HELIX-LOOP-HELIX PROTEINS IN GROWTH AND DEVELOPMENT

一些即刻早期(IE)基因，它们构成了初级 已经确定了基因组对生长刺激的反应，包括 新基因HLH462。HLH462编码一种含有 螺旋-环-螺旋(HLH)结构域类似于在一类 转录调控分子在细胞中的重要作用 分化和胚胎发育。这一假设认为 HLH462基因蛋白产物在调控中起重要作用 将在四个具体目标中对增长和发展进行测试。第一, HLH462基因和蛋白表达的时空格局 小鼠胚胎将使用原位检测的方法进行检测 用HLH462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，以及 HLH462蛋白的生化和生物学特性将是 调查过了。由于HLH462基因结构预测蛋白质 该产品含有HLH域，该结构域对 蛋白质之间的相互作用，这一提议的第三个目标是 识别其他已知或未知的能够相互作用的HLH蛋白 与HLH462蛋白有关。来自其他来源的体外翻译蛋白 克隆的人促黄体生成素基因将被测试其与 纯化的重组HLH462蛋白及其结合能力的研究 HLH462抗体免疫沉淀。与之相互作用的蛋白质 HLH462在这些检测中将得到进一步的研究。编码基因 可以通过以下方法克隆与HLH462相互作用的未知蛋白质 用标记的重组HLH462蛋白作为分子探针。这个 拟议研究的第四个目标是检验HLH462的假设 蛋白质是培养细胞生长调节所必需的。正常的 HLH462在这些细胞中的表达模式将被有序扰乱 分析其对生长的影响及对生长刺激的反应。 为了抑制HLH462的诱导，构建了反义RNA表达载体 并将使用反义寡核苷酸。此外, 将抗HLH462抗体注射到细胞内以中和 蛋白质的活性。高表达HLH462蛋白的能力 赋予成纤维细胞生长的优势或劣势将 也要做化验。预计这些实验将确定 是否需要HLH462(生长相关基因)的诱导 用于细胞生长。HLH462表达位点的确定， 蛋白质性质的表征、蛋白质的鉴定 HLH462的配对及其对细胞生长影响的测定 监管对于理解HLH462在人类免疫系统中的作用非常重要 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监管。

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.