JACOBSHAGEN POST-DOC/TECHNICIAN SUPPORT

JACOBSHAGEN 博士后/技术员支持

• 批准号: 8168287
• 负责人: SIGRID JACOBSHAGEN
• 金额: $ 6.45万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168287
• 关键词: Amino Acid Sequence; Animal Model; Animals; Antibodies; Circadian Rhythms; Cloning; Code; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA Photolyase; Funding; Grant; Guanine + Cytosine Composition; Institution; Light; Organism; Peptide Sequence Determination; Phase; Photoreceptors; Physiologic pulse; Plants; Postdoctoral Fellow; Proteins; RNA Interference; Reporting; Research; Research Personnel; Resources; Sampling; Secure; Source; Testing; Time; United States National Institutes of Health; Western Blotting; base; cryptochrome; light entrainment; mutant; phototropin; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. We were able to determine the optimal time of giving a light pulse. Greatest phase shifts can be observed 7 h into the dark phase of a 12 h light/12 h dark cycle. We have not optimized the duration of the light pulse yet. However, since two of the photoreceptors we are investigating for their possible involvement in circadian entrainment by light in our model organism absorb in the blue region of the light spectrum, we already tested blue light. We have preliminary evidence that blue light is indeed effective. This is contrary to previously reported findings that were based on a particular mutant of the organism and obtained under ambiguous conditions. Plant-like cryptochrome: We succeeded in identifying several of our transformants with the RNA interference construct for this particular photoreceptor as having indeed reduced amounts of the protein. They will now provide the basis for experiments that test, whether these strains also show reduced abilities to phase shift upon blue light pulses. Animal-like cryptochrome: We did not succeed in cloning the coding sequence for this protein, because we were not able to amplify it by PCR. We only succeeded in amplifying shorter fragments, most likely due to the high GC content of the DNA. Because of our difficulties, and because the protein is most likely a DNA photolyase rather than a photoreceptor as previously suggested, we have abandoned this part of the project. Chlamyopsin: Experiments are currently underway to clone a marker into an RNA interference construct received by a collaborator, so that strains with reduced amounts of this photoreceptor can be created. Phototropin: When testing RNA interference strains for this photoreceptor that we received from a colleague, we did not succeed in detecting the protein in western blots. There is some evidence that the problem arises from degradation problems with the antibodies during shipment and subsequent storage. Our collaborator has kindly agreed to send us another sample in a more secure fashion.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 我们能够确定发出光脉冲的最佳时间。在 12 小时光照/12 小时黑暗循环的黑暗阶段 7 小时可以观察到最大的相移。我们尚未优化光脉冲的持续时间。然而，由于我们正在研究的两个光感受器可能参与我们模型生物中光的昼夜节律夹带，吸收光谱的蓝色区域，因此我们已经测试了蓝光。我们有初步证据表明蓝光确实有效。这与之前报道的基于该生物体的特定突变体并在模糊条件下获得的发现相反。植物样隐花色素：我们成功地鉴定了几个具有针对这种特定光感受器的RNA干扰构建体的转化体，其蛋白质含量确实减少了。他们现在将为测试这些菌株是否也表现出蓝光脉冲相移能力降低的实验提供基础。动物样隐花色素：我们未能成功克隆该蛋白质的编码序列，因为我们无法通过 PCR 对其进行扩增。我们只成功扩增了较短的片段，很可能是由于 DNA 的 GC 含量较高。由于我们的困难，并且因为该蛋白质很可能是 DNA 光裂合酶而不是之前建议的光感受器，所以我们放弃了该项目的这一部分。衣藻蛋白：目前正在进行实验，将标记克隆到合作者收到的 RNA 干扰构建体中，以便可以创建这种光感受器数量减少的菌株。向光素：当我们测试同事提供的这种光感受器的 RNA 干扰菌株时，我们没有成功地在蛋白质印迹中检测到该蛋白质。有一些证据表明，该问题是由抗体在运输和随后储存过程中的降解问题引起的。我们的合作者善意地同意以更安全的方式向我们发送另一个样本。