Nuclease digestion, resistance

PNA oligomer Peptide nucleic acid oligo (Fig. ID) manufacturers include Boston Probes (www.bostonprobes.com) the Danish company Pantheco (www.pantheco.com), and Research Genetics (www.resgen.com). These, like morpholino analogs, resist nuclease digestion. 

At this point, investigators have two alternatives. If nuclease digestion is performed at 37°C, Drosophila karyoskeletal elements are effectively heat stabilized (also see below) and karyoskeletal proteins become at least partially resistant to extraction with 2% (v/v) Triton X-100 and 1 M NaCI. If nuclease digestion is performed at 23°C, heat stabilization aj arently does not occur (McConnell et al., 1987). Karyoskeletal proteins still resist extraction with 2% Triton X-100 but can be largely or completely solubilized with solutions of high NaCl concentration (McConnell et aL, 1987 also see Lin and Fisher, 1990). In either case (23 or 37°C), incubation should be for IS min. 

Nuclei prepared by these methods vary in susceptibility to nuclease digestion and chromatin release, those exposed to polyamines being the most resistant. In choosing a buffering agent, it should be borne in mind that, as mentioned above, Tris interferes with aldehyde fixation and should be avoided for most applications involving subsequent imaging steps. PIPES has been the most suitable buffer in our hands. 

Are ADP-ribose polymers randomly or non randomly distributed in chromatin Isolated nuclei from hyperthermia-MNNG-treated ceUs were digested with MNase and the DNA and poly(ADP-ribose) released into the digestion supernatant were determined. While 80% of the total DNA was rendered soluble by the nuclease, 90% of the total ADP-ribose residues remained with the MNase resistant chromatin (Fig 1). These data show that ADP-ribose polymers are not randomly distributed in chromatin but rather they exhibit a predominant association with the chromatin fraction which was resistant to MNase digestion. However the possibility that these polymers confer resistance to the nuclease digestion can not be ruled out at this point. The MNase resistant chromatin fraction isolated in these experiments contained approximately 20% of the total DNA and proteins that isolate with the nuclear matrix. TTius, the possibility of the association of ADP-ribose polymers with the nuclear matrix was examined. 

As to the stoichiometry of the H3-H4-DNA particle, two complexes were identified an H3-H4 tetramer and an H3-H4 octamer, each associated with about 140 base pairs of DNA. The complexing of 140 base pairs of DNA with H3 and H4 resulted in the formation of nucleosome-like particles, as observed by the EM, and reported to have an s20base pairs (Bina-Stein and Simpson, 1977 Bina-Stein, 1978). These results differ from those of Simon et al. (1978) who report that at least two complexes of H3 H4-DNA can be obtained upon reconstitution of H3, H4, and 150 bp DNA. In this experiment both an octamer and a tetramer of H3-H4 were found bound to 150 base pairs of DNA, having sM,w equal to 10.4 and 7.5 for the octamer and tetramer, respectively. The stoichiometry of the complexes obtained is dependent on the histone-to-DNA ratio. At low ratios of histone to DNA the predominant species contains an H3-H4 tetramer per 150 base pairs of DNA. At a histone-to-DNA ratio of 1 1 the octamer prevails. The nuclease and protease digestion experiments (Camerini-Otero et al., 1976 Sollner-Webb et al., 1976) were performed at a histone-to-DNA ratio of 0.5, conditions which for 140-base-pair DNA would lead primarily to a tetrameric complex. Therefore, it seems that a tetramer of H3 H4 is sufficient for the generation of nuclease-resistant fragments similar to those of complete nucleosomes. Upon addition of H2A and H2B to the tetrameric complex, nucleosomes are formed. Addition of H3-H4 to the tetrameric complex resulted in an octameric complex which is similar in compaction to nucleosomes. H3-H4 tetramers and octamers were similarly found complexed with about 140 base pairs of DNA upon reconstitution of H3-H4 with SV40 DNA. Both complexes were reported to be able to fold 140 base pairs of DNA (Thomas and Oudet, 1979). 

Fig. 10. Incremental truncation libraries (Ostermeier et al., 1999b). Plasmid DNA is digested with two restriction enzymes one that produces a 3 recessed end (A which is susceptible to Exo III digestion) and the other that produces a 5 recessed end (B which is resistant to Exo III digestion). Digestion with Exonuclease III proceeds under conditions in which the digestion rate is slow enough so that the removal of aliquots at frequent intervals results in a library of deletions of all possible lengths from one end of the fragment. The ends of the DNA can be blunted by treatment with SI nuclease and Klenow so that unimolecular ligation results in the desired incremental truncation library.

Contrary to information that LNA oligonucleotides are resistant to digestion with nucleases [67], we have observed that diastereomer 45a of LNA dinucleoside phosphorothioate (presumably RP), obtained from/asf-44, was readily digested by snake venom phosphodiesterase. 

The 2 -deoxynucleoside 5 -a-[P-borano]-triphosphates 118 have been used for PCR-based DNA sequencing.The method relies on the resistance of borano-phosphate linkages to nucleases, thus the positions of the boranophosphates can be revealed by exonuclease digestion, thereby generating a set of fragments that defines the DNA sequence. An abstract has also described the use of fluorescently labelled 2 -deoxynucleoside 5 -a-[P-borano]-triphosphates for DNA sequencing. 

Instead of SI nuclease, mung bean nuclease (cleaner results with the same enzyme activities but much more expensive) or exonuclease VII can be used. Exonuclease VII is interesting since it requires a free 3 - or 5 -end for digestion and introns will be resistant. However, exonuclease VII is useful in combination with SI nuclease, particularly for short introns. 

The incorporation of the 5R and 5S diastereoisomers of the 5-hydroxyhydan-toin (derived from oxidative damage of dC) derivative (89) has been achieved using phosphoramidite chemistry and mild deprotection. Whilst the ODNs containing either diastereoisomer were digested with nuclease Pi, the hydantoin derivatives were resistant to digestion by calf spleen and bovine intestinal mucosa phosphodiesterases. 

A fair comparison should also mention, however, the disadvantages of aptamers DNA and especially RNA are very sensitive to hydrolytic digestion by nucleases, thus requiring highly pure environments for their applications. Several solutions to transform aptamers into nnclease-resistant moieties by the modification of a ribose ring at the 2 -position (Pieken et al., 1991) or by the specific modification of the pyrimidine nncleotide were reported (Heidenreich and Eckstein, 1992 Kusser, 2000), and snch hybrid nucleic acid are anticipated to provide the stabihties requested. 

The nudeo-proteins under the influence of enzymes are successively transformed into nucleins, into nudeic adds, then into xanthin bases, into phosphoric acid, etc. But only the first phase of the transformation, that which corresponds to the formation of protein and nudein, can be realized by p>epsin and trypsin. The nudeins resist the action of the digestive juices, botii gastric arid pancreatic. The transformation of the nucleins first into nucldc adds, then into phosphoric add and into purin bas, is induced in living cells, on the contrary, by the endo-cellular enzymes, which bear the name of nucleases. 

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