| Polysome Analysis
|
|
Author:
Date:
2017-03-20
[Abstract] Polysome analysis is a method to separate mRNAs from a cell into actively translating and non-translating fractions depending on their association with polysomes. By this protocol, cell lysates are fractionated by sucrose density gradient ultracentrifugation. Free mRNA fraction and various ribosomal fractions, such as 40S, 60S, monosomes and polysomes are collected by fractionation. Association of particular mRNAs with these fractions is detected by reverse transcription – PCR to investigate the translational state of the mRNA.
[摘要] 多聚赖氨酸分析是一种将mRNA从细胞分离为主动翻译和非翻译部分的方法，这取决于它们与多核糖体的关联。通过该方案，通过蔗糖密度梯度超速离心分离细胞裂解物。通过分级收集游离mRNA级分和各种核糖体级分，例如40S，60S，单体和多核糖体。通过逆转录PCR检测特异性mRNA与这些级分的关联，以研究mRNA的翻译状态。
背景 细胞mRNA在任何时间点分布到主动翻译和非翻译池中，并可响应于各种刺激而在这些池之间动态地重新分布。主动翻译的mRNA具有较高数量的与它们相关的核糖体，与mRNA相关的核糖体数量是mRNA的翻译状态的量度。因此，从细胞中分离核糖体时，会在多核糖体组分中发现主要转录的mRNA，而在游离mRNA部分或与40S核糖体亚基相关的非翻译/不良翻译mRNAs。因此，多聚赖氨酸分析是根据其与多核糖体的关联将mRNA从细胞分离为主动翻译和非翻译部分的方法（Ray et al。，2009; Poria等人， >。，2016）。可以通过RT-PCR检测单个mRNA与翻译/非翻译级分的关联，而通过RNA测序或微阵列分析可以鉴定mRNA的整个翻译或非翻译池。
|
|
|
| Purification of Rubisco from Chlamydomonas reinhardtii
|
|
Author:
Date:
2015-12-05
[Abstract] Chlamydomonas reinhardtii is a model organism for chloroplast studies. Besides other convenient features, the feasibility of chloroplast genome transformation distinguishes this unicellular alga as ideal for the manipulation of chloroplastic gene expression aiming biotechnological goals, such as improved biofuel and biomass production. Ribulose 1, 5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39, Rubisco) is the photosynthetic carbon-fixing enzyme which is considered crucial for biomass accumulation in algal cultures. Purification of wild type and site-directed mutants of Rubisco in C. reinhardtii is usually performed to study its catalytic properties and assess the carbon-fixing potential of the strains. In this protocol Rubisco is extracted through sonication of cell ...
[摘要] 衣藻衣原体是叶绿体研究的模式生物体。 除了其他方便的特点，叶绿体基因组转化的可行性区分这种单细胞藻理想的操纵叶绿体基因表达瞄准生物技术目标，如改善生物燃料和生物质生产。 核酮糖1,5-二磷酸羧化酶/加氧酶（EC 4.1.1.39，Rubisco）是光合作用碳固定酶，其被认为对藻类培养物中的生物量累积是至关重要的。 Rubisco的野生型和定点突变体的纯化。 通常进行研究其催化性质并评估菌株的碳固定潜力。 在该方案中，通过细胞沉淀的超声处理提取Rubisco，并通过硫酸铵沉淀，蔗糖梯度离心（Goldwaithe和Bogorad，1975）和阴离子交换层析纯化。
|
|
|
| Polysome Preparation, RNA Isolation and Analysis
|
|
Author:
Date:
2012-11-05
[Abstract] During mRNA translation, 40S and 60S ribosomal subunits bind to target mRNA forming into an 80S complex (monosome). This ribosome moves along the mRNA during translational elongation to facilitate tRNA reading codon, where translation is activated and many monosomes can bind the same mRNA simutaneously, which forms polysomes. Polysomes can be size-fractionated by sucrose density gradient centrifugation. The more specific mRNA in polysomes implies more active translational status of the mRNA.
[摘要] 在mRNA翻译期间，40S和60S核糖体亚基结合靶mRNA形成80S复合物（单体）。 这种核糖体在翻译延长过程中沿着mRNA移动以促进tRNA阅读密码子，其中翻译被激活并且许多单体可以同时结合相同的mRNA，其形成多核糖体。 多聚体可以通过蔗糖密度梯度离心进行大小分级。 多核糖体中更特异的mRNA意味着mRNA的更有活性的翻译状态。
|
|
|