Wei S, Charmley P, Robinson M A, Concannon P
Virginia Mason Research Center, Seattle, WA 98101.
Immunogenetics. 1994;40(1):27-36. doi: 10.1007/BF00163961.
An assessment of the size of the human TCRBV gene segment repertoire based on the identification of TCRBV gene segments in genomic DNA was undertaken. PCR amplification from cloned and uncloned genomic DNA sources, nucleotide sequencing, Southern blot hybridization, and cosmid cloning were used to identify TCRBV gene segments in multiple unrelated individuals. The key advantages to this approach were: 1) TCRBV gene segments which are expressed only at very low levels in cDNA libraries were still detectable, and 2) it was possible to discriminate between alleles at the same locus vs products of different loci. A total of 63 unique TCRBV gene segments were identified and sequenced. Six of these TCRBV gene segments had not been previously described. Thirty-four cosmid clones containing 51 of the 63 identified TCRBV gene segments were isolated and screened for the presence of additional novel TCRBV subfamily members. These results, obtained by a variety of complementary approaches, indicate that the human TCRBV germline repertoire encodes at least 63 TCRBV gene segments of which 52 are functional. The availability of the majority of these TCRBV gene segments on cosmid clones should facilitate further investigation of germline TCRBV gene segment polymorphism and putative disease associations.
基于在基因组DNA中鉴定TCRBV基因片段，对人类TCRBV基因片段库的大小进行了评估。使用来自克隆和未克隆基因组DNA来源的PCR扩增、核苷酸测序、Southern印迹杂交和黏粒克隆来鉴定多个无关个体中的TCRBV基因片段。该方法的主要优点是：1）在cDNA文库中仅以非常低水平表达的TCRBV基因片段仍然可检测到，并且2）能够区分同一基因座的等位基因与不同基因座的产物。总共鉴定并测序了63个独特的TCRBV基因片段。其中6个TCRBV基因片段以前未被描述过。分离出34个包含63个已鉴定TCRBV基因片段中的51个的黏粒克隆，并筛选是否存在其他新的TCRBV亚家族成员。通过多种互补方法获得的这些结果表明，人类TCRBV种系库编码至少63个TCRBV基因片段，其中52个是有功能的。黏粒克隆上大多数这些TCRBV基因片段的可得性应有助于进一步研究种系TCRBV基因片段多态性和假定的疾病关联。
