Systematic isolation of genes encoding candidate proteins for human melanoma antigens using serial analysis of gene expression (SAGE) and EST database

为了确定优先表达的基因在黑色素细胞/黑色素瘤，我们采用了两种方法;比较基因表达（SAGE）和EST数据库从各种组织的系列分析产生的cDNA图谱。首先使用SAGE评估高度色素性黑素瘤细胞系SKme 123的AcDNA谱。对由10，382个转录物组成的25，997个标签进行测序。将该黑色素瘤SAGE文库与来自正常组织（包括脑、结肠和睾丸、脑和结肠癌）的SAGE数据库进行比较，并且还与黑色素细胞cDNA文库进行比较。通过RT-PCR和北方印迹分析，确定了两个可能编码新的黑素细胞特异性基因的标签，并证实了它们的特异性表达。从SKme 123 λ噬菌体分离这些序列的全长克隆。一个是TRP 2的剪接变体，另一个是功能未知的新基因。在接下来的研究中，通过计算机比较来自不同组织的EST数据库，选择了80个可能的黑素细胞特异性基因，然后使用RT-PCR评估它们的表达。发现7个序列仅在黑素细胞或黑素瘤中表达，并尝试分离全长cDNA。M43是AMI的剪接变体，AMI是先前鉴定的由T细胞识别的黑色素瘤抗原。M44编码一种新的黑素细胞特异性分子。M125是Melastatin 1的剪接变体，Melastatin 1可能是黑色素瘤的肿瘤标志物。M40、M109和M674为黑素细胞特异性新基因的3 '端序列。M216的5 ′上游可能还含有一个新的黑素细胞特异性基因。因此，黑素细胞/黑色素瘤特异性基因可以使用SAGE和EST数据库系统地分离。这些黑素细胞特异性基因可能对黑素细胞生物学的研究以及色素沉着和黑色素瘤的诊断和治疗方法的发展有帮助。

To identify genes preferentially expressed in melanocyte / melanoma, we have applied two methods; comparison of cDNA profiles generated by serial analysis of gene expression (SAGE) and of EST databases from various tissues. AcDNA profile of highly pigmented melanoma cell line SKme123 was first evaluated using SAGE. 25,997 tags consisting of 10,382 transcripts were sequenced. This melanoma SAGE library was compared to SAGE databases from normal tissues including brain, colon and testis, brain and colon cancers, and also compared to the melanocyte cDNA library. Two tags possibly encoding new melanocyte specific genes with relatively high frequency were identified, and their specific expression was confirmed by RT-PCR and Northern blot analysis. Full length clones of these sequences were isolated from the SKme123 λ phage. One was a splicing variant of TRP2; the other was a novel gene with unknown function. In the next study, by comparing EST databases from various tissues using computer, 80 possibly melanocyte specific genes were selected, then, their expression was evaluated using RT-PCR. Seven sequences were found to express only in melanocytes or melanoma, and the isolation of the full length cDNA were attempted. M43 was a splicing variant of AMI, previously identified melanoma antigen recognized by T cells. M44 encoded a novel melanocyte specific molecule. M125 was a splicing variant of Melastatin 1, a possible tumor marker for melanoma. M40, M109 and M674 were found to be a3'-sequence of the novel melanocyte specific gene. M216 may also have a novel melanocyte specific gene at its 5'-upstream. Therefore, melanocyte / melanoma specific genes can be systematically isolated using SAGE and EST databases. These melanocyte specific genes may be useful for research on biology of melanocytes as well as development of diagnostic and therapeutic methods for disorder of pigmentation and melanoma.