Endogenous Nuclease Research Articles

2',5'-Oligoadenylates and related 2',5'-oligonucleotide analogues. 2. Effect on cellular proliferation, protein synthesis, and endoribonuclease activity.

A number of the new enzymatically synthesized 2',5'-oligonucleotide trimers, namely, those containing the nucleosides 8-azaadenosine, toyocamycin, sangivamycin, formycin, 8-bromoadenosine, tubercidin, and guanosine, were found to inhibit protein synthesis and cellular proliferation after uptake into intact L and HeLa cells. 2',5'-Oligonucleotide trimers containing cytidine, inosine, uridine, and 1,N6-ethenoadenosine had some effect while those containing 2-chloroadenosine, 3-ribosyladenine, ribavirin, and 2-beta-D-ribofuranosylthiazole-4-carboxamide had no detectable effect on protein synthesis or cellular proliferation after uptake into L or HeLa cells. All of these 2',5'-oligonucleotide analogues inhibited protein synthesis in the in vitro rabbit reticulocyte lysate system except for the trimer containing ribavirin. Such nucleoside substitutions have further defined the substrate-specificity requirements for the endoribonuclease and/or the inhibitors for the 2',5'-phosphodiesterase. Most of the 2',5'-analogues were degraded in L-cell extracts so the endogenous nucleases are not very specific. The 2',5'-trimers containing tubercidin and 2-beta-D-ribofuranosylthiazole-4-carboxamide were quite stable in comparison to the 2',5'-A trimer. The inhibition of protein synthesis and cellular proliferation observed correlated well with the degradation of rRNA and polyadenylated mRNA observed after uptake of the 2',5'-analogues into intact L cells. The degradation of the polyadenylated mRNA appeared to be a more sensitive test than inhibition of cellular protein synthesis for determining biological activities of the 2',5'-oligonucleotide analogues.

DNA excision repair in permeable human fibroblasts.

U.v. irradiation of confluent human fibroblasts activated DNA repair, aspects of which were characterized in the cells after they were permeabilized. Incubation of intact cells for 20 min between irradiation and harvesting was necessary to obtain a maximum rate of reparative DNA synthesis. Cells harvested immediately after irradiation before repair was initiated displayed only a small stimulation of DNA synthesis, indicating that permeable cells have a reduced capacity to recognize pyrimidine dimers and activate repair. The distribution of sizes of DNA strands labeled during 10 min of reparative DNA synthesis resembled that of parental DNA. However, during a 60-min incubation of permeable cells at 37 degrees C, parental DNA and DNA labeled by reparative DNA synthesis were both cleaved to smaller sizes. Cleavage also occurred in unirradiated cells, indicating that endogenous nuclease was active during incubation. Repair patches synthesized in permeable cells displayed increased sensitivity to digestion by micrococcal nuclease. However, the change in sensitivity during a chase with unlabeled DNA precursors was small, suggesting that reassembly of nucleosome structure at sites of repair was impaired. To examine whether this deficiency was due to a preponderance of incomplete or unligated repair patches, 3H-labeled (repaired) DNA was purified, then digested with exonuclease III and nuclease S1 to probe for free 3' ends and single-stranded regions. About 85% of the [3H]DNA synthesized during a 10-min pulse resisted digestion, suggesting that a major fraction of the repair patches that were filled were also ligated. U.v. light-activated DNA synthesis in permeable cells, therefore, appears to represent the continuation of reparative gap-filling at sites of excision repair activated within intact cells. Gap-filling and ligation were comparatively efficient processes in permeable cells, whereas activation of reparative DNA synthesis and reassembly of native chromatin structure upon completion of repair were not.

Different RNA patterns of globin and non-globin 40S heterogeneous nuclear RNA-protein complexes in chicken reticulocyte nuclei.

40s heterogeneous nuclear RNA-protein complex (HnRNP) was isolated from chicken reticulocyte nuclei after digestion of RNA by the endogenous nuclease. The size of the total RNA and beta globin RNA was compared by agarose and high resolution acrylamide gel electrophoresis. The results show that size of the total RNA ranges between 20 to 650 bases and the most prominent sizes are between 20 and 70-80 bases. The size of the RNA increases at roughly 10 base intervals between 40 and 70 bases on an acrylamide gel. The size of the beta globin RNA is smaller (25-50 bases) than the total RNA and also the RNA pattern is considerably different from that of total RNA. This suggests that, although the overall structure of 40s HnRNP might be the same, there is significant differences in the interaction of globin RNA with HnRNP protein and that of non-globin RNA with HnRNP protein in chicken reticulocyte nuclei.

Nuclease-hypersensitive sites in the chromatin domain of the chicken lysozyme gene.

