Nuclease sources

It should be pointed out that when using ethidium bromide the sensitivity of the assays varies depending on the physical state of the nucleic acids (see Table I). Ethidium does not discriminate between RNA and DNA, although dyes are available which bind DNA exclusively, so the relative amounts of each may be determined by taking two sets of measurements. Alternatively, nucleases (DNA-ase or RNA-ase) can be used to exclusively remove one or the other in a mixture. Nucleic acids from different sources (see Table II) also show a variation in sensitivity, and the fluorescence assay lacks the selectivity of the hybridization technique. Nevertheless, for rapid screening or quality-control applications the fluorescence assay is still the method of choice. 

Endotoxins are found in some bacterial sources, such as E. coli. For other products they are considered a contaminant that should not be present and can be controlled by adherence to good manufacturing practices (GMPs). Nucleic acids, once considered a significant risk, are now thought of as cellular impurities, and their removal should be validated [2,3]. Proteins that pose a potential risk (e.g., immunogenicity) include host cell proteins, aberrant protein product, proteins used in cell culture, and those associated with the process (e.g., protein A affinity ligands or nucleases employed to reduce viscosity). 

The reader is referred to the following sources of further information on the specific cocatalytic enzymes superoxide dismutase, and the reduced form, alkahne phosphatases, nuclease Pl, purple acid phosphatase, amidohydrolase, leucine amtnopeptidase, general comments on the mechanisms of the phosphatases and aminopeptidases, and other cocatalytic zinc enzymes. ... 

Branum ME, Reardon JT, Sancar A. DNA repair excision nuclease attacks undamaged DNA. A potential source of spontaneous mutations. J. Biol. Chem. 2001 276(27) 25421-426. 

The first activity of DNA topoisomerases to be described was the relaxation of supercoiled closed-circular DNA, i.e., conversion to a less supercoiled form (Wang, 1971). This activity was clearly distinct from that of nucleases since the products were covalently closed and relaxation could occur in a stepwise fashion. A dependence on DNA ligase was ruled out, since no energy source was required for this reaction. All topoisomerases discovered subsequently can relax negatively supercoiled DNA the ability to relax positively supercoiled DNA is less gen-... 

Several kits are commercially available for the extraction of RNA from plant, bacteria, or mammalian sources. We have used the RNeasy Mini Kit (Qiagen, Valencia, CA) for the extraction of RNA from bacteria following the procedure recommended by the manufacturer (92). This kit relies on guanidine thiocyanate-silica isolation of RNA. Here, guanidine thiocyanate serves as a chaotropic agent, which both lyses cells and inactivates nucleases. In its presence, the released nucleic acids bind to silica particles, which provide a solid phase from which the collected RNA can later be eluted using water (93). This kit is suitable for the isolation and purification of up to 100 pg RNA molecules >200... 

When chemical and enzymatic methods for NTP synthesis are compared[941, enzymatic techniques provide the most convenient route to CTP and GTP, whereas chemical deamination of CTP is the best method for preparing UTP 94L ATP is relatively inexpensive from commercial sources, although it has been synthesized enzymatically from AMP on 50 mmol scale. Mixtures of NTPs can be prepared from RNA by sequential nuclease Pi, polynucleotide phosphorylase, and pyruvate kinase-catalyzed reactions11101. This mixture can be selectively converted to a sugar nucleotide using a particular sugar nucleoside diphosphate pyrophosphorylase11101. 

Figure 6.S. Fluorescence emission specUa of native (N) (pH 7.5) and acid-unfolded (Ua) (pH 2.0) staphylococcal nuclease K46C labeled with lAEDANS. Excitation of tryptophan residue ( e = 295 nm) meteases the emission ai 485 nm of lAEDANS as the result of Trp I.AEDANS energy transfer. This increase k less import art in the nnfolded slate than in llie folded one Source Nishhnura. C. Ril. R., Eastman. P. and Fkik. A. L. 2000, Journal of Molecular Biokigy. 299,1133 1146.

Lin and Fisher, 1990) and as detailed below. All procedures should be performed at 4°C unless otherwise indicated. To purify interphase lamins Dmi and Dm2, it is first necessary to solubilize them from nuclei under nondenaturing conditions. To accomplish this, nuclei should be purified according to standard procedures (see chapter by Fisher, this volume see also Fisher et ai, 1982, 1989). Purified nuclei should be treated with DNase I (10 jug/ml) and RNase A (8 jug/ml) nuclease treatment must be performed at 23°C rather than at 37°C (McConnell et al., 1987). Residual nuclei (after nuclease treatment) should be extracted first with 2% (v/v) Triton X-100 (no lamin solubilized) and then with 0.5 M NaCl (instead of 1 M NaCl) essentially as detailed previously (Fisher et ai, 1982 McConnell et ai, 1987 Lin and Fisher, 1990). This will result in about 75% of the interphase nuclear lamin being recovered in the 0.5 M NaCl solution this solution should be used immediately as the source of lamins Dmi and Dm2 for further purification. 

Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter, W. J. (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribo-nuclease. Biochemistry 18,5294-5299. 

Sources of chemicals, method of RNA preparation, making of single stranded probes, mapping with SI nuclease and primer extension analysis have been described (5). 

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