Nuclease, intracellular

Another new modification is the 2 -deoxy-2 flouro-Darabinonucleic acid (2 F-ANA), which increases the strength of the oligonucleotide-mRNA hybrids, elicits efficient RNaseH-mediated degradation of the target, is more nuclease resistant and reaches high intracellular concentrations for prolonged time. Similar results could be obtained with oxetane modified ASONs. 

Ribozymes are small RNA molecules with endoribo-nuclease activity. Under appropriate conditions, ribozymes exhibit sequence-specific cleavage of the target. The cleaved mRNA is destabilized and subject to intracellular degradation. 

The mature red blood cell cannot synthesize protein. Reticulocytes are active in protein synthesis. Once reticulocytes enter the circulation, they lose their intracellular organelles (ribosomes, mitochondria, etc) within about 24 hours, becoming young red blood cells and concomitandy losing their ability to synthesize protein. Extracts of rabbit reticulocytes (obtained by injecting rabbits with a chemical—phenylhydrazine—that causes a severe hemolytic anemia, so that the red cells are almost completely replaced by reticulocytes) are widely used as an in vitro system for synthesizing proteins. Endogenous mRNAs present in these reticulocytes are destroyed by use of a nuclease, whose activity can be inhibited by addition of Ca +. The system is then pro-... 

A second explanation of the ability of oxidative stress to cause DNA damage is that the stress tri ers a series of metabolic events within the cell that lead to activation of nuclease enzymes, which cleave the DNA backbone. Oxidative stress causes rises in intracellular free Ca, which can fiagment DNA by activating Ca -dependent endonucleases (Orrenius etal., 1989 Farber, 1990 Ueda and Shah, 1992) in a mechanism with some of the features of apoptosis (see Wyllie, 1980). An example of apoptosis is the killing of immature thymocytes by glucocorticoid hormones, which activate a cell-destructive process that apparently involves DNA fragmentation by a Ca -dependent nuclease. 

Endogenous microbial enzymes are sometimes utilized to break down their parent cells, and thus extract valuable intracellular materials. For instance, in the production of yeast extract, cells are allowed to autolyse at about pH 5 and 55 0. Proteases are probably the most important class of enzymes involved in autolysis, although others such as glucanases, lipases and nucleases also have... 

Successful gene delivery by use of cationic liposomes requires the following conditions (134) (1) condensation of DNA into the complex and its protection from degradation by intracellular nucleases (2) adhesion of DNA-lipid complex onto the cellular surface (3) complex internalization (4) fusion of an internalized DNA-cationic liposome complex with the endosome membrane (5) escape of DNA from the endosome (6) entry of DNA into the nucleus followed by gene expression. 

The leader sequence of the Serratia marcescens extracellular nuclease has been removed and the gene for the resulting nontransportable protein cloned behind the leftward promoter (PL) of lambda (Ahrenholtz, Lorenz Wackernagel, 1994). In the presence of a temperature-sensitive repressor (cl857), the cells can be induced to produce this altered enzyme by an increase in temperature. This produces the nuclease within the cell and, because it cannot be exported, its intracellular concentrations rise, rapidly degrading the cellular genome. Limitations to this system are that cell survival is reduced to only 2 x 10 5. 

Uncoating and eclipse. Uneoating is detected by the lability of viral nucleic acids to nuclease after the artificial disruption of the cell. Eclipse is recognized by loss of infectivity of intracellular virions recovered from disrupted cells. 

The first step in the extraction of nucleic acids may require the lysis of cells and the inactivation of cellular nucleases. These two steps may be combined into one. For instance, a single solution may contain detergents to solubilize the cell membrane and strong chaotropic salts to inactivate the intracellular... 

Calcium phosphate co-precipitation, first developed by Graham and van der Eb (9), is popularly used for both stable and transient transfections in many different cell lines due to easy availability of component materials and their low cost. DNA when mixed with calcium chloride and added in a controlled manner to a buffered saline-phosphate solution at room temperature (RT) results in the formation of a precipitate that is dispersed on the attached cells (10). The precipitate is presumably taken up by the cells by endocytosis. Calcium phosphate, like several other nucleic acid delivery reagents, protects the nucleic acid in the complex from the action of intracellular and serum nucleases. 

The extreme sensitivity of RNA to the ubiquitous inter- and intracellular nudeases (for example on the skin of the investigator) makes special precautions necessary for effective RNA preparations. The use of disposable containers is recommended, or glassware that has been soaked in dilute hydrochloric acid and rinsed with autoclaved distilled water. Disposable gloves must be worn in all procedures where RNA is handled, or is likely to come into contact with RNA, such as solutions, chemicals, glassware, spatulas etc. Buffers for RNA work should be prepared from reagents reserved for this purpose, and stored separately. Buffers can be treated with 0.2 % (v/v) diethylpyrocarbonate (care - this is carcinogenic) and autodaved to inactivate RNases, or at least those with adive site histidines. Since most nucleases require Mg2 for activity, the addition of EDTA in mM concentrations to solutions is also recommended. 

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