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1.
《Insect Biochemistry》1976,6(3):333-337
Thymidylate synthetase activity (as expressed per mg of protein and per the whole insect) in extracts from Bombyx mori changes considerably during development. The enzyme is present in unfertilized eggs. After fertilization its activity initially increases but then decreases in parallel with the beginning of diapause. During postdiapause embryonic development the level of the enzyme quickly increases, but decreases again after hatching. In larvae the activity is low during the first instars but rises dramatically after the 4th ecdysis. The whole period of pharate pupal, pupal, and pharate adult development is characterized by a U-shaped pattern of the thymidylate synthetase activity.In order to explain some aspects of this pattern the activity of the thymidylate synthetase in extracts from some organs of the silkworm was also examined. 相似文献
2.
Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs. 相似文献
3.
A procedure is described for the isolation and crystallization of thymidylate synthetase from a dichloromethotrexate resistant strain of Lactobacillus casei. The recrystallized enzyme shows one band on polyacrylamide gel electrophoresis. The specific activity of the recrystallized enzyme is 216 μmoles of thymidylate formed per hour per mg. at30°C. 相似文献
4.
Thymidine 5′-Monophosphate-Requiring Mutants of Saccharomyces cerevisiae Are Deficient in Thymidylate Synthetase
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Thymidylate synthetase activity was measured in crude extracts of the yeast Saccharomyces cerevisiae by a sensitive radiochemical assay. Spontaneous non-conditional mutants auxotrophic for thymidine 5'-monophosphate (tmp1) lacked detectable thymidylate synthetase activity in cell-free extracts. In contrast, the parent strains (tup1, -2, or -4), which were permeable to thymidine 5'-monophosphate, contained levels of activity similar to those found in wild-type cells. Specific activity of thymidylate synthetase in crude extracts of normal cells or of cells carrying tup mutations was essentially unaffected by the ploidy or mating type of the cells, by the medium used for growth, by the respiratory capacity of the cells, by concentrations of exogenous thymidine 5'-monophosphate as high as 50 mug/ml, or by subsequent removal of thymidine 5'-monophosphate from the medium. Extracts of a strain bearing the temperature-sensitive cell division cycle mutation cdc21 lacked detectable thymidylate synthetase activity under all conditions tested. Its parent and another mutant (cdc8), which arrests with the same terminal phenotype under restrictive conditions, had normal levels of the enzyme. Cells of a temperature-sensitive thymidine 5'-monophosphate auxotroph arrested with a morphology identical to the cdc21 strain at the nonpermissive temperature and contained demonstrably thermolabile thymidylate synthetase activity. Tetrad analysis and the properties of revertants showed that the thymidylate synthetase defects were a consequence of the same mutation causing, in the auxotrophs, a requirement for thymidine 5'-monophosphate and, in the conditional mutants, temperature sensitivity. Complementation tests indicated that tmp1 and cdc21 are the same locus. These results identify tmp1 as the structural gene for yeast thymidylate synthetase. 相似文献
5.
Two asparagine auxotrophic mutants (N3, N4) were isolated from the Gat- strain of Chinese hamster ovary cells, using a selection procedure modified from that of Goldfarb et al. (1). The defect in these mutants is due to a deficiency in asparagine synthetase activity. N3, in particular, had no measurable enzyme activity. Complementation analysis by PEG-mediated cell fusion showed that the auxotrophic phenotype behaved as a recessive trait; complementation was obtained between N3 or N4 and the pseudoauxotroph, Asn3, which has a temperature-sensitive asparagyl-tRNA synthetase activity. Revertants obtained by plating N3 or N4 in asparagine-free medium had about normal levels of asparagine synthetase activity and were produced with a probability of about 10(-6) per cell per generation. Three particular revertants of N3 and one revertant of N4 were shown to have asparagine synthetase activities that were different in thermolability from that of the wild type. This observation is consistent with the suggestion that N3 and N4 have defective structural genes rather than defective regulatory genes for asparagine synthetase. 相似文献
6.
Immunochemical characterization of glutamine synthetase from Neurospora crassa glutamine auxotrophs.
