ABSTRACT
PURPOSE: Increasing the sensitivity of tumor cells to radiation is a major goal of radiotherapy. The present study investigated the radiosensitizing effects of andrographolide and examined the molecular mechanisms of andrographolide-mediated radiosensitization. METHODS AND MATERIALS: An H-ras-transformed rat kidney epithelial (RK3E) cell line was used to measure the radiosensitizing effects of andrographolide in clonogenic assays, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide assays, and a xenograft tumor growth model. The mechanism of andrographolide-sensitized cell death was analyzed using annexin V staining, caspase 3 activity assays, and terminal transferase uridyl nick end labeling assays. The roles of nuclear factor kappa B (NF-kappaB) and Akt in andrographolide-mediated sensitization were examined using reporter assays, electrophoretic mobility shift assays, and Western blotting. RESULTS: Concurrent andrographolide treatment (10 microM, 3 h) sensitized Ras-transformed cells to radiation in vitro (sensitizer enhancement ratio, 1.73). Andrographolide plus radiation (one dose of 300 mg/kg peritumor andrographolide and one dose of 6 Gy radiation) resulted in significant tumor growth delay (27 +/- 2.5 days) compared with radiation alone (22 +/- 1.5 days; p <.05). Radiation induced apoptotic markers (e.g., caspase-3, membrane reversion, DNA fragmentation), and andrographolide treatment did not promote radiation-induced apoptosis. However, the protein level of activated Akt was significantly reduced by andrographolide. NF-kappaB activity was elevated in irradiated Ras-transformed cells, and andrographolide treatment significantly reduced radiation-induced NF-kappaB activity. CONCLUSION: Andrographolide sensitized Ras-transformed cells to radiation both in vitro and in vivo. Andrographolide-mediated radiosensitization was associated with downregulation of Akt and NF-kappaB activity. These observations indicate that andrographolide is a novel radiosensitizing agent with potential application in cancer radiotherapy.
Subject(s)
Apoptosis , Diterpenes/pharmacology , Genes, ras , Radiation Tolerance/drug effects , Radiation-Sensitizing Agents/pharmacology , Animals , Annexin A5/analysis , Apoptosis/drug effects , Apoptosis/physiology , Apoptosis/radiation effects , Caspase 3/metabolism , Cell Line, Transformed , Coloring Agents , DNA Fragmentation , Down-Regulation , Mice , Mice, Nude , NF-kappa B/metabolism , Proto-Oncogene Proteins c-akt/metabolism , RNA Nucleotidyltransferases/analysis , Radiation Tolerance/genetics , Rats , Tetrazolium Salts , Thiazoles , Transplantation, Heterologous , ras Proteins/metabolismABSTRACT
Binding of a pre-mRNA substrate triggers spliceosome activation, whereas the release of the mRNA product triggers spliceosome disassembly. The mechanisms that underlie the regulation of these rearrangements remain unclear. We find evidence that the GTPase Snu114p mediates the regulation of spliceosome activation and disassembly. Specifically, both unwinding of U4/U6, required for spliceosome activation, and disassembly of the postsplicing U2/U6.U5.intron complex are repressed by Snu114p bound to GDP and derepressed by Snu114p bound to GTP or nonhydrolyzable GTP analogs. Further, similar to U4/U6 unwinding, spliceosome disassembly requires the DExD/H box ATPase Brr2p. Together, our data define a common mechanism for regulating and executing spliceosome activation and disassembly. Although sequence similarity with EF-G suggests Snu114p functions as a molecular motor, our findings indicate that Snu114p functions as a classic regulatory G protein. We propose that Snu114p serves as a signal-dependent switch that transduces signals to Brr2p to control spliceosome dynamics.
