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1.
SANDRA J. SMITH-GILL J. GARRETT REILLY ELISABETH M. WEBER 《Development, growth & differentiation》1979,21(4):281-290
The relationship of DNA synthesis and cellular turnover to biochemical differentiation during metamorphosis of R. pipiens liver was investigated. Average DNA/cell was constant at 11.6 pg/ nucleus through stage XXV; but increased during juvenile growth; during metamorphosis stages, changes in total DNA content must correspond to changes in cell number. Rates of DNA synthesis were estimated by rates of 3H-thymidine incorporated into the acid-precipitable fractions, corrected for both precursor uptake into the acid-soluble pool, and for endogenous thymine pool size. DNA content increased steadily from premetamorphosis until late prometamorphosis; at preclimax stages XVIII and XX there were two successive decreases in DNA content of approximately 30%. Fluctuations in synthesis rates preceded corresponding fluctuations in content; DNA synthesis was maximal at stages XVI and XVIII, decreased nearly ten-fold at metamorphic climax, and then gradually rose again during late climax stages. The size of the endogenous thymine pool increased transitorily during spontaneous metamorphosis corresponding to a stage of maximal DNA synthesis. These results indicate that both DNA synthesis and cellular turnover play a significant role in determining net DNA synthesis rates and content during metamorphosis. Metamorphosis of the tadpole liver appears to be associated with both proliferation and cellular death, perhaps a replacement of “larval” by “adult” cells. Metamorphosis of the liver cannot be occuring in a “fixed population of cells” as is commonly assumed. An interpretation of the population dynamics of the metamorphic liver is presented. 相似文献
2.
A previous paper in this series (C. K. Mathews, (1972) J. Biol. Chem.247, 7430) showed that deoxynucleoside triphosphate pools expand manyfold when DNA synthesis is blocked genetically in infection by bacteriophage T4. This paper describes a more detailed analysis of this phenomenon. The key approach involves labeling with thymine or thymidine under conditions of infection where both phage and host bear mutations that inactivate thymidylate synthetase. Principal findings include the following: (1) Nucleotides in the expanded pools are derived in roughly equal measure from breakdown of host cell DNA and from nucleotide synthesis de novo after infection. (2) Thymidine diphosphate pool expansion is comparable, in rate and extent, to thymidine triphosphate pool expansion, but thymidine monophosphate pools accumulate much less. (3) The rate of expansion of the total thymine nucleotide pool following temperature upshift in infection by a temperature-sensitive gene 45 mutant is approximately equal to the rate of thymine incorporation into DNA immediately preceding the upshift. (4) Similarly, when DNA synthesis is restored by a downshift, the total thymine nucleotide pool drains at a rate commensurate with that of thymine incorporation into DNA. (5) Under these latter conditions the dTTP pool begins to drain earlier than the dTDP pool, suggesting that dTTP is the more proximal DNA precursor in this system. 相似文献
3.
DNA synthesis in regenerating liver was studied to determine whether the onset of stimulated DNA synthesis preceded the onset of increased incorporation of thymidine into DNA. Thymidine incorporation into hepatic DNA was not stimulated 15 h after operation, but was stimulated after 18 h; peak stimulation occurred 30 h after operation. Thymidine kinase activity was stimulated 24 h after operation; highest kinase activity was observed at 36 h. The onset of stimulated DNA synthesis was estimated by following the incorporation of labeled aspartic acid, sodium formate, adenine or orotic acid into appropriate DNA bases, viz., thymine, adenine, adenine or cytosine, respectively. Incorporation of adenine and orotic acid was stimulated between 15 h and 18 h after operation; incorporation of aspartic acid and sodium formate was stimulated between 18 h and 21 h after operation.The incorporation of thymidine into DNA was accelerated by stress stimulus and was inhibited by hydrocortisone. Changes in thymidine kinase activity also were correspondingly accelerated or delayed. Incorporation of labeled thymidine, adenine, formate, orotic acid or thymine into appropriate DNA bases, viz., thymine, adenine, adenine, cytosine or thymine, respectively, was stimulated by stress stimulus or was inhibited by hydrocortisone.It was concluded from these data that stimulation of DNA synthesis and of thymidine incorporation into DNA was essentially synchronized in regenerating rat liver. Results from this study were compared with results from similar studies in 2 other tissues, and the limitations, attendant with using thymidine incorporation into DNA as an indicator of stimulated DNA synthesis, were discussed. 相似文献
4.
