Advanced

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

Lundwall, Åke LU ; Wetsel, R. A.; Kristensen, T.; Whitehead, A. S.; Woods, D. E.; Ogden, R. C.; Colten, H. R. and Tack, B. F. (1985) In J Biol Chem 260(4). p.12-2108
Abstract
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... (More)
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. (Less)
Please use this url to cite or link to this publication:
author
publishing date
type
Contribution to journal
publication status
published
subject
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
in
J Biol Chem
volume
260
issue
4
pages
12 - 2108
external identifiers
  • Scopus:0021923201
language
English
LU publication?
no
id
03da56a5-823a-40b0-9dd1-78b61cc21dff (old id 3965102)
alternative location
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=2579066
date added to LUP
2013-08-11 18:28:00
date last changed
2016-06-29 08:58:55
@misc{03da56a5-823a-40b0-9dd1-78b61cc21dff,
  abstract     = {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.},
  author       = {Lundwall, Åke and Wetsel, R. A. and Kristensen, T. and Whitehead, A. S. and Woods, D. E. and Ogden, R. C. and Colten, H. R. and Tack, B. F.},
  keyword      = {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},
  language     = {eng},
  number       = {4},
  pages        = {12--2108},
  series       = {J Biol Chem},
  title        = {Isolation and sequence analysis of a cDNA clone encoding the fifth complement component},
  volume       = {260},
  year         = {1985},
}