Schuchman E H, Astrin K H, Aula P, Desnick R J
Proc Natl Acad Sci U S A. 1984 Feb;81(4):1169-73. doi: 10.1073/pnas.81.4.1169.
The structural gene encoding human alpha-L-iduronidase has been assigned to chromosome 22 by using immunologic, electrophoretic, and somatic cell hybridization techniques. Polyclonal rabbit antibodies raised against purified human low-uptake alpha-L-iduronidase were used to discriminate the human and murine isozymes by a sensitive immuno-precipitation assay. The human chromosome constitution of each clone was determined by cytogenetic and enzyme marker electrophoretic techniques. In 65 human (fibroblast)-mouse (RAG) somatic cell hybrids (from four independent fusions), the expression of human alpha-L-iduronidase was 100% concordant with the presence of human chromosome 22; the assignment was confirmed by the demonstration of the human enzyme in tertiary somatic cell hybrids containing only chromosome 22. Further verification of the gene assignment was made by detection of the human enzyme in tertiary chromosome 22 positive hybrids by Ouchterlony immunodiffusion and rocket immunoelectrophoretic experiments with polyclonal anti-human alpha-L-iduronidase antibodies that were monospecific for the human enzyme. Expression of human enzymatic activity in chromosome 22 positive hybrid lines was markedly reduced; for example, a tertiary hybrid (R-G21-J-15), which contained an average of 1.7 chromosome 22s per cell, only had about 15% of the activity detected in normal diploid fibroblasts. Immunologic studies suggested that the reduced expression was due to abnormal post-translational processing or aggregation (or both) of the human and murine isozymes in these hybrids. Regional assignment of the human structural gene to 22pter----q11 was accomplished by gene dosage studies using diploid human fibroblast lines that were partially monosomic or trisomic for chromosome 22.
通过免疫、电泳和体细胞杂交技术，已将编码人类α-L-艾杜糖醛酸酶的结构基因定位于22号染色体。用针对纯化的人类低摄取α-L-艾杜糖醛酸酶产生的多克隆兔抗体，通过灵敏的免疫沉淀测定法区分人类和鼠类同工酶。通过细胞遗传学和酶标记电泳技术确定每个克隆的人类染色体组成。在65个人（成纤维细胞）-小鼠（RAG）体细胞杂种（来自四次独立融合）中，人类α-L-艾杜糖醛酸酶的表达与人类22号染色体的存在100%一致；通过在仅含22号染色体的第三代体细胞杂种中检测到人类酶，证实了该定位。通过用对人类酶具有单特异性的多克隆抗人类α-L-艾杜糖醛酸酶抗体进行双向免疫扩散和火箭免疫电泳实验，在第三代22号染色体阳性杂种中检测人类酶，进一步验证了基因定位。在22号染色体阳性杂种细胞系中人类酶活性的表达明显降低；例如，一个第三代杂种（R-G21-J-15），每个细胞平均含有1.7条22号染色体，其活性仅为正常二倍体成纤维细胞中检测到的活性的约15%。免疫学研究表明，表达降低是由于这些杂种中人类和鼠类同工酶的异常翻译后加工或聚集（或两者兼有）所致。通过使用对22号染色体部分单体或三体的二倍体人类成纤维细胞系进行基因剂量研究，将人类结构基因定位到22pter----q11区域。
