细胞功能测定

Many important cellular processes are mediated by sequence-specific RNA binding proteins, and it is often necessary to purify these proteins. When the RNA binding site is known, it is convenient to use this RNA as a matrix for affinity purification. The intronic splicing silencer (ISS) element ...

The understanding of physiology and pathology requires accurate quantification of intracellular concentrations of important molecules such as unique RNA species. Accurate quantification of highly homologous messenger RNAs (mRNAs) (1–3), alternatively spliced mRNAs (4 ...

RNA helicases are essential for the adenosine 5′-triphosphate (ATP)-driven rearrangement of many RNAs and RNA-protein complexes (ribonucleoproteins, RNPs) throughout RNA metabolism. We describe assays to measure RNA and RNP remodeling by RNA helicases in vitro. We show how to prepa ...

Terminal transferase-dependent polymerase chain reaction (TDPCR) can be used after reverse transcription (RT) to analyze RNA. This method (RT-TDPCR) has been used for study of RNA structure and RNA-protein interactions at nucleotide-level resolution. A detailed protocol of RT-TD ...

Small interfering RNA (siRNA)-mediated RNA interference (RNAi) is a very powerful tool for triggering posttranscriptional gene silencing in several organisms. We discuss the improvement of two different sources of siRNAs synthesized either chemically or by an enzymatic method. ...

Pre-mRNA (messenger RNA) splicing is an essential step for gene expression in higher eukaryotes. Splicing reactions have been well studied in vitro using extracts prepared from cultured cells. We describe protocols for the preparation of splicing-competent extracts from whole cel ...

Biochemical assay of proteomic libraries derived from the Saccharomyces cerevisiae genome provides a powerful new tool for the assignment of activities to proteins. Particular advantages of this approach include the speed with which a protein can be identified and the generality for ...

Antisense oligonucleotides have been used to study the structure and function of small nuclear ribonucleoprotein (snRNP) complexes and were adapted and modified for the purification of a variety of RNPs. We describe methods for recombinant expression and reconstitution of cataly ...

Isolation of ribonucleoprotein particles from living cells and cell lysates has allowed the identification of both simple bimolecular interactions and the members of large, extended complexes. A number of different strategies have been devised to isolate these complexes by using a ...

Studies of RNA—protein interactions often require assembly of the RNA—protein complex using in vitro synthesized RNA or recombinant protein. Here, we describe a protocol to assemble a functional spliceosome in yeast extracts using transcribed or synthetic RNAs. The in vitro assembl ...

Recently developed affinity purification methods have revolutionized our understanding of the higher-ordered structures of multisubunit, often low-abundance macromolecular complexes, including ribonucleoproteins (RNPs). Often, purification by classical, no ...

We present a newly developed method for fixing RNA-protein complexes in situ in living cells and the subsequent purification of the RNA targets. Using this approach, complex tissue such as mouse brain can be ultraviolet (UV) irradiated to covalently crosslink RNA-protein complexes. Once c ...

Isothermal titration calorimetry (ITC) is a useful technique to study RNA-protein interactions as it provides the only method by which the thermodynamic parameters of free energy, enthalpy, and entropy can be directly determined. This chapter presents a general procedure for studyi ...

The gel mobility shift assay is routinely used to visualize protein—RNA interactions. Its power resides in the ability to resolve free from bound RNA with high resolution in a gel matrix. We review the quantitative application of this approach to elucidate thermodynamic properties of prot ...

Analogs of naturally occurring substances obtained by chemical modifications are powerful tools to study intra- and intermolecular interactions. We have used the phosphorothioate technique to analyze RNA-protein interactions, here the interactions of transfer RNAs (tRNA ...

Mammalian fertilization is traditionally viewed as a process during which the fertilizing spermatozoon penetrates the egg vestments to plant its chromosomes into the fertile environment of oocyte cytoplasm. Much less attention has been paid to the events that occur between gamete f ...

Conventional PCR screens of genomic DNA will often yield a substantial fragment of the gene of interest. However, identification of flanking sequences on either side of a known sequence can be problematic with conventional PCR. in which primers extend a complementary chain in a 5′ → 3′ direction. H ...

Their large size and their relative resistance to proteolytic cleavage (1) make myosins particularly difficult substrates for the acquisition of their peptide sequences by standard protocols. For this reason, instead of identifying myosins first according to their biochemical ...

In this chapter, we describe our methods to evaluate the dynein heavy chain genes expressed in a cell. Dynein is the high molecular weight molecular motor that produces directed movement along microtubules. All known dyneins travel to the proximal or minus end of the microtubule. The large dynein ...

The cytoskeleton is a dynamic network of filaments in the cytoplasm of cells and functions as the roadways for vesicular transport. Of the three types of cytoskeletal filaments, both microtubules and actin filaments have been shown to support vesicle transport. The transport of vesicles is ...