细胞功能测定

The MCF-7 cell line was isolated from a pleural metastasis of a human breast adenocarcinoma, and, when grown on plastic substrates, typically forms a continuous cell monolayer at confluence (1). MCF-7 cell cultures respond to 17β-estradiol (E2) by increases in the expression of a number of genes (( ...

The yeast two-hybrid system is a powerful tool for the isolation and characterization of nuclear receptor interacting proteins such as coactivator and corepressor proteins. Generally coactivators associate with the nuclear receptors in an agonist-dependent manner and this sp ...

The cell cycles of the budding yeast Saccharomyces cerevisiae and the fission yeast, Schizosaccharomyces pombe are currently the best understood of all eukaryotes. Studies in these two evolutionarily divergent organisms have identified common control mechanisms, which have p ...

This chapter reviews the basic features of plant cell proliferation. Although plant cell division seems to be largely similar to animals and fungi, there are a number of peculiarities that are perhaps related to their lifestyle and development. Initial comparisons of animal, fungal, and pl ...

Drosophila melanogaster provides an outstanding experimental system to study the regulation of cell cycle progression during animal development. Sophisticated forward and reverse genetic techniques and the ability to observe detailed cell biological phenomena in vivo ha ...

The nematode C. elegans provides an animal model that allows for genetic dissection of cell cycle regulation in the context of development. Processes such as progression through meiotic prophase, spindle function, chromosome segregation, and cytokinesis are attractive topics to s ...

Oocytes, eggs, and embryos from the frog Xenopus laevis have been an important model system for studying cell cycle regulation for several decades. First, progression through meiosis in the oocyte has been extensively investigated. Oocyte maturation has been shown to involve complex ne ...

Short interfering (si) RNAs are commonly used to knock down expression of proteins in mammalian cells and thereby investigate protein function. siRNAs were originally introduced into mammalian cells by transient transfection of short, synthetic, double-stranded RNA oligo nucl ...

In recent years, we have witnessed major advances in our understanding of the mammalian cell cycle and how it is regulated. Normal mammalian cellular proliferation is tightly regulated at each phase of the cell cycle by the activation and deactivation of a series of proteins that constitute the c ...

A number of different viruses interact with the cell cycle in order to subvert host-cell function and increase the efficiency of virus replication; examples can be found from DNA, retro, and RNA viruses. The majority of studies have been conducted on DNA and retroviruses whose primary site of repl ...

The method described in the following chapter utilizes a double thymidine block (an inhibitor of DNA synthesis) followed by treatment of cells with nocodazole (a mitotic inhibitor) to obtain large cell populations at distinct phases of the cell cycle. Treatment with double thymidine res ...

The determination of structures for proteins that control the eukaryotic cell cycle by nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography has made a significant contribution to our understanding of the molecular mechanisms that control cell cycle progres ...

Methods are described here to map an origin of replication in eukaryotes. Replicating DNA is enriched by BND cellulose column chromatography and by λ-exonuclease digestion; this approach has largely superceded enrichment by BrdU incorporation. The general area in which replication ...

An in situ technique for studying the chromatin binding of proteins in single fission yeast cells (Schizosaccharomyces pombe) is described. Cells are permeabilized by enzymatic digestion and extracted with a detergent-containing buffer. This procedure removes soluble protei ...

The E2F-family of transcripion factors exerts fascinating and contrasting functions in tran-scriptional repression and activation of genes regulating proliferation, apoptosis, and differentiation. E2F is principally regulated by its temporal association with retin ...

Geminin is an unstable protein that inhibits DNA replication by interfering with the assembly of prereplication complex at replication origins. Geminin is thought to prevent a second round of replication during late S- or G2-phase. The protein is destroyed by ubiquitin-dependent prot ...

Certain forkhead (FOX) transcription factors have been shown to play an intrinsic role in controlling cell cycle progression. In particular, the FoxO subclass has been shown to regulate cell cycle entry and exit, whereas the expression and activity of FoxM 1 is important for the correct coupli ...

All cyclin-dependent kinases (CDKs) involved in eukaryotic cell cycle control require phosphorylation at a conserved threonine (or serine) residue within the activation- or T-loop to attain full enzymatic activity. The enzyme responsible for this activating phosphorylation, ...

The cyclin/cyclin-dependent kinase (Cdk) complexes and the Cdk inhibitors (CDKI) are crucial regulators of cell cycle progression in all eukaryotic cells. Using rat cardiac myocytes as a model system, this chapter provides a detailed account of methods that can be employed to measure both c ...

The Wee kinases (Wee1, Wee2, and Myt1) are major regulators of mitotic entry. They function by phosphorylating Cdc2 and related Cdks on conserved tyrosine and threonine residues. This phosphorylation blocks the activity of the Cdc2 and prevents entry into mitosis. The abundance and activi ...