Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.21.1 (DNase)
7,655 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Intact nuclei derived from poorly or highly liver-metastatic murine large-cell lymphoma cell line RAW117 were digested to discrete subchromatin deoxyribonucleoprotein/ribonucleoprotein (DNP/RNP) complexes with Msp-I. The DNP/RNP complexes were composed of DNP/RNPs which were derived from the DNP/RNP complexes by incubation in the presence or absence of DNase-I and subsequent isolation by two-dimensional [isoelectric focusing/sodium dodecylsulfate (SDS)] polyacrylamide gel electrophoresis (PAGE), electroelution from the gel, and removal of SDS. Approximately 450 DNP/RNPs in the two-dimensional gels corresponding to discrete spots or in some cases streaks were analyzed for the presence of v-abl, p53, c-neu, c-H-ras, beta-casein, 18s rDNA, and mu-chain immunoglobulin genes using a hybridization technique. Ten DNP/RNP complexes contained tightly associated p53 DNA, whereas six contained c- or v-abl, four contained mu-chain gene, two contained c-H-ras, one contained dot-blot beta-casein, two contained 18s rDNA, and c-neu was found in one of the DNP/RNPs. The DNP/RNPs were also analyzed for in vitro RNA polymerase and primase activities. To assess the potential transcription abilities of the isolated DNP/RNPs, individual DNP/RNPs or DNP/RNP mixtures (reconstituted after SDS-PAGE separation) were examined for RNA polymerase initiation and synthesis. When RNA products were formed, these were purified by extracellulose chromatography and used as back-hybridization probes for the genes of interest. The RNA products were also analyzed by RNA gel electrophoresis. RNA formation was inhibitable by actinomycin D, and the RNAs formed ranged in size from approximately 80 kbp to approximately 1 kbp. By mixing various DNP/RNP complexes together, different patterns of RNA synthesis were found. For example, one DNP/RNP of M(r) approximately 140,000, isoelectric point (pl) approximately 5.8 synthesized a high molecular weight RNA in vitro that hybridized with beta-casein cDNA, but beta-casein is not expressed in RAW117 cells, suggesting that the silencing of the beta-casein gene was negated by isolation of the DNP/RNP. Mixing this DNP/RNP with two other specific DNP/RNPs again inhibited the synthesis of beta-casein RNA, suggesting that interactions between DNP/RNP complexes can result in differential RNA expression or regulation of RNA polymerases in vitro.
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PMID:Nucleoproteins derived from subnuclear RNA polymerase complexes of metastatic large-cell lymphoma cells possess transcription activities and regulatory properties in vitro. 146 66

The DNA from a wide variety of human tumors has sustained mutations within the conserved p53 coding regions. We have purified wild-type and tumor-derived mutant p53 proteins expressed from baculovirus vectors and examined their interactions with SV40 DNA. Using DNAase I footprinting assays, we observed that both human and murine wild-type p53 proteins bind specifically to sequences adjacent to the late border of the viral replication origin. By contrast, mutant p53 proteins failed to bind specifically to these sequences. SV40 T antigen prevented wild-type p53 from interacting with this region. These data show that normal but not oncogenic forms of p53 are capable of sequence-specific interactions with viral DNA. Furthermore, they provide insights into the mechanisms by which viral proteins might regulate the control of viral growth and cell division.
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PMID:Wild-type but not mutant p53 immunopurified proteins bind to sequences adjacent to the SV40 origin of replication. 164 78

