Nuclease treatment

Bacterial nucleic acids were hydrolyzed using bovine pancreatic ribonuclease and deoxyribonuclease.37 Deoxyribonuclease treatment of disrupted bacterial suspensions has also been reported.38 

Melling and Atkinson29 investigated nuclease treatment as a method for the removal of nucleic acids from bacterial suspensions. Two strains of E. coli were used and for both the strains the nuclease treatment was effective in depolymerizing nucleic acids and, hence, in recovery of supernatant after centrifugation to remove cell debris from disrupted cells. The nucleotide content in the supernatant was found to be 15-20% of total proteins and nucleic acids. The nucleotide content in the supernatant was reduced to a very low level by ammonium sulfate precipitation followed by dialysis of the redissolved precipitate. As stated earlier, direct precipitation of nucleotides resulted in significant residual nucleotides in the proteins. 

For effective removal of nucleic acids, both ribonuclease and deoxyribonuclease need to be used.29 The cost of nucleases is a major hindrance in use of this method in removal of nucleic acids from mixtures during protein purification. An organism s own nucleases may provide an often-unrecog-nized nuclease treatment. 

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Nucleic acids may also be removed by treatment with nucleases, which catalyse the enzymatic degradation of these biomolecules. Indeed, nuclease treatment is quickly becoming the most popular method of nucleic acid removal during protein purification. This treatment is efficient, inexpensive and, unlike many of the chemical precipitants used, nuclease preparations themselves are innocuous and do not compromise the final protein product. 

Limited nuclease treatment of 30 S subunits yields two main fragments of unequal size one containing proteins S4, S5, S6, S8, S15, S17, S20, and possibly S13 or S14, and the other S7, S9, SIO, S19, and S13 or S14. Various smaller fragments have been isolated consisting of subsets of these two groups of proteins, the smallest one containing S8 and S15 and another one S7 and S19 (Morgan and Brimacombe, 1972, 1973 Roth and Nierhaus, 1973 Yuki and Brimacombe, 1975). 

The structure of the condensed chromatin fiber is still under discussion [1,23,54], with two competing models the original solenoid model of Finch and Klug [16], and the straight-linker model [12,14,55]. Assessing the structure in vivo or in situ has proven impossible thus far, due to technical limitations. Chromatin fibers released from nuclei into solution by nuclease treatment have been widely used as models for fiber structure such fibers are extended at low ionic strength and condensed at ionic strengths believed to be close to those found in vivo ( 150 mM Na" " or 0.35 mM Mg " "). The salt-induced fiber compaction has been extensively studied in the past but is still poorly understood in terms not only of the details of the structure but also in terms of the molecular mechanisms of the compaction process. 

Precipitation of protein may also occnr in some cases, resnlting in loss of enzyme activity. Nuclease treatment is a very effective method of nncleic acid removal (Melling and Phillips, 1975), bnt of limited nse for enzymes which ate intended for apphcations in molecular biology. 

The histones present in chromatin are of five major types HI, H2a, H2b, H3, and H4 (table 25.2). The lysine-rich histone HI is not present in the nucleosome core particles, as evidenced by its release on extensive nuclease treatment and the finding that HI is the only histone that readily exchanges between free and DNA-bound histone. HI may play a key role in the conversion of chromatin to the highly compacted chromosome that occurs immediately before cell division. The other eight histones, two each of the other four histones, form the protein core of the nucleosome. These protein octamers do not come apart even when chromosomes duplicate. 

Incorporation of bromodeoxyuridine is enhanced by incubating in the presence of fluorodeoxyuridine (to block endogenous synthesis of thymidine — 11.8.2) and access of the antibody to the fixed cells is ensured by partial denaturation or brief nuclease treatment (Goncharoff et al., 1986). After development of the peroxidase reaction cells can be counterstained, for example, with neutral red. 

Structure of Nucleosomes The DNA component of nucleosomes Is much less susceptible to nuclease digestion than Is the linker DNA between them. If nuclease treatment Is carefully controlled, all the linker DNA can be digested, releasing individual nucleosomes with their DNA component. A nucleosome consists of a protein core with DNA wound around its surface like thread around a spool. The core is an octamer containing two copies each of histones H2A, H2B, H3, and H4. X-ray crystallography has shown that the octameric histone core is a roughly disk-shaped molecule made of interlocking histone subunits... 

