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Isolation and sequence analysis of a cDNA clone encoding the fifth complement component

På svenska

Author

  • Åke Lundwall
  • R. A. Wetsel
  • T. Kristensen
  • A. S. Whitehead
  • D. E. Woods
  • R. C. Ogden
  • H. R. Colten
  • B. F. Tack

Summary, in English

We have used available protein sequence data for the anaphylatoxin (C5a) portion of the fifth component of human complement (residues 19-25) to synthesize a mixed-sequence oligonucleotide probe. The labeled oligonucleotide was then used to screen a human liver cDNA library, and a single candidate cDNA clone of 1.85 kilobase pairs was isolated. Hybridization of the mixed-sequence probe to the complementary strand of the plasmid insert and sequence analysis by the dideoxy method predicted the expected protein sequence of C5a (positions 1-12), amino-terminal to the anticipated priming site. The sequence obtained further predicted an arginine-rich sequence (RPRR) immediately upstream of the N-terminal threonine of C5a, indicating that the promolecule form of C5 is synthesized with a beta alpha-chain orientation as previously shown for pro-C3 and pro-C4. The C5 cDNA clone was sheared randomly by sonication, subcloned into M13 mp8, and sequenced at random by the dideoxy technique, thereby generating a contiguous sequence of 1703 base pairs. This clone contained coding sequence for the C-terminal 262 amino acid residues of the beta-chain, the entire C5a fragment, and the N-terminal 98 residues of the alpha'-chain. The 3' end of the clone had a polyadenylated tail preceded by a polyadenylation recognition site, a 3'-untranslated region, and base pairs homologous to the human Alu concensus sequence. Comparison of the derived partial human C5 protein sequence with that previously determined for murine C3 and human alpha 2-macroglobulin has indicated regions of pronounced sequence similarity. Examination of cytoplasmic RNA prepared from human liver and the human hepatoma cell line Hep G2 by Northern transfer has indicated a C5 mRNA species of about 5.2 kilobase pairs.

Publishing year

1985

Language

English

Pages

12-2108

Publication/Series

Journal of Biological Chemistry

Volume

260

Issue

4

Document type

Journal article

Publisher

American Society for Biochemistry and Molecular Biology

Topic

  • Medicinal Chemistry

Keywords

  • Humans
  • Deoxyribonucleotides
  • Recombinant/isolation & purification
  • DNA
  • DNA/*isolation & purification
  • Complement C5a
  • Complement C5/*genetics
  • Complement C3
  • Comparative Study
  • Cell Line
  • Hepatocellular/analysis
  • Carcinoma
  • Base Sequence
  • Animals
  • Anaphylatoxins/genetics
  • Adult
  • Amino Acid Sequence
  • Liver/analysis
  • Liver Neoplasms
  • Mice
  • Nucleic Acid Hybridization
  • RNA
  • Messenger/genetics
  • Research Support
  • Non-U.S. Gov't
  • U.S. Gov't
  • P.H.S.
  • alpha-Macroglobulins

Status

Published

ISBN/ISSN/Other

  • ISSN: 0021-9258