细胞功能测定

Methods of iterative nucleic acid selection and amplification were enabled by the invention of the polymerase chain reaction (PCR). Thus, the ability to amplify as few as a single DNA or RNA molecule made it possible to diversify sequences and to partition the desirable from the undesirable sub ...

A number of genetic assays have recently been developed for detecting RNA-protein interactions, several of which have been applied to screen cDNA or combinatorial libraries for RNA-binding peptides and proteins (1–5) (see Chapters 13 and 15). These methods are useful for understanding t ...

In recent years, interest in RNA-binding proteins (RNA-BPs) and RNA-protein interactions has grown considerably. An important reason for this interest is the increased recognition that RNA-BPs are involved in a wide variety of critical viral and cellular processes including transc ...

Several different methods can be used for detection of interactions between RNA and other molecules (e.g., proteins and other nucleic acids). Among these are: (1) enzymatic assays in which there is a measurable change in the property of the substrate such as an increase or decrease in size (mobility ...

The PACE assay is a relatively new addition to the arsenal of techniques used to examine quantitatively the interactions of proteins and peptides with DNA and RNA (1). Polyacrylamide coelectrophoresis (PACE) involves electrophoresis of a labeled nucleic acid through a gel medium that co ...

Nitrocellulose filter binding has been used to measure the association of proteins to both DNA and RNA for many years. Yarus and Berg (1) first described the application of the method for characterization of aminoacyl-tRNA synthetase:tRNA association; other RNA applications include t ...

When analyzing ribonucleoprotein complexes, it is important to keep in mind that any such complex in solution must coexist with some concentration of the free (uncomplexed) RNA and protein components of the RNP. This can be formalized by the equilibrium equation: where R represents the free RN ...

This chapter focuses on the analysis of RNA-protein complexes (RNPs, ribonucleoprotein particles) by oligonucleotide-targeted RNase H digestion, a powerful approach to probe the domain structure of an RNP, both in crude extracts and in purified preparations. RNase H requires DNA-RNA ...

RNA plays a central role in a wide range of processes within the cell. In some organisms RNA replaces DNA as the genetic material. In all organisms RNA is essential at all stages in the translation of genetic information to protein (1–4). RNA-protein interactions have a key role in almost all of these biolog ...

Crosslinking techniques have been used widely to obtain meaningful structural information on RNA-protein interactions. Exact data on the contact sites of RNA-protein complexes at the molecular level are required for a detailed modeling of three-dimensional structures within t ...

RNA molecules can fold into extensive structures containing regions of double-stranded duplex, hairpins, internal loops, bulged bases, and pseudo-knotted structures (1). Owing to the complexity of RNA structure, the rules governing sequence-specific RNA-protein recogniti ...

Photochemical crosslinking is an approach that is widely used to detect and analyze interactions between proteins and nucleic acids. The approach involves illuminating a mixture of protein and nucleic acid with light of a suitable wavelength to induce photochemical reactions that r ...

Photochemical crosslinking is a powerful technique for characterization of RNA-protein interactions in ribonucleoprotein complexes. Intermolecular crosslinks can be generated without chemical modification of either the RNA or the protein by irradiation of native comp ...

Two groups of observations support the notion that mRNA turnover influences gene expression in virtually all cells (1–3). (1) The steady-state levels of many mRNAs are determined more by their half-lives than by their gene transcription rates. In other words, mRNA levels often fluctuate with ...

The in vitro translation system provides an important means of identifying mRNA species of a gene of interest, characterizing the protein products, and investigating translational control. Rabbit reticulocyte lysate (RRL) or wheat germ lysate have been used to successfully transl ...

The procedure described here, the PCR poly(A) test (PAT), allows the analysis of the poly(A) tail on a specific mRNA within a pool of total RNA in a rapid (1 day) and sensitive (subnanogram total RNA) fashion. The assay also provides quantitative estimates of the RNAs poly(A) tail length (1). RNA manipulatio ...

The 3′ ends of most nonhistone mRNAs in mammalian cells are generated by the endonucleolytic cleavage of an mRNA precursor (pre-mRNA) followed by the addition of a polyadenylate (poly) tail (see ref. 1 for a recent review on mRNA 3′ end processing). The ability to process pre-mRNAs in vitro has contribu ...

In recent years, the SR protein family of precursor messenger RNA splicing factors has emerged as a key player in the assembly of the spliceosomal machinery onto pre-mRNA. The SR proteins are essential splicing factors and different family members can direct usage of alternative splice sites ...

Pluripotent embryonic stem (ES) cell lines were first isolated over 25 years ago and remain an essential tool in molecular and developmental biology to this day. In particular, the use of homologous recombination and subsequent generation of ES-derived mice has greatly facilitated rese ...

Human embryonic stem cells (hESCs) represent a powerful platform to study human development and its dysfunction in human disease. However, certain biological properties have hampered the application of standard gain of function and loss of function tools to these cells. For example, wh ...