Subject(s)
RNA, Small Nuclear/metabolism , Repressor Proteins/physiology , Ribonucleoprotein, U5 Small Nuclear/physiology , Saccharomyces cerevisiae Proteins/physiology , Spliceosomes/metabolism , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , DEAD-box RNA Helicases , Guanosine Diphosphate/metabolism , Guanosine Diphosphate/pharmacology , Guanosine Triphosphate/metabolism , Guanosine Triphosphate/pharmacology , Introns/genetics , Models, Biological , Mutation/genetics , Nucleic Acid Conformation/drug effects , RNA Helicases/analysis , RNA Nucleotidyltransferases/analysis , RNA Precursors/metabolism , RNA Splicing/genetics , RNA Splicing Factors , RNA, Small Nuclear/analysis , RNA, Small Nuclear/chemistry , Repressor Proteins/genetics , Ribonucleoprotein, U5 Small Nuclear/genetics , Ribonucleotides/pharmacology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/physiology , Saccharomyces cerevisiae Proteins/analysis , Saccharomyces cerevisiae Proteins/genetics , Spliceosomes/chemistry , Spliceosomes/drug effectsABSTRACT
Spb4p is a putative ATP-dependent RNA helicase that is required for synthesis of 60S ribosomal subunits. Polysome analyses of strains genetically depleted of Spb4p or carrying the cold-sensitive spb4-1 mutation revealed an underaccumulation of 60S ribosomal subunits. Analysis of pre-rRNA processing by pulse-chase labeling, northern hybridization, and primer extension indicated that these strains exhibited a reduced synthesis of the 25S/5.8S rRNAs, due to inhibition of processing of the 27SB pre-rRNAs. At later times of depletion of Spb4p or following transfer of the spb4-1 strain to more restrictive temperatures, the early pre-rRNA processing steps at sites A0, Al, and A2 were also inhibited. Sucrose gradient fractionation showed that the accumulated 27SB pre-rRNAs are associated with a high-molecular-weight complex, most likely the 66S pre-ribosomal particle. An HA epitope-tagged Spb4p is localized to the nucleolus and the adjacent nucleoplasmic area. On sucrose gradients, HA-Spb4p was found almost exclusively in rapidly sedimenting complexes and showed a peak in the fractions containing the 66S pre-ribosomes. We propose that Spb4p is involved directly in a late and essential step during assembly of 60S ribosomal subunits, presumably by acting as an rRNA helicase.
Subject(s)
Polyribosomes/metabolism , RNA Nucleotidyltransferases/physiology , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/enzymology , Cell Nucleolus/chemistry , DEAD-box RNA Helicases , RNA Nucleotidyltransferases/analysis , RNA Nucleotidyltransferases/genetics , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional , RNA, Ribosomal/biosynthesis , RNA, Ribosomal, 5.8S/biosynthesis , Recombinant Fusion ProteinsABSTRACT
Vasa, a DEAD box mRNA helicase similar to eIF4A, is involved in pole plasm assembly in the Drosophila oocyte and appears to regulate translation of oskar and nanos mRNAs. However, several vasa alleles exhibit a wide range of early oogenesis phenotypes. Here we report a detailed analysis of Vasa function during early oogenesis using novel as well as previously identified hypomorphic vasa alleles. We find that vasa is required for the establishment of both anterior-posterior and dorsal-ventral polarity of the oocyte. The polarity defects of vasa mutants appear to be caused by a reduction in the amount of Gurken protein at stages of oogenesis critical for the establishment of polarity. Vasa is required for translation of gurken mRNA during early oogenesis and for achieving wild-type levels of gurken mRNA expression later in oogenesis. A variety of early oogenesis phenotypes observed in vasa ovaries, which cannot be attributed to the defect in gurken expression, suggest that vasa also affects expression of other mRNAs.
Subject(s)
Cell Polarity/physiology , Drosophila Proteins , Drosophila/cytology , Insect Proteins/genetics , Oocytes/cytology , RNA Helicases , RNA Nucleotidyltransferases/physiology , Transforming Growth Factor alpha , Transforming Growth Factors/genetics , Animals , DEAD-box RNA Helicases , Drosophila/genetics , Female , Gene Expression Regulation, Developmental/physiology , Mutation , Oocytes/chemistry , Oogenesis/genetics , Ovary/chemistry , Protein Biosynthesis/physiology , RNA Nucleotidyltransferases/analysis , RNA, Messenger/metabolismABSTRACT
Putative ATP-dependent RNA helicases are ubiquitous, highly conserved proteins that are found in most organisms and they are implicated in all aspects of cellular RNA metabolism. Here we present the functional characterization of the Dbp7 protein, a putative ATP-dependent RNA helicase of the DEAD-box protein family from Saccharomyces cerevisiae. The complete deletion of the DBP7 ORF causes a severe slow-growth phenotype. In addition, the absence of Dbp7p results in a reduced amount of 60S ribosomal subunits and an accumulation of halfmer polysomes. Subsequent analysis of pre-rRNA processing indicates that this 60S ribosomal subunit deficit is due to a strong decrease in the production of 27S and 7S precursor rRNAs, which leads to reduced levels of the mature 25S and 5.8S rRNAs. Noticeably, the overall decrease of the 27S pre-rRNA species is neither associated with the accumulation of preceding precursors nor with the emergence of abnormal processing intermediates, suggesting that these 27S pre-rRNA species are degraded rapidly in the absence of Dbp7p. Finally, an HA epitope-tagged Dbp7 protein is localized in the nucleolus. We propose that Dbp7p is involved in the assembly of the pre-ribosomal particle during the biogenesis of the 60S ribosomal subunit.
