RESUMO
SUMMARY: BioCichlid is a 3D visualization system of time-course microarray data on molecular networks, aiming at interpretation of gene expression data by transcriptional relationships based on the central dogma with physical and genetic interactions. BioCichlid visualizes both physical (protein) and genetic (regulatory) network layers, and provides animation of time-course gene expression data on the genetic network layer. Transcriptional regulations are represented to bridge the physical network (transcription factors) and genetic network (regulated genes) layers, thus integrating promoter analysis into the pathway mapping. BioCichlid enhances the interpretation of microarray data and allows for revealing the underlying mechanisms causing differential gene expressions. AVAILABILITY: BioCichlid is freely available and can be accessed at http://newton.tmd.ac.jp/. Source codes for both biocichlid server and client are also available.
Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Software , Gráficos por Computador , Modelos Biológicos , Interface Usuário-ComputadorRESUMO
Density distributions of negative hydrogen (H-) ions and negative deuterium (D-) ions were measured with the laser photodetachment method in the extraction region of the negative ion source. The distribution of H- ion density peaks at the center of the ion source, while that of the D- ion shows a flatter profile in the direction parallel to the plasma grid. The positive ion densities of hydrogen and deuterium estimated from the positive saturation current indicate similar profiles with different amounts close to the grid. The difference in the H- ion and D- ion distributions can be explained by the difference in the negative ion yield and the survival probability of the ions due to the isotope effect.
RESUMO
Simultaneous measurement of negative ion source plasma and extracted beam is carried out in order to clarify a key plasma parameter governing the meniscus formation in negative ion sources for fusion. The plasma discharge is performed with various discharge powers at different bias voltages in order to vary the plasma parameters. It is shown that the beam width changes along the same curve with respect to the negative ion density at any bias voltage while it varies along different curves with other plasma parameters depending on the bias voltage. This implies that the mechanism of meniscus formation in negative ion sources could be described along the similar manner as positive ion sources.
RESUMO
We partially characterized the transferrin-independent iron uptake (Tf-IU) of neuronal and glial cells in the previous report. In the present study, we further examined a mechanism of which glial cells protect neuronal cells against iron stress using neuron-microglia (N-MG) and neuron-astrocyte (N-AS) co-cultures. When each solely purified cell was treated with iron citrate, cell death occurred in N and MG. However, AS proliferated under the same condition. Both N-MG and N-AS co-cultures were effective in resistance to excessive iron. The total and specific Tf-IU activities of N-MG co-cultures similar to those of N did not increase in a density-dependent manner. Contrarily, the total activity of AS was extremely high and the specific activity was extremely low as a result of proliferation. Regarding of effect of co-cultures on H(2)O(2)-induced cell death, N-MG co-cultures were less effective, but N-AS co-cultures were more effective in protecting N from the oxidative stress. These results suggest that N-MG co-cultures suppress the Tf-IU and N-AS co-cultures stimulate AS proliferation to protect neuronal cells. Brain cells from aceruloplasminemia with mutations in the ceruloplasmin gene take up iron by Tf-IU. Therefore, the different mechanisms of neuronal cell protection by MG and AS may explain the pathophysiological observations in the brains of patient with aceruloplasminemia.
Assuntos
Astrócitos/fisiologia , Ceruloplasmina/deficiência , Microglia/fisiologia , Degeneração Neural/prevenção & controle , Doenças Neurodegenerativas/etiologia , Neurônios/fisiologia , Estresse Oxidativo/fisiologia , Animais , Animais Recém-Nascidos , Morte Celular , Células Cultivadas , Ceruloplasmina/genética , Técnicas de Cocultura , Citoproteção/fisiologia , Embrião de Mamíferos , Ferro/efeitos adversos , Ferro/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Neurônios/patologia , RatosRESUMO
Second deuterium operation of the negative ion based neutral beam injector was performed in 2018 in the large helical device. The electron and ion current ratio improves to Ie/Iacc(D) = 0.31 using the short extraction gap distance of 7 mm between the plasma grid (PG) and the extraction grid (EG). The strength of the magnetic field by the electron deflection magnet installed in the EG increases by 17% at the PG ingress surface, which effectively reduces the electron component in the negative ion rich plasma in the vicinity of PG apertures. The reduction of the electron current made it possible to operate at a high power arc discharge and beam extraction. Then, the deuterium negative ion current increases to 55.4 A with the averaged current density of 233 A/m2. The thermal load on the EG using 7 mm gap distance is 0.6 times smaller than the thermal load using a 8 mm gap caused by the reduction of coextracted electron current. The injection beam power increases to 2.9 MW in the beam line BL3, and the total beam injection power increases to 7 MW by three beam lines in the second deuterium campaign.
