生物化学技术

Both RNA and DNA viruses have been engineered to serve as vectors for transient silencing in intact plants. Host gene sequences carried by the virus are seen by the plant as “foreign,” and homologous gene-silencing machinery acts on both the viral vector RNA and the endogenous host gene mRNA. DNA viru ...

Double-stranded RNA when introduced into cells results in severe reduction of the target mRNA. This phenomenon is known as posttranscriptional gene silencing in plants and RNA interference in animals. Hairpin RNA-mediated gene silencing exploits this cellular mechanism. A conve ...

MicroRNAs (miRNAs) and other small RNAs can be identified by cloning and sequencing cDNAs prepared from the ∼22-nt fraction of total RNA. Methods are described for the construction of cDNA libraries from small noncoding RNAs through the use of T4 RNA ligase, reverse transcriptase, and polyme ...

Adenosine deaminases that acts on RNA (ADARs) are RNA-editing enzymes that convert adenosine to inosine in double-stranded RNA. This chapter provides a detailed protocol for identifying inosine-containing RNAs. Candidate ADAR substrates are identified by cleaving poly (A)+ RNA sp ...

This chapter describes the technique of RNA affinity chromatography, which is a powerful approach for isolating RNA-binding proteins. This method takes advantage of the fact that sequence-specific RNA-binding proteins often bind their targets with high affinity. Here we outline a pr ...

ADARs are found in Metazoans but are not present in yeasts. We have found that the methanol-utilizing yeast Pichia pastoris can be used to efficiently express enzymatically active epitope-tagged ADARs. We describe plasmid construction and protein expression procedures for produci ...

This chapter describes the methods used to purify the RNA-editing complex, to identify the proteins by mass spectrometry, and to demonstrate the functions for some of the proteins.

This chapter describes biochemical assays that have been used in analyzing RNA editing in kinetoplastid mitochondria and to characterize the general mechanism of editing by the editosome. Studies using these assays have shown that the characteristics of each activity contribute to ...

RNAs made in the mitochondrion of Physarum polycephalum are edited relative to their template by the precise addition of nonencoded nucleotides, while they are being synthesized. This insertional editing has been reproduced in vitro during run-on extension of RNAs initiated in vivo, wi ...

We present here methodology for assaying 5′-terminal editing of mitochondrial tRNAs in the amoeboid protist Acanthamoeba castellanii. This type of editing involves replacement of one or more nucleotides within the first three positions at the 5′ end of a tRNA substrate. The assay procedu ...

RNA editing in plant cell organelles is a posttranscriptional process that changes the identity of individual nucleotides by pyrimidine transitions. C-to-U conversions are the predominantly found modifications in both mitochondrial and chloroplast transcripts of vascul ...

RNA editing in higher plant chloroplasts involves C-to-U conversion at ∼30 specific sites. In vitro systems supporting accurate editing have been developed from tobacco and pea chloroplasts. mRNA substrates labeled with 32P at C residues to be edited provide sensitive detection of edit ...

Identification of a modified nucleotide and its localization within an RNA molecule is a difficult task. Only direct sequencing of purified RNA molecules and high-performance liquid chromatography mass spectrometry analysis of purified RNA fragments allow determination of bo ...

A protocol is described using lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by the substituted-cysteine accessibility method as applied to transmembrane domains (SCAM™). SCAM™ is adapted to follow ...

Integral membrane proteins typically span the lipid bilayer with hydrophobic α helices. These proteins can span the membrane once or multiple times with hydrophilic domains facing both sides of the membrane. In Escherichia coli, the insertion of proteins into the membrane is catalyzed by ...

Non-messenger RNAs (nmRNAs) play a wide and essential role in cellular functions. Computational identification of novel nmRNAs in genomes of model organisms is severely restricted owing to their lack of an open reading frame. Hence, we describe experimental approaches for their ident ...

The known examples of RNA editing now encompass a variety of alterations of RNA primary sequence that arise from base modifications, nucleotide insertions or deletions, and nucleotide replacements. Hence, the definition of RNA editing has evolved as new systems have been described. This ...

There have been many major advances in the field of liver transplantation in the past 30 yr. Orthotopic liver transplantation is currently the only established successful treatment for end-stage liver disease, with over 3000 liver transplantations being performed each year in more than ...

Of the 11 families of mammalian cyclic nucleotide phosphodiesterases (PDEs), 5 contain regulatory domains capable of binding cyclic guanosine 5′-monophosphate (cGMP). The best understood of the GAF-containing PDEs is the family of rod (PDE6R) and cone (PDE6C) photoreceptor PDEs. Bin ...

An in vitro selection procedure for identifying highly efficient RNase P ribozyme (M1GS RNA) variants is presented as a model system for engineering ribozymes to improve their catalytic efficiency. Detailed protocols as well as the rationale for setting up such a system are included, using ...