Subject(s)
RNA Helicases , RNA Nucleotidyltransferases/physiology , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Cell Nucleolus/chemistry , Cloning, Molecular , DEAD-box RNA Helicases , Molecular Sequence Data , Mutation , Open Reading Frames/genetics , RNA Nucleotidyltransferases/analysis , RNA Nucleotidyltransferases/genetics , RNA Precursors/genetics , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional/genetics , RNA, Ribosomal/biosynthesis , RNA, Ribosomal, 5.8S/biosynthesis , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/growth & developmentABSTRACT
Identification and manipulation of the germ line are important to the study of model organisms. Although zebrafish has recently emerged as a model for vertebrate development, the primordial germ cells (PGCs) in this organism have not been previously described. To identify a molecular marker for the zebrafish PGCs, we cloned the zebrafish homologue of the Drosophila vasa gene, which, in the fly, encodes a germ-cell-specific protein. Northern blotting revealed that zebrafish vasa homologue (vas) transcript is present in embryos just after fertilization, and hence it is probably maternally supplied. Using whole-mount in situ hybridization, we investigated the expression pattern of vas RNA in zebrafish embryos from the 1-cell stage to 10 days of development. Here we present evidence that vas RNA is a germ-cell-specific marker, allowing a description of the zebrafish PGCs for the first time. Furthermore, vas transcript was detected in a novel pattern, localized to the cleavage planes in 2- and 4-cell-stage embryos. During subsequent cleavages, the RNA is segregated as subcellular clumps to a small number of cells that may be the future germ cells. These results suggest new ways in which one might develop techniques for the genetic manipulation of zebrafish. Furthermore, they provide the basis for further studies on this novel RNA localization pattern and on germ-line development in general.
Subject(s)
Cleavage Stage, Ovum/chemistry , Gene Expression Regulation, Developmental/physiology , Germ Cells/chemistry , RNA Helicases , RNA Nucleotidyltransferases/genetics , RNA, Messenger/analysis , Zebrafish/genetics , Amino Acid Sequence , Animals , Cloning, Molecular , DEAD-box RNA Helicases , DNA, Complementary/genetics , Genetic Markers , Molecular Sequence Data , RNA Nucleotidyltransferases/analysis , Sequence Homology, Amino Acid , Somites/chemistry , Zebrafish ProteinsABSTRACT
Two polypeptides of 50 and 45 kDa were adenylated by incubation of a mitochondrial extract from Leishmania tarentolae with [alpha-32P]ATP. These proteins were components of a complex that sedimented at 20S in glycerol gradients and migrated as a single band of approximately 1800 kDa in a native gel. The facts that RNA ligase activity cosedimented at 20S and that the ATP-labeled p45 and p50 polypeptides were deadenylated upon incubation with a ligatable RNA substrate suggested that these proteins may represent charged intermediates of a mitochondrial RNA ligase. Hybridization of native gel blots with guide RNA (gRNA) probes showed the presence of gRNA in the previously identified T-IV complexes that sedimented in glycerol at 10S and contained terminal uridylyl transferase (TUTase) activity, and also in a previously unidentified class of heterodisperse complexes that sedimented throughout the gradient. gRNAs were not detected in the p45 + p50-containing 1800 kDa complex. The heterodisperse gRNA-containing complexes were sensitive to incubation at 27 degrees C and appear to represent complexes of T-IV subunits with mRNA. Polyclonal antiserum to a 70 kDa protein that purified with terminal uridylyl transferase activity was generated, and the antiserum was used to show that this p70 polypeptide was a component of both the T-IV and the heterodisperse gRNA-containing complexes. We propose that the p45 + p50-containing 1800 kDa complex and the p70 + gRNA-containing heterodisperse complexes interact in the editing process. Further characterization of these various complexes should increase our knowledge of the biochemical mechanisms involved in RNA editing.