RESUMO
Chemically synthesized beta-2'-deoxy-6-thioguanosine 5'-triphosphate, a putative active form of beta-2'-deoxy-6-thioguanosine, was used efficiently as a substrate for DNA synthesis catalyzed by DNA polymerase alpha from calf thymus. The deoxythioguanylate was incorporated into DNA by replacing deoxyguanylate and supported the further elongation of DNA chains on activated calf thymus DNA. In contrast, DNA polymerase beta used beta-2'-deoxy-6-thioguanosine 5'-triphosphate at a much lower rate. The reaction product of DNA polymerase alpha, i.e., 6-thioguanine-containing DNA, adsorbed specifically to the organomercurial agarose column, and showed a peak of UV absorption at 342 nm, which is characteristic of thioguanine.
Assuntos
DNA Polimerase II/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Nucleotídeos de Desoxiguanina/metabolismo , Animais , Ligação Competitiva , Bovinos , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Técnicas In Vitro , Especificidade por Substrato , Tionucleosídeos/metabolismo , Tionucleotídeos/metabolismoRESUMO
A novel DNA polymerase, which could use both poly(rA) . oligo(dT) and activated calf thymus DNA efficiently as template-primers, was purified 20 000-fold from calf thymus extract. These activities were co-purified throughout successive column chromatographies and banded at the same position in either electrofocussing (pI = 6.5--7.0) or sucrose rate-zonal centrifugation (10--10.5 S). The most purified fraction (DNA-cellulose fraction) possessed specific activities of 3900 units/mg of protein with poly(rA) . oligo(dT) and 32 000 units/mg of protein with activated DNA. The poly(rA) . oligo(dT)-dependent activity differed from the previously described DNA polymerase gamma from other sources in the following ways: 1. The activity was inhibited by 100--300 mM KCl and and 80 mM potassium phosphate buffer. 2. The activity was 4-fold higher at 26 degrees C than at 37 degrees C. 3. The Km value for dTTP was 2.6--3.0 . 10(-4) M, which is several hundred-fold greater than that of DNA polymerase gamma. 4. Mn2+ was essential for the reaction and could not be replaced by Mg2+. The activated DNA-dependent activity shared many properties with DNA polymerase alpha, except that it was less sensitive to N-ethylmaleimide and anti-alpha polymerase immunoglobulin G. The 10-S DNA polymerase was dissociated into 8.5-S and 3.3-S by treatment with Triton X-100.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , Oligodesoxirribonucleotídeos , Oligonucleotídeos , Poli A , Timo/enzimologia , Animais , Bovinos , DNA Polimerase Dirigida por DNA/isolamento & purificação , Cinética , Relação Estrutura-Atividade , Moldes Genéticos , Nucleotídeos de TiminaRESUMO
DNA polymerases alpha and beta (EC 2.7.7.7.) from calf thymus could utilize dUTP as a substrate for DNA synthesis as well as DNA polymerase I of Escherichia coli. Deoxyuridylate was incorporated into DNA by replacing deoxythymidylate and supported the further elongation of DNA chains on activated DNA or on the intiated homopolymers, poly(dA) . (dT)10 and poly(rA) . (dT)10. The rate of the incorporation of deoxyuridylate into DNA varied from 50 to 160% of that of deoxythymidylate, depending on the nature of the template primers and the species of DNA polymerase used. The apparent Km values for dUTP were very similar to those for dTTP. Uracil DNA-glycosylase excised efficiently the uracil residues in products of DNA polymerase reactions with either activated calf thymus DNA or initiated homopolymers.
