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.

Bioinformatic Characterization of Genes and Proteins Involved in Blood Clotting in Lampreys

“…In this regard, it was disconcerting to read in the report covering the 2012 assembly (Smith et al 2013) that the sea lamprey had lost clotting genes, in contrast to our earlier (and current) findings that several clotting genes had never been there to lose (Doolittle 2009(Doolittle , 2012. It was also dispiriting to read of the discovery that sea lamprey codon usage is greatly biased in favor of G and C at the third position, an observation reported three decades ago (Strong et al 1985;Bohonus et al 1986;Pontes et al 1988, inter alia) and rediscovered in 2011 (Qiu et al 2011). …”

Crystal structure of a recombinant α E C domain from human fibrinogen-420

“…In addition to all the other known ␣ E C domains in mammals and chicken and the equivalent domain in lamprey, putative carbohydrate-binding sites occur at this position in the Drosophila scabrous protein (27), in the lamprey ␥ chain (28), and in the mammalian fibrinogen-related protein known as PT49 (29,30). …”

Characterization, primary structure, and evolution of lamprey plasma albumin

“…Blood plasma was collected streamside, frozen, and processed as described previously (Cottrell & Doolittle, 1976). Lamprey livers were excised on location and immediately frozen in liquid nitrogen (Strong et al, 1985). Restriction endonucleases and all other enzymes were purchased from Bethesda Research Laboratories (BRL), Grand Island, New York.…”