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Glutamine synthetase derived from two Neurospora crassa glutamine auxotrophs was characterized. Previous genetic studies indicated that the mutations responsible for the glutamine auxotrophy are allelic and map in chromosome V. When measured in crude extracts, both mutant strains had lower glutamine synthetase specific activity than that found in the wild-type strain. The enzyme from both auxotrophs and the wild-type strain was partially purified from cultures grown on glutamine as the sole nitrogen source, and immunochemical studies were performed in crude extracts and purified fractions. Quantitative rocket immunoelectrophoresis indicated that the activity per enzyme molecule is lower in the mutants than in the wild-type strain; immunoelectrophoresis and immunochemical titration of enzyme activity demonstrated structural differences between the enzymes from both auxotrophs. On the other hand, the monomer of glutamine synthetase of both mutants was found to be of a molecular weight similar to that of the wild-type strain. These data indicate that the mutations are located in the structural gene of N. crassa glutamine synthetase. 相似文献
7.
Summary The assignment of the knownade genes to steps in purine biosynthesis inSchizosaccharomyces pombe has been completed with the demonstration that anade3 mutants lacks FGAR amidotransferase,ade1A mutants lack GAR synthetase andade1B mutants lack AIR synthetase. A comparison of enzyme activity with map position forade1 mutants shows that (1) complementingade1A mutants lack GAR synthetase but possess wild type amounts of AIR synthetase, (2) complementingade1B mutants lack AIR synthetase but posses variable amounts of GAR synthetase, (3) non-complementing mutants lack both activities. In wild type strains the two activities fractionate together throughout a hundred-fold purification. Hence theade1 gene appears to code for a bifunctional enzyme catalysing two distinct steps in purine biosynthesis. The two activities are catalysed by two different regions of the polypeptide chain which can be altered independendently by mutation. Gel filtration studies on partially purified enzymes from wild type and various complementing mutant strains, indicate that the bifunctional enzyme is a multimer consisting of between four and six sub-units of 40,000 daltons each. GAR synthetase activity is associated with both the monomeric and multimeric forms but AIR synthetase is only associated with the multimer. A comparison of enzyme levels between diploids and their original complementing haploid strains suggests that complementation is due to hybrid enzyme formation. 相似文献
8.
Genetic and Immunological Studies of Bacteriophage T4 Thymidylate Synthetase 总被引:3,自引:3,他引:0
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Thymidylate synthetase, which appears after infection of Escherichia coli with bacteriophage T4, has been partially purified. The phage enzyme is immunologically distinct from the host enzyme and has a molecular weight of 50,000 in comparison to 68,000 for the host enzyme. A system has been developed to characterize T4 td mutants previously known to have impaired expression of phage thymidylate synthetase. For this system, an E. coli host lacking thymidylate synthetase was isolated. Known genetic suppressors were transduced into this host. The resulting isogenic hosts were infected with phage T4 td mutants. The specific activities and amounts of cross-reacting material induced by several different types of phage mutants under conditions of suppression or non-suppression have been examined. The results show that the phage carries the structural gene specifying the thymidylate synthetase which appears after phage infection, and that the combination of plaque morphology, enzyme activity assays, and an assay for immunologically cross-reacting material provides a means for identifying true amber mutants of the phage gene. 相似文献
9.
P. Gröbner P. Loidl 《Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression》1982,697(1):83-88
Thymidylate synthetase and thymidine kinase activities in wild type strain M3b and in thymidine kinase-deficient mutant TU63 of Physarum polycephalum are studied. Whenever nuclear division occurs in macroplasmodia of wild type, thymidine kinase and thymidylate synthetase activities sharply increase, although the increase of thymidylate synthetase activity is less pronounced than thymidine kinase activity. This is also true for other investigated nuclear divisions during the life cycle of P. polycephalum. It is shown for the first time that thymidylate synthetase is a periodically fluctuating enzyme during the naturally synchronous nuclear division cycle of P. polycephalum with a peak of specific activity in the S phase. In macroplasmodia, as well as after germination of microsclerotia of M3b, thymidine kinase is the dominant enzyme, whereas at the time of the precleavage mitosis in sporulating macroplasmodia thymidylate synthetase is the predominant enzyme. This study describes and compares both dTMP-synthesizing enzymes during proliferation and differentiation of the same organism. 相似文献
10.
