Willems van Dijk K, Schroeder H W, Perlmutter R M, Milner E C
Virginia Mason Research Center, Seattle, WA 98101.
J Immunol. 1989 Apr 1;142(7):2547-54.
We have used synthetic oligonucleotides corresponding to human VH sequences to analyze sequence variation in human genomic DNA. By using probes 20 to 24 bp long and conditions of hybridization and washing under which identity in 17 to 21 consecutive bp is required for hybridization, it has been possible to dramatically reduce the complexity of hybridization patterns. We have been able to identify unambiguously individual VH elements. Concomitant with the reduction in overall complexity of hybridization patterns has been a marked increase in the variation between hybridization patterns when different individuals are compared. Variation between individuals was detected using probes corresponding to both framework and complementarity determining regions and depended in part on the complexity of the corresponding VH gene family. Probes corresponding to a cDNA clone belonging to the single-member VH6 family, hybridized to a single, invariant, band in all individuals tested. An oligonucleotide probe corresponding to CDR2 of one member of the VH3 family also detected a single, invariant, band in all individuals tested. However, an oligonucleotide probe corresponding to framework region 2 revealed variants of more than 40% of the 22 VH elements it detects. In addition, a panel of 5 oligonucleotide probes corresponding to a second member of the VH3 family revealed variants of 10 of 14 elements detected. The patterns of variation suggest that some VH elements have multiple alleles, whereas some elements are remarkably conserved. The number of variant elements we have detected is evidence that the haplotype arrangement of the human VH locus is probably extremely complex. Importantly, this heterogeneity may contribute directly to disease susceptibility in man.
我们已使用与人类VH序列对应的合成寡核苷酸来分析人类基因组DNA中的序列变异。通过使用20至24个碱基对长的探针，并在杂交和洗涤条件下，杂交需要17至21个连续碱基对的同一性，从而有可能显著降低杂交模式的复杂性。我们已经能够明确识别单个VH元件。与杂交模式总体复杂性的降低相伴的是，当比较不同个体时，杂交模式之间的差异显著增加。使用对应于框架区和互补决定区的探针检测个体间的差异，部分取决于相应VH基因家族的复杂性。对应于属于单成员VH6家族的cDNA克隆的探针，在所有测试个体中杂交到单一的、不变的条带。对应于VH3家族一个成员的CDR2的寡核苷酸探针，在所有测试个体中也检测到单一的、不变的条带。然而，对应于框架区2的寡核苷酸探针在其检测的22个VH元件中，揭示了超过40%的变体。此外，一组对应于VH3家族另一个成员的5个寡核苷酸探针，在检测的14个元件中有10个揭示了变体。变异模式表明，一些VH元件有多个等位基因，而一些元件则非常保守。我们检测到的变异元件数量证明，人类VH基因座的单倍型排列可能极其复杂。重要的是，这种异质性可能直接导致人类的疾病易感性。
