细胞功能测定

Mammalian target of rapamycin (mTOR) is a giant protein kinase that controls cell proliferation, growth, and metabolism. mTOR is regulated by nutrient availability, by mitogens, and by stress, and operates through two independently regulated hetero-oligomeric complexes. We have a ...

mTOR, the mammalian target of rapamycin, regulates protein synthesis (mRNA translation) by affecting the phosphorylation or activity of several translation factors. Here, we describe methods for studying the impact of mTOR signalling on protein synthesis, using inhibitors of mTOR ...

Specific and sensitive cellular markers are necessary for the detection and quantitative analysis of apoptosis. Identification of apoptotic cells by specific markers in histological sections is especially important for heterogenous cell populations, such as occurs in normal ...

The presence of DNA strand breaks resulting from the cleavage of nuclear DNA by the apoptosis-associated endonuclease(s) is one of the most characteristic features of apoptotic cells (1,2). A widely used methodology to detect apoptotic cells thus relies on labeling DNA strand breaks in situ e ...

A characteristic feature of apoptosis is activation of an endonuclease(s), which has preference for internucleosomal DNA (reviewed in 1–5). As a result, the products of DNA cleavage during apoptosis are discontinuous DNA sections of mono- and oligonucleosome size, which generate a typi ...

p53 protein is a key regulatory component of a stress-inducible cell-cycle checkpoint pathway in mammalian cells, which can promote either cell-growth arrest or apoptosis, depending on the type of cell and damaging agent utilized. Environmental insults that can activate the p53 pathway ...

The use of ionizing radiation as a therapeutic agent has been recognized for almost a century, and continues to be widely used for the treatment and palliation of many human cancers. Ionizing radiation can also be mutagenic or lethal to individual cells, thus a critical balance must be achieved when ...

It has been a decade since proliferating cell nuclear antigen (PCNA) was found to be essential for DNA replication as an auxiliary protein of DNA polymerase δ (1,2; reviewed in 3,4). There is compelling evidence that a homotrimer of PCNA forms a toroidal clamp around duplex DNA (5–7), an association cat ...

The genotoxic consequences of DNA damage in living organisms include short-term genetic instability and programmed cell death, as well as long-term inheritance of mutations and somatically acquired cancer. To respond to such constant genotoxic insults, living creatures from vir ...

Inhibition of DNA replication in eukaryotic cells was one of the earliest effects of radiation to be reported and quantitated. The elucidation of the mechanism of this inhibition has been the focus of research in several laboratories for four decades. A significant development in recent yea ...

In mammalian cells, the rate of DNA synthesis decreases after X-ray exposure. The dose-response curve indicates a biphasic kinetics of inhibition (see Fig. 1). The initial, steep component of the curve represents inhibition of initiation of new replicons, whereas the shallow component is a m ...

Early Drosophila development begins as a syncytium with 13 rapid nuclear divisions without cytokinesis (1). These divisions provide an excellent system in which to study the effects of DNA damage on the mitotic cycle. Because the effects of ionizing radiation can be assessed in both wild-type ...

The organization of eukaryotic DNA is increasingly well understood at each extreme of chromatin organization. Advances in understanding the organization of DNA at the level of nucleosome and chromosome have outstripped knowledge of the intermediate states and function of DNA. Most w ...

Gene targeting, defined as homologous recombination or genetic exchange between an introduced DNA sequence and its endogenous chromosomal locus, or “target,” is a powerful approach for genetic manipulation. Gene-targeting strategies for both yeast (1) and mammalian cells (2–4) ha ...

DNA double-strand break (DSB) repair in mammalian cells has been demonstrated to be complex, involving both homologous and nonhomologous processes. Although manipulation of chromosomal DSBs and analysis of their repair are possible (1; see Chapters 37–39), this is usually time-cons ...

A large number of studies suggests that double-strand breaks (DSBs) induced in DNA by ionizing radiation or chemical agents are critical lesions, which if unrepaired or misrepaired may kill a cell, or cause its transformation to a cancer cell. Cells have developed efficient repair mechanis ...

The introduction of restriction endonucleases into mammalian cells in culture provides a unique method for introducing double-strand breaks (DSBs) into the DNA of the host cell. Restriction enzymes recognize, bind, and cleave specific DNA sequences to produce a DNA DSB in the absence of ot ...

Until recently, investigators interested in analyzing the repair of chromosomal double-strand breaks (DSBs) in mammalian cells have been limited by the inability to introduce defined DSBs within the genome. Traditional methods of introducing breaks have included irradiation or ...

Double strand-breaks (DSBs) are key intermediates in DNA recombination reactions. The possibility of inducing DSBs at specific sites in the genome by the expression of rare-cutting endonucleases has resulted in a tremendous increase in our knowledge on the mechanisms of DSB repair, esp ...

Generation of double-strand breaks (DSBs) in chromosomal DNA induces repair machinery of a cell, and is also a necessary step for recombination events. A system for the directed introduction of DSBs into a genome could substantially facilitate progress in understanding DSB repair mecha ...