We have compared the ATPase, DNA-binding, and helicase activities of free simian virus 40 (SV40) large T antigen (To) and T antigen complexed with cellular p53 (T+p53). Each activity is essential for productive viral infection. The T+p53 and To fractions were prepared by sequential immunosorption of infected monkey cells with monoclonal antibodies specific for p53 and T antigen. The immune-complexed T fractions were then assayed in parallel. For ATP hydrolysis, the Vmax for T+p53 was 143 nmol of ADP per min per mg of protein, or 18-fold greater than for To. ATP had no effect on the stability of the T+p53 complex. The T+p53 complex was significantly more active than To in hydrolyzing dATP, dGTP, GTP, and UTP. Of the nucleotide substrates tested, the greatest relative increase (T+p53/To) was in hydrolyzing dGTP and GTP. In DNase footprinting assays performed under replication conditions, the T+p53 complex protected regions I, II, and III of origin DNA while equivalent amounts of To protected only regions I and II. Region III is known to contribute to the efficiency of DNA replication and contains the SP1-binding sites of the early viral promoter. The T+p53 fraction was also a more efficient helicase than To, especially with a GC-rich primer and template. Thus, the T+p53 complex has enhanced ATPase, GTPase, DNA-binding, and helicase activities. These findings imply that complex formation between cellular monkey p53 and SV40 T antigen modulates a number of essential activities of T in SV40 productive infection.
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PMID:The p53 complex from monkey cells modulates the biochemical activities of simian virus 40 large T antigen. 252 75

p53 is a nuclear protein associated with cellular transformation and normal cellular proliferation. Some transformed cells have been found to have one or several quantitative or qualitative abnormalities of p53. We studied expression, kinetics, phosphorylation, DNA methylation and chromatin structure of p53 in resting and proliferating untransformed T-lymphocytes and in human T-cell leukemia virus type I transformed T-lymphocytes from the same individuals. p53 expression is indistinguishable in transformed compared to untransformed proliferating T-lymphocytes by: (1) p53 mRNA levels, (2) rate of synthesis and stability of p53 protein, (3) change in protein stability after exposure to an inhibitor of protein synthesis, (4) presence of phosphorylation of the p53 protein. Resting T-lymphocytes from these same individuals did not express p53. No difference in DNA methylation and chromatin structure of the p53 gene was observed in either resting or proliferating untransformed, or virally transformed T-lymphocytes. The gene was fully methylated and resistant to DNAase I over its entire coding region but was demethylated and contained DNAase I hypersensitive sites in a distinct region 5' of the site of initiation of transcription.
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PMID:Expression, methylation and chromatin structure of the p53 gene in untransformed and human T-cell leukemia virus type I-transformed human T-lymphocytes. 265 79

The intracellular distribution of the transformation-associated cellular protein p53 was studied by indirect immunofluorescence in a series of adenovirus-transformed rodent cells. In most adenovirus 2 or 5 (group C) transformed cell lines p53 was detected in discrete areas in nuclei and in all cell lines p53 was also present in a perinuclear structure. The adenovirus 2 or 5 E1B-58 kD protein, previously found to form molecular complexes with p53 in group C transformed cells, colocalized with p53 in both intracellular locations. Further studies on the region of the cell corresponding to the perinuclear body containing p53 showed that it frequently included the centrosomes of the transformed cell. The intranuclear p53 was released by mild DNAase I digestion.
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PMID:The intracellular distribution of the transformation-associated protein p53 in adenovirus-transformed rodent cells. 329 Aug 6

Using a serum from a patient with an autoimmune disease, we have recently described a novel 55 000-dalton antigen (p55) in the nucleus of several animal cells including human ones. This antigen, designated PSL, was not related to the previously defined antigens recognized by sera from patients with systemic rheumatic diseases (Sm, n-RNP, SS-B, Scl-70). We have now found that p55 is associated with chromatin structures as it is released from the nucleus of mink cell fibroblasts by saline + DNase treatments. Analysis by sucrose gradient centrifugation of the nuclear material released in these conditions indicated that p55 co-migrated with core histones. Meanwhile, p55 was absent from the residual nuclear matrices (achromatinic nuclei). Localization of p55 in synchronized cells was performed by indirect immunofluorescence and immunoprecipitation. P55 appeared to accumulate in the nucleus during the S phase. Finally, it was not recognized by an anti-SV40 tumor serum that specifically precipitated the protein p53, which has been recently related to cell proliferation. Thus, PSL an p53, although apparently not antigenically related, appear to be implicated in the same step of the cell cycle.
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PMID:Characterization by human autoantibody of a nuclear antigen related to the cell cycle. 635 85