Subject(s)
Leishmania/chemistry , Mitochondria/chemistry , Protozoan Proteins/chemistry , Ribonucleoproteins/chemistry , Adenine Nucleotides/metabolism , Animals , RNA Editing , RNA Ligase (ATP)/analysis , RNA Nucleotidyltransferases/analysis , RNA, Guide, Kinetoplastida/analysis , RNA, Messenger/analysis , RNA, Protozoan/analysisABSTRACT
In investigating the composition of stored (maternal) mRNP particles in Xenopus oocytes, attention has focussed primarily on the phosphoproteins pp60/56, which are Y-box proteins involved in a general packaging of mRNA. We now identify a third, abundant, integral component of stored mRNP particles, Xp54, which belongs to the family of DEAD-box RNA helicases. Xp54 was first detected by its ability to photocrosslink ATP. Subsequent sequence analysis identifies Xp54 as a member of a helicase subfamily which includes: human p54, encoded at a chromosomal breakpoint in the B-cell lymphoma cell line, RC-K8; Drosophila ME31B, encoded by a maternally-expressed gene, and Saccharomyces pombe Ste13, cloned by complementation of the sterility mutant ste13. Expression studies reveal that the gene encoding Xp54 is transcribed maximally at early oogenesis: no transcripts are detected in adult tissues, other than ovary. Using a monospecific antibody raised against native Xp54, its presence in mRNP particles is confirmed by immunoblotting fractions bound to oligo(dT)-cellulose and separated by rate sedimentation and buoyant density. On isolating Xp54 from mRNP particles, it is shown to possess an ATP-dependent RNA helicase activity. Possible functions of Xp54 are discussed in relation to the assembly and utilization of mRNP particles.
Subject(s)
Oocytes/enzymology , RNA Nucleotidyltransferases/chemistry , Ribonucleoproteins/chemistry , Amino Acid Sequence , Animals , Base Sequence , Cells, Cultured , Centrifugation, Density Gradient , Cloning, Molecular , DNA, Complementary , Embryo, Nonmammalian/metabolism , Female , Humans , Molecular Sequence Data , Polymerase Chain Reaction , Protein Conformation , RNA Helicases , RNA Nucleotidyltransferases/analysis , RNA Nucleotidyltransferases/genetics , RNA Nucleotidyltransferases/immunology , RNA, Messenger , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Xenopus laevisABSTRACT
In Saccharomyces cerevisiae, ribosomal biogenesis takes place primarily in the nucleolus, in which a single 35S precursor rRNA (pre-rRNA) is first transcribed and sequentially processed into 25S, 5.8S, and 18S mature rRNAs, leading to the formation of the 40S and 60S ribosomal subunits. Although many components involved in this process have been identified, our understanding of this important cellular process remains limited. Here we report that one of the evolutionarily conserved DEAD-box protein genes in yeast, DBP3, is required for optimal ribosomal biogenesis. DBP3 encodes a putative RNA helicase, Dbp3p, of 523 amino acids in length, which bears a highly charged amino terminus consisting of 10 tandem lysine-lysine-X repeats ([KKX] repeats). Disruption of DBP3 is not lethal but yields a slow-growth phenotype. This genetic depletion of Dbp3p results in a deficiency of 60S ribosomal subunits and a delayed synthesis of the mature 25S rRNA, which is caused by a prominent kinetic delay in pre-rRNA processing at site A3 and to a lesser extent at sites A2 and A0. These data suggest that Dbp3p may directly or indirectly facilitate RNase MRP cleavage at site A3. The direct involvement of Dbp3p in ribosomal biogenesis is supported by the finding that Dbp3p is localized predominantly in the nucleolus. In addition, we show that the [KKX] repeats are dispensable for Dbp3p's function in ribosomal biogenesis but are required for its proper localization. The [KKX] repeats thus represent a novel signaling motif for nuclear localization and/or retention.
