IDENTIFICATION OF NOVEL METASTASIS ASSOCIATED GENE PRODUCTS

我们已经使用合成的寡核苷酸探针来筛选人类黑色素瘤 cDNA文库含有与包含在DNA中的序列相似的序列的探针 转移相关金属蛋白酶用于筛选该文库。 筛选产生了几个cDNA克隆。其中两个克隆人已经完全 测序 第一个克隆，称为C1114.12，含有1.7 kB插入片段。的 该克隆的测序显示，它含有一个新的序列， 编码独特肽序列的单个开放阅读框。引物 扩展数据与表示全长的此克隆一致 留言该北方印迹分析表明，信息大小取决于 数量级为1.9-2.0 kB，这是一个相对较低的转录本， 丰饶。北方印迹分析还显示，这一信息更多的是 与转录水平相比， 在结肠肿瘤中可见。这表明C1114.12转录本可能 水平在肿瘤细胞中下调。这种可能性正在 进一步探索。 第二个克隆被称为pM 5。该克隆含有一个4086 bp的 插入物包含单个开放阅读帧，其编码 1144个氨基酸残基的多肽。计算机同源性搜索 显示与任何已知序列都没有同源性的蛋白质和 核苷酸水平。这表明pM 5是一种独特的基因产物。 来自pM 5蛋白质序列的特异性亲水结构域已经被发现。 选择用于产生抗肽抗体。这些将用于 确定pM 5基因产物的亚细胞定位， 用免疫组织化学技术确定组织定位。

We have used synthetic oligonucleotide probes to screen a human melanoma cDNA library. Probes containing sequences similar to those contained in metastasis associated metalloproteinases were used to screen this library. Screening yielded several cDNA clones. Two of these clones have been fully sequenced. The first clone, referred to as C1114.12, contains a 1.7 kB insert. The sequencing of this clone revealed that it contained a novel sequence with a single open reading frame encoding for a unique peptide sequence. Primer extension data are consistent with this clone representing a full-length message. Northern blot analysis suggests that the message size is on the order of 1.9-2.0 kB and that this is a transcript of relatively low abundance. Northern blot analysis also revealed that this message was more abundant in normal mucosal tissue when compared with the transcript levels seen in colon tumors. This suggests that possibly the C1114.12 transcript levels are down regulated in tumor cells. This possibility is being explored further. The second clone is referred to as pM5. This clone contained a 4086 bp insert which contained a single open reading frame which encoded a polypeptide of 1144 amino acid residues. Computerized homology searches revealed no homology with any known sequences both at the protein and at the nucleotide level. This suggests that pM5 is a unique gene product. Specific hydrophilic domains from the pM5 protein sequences have been selected for the generation of antipeptide antibodies. These will be used to determine the subcellular localization of the pM5 gene product and to determine the tissue localization using immunohistochemical techniques.