细胞功能测定

As the molecular mechanism of circadian clocks has reached high complexity, the fungal model system, Neurospora crassa, is increasingly important for clock research. It offers the possibility of extensive biochemical experimentation and thorough description of circadian pro ...

Forward genetics in Drosophila has profoundly affected our understanding of circadian rhythms in this organism and, more generally, in the animal kingdom. Most Drosophila pacemaker genes were discovered through the isolation of gene variants affecting the free-running period of t ...

Ethyl methane sulfonate (EMS) mutagenesis in Arabidopsis is the most widely used mutagenesis technique. EMS has high mutagenicity and low mortality and can be used in any laboratory with a fume hood. The chemical principle of EMS mutagenesis is simple; it is based on the ability of EMS to alkylate gua ...

Yeast two-hybrid screening represents a sensitive in vivo method for the identification and analysis of protein-protein interactions. The principle is based on the ability of a separate DNA-binding domain (DNA-BD) and activation domain (AD) to reconstitute a functional transacti ...

Proteomics is the study of the complete set of proteins encoded by the genome. The study of the proteome involves the investigation of changes in protein abundance, localization, involvement in multiprotein complexes, and detection of different protein isoforms and posttranslatio ...

In filamentous fungi, including the model organism Neurospora crassa, plentiful biological tissue from which RNA can be extracted may be obtained by allowing fungal spores to germinate and form a mycelium in liquid culture. The mycelium constitutes a mosaic of multinuclear, tubular fil ...

In Drosophila, input, pacemaker, and output genes are expressed circadianly. mRNA oscillations contribute largely to these rhythms. Determining RNA levels of circadian genes is thus frequently necessary to understand their regulation, or the effect of mutations and genetic manip ...

Purification of intact RNA is the primary step of many molecular biology techniques, including Northern blotting, RNase protection, quantitative polymerase chain reaction, and microarray assays. RNA extraction is typically conducted using either a phenol-choloroform or a sol ...

This protocol details an RNA preparation for medium-scale, high-purity RNA production from higher plants. It uses hot acid phenol with standard sodium acetate ethanol precipitation and is suitable for producing RNA for both Northern blotting and enzyme-based downstream applicat ...

In Northern analysis the presence of specific RNA transcripts is detected and their quantity can be estimated. RNA is separated using denaturing agarose gel electrophoresis and is subsequently transferred and fixed to a solid support, such as a nitrocellulose filter. When labeled prob ...

Quantitative PCR (qPCR) has entered widespread use with the increasing availability of real-time PCR. By the incorporation of fluorescent dyes in the reaction mixture, increases in amplification products can be monitored throughout the reaction, enabling measurements to be taken ...

This chapter deals with methods of protein extraction from cyanobacterial cells based on work in the circadian model organism Synechococcus elongatus PCC 7942. Some of these techniques have already been used successfully for analysis of circadian rhythms in cyanobacteria, whereas ...

A method is presented for the extraction of total protein from Arabidopsis thaliana tissue. The protocol was designed for the solubilization of a range of proteins and their efficient and quantitative recovery. It is especially compatible with the small quantities of available tissue of ...

In Drosophila, the concentration and phosphorylation levels of several important circadian proteins (e.g., PERIOD, TIMELESS) oscillate on a 24-h basis. A simple and rapid method for extracting proteins from fly heads is presented here. The extracts can immediately be loaded onto an sodium ...

For Western blotting and coimmunoprecipitation (coIP), protein samples must be extracted from tissues. The protocol described in this chapter has been used to extract clock proteins from mammalian tissues as diverse as liver, kidney, and brain. The extraction protocol is mild enough to be u ...

As with most other proteins, clock proteins physically interact with one another. Coimmunoprecipitation (coIP) is the most straightforward technique to study protein-protein interactions in vivo, if antibodies against the proteins of interest are available. To perform coIP, fi ...

Phosphorylation assay is a widespread technique usually necessary for the identification of a specific kinase substrate and/or for the measurement of kinase activity. As an example of the technique, here we describe an assay aimed to test the phosphorylation of the myelin basic protein (M ...

Coimmunoprecipitation (coIP) provides evidence that two or more proteins can be found in the same complex. It can be performed in vitro (employing in vitro transcribed and translated proteins, or proteins expressed in Escherichia coli) or from transfected cells, which assess whether the ...

In mammals, the central circadian pacemaker resides in the hypothalamic suprachiasmatic nucleus (SCN), but circadian oscillators also exist in peripheral tissues. We have used wild-type and cryptochrome (mCry)-deficient mouse embryonic fibroblasts (MEFs) to demonstrate th ...

Circadian rhythms in metabolic, physiological, and behavioral processes are regulated by biological clocks. Many of these rhythmic processes can be measured over many days or weeks using automated recording devices, thus making it possible to precisely calculate period, phase, and a ...