that covered the carboxy-terminal third of the chain and another with a 1950-base insert that stretched more than full length. The two inserts were sequenced by the Maxam-Gilbert procedure. The DNA sequencing was corroborated by reference to the amino acid sequences of five cyanogen bromide peptides that compose the carboxy-terminal 130 amino acids, as well as to a number of tryptic peptides from elsewhere in the molecule. The clone with the smaller insert (6G) contained 594 nucleotides (not counting G and C tails), 435 of which are coding and correspond to residues 264-408 of the gamma chain. The remaining 159 nucleotides included the terminator codon followed by a noncoding segment. The larger clone (2E) coded for 408 amino acids that could be readily aligned with the 411-residue human gamma chain. A 24-residue signal peptide adjacent to the proposed amino terminal was also inferred. The amino acid sequence of the fibrinogen gamma chain has been differentially conserved during evolution, the lamprey and human sequences being more than 70% identical in certain key regions but dropping to less than 25% in other sections, including the segment thought to be a part of the "coiled coils". Overall, the resemblance amounts to 50% identity. Of the 10 cysteines found in mammalian chains, 9 are at identical positions, but the tenth, which in mammalian fibrinogens is a part of the interdimeric bridging, is absent in the lamprey.