Cloning and purification of the rainbow trout fifth component of complement (C5)

Dev Comp Immunol. Jun-Jul 2001;25(5-6):419-30. doi: 10.1016/s0145-305x(01)00010-6.

Abstract

To gain further insight into the evolutionary history of the complement proteins C3, C4, and C5 we have now cloned the fifth component of complement from a rainbow trout (Oncorhynchus mykiss) liver cDNA library; this is the first report of C5 cloning in a species other than human and mouse. The deduced amino acid sequence of a partial cDNA clone (2.25kb), representing approximately 44% of the coding sequence, showed 60 and 58% similarity to human and mouse C5, respectively. To validate the molecular information derived from the cloning we developed an improved purification protocol. Mass spectrometric analysis of C5 tryptic digests yielded peptide signals that matched theoretical protein sequence derived from the partial cDNA. Northern blot analysis of RNA from various tissues showed the presence of a single mRNA transcript in trout liver and Southern blot analysis indicated that the gene coding for C5 is present as a single copy in the trout genome. The presence of C5 in trout suggests that C3, C4, and C5 must have diverged before the appearance of teleost fish.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Blotting, Northern
  • Blotting, Southern
  • Cloning, Molecular
  • Complement C3 / genetics
  • Complement C4 / genetics
  • Complement C5 / classification
  • Complement C5 / genetics*
  • Complement C5 / immunology
  • DNA, Complementary
  • Humans
  • Molecular Sequence Data
  • Oncorhynchus mykiss / genetics*
  • Oncorhynchus mykiss / immunology
  • Sequence Analysis, DNA
  • Sequence Homology, Amino Acid
  • alpha-Macroglobulins / genetics

Substances

  • Complement C3
  • Complement C4
  • Complement C5
  • DNA, Complementary
  • alpha-Macroglobulins

Associated data

  • GENBANK/AF349001