125I-labeled deoxyuridine (IdUrd) has been used to estimate the turnover of DNA in liver, tail, and hind limb during spontaneous and triiodothyronine-induced metamorphosis. It was found that the total amount of liver DNA remained constant and there was no significant loss of the label from the liver DNA, which would be expected if there was an increase in DNA turnover during metamorphosis. Also, the change in specific activity of liver DNA parallels that of tail DNA during spontaneous metamorphosis. These data suggest that metamorphic transitions in the tadpole liver do not involve significant changes in DNA turnover. It was observed that the incorporation of label into hind limb DNA showed a high variability among individual animals as compared to liver and tail tissue. The data presented suggest that the observed variability is not a random phenomenon but related directly to the rate at which animals will metamorphose. 相似文献
5.
Prostaglandin F2 alpha stimulates phosphatidylinositol turnover and increases the cellular content of 1,2-diacylglycerol in confluent resting Swiss 3T3 cells 总被引:10,自引:0,他引:10
C H Macphee A H Drummond A M Otto L Jimenez de Asua 《Journal of cellular physiology》1984,119(1):35-40
Prostaglandin F2 alpha (PGF2 alpha); which stimulates DNA synthesis in resting 3T3 cells, also stimulates the incorporation of [32P]PO4 into phosphatidylinositol. The effect is selective for PGF2 alpha when compared with PGE1, PGE2, and PGF2 beta. Epidermal growth factor (EGF) also stimulates DNA synthesis but does not affect phosphatidylinositol turnover. PGE1, which acts synergistically with PGF2 alpha to enhance DNA synthesis, does not affect the ability of PGF2 alpha, to enhance the incorporation of [32P]PO4 into phosphatidylinositol. PGF2 alpha, also causes a small increase in the cellular content of 1,2-diacylglycerol. This effect is not shared by EGF or PGE1. Stimulation of phosphatidylinositol metabolism resulting in an increase in the cellular content of 1,2-diacylglycerol may thus constitute an event in the pathway leading to the initiation of DNA synthesis in which PGF2 alpha differs in its action from EGF. 相似文献
6.
7.
The influence of orotic acid on the incorporation of precursors into nucleic acids was studied in mice and rats and in isolated cells. In vivo, orotate levels were modified by two diets which are known to increase the rate of pyrimidine nucleotide synthesis in rat liver. Of these diets, a 1% orotate diet had greater inhibitory effects than an arginine-deficient diet on the incorporation of [3H]orotate into RNA of mouse kidney than mouse liver. This contrasted with the situation in the rat where there was a greater effect in the liver than the kidney. The situation in the rat was more readily interpreted than in the mouse in terms of previously established effects of these diets on ribonucleotide pool sizes. However, studies using [3H]adenosine as a precursor for incorporation into RNA suggested that even in the mouse the effects of orotate were on pool sizes rather than an inhibitory effect on RNA synthesis. The incorporation of [3H]thymidine into DNA was inhibited by orotate to a similar degree in cultured HTC hepatoma cells and a line of rat liver epithelial cells. An effect on DNA synthesis rather than solely on pool sizes was suggested by the observation that the pool size of dTTP was not increased by 5 mM orotate under conditions in which there was a four-fold increase in the level of UTP in HTC cells. An inhibitory effect of orotate on DNA synthesis was further supported by an observation of decreased incorporation of [3H]deoxyadenosine into DNA and a lower rate of cellular proliferation. 相似文献
8.