Relationship Between Escherichia coli B Titer and the Level of Deoxycytidylate Deaminase Activity Induced on Bacteriophage T2r+ Infection
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The activities of six bacteriophage T2r(+)-induced enzymes (thymidylate synthetase, deoxycytidylate deaminase, thymidylate kinase, deoxycytidylate hydroxymethylase, deoxycytidine pyrophosphatase, and dihydrofolate reductase) were measured after dilution of phage-infected Escherichia coli B from 8 x 10(8) to 2 x 10(8) cells per ml. The only enzyme activity altered was that of deoxycytidylate deaminase, which increased three- to fourfold. Conversely, the rapid concentration of cells from 2 x 10(8) to 8 x 10(8) per ml did not result in a reduction in deaminase activity. Although an enhancement in aeration reduced the response of deoxycytidylate deaminase to cellular dilution, the influence of potential metabolic inhibitors or activators could not be shown. The change in deoxycytidylate deaminase activity appeared to be associated with an altered translational event, since the increase could not be prevented by rifampin but was blocked effectively by chloramphenicol and hydroxylamine. In addition, antibody to the T2 phage-induced deoxycytidylate deaminase demonstrated that the increase in enzyme activity was associated with a corresponding increase in radioactive leucine incorporated into the enzyme antigen. 相似文献
11.
B N Mishan'kan A N Kravchenko V G Ma?ski? 《Zhurnal mikrobiologii, epidemiologii, i immunobiologii》1989,(2):24-29
Thymine- and thymidine-dependent mutants of Y. pestis strain EV-76 have been isolated and characterized. Obtaining Y. pestis thymine-dependent mutants in trimethoprim-containing media with full nutritional value in the presence of thymine and thymidine and the capacity of natural strains from the foci of infection in Transcaucasia and Mongolia to grow in such media indicate that Y. pestis has gene tpp controlling thymidine phosphorylase, but this enzyme is strongly suppressed under normal conditions. The capacity for its suppression under definite conditions and the degree of the activation of thymidine phosphorylase determine the realization of Thy and Thyd phenotypes in Y. pestis mutants under study, though both types of these mutants have a mutation damage of gene thy A coding the synthesis of thymidylate synthetase. 相似文献
12.
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
13.
Valine accumulation by alpha-aminobutyric acid-resistant mutants of Serratia marcescens 总被引:6,自引:2,他引:4
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alpha-Aminobutyric acid, norvaline, and norleucine, which are analogues of branched-chain amino acids, inhibited the growth of Serratia marcescens. The inhibitory effect of these three analogues was counteracted by branched-chain amino acids. A number of mutants resistant to these analogues were isolated. alpha-Aminobutyric acid-resistant (abu-r) mutants markedly accumulated l-valine in the culture medium, but the other analogue-resistant mutants did not. Acetohydroxy acid synthetase, which seems to be rate-limiting for the biosynthesis of l-valine, was derepressed in abu-r mutants. One of the abu-r mutants, no. 140, which accumulated over 8 mg of l-valine per ml, had about a 20-fold increase in the enzyme level. Most of the abu-r mutants had acetohydroxy acid synthetase activity which was sensitive to feedback inhibition by l-valine to the same extent as in the parent strain. However, the enzyme of two of abu-r mutants was less sensitive to l-valine, and one of the two was the best valine accumulator. 相似文献
14.
L-Methionine SR-sulfoximine-resistant glutamine synthetase from mutants of Salmonella typhimurium 总被引:3,自引:0,他引:3
Two mutants of Salmonella typhimurium resistant to growth inhibition by the glutamine synthetase transition state analog, L-methionine SR-sulfoximine, were isolated and characterized. These mutants are glutamine bradytrophs and cannot use growth rate-limiting nitrogen sources. Although this phenotype resembles that of mutants with lesions in the regulatory gene for glutamine synthetase, glnG, these mutations do not lie in the glnG gene. Purification and characterization of the glutamine synthetase from one of the mutants and a control strain demonstrated that the mutant enzyme is defective in the reverse gamma-glutamyltransferase activity but has biosynthetic activity that is resistant to inhibition by L-methionine SR-sulfoximine. The mutant enzyme also has a 4.4-fold higher apparent Km for glutamate (0.2 mM versus 2.1 mM, respectively) and a 13.8-fold higher Km for NH3 (6.4 mM versus 0.46 mM) than the enzyme from the control. These data show that the glutamine synthetase protein has been altered by this mutation, designated as glnA982, and suggest that the L-methionine SR-sulfoximine resistance is conferred by a change in the NH3 binding domain of the enzyme. 相似文献
15.
