JACOBSHAGEN POST-DOC/TECHNICIAN SUPPORT

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 我们能够确定发出光脉冲的最佳时间。在12h亮/12h暗周期中，最大相移可观察到7h。我们还没有优化光脉冲的持续时间。然而，由于我们正在调查的两个光感受器可能参与了我们的模式生物中的光的昼夜携带，吸收了光谱的蓝色区域，我们已经测试了蓝光。我们有初步证据表明蓝光确实有效。这与之前报道的发现相反，这些发现是基于这种有机体的特定突变，并在不明确的条件下获得的。植物样隐花色素：我们成功地用针对这种特定感光细胞的RNA干扰结构鉴定了我们的几个转化子，发现它们确实减少了蛋白质的含量。它们现在将为测试这些菌株是否也表现出对蓝光脉冲的相移能力降低的实验提供基础。类动物隐色素：我们没有成功克隆这种蛋白的编码序列，因为我们不能通过聚合酶链式反应扩增它。我们只成功地扩增了较短的片段，很可能是因为DNA的GC含量很高。由于我们的困难，而且因为蛋白质很可能是DNA光解酶，而不是像之前建议的那样是光感受器，我们放弃了这一部分的项目。Chlamyopsin：目前正在进行实验，将一个标记克隆到合作者收到的RNA干扰结构中，以便创建这种光感受器数量减少的菌株。趋光素：当我们测试从同事那里收到的这种光感受器的RNA干扰菌株时，我们没有成功地在蛋白质印迹中检测到这种蛋白质。有一些证据表明，这个问题是由于抗体在运输和随后储存过程中的降解问题引起的。我们的合作者好心地同意以更安全的方式再寄给我们一份样品。

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.