BIOCHEMICAL STUDIES OF SILICEOUS SKELETAL FORMATION

硅质骨骼形成的生物化学研究

• 批准号: 3267994
• 负责人: BENJAMIN E VOLCANI
• 金额: $ 17.27万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1975
• 资助国家: 美国
• 起止时间: 1975-05-01 至 1991-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3267994
• 关键词: DNA directed DNA polymerase; Escherichia coli; autoradiography; chromosome deletion; complementary DNA; cyclic AMP; electron microscopy; gene expression; genetic manipulation; genetic regulation; genetic transcription; genetically modified plants; marine biology; messenger RNA; molecular cloning; nucleic acid sequence; plant genetics; plasmids; radiotracer; regulatory gene; silicates; yeasts

It is now known that silicon (Si) is an essential trace element in animals for the formation of bone, cartilage, and connective tissue; it participates in female hormonal balance (in rats) and is a pathogenic agent in fibrotic lung diseases and cancers. It is interrelated with calcium and hormones in atherosclerosis and aging, and with aluminum in Alzheimer's disease. Though Si is clearly implicated in a variety of health-related questions, relatively little is known of its mode of action in mammals, due in part to inherent methodological difficulties. Many of these obstacles can be avoided by using the diatom as an experimental system, due to this organism's absolute dependence in Si. In the diatom, Si is required not only for cell wall formatin, but also for DNA synthesis and for net synthesis of cAMP. Si induces a number of mRNAs and proteins, including two nuclear DNA polymerases and thymidylate kinase, whose appearance coincides with DNA synthesis. Si may thus be a major regulator of diatom proliferation at the level of gene expression. The long term objective of our research is to understand how Si affects cellular metabolism in the diatom and other organisms; in particular whether it regulates gene expression through direct interaction with a regulatory molecule or indirectly by altering cellular metabolism. The specific goal of the proposed research is to characterize gene sequences which are responsive, either through direct or indirect effects, to Si. This will be accomplished in three studies, using Cylindrotheca fusiformis. In Study I, - "Determination of the Si-responsive Regulatory Point in Gene Expression" - (1) cDNA libraries representing the distinctive mRNA populations from Si-starved and Si-replenished diatoms will be screened to identify cDNA clones derived from genes with Si-responsive mRNAs; (2) cDNA clones will be identified from particualr genes whose gene products, thymidylate kinase and nuclear DNA polymerases, are Si-responsive; (3) these cDNAs will be sequenced; and (4) hybridization probes derived from these clones will be used to determine the points in gene expression and the point(s) in the cell cycle at which Si-responsive genes are regulated. In Study II, -"Development of a Diatom Transformation System" - a transformation system will be developed to re-introduce Si-responsive genes into the diatom. Based on the foundation laid in Studies I and II, in Study III - "Determination of Diatom Si-responsive Regulatory Sequences" - specific Si-responsive regulatory sequences within Si-responsive genes will be identified, by fusing putative regulatory sequences appropriately to an assayable marker gene, transforming diatoms with these fusions, and determining the response of the marker gene to Si. The isolation of such sequences is an essential step in elucidating the mechanism(s) involved in Si regulation of gene expression and cell proliferation. In view of Si's importance both as a metabolite and as a pathogen, a contribution to understanding its mode of action at the molecular level is most germane to health related research.

现在众所周知，硅（SI）是 动物形成骨骼，软骨和结缔组织 组织;它参与女性荷尔蒙平衡（在大鼠中），是 纤维化肺部疾病和癌症中的致病剂。 这是 在动脉粥样硬化中与钙和激素相互关联 老化，以及阿尔茨海默氏病的铝。 虽然是 明确涉及各种与健康有关的问题， 对哺乳动物中的作用方式相对鲜为人知，在 固有的方法论困难的一部分。 其中许多 可以使用硅藻作为实验，可以避免障碍 系统，由于这种生物在Si中的绝对依赖性。 在 硅藻不仅需要细胞壁格式，而且还需要 DNA合成和CAMP的净合成。 SI诱导a mRNA和蛋白质的数量，包括两个核DNA 聚合酶和胸苷酸激酶，其外观重合 与DNA合成。 因此，SI可能是硅藻的主要调节剂 基因表达水平的增殖。 长期 我们研究的目的是了解SI如何影响细胞 硅藻和其他生物的代谢；尤其 它是否通过直接相互作用调节基因表达 通过调节分子或间接改变细胞 代谢。 拟议研究的具体目标是 表征反应灵敏的基因序列 直接或间接效应，to si。 这将在 三项研究，使用圆锥形叶叶霉菌。 在研究i中， - “基因中的Si响应调节点的确定 表达“ - （1）代表独特性的cDNA库 来自SI饥饿和Si-Replened硅藻的mRNA种群 将筛选以识别从具有的基因得出的cDNA克隆 Si响应性mRNA； （2）将从 特有基因的基因，其基因产物，胸苷激酶和 核DNA聚合酶具有SI响应性； （3）这些cDNA将会 被测序； （4）从中得出的杂交探针 克隆将用于确定基因表达的点和 Si响应基因的细胞周期中的点是 受监管。 在研究II中 - “硅藻的发展 转换系统” - 转换系统将是 开发为将Si响应基因重新引入硅藻。 基于研究I和II的基础，研究III- “确定硅藻响应性调节序列” - SI响应中的特定Si响应性调节序列 将通过融合推定的调节序列来鉴定基因 适当地转化为可分析的标记基因，转化硅藻 使用这些融合，并确定标记的响应 基因到si。 这种序列的隔离是必不可少的一步 阐明基因调节涉及的机制 表达和细胞增殖。 鉴于SI的重要性 作为代谢产物和病原体，对理解的贡献 它在分子水平上的作用方式最为隐理 健康相关研究。