Assuntos
DNA Polimerase II/metabolismo , DNA Polimerase I/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Nucleotídeos de Desoxiuracil/metabolismo , Timo/enzimologia , Animais , Bovinos , Cinética , Especificidade por SubstratoRESUMO
DNA polymerase alpha (EC 2.7.7.7) from calf thymus has been separated into three molecular species, i.e., 10 S DNA polymerase alpha, 6.5 S DNA polymerase alpha-1 and 6.5 S DNA polymerase alpha-2 (Masaki, S. and Yoshida, S. (1978) Biochim, Biophys. Acta 531, 74-88; Yoshida, S., Yamada, M., Masaki S. and Seneyoshi, M. (1979) Cancer Res. 39, 3955-3958). Among these three, 10 S DNA polymerase alpha and 6.5 S DNA polymerase alpha-2 were found to copy efficiently poly(rA) . oligo(dT), a template-primer, which was thought to be specific for DNA polymerase gamma or beta. 6.5 S DNA polymerase alpha-1, however, could not use the ribopolymer as a template. The poly(rA) . oligo(dT)-dependent activities of DNA polymerase alpha species differed markedly from those with activated calf thymus DNA in sensitivity to various reagents: the former was inhibited more than 80% by 80 mM KCl, while the latter was stimulated somewhat. Furthermore, aphidicolin, a specific inhibitor of DNA polymerase alpha, did not inhibit the poly(rA) . oligo(dT)-dependent activity. 2',3'-DideoxyTTP, a potent inhibitor of DNA polymerase beta or gamma, slightly inhibited the reactions with poly(rA) . oligo(dT), while it did not inhibit the reactions with activated DNA. The apparent Km values for dTTP on poly(rA) . oligo(dT) template were 260 and 70 microM for 10 S alpha and 6.5 S alpha-2, respectively; these values were much higher than those obtained on activated DNA template (8-10 microM).
Assuntos
DNA Polimerase II/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Oligodesoxirribonucleotídeos/farmacologia , Oligonucleotídeos/farmacologia , Poli A/farmacologia , Timo/enzimologia , Animais , Bovinos , DNA Polimerase II/antagonistas & inibidores , Didesoxinucleotídeos , Cinética , Cloreto de Potássio/farmacologia , Especificidade por Substrato , Nucleotídeos de TiminaRESUMO
1-beta-D-Arabinofuranosylcytosine 5'-triphosphate (araCTP), an active form of a inhibitor of DNA replication, 1-beta-D-arabinofuranosylcytosine (araC) was tested for its inhibitory action on the DNA polymerase-alpha and -beta (EC 2.7.7.7) purified from calf thymus. The reaction of DNA polymerase-alpha was shown to be more sensitive to the inhibition by araCTP than that of DNA polymerase-beta. The mode of the inhibition by araCTP was competitive to dCTP in the reaction catalysed by either DNA polymerase-alpha or -beta. The Ki value of DNA polymerase-beta for araCTP was 32 micron; eight times higher than that of DNA polymerase-alpha (4 micron) for this inhibition.
Assuntos
Citarabina/análogos & derivados , DNA Polimerase II/antagonistas & inibidores , DNA Polimerase I/antagonistas & inibidores , Inibidores da Síntese de Ácido Nucleico , Timo/enzimologia , Animais , Ligação Competitiva , Bovinos , Citarabina/farmacologia , Escherichia coli/enzimologia , Cinética , Relação Estrutura-AtividadeRESUMO
The amount of DNA synthesis in vitro with the ultraviolet-irradiated poly-(dT) . oligo(rA) template initiators catalysed by DNA polymerase alpha (Masaki, S. and Yoshida, S., Biochim. Biophys. Acta 521, 74--88) decreased with the dose of ultraviolet-irradiation. The ultraviolet irradiation to the template, however did not affect the rate of incorporation of incorrect deoxynucleotides into the newly synthesized poly(dA). The addition of terminal deoxynucleotidyl transferase to this system enhanced the DNA synthesis to a level which is comparable to that of the control and it concomitantly increased the incorporation of the mismatched deoxynucleotide into the newly synthetized poly(dA) strands. On the other hand, with an unirradiated template initiator, the misincorporation was only slightly enhanced by the addition of terminal deoxynucleotidyl transferase. The sizes of newly synthetized DNA measured by sedimentation velocities were found to be smaller with the ultraviolet-irradiated templates but they increased to the control level with the addition of terminal deoxynucleotidyl transferase to the systems. These results suggest that terminal deoxynucleotidyl transferase can help DNA polymerase alpha to "bypass" thymine dimers in vitro by the formation of mismatched regions at the positions opposite to pyrimidine dimers on the template.
