HELIX-LOOP-HELIX PROTEINS IN GROWTH AND DEVELOPMENT

一些构成主要的早期（IE）基因， 已经确定了对生长刺激的基因组反应，包括 新基因HLH 462。 HLH 462编码一种蛋白质， 螺旋-环-螺旋（HLH）结构域类似于在一类 转录调节分子在细胞内重要 分化和胚胎发育。 的假设 HLH 462基因的蛋白质产物在控制 将在四个具体目标中进行测试。 第一、 HLH 462基因和蛋白表达的时空分布 小鼠胚胎将使用原位方法确定 用HLH 462特异性探针进行杂交和免疫组织化学。 第二，将产生和纯化特异性抗体试剂， HLH 462蛋白的生物化学和生物学特性将是 研究了 由于HLH 462基因结构预测， 产品含有HLH结构域，已知其对于 蛋白质-蛋白质相互作用，本提案的第三个目的是 鉴定能够相互作用的其他已知或未知HLH蛋白 HLH 462蛋白质 体外翻译的蛋白质来源于其他 克隆的HLH基因将被测试它们与 纯化的重组HLH 462蛋白，并对它们与重组HLH 462蛋白共表达的能力进行了研究。 用HLH 462抗体免疫沉淀。 蛋白质与 将进一步研究这些试验中的HLH 462。 基因编码 可以通过以下方法克隆可以与HLH 462相互作用的未表征的蛋白质： 以标记的重组HLH 462蛋白为分子探针， 的 本研究的第四个目的是检验HLH 462 蛋白质是培养细胞生长调节所必需的。 正常 这些细胞中HLH 462的表达模式将被打乱， 以测定对生长的影响和对生长刺激的反应。 为了抑制HLH 462的诱导，反义RNA表达构建物 并且将使用反义寡核苷酸。 此外，本发明还提供了一种方法， 将抗HLH 462抗体注射到细胞中以中和 蛋白质的活性。 过表达HLH 462蛋白 赋予成纤维细胞生长优势或劣势将 也可以化验。 预计这些实验将确定 是否需要诱导HLH 462（生长相关基因） for cell细胞growth生长. HLH 462表达位点的确定， 蛋白质性质的表征，蛋白质的鉴定 测定HLH 462对细胞生长的影响 调节对于理解HLH 462在 控制正常和异常的生长发育和疾病状态 这涉及到对这些过程的调节。

A number of immediate-early (IE) genes which constitute the primary genomic response to growth stimuli have been identified, including a novel gene, HLH462. HLH462 encodes a protein containing a helix-loop-helix (HLH) domain similar to that found in a class of transcriptional regulatory molecules important in cellular differentiation and embryonic development. The hypothesis that the protein product of the HLH462 gene plays an important role in the control of growth and development will be tested in four specific aims. First, the temporal and spatial pattern of HLH462 gene and protein expression in mouse embryos will be determined using the methods of in situ hybridization and immunohistochemistry with probes specific for HLH462. Second, specific antibody reagents will be generated and purified, and the biochemical and biological properties of the HLH462 protein will be investigated. Since the HLH462 gene structure predicts that the protein product contains an HLH domain which is known to be important for protein-protein interactions, the third aim of this proposal is to identify other known or unknown HLH proteins which are able to interact with the HLH462 protein. In vitro translated proteins derived from other cloned HLH genes will be tested for their ability to interact with purified recombinant HLH462 protein and for their ability to be co- immunoprecipitated with HLH462 antibodies. Proteins which interact with HLH462 in these assays will be further investigated. Genes encoding uncharacterized proteins which can interact with HLH462 can be cloned by using labeled recombinant HLH462 protein as a molecular probe. The fourth aim of the proposed research is to test the hypothesis that HLH462 protein is necessary for growth regulation in cultured cells. The normal pattern of expression of HLH462 in these cells will be disturbed in order to assay the effects on growth and the response to growth stimulation. To inhibit the induction of HLH462, antisense RNA expression constructs and antisense oligonucleotides will be employed. In addition, anti-HLH462 antibodies will be injected into cells in order to neutralize the activity of the protein. The ability of overexpressed HLH462 protein to confer a growth advantage or disadvantage on fibroblast cells will also be assayed. It is expected that these experiments will determine whether the induction of HLH462 (a growth-associated gene) is necessary for cell growth. The determination of the sites of HLH462 expression, characterization of the protein's properties, identification of protein partners and determination of the effects of HLH462 on cell growth regulation will be important for understanding the role of HLH462 in the control of normal and abnormal growth and development and disease states which involve regulation of these processes.