The Ku autoantigen is a heterodimer of 70 kDa (p70) and -80 kDa (p80) subunits that is the DNA-binding component of a DNA-dependent protein kinase (DNA-PK). The 350 kDa (p350) catalytic subunit of DNA-PK phosphorylates Sp-1, Oct-1, p53 and RNA polymerase II in vitro, but the precise cellular role of DNA-PK remains unclear. In the present studies, the assembly of p70/p80 heterodimers and the interaction of Ku with DNA was investigated using recombinant vaccinia viruses directing the synthesis of human p70 (p70-vacc) and p80 (p80-vacc), and monoclonal antibodies (mAbs). Expression of human Ku antigens in rabbit kidney (RK13) cells could be demonstrated by immunofluorescent staining because this cell line contains little endogenous Ku. A novel mAb designated 162 stained the nuclei of RK13 cells coinfected with p70-vacc and p80-vacc, but not cells that were infected with either virus alone, suggesting that it recognized the p70/p80 heterodimer but not monomeric p70 or p80. In agreement with the immunofluorescence data, 162 immunoprecipitated both p70 and p80 from extracts of coinfected cells, but did not immunoprecipitate either subunit by itself from extracts of cells infected with p70-vacc or p80-vacc, respectively. Conversely, the binding of 162 to Ku isolated from human K562 cells stabilized the p70/p80 heterodimer under conditions that normally dissociate p70 from p80. The nuclei of cells infected with p70-vacc alone could be stained with mAb N3H10 (anti-p70) and cells infected with p80-vacc alone could be stained with mAb 111 (anti-p80), indicating that the formation of p70/p80 heterodimers was not required for nuclear transport. Finally, free recombinant and cellular p70 both bound to DNA efficiently in vitro, suggesting that free p70, like the p70/p80 heterodimer, serves as a DNA-binding factor. Moreover, free human p70 could be released from the nuclei of p70-vacc-infected RK13 cells by deoxyribonuclease I treatment, suggesting that it was associated with chromatin in vivo. The nuclear transport of free p70 and the association of free p70 with chromatin in vivo raise the possibility that newly synthesized cellular p70 might undergo nuclear transport and DNA-binding prior to dimerization with p80 or assembly with p350.
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PMID:Assembly and DNA binding of recombinant Ku (p70/p80) autoantigen defined by a novel monoclonal antibody specific for p70/p80 heterodimers. 769 19

Apoptosis, or programmed cell death, is defined by morphologic change resulting in nonpathologic cell loss and is relevant to a wide spectrum of biology. The process is best characterized in the nematode Caenorhabditis elegans where ced genes mediate the death of specific cells during development. Some corresponding genes have been identified in mammalian cells. Expression of the mammalian bcl-2 gene (homologous to ced-9) suppresses apoptosis in many systems. The ced-3 gene is homologous to a mammalian protease. Increased levels of the tumor suppressor p53 due to DNA damage may result in either blockage of the cell cycle at G1 or apoptosis. Mutation of p53 is associated with decreased cell death from radiation and cytotoxic drugs. Initiation of the apoptotic pathway may occur as a consequence of conflicting growth signals. Hierarchical relationships variously between bcl-2, p53, myc, and other genes indicate a complex pattern of regulation. Stimuli resulting in apoptosis may cause production of free radicals and increased intracellular calcium concentration. The relationship of these changes to the hallmark of apoptosis, internucleosomal fragmentation of DNA, is unclear, and "laddering" of DNA is not always evident. Apoptotic DNA degradation probably occurs sequentially, initially involving breakage into 50 kilobases or larger fragments. The nuclease(s) responsible have not been identified, but deoxyribonuclease I is implicated. The association between nuclease activation and chromatin condensation is complex, and programmed cell death may be subject to cytoplasmic regulation. Available data suggest that clearer understanding of apoptosis will result in better cancer therapy.
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PMID:Mechanisms of apoptosis: integration of genetic, biochemical, and cellular indicators. 806 87