We have examined the chromatin structure of a 22 kilobase-pair chromosomal region containing the lysozyme gene in laying hen. Nuclease-hypersensitive sites were probed with DNAase I by using an indirect end-labeling technique. Eight DNAase I-hypersensitive sites could be mapped in the flanking regions of the gene in oviduct cells, in which the gene is expressed. The same sensitive sites were detected by utilization of an endogenous nuclease activity present in oviduct nuclei. Only one hypersensitive site was detected in the chromatin from erythrocytes, in which the gene is not expressed. The 3'-terminus of the lysozyme gene is highly exposed in nuclei from both tissues. Of special interest is the hypersensitive site at the 5'-terminus of the actively transcribed gene since it maps at the region of multiple initiation sites of transcription and the putative control regions of steroid hormones. DNAase I-hypersensitive chromatin structures also at a greater distance from the gene may take part in the control of gene expression.

A site and strand spedfic nuciease activity with analogies to topoisomerase I frames the rRNA gene ofTetrahymena

Exposure of macronuclear chromatin from Tetrahymena thermophila to sodium dodecyl sulfate causes an endogenous nuclease to cleave the extra-chromosomal rDNA at specific sites. All cuts are single-strand cleavages specific to the non-coding strand. Three cleavages map in the central non-transcribed spacer of the palindromic molecule at positions -1000, -600 and -150 bp with respect to the transcription initiation point. A fourth site is located close to the transcription termination point, while no cleavage is observed in the coding region. The position of each cleavage is in the immediate neighbourhood of DNAse I hypersensitive sites. Additionally, certain DNA sequence motifs are repeated in the region around the cleavages. Upon cleavage induction a protein becomes attached to the rDNA. Our results indicate covalent binding to the generated 3' end, in analogy to the aborted reaction of topoisomerase I.

Nuclear-hormone mediated changes in chromatin solubility

Rat liver nuclei were incubated with either thyroid hormone or angiotensin (AII) at varying concentrations or with buffer (control) prior to digestion with micrococcal nuclease. Concentrations of hormones greater than 10 −10M were effective in increasing the solubilization of chromatin with physiological levels (10 −9M) of AII showing an approximate 2.4 fold increase over control. Nuclei were also isolated from animals treated in- vivo with either AII or buffer (control) and chromatin solubility was increased in the AII treated nuclei even prior to the addition of exogenous nuclease, presumably from the action of endogenous nucleases. The data suggest that hormone-induced increases in solubility are a reflection of structural changes in chromatin which enhance the accessibility of DNA to endonuclease attack.

Influence of disulfide-reducing agents on fractionation of the chromatin complex by endogenous nucleases and deoxyribonuclease I in aging mice.

Age-related alterations in chromatin were evaluated by a fractionation procedure involving limited digestion of liver cell nuclei from young adult and old mice with endogenous nucleases and deoxyribonuclease I. The results suggest that in the chromatin the bulk of the nuclear deoxyribonucleic acid (DNA) may be folded or compacted into domains or loops and associated at a few points with a nuclear protein skeleton structure represented by the final pellet fraction which is resistant to deoxyribonuclease I. Disulfide bonds appear to play a role in the linkage of regions of DNA with this nuclear protein skeleton structure. The amount of DNA that could be released from the pellet fraction by disulfide-reducing agents was significantly greater with old than with young adult mice. In addition, the amount of chromatin material released into the first soluble fraction decreased and that in the second soluble fraction increased with age. Treatment of the nuclei with disulfide-reducing agents did not correct this particular age-related change. Previous results had shown a similar alteration with age of chromatin subjected to digestion by micrococcal nuclease. The combined results suggest the existence of a supranucleosomal alteration in chromatin structure during aging.

Endogenous nuclease. Properties and effects on transcribed genes in chromatin.

Endogenous nuclease. Properties and effects on transcribed genes in chromatin.

Reformation of the nuclear envelope in melanophores during recovery from a hormone plus puromycin treatment

Previous experiments have shown that treatment of the melanophores of Pachymedusa (Agalychnis) dacnicolor with Melanophore-Stimulating Hormone (MSH) + puromycin causes the nuclear envelope to breakdown leading to the formation of discontinuous vesicles and the hyperdispersion of chromatin. We show here that these cells recover, reform their nuclear envelopes, and recondense their chromatin, both in the presence and in the absence of actinomycin D (actD). After recovery, these cells respond to MSH by melanosome dispersion. From these results, the following conclusions or observations are drawn: 1. 1, Reformation of nuclear envelope does not require that the chromatin be condensed into chromosomes as in mitosis. 2. 2, The new nuclear envelope is derived primarily from reutilization of the membrane vesicles produced during nuclear envelope breakdown, somewhat similar to mitosis. There may also be contributions from other membranous organelles. 3. 3, The hyperdispersed chromatin appears not to be subject to extensive attack by endogenous nucleases as the recovered cells are of good ultrastructure and can respond tropically to MSH. 4. 4, The presence of actD appears not to prevent the conversion of the hyperdispersed chromatin into the normal pattern.

