细胞技术

Cell synchronization is often achieved by inhibition of DNA replication. The cells cultured in the presence of such inhibitors as hydroxyurea, aphidicolin, or thymidine become arrested at the entrance to S phase and upon release from the block they synchronously progress through S, G2, and M. We ...

Mammalian cells are amenable to study the regulation of cell cycle progression in vitro by shifting them into the same phase of the cycle. Procedures to arrest cultured cells in specific phases of the cell cycle may be termed in vitro synchronization. The procedure described here was developed for ...

DNA interstrand crosslinks (ICLs) are lesions that covalently link the two strands of DNA. This type of DNA damage represents one of the most complex DNA lesions whose repair mechanisms remain largely unclear. Uncovering proteins involved in the processing of ICLs and understand how they in ...

In yeast like all eukaryotes, microtubules are a crucial element of the mitotic spindle that separates the genetic material during cell division. The assembly status and position of the mitotic spindle, as well as cytoplasmic microtubules, can be monitored easily using indirect immunof ...

Maintenance of genomic integrity is critical for the survival of organisms. Thus, mammalian cells employ a complex DNA damage response that can sense and repair DNA damage. One important aspect of the cellular DNA damage response is the activation of checkpoints that result in cell cycle arre ...

The most critical feature of the cellular response to DNA damage is the ability of the cell to pause and repair the damage so that detrimental mutations will not be passed along to future generations of cells. The cell cycle of mammalian cells is equipped with checkpoints that can prevent cell cycle pro ...

Cell proliferation depends on the timely synthesis and destruction of proteins at specific phases of the cell cycle. Recently it was discovered that the destruction of several key cell cycle regulatory proteins during S phase is coupled directly to DNA replication. These proteins harbor a m ...

The Ataxia telangiectasia-mutated (ATM) and the ATM-Rad3-related (ATR) kinases are master regulators of the DNA damage-signaling pathways that respond to a wide variety of DNA damage. In this chapter, we describe an in vitro biochemical assay to study the activation of ATM and ATR by double-st ...

Attempts to passage through mitosis with unrepaired DNA damage or incompletely replicated DNA leads to genome instability and/or cell death. To prevent this from occurring, an ancient checkpoint (known as the G2 DNA damage checkpoint) that inhibits the activation of the mitotic cyclin- ...

Following acquisition of DNA damage S-phase progression may potentially be affected via multiple mechanisms. For example DNA damage-activated signal transduction pathways negatively regulate the initiation of DNA synthesis at unfired origins of replication, a process term ...

Cell cycle checkpoints are involved in the coordinated response to DNA damage and thus play a key role in maintaining genome integrity. Several model systems have been developed to study the mechanisms and complexity of checkpoint function. Here we describe the application of cell-free ex ...

Cell cycle checkpoints operating through a network of multiple signaling pathways provide a key mechanism for self-defense of cells against DNA damage caused by various endogenous or environmental stresses. In cancer treatment, checkpoints are activated in response to diverse DNA ...

Slowing of replication in response to DNA damage is a universal response to DNA damage during S-phase. Originally discovered to be defective in checkpoint mutant cells in metazoans, this S-phase DNA damage checkpoint response has been extensively studied in yeast. Unlike other checkpoi ...

Using synchronized cells, one can directly measure delay in mitosis brought about by the G2 DNA damage checkpoint in response to exposure to exogenous DNA damaging agents. Scoring mitosis in the fission yeast Schizosaccharomyces pombe is relatively simple. Many techniques exist for sy ...

In response to post-replicative DNA damage, cells activate the G2 DNA damage checkpoint to ensure mitosis is not attempted until the damage has been repaired. This is a common response to a variety of DNA damaging agents, including ionizing radiation and many chemotherapeutic agents used in the ...

The spindle assembly checkpoint (SAC) is a quality control mechanism for overseeing the fidelity of chromosome segregation. By modulating the activity of the anaphase-promoting complex or cyclosome (APC/C), the SAC sets the timing of anaphase-onset by co-ordinating the timely destr ...

Methods are described here to monitor changes in protein level and subcellular localization during the cell cycle progression in the budding yeast Saccharomyces cerevisiae. Cell synchronization is achieved by an α-factor-mediated block-and-release protocol. Cells are colle ...

Drosophila cell lines are valuable tools to study a number of cellular processes, including DNA damage responses and cell cycle checkpoint control. Using an in vitro system instead of a whole organism has two main advantages: it saves time and simple and effective molecular techniques are ava ...

Live-imaging of cells has been an excellent technique to provide us with highly accurate and valuable information about cell cycle checkpoint regulation and DNA damage responses. Early stage Drosophila embryos have several advantages to be studied by live-imaging. Fly embryos are much ...

Under genotoxic stress, activation of cell cycle checkpoint responses leads to cell cycle arrest, which allows cells to repair DNA damage before continuing to cycle. Drosophila larval epithelial sacs, called imaginal discs, are an excellent in vivo model system for studying radiation- ...