Cloning, sequencing and expression of the bovine CD3 epsilon and TCR-zeta chains, two invariant components of the T-cell receptor complex.
CD3 epsilon and the zeta-chain of the bovine T-cell receptor (TCR) are two invariant molecules with an important role in signal transduction via the TCR/CD3 complex. The nucleotide sequence of a bovine CD3 epsilon cDNA clone containing the complete coding sequence was determined and the deduced amino acid (aa) sequence compared to that of other species. The cytoplasmic domains of the different CD3 epsilon clearly show a higher degree of conservation than the extracellular domains. Bovine CD3 epsilon produced in Escherichia coli using different bacterial expression vectors was recognised by antibodies (Ab) directed against the intracytoplasmic domain of human CD3 epsilon. A partial bovine TCR zeta-chain cDNA was generated by the polymerase chain reaction (PCR) using primers that were based on sequences that are conserved between different species; 3' and 5' RACE-PCR were carried out to obtain the complete TCR zeta-chain cDNA sequence. A comparison of the predicted TCR zeta-chain aa sequence reveals that the GDP/GTP-binding motif, which is conserved in other species, shows marked differences in the bovine and ovine TCR zeta-chains. In contrast to CD3 epsilon, the short extracellular domain of the TCR zeta-chain is 100% conserved between the different species and the transmembrane domain also shows a high degree of identity. Ab were raised against the TCR zeta-chain, produced as a glutathione S-transferase fusion protein in E. coli, and were used in Western blot analysis to further characterise TCR zeta-chain expression in T-cells. The regents provide valuable tools for the study of signal transduction pathways in normal and transformed bovine T-cells.
Hagens
G
G
Institute of Animal Pathology, University of Berne, Switzerland.
Galley
Y
Y
Glaser
I
I
Davis
W C
WC
Baldwin
C L
CL
Clevers
H
H
Dobbelaere
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001483:Base Sequence; D017252:CD3 Complex / Q000737:chemistry / Q000235:genetics*; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D017260:Receptor-CD3 Complex, Antigen, T-Cell / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017421:Sequence Analysis; D013601:T-Lymphocytes / Q000166:cytology
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; C099696:CD3epsilon protein, bovine; D018076:DNA, Complementary; D008565:Membrane Proteins; D012333:RNA, Messenger; D017260:Receptor-CD3 Complex, Antigen, T-Cell; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C099698:TCRzeta protein, Bos taurus; C054979:antigen T cell receptor, zeta chain