Subject(s)
RNA Helicases , RNA Nucleotidyltransferases/genetics , RNA Precursors/metabolism , RNA Processing, Post-Transcriptional/physiology , RNA, Ribosomal/metabolism , RNA-Binding Proteins , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Cell Nucleolus/chemistry , DEAD-box RNA Helicases , Molecular Sequence Data , RNA Nucleotidyltransferases/analysis , RNA Nucleotidyltransferases/physiology , RNA, Fungal/metabolism , Repetitive Sequences, Nucleic Acid , Restriction Mapping , Ribosomes/metabolism , Saccharomyces cerevisiae/growth & development , Sequence DeletionABSTRACT
We have previously purified and characterized wheat germ DNA polymerases A and B. To determine the role played by DNA polymerases A and B in DNA replication, we have measured the level of their activities during wheat embryo germination. The level of cellular proteins known to be associated with DNA synthesis such as PCNA and DNA primase were also investigated. The activity of DNA polymerase A gradually increased reaching a maximal level at 12 h after germination. Three days later, only a residual activity was detected. DNA polymerase B showed the same pattern during germination with very similar changes in activity. Our results indicate a striking correlation between maximal activities of DNA polymerase A, DNA polymerase B and optimal levels of DNA synthesis. These results support a replicative role of these enzymes. The activity of wheat DNA primase that copurifies with DNA polymerase A also increases during wheat germination. Taking together all its properties, and in spite of its behaviour with some inhibitors. DNA polymerase A may be considered as the plant counterpart of animal DNA polymerase alpha. Concerning DNA polymerase B we have previously shown that PCNA stimulates its processivity. Besides studying the changes of DNA polymerases A and B and DNA primase we have also studied changes in PCNA during germination. We show that PCNA is present in wheat embryos at a constant relatively high level during the first 24 h of germination. After 48 h, the absence of PCNA is concomitant with an important decrease in DNA polymerase B activity. In this report we confirm the behaviour of DNA polymerase B as a delta-like activity.
Subject(s)
DNA Replication , DNA, Plant/biosynthesis , DNA-Directed DNA Polymerase/metabolism , Germination/physiology , Seeds/metabolism , Animals , Aphidicolin/pharmacology , Cattle , DNA Polymerase I/metabolism , DNA Polymerase III/metabolism , DNA Primase , Dideoxynucleotides , Enzyme Inhibitors/pharmacology , Proliferating Cell Nuclear Antigen/analysis , Proliferating Cell Nuclear Antigen/immunology , RNA Nucleotidyltransferases/analysis , RNA Nucleotidyltransferases/immunology , Seeds/enzymology , Thymine Nucleotides/pharmacology , Thymus Gland/chemistry , Time Factors , Triticum/embryology , Triticum/enzymology , Triticum/metabolismABSTRACT
CCA1 codes for mitochondrial, cytosolic, and nuclear ATP(CTP):tRNA nucleotidyltransferase. Studies reported here examine the mechanisms leading to and the consequences of altering the distribution of this important tRNA processing enzyme. We show that the majority of Cca1p-I, translated from the first in-frame ATG, is in mitochondria but surprisingly, there is a small contribution to nuclear and cytosolic tRNA processing by this form as well. The majority of Cca1p-II and Cca1p-III, translated from ATG2 and ATG3, respectively, is in the cytosol but both are also located in the nucleus for processing precursors. Altering the cytosolic/nuclear distribution of Cca1p by fusing the SV40 nuclear localization signal to the 5' end of CCA1 causes a growth defect and results in the accumulation of end-shortened tRNAs in the cytosol. These results suggest an important role for Cca1p in the cytosol of eukaryotes, presumably in the repair of 3' CCA termini. These experiments also demonstrate that individual tRNAs are affected differently by reduced cytosolic nucleotidyltransferase and that cells resuming exponential growth are more severely affected than those continuing exponential growth.