A method has been developed for the measurement of DNA synthesis in vivo using the incorporation of multilabeled, non-radioactive thymidine. Simultaneous intraperitoneal injection of hexalabeled thymidine and tritiated thymidine into a normal adult rat resulted in the incorporation of both labeled nucleosides into the DNA of cells undergoing replication. The DNA of several tissues and organs was analysed, including liver, thymus, spleen, bone marrow, and small intestine. Following extraction with hot trichloroacetic acid, acid hydrolysis, and thin-layer chromatography of the hydrolysates, the isotopic compositions of the thymine products were determined by field ionization mass spectrometry and by scintillation counting. The relative incorporation of radioactive and stable isotope-labeled thymidine was similar in all tissues, and corresponded to the ratio of the two labeled nucleosides in the injected material. These results indicate the feasibility of utilizing thymidine multilabeled with stable isotopes for measurement of cellular proliferation rates in conjunction with cancer therapy. 相似文献
9.
The formation, distribution and function of ribosomes and microsomal membranes during induced amphibian metamorphosis 总被引:13,自引:10,他引:3
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J. R. Tata 《The Biochemical journal》1967,105(2):783-801
1. A lag period of about 4 days preceded the onset of metamorphosis precociously induced by tri-iodothyronine in tadpoles of the giant American bullfrog (Rana catesbeiana). It was established by the accelerated synthesis or induction of carbamoyl phosphate synthetase and cytochrome oxidase in the liver, serum albumin and adult haemoglobin in the blood, acid phosphatase in the tail, and the increase in the hindleg/tail length ratio. 2. A 4- to 6-fold stimulation, 2 days after the induction of metamorphosis, of the rate of synthesis of rapidly labelled nuclear RNA in liver cells was followed by an increasing amount of RNA appearing in the cytoplasm. Most of the newly formed RNA on induction of metamorphosis was of the ribosomal type. An accelerated turnover at early stages of development preceded a net accumulation of RNA in the cytoplasm, with no change in the amount of DNA per liver. 3. Most hepatic ribosomes of the pre-metamorphic tadpoles were present as 78s monomers and 100s dimers; metamorphosis caused a shift towards larger polysomal aggregates with newly formed ribosomes that were relatively more tightly bound to membranes of the endoplasmic reticulum. 4. The appearance of new polyribosomes in the cytoplasm on induction of metamorphosis was co-ordinated in time with a stimulation of synthesis of phospholipids of the smooth and rough endoplasmic reticulum, followed by a gradual shift in preponderance from the smooth to the rough type of microsomal membranes. 5. Electron- and optical-microscopic examination of intact hepatocytes revealed a striking change in the distribution and nature of ribosomes and microsomal membranes during metamorphosis. 6. Ribosomes prepared from non-metamorphosing and metamorphosing animals were identical in their sedimentation coefficients and in the structural ribosomal proteins. The base composition and sedimentation coefficients of ribosomal RNA were also identical. Induction of metamorphosis also did not alter the incorporation of (32)P into the different phospholipid constituents of microsomal membranes. 7. Nascent (14)C-labelled protein with the highest specific activity was recovered in the ;heavy' rough membrane fraction of microsomes, whereas little (14)C was associated with ;free' polysomes. Protein synthesis in vivo was most markedly stimulated during metamorphosis in the tightly membrane-bound ribosomal fraction after the appearance of new ribosomes. 8. The rate of synthesis of macromolecules in vivo could not be followed beyond 7-8 days after induction because of variable shifts in precursor pools due to regression of larval tissues. 9. The stimulation of RNA and ribosome formation was specifically associated with the process of metamorphosis since no similar response to thyroid hormones occurred in those species (Axolotl and Necturus) in which the hormones failed to induce metamorphosis. 相似文献
10.