Assuntos
DNA Nucleotidiltransferases/metabolismo , DNA Polimerase II/metabolismo , Replicação do DNA/efeitos da radiação , DNA Polimerase Dirigida por DNA/metabolismo , DNA/efeitos da radiação , Raios Ultravioleta , Animais , Bovinos , Poli T , Moldes Genéticos , TimoRESUMO
Among multiple subspecies of DNA polymerase alpha of calf thymus, only 10 S DNA polymerase alpha had a capacity to initiate DNA synthesis on an unprimed single-stranded, circular M13 phage DNA in the presence of ribonucleoside triphosphates (DNA primase activity). The primase was copurified with 10 S DNA polymerase alpha through the purification and both activities cosedimented at 10 S through gradients of either sucrose or glycerol. Furthermore, these two activities were immunoprecipitated at a similar efficiency by a monoclonal antibody directed against calf thymus DNA polymerase alpha. These results indicate that the primase is tightly bound to 10 S DNA polymerase alpha. The RNA polymerizing activity was resistant to alpha-amanitin, required high concentration of all four ribonucleoside triphosphates (800 microM) for its maximal activity, and produced the limited length of oligonucleotides (around 10 nucleotides long) which were necessary to serve as a primer for DNA synthesis. Covalent bonding to RNA to DNA was strongly suggested by the nearest neighbour frequency analysis and the DNAase treatment. The DNA synthesis primed by the RNA oligomers may be carried out by the associating DNA polymerase alpha because it was strongly inhibited by araCTP, resistant to d2TTP, and was also inhibited by aphidicolin but at relatively high concentration. The primase preferred single-stranded DNA as a template, but it also showed an activity on the double-stranded DNA from calf thymus at an efficiency of approx. 10% of that with single-stranded DNA.
Assuntos
DNA Polimerase II/isolamento & purificação , Replicação do DNA , RNA Nucleotidiltransferases/isolamento & purificação , Timo/enzimologia , Animais , Bovinos , DNA Polimerase I/metabolismo , DNA Polimerase II/metabolismo , DNA Primase , Escherichia coli/enzimologia , Escherichia coli/genética , Plasmídeos , RNA Nucleotidiltransferases/metabolismo , Moldes GenéticosRESUMO
The DNA polymerase alpha-DNA primase complex was purified over 17,000-fold to near homogeneity from calf thymus using an immunoaffinity column. Sodium dodecyl sulfate gel electrophoresis revealed three polypeptides with molecular weights of 140, 50 and 47 kDa, in a ratio of 1:2:0.25. The complex showed a sedimentation coefficient of 9.7 S, a Stokes radius of 56 A and a native molecular weight of 250-260 kDa. Taken together, the data suggest that the calf thymus dNA polymerase alpha-DNA primase complex is essentially a heterotrimer of large (140 kDa) and small (50 kDa) subunits in a ratio of 1:2, with a globular conformation. Electron-microscopic studies of the complex revealed a spherical particle of 120 A in diameter, in agreement with the physiochemical results. The binding of the complex to DNA was also demonstrated.
Assuntos
RNA Nucleotidiltransferases/isolamento & purificação , Timo/enzimologia , Animais , Bovinos , Cromatografia de Afinidade , DNA Primase , Substâncias Macromoleculares , Microscopia Eletrônica , Peso Molecular , Conformação Proteica , RNA Nucleotidiltransferases/metabolismoRESUMO
The full length cDNA sequences of rat and mouse filensin are presented, as well as the structure of the rat filensin gene. This gene spanned 31 kb and included seven introns. The first six introns were conserved in position and phase with those found in the intermediate filament (IF) protein genes of the type II (type II keratin), type III (vimentin) and type V (lamin). The last intron of the filensin was unique. As none of the filensin intron positions coincided with those unique to type I, II or IV genes, it appears that filensin is most similar to type III genes. Comparison of the deduced amino acid sequences for rat and mouse filensin with those of cow and chick, and with other species of IF proteins, indicated the C-terminal non-alpha-helical tail domain of filensin to be one of the most divergent yet found in the vertebrate IF family. The tail domain had three conserved regions which are interrupted with two regions with lower identity. Two motifs, (1) PGDVPDGxxISKAF; and (2) KVEVVESIEKxxxxxIQTYEETxxIVET, were identified as sequences which were particularly highly conserved across species. Coassembly studies using CP49 and a physiologically derived 53 kDa-fragment of filensin showed the motif (2) was not required for filament assembly in vitro. These data strengthen the view that the C-terminal non-alpha-helical domain of filensin contributes in more than one way to filensin function in the lens.