Subject(s)
RNA Nucleotidyltransferases/metabolism , Saccharomyces cerevisiae/enzymology , Base Sequence , Blotting, Northern , Cell Nucleus/enzymology , Cytosol/enzymology , Fluorescent Antibody Technique, Indirect , Gene Expression , Mitochondria/enzymology , Molecular Sequence Data , Mutagenesis, Site-Directed , RNA Nucleotidyltransferases/analysis , RNA Nucleotidyltransferases/biosynthesis , RNA, Fungal/analysis , RNA, Fungal/biosynthesis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & developmentABSTRACT
Leukophysin (LKP) is a 28-kDa protein of CTL and U937 monocytic cells that is located in the membrane of high density granules as well as lighter cytoplasmic granules or vesicles. mAbs to KLP were used to clone a full length cDNA clone with an open reading frame coding for a 235-amino acid polypeptide with a molecular mass of 24.3 kDa and two potential transmembrane regions. The nucleotide sequence was highly homologous to the 3' end of human RNA helicase A. Expression of the LKP was confirmed as a reverse transcriptase-PCR product that may be an alternately spliced product of RNA helicase A. The cDNA contained a repetitive motif that was similar to synaptophysin 1, a protein that is important for synaptic vesicle exocytosis. A polyclonal Ab directed against the 17 carboxyl-terminal amino acids of LKP detected the same 28-kDa granule membrane protein as the D545, one of the mAbs used to clone the cDNA. In addition, the D545 mAb reacted strongly with the GST fusion protein of the bacterially expressed LKP cDNA. In confocal immunofluorescence studies, the anti-LKP peptide Ab reacted with granzyme A-negative granules and vesicles in CD8+ CTL lymphocytes from normal and Chediak-Higashi patients. Thus, based on the expression of the C-terminal LKP epitope, vesicular structures an granules have been detected in CTL that are distinct from classical granzyme-containing cytolytic granules.
Subject(s)
Cytoplasmic Granules/enzymology , Intracellular Membranes/enzymology , Membrane Proteins/isolation & purification , Neoplasm Proteins/isolation & purification , RNA Nucleotidyltransferases/genetics , T-Lymphocytes, Cytotoxic/enzymology , Amino Acid Sequence , Base Sequence , Blotting, Southern , Cloning, Molecular , DEAD-box RNA Helicases , DNA, Complementary/isolation & purification , Epitopes/analysis , Granzymes , Humans , Leukemia, T-Cell , Lymphoma, Large B-Cell, Diffuse , Membrane Proteins/genetics , Molecular Sequence Data , Neoplasm Proteins/genetics , RNA Helicases , RNA Nucleotidyltransferases/analysis , Sequence Homology, Nucleic Acid , Serine Endopeptidases/analysis , Tumor Cells, CulturedABSTRACT
In this report, we describe for the first time the isolation and purification of a multiprotein complex for DNA replication from MDA MB-468 human breast cancer cells. This complex, which we designate the DNA synthesome, fully supports the in vitro replication of simian virus 40 (SV40) origin-containing DNA in the presence of the viral large T-antigen. Since the SV40 virus utilizes the host's cellular proteins for its own DNA replication, our results indicate that the DNA synthesome may play a role not only in viral DNA synthesis but in human breast cell DNA replication as well. Our studies demonstrate that the following DNA replication proteins constitute the DNA synthesome: DNA polymerase alpha, DNA primase, DNA polymerase delta, proliferating cell nuclear antigen, replication protein A, replication factor C, DNA topoisomerases I, II, and DNA polymerase epsilon. In addition, we successfully isolated the DNA synthesome from human breast tumor tissue as well as from xenografts from nude mice injected with the human breast cancer cell line MCF-7. The DNA synthesome purified from the breast cancer tissues fully supports SV40 DNA replication in vitro. Furthermore, our results obtained from a novel forward mutagenesis assay suggest that the DNA synthesome isolated from a nonmalignant breast cell line mediates SV40 DNA replication by an error-resistant mechanism. In contrast, the DNA synthesome derived from malignant breast cells and tissue exhibited a lower fidelity for DNA synthesis in vitro. Overall, our data support the role of the DNA synthesome as mediating breast cell DNA replication in vitro and in vivo.
Subject(s)
Breast Neoplasms/metabolism , Carcinoma, Ductal, Breast/metabolism , DNA Replication/physiology , DNA, Neoplasm/biosynthesis , Animals , Antigens, Polyomavirus Transforming/isolation & purification , Breast Neoplasms/chemistry , Carcinoma, Ductal, Breast/chemistry , DNA Primase , DNA-Directed DNA Polymerase/analysis , Female , Humans , Immunohistochemistry , Macromolecular Substances , Mice , Mice, Nude , Multiprotein Complexes , Neoplasm Transplantation , Proliferating Cell Nuclear Antigen/analysis , RNA Nucleotidyltransferases/analysis , Transplantation, Heterologous , Tumor Cells, CulturedABSTRACT
In the present review, we report on recent results obtained by in situ transferase-immunogold techniques as to the ultrastructural distribution of DNA and RNA within the cell nucleus. Special emphasis is placed on the various nucleolar components and the various enigmatic structures of the extranucleolar region: interchromatin granules, coiled bodies, and simple nuclear bodies. These data are discussed in the light of our current understanding of the functional organization of the cell nucleus.
