GLYCOSYLTRANSFERASE GENE EXPRESSION IN TRANSFORMED CELLS

转化细胞中的糖基转移酶基因表达

• 批准号: 3194272
• 负责人: NEVIS L FREGIEN
• 金额: $ 13.15万
• 依托单位: UNIVERSITY OF MIAMI SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3194272
• 关键词: RNA; carcinogenesis; cell differentiation; complementary DNA; concanavalin A; gene expression; genetic manipulation; genetic promoter element; genetic regulation; glycosyltransferase; membrane activity; molecular cloning; molecular genetics; molecular oncology; nucleic acid sequence; oligosaccharides; oncogenes; oncoproteins; posttranslational modifications; protein sequence; protein structure function; surface antigens; tissue /cell culture

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.

大多数哺乳动物细胞的细胞表面蛋白是翻译后蛋白 修饰以表达支链寡糖。 特色之一 肿瘤转化过程中细胞病理学的变化是 ASN 连接寡糖的支化增加。 此外， 分支模式的这些变化与转移相关 恶性细胞的潜能。 例如，增加 2 倍 [GlcNAcbeta(1,6)Man]序列在成纤维细胞转化时出现 肿瘤病毒或单独由ras癌基因引起。 添加这些分支 通过特定糖基转移酶的连续作用来执行。 特定糖基转移酶的调节和表达。 这 编码这些酶的基因的调节和表达 正常细胞和恶性细胞的特征尚不明确。 更好的 了解这些基因的控制是必要的 细节、它们在调节肿瘤病理学中的作用及其后果 它们引起的细胞表面变化。 该项目的目标是通过分子克隆来识别和分离 编码N-乙酰葡糖胺基转移酶I和V的基因和cDNA是 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体 人类基因组 DNA 介导的转化结合筛选 差异凝集素结合。 我们现在正在克隆人类 DNA 序列 从这些回复体中确定它们是否含有人类 糖基转移酶基因。 我们还将使用补充方法 使用真核表达直接克隆这些基因的 cDNA 向量和瞬态平移选择方案，这将是很多 快点。 这些序列被克隆、表征和鉴定后 测序后，它们将用作探针来研究这些基因的表达 肿瘤转化和体外转化过程中的基因 HL60细胞的分化。 这些探针也将用于确定 突变细胞系病变的分子基础。

项目成果

Isolation, characterization, and expression of a cDNA encoding N-acetylglucosaminyltransferase V.

编码 N-乙酰葡糖胺基转移酶 V 的 cDNA 的分离、表征和表达。

• 发表时间: 1993
• 期刊: The Journal of biological chemistry
• 作者: Shoreibah,M;Perng,GS;Adler,B;Weinstein,J;Basu,R;Cupples,R;Wen,D;Browne,JK;Buckhaults,P;Fregien,N;Pierce,M
• 通讯作者: Pierce,M