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)

The presence of actin in eukaryotic nuclei, and, especially, its functional significance has not been well established. We have found that under routine immunocytochemical conditions, no actin can be detected in insect follicle cell nuclei by means of antibody (both mono- and polyclonal) or phalloidin staining. However, a pretreatment of nuclear preparations with two different endonucleases (deoxyribonuclease I or micrococcal nuclease) to remove a substantial amount of chromosomal DNA uncovers the presence of nuclear actin for both antibody and phalloidin detection. Employing the same nuclease digestion followed by antibody or phalloidin staining with squash preparations of Drosophila polytene chromosomes revealed that the nuclear actin is directly associated with the chromosomes. A strong positive signal in the polytene chromosomes obtained with phalloidin labeling not only confirmed the presence of actin in the chromosomes, but indicates that a considerable amount of nuclear actin is present in filamentous form (F-actin) rather than monomeric (G-actin). The detection of actin associated with Xenopus embryo chromosomes suggests the significance of chromosomal actin for diploid vertebrate cells. Using the specific actin disrupting agent cytochalasin D, we have demonstrated the structural significance of nuclear actin in maintaining the linear integrity of polytene chromosomes. Further, we present evidence that RNA polymerase II closely interacts with the chromosomal actin scaffold, and that its association with chromosomes does not require the presence of DNA.
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PMID:An actin infrastructure is associated with eukaryotic chromosomes: structural and functional significance. 752 80

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

The Rad2, Rad3, Rad4, and Ss12 proteins are required for nucleotide excision repair in yeast cells and are homologs of four human proteins which are involved in a group of hereditary repair-defective diseases. We have previously shown that Rad3 protein is one of the five subunits of purified RNA polymerase II basal transcription initiation factor b (TFIIH) and that Ss12 protein physically associates with factor b (W.J. Feaver, J.Q. Svejstrup, L. Bardwell, A.J. Bardwell, S. Buratowski, K.D. Gulyas, T.F. Donahue, E.C. Friedberg, and R.D. Kornberg, Cell 75:1379-1387, 1993). Here we show that the Rad2 and Rad4 proteins interact with purified factor b in vitro. Rad2 (a single-stranded DNA endonuclease) specifically interacts with the Tfb1 subunit of factor b, and we have mapped a limited region of the Rad2 polypeptide which is sufficient for this interaction. Rad2 also interacts directly with Ss12 protein (a putative DNA helicase). The binding of Rad2 and Rad4 proteins to factor b may define intermediates in the assembly of the nucleotide excision repair repairosome. Furthermore, the loading of factor b (or such intermediates) onto promoters during transcription initiation provides a mechanism for the preferential targeting of repair proteins to actively transcribing genes.
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PMID:Yeast nucleotide excision repair proteins Rad2 and Rad4 interact with RNA polymerase II basal transcription factor b (TFIIH). 819 2

A phage T7 class-III promoter (pT7), which is highly specific for T7 RNA polymerase in bacteria, was tested in mammalian cells for its specificity. After having shown that T7 RNA polymerase can transcribe from pT7 in the nucleus of stably transformed cells [Lieber et al., Nucleic Acids Res. 17 (1989) 8485-8493], we describe here that pT7 could also direct efficient intracellular gene expression in the absence of T7 RNA polymerase. Using the genomic human growth hormone-encoding gene and the firefly luciferase-encoding gene as reporters, we found expression levels comparable with those obtained with the Rous sarcoma viral promoter. Inhibition of expression with alpha-amanitin suggests that transcription is by RNA polymerase II. Binding studies with HeLa cell extracts clearly show that synthetic pT7 sequences are specifically bound (gel retardation) and that the promoter region is protected from DNase degradation. The experimental data, as well as the nucleotide sequence, suggest that pT7 has properties of an initiator element. Indeed, the activity of pT7 can be stimulated by the presence of an upstream element or an enhancer. These results have practical implications for the use of pT7 in mammalian expression vectors. Commercial pT7 plasmids can be used for both prokaryotic and eukaryotic expression systems.
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PMID:A phage T7 class-III promoter functions as a polymerase II promoter in mammalian cells. 840 19

Elongation complexes of RNA polymerase II, RNA-DNA-enzyme ternary complexes, are intermediates in the synthesis of all eukaryotic mRNAs and are potential regulatory targets for factors controlling RNA chain elongation and termination. Analysis of such complexes can provide information concerning the structure of the catalytic core of the RNA polymerase and its interactions with the DNA template and RNA transcript. Knowledge of the structure of such complexes is essential in understanding the catalytic and regulatory properties of RNA polymerase. We have prepared and isolated complexes of purified RNA polymerase II halted at defined positions along a DNA template, and we have used deoxyribonuclease I (DNAse I) to map the interactions of the polymerase with the DNA template. DNAse I footprints of three specific ternary complexes reveal that the enzyme-template interactions of individual elongation complexes are not identical. The size of the protected region is distinct for each complex and varies from 48 to 55 bp between different complexes. Additionally, the positioning of the protected region relative to the active site varies in different complexes. Our results suggest that RNA polymerase II is a dynamic molecule and undergoes continual conformational transitions during elongation. These transitions are likely to be important in the processes of transcript elongation and termination and their regulation.
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PMID:Contacts between mammalian RNA polymerase II and the template DNA in a ternary elongation complex. 844 6

