Conserved nucleotide sequences at the 5' end of T cell receptor variable genes facilitate polymerase chain reaction amplification.
Alignment of all available nucleotide sequences of mouse and rat alpha/beta T cell receptor (TcR) variable (V) regions revealed the presence of relatively conserved sequences at the 5' end of the V gene segments. Based on these conserved sequences, degenerate primers were developed for use in the polymerase chain reaction (PCR). The degenerate primers developed on the basis of the conserved sequences at the 5' end of rat and mouse V gene segments are expected to enable the amplification of all mouse and rat TcR alpha/beta chain V regions. To test their applicability, the primers were used for the amplification of the V region of the TcR alpha/beta expressed by rat T cell lines. After amplification, the TcR V regions expressed were cloned and sequenced. The Z1a T cell line was shown to use the same TcR V gene segments (V alpha 2 and V beta 8.2), as most other experimental allergic encephalomyelitis associated T cell lines, but had different D and J segments. In spite of these differences at the nucleotide level, a remarkable conservation of the amino acid sequence at the V beta D beta J beta junction was found. Alignment of a large number of human V alpha and V beta gene segments revealed the presence of similarly conserved sequences. Degenerate primers based on these conserved sequences enabled the amplification of TcR V regions of human T cell lines.
Broeren
C P
CP
Institute of Infectious Diseases and Immunology, School of Veterinary Medicine, University of Utrecht, The Netherlands.
Verjans
G M
GM
Van Eden
W
W
Kusters
J G
JG
Lenstra
J A
JA
Logtenberg
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D009841:Oligonucleotides; D011948:Receptors, Antigen, T-Cell