Assuntos
Proteínas do Olho/genética , Proteínas de Filamentos Intermediários/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , DNA Complementar , Proteínas do Olho/classificação , Proteínas do Olho/metabolismo , Humanos , Proteínas de Filamentos Intermediários/classificação , Proteínas de Filamentos Intermediários/metabolismo , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
Filensin (also called CP94; CP95; CP97; 115kDa protein) is a component of the lens-specific beaded filament which is believed to be functionally important in lens fiber cell differentiation and in maintaining lens fiber cell conformation and transparency. A 17.2kb fragment containing the 5'-upstream sequence of the filensin gene was isolated. S1-mapping analysis determined the transcription start point (tsp; +1) which locates at 94base pairs upstream from the initiating ATG on the filensin gene. In addition to a major tsp, a minor tsp (-136) was observed. DNA sequence of the fragment around the tsp (-2144 to +155) was identified. Analysis of the DNA sequence of the promoter region around tsp revealed two motifs with sequence homology to Sox2 and Maf recognition sequences in addition to one GATA-1 site, two Sp1 binding sites, and three AP-2 binding motifs. No TATA-box or CCAAT-motif was found around the tsp region. A series of sequentially deleted fragments of (-2144 to +40) were fused to firefly luciferase reporter plasmid pGL2 and tested for activity in chicken embryonic lens explants. A minimal promoter region for mouse filensin of (-70 to +40) was identified. The lens-specific promoter activity was detected using lens explants cultured within 12h after dissection. The activity was remarkably enhanced by culture in the presence of 5ng/ml of basic fibroblast growth factor. Each one of the Sp1 and AP-2 binding motifs was localized to the fragment of (-27 to +40) using electrophoretic mobility shift assays. These are the first data to identify the basic elements to the 5'-upstream sequences of the filensin gene, namely the tsp and the minimal filensin promoter.
Assuntos
Proteínas do Olho/genética , Proteínas de Filamentos Intermediários/genética , Regiões Promotoras Genéticas , Animais , Sequência de Bases , Sítios de Ligação/genética , Embrião de Galinha , DNA/genética , DNA/metabolismo , Primers do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Genes Reporter , Cristalino/citologia , Cristalino/metabolismo , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/efeitos dos fármacos , Mapeamento por Restrição , Deleção de Sequência , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição AP-2 , Fatores de Transcrição/metabolismoRESUMO
Studies in Western cultures have indicated significant sex differences in certain cognitive abilities. To determine whether similar differences occur in a non-Western culture, this study administered a cross-linguistic battery of tests to high school students in Japan and America. In both cultures, girls averaged significantly higher scores on a Story Recall test, the Digit-Symbol test and a Word Fluency test whereas boys achieved significantly higher scores on a Mental Rotation test. The analysis of standardized test scores further indicated that the size of the sex difference was culture-independent in three out of these four cases. These results are discussed in the context of the GESCHWIND and GALABURDA [Cerebral Lateralization, Biological Mechanisms, Associations and Pathology, Bradford Books, Cambridge, Massachusetts] account of the contribution of testosterone to left-right asymmetries in early cerebral development.
Assuntos
Aptidão , Comparação Transcultural , Identidade de Gênero , Inteligência , Adolescente , Feminino , Florida , Humanos , Japão , Masculino , Rememoração Mental , Orientação , Escalas de Wechsler , Testes de Associação de PalavrasRESUMO
Purified DNA polymerase beta of calf thymus can utilize poly(rA).oligo(dT) as efficiently as poly(dA).oligo(dT) or activated DNA as a template primer. The poly(rA).oligo(dT)-dependent activity of DNA polymerase beta was found to differ markedly from the DNA-dependent activity of the same enzyme (with either activated calf thymus DNA or poly(dA).(dT)10) in the following respects. 1) Poly(rA)-dependent activity was strongly inhibited by natural DNA from various sources or synthetic deoxypolymer duplexes at very low concentrations (less than 0.5 microgram/ml) at which the DNA-dependent activity was affected to a much smaller extent, if at all. 2) Poly(rA)-dependent activity was inhibited by N-ethylmaleimide more strongly than DNA-dependent activity measured at 37 degrees C, while it was resistant to this reagent at 26 degrees C. 3) The curves of the activity versus substrate concentration were sigmoidal in the poly(rA)-dependent reaction but hyperbolic in the activated DNA-dependent reaction. A kinetic study suggested that the association of beta-enzyme protomers may be required to copy the poly(rA) strand.