Glucocorticoids inhibit transcription of the murine cytoplasmic thymidine kinase gene (Tk-1). Glucocorticoid regulation of Tk-1 transcription can be demonstrated in cells that are arrested in late G1. This observation indicates that inhibition of Tk-1 expression is not dependent upon redistribution within the cell cycle but is due to glucocorticoid regulation of this gene. Transfection studies have been carried out using chimeric genes in which restriction fragments of the Tk-1 promoter were fused to chloramphenicol acetyltransferase or neomycin phosphotransferase. These chimeric reporters were assayed for stable expression and glucocorticoid regulation in P1798 lymphoma cells. A 140-bp fragment, extending from -143 to -3 bp with respect to the thymidine kinase translational start site, was capable of both basal and glucocorticoid-regulated transcription of reporter genes. The extent of inhibition by glucocorticoids was similar to that observed for the endogenous gene, and no increase in basal expression or the extent of inhibition was observed with constructs containing additional 5'-flanking DNA. The 140-bp Tk-1 core promoter fragment binds to transcription factors in extracts from P1798 cells. Control cell extracts contain factors that bind to and protect (from deoxyribonuclease I) a distal promoter element from -106 to -87 bp, relative to the translational start site. A second, proximal element was protected at -43 to -36 bp. The proximal element of the Tk-1 promoter resembles an RNA polymerase II initiator element. No other elements were protected. Glucocorticoids inhibit the amount or activity of the transcription factor that binds to this initiator-like element within the Tk-1 promoter. This element, when fused to upstream activation sequences from the herpes simplex virus thymidine kinase promoter, conveys glucocorticoid sensitivity in cis.
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PMID:Glucocorticoid regulation of a transcription factor that binds an initiator-like element in the murine thymidine kinase (Tk-1) promoter. 896 Dec 64

Sen1p in Saccharomyces cerevisiae is a Type I DNA/RNA helicase. Mutations in the helicase domain perturb accumulation of diverse RNA classes, and Sen1p has been implicated in 3' end formation of non-coding RNAs. Using a combination of global and candidate-specific two hybrid screens, eight proteins were identified that interact with Sen1p. Interactions with three of the proteins were analyzed further: Rpo21p(Rpb1p), a subunit of RNA polymerase II, Rad2p, a deoxyribonuclease required in DNA repair, and Rnt1p (RNase III), an endoribonuclease required for RNA maturation. For all three interactions, the two-hybrid results were confirmed by co-immunoprecipitation experiments. Genetic tests designed to assess the biological significance of the interactions indicate that Sen1p plays functionally significant roles in transcription and transcription-coupled DNA repair. To investigate the potential role of Sen1p in RNA processing and to assess the functional significance of the Sen1p/Rnt1p interaction, we examined U5 snRNA biogenesis. We provide evidence that Sen1p functions in concert with Rnt1p and the exosome at a late step in 3' end formation of one of the two mature forms of U5 snRNA but not the other. The protein-protein and protein-RNA interactions reported here suggest that the DNA/RNA helicase activity of Sen1p is utilized for several different purposes in multiple gene expression pathways.
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PMID:Multiple protein/protein and protein/RNA interactions suggest roles for yeast DNA/RNA helicase Sen1p in transcription, transcription-coupled DNA repair and RNA processing. 1512 1

