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1.
ACQUISITION OF CHICK CYTOSOL THYMIDINE KINASE ACTIVITY BY THYMIDINE KINASE-DEFICIENT MOUSE FIBROBLAST CELLS AFTER FUSION WITH CHICK ERYTHROCYTES 总被引:3,自引:1,他引:2
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Saul Kit Wai-Choi Leung George Jorgensen David Trkula Del Rose Dubbs 《The Journal of cell biology》1974,63(2):505-514
Chick-mouse heterokaryons were obtained by UV-Sendai virus-induced fusion of chick erythrocytes with thymidine (dT) kinase-deficient mouse fibroblast [LM(TK-)] cells. Autoradiographic studies demonstrated that 1 day after fusion, [3H]dT was incorporated into both red blood cell and LM(TK-) nuclei of 23% of the heterokaryons. Self-fused LM(TK-) cells failed to incorporate [3H]dT into nuclear DNA. 15 clonal lines of chick-mouse somatic cell hybrids [LM(TK-)/CRB] were isolated from the heterokaryons by cultivating them in selective hypoxanthine-aminopterin-thymidine-glycine medium. LM(TK-) and chick erythrocytes exhibited little, if any, cytosol dT kinase activity. In contrast, all 15 LM(TK-)/CRB lines contained levels of cytosol dT kinase activity comparable to that found in chick embryo cells. Disk polyacrylamide gel electrophoresis and isoelectric focusing analyses demonstrated that the LM(TK-)/CRB cells contained chick cytosol, but not mouse cytosol dT kinase. The LM(TK-)/CRB cells also contained mouse mitochondrial, but not chick mitochondrial dT kinase. Hence, the clonal lines were somatic cell hybrids and not LM(TK-) cell revertants. The experiments demonstrate that chick erythrocyte cytosol dT kinase can be activated in heterokaryons and in hybrid cells, most likely as a result of functions supplied by mouse fibroblast cells. 相似文献
2.
—The effects of hypothyroidism and several degrees of undernutrition on the development of cerebellar weight, DNA, and thymidine kinase activity were studied in young rats ranging in age from 2 to 22 days. Early propylthiouracil treatment caused a delayed cerebellar cell multiplication. The activity of cerebellar thymidine kinase was suppressed at ages 2 and 5 days and was in excess of control values on days 15 and 22, thus resulting in a delay in the developmental spectrum for thymidine kinase, and extending the time span of activity beyond that of controls. Undernutrition led to varying degrees of reduced cell proliferation at experimental ages 5, 12, and 19 days. Cerebella from the most undernourished animals showed significant differences from controls in thymidine kinase activity at ages 5 and 12 days. Comparisons between sub-groups from within the oversized litters at 5 and 12 days suggested that changes in thymidine kinase activity relate to the degree of undernutrition to which the sub-group is subjected and that during development there may be a critical degree of undernutrition at which a particular essential enzyme becomes affected. This study emphasizes the biochemical similarities and differences between neonatal hypothyroidism and undernutrition, while pointing out the difficulties which exist in biochemical separation of components of the two conditions. Further evidence is presented that thymidine kinase is responsive to hormonal stimuli during cerebellar development and may play an important role in the regulation of DNA biosynthesis in brain as well as other organs. 相似文献
3.
CHANGES OF THYMIDINE KINASE IN THE DEVELOPING RAT BRAIN 总被引:4,自引:1,他引:3
Abstract— Thymidine kinase (ATP: thymidine-5'-phosphotransferase EC 2.7.1.21) of the supernatant fraction from 6-day-old rat brain possessed a pH optimum of 8.0 and required the presence of 5mM-ATP and 2.5 mM-MgCl2 for maximum activity. The activity was completely inhibited by addition of 1.8 mM-TTP. The enzyme activity was lost if the same supernatant fraction was refrozen and thawed. Km was 2.8 × 10−6 M for [6-3 H]thymidine.