The testis is a tissue of high proliferative activity. In this organ, sperm cells (spermatozoa) are produced from stem cells (spermatogonia) by two consecutive steps of cell multiplication and spermatid cytodifferentiation. Mitotic proliferation of spermatogonia generates primary spermatocytes which enter meiosis, leading to the generation of spermatids. The number of cells entering meiosis is held constant, since outnumbering spermatogonia or premeiotic spermatocytes are eliminated by apoptosis (programmed cell death). During apoptosis, the nuclear chromatin is internucleosomally degraded by the activity of a Ca2+, Mg2+-dependent endonuclease. Recent data indicate that deoxyribonuclease I (DNase I) is identical to the apoptotic endonuclease responsible for the internucleosomal DNA degradation. Previous results using primers specific for rat parotid DNase I in a polymerase chain reaction have demonstrated the presence of DNase I-specific gene transcripts in rat testis. We have therefore analysed the presence of DNase I in rat testis by immunohistochemistry and biochemical procedures. The presence of DNase I-like endonucleolytic activity was verified enzymatically. DNase I immunoreactivity was detected in the nuclei of a few spermatogonia and premeiotic spermatocytes, but within the acrosomic vesicle of all spermatids and spermatozoa. In situ hybridisation revealed the accumulation of DNase I-specific gene transcripts in a small number of spermatogonia and/or premeiotic spermatocytes, but in a large number of spermatids. The occurrence of apoptotic DNA fragmentation was investigated by in situ end-labelling (ISEL) of free 3'-OH DNA ends and gave positive nuclear staining of only very few spermatogonia. No positive ISEL staining was observed in maturing spermatids and/or spermatozoa. These data support the notion that, within the seminiferous epithelium, the number of primary spermatocytes entering meiosis is controlled by apoptosis. In addition, they demonstrated that mature sperm cells are equipped with an endonuclease that might be used for DNA degradation during their elimination at later stages of their life span. The expression and distribution of the tumour suppressor gene product, p53, was analysed by immunostaining. Strong p53 immunoreactivity was observed in the nuclei of a number of spermatogonia, of some premeiotic spermatocytes and probably in all spermatids. Thus, p53 expression appeared to parallel that of DNase I. In contrast, p53 immunoreactivity was absent in mature spermatozoa present in the lumen of the testicular tubules or the ductus epididymidis. It is therefore proposed that at later stages of spermatid maturation most probably before their release as mature spermatozoa-the p53 gene product was either degraded or retained in residual bodies, since p53 immunoreactivity was found to be concentrated within these organelles.
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PMID:Distribution of deoxyribonuclease I (DNase I) and p53 in rat testis and their correlation with apoptosis. 891 66

A detailed investigation of how nucleosomes are formed and arranged on the DNA sequence is a prerequisite to understanding the molecular mechanisms of DNA-dependent processes such as transcription, replication, DNA repair, and mutagenesis. In this report we analyzed the chromatin structure of exons 5-8 of the p53 gene in human fibroblasts. We mapped at the nucleotide level the positions of DNase I and micrococcal nuclease cleavage sites in permeabilized cells. Areas of clear DNase I protection, which would be indicative of the binding of sequence-specific proteins, were not detected. Instead, the micrococcal nuclease and DNase digestion patterns suggested that this region was covered by nucleosomes and that two areas spanning exons 5 and 6 are occupied preferentially. These nucleosomes could influence DNA damage distribution, repair of certain lesions, and other aspects of the mutagenesis process in p53 sequences.
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PMID:A high-resolution analysis of chromatin structure along p53 sequences. 898 12


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