Mutants of Escherichia coli B that contain essentially no detectable glutathione were isolated. These mutants had a very low activity of gamma-glutamylcysteine synthetase or glutathione synthetase. No significant differences in growth in minimal medium were observed between the mutants and the parental strain. The mutants lacking gamma-glutamylcysteine synthetase activity were more susceptible to toxic compounds than either the parental strain or a glutathione synthetase-deficient strain. The mutants lacking gamma-glutamylcysteine synthetase activity were also susceptible to oxygen. 相似文献
16.
Early Biochemical Events Occurring After Infection of Bacillus cereus 569-SP1 with Bacteriophage GSW
After infection of Bacillus cereus 569-SP1 with the 5-hydroxymethyluracil-containing phage GSW, new dTTPase, dUTPase, and dUMP-hydroxymethylase activities appear. No significant changes in activities of other pyrimidine ribonucleoside or 2'-deoxyribonucleoside triphosphate nucleotidohydrolases were detected. dUTP and dUMP inhibit the dTTPase activity, whereas dTTP failed to inhibit dUTPase activity. The K(m) value for the substrate dUTP is 10(-4) M and for dTTP is 4.85 x 10(-4) M. Thymidylate synthetase activity is inhibited only when cells are infected during the late lag or very early log phases of growth; when cells are infected with phage during mid-log, thymidylate synthetase activity is unaffected. The data support the suggestion that, although phage GSW may inhibit an otherwise expected increase in activity of thymidylate synthetase, it fails to affect the already existing activity. The data presented do not allow discrimination as to whether the phage specifies inhibition of de novo synthesis of thymidylate synthetase or the increase in activity of already existing but not fully expressed enzyme. 相似文献
17.
Human leukemic cells resistant to 5-fluoro-2'-deoxyuridine contain a thymidylate synthetase with lower affinity for nucleotides 总被引:4,自引:0,他引:4
A line of human lymphocytic leukemia cells (CCRF-CEM) has been obtained which is 140-fold resistant to the potent cell growth inhibitor 5-fluoro-2'-deoxyuridine (FdUrd). The cells were also 11-fold cross-resistant to 5-fluorouracil. In contrast to several previous studies involving FdUrd-resistant mouse cells, thymidylate synthetase levels were not substantially elevated in these FdUrd-resistant human leukemic cells. Thymidine kinase activity was also unchanged in the resistant cells, although the levels of 5-fluoro-2'-deoxyuridylate (FdUMP), the potent inhibitor of thymidylate synthetase, generated at equimolar doses of FdUrd were about 40% lower than in the sensitive cells. Studies of the kinetics of FdUMP binding to thymidylate synthetase isolated from the FdUrd-resistant cells disclosed a considerably higher dissociation constant (Kd = 1.0 X 10(-9) M) for the ternary covalent enzyme . FdUMP . 5,10-methylene tetrahydrofolate complex compared to the value obtained with enzyme from sensitive cells (Kd = 4.4 X 10(-11) M). The thymidylate synthetase from the FdUrd-resistant cells also showed 17-fold weaker binding of 2'-deoxyuridylate, even though the Km value for 2'-deoxyuridylate was 3-fold lower compared to the enzyme from FdUrd-sensitive cells. The turnover number of the altered enzyme was 1.8-fold higher than that for the normal enzyme but the rate constants for the release of FdUMP from the ternary complex, which is also an enzyme-catalyzed reaction, were identical for both enzymes. Electrophoresis of the radiolabeled ternary complexes on nondenaturing gels showed small but reproducible differences in migration rates. These results demonstrate that the mechanism of resistance to FdUrd in this cell line involves an alteration in the target enzyme, thymidylate synthetase, which causes it have a lower affinity for nucleotides. 相似文献
18.
The addition of methotrexate to the assay system of thymidylate synthetase caused a reduction in the activity of the enzyme but addition of methotrexate to the culture of phytohemagglutinin stimulated normal human lymphocytes caused an increase in the activity of the enzyme which was abolished by the addition of actinomycin D or cycloheximide. These studies suggest that the antimetabolite augmented the enzyme activity by modulating the gene for the enzyme. This modulation of the gene could have been achieved by the thymineless state brought about by methotrexate or the antimetabolite could have affected gene reodont or brought about amplification of the gene. The results of the nucleoside incorporation were consistent with a thymidylate synthetase block; however, other explanations are offered. 相似文献
19.
M Gálová D Koptidesová D Rusznáková P Racay M Kollárová 《Archives of biochemistry and biophysics》1992,296(1):81-87
NADPH-dependent thