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.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 我们能够确定给出轻脉冲的最佳时间。最大的相移可观察到7小时的光线/12小时黑暗周期的黑暗相。我们尚未优化光脉冲的持续时间。但是，由于我们正在研究其中两个感光体是否可能参与光谱蓝色区域中的模型有机体中的昼夜节律夹带，因此我们已经测试了蓝光。我们有初步证据表明蓝光确实是有效的。这与先前报道的发现基于生物体的特定突变体并在模棱两可的条件下获得的发现相反。类似植物的加密色素：我们成功地鉴定出了几种转化物，它使用该特定感光器的RNA干扰构建体确实减少了蛋白质的量。现在，它们将为测试的实验提供基础，这些菌株是否还显示出蓝光脉冲上相移的能力。类似动物样的隐性色素：我们没有成功克隆该蛋白的编码顺序，因为我们无法通过PCR扩大它。我们只能成功放大较短的片段，这很可能是由于DNA的GC含量很高。由于我们的困难，并且由于蛋白质很可能是DNA光溶解酶，而不是先前建议的光感受器，因此我们放弃了该项目的这一部分。衣原体：目前正在进行实验，将标记物克隆到合作者接收的RNA干扰构建体中，因此可以创建减少该光感受器量的菌株。光蛋白：在我们从同事那里收到的这种光感受器的RNA干扰菌株测试时，我们没有成功地检测Western印迹中的蛋白质。有证据表明，该问题是由于抗体和随后存储期间的抗体降解问题引起的。我们的合作者友好同意以更安全的方式向我们发送另一个样本。