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

使用基因表达系列分析 (SAGE) 和 EST 数据库系统分离编码人类黑色素瘤抗原候选蛋白的基因

基本信息

批准号：
12470180
负责人：
KAWAKAMI Yutaka
金额：
$ 9.22万
依托单位：
Keio University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2001
项目状态：
已结题

项目摘要

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.

为了鉴定在黑素细胞 /黑素细胞中最好表达的基因，我们采用了两种方法。通过对各种组织的基因表达（SAGE）和EST数据库产生的cDNA谱的比较。首先使用SAGE评估了高度猪黑色素瘤细胞系SKME123的ACDNA谱。测序由10,382个转录本组成的25,997个标签。将这个黑色素瘤鼠尾草文库与来自脑，结肠和睾丸，脑和结肠癌在内的正常组织的SAGE数据库进行了比较，并将其与黑素细胞cDNA文库进行了比较。确定了两个可能编码具有相对高频的新型黑素细胞特异性基因的标签，并通过RT-PCR和Northern blot分析证实了它们的特定表达。这些序列的全长克隆从SKME123λ噬菌体中分离出来。一个是TRP2的剪接变体；另一个是一个具有未知功能的新基因。在下一项研究中，通过使用计算机比较来自各个时间的EST数据库，选择了80个可能的黑素细胞特定基因，然后使用RT-PCR评估它们的表达。发现仅在黑素细胞或黑素细胞中表达七个序列，并尝试了全长cDNA的分离。 M43是AMI的剪接变体，以前被T细胞识别的黑素细胞抗原。 M44编码了一种新型黑素细胞特异性分子。 M125是Melastatin 1的剪接变体，这是黑色素瘤可能的肿瘤标记。发现M40，M109和M674是新型黑素细胞特异性基因的A3'-序列。 M216在其5'上游也可能有一个新型的黑素细胞特异性基因。因此，可以使用SAGE和EST数据库系统地分离黑色素 /黑素细胞特定基因。这些黑素细胞特异性基因可能有助于研究黑素细胞生物学以及用于色素沉着和黑色素瘤疾病的诊断和治疗方法的开发。