CARBOHYDRATE-BINDING PROTEIN 35

碳水化合物结合蛋白 35

• 批准号: 2179500
• 负责人: JOHN L WANG
• 金额: $ 12.38万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-04-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179500
• 关键词: RNA splicing; binding proteins; galactose; genetic transcription; glycolipids; glycoproteins; growth media; laboratory rabbit; lectin; molecular cloning; nucleic acid sequence; ribonucleoproteins; tissue /cell culture; transfection; yeasts

This project proposes to continue our studies on Carbohydrate Binding Protein 35 (CBP35), a predominantly intracellular lectin that binds to galactose-containing glycoconjugates. The polypeptide (M/T about 35,000) consists of two domains: a proline- and glycine-rich domain at the NH2- terminal half and a carbohydrate recognition domain at the COOH-terminal half, with extensive homology to other S-type lectins. The addition of serum growth factors to quiescent cultures of mouse 3T3 fibroblasts increased the expression of CBP35, in terms of elevated transcription rate of the gene, increased accumulation of mRNA, and increased amount of the protein that is translocated to the nucleus. Analysis of CBP35 in the cytoplasm and nucleoplasm suggests that the lectin is associated with a ribonucleoprotein complex, as indicated by its position of sedimentation on sucrose and cesium sulfate gradients. Using a cell-free assay for the splicing of intervening sequences from pre-mRNA, we found that saccharides and antibodies that bind to CBP35 perturb the splicing reaction, whereas control reagents failed to yield the same effort. On the basis of these results, the specific aim of the proposed research includes: (1) to determine the effect on cell-free splicing activity when a nuclear extract is depleted of CBP35 and to attempt to reconstitute the system by readdition of CBP35; (2) to transfect cells with CBP35 cDNA in the sense and antisense orientations and to monitor the growth and the status of mRNA in these cells; (3) to identify the RNA and protein components of the CBP35- ribonucleoprotein complex; and (4) to search for a homolog of CBP35 in yeasts and to study the in vivo consequences of a gene knockout via integrative disruption.

本项目建议继续我们对碳水化合物结合的研究。 蛋白35(CBP35)，一种主要在细胞内结合的凝集素 含半乳糖的糖共轭化合物。多肽(M/T约35,000) 由两个结构域组成：在NH2-上有一个富含脯氨酸和甘氨酸的结构域。 末端半部分和COOH-末端的碳水化合物识别结构域 一半，与其他S类凝集素具有广泛的同源性。增加了 血清生长因子对小鼠3T3成纤维细胞静止培养的影响 通过提高转录水平，增加CBP35的表达 基因的速率、mRNA的积累和数量的增加 转移到细胞核的蛋白质。对CBP35的分析 在细胞质和核质中表明凝集素与 与核糖核蛋白复合体，如其位置所示 蔗糖和硫酸铯梯度上的沉淀。使用无单元格 对来自前-信使核糖核酸的干涉序列的剪接检测，我们发现 与CBP35结合的糖类和抗体干扰了剪接 反应，而对照试剂没有产生同样的努力。在……上面 在这些研究成果的基础上，提出研究的具体目的 包括： (1)为了确定当细胞外剪接活性 核提取液中CBP35耗尽，并试图重建 该系统由CBP35重新解读而成； (2)将CBP35正、反义基因导入细胞 定位，并监测细胞的生长和mRNA的状态 这些细胞； (3)鉴定CBP35的RNA和蛋白质组分- 核糖核蛋白复合体；以及 (4)在酵母中寻找CBP35的同源物，并进行体内研究。 通过整合破坏进行基因敲除的后果。