Replication of DNA templates containing 5-formyluracil, a major oxidative lesion of thymine in DNA. 总被引:5,自引:4,他引:1
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Q M Zhang H Sugiyama I Miyabe S Matsuda I Saito S Yonei 《Nucleic acids research》1997,25(20):3969-3973
5-Formyluracil (5-foU) is a major lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. To assess its biochemical effects on DNA replication, 22mer oligonucleotide templates containing an internal 5-foU at defined sites were synthesized by the phosphoramidite method and examined for ability to serve as a template for various DNA polymerases in vitro . Klenow fragments with and without 3'-->5'exonuclease of DNA polymerase I, Thermus thermophilus DNA polymerase (exonuclease-deficient) and Pyrococcus furiosus DNA polymerase (exonuclease-proficient) read through the site of 5-foU in the template. Primer extension assays revealed that the 5-foU directed not only incorporation of dAMP but also dCMP opposite the lesion during DNA synthesis. Misincorporation opposite 5-foU was unaffected by 3'-->5' exonuclease activity. DNA polymerases had different dissociation rates from a dCMP/T mispair and from a dCMP/5-foU mispair. The incorporation of an 'incorrect' nucleotide was dependent on the sequence context and DNA polymerase used. These results suggest that 5-foU produced in DNA has mutagenic potential leading to T-->G transversions during DNA synthesis. 相似文献
11.
12.
Michael A. Carpenter Ming Li Anurag Rathore Lela Lackey Emily K. Law Allison M. Land 《Journal of biomolecular structure & dynamics》2013,31(1)
Multiple studies have indicated that the TET oxidases and, more controversially, the AID/APOBEC deaminases have the capacity to convert genomic DNA 5-methyl-cytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC-to-T activity and 10-fold less C-to-U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered whereas both MeC and C nucleobases in transfected plasmid DNA substrates are highly susceptible to editing. Knockdown experiments show that endogenous A3A is the source of both of these cellular DNA deaminase activities. This is the first evidence for non-chromosomal DNA MeC-to-T editing in human cells. These biochemical and cellular data combine to suggest a model in which the expanded substrate versatility of A3A may be an evolutionary adaptation that occurred to fortify its innate immune function in foreign DNA clearance by myeloid lineage cell types. 相似文献
13.
Giorgio Mastromei Silvano Riva 《Biochemical and biophysical research communications》1975,63(2):522-528
In a Thy? strain, thymidine is rapidly converted into thymine and, at the steady state, the pool size of thymidine is very small as compared to that of thymine. Consequently when such strain is used for pulse incorporation experiments with labelled thymidine paradoxical results are obtained. A quantitative estimation of the rate of DNA synthesis can only be obtained by thymine pulses or by cumulative incorporation experiments. We also pr sent evidence that, during a short pulse, thymidine is mainly utilized for replicative DNA synthesis. 相似文献
14.
Differentiation of confluent 3T3-L1 preadipocytes to adipocytes in the presence of dexamethasone and 1-methyl-3-isobutylxanthine for 7 days resulted in a 4-fold increase in the incorporation of acetoacetate-carbon into fatty acids and in the activity of 3-oxoacid CoA-transferase, which catalyzes the first committed step in the conversion of acetoacetate to acetoacetyl-CoA. The increase in enzyme activity was due to an increase in the cellular content of the enzyme, as determined by immunoprecipitation of 3-oxoacid CoA-transferase from 3T3-L1 preadipocytes and adipocytes with rabbit antiserum specific for the rat brain enzyme. The 4-fold increase in enzyme activity was accompanied by a 2.7-fold increase in the average relative rate of synthesis of 3-oxoacid CoA-transferase (between Days 4 and 7). Additionally, the half-life of the enzyme increased 1.9-fold relative to the half-life of total protein, indicating that changes in both synthesis and degradation of 3-oxoacid CoA-transferase are responsible for alterations in its activity. Previous studies on the turnover of other enzymes that are induced during differentiation of 3T3-L1 cells have assigned changes in enzyme synthesis as the primary or sole mechanism for changes in enzyme activity. This report provides the first documentation that both enzyme synthesis and degradation play a role in regulating the enzyme activity of an enzyme during differentiation of 3T3-L1 cells. 相似文献
15.
