Hotta H, Ross A H, Huebner K, Isobe M, Wendeborn S, Chao M V, Ricciardi R P, Tsujimoto Y, Croce C M, Koprowski H
Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104.
Cancer Res. 1988 Jun 1;48(11):2955-62.
The melanoma-associated antigen ME491 is expressed strongly during the early stages of tumor progression. The ME491 gene was molecularly cloned by means of DNA-mediated gene transfer followed by screening a lambda genomic library with human repetitive Alu sequences as a probe. The cloned DNA, after transfection into mouse L-cells, generated a protein with characteristics that were indistinguishable in Western blot analysis from the ME491 antigen expressed by human melanoma cells. Repeat-free subfragments of the cloned DNA were used for further studies. By Northern blot analysis, the subfragments detected a single 1.2-kilobase mRNA in the transformants and various human melanoma cell lines. ME491 complementary DNA clones were then obtained by probing a melanoma complementary DNA library with the genomic subfragments. Nucleotide sequence analysis of the cloned complementary DNA indicated that the ME491 antigen consists of 237 amino acids (Mr 25,475) with four transmembrane regions and three putative N-glycosylation sites. No significant structural homology was observed with other proteins thus far reported. We observed that the amounts of mRNA varied greatly with different melanoma cell lines. Southern blot analysis revealed no amplification or rearrangement of the ME491 gene in the human melanoma cell lines tested, including both high and low expressors of this antigen. The ME491 gene has been mapped to chromosome region 12p12----12q13 by somatic cell hybrid analysis and more narrowly localized to 12q12----12q14 by in situ hybridization.
黑色素瘤相关抗原ME491在肿瘤进展的早期阶段强烈表达。通过DNA介导的基因转移，随后以人重复Alu序列为探针筛选λ基因组文库，对ME491基因进行了分子克隆。将克隆的DNA转染到小鼠L细胞后，产生了一种蛋白质，其特性在蛋白质印迹分析中与人黑色素瘤细胞表达的ME491抗原无法区分。克隆DNA的无重复亚片段用于进一步研究。通过Northern印迹分析，这些亚片段在转化体和各种人黑色素瘤细胞系中检测到一条单一的1.2千碱基mRNA。然后用基因组亚片段探测黑色素瘤互补DNA文库，获得了ME491互补DNA克隆。对克隆的互补DNA进行核苷酸序列分析表明，ME491抗原由237个氨基酸（Mr 25,475）组成，有四个跨膜区和三个推定的N-糖基化位点。与迄今报道的其他蛋白质未观察到明显的结构同源性。我们观察到，不同黑色素瘤细胞系中mRNA的量差异很大。Southern印迹分析显示，在所测试的人黑色素瘤细胞系中，包括该抗原的高表达和低表达细胞系，ME491基因均未发生扩增或重排。通过体细胞杂交分析，ME491基因已被定位到染色体区域12p12----12q13，通过原位杂交更精确地定位到12q12----12q14。
