MOLECULAR BIOLOGY OF ACTIN-BINDING PROTEIN

肌动蛋白结合蛋白(ABP)，有时称为丝胺，是一种交联体 肌动蛋白细丝在皮质细胞质中形成一个正交阵， 参与动态变化的细胞器排除区域 细胞运动性。ABP存在于白细胞、血小板、成纤维细胞、 上皮细胞、两栖虫卵、心肌和平滑肌。 尽管已经有了重要的功能和形态 鉴定，这种蛋白质的一级结构还没有 已经确定了。总部基地由两个明显相同的 270 kDa亚基，每个亚基包含用于自结合的结构域， 肌动蛋白结合和跨膜蛋白结合(血小板GP1b)。 对这种细胞骨架蛋白结构的阐明可能 促进我们对细胞质结构和 能动性。 该项目首先从ABP基因的克隆和测序开始。 克隆是从内皮细胞表达中挑选出来的 用抗ABP的单抗构建文库。限制 ABP cDNA内的位点和序列缺失将被用于 亚克隆到载体上进行测序。因为ABP的基因应该是 大小至少为8.1kb，获得的大多数克隆将不包含 完整的总部基地消息。用DNA探针重新筛选文库 从先前识别的克隆的末端或合成 可能需要特定的引物扩展文库。组织和 ABP的种间分布将通过探测进行调查 用于细胞提取的RNA中的ABP信息。初级阶段 将对蛋白质的结构进行分析以确定其结构 功能域的相关性，并将与 其他肌动蛋白结合蛋白的已知序列。第二阶段 该项目将使用ABP cDNA克隆来进一步表征 功能域结构。具有结构域的单抗 专一性将被用来将结构与功能联系起来。 将编码ABP基因片段的独特构建体介导入 细胞(即，使细胞制造ABP亚单位片段 将与产生非活性二聚体的天然亚基相关联) 可以进一步阐明ABP在体内的作用(例如，自我表达 关联域)。 编码ABP的基因的特征将包括 染色体定位和定位。初步研究： 体细胞杂交系提示ABP基因的定位 X染色体，位于X染色体脆性部位的远端。 染色体。将进行原位杂交研究。 将建立限制片段长度多态以 帮助绘制基因图谱。

Actin-binding protein (ABP), sometimes called filamin, crosslinks actin filaments into an orthogonal array in the cortical cytoplasm, an organelle-excluding area involved in dynamic changes during cell motility. ABP is found in leukocytes, platelets, fibroblasts, epithelial cells, amphibian eggs and cardiac and smooth muscle. Although there has been significant functional and morphologic characterization, the primary structure of this protein has not been determined. ABP is composed of two apparently identical 270 kDa subunits, each containing domains for self-association, actin-binding and transmembrane protein binding (platelet GP1b). The elucidation of the structure of this cytoskeletal protein may facilitate our understanding of cytoplasmic structure and motility. The project begins with the cloning and sequencing of ABP cDNA. Clones are being selected from an endothelial cell expression library using monoclonal antibodies against ABP. Restriction sites within the ABP cDNA and serial deletions will be used to subclone into vectors for sequencing. Since the ABP cDNA should be at least 8.1 kb, most clones obtained will not encompass the entire ABP message. Rescreening the library with DNA probes from the ends of previously identified clones or synthesis of a specific primer-extended library may be required. Tissue and interspecies distribution of ABP will be investigated by probing for ABP message in cellular extracts of RNA. The primary structure of the protein will be analysed to identify structural correlation of functional domains and will be compared with known sequence of other actin binding proteins. Phase two of the project will use ABP cDNA clones to further characterize functional domain structure. Monoclonal antibodies with domain specificity will be used to correlate structure to function. Transfection of unique constructs encoding fragments of ABP into cells (i.e. cause the cell to manufacture ABP subunit pieces that will associate with native subunits generating inactive dimers) may further clarify ABP's role in vivo (e.g. expression of the self- association domain). Characterization of the gene coding for ABP will include chromosomal localization and mapping. Preliminary studies with somatic cell hybrid lines suggest localization of the ABP gene to the X-chromosome, distal to the fragile site of the X- chromosome. In situ hybridization studies will be performed. Restriction fragment length polymorphisms will be established to aid in mapping the gene.