Error induction and correction by mutant and wild type T4 DNA polymerases. Kinetic error discrimination mechanisms. 总被引:15,自引:0,他引:15
L K Clayton M F Goodman E W Branscomb D J Galas 《The Journal of biological chemistry》1979,254(6):1902-1912
The fidelity of DNA synthesis as determined by the misincorporation of the base analogue 2-aminopurine in competition with adenine has been measured as a function of deoxynucleoside triphosphate substrate concentrations using purified mutator (L56), antimutator (L141), and wild type (T4D) T4 DNA polymerases. Although the rates of both incorporation and turnover of aminopurine and adenine decrease as substrate concentrations are decreased, the ratio of turnover/polymerase activity is increased. Thus, the nuclease/polymerase ratio of each of these three DNA polymerases can be controlled. The misincorporation of aminopurine decreases with decreasing substrate concentrations such that all three enzymes approach nearly identical misincorporation frequencies at the lowest substrate concentration. The increased accuracy of DNA synthesis corresponds to conditions producing a high nuclease/polymerase ratio. The misinsertion frequency for aminopurine is independent of substrate concentrations and enzyme phenotype; therefore, the increased accuracy of DNA synthesis with decreasing substrate concentrations is shown to be a result of increased nuclease activity and not increased polymerase or nuclease specificity. The data are analyzed in terms of a kinetic model of DNA polymerase accuracy which proposes that discrimination in nucleotide insertion and removal is based on the free energy difference between matched and mismatched base pairs. A value of 1.1 kcal/mol free energy difference, delta G, between adenine: thymine and aminopurine:thymine base pairs is predicted by model analysis of the cocentration dependence of aminopurine misincorporation and removal frequencies. An independent estimate of this free energy difference based on the 6-fold higher apparent Km of T4 DNA polymerase for aminopurine compared to adenine also gives a value of 1.1 kcal/mol. It is shown that the aminopurine misinsertion frequency for an enzyme having either extremely low 3'-exonuclease activity, Escherichia coli DNA polymerase I, or no measurable exonuclease activity, calf thymus DNA polymerase alpha, is 12 to 15%, which is similar to that for the T4 polymerases and consistent with delta G approximately 1.1 kcal/mol. 相似文献
16.
Effect of cAMP on nucleoside metabolism. I. Effect on thymidine transport and incorporation in monkey cells (CV-1) 总被引:2,自引:0,他引:2
The effect of cAMP on a monkey kidney cell line (CV-1) noninfected and infected by SV40 was studied. No effect was found on either growth rate or cell morphology when concentrations up to 1 mM of 3′5′ cAMP were used. However, cAMP was found to increase the incorporation of 3H-thymidine into both cellular and viral DNA without a net increase in DNA synthesis. This increased incorporation was found to be related to an enhanced uptake of thymidine into the nucleotide pool which is reflected in an increase of phosphorylated nucleotides. This, coupled with a lack of effect of cAMP on endogenous deoxyribonucleotide production, produces an increased specific activity of the deoxyribonucleotide triphosphates, with a resultant increase in specific activity of DNA. 相似文献
17.
Guanine for DNA synthesis. A compulsory route through ribonucleotide reductase. 总被引:1,自引:1,他引:0
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Two alternative pathways for the synthesis of dGTP and its incorporation into DNA were studied: guanine (Gua)----GMP----GDP----dGDP----dGTP----DNA and dG----dGMP----dGDP----dGTP----DNA. To determine the contribution of each pathway to DNA synthesis independently of each other, [14C]Gua and [3H]dG tracer experiments were performed in a double-mutant S-49 mouse T-lymphoma cell line, dGuo-L, with purine nucleoside phosphorylase (EC 2.4.2.1)-deficiency and dGTP-feedback-resistant ribonucleotide reductase (RR, EC 1.17.4.1). In this cell line, dGTP pools can be selectively elevated by exogenous dG without affect RR and DNA synthesis. Although [3H]dG, but not [14C]Gua (up to 200 microM), readily expanded the cellular dGTP pool in a dose-dependent fashion in asynchronous cells, only a small fraction of the Gua flux into DNA was derived from [3H]dG, with the major fraction coming from [14C]Gua. H.p.l.c. analysis of G1- and partially enriched S-phase cells revealed that [3H]dGTP only accumulates in G1- but not in S-phase cells because of a rapid turnover of the dGTP pool during DNA synthesis. These results fail to provide evidence for cellular dGTP compartmentation and suggest that the pathway dG----dGMP----dGDP----dGTP alone has insufficient capacity to maintain DNA synthesis. 相似文献
18.
