THE CIRCADIAN TIMING SYSTEM IN THE MODEL ORGANISM CHLAMYDOMONAS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们能够确定发出光脉冲的最佳时间。最大的相移可以观察到7小时进入黑暗阶段的12小时的光/12小时的黑暗周期。我们还没有优化光脉冲的持续时间。然而，由于我们正在研究的两种光感受器可能参与我们的模式生物中的光的昼夜节律夹带，因此在光谱的蓝色区域吸收，我们已经测试了蓝光。我们有初步证据表明蓝光确实有效。这与先前报道的基于生物体的特定突变体并在模糊条件下获得的发现相反。植物状隐花色素：我们成功地鉴定了我们的几个转化体与RNA干扰构建体的这种特定的光感受器，因为确实减少了蛋白质的量。他们现在将为实验提供基础，测试这些菌株是否也显示出降低蓝光脉冲相移的能力。类动物隐花色素：我们没有成功克隆这种蛋白质的编码序列，因为我们不能通过PCR扩增它。我们只成功扩增了较短的片段，很可能是由于DNA中GC含量高。由于我们的困难，并且因为蛋白质最有可能是DNA光裂合酶而不是之前建议的感光体，我们放弃了这部分项目。衣原体视蛋白：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受体数量减少的菌株。趋光素：当我们从一位同事那里收到这种感光细胞的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，该问题是由抗体在运输和随后的储存过程中的降解问题引起的。我们的合作者已同意以更安全的方式再寄一份样品给我们。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.