Fractionation of mechanically sheared chromatin on ECTHAM-cellulose

Chromatography of chromatin on the weak ion-exchange resin ECTHAM-cellulose was re-examined using the combined salt-pH elution conditions of Stratling, W.H., Van, N.T. and O'Malley, B.W. (1976) Eur. J. Biochem. 66, 423–433. When mechanically sheared rat liver chromatin was chromatographed on ECTHAM-cellulose the histone composition of eluted fractions was very similar, whereas early eluting fractions were enriched in non-histone proteins, including certain high mobility group proteins, and in hnRNP particles, containing newly synthesised RNA. Later eluting fractions were depleted in all of these components. The majority of hnRNP particles in early eluting chromatin were shown to be physically associated with chromatin by centrifugation in metrizamide. Hen erythrocyte chromatin contained no early eluting material. Size of DNA fragments was not a significant factor in determining the elution position of chromatin fragments. Early eluting material was not generated by endogenous nuclease and protease action. The conditions of chromatin preparation, and of elution of early chromatin fractions caused no gross disruption of chromatin structure, or dissociation of chromatin proteins, although some nucleosome sliding may have occurred. The conditions required for elution of some of the later fractions are sufficient to cause dissociation of protein, and alteration of chromatin conformation.

Endogenous nuclease. Properties and effects on transcribed genes in chromatin

Endogenous nuclease. Properties and effects on transcribed genes in chromatin

DNase I hypersensitive regions correlate with a site-specific endogenous nuclease activity on the r-chromatin of Tetrahymena.

A novel nuclease activity have been detected at three specific sites in the chromatin of the spacer region flanking the 5'-end of the ribosomal RNA gene from Tetrahymena. The endogenous nuclease does not function catalytically in vitro, but is in analogy with the DNA topoisomerases activated by strong denaturants to cleave DNA at specific sites. The endogenous cleavages have been mapped at positions +50, -650 and -1100 relative to the 5'-end of the pre-35S rRNA. The endogenous cleavage sites are associated with micrococcal nuclease hypersensitive sites and DNase I hypersensitive regions. Thus, a single well-defined micrococcal nuclease hypersensitive site is found approximately 130 bp upstream from each of the endogenous cleavages. Clusters of defined sites, the majority of which fall within the 130 bp regions defined by vicinal micrococcal nuclease and endogenous cleavages, constitute the DNase I hypersensitive regions.

Eukaryotic ternary transcription complexes. II. An approach to the determination of chromatin conformation at the site of transcription.

Digestion of rat liver nuclei by endogenous nucleases or micrococcal nuclease releases a chromatin fraction containing RNA polymerases I and II bound to DNA fragments in ternary transcription complexes. To label the DNA in these transcription complexes, the polymerases were allowed to add radioactively labelled ribonucleotides in vitro to in vivo-initiated RNA chains. During this transcription step, nucleic acids were photochemically cross-linked using 8-methoxypsoralen. Nucleic acids in transcription complexes were then sized by gel electrophoresis. Under conditions where RNA polymerases I and II were active in vitro, most of the labelled DNA was found in a series of fragments of sizes which were multiples of approximately 200 base-pairs. When polymerase I alone was active, the smallest member of this series carried the bulk of the label; when polymerase II also was active, a significant proportion of the label was carried on the dimer and higher oligomers. Proteins other than polymerase alone are shown to be responsible for the pattern of DNA fragments protected from nucleases. Therefore active RNA polymerases I and II in vivo are in close proximity to structures protecting DNA fragments, the sizes of which are similar to those found in nucleosomes. We have yet to establish that these structures are composed of histones.

Eukaryotic ternary transcription complexes. I. The release of ternary transcription complexes of RNA polymerases I and II by the endogenous nucleases of rat liver nuclei.

Autodigestion of rat liver nuclei in magnesium-containing buffers leads to the release of about 80% of DNA-dependent RNA polymerases I and II, together with 4 to 8% of the DNA. The RNA polymerases are at least partially DNA bound as judged by the effect on in vitro transcription of (1) Actinomycin D, (2) preirradiation of the enzymes in the presence of 8-methoxypsoralen and (3) heparin. The released DNA migrates as ladders of nicked nucleosome-size fragments on electrophoresis. On sucrose gradients, most RNA polymerase activity sediments as two peaks: one slightly smaller than the 11S mononucleosome and the other with the dinucleosome. The released material can act as a source of ternary transcription complexes for further structural studies.

Isolation of a mouse DNA fraction which encodes more informational than non informational RNA sequences.