Following subcellular fractionation of rat brain, the greatest proportion and highest specific activity of thymidine kinase was found in the supernatant fraction. Thymidine kinase activities reached a maximum at 6 days of age and then dropped sharply during maturation. Comparative studies of thymidine kinase activities of cerebrum, cerebellum and the remainder of the brain during growth indicated that the activity in the cerebellum was usually higher than those in the cerebrum and the remainder, and the biggest differences obtained at 6 days after birth corresponded with the peak in cerebellar activity. 相似文献
Following subcellular fractionation of rat brain, the greatest proportion and highest specific activity of thymidine kinase was found in the supernatant fraction. Thymidine kinase activities reached a maximum at 6 days of age and then dropped sharply during maturation. Comparative studies of thymidine kinase activities of cerebrum, cerebellum and the remainder of the brain during growth indicated that the activity in the cerebellum was usually higher than those in the cerebrum and the remainder, and the biggest differences obtained at 6 days after birth corresponded with the peak in cerebellar activity. 相似文献
4.
Abstract— Using the method of least squares, a logistic curve was fitted to the data points for DNA content in neonatal rat cerebellum versus postnatal age (day 0 is the day of birth). The resultant equation was differentiated to give an expression for the rate of cerebellar DNA accumulation in units of ng/h per mg wet cerebellum. The DNA accumulation rate in control rats increased from 77.0 at 2 days of age to a maximum of 108 at 7 days of age and declined thereafter to a minimum of 16.3 on day 15. Thyroxine treatment significantly (P < 0.05) increased the rate to 89.8 (117% of control) at 2 days of age, and a significant elevation was maintained to 6 days of age at which time a maximum rate of 115 (114% of control) was attained. The rate was significantly decreased below control at 9 and 12 days of age, and reached a minimum of 9.22 on day 15. The developmental pattern for the activity of cerebellar thymidylate synthetase (EC 2.1.1.6), in units of pmol/h per mg wet cerebellum, closely paralleled the pattern for DNA accumulation rate in both control and thyroxine-treated animals. In controls, thymidylate synthetase activity increased from 98.6 at 2 days of age to a maximum of 125 at 7 days of age and declined thereafter to a minimum of 30.0 at 15 days of age. In thyroxine-treated animals, the activity was significantly increased to 118 (122% of control) at 4 days of age and remained significantly elevated through 6 days of age at which time a maximum activity of 154 (115% of control) was attained; thereafter, the activity was significantly decreased below control and reached a minimum of 16.9 (56.3% of control) on day 15. The developmental pattern for the activity of cerebellar thymidine kinase (EC 2.7.1.21) did not parallel the DNA accumulation rate quite so closely, in neither treated nor control animals, as did the pattern for thymidylate synthetase activity. These data suggest that thymidylate synthetase activity in the developing rat cerebellum may be more important for maintenance of replicative DNA synthesis than is thymidine kinase activity. In addition, the thyroxine-induced acceleration of the increase and subsequent decline in rate of DNA accumulation and in the activities of thymidylate synthetase and thymidine kinase in developing rat cerebella is probably the result of alterations in the number of external granular cells undergoing replicative DNA synthesis. 相似文献
5.
GENETIC CONTROL OF MITOCHONDRIAL THYMIDINE KINASE IN HUMAN-MOUSE AND MONKEY-MOUSE SOMATIC CELL HYBRIDS 总被引:4,自引:1,他引:3
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Distinctive thymidine (dT) kinase molecular forms are present in mouse, human, and monkey mitochondria. Disk polyacrylamide gel electrophoresis (disk PAGE) analyses have shown that the mitochondrial-specific dT kinases differ from cytosol dT kinases in relative electrophoretic mobilities (Rm). Furthermore, the mouse mitochondrial dT kinase differs in Rm value from primate mitochondrial dT kinases. The mouse and primate cytosol dT kinases can also be distinguished. Disk PAGE analyses have been carried out on the cytosol and mitochondrial dT kinases of human-mouse (WIL-8) and monkey-mouse (mK·CVIII) somatic cell hybrids in order to learn whether the mitochondria of the hybrid cells contained murine mitochondrial-specific, primate mitochondrial-specific, or both dT kinases. WIL-8 cells were derived from cytosol dT kinase-negative, mitochondrial dT kinase-positive mouse fibro blasts and from cytosol dT kinase-positive, mitochondrial dT kinase-positive human embryonic lung cells; they contained mostly mouse chromosomes and a few human chromosomes, including the determinant for human cytosol dT kinase. The mK·CVIII cells were derived from cytosol dT kinase-negative, mitochondrial dT kinase-positive mouse kidney cells and from cytosol dT kinase-positive, mitochondrial dT kinase-positive monkey kidney cells; they contained mostly mouse chromosomes and a few monkey chromosomes, including the determinant for monkey cytosol dT kinase. Disk PAGE analyses demonstrated that the mitochondria of human-mouse and monkey-mouse somatic cell hybrids contained the mouse-specific mitochondrial dT kinase but not the human- or monkey-specific mitochondrial dT kinase. These findings suggest that primate cytosol and mitochondrial thymidine kinase genes are coded on different chromosomes. 相似文献
6.
