Detection of human T cell receptor cDNAs (alpha, beta, gamma and delta) by ligation of a universal adaptor to variable region.
The study of T cell receptor (TCR) genes has been hampered by their large repertoires and elusive methods for gene amplification. We have developed a new method for amplification of all human TCR genes (alpha, beta, gamma, and delta) with the ligation of a universal adaptor to the leader sequence of variable (V) regions, which permitted effective and reproducible amplification of all four types of TCR genes. cDNA sequencing of TCR-gamma, -delta, -alpha, -beta was carried out in respectively 15, 13, 28, and 26 T cell clones from human peripheral blood T cells using a newly developed universal adaptor and these methods. TCR-gamma V-II (V gamma 9) was a major population, and V-I (V gamma 2 and 3) and V-III (V gamma 10) were next major populations among TCR-gamma subfamilies, and confirmed the previous observations determined using mAbs specific to TCR-gamma. All five clones of TCR-gamma V-II and three of five clones of TCR-gamma V-I subfamilies had in-frame V-N-J junctions. In contrast, sequences from both TCR-gamma V-III (4/4 clones) and V-IV (1/1 clones) subfamilies had intron-like regions that caused out-of-frame cDNA, suggesting that most of TCR-gamma V-III and V-IV in PBL are not functional. V delta 2 was a major population and V delta 1 was a next predominant population among TCR-delta subfamilies, also confirming the previous observations determined using mAbs to TCR-delta. With regards to TCR-alpha and -beta, this new method randomly amplified TCR cDNAs. In addition, the sequences of 5' portions of three TCR-V-alpha and one TCR-V beta were extended. Two new TCR-alpha subfamilies and one new TCR-beta family were also identified. In summary, this new method will provide a scientific tool for understanding structures of the human TCR genes involved in specific immune responses.
Tsuruta
Y
Y
Shionogi Research Laboratories, Shionogi & Co., Ltd., Osaka, Japan.
Iwagami
S
S
Furue
S
S
Teraoka
H
H
Yoshida
T
T
Sakata
T
T
Suzuki
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics*; D016692:Receptors, Antigen, T-Cell, gamma-delta / Q000235:genetics*; D013601:T-Lymphocytes / Q000378:metabolism
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D016693:Receptors, Antigen, T-Cell, alpha-beta; D016692:Receptors, Antigen, T-Cell, gamma-delta; D004247:DNA