CARBOHYDRATE-BINDING PROTEIN 35

本计画将继续进行碳水化合物结合的研究 蛋白35（CBP 35），一种主要结合至 含半乳糖的糖缀合物。 多肽（M/T约35，000） 由两个结构域组成：位于NH 2-的富含脯氨酸和甘氨酸的结构域， 末端的一半和碳水化合物识别结构域在COOH-末端 一半，与其他S型凝集素具有广泛的同源性。 添加 血清生长因子对小鼠3 T3成纤维细胞静止培养的影响 增加了CBP 35的表达， 基因的速率，mRNA的积累增加， 转移到细胞核中的蛋白质。 CBP 35分析 在细胞质和核质中表明凝集素与 与核糖核蛋白复合物，如其位置所示 在蔗糖和硫酸铯梯度上沉淀。 使用无细胞 通过对前体mRNA间插序列剪接的分析，我们发现 与CBP 35结合的抗体干扰了 反应，而对照试剂未能产生相同的效果。 对 在这些结果的基础上，提出了研究的具体目标 包括以下步骤： (1)为了确定当使用一种 细胞核提取物中的CBP 35被耗尽，并试图重建 系统采用CBP 35; (2)用CBP 35 cDNA的正义和反义转染细胞， 方向，并监测生长和mRNA的状态， 这些细胞; (3)来鉴定CBP 35的RNA和蛋白质成分- 核糖核蛋白复合物;和 (4)在酵母中寻找CBP 35的同源物，并研究其在体内的表达。 通过整合破坏基因敲除的后果。

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.