As an alternative to nuclease digestion, cationic detergents can be added during lysis to selectively precipitate cellular DNA. Recent experiments with the detergent domiphen bromide have shown that it is possible to obtain three logs of DNA clearance without losses in the infectivity of purified viral particles [107]. Thus, the use of DNA removal operations such as nuclease treatment and anion exchange chromatography may be eliminated or reduced. 

The viral particle-containing solutions resulting from nuclease treatment and cationic detergent precipitation are typically filtered, concentrated, and conditioned before chromatographic purification [40, 80, 101, 106, 107]. The inclusion of a solvent/detergent step at this stage may be included to inactivate potential enveloped viruses that could have been coamplified [32]. 

Fig. 5 Nucleic acid solubilization during nuclease treatment of isolated liver nuclei. Nuclei isolated from 3-day-old chicks (cockerels) were digested with DNase I and RNase A (4 ug/ml each) for 40 min at 37°C. At each time point, cold trichloroacetic acid (TCA)-soluble nucleotides were measured using absorbance at 260 nm [see also Fisher et al. (1982)].

Fig. 6 Nuclease treatment and subfractionation of nuclei isolated from vertebrate livers. Flow chart showing nuclease digestion of isolated nuclei as well as fractionation steps leading to a karyoskeletal protein-enriched fraction.

Nuclease Treatment of Drosophila Nuclei Subffactionation of Nuclei... 

Figure 2 is a transmission electron micrograph of a nuclease-treated, Triton X-lOO-extracted, twice NaCl-extracted karyoskeletal protein-enriched fraction derived from Drosophila embryos. Nuclease treatment was at 37°C. Identifiable karyoskeletal elements are labeled. The SDS-PAGE profiles of the final karyoskeletal protein-enriched pellet fraction generated after subfractionation of nuclei treated with nucleases at 37°C are shown in Fig. 3 (lane 1) as well as the first 1 M NaCl extract generated after subfractionation of nuclei treated with nucleases at 23°C (also highly enriched for Drosophila karyoskeletal proteins but in soluble form) (lane 2). 

Fractions are as follows 1, filtered crude homogenate 2, postnuclear supernatant 3, supernatant from the first wash of the nuclei 4, supernatant from the second wash of the nuclei 5, resuspended nuclei 6, resuspended nuclei after treatment with nucleases 7, supernatant from resupended nuclei after treatment with nucleases and centrifugation 8, supernatant from nuclease-treated nuclear pellet after extraction with Triton X-lOO and centrifugation 9, supernatant from nuclease-treated, Triton X-lOO-treated nuclear pellet after extraction with NaCI and centrifugation 10, supernatant from nuclease-treated, Triton X-lOO-treated, NaCl-treated nuclear pellet after repeat extraction with NaO and centrifugation 11, final karyoskeletal protein-enriched fraction after purified nuclei were subjected to nuclease treatment, Triton X-100 treatment, and two NaCI treatments, all performed sequentially. 

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. 

Table 1 shows the distribution of poly(ADP-ribose) synthetase activity in the different subnuclear fractions. The finding that the bulk of the poly(ADP-ribose) synthetase is released upon nuclease treatment of the nuclei, conforms with the previous observa-... 

Razin SV, Mantieva VL, Georgiev GP (1979) The similarity of DNA sequences remaining bound to scaffold upon nuclease treatment of interphase nuclei and metaphase chromosomes. Nucleic Acids Res 7 1713-1735... 

Cloning of the "140 bp DNA" in M13mp8 vector. DNA from the "140 bp DNA" was subjected to SI nuclease treatment followed by a dephosphorylation using bacterial alkaline phosphatase. The DNA was en treated with T4 polynucleotide kinase and the ends repaired with T4 DNA polymerase. After each enzymatic step, the solutions were extracted with phenol and chloroform and the DNA recovered by ethanol precipitation. The blunt-ended DNA was cloned by blunt-end ligation in the Hindi site of M13mp8 vector. All these steps were achieved according to standard procedures (15). Ml 3 clones (26 total) were obtained. 

The kinetics of translation vary depending on the nature of the reaction being performed. In the absence of any additions except [ S]methionine, whole extract (before nuclease treatment) shows approximately linear incorporation over time for a number of hours. When creatine phosphate and reticulocyte lysate are present, the rate of incorporation is enhanced such that a plateau is reached after around 45 min. After nuclease treatment, the rate of reaction is often a function of the activity of the mRNA added. Reactions programmed with Xenopus poly(A)+ RNA... 

Provided that the eggs used are of reasonable quality, preparation of the basic extract gives reliable results. The two points where the extract can be compromised are nuclease treatment and recovery from freezing. 

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