GLYCOSYLTRANSFERASE GENE EXPRESSION IN TRANSFORMED CELLS

哺乳动物细胞的大多数细胞表面蛋白质都是后天性的。 被修饰以表达支链寡糖。 特征之一 肿瘤转化过程中细胞病理学的变化是 ASN连接的寡糖的分支增加。 此外，委员会认为， 这些分支模式的变化与转移性 潜在的恶性细胞。 例如， [GlcNAc β（1，6）Man]序列在成纤维细胞被转化成纤维细胞时发生。 肿瘤病毒或单独的ras癌基因。 这些分支的加入 是通过特异性糖基转移酶的连续作用进行的。 特异性糖基转移酶的调控和表达。 的 调控和表达的基因编码这些酶在这两个 正常和恶性细胞的特征还不清楚。 更好的 了解这些基因的控制是必要的， 详细说明，它们在调节肿瘤病理学中的作用及其后果 它们所引起的细胞表面变化。 本项目的目标是通过分子克隆技术鉴定和分离 编码N-乙酰葡糖胺基转移酶I和V的基因和cDNA被 用作分析这些基因表达的探针。 我们 描述初步数据，以表明我们可以生成，识别和 使用DNA分离突变体、酶缺陷细胞系的回复突变体， 用人基因组DNA介导的转化结合筛选 差异凝集素结合。 我们正在克隆人类的DNA序列 以确定它们是否含有人类 糖基转移酶基因 我们还将采用一种互补的方法， 利用真核表达直接克隆这些基因的cDNA 矢量和一个短暂的，平移选择方案，这将是很多 快 在这些序列被克隆、表征并 测序后，它们将被用作探针来研究这些基因的表达。 在肿瘤转化期间和在体外转化期间， HL 60细胞的分化。 这些探针也将用于确定 突变细胞系中病变的分子基础。

Most cell surface proteins of mammalian cells are posttranslationally modified to express branched oligosaccharides. One of the characteristic changes in cellular pathology during neoplastic transformation is the increase in the branching of ASN-linked oligosaccharides. Furthermore, these changes in branching patterns have been correlated with metastatic potential of malignant cells. For example, a 2-fold increase in [GlcNAcbeta(1 ,6)Man] sequences occurs when fibroblasts are transformed by tumor viruses or by the ras oncogene alone. The addition of these branches is performed by the sequential action of specific glycosyltransferases. The regulation and expression of the specific glycosyltransferases. the regulation and expression of the genes encoding these enzymes in both normal and malignant cells are not well characterized. A better understanding of the control of these genes is necessary to understand, in detail, their role in regulating oncologic pathology and the consequences of the cell surface changes which they cause. The goals of this project are to identify and isolate by molecular cloning the genes and cDNAs encoding N-acetylglucosaminyltransferases I and V to be used as probes for the analysis of the expression of these genes. We describe preliminary data to show that we can generate, identify and isolate revertants of mutant, enzyme-deficient cell lines using DNA- mediated transformation with human genomic DNA combined with screening by differential lectin binding. We are now cloning the human DNA sequences from these revertants to determine whether they contain the human glycosyltransferase gene. We also will use a complementary approach to directly clone the cDNAs for these genes using eucaryotic expression vectors and a transient, panning selection scheme which will be much faster. After these sequences have been cloned, characterized and sequenced, they will be used as probes to study the expression of these genes during Neoplastic transformation and during the in vitro differentiation of HL60 cells. These probe will also be used to determine the molecular basis of the lesions in the mutant cells lines.