细胞技术

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 ...

Transcriptional feedback loops are at the core of the molecular clockworks. As single clock genes were cloned it was compelling to develop an assay that allowed simple and direct functional testing of putative activators or repressors of transcription. This chapter includes a general de ...

Brain slices prepared from early postnatal rodents can be maintained in culture from many weeks to months. In culture, brain slices retain their original characteristic cytoarchitecture (organotypic) and continue to differentiate and mature in vitro resembling the characteri ...

RNA in situ hybridization is a commonly used technique to achieve spatiotemporal detection of transcripts in tissues. This chapter gives an overview of novel techniques using fluorescent dyes, signal amplification methods, and confocal microscopy in regard to chronobiological ...

The progress in the understanding of the molecular machinery of mammalian circadian clocks, in combination with the well-established role of the hypothalamic suprachiasmatic nucleus (SCN) as a master circadian clock, has provided an invaluable system for the study of the molecular ba ...

Immunocytochemistry (ICC) is a sensitive and powerful method that is used to localize and identify cells containing a particular antigen. This chapter is dedicated to ICC of suprachiasmatic nucleus (SCN) slices. After a brief introduction to the technique, the materials and methods sec ...

The timing of both entry and permanence of core-clock proteins in the nucleus is critical to maintain the correct pace of the clock mechanism. Several such proteins, namely CRYPTOCHROMEs (CRY), PERIODs (PER), and BMAL1, were recently shown to contain nuclear transport signals that facilita ...

The author has developed an ensemble of digital signal analysis techniques applicable to biological time series containing circadian and ultradian periodicities that is of very high resolution and functions well even in the presence of extreme noise and trend. A method for quantifying ...

Arabidopsis thaliana is internationally adopted as the model plant species for molecular genetics. As such a huge range of resources are available for its study. Arabidopsis does not display any obvious circadian rhythms; however, rhythms in gene expression can be readily detected. For t ...

In the filamentous fungus Neurospora crassa the production of asexual spores (conidia) is regulated by its circadian clock. When the fungus is grown on a thin layer of agar medium in long growth tubes (so-called “race tubes”), restricting its growth to one direction only, bright orange bands are cl ...

In the 1970s, the intriguing discovery of autonomous circadian rhythmicity at the behavioral level in Drosophila set the starting point for one of the most remarkably rapid advancements in the understanding of the genetic and molecular bases of a complex behavioral trait. To this end, the des ...

Oxidative damage to cellular macromolecules is believed to underlie the development of many pathological states and aging. The agents responsible for this damage are generally thought to be reactive oxygen species, such as superoxide, hydrogen peroxide, and hydroxyl radical. The ma ...