The amount of thymidine-H3 converted to thymidine-H3 monophosphate in 30 min formed the basis for assays of thymidine kinase in cell extracts from Tetrahymena pyriformis. The optimal concentration of adenosine triphosphate is lower than that required by other cell types. Thymidine triphosphate does not exercise any feedback control of the enzyme. Other deoxyprimidine nucleotides were tested, but these also failed to exhibit any feedback inhibition. At suboptimal adenosine triphosphate levels, thymidine triphosphate and other deoxypyrimidine nucleotides stimulate the reaction, suggesting that these nucleotides may act either directly or indirectly as phosphate donors in the crude enzyme preparations. This possibility was affirmed when thymidine triphosphate and deoxycytidine triphosphate were shown to be capable of limited phosphorylation of thymidine. Comparison of enzymatic activities in logarithmically growing culture and stationary phase culture, in which nuclear DNA synthesis has virtually ceased, reveals no change in enzymatic activity. The results suggest that thymidine kinase is a constitutive enzyme in Tetrahymena. 相似文献
7.
We have investigated the relationship between cell numbers and the amount of tritiated thymidine ([3H]TdR) taken up by stimulated human peripheral lymphocytes, as a function both of labeling time and of the specific activity of the thymidine. Cells responding either to mitogens or to allogenic cells show simple first order kinetics for the uptake of thymidine. Fitting the data to a Michaelis-Menten type of model, we observe for labeling times of 12 hr and longer, non-competitive inhibition of thymidine uptake by increased specific activity of tritium label, regardless of the mode of stimulation. However, for an individual responder in MLC at any arbitrary but fixed specific activity, dose of [3H]TdR and labeling interval, we still observe a linear relationship between cell mass and incorporated label. In contrast, if specific responding combinations in mixed lymphocyte culture are compared, the inhibition by specific activity at longer time intervals becomes significant and influences the quantitative interpretation of results. Specific activities of less than 10 Ci/mmole and labeling times of 6 hr or less avoid inhibition and ensure a linear relationship between dividing cell number and CPM (counts per minute recorded) of incorporated label. 相似文献
8.
《Cell biology international reports》1981,5(6):555-561
Wild type Friend erythroleukaemia cells (clone 707) and thymidine kinase (EC. 2.7.1.2.1) deficient derivatives were examined for sensitivity to killing by ultra-violet (UV) irradiation. Deficiency of thymidine kinase leads to increased cell killing as evidenced in all of twelve thymidine kinase deficient clones examined. A revertant thymidine kinase positive clone was found to have a level of thymidine kinase activity and UV sensitivity intermediate between wild type cells and its thymidine kinase deficient parent clone. The increased cell killing in thymidine kinase deficient clones is also reflected in increased mutagenesis by UV to 6-thioguanine resistance. It is suggested that the increased mutagenesis and sensitivity is due to defective DNA repair as a result of an imbalance in deoxyribonucleoside triphosphate pools in thymidine kinase deficient cells. 相似文献
9.
Margaret R. Hummon 《American journal of botany》1962,49(10):1038-1046
Hummon , Margaret R. (Montana State U., Missoula.) The effects of tritiated thymidine incorporation on secondary root production by Pisum sativum. Amer. Jour. Bot. 49(10): 1038–1046. Illus. 1962.—Most studies of effects of radiation on plants have involved a general exposure of all the cells and tissues of an organ or entire plant. Tritiated thymidine offers a tool for selective irradiation of the nucleus with little effect on the cytoplasm of a cell. Furthermore, differential incorporation due to variation in the pattern of DNA synthesis permits selective irradiation of cells and tissues. In this study, developing primary roots of Pisum sativum were submerged for brief periods in a solution of tritiated thymidine. This resulted in an alteration of the lateral root pattern. In the area corresponding to the region of elongation during treatment, subsequent lateral root production was suppressed. This correlated with the portion of the root in which there was incorporation of tritiated thymidine into a high percentage of pericycle nuclei. Abnormal development of vascular tissues also occurred, with evidence of altered polarity in the xylem. Although incorporation also occurred in the apical meristem, the latter was not affected at this level of exposure. Thus, differential sensitivity as well as differential incorporation may have been involved in producing the temporary alteration of lateral root production. 相似文献
10.
