细胞功能测定

Autophagy is a cellular homeostasis pathway used to sustain cellular anabolic needs during times of nutrient or energy deprivation. Autophagosomes sequester cytoplasmic constituents, including macromolecules such as long-lived proteins. Upon fusion of autophagosomes w ...

Insects such as the fruit fly Drosophila melanogaster, which fundamentally reorganize their body plan during metamorphosis, make extensive use of autophagy for their normal development and physiology. In the fruit fly, the hepatic/adipose organ known as the fat body accumulates nut ...

Recent studies of the molecular mechanism of autophagy have made available several marker proteins for autophagosomes. These marker proteins allow us to identify autophagic structures easily and accurately by fluorescent microscopy. The most widely used marker for autophagos ...

Autophagy is a physiological process functionally linked to cellular dynamics during starvation, cardiomyopathies, neurodegeneration, cellular immunity, and certain cancers. Although nearly 30 autophagy-related (ATG) genes have been identified and characterized, t ...

In this chapter, we explain different strategies to analyze the extracellular Hedgehog (Hh) morphogen distribution and Hh intracellular trafficking by immunohistochemistry techniques. For this purpose, it has been very useful to have a transgenic fly line that expresses a Hh-green ...

Hedgehog (Hh) family members are secreted proteins that can act at short and long range to direct cell fate decisions during developmental processes. In both Drosophila and vertebrates, the morphogenetic gradient of Hh must be tightly regulated for correct patterning. The posttransla ...

The identification of protein domains required for function is an important means of defining biochemical roles for a polypeptide. Our studies on regulatory proteins that function during the transition between mitosis and meiosis have extensively relied on targeted in vitro mutag ...

Genetic screens have been extraordinarily useful for the identification of protein components that function in a variety of cellular processes. For example, a number of proteins that are necessary for responding to deoxyribonucleic acid (DNA) damage were found in screens for loss-of- ...

The technique described in this chapter—gene targeting in cultured human cancer cells—brings a powerful tool to scientists studying the function of cell cycle control genes (1). This technology allows scientists to knock out genes in cultured human cells in an analogous fashion to the crea ...

The generation and analysis of mutants has had an essential role in the identification of genes involved in cell cycle control (1–3). In this regard, the genetic analysis of mutations has helped reveal the normal function of wild-type gene products as well as provided powerful insights into the in ...

Cell cycle phase-specific regulation of transcription is a major mechanism for the regulation of progress through the eukaryotic cell cycle. Hundreds of genes are known to be cell cycle regulated, including histones, cyclins, transcription factors, and genes for such cycle-specific ...

Fission yeast is a popular model organism for the study of the cell cycle. It grows quickly compared with other eukaryotic species; under normal conditions, a wild-type cell takes about 2.5–3 h to complete a cell cycle. A wild-type S. pombe cell has a rod shape, grows by elongation, and divides by medial fissi ...

The budding yeast Saccharomyces cerevisiae represents the eukaryotic model system in which the checkpoint concepts were initially developed (1). Whereas many arguments can be made in favor of the continued use of S. cerevisiae as a model organism for checkpoint studies, the ease of disting ...

Saccharomyces cerevisiae, the budding yeast, is widely used as a model eukaryote to study a large number of cellular processes including cell cycle regulation (1–4). Extensive genetic research in the last two decades has revealed that the basic mechanism of cell cycle control is highly conse ...

S phase is that period of time in the cell-division cycle during which nuclear chromosomal deoxyribonucleic acid (DNA) is replicated (1,2). The time required for S phase depends on the size of the genome, the organism, and its developmental state. DNA replication requires only 15 to 20 min in budding ye ...

One of the most common uses of flow cytometry is to analyze the cell cycle of mammalian cells. Flow cytometry can measure the deoxyribonucleic acid (DNA) content of individual cells at a rate of several thousand cells per second and thus conveniently reveals the distribution of cells through the ce ...

Understanding the molecular and biochemical basis of cellular functions involved in growth and proliferation requires the investigation of regulatory events that most often occur in a cell cycle phase-dependent fashion. Studies involving cell cycle regulatory mechanisms and ...

When studying cell cycle checkpoints, it is often very useful to have large numbers of cells that are synchronized in various stages of the cell cycle. A variety of methods have been developed to obtain synchronous (or partially synchronous) cells, all of which have some drawbacks. Many cell types t ...

The way cells respond to radiation or chemical exposure that damages deoxyribonucleic acid (DNA) is important because induced lesions left unrepaired, or those that are misrepaired, can lead to mutation, cancer, or lethality. Prokaryotic and eukaryotic cells have evolved mechanisms ...

Omega-3 polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) display beneficial actions in human diseases. The molecular basis for these actions remains of interest. We recently identified novel mediators generated from ω-3 PUFA ...