A V Alessenko L V Platonova G R Sakevarashvili A V Khrenov L N Shingarova N I Shono E I Galperin 《Biochemistry. Biokhimii?a》1999,64(8):890-895
Cytokine-stimulated metabolism of sphingomyelin results in the accumulation of ceramide and sphingosine which play a part in the regulation of cell proliferation, differentiation, and reception, as well as in oncogenesis. Formation of TNF-alpha (a member of the cytokine family), accumulation of sphingosine, and DNA synthesis (measured by immunoblotting, HPLC, and [3H]thymidine incorporation, respectively) were studied in rat liver after partial hepatectomy. The content of TNF-alpha was found to increase during 12 h following hepatectomy. The maximum of sphingomyelinase activity and accumulation of sphingosine precede the maximum of DNA synthesis. Sphingosine is known to inhibit protein kinase C. On the other hand, it stimulates the metabolism of phosphatidylinositol, thus causing accumulation of diacylglycerol and inositol-1,4,5-triphosphate, which in turn activate protein kinase C. Hence, the release of TNF-alpha in regenerating liver may modulate DNA synthesis through the accumulation of sphingosine which is involved in regulation of protein kinase C activity and of phosphatidylinositol turnover. 相似文献
19.
Jay I. Goodman 《Experimental cell research》1974,85(2)
A comparison was made between the utilization of thymine and thymidine for the synthesis of DNA in Novikoff hepatoma cells growing in suspension culture. When the cell cultures were switched from exponential growth to a relatively non-growing condition, by resuspending them in culture media minus serum for 18 h, there was an 85% decrease in the rate of thymidine incorporation but only a 15% decrease in the rate of thymine incorporation. Exposure to an alkylating agent (methyl methane sulfonate) resulted in a 79% decrease in thymidine incorporation, while thymine incorporation was decreased only 35%. Thymidine at a concentration equal to its Km for incorporation into DNA (4 × 10−7 M) had virtually no effect on thymine incorporation. It was not until a thymidine concentration of ten times the Km was employed that appreciable (40%) decreases in the rate of thymine incorporation were observed. Examination of total cellular DNA or nuclear DNA gave similar results. These studies are interpreted as indicating the presence of multiple precursor pools for the synthesis of DNA-thymine in Novikoff hepatoma cells. 相似文献
20.
Shimizu M Gruz P Kamiya H Masutani C Xu Y Usui Y Sugiyama H Harashima H Hanaoka F Nohmi T 《Biochemistry》2007,46(18):5515-5522
Altered oxidative metabolism is a property of many tumor cells. Oxidation of DNA precursors, i.e., dNTP pool, as well as DNA is a major source of mutagenesis and carcinogenesis. Here, we report the remarkable nature of human DNA polymerase eta that incorporates oxidized dNTPs into a nascent DNA strand in an efficient and erroneous manner. The polymerase almost exclusively incorporated 8-hydroxy-dGTP (8-OH-dGTP) opposite template adenine (A) at 60% efficiency of normal dTTP incorporation, and incorporated 2-hydroxy-dATP (2-OH-dATP) opposite template thymine (T), guanine (G), or cytosine (C) at substantial rates. The synthetic primers having 8-hydroxy-G paired with template A or 2-hydroxy-A paired with template T, G, or C at the termini were efficiently extended. In contrast, human DNA polymerase iota incorporated 8-OH-dGTP opposite template A with much lower efficiency and did not incorporate 2-OH-dATP opposite any of the template bases. It did not extend the primers having the oxidized bases at the termini either. We propose that human DNA polymerase eta may participate in oxidative mutagenesis through the efficient and erroneous incorporation of oxidized dNTPs during DNA synthesis. 相似文献