Deoxypyrimidine nucleoside metabolism in varicella-zoster virus-infected cells. 总被引:2,自引:2,他引:0
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Noninfected and varicella-zoster virus (VZV)-infected human foreskin fibroblasts were examined for thymidine kinase activity. The specific activity of VZV-infected cell extracts was approximately 7.5-fold greater than that of mock-infected cells and 3-fold greater than that of actively growing cells. The pH optimum of VZV-infected cell thymidine kinase activity was found to be 8.0, whereas thymidine kinase activity in noninfected cells exhibited a sharp pH optimum at 7.4. Electrophoretic analysis of cellular enzymes involved in pyrimidine nucleoside phosphorylation revealed at least three enzymes distinguishable by electrophoretic mobility and substrates used. These enzymes were presumed to be thymidine kinase, deoxycytidine kinase, and uridine kinase. The relative mobilities of these enzymes on 5% polyacrylamide gels were 0.18, 0.91, and 0.54, respectively. In VZV-infected cells, a single band of activity catalyzing the phosphorylation of thymidine, deoxyuridine, deoxycytidine, and cytidine was observed with a relative mobility of 0.48. Cellular pyrimidine-phosphorylating enzymes were not detected in VZV-infected cells. The molecular weight of the VZV-induced enzyme was determined to be 72,000 +/- 7%. 相似文献
11.
A M Peterson 《Cell differentiation》1976,5(1):37-43
Proliferative and mature intestinal cells of the jejunum and colon of rat, colon of man, and the surface cells of neoplastic colon lesions of man were assayed for thymidylate synthetase and thymidine kinase activities. Cells from the proliferative region of rat jejunal mucosa were found to have higher enzyme activities than cells from the non-proliferative region. Thymidylate synthetase activity was observed to decrease as cells migrated from base to upper crypt, whereas thymidine kinase activity increased during crypt migration and then declined as cells migrated onto villi. Thymidine kinase activity also remained elevated longer than thymidylate synthetase during cell migration in colonic mucosa of rat and man. High thymidine kinase: thymidylate synthetase ratios similar to those observed in flat mucosa before cells become fully mature were found in cells removed from expanding neoplastic lesions of man. 相似文献
12.
Cells of the Friend erythroleukemia cell line show a high frequency of variants which have lost thymidine kinase activity. We have exposed a thymidine kinase-deficient clone of this cell line to a series of concentrations of azacytidine and found a dose-dependent induction of thymidine kinase-positive revertants. Maximum reversion occurred with 0.9 micrograms/ml azacytidine where the frequency of revertants amongst survivors was increased by a factor of 10(6) compared to that of control cultures. Revertants were found to have varying levels of thymidine kinase activity. We conclude that DNA methylation of one or both alleles of the thymidine kinase gene is largely responsible for the instability of this gene in Friend erythroleukemia cells. 相似文献
13.
用磷酸钙沉淀法,我们把带有人体TK基因片段的重组噬菌体DNA共转染小鼠Ltk~-细胞,得到TK~+转化细胞克隆。同时用HeLa细胞DNA转染Ltk~-细胞,得到第一代TK~+转化细胞,再进行第二轮、第三轮转染,得到第二代、第三代TK~+转化细胞。比较其转化效率,结果基因组DNA转化率大于基因两个片段的共转化率,更大于不加携带者DNA的共转化率。限制性内切酶消化各种TK~+转化细胞的DNA,与TK基因探针作Southern印迹杂交,结果表明两个TK基因片段共转染Ltk~-细胞时,它们可以在受体细胞里重建成一个具有完整功能的遗传单位,但在连接过程中结构可以发生改变。当用HeLa纽胞DNA转染Ltk~-细胞时,虽然连续三代转染,每一代TK~+转化细胞中人TK基因的结构未发现变化。但也不能排除基因结构改变的频率很低未能有效检出的可能性。 相似文献
14.
Regulation of human thymidine kinase during the cell cycle 总被引:42,自引:0,他引:42
15.