A small fraction of single-stranded DNA (ssDNA), amounting to 1.5--2% of the total DNA, was isolated from native nuclear DNA of mouse Ascites tumour cells. In RNA-driven annealing reactions, 40--42% of the ssDNA, (labelled with [3H]-thymidine or 125I) could be hybridized to cytoplasmic RNA was compared with 4--4.5% for the non repetitious component of bulk DNA and 60--63% of the ssDNA could be hybridized to nuclear RNA as compared with 20--22% for bulk DNA. It was also found that most of the ssDNA sequences which are hybridizable to homologous RNA, consisted of non self-reassociating DNA regions (which can be reassociated with non repetitious bulk DNA). These findings complete earlier data obtained in other cell systems and demonstrate that ssDNA mainly originates from active DNA transcription sites which encode informational RNA sequences. It is proposed that ssDNA is formed via selective endogenous nuclease attacks presumably at an early stage of the DNA purification procedure.

Macronuclear chromatin organization in Paramecium primaurelia.

Chromatin organization and transcriptional activity in the macronucleus of the ciliate Paramecium primaurelia have been studied by electron microscopic examination of spread chromatin preparations. Under spreading conditions which inhibit the activity of endogenous nucleases and proteases, various morphologically distinct types of chromatin are seen: (1) Largely condensed chromatin in the form of 30-40 nm supranucleosomal globules which aggregate to form knobbly 40-60 nm fibres and unravel into 12 nm nucleosomal filaments; (2) In rapidly growing cells, extended chromatin arranged groups of 4-8 adjacent nucleofilaments, each bearing numerous and very densely packed lateral fibrils. These fibrils are 20 nm thick, have a smooth outline and extend to lengths of up to 3 micron. Such transcripts are grouped in units along the chromatin axis and are apparently organized in register in adjacent filaments; (3) In starved cells, extended nucleofilaments bearing only occasional RNP fibrils. These various morphological characteristics are discussed in terms of macronuclear transcriptional activity and the polyploid nature of macronuclei.

Chromatin changes in apoptosis.

Murine lymphoid cell lines and rat thymocytes treated in vitro with glucocorticoid hormones provide a convenient system for studying the nuclear changes in apoptosis. Morphologically the nucleolus disintegrates and chromatin undergoes an unusual generalized condensation. This is associated with excision of most of the nuclear DNA to short but well-organized chains of nucleosomes, apparently by an endogenous non-lysosomal nuclease. The process is dependent upon macromolecular synthesis and probably is mediated, at least remotely, by the classical steroid-receptor-gene activation pathway. A similar process of chromatin condensation and excision can be produced by the calcium-magnesium ionophore A23187. In other circumstances of 'programmed cell death', analogous chromatin condensation, excision and requirements for macromolecular synthesis have been documented.

Messenger RNA sequences in nuclear ribonucleoprotein particles are complexed with protein as shown by nuclease protection

Soon after their transcription, eukaryotic messenger RNA precursors (heterogeneous nuclear RNA) are assembled into ribonucleoprotein particles (hnRNP). In the present experiments we have asked whether the mRNA-homologous components of hnRNA are complexed with proteins in hnRNP or, alternatively, exist as stretches of naked RNA in the particles. Mild digestion of HeLa cell hnRNP particles by micrococcal nuclease, pancreatic RNAase or an endogenous particle-bound nuclease, followed by hybridization of the surviving RNA with a complementary DNA probe for poly(A) + mRNA sequences reveals that messengerhomologous sequences are protected from nuclease attack in hnRNP.

Isolation of polysomes from larval and adult Drosophila melanogaster

A method has been developed for the isolation of polysomes from adult Drosophila melanogaster. The profiles are not degraded by endogenous nucleases at 4°C, but are sensitive to added ribonuclease A. Cycloheximide added to the preparation does not alter the profile, indicating that chain elongation leading to loss of polysomes is not occurring under these conditions. Resuspension of the polysome pellets in high-salt buffer leads to dissociation of inactive ribosomes which is reversible. The addition of N-ethylmaleiide, cytidine 5′-monophosphoric acid, diethyl sulfate, and polyvinylsulfate does not improve the profiles. The profiles are dependent on added magnesium ion. Polyadenylated RNA was extracted from the polysomes using the guanidinium chloride method and oligo(dT)-cellulose chromatography. The RNA was translated in a rabbit reticulocyte lysate cell-free system and the translation products were analyzed by polyacrylamide gel electrophoresis.

A transcriptionally enriched chromatin fraction released by a Mg ++-dependent endogenous nuclease from rat liver

When rat liver nuclei are subjected to endogenous nuclease digestion in the Presence of 5 mM Mg ++, they release a chromatin fraction containing DNA of well defined fragment lengths and DNA-dependent RNA polymerases. The DNA is enriched 4.4-fold for sequences complementary to cDNA prepared to cytoplasmic poly-A-containing RNA when compared with whole cell DNA. This method of obtaining a transcriptionally enriched chromatin fraction is compared with other reported nuclease methods.