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Analysis by HPLC of Distributive Activities and the Synthetic (Back) Reaction of Pancreatic-Type Ribonucleases

Nucleic-acid cleavage can be processive, with the enzyme moving from one site to the next in the polymer before dissociating from the substrate, or distributive, with partially cleaved substrates released to the medium after the initial reaction. Kinetics indicate that either mechani ...

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Ier1 p: A Kinase and Site-Specific Endoribonuclease

The lumen of the endoplasmic reticulum (ER) is a highly specialized compartment in eukaryotic cells. Here, secretory and most membrane proteins are folded, covalently modified, and oligomerized with the assistance of specialized ER resident proteins (1). Perturbation of the ER lumen i ...

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Microtiter-Plate Assay and Related Assays for Nonspecific Endonucleases

Nucleases catalyze the cleavage of phosphodiester bonds in nucleic acids. They range in size from divalent cations, such as Pb2+, which specifically cleaves tRNAPhe from yeast (1), and Mg2+, which specifically acts on squid tRNALys (2) to divalent cations complexed to chelating agents, for ex ...

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Quantitating mRNAs with Relative and Competitive RT-PCR

Reverse transcription coupled with the polymerase chain reaction (RT-PCR), permits amplification of cellular RNA. Gene expression can be measured even when the message copy number is very low (1–10 copies per cell) (1) or the sample size is very small (1–1000 cells) (2,3). In RT-PCR, an RNA sample is pri ...

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Expressing Self-Incompatibility RNases (S-RNases) in Transgenic Plants

The most obvious functions of nucleases are nucleic-acid turnover and phosphate mobilization, but they also have more subtle roles. By its nature, nuclease activity may disrupt cellular information flow. Familiar examples include degradation of specific mRNAs, and bacterial rest ...

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Molecular Cloning, Tissue Distribution, and Chromosomal Localization of the Human Homolog of the R2/Th/Stylar Ribonuclease Gene Family

Allelic losses in the peritelomeric region of the long arm of human chromosome 6 (6q26–27) are associated with several human malignancies. These range from solid neoplasms such as ovarian carcinomas (1–8) to hematological cancers, among which B-cell non-Hodgkin’s lymphomas and acute ly ...

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Assays for the Evaluation of HIV-1 Integrase Inhibitors

Integration is an essential step in the replication cycle of a retrovirus, such as the human immunodeficiency virus type 1 (HIV-1) (for a review, see ref. 1). After reverse transcription of the RNA genome, the DNA copy is transported into the nucleus and integrated in the host chromosome. The only viral e ...

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The 25A/RNase L Pathway and Inhibition by RNase L Inhibitor (RLI)

Interferons (IFN) belong to a multigenic family of cytokines that activate the transcription of several genes through a signaling pathway involving cell-surface receptors (IFNAR), Janus kinases (JAK), and signal-transducing proteins (STAT), as comprehensively reviewed by St ...

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The Direction of Ribonucleases H by Antisense Oligodeoxynucleotides

Ribonuclease H (RNase H), first observed in extracts of calf thymus, is an apparently ubiquitous enzyme activity (1), which catalyzes the hydrolysis of RNA in RNA-DNA heteroduplexes (2). The two main classes (Type I and II) of eukaryotic RNase H have different biochemical requirements for maxi ...

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Ribonuclease Inhibitors

RNA analysis and quantification require completely intact, nondegraded RNA samples to produce optimal results. Although nonenzymatic hydrolysis of phosphodiester bonds is favored by high temperature or pH and the presence of divalent cations (Mg2+, Mn2+), an RNA sample is most likely ...

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Producing Soluble Recombinant RNases and Assays to Measure Their Interaction with Interferon- In Vitro

Many cytokines alter the half-lives and stabilities of mRNAs for other cytokines (1) and enzymes that may be related to disease progression. For example, IL-1β stabilizes the mRNA for human interstitial collagenase-1 in normal fibroblasts, but not in breast cancer cells (2). Altering mRNA st ...

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Crystallization and Crystal Structure Determination of Ribonuclease A-Ribonuclease Inhibitor Protein Complex

Ribonuclease inhibitor (RI) is a cytoplasmic protein of ∼50 kDa that tightly binds and inhibits ribonucleases (RNases) from the pancreatic superfamily (1). Diverse RNases with very limited sequence similarities, including RNase A, angiogenin, RNase-2 (also known as eosinophil-d ...

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Methods for Studying the Interaction of Barnase with Its Inhibitor Barstar

Barnase is a 110-residue extracellular ribonuclease produced by Bacillus amyloliquefaciens. The same organism produces an 89-residue polypeptide, barstar, which binds tightly to barnase and inhibits its potentially lethal RNase activity inside the cell. The three-dimenti ...

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Dissecting Nucleases into Their Structural and Functional Domains: Mapping the RNA-Binding Surface of RNase III by NMR

The enzyme RNase III catalyzes the hydrolysis of the phosphodiester bond between two nucleotides. The protein was discovered in 1968, when Robertson and coworkers isolated a double-stranded RNA (dsRNA) cleaving activity in Escherichia coli cells (1). A few years later, specific targets ...

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Assaying In Vitro Refolding of RNases by Mass Spectrometry

Although evidence has been mounting since the 1930s that proteins could be reversibly denatured, it was not until the 1960s that the elegant experiments of Anfinsen on bovine pancreatic Ribonuclease A (RNase A) established that all the information needed for a protein to acquire three-dim ...

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Transmembrane Signaling by G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) make up the largest and most diverse family of membrane receptors in the human genome, relaying information about the presence of diverse extracellular stimuli to the cell interior. All known GPCRs share a common architecture of seven membrane-span ...

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Crosstalk Coregulation Mechanisms of G Protein- Coupled Receptors and Receptor Tyrosine Kinases

G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) are transmembrane receptors that initiate intracellular signaling cascades in response to a diverse array of ligands. Recent studies have shown that signal transduction initiated by GPCRs and RTKs is not org ...

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Protein-Protein Interactions in Signaling Cascades

The process of signal transduction is dependent on specific protein-protein interactions. In many cases, these interactions are mediated by modular protein domains that confer specific binding activity to the proteins in which they are found. Rapid progress has been made in the biochem ...

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Methods for the Study of Signaling Molecules in Membrane Lipid Rafts and Caveolae

Lipid rafts and caveolae are cholesterol- and sphingolipid-rich microdomains of the plasma membrane that concentrate components of certain signal transduction pathways. Interest in and exploration of these microdomains has grown in recent years, especially after the discove ...

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Bioluminescence Resonance Energy Transfer to Monitor Protein-Protein Interactions

The bioluminescence resonance energy transfer (BRET) methodology allows for the study of protein-protein interactions as well as conformational changes within proteins or molecular complexes. BRET is a highly versatile technique that can be applied to in vitro studies using puri ...

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