The presence of phospholipids as a component of chromatin is now well documented and many enzymes such as sphingomyelinase, sphingomyelin-synthase, reverse sphingomyelin-synthase and phosphatidylcholine-dependent phospholipase C have been described and characterised. Other lipids were demonstrated inside the nucleus especially plasmalogens and cholesterol. The chromatin phospholipids, comprising 10% of that present in the nucleus, show a different metabolism with respect to those present in either microsomes or in nuclear membranes; they increase also during the DNA duplication as shown during both liver regeneration and cell maturation. They appear localised near newly synthesized RNA in decondensed chromatin. Digestion of chromatin with RNase, but not with DNase, causes a loss of phospholipids. The composition of the chromatin phospholipid fraction shows an enrichment in sphingomyelin and phosphatidylserine. In this review the behaviour of single lipids in relation to cell proliferation, cell differentiation and apoptosis is described. Sphingomyelin, the lipid most represented in chromatin with respect to microsomes and nuclear membranes, is localised near to newly synthesized RNA, its presence appearing to protect RNA from RNase digestion. This effect is reversed by sphingomyelinase which digests sphingomyelin and, as a consequence, RNA may be hydrolysed. The amount of sphingomyelin is restored by sphingomyelin-synthase. Sphingomyelin increases during the differentiation process and apoptosis. An increase of sphingomyelinase with consequent decrease in sphingomyelin is observed at the beginning of S-phase of the cell cycle. A possible role in stabilising the DNA double helix is indicated. Phosphatidylserine behaves similarly during differentiation and appears to stimulate both RNA and DNA polymerases. Phosphatidylcholine is implicated in cell proliferation through the activation of intranuclear phosphatidylcholine-dependent phospholipase C and diacylglycerol production. The increase in diacylglycerol stimulates phosphatidylcholine synthesis through the major pathway from cytidyltriphosphate. An inhibition of phosphatidylcholine synthesis is responsible for the initiation of apoptosis. The presence of reverse sphingomyelin-synthase favours the formation of phosphatidylcholine, the donor of phosphorylcholine, from sphingomyelin. Little information has been reported for phospatidylethanolamine, but phosphtidylinositol appears to influence cell differentiation and proliferation. This last effect is due to the action of two enzymes: PI-PLCss1 having a role in the onset of DNA synthesis and PC-PLCgamma1 acting in G2 transit. Phosphoinositides also may have an important role: in membrane-stripped nuclei isolated from mitogen stimulated cells a decrease in PIP and PIP2 followed by an increase in diacylglycerol and a translocation of protein kinase C inside the nucleus is observed. On the other hand, overexpression of the enzyme inositol polysphosphate-1-phosphatase reduced DNA synthesis by 50%. Nevertheless, an enhanced rate of phosphorylation has been demonstrated in cells induced to differentiate. These molecules probably favour RNA transcription, counteracting the inhibition of H1 on RNA polymerase II. Plasmalogens were demonstrated in the nucleus and their increase favours the increased activity of phosphatidylcholine-dependent phospholipase C when DNA synthesis starts. Moreover, two forms of cholesterol has been described in chromatin: one, a less soluble sphingomyelin-linked form and a free fraction. Cholesterol increases during liver regeneration, first as a linked fraction and then, when DNA synthesis starts, as a free fraction. The changes of these components have been summarised in relation to cell function in order to give an overview of their possible roles in the different phases of cell duplication and their influence on cell differentiation and during apoptosis. Finally, the relevance of these molecules as intranuclear signals is discussed and future directions are indicated in clarifying pathological process such as tumour cell transformation and the possibility in finding new therapeutic tools.
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PMID:The role of intranuclear lipids. 1551 99

The global inhibition of transcription at the mitotic phase of the cell cycle occurs together with the general displacement of transcription factors from the mitotic chromatin. Nevertheless, the DNase- and potassium permanganate-hypersensitive sites are maintained on potentially active promoters during mitosis, helping to mark active genes at this stage of the cell cycle. Our study focuses on the role of histone acetylation and H3 (Lys-4) methylation in the maintenance of the competency of these active genes during mitosis. To this end we have analyzed histone modifications across the promoters and coding regions of constitutively active, inducible, and inactive genes in mitotic arrested cells. Our results show that basal histone modifications are maintained during mitosis at promoters and coding regions of the active and inducible RNA polymerase II-transcribed genes. In addition we have demonstrated that, together with H3 acetylation and H3 (Lys-4) methylation, H4 (Lys-12) acetylation at the coding regions contributes to the formation of a stable mark on active genes at this stage of the cell cycle. Finally, analysis of cyclin B1 gene activation during mitosis revealed that the former occurs with a strong increase of H3 (Lys-4) trimethylation but not H3 or H4 acetylation, suggesting that histone methyltransferases are active during this stage. These data demonstrate a critical role of histone acetylation and H3 (Lys-4) methylation during mitosis in marking and activating genes during the mitotic stage of the cell cycle.
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PMID:Role of histone modifications in marking and activating genes through mitosis. 1619 28

Basonuclin (Bnc 1) is a transcription factor that has an unusual ability to interact with promoters of both RNA polymerases I and II. The action of basonuclin is mediated through three pairs of evolutionarily conserved zinc fingers, which produce three DNase I footprints on the promoters of rDNA and the basonuclin gene. Using these DNase footprints, we built a computational model for the basonuclin DNA-binding module, which was used to identify in silico potential RNA polymerase II target genes in the human and mouse promoter databases. The target genes of basonuclin show that it regulates the expression of proteins involved in chromatin structure, transcription/DNA-binding, ion-channels, adhesion/cell-cell junction, signal transduction, and intracellular transport. Our results suggest that basonuclin, like MYC, may coordinate transcriptional activities among the three RNA polymerases. But basonuclin regulates a distinctive set of pathways, which differ from that regulated by MYC.
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PMID:Search for basonuclin target genes. 1691 36


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