Thymidine kinase activity was found in whole cell extracts of growing and stationary mouse embryo fibroblast cells after infection with murine cytomegalovirus. Determination of the kinetic constants and heat stability characteristics indicated that the enzyme activity from infected cells was different to that found in uninfected cells in the growth phase. The expression of thymidine kinase activity during virus replication was reflected by the incorporation of (6-3H) thymidine into acid precipitable fractions of infected cell cultures. Preliminary data from kinetic studies showed a reduction in the phosphorylation of thymidine by this enzyme activity in the presence of Acyclovir, a potent inhibitor of herpes virus replication. 相似文献
16.
The validity of using the incorporation of [3H]thymidine into DNA as an indicator of epidermal keratinocyte proliferation in vitro has been investigated. Other parameters of cell proliferation, direct count of cell number and measurement of DNA content, consistently fail to correlate with changes in [3H]thymidine incorporation into DNA in primary and first passage cultures of rabbit and human epidermal keratinocytes. Maximum incorporation of [3H]thymidine precedes the active growth period by three days. Incorporation declines markedly during the proliferative period. Thymidine kinase activity decreases during the proliferative growth phase. Incorporation of another pyrimidine nucleotide precursor, [14C]aspartic acid, suggests that in epidermal keratinocytes in vitro the extent of utilization of the salvage and the de novo pathways may be inversely related. In such cases [3H]thymidine incorporation into TCA precipitable material fails to reflect accurately cell proliferation. 相似文献
17.
Aanlysis of dCMP deaminase and CDP reductase levels in hamster cells infected by herpes simplex virus. 总被引:7,自引:7,他引:0
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Several enzymatic activities involved in the biosynthetic pathways of nucleotides, including thymidine kinase, which has been used as a biochemical marker in studies of gene transfer, are induced by herpes simplex virus (HSV). The utility of additional markers prompted us to reanalyze the effects of HSV infection on the activities of two other enzymes for which direct selective methods can be devised: dCMP deaminase and CDP reductase. For this purpose, mutant Chinese hamster (lA1) cells devoid of dCMP deaminase activity or Syrian hamster (BHK-21/C13) cells were infected by HSV type 1 or 2, and the activities of thymidine kinase, dCMP deaminase, and CDP reductase were measured in the cell extracts. The reported induction of thymidine kinase and CDP reductase by HSV was confirmed, whereas the stimulation of dCMP deaminase activity could not be observed. For both cell lines, the HSV-induced CDP reductase differed from the host enzyme by sensitivity to inhibition by both dTTP and dATP. This property should be helpful in developing a selection system for this activity. 相似文献
18.
Experimental evidence presented suggests that [3H]TdR can be rapidly and efficiently transported from the intestine to the systemic circulation. This pathway for thymidine transport may be physiologically important since administration of cold thymidine in the drinking water enhances the utilization of a parenterally injected dose of [3H]TdR in several body tissues of the mouse. 相似文献
19.
B. J. Bryant 《The Journal of cell biology》1966,29(1):29-36
Tritium from methyl-H3-thymidine was found to be incorporated into proteins in mice. This incorporation in the mouse as a whole represented between 1 and 10% of the injected tritium. Tritiated water was not an intermediate. Transmethylation reactions are proposed as a means whereby certain amino acids might have acquired the tritium from thymidine at some stage of its catabolism. The initial (2 hr) ratios of DNA to protein tritium activities per milligram of wet tissue ranged from 5 in two tissues of low DNA synthetic activity (pancreas, liver) to 35 to 40 in two tissues of high DNA synthetic activity (spleen, small intestine). Labeled nuclear protein was coincident with labeled DNA in nuclei, where it constituted less than 2.5% of the total tritium. The significance of the findings is discussed. 相似文献
20.
To determine the respective role of thymidine kinase and thymidylate synthase activities in the hyperoxia-induced decrease in DNA synthesis and their relationship with cell replication, we measured these two enzyme activities in primary cultures of porcine aortic endothelial cells under different O2 concentrations for various durations. In confluent cells, exposure to 95% O2 for 5 days reduced thymidine kinase activity to 15% of control values; thymidylate synthase activity was unaffected. In preconfluent cells exposed to 95% O2 for 2 days, similar results were obtained, together with evidence for arrest in cell proliferation. Thymidylate synthase activity could therefore not be related to decreased cell proliferation under hyperoxia. [3H]thymidine incorporation into DNA, thymidine kinase activity, and cell proliferation were all similarly affected under exposure to graded O2 concentration for 2 days. Thymidine kinase appears to be a key enzyme in the modulation of DNA synthesis from thymidine and in its replication in endothelial cells. 相似文献