Subject(s)
Cell Nucleus/chemistry , DNA/analysis , Immunohistochemistry/methods , RNA/analysis , Cell Compartmentation , Cell Line , Cell Nucleus/ultrastructure , DNA/ultrastructure , DNA Nucleotidylexotransferase/analysis , DNA Nucleotidylexotransferase/ultrastructure , Humans , In Situ Hybridization , Microscopy, Immunoelectron/methods , RNA/ultrastructure , RNA Nucleotidyltransferases/analysis , RNA Nucleotidyltransferases/ultrastructure , Tumor Cells, CulturedABSTRACT
An immunocytochemical method using specific antibodies was employed to detect DNA polymerase alpha-primase complex in Drosophila melanogaster embryos during the first 13 nuclear division cycles. A monoclonal antibody specific to the 72 kDa polypeptide stained interphase nuclei, but not metaphase chromosome, while at late anaphase and thereafter staining of the chromosome was regained. On the other hand, a polyclonal antibody specific to the 180 kDa polypeptide stained not only the interphase nuclei but also the cytoplasmic regions surrounding interphase nuclei. These results suggest that the distributions of the 180 kDa and the 72 kDa polypeptides of DNA polymerase alpha-primase complex are different. We detected the 180 kDa and the 72 kDa polypeptides in the extract prepared from a single Drosophila embryo by Western blotting, and a 130 kDa polypeptide immunologically related to the 180 kDa polypeptide was also detected in the extract. These polypeptides (180, 130, and 72 kDa) in the embryos were detected at similar levels at interphase and at the mitotic phase. These three polypeptides were also detected in unfertilized eggs, showing that they were maternally stored. The 130 kDa polypeptide was detected till cycle 10, then began to decrease, and finally disappeared at cycle 14, whereas the 180 kDa and the 72 kDa polypeptides were present without marked fluctuation in quantity throughout the developmental stages. Even in unfertilized eggs, the level of the 130 kDa polypeptide decreased gradually with a similar time course to that in fertilized ones, but the levels of the 180 kDa and the 72 kDa polypeptides remained unchanged. This is the first report suggesting the existence of the 130 kDa polypeptide in vivo in the early embryos of Drosophila. The significance of the 130 kDa polypeptide is discussed.
Subject(s)
Drosophila melanogaster/enzymology , Peptides/analysis , Peptides/immunology , RNA Nucleotidyltransferases/analysis , RNA Nucleotidyltransferases/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Antibody Specificity , Blotting, Western , Cell Division/physiology , Cell Nucleus/enzymology , Cell Nucleus/physiology , Chromatography , DNA Primase , Drosophila melanogaster/embryology , Immunohistochemistry , Interphase , Macromolecular Substances , Molecular Sequence Data , Molecular Weight , Staining and Labeling/methodsSubject(s)
DNA Replication , RNA Nucleotidyltransferases/analysis , RNA Nucleotidyltransferases/metabolism , Animals , Bacteriophage T7/enzymology , Base Sequence , Binding Sites , Cattle , DNA Primase , DNA Primers/biosynthesis , DNA-Directed DNA Polymerase/analysis , DNA-Directed DNA Polymerase/metabolism , Indicators and Reagents , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphorus Radioisotopes , Radioisotope Dilution Technique , Recombinant Proteins/analysis , Recombinant Proteins/metabolism , Thymine Nucleotides/metabolism , Thymus Gland/enzymology , Tritium , Zinc/metabolismSubject(s)
DNA Polymerase II/isolation & purification , DNA-Directed DNA Polymerase/isolation & purification , Drosophila melanogaster/enzymology , Embryo, Nonmammalian/enzymology , RNA Nucleotidyltransferases/isolation & purification , Animals , Centrifugation, Density Gradient/methods , Chromatography/methods , Chromatography, Affinity/methods , Chromatography, Gel/methods , Chromatography, Ion Exchange/methods , DNA Polymerase II/analysis , DNA Polymerase II/metabolism , DNA Polymerase III , DNA Primase , DNA-Directed DNA Polymerase/analysis , DNA-Directed DNA Polymerase/metabolism , Deoxyadenine Nucleotides/metabolism , Drosophila melanogaster/embryology , Durapatite , Electrophoresis, Polyacrylamide Gel/methods , Enzyme Inhibitors/pharmacology , Indicators and Reagents , Kinetics , Macromolecular Substances , Molecular Weight , RNA Nucleotidyltransferases/analysis , RNA Nucleotidyltransferases/metabolism , Radioisotope Dilution Technique , TritiumABSTRACT
