MOLECULAR GENETIC BASIC FOR HLA POLYMOPHISM

这项建议的目的是研究分子遗传学 人类白细胞抗原II类广泛多态的基础 组织相容性抗原。因为这些基因产物表达在 EB病毒转化的B淋巴母细胞系(B-LCL)， 方法将是确定克隆的cDNA的核苷酸序列 由B-LCL的RNA构建。因为基因具有广泛的多态 在13岁以上人群中观察到的人类白细胞抗原II类决定因素 血清学可检测到的HLA-DR单倍型，将有必要 对每个超型DR特异性的几个基因进行测序(例如，几个DR1 单倍型、几种DR2单倍型等)。来自以下个人的B-LCL 由于来自第一代的后代，导致人类白细胞抗原纯合子 表亲婚姻将从合作实验室获得 作为正在进行的第十届世界经济论坛筹备工作的一部分 国际组织相容性研讨会。来自20个国家的cDNA文库 代表性的B-LCL将在Lambdagt 10中准备。图书馆将 用人类白细胞抗原II类DNA探针进行筛选，适当克隆的基因将 为核苷酸测序做好准备。克隆将分发到 参与实验室将其插入到载体pUC12中。这个 克隆人将在1986年秋季晚些时候推出。按顺序排列 克隆人将由参与此次活动的调查人员进行 第十届国际组织相容性研讨会的组成部分。每个人 实验室将负责将插入片段亚克隆到 M13。预计这一关于cdna核苷酸的集体努力 人类白细胞抗原II类基因的测序将大大有助于 基因和等位基因特异性核苷酸序列的鉴定。

The objective for this proposal is to investigate the molecular genetic basis for the extensive polymorphism which exists for the HLA Class II histocompatibility antigens. Since these gene products are expressed on Epstein-Barr virus transformed B-lymphoblastoid cell lines (B-LCL), the approach will be to determine the nucleotide sequence of cloned cDNAs constructed from RNA of B-LCL. Because of the extensive polymorphism of the HLA-Class II determinants observed in the population with at least 13 serologically detectable HLA-DR haplotypes, it will be necessary to sequence several genes for each supertypic DR specificity (e.g. several DR1 haplotypes, several DR2 haplotypes, etc.). B-LCL from individuals who are homozygous for HLA as a result of being derived from offspring of first cousin marriages will be obtained from collaborating laboratories throughout the world as part of the ongoing preparations for the Xth International Histocompatibility Workshop. cDNA libraries from 20 representative B-LCL will be prepared in Lambdagt 10. The libraries will be screened with HLA Class II DNA probes and appropriate cloned genes will be prepared for nucleotide sequencing. The clones will be distributed to participating laboratories as inserts in the plasmid vector, pUC12. The clones will be made available during late Fall of 1986. The sequencing of the clones will be performed by the investigators who participate in this component of the Xth International Histocompatibility Workshop. Each laboratory will be responsible for subcloning the insert fragment into M13. It is anticipated that this collective effort on cDNA nucleotide sequencing of HLA class II genes will contribute substantially to the identification of gene and allele specific nucleotide sequences.