Assuntos
DNA Polimerase II/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Oligodesoxirribonucleotídeos , Oligonucleotídeos , Poli A , Animais , Bovinos , Cinética , Relação Estrutura-Atividade , Reagentes de Sulfidrila/farmacologia , Moldes Genéticos , Timo/enzimologiaRESUMO
DNA synthesizing reactions catalyzed both large and small species of calf thymus DNA polymerase (DNA polymerase-alpha and -beta) [EC 2.7.7.7] were stimulated to comparable extents by the presence of spermidine or spermine, and it was not possible to differentiate these two species in terms of their sensitivities to polyamines. Optimal concentrations for stimulation were 0.5-1.0 mM for spermidine and 2-10 mjM for spermine. Excess polyamines strongly inhibited the reactions. The modes of stimulation were as follows: 1) Stimulation was observed with templates bearing long single-stranded sections when either natural DNA or synthetic homopolymer-oligomer duplex was used. 2) The nautral DNA-dependent reaction was stimulated by polyamines at suboptimal concentrations of Mg2+; the apparent Km value for Mg2+ was lowered on adding polyamines, while the Vmax value was unchanged. When synthetic homopolymer-oligomer duplex was used as a template, the reaction was stimulated spermidine even at the optimal concentration of Mn2+. 3) Polyamines markedly influenced the salt requirements of the reactions of DNA polymerase. Spermidine could replace salts such as KC1 or NaC1 at concentrations less than 1/100 of those of salts.
Assuntos
DNA Nucleotidiltransferases/metabolismo , Replicação do DNA/efeitos dos fármacos , Putrescina/farmacologia , Espermidina/análogos & derivados , Espermidina/farmacologia , Espermina/farmacologia , Timo/enzimologia , Animais , Bovinos , Ativação Enzimática/efeitos dos fármacos , Cinética , Magnésio/farmacologia , Manganês/farmacologia , Relação Estrutura-Atividade , Timo/efeitos dos fármacosRESUMO
Calf thymus DNA polymerases alpha and beta [EC 2.7.7.7] and terminal deoxynucleotidyl transferase [EC 2.7.7.31] were analyzed on two-dimensional gel slabs. DNA polymerase beta appeared as a single spot on two-dimensional gel at the position of 40,000 daltons and pI 8.0 using non-equilibrium pH gradient gel electrophoresis for the first-dimensional run. By overlapping gel slabs, it was possible to identify the distinct spot of DNA polymerase beta among many polypeptide spots of a crude enzyme fraction. 10S DNA polymerase alpha showed two clusters of polypeptide spots on two-dimensional gel slab. One cluster was composed of three large polypeptides of 140,000-150,000 daltons and another was composed of four smaller polypeptides of 46,000-50,000 daltons. All these spots were arranged in a narrow pI range (6.5-6.8) although each spot showed a distinct pI value. Purified terminal deoxynucleotidyl transferase showed three polypeptides of 57,000, 42,000, and 33,000 daltons at similar pI values (7.0-7.2). Each polypeptide consisted of plural spots which differed slightly in pI but were the same in molecular weight. These results suggest a microheterogeneity of polypeptides of terminal deoxynucleotidyl transferase as well as those of 10S DNA polymerase alpha.
Assuntos
DNA Nucleotidilexotransferase/análise , DNA Nucleotidiltransferases/análise , DNA Polimerase II/análise , DNA Polimerase I/análise , DNA Polimerase Dirigida por DNA/análise , Timo/enzimologia , Animais , Bovinos , Eletroforese em Gel de Poliacrilamida/métodos , Focalização IsoelétricaRESUMO
It has been shown that DNA primase activity is tightly associated with 10S DNA polymerase alpha from calf thymus and that the ribonucleotide-dependent DNA synthesis is more sensitive to araCTP than DNA-primed DNA synthesis (Yoshida, S., et al. (1983) Biochim. Biophys. Acta 741, 348-357). Here we measured DNA primase activity using poly(dT) template or M13 bacteriophage single-stranded DNA template and primer RNA synthesis was coupled to the reaction by Escherichia coli DNA polymerase I Klenow fragment. By this method, the primer RNA synthesis can be measured independently of the associating DNA polymerase alpha. Using poly(dT) template, it was found that arabinosyladenine 5'-triphosphate (araATP) strongly inhibited DNA primase in competition with rATP. The apparent Ki for araATP was 21 microM and the ratio of Ki/Km (for rATP) was as low as 0.015. With poly(dI, dT) or M13 DNA, it was shown that araCTP also inhibited DNA primase in the similar manner. Product analysis using [alpha-32P]rATP showed that araATP inhibited the elongation of primer RNA. However, it is not likely that arabinosylnucleotides act as chain-terminators, since incubation of primer RNA with araATP did not abolish its priming activity. From these results, it is suggested that arabinosylnucleotide inhibits the initiation as well as elongation of Okazaki fragments in mammalian cells.