DNA polymerase-alpha and primase activities present in a complex, have been isolated, partially purified, and characterized from embryonic chicken brain. DNA polymerase-alpha activity, characterized by its sensitivity to N-ethyl-maleimide, high sedimentation coefficient (11.3 S), and acidic isoelectric point (5-5.5) was found in all embryonic ages. Primase activity, the enzyme responsible for the initiation of DNA synthesis, co-sedimented with DNA polymerase-alpha activity on a continuous glycerol velocity gradient. A complex containing both DNA polymerase-alpha and primase activities was isolated by DE-23 cellulose column chromatography of cell-free extracts of different embryonic ages of chicken brain. In addition to the primase complexed with DNA polymerase-alpha, a free primase activity was isolated by DE-23 cellulose column chromatography of an ammonium sulfate (0-45%; w/v) precipitated fraction of embryonic chicken brain cell-free extract. DNA polymerase-alpha activity from developing chicken brains in the embryonic stage was purified by immuno-affinity column chromatography. Of all the single-stranded DNA templates tested, primase activity was found to be maximally active with poly dC. Primase activity was not inhibited by a high concentration of alpha-amanitin. The results obtained may provide insight into further understanding of regulation of chromosomal DNA replication in developing tissues.
Subject(s)
Brain/enzymology , DNA Replication , RNA Nucleotidyltransferases/analysis , Animals , Brain/embryology , Chick Embryo , DNA PrimaseABSTRACT
The molecular mechanism of RNA editing in trypanosomatid mitochondria is an unsolved problem. We show that two classes of ribonucleoprotein complexes exist in a mitochondrial extract from Leishmania tarentolae and appear to be involved in RNA editing. The 'G' class of RNP complexes consists of 170-300 A particles which contain guide RNAs and proteins, show little terminal uridylyl transferase (TUTase) activity and exhibit an in vitro RNA editing-like activity. The 'T' class consists of approximately six RNP complexes, the endogenous RNA of which can be self-labeled with [alpha-32P]UTP. The most abundant T complex, T-IV, is visualized by electron microscopy as 80-140 A particles. This complex exhibits TUTase activity in the native gel and contains guide RNAs. Both G and T complexes are possibly involved with RNA editing in vivo. These results are a starting point for the analysis of the biochemistry of RNA editing.
Subject(s)
Leishmania/genetics , Mitochondria/physiology , RNA Editing , RNA, Protozoan/genetics , Ribonucleoproteins/chemistry , Animals , Base Sequence , Molecular Sequence Data , RNA , RNA Nucleotidyltransferases/analysis , RNA, Guide, Kinetoplastida/analysis , RNA, Mitochondrial , Ribonucleoproteins/ultrastructure , Uridine Triphosphate/metabolismABSTRACT
The four-subunit DNA polymerase alpha-primase complex is unique in its ability to synthesize DNA chains de novo, and some in vitro data suggest its involvement in initiation and elongation of chromosomal DNA replication, although direct in vivo evidence for a role in the initiation reaction is still lacking. The function of the B subunit of the complex is unknown, but the Saccharomyces cerevisiae POL12 gene, which encodes this protein, is essential for cell viability. We have produced different pol12 alleles by in vitro mutagenesis of the cloned gene. The in vivo analysis of our 18 pol12 alleles indicates that the conserved carboxy-terminal two-thirds of the protein contains regions that are essential for cell viability, while the more divergent NH2-terminal portion is partially dispensable. The characterization of the temperature-sensitive pol12-T9 mutant allele demonstrates that the B subunit is required for in vivo DNA synthesis and correct progression through S phase. Moreover, reciprocal shift experiments indicate that the POL12 gene product plays an essential role at the early stage of chromosomal DNA replication, before the hydroxyurea-sensitive step. A model for the role of the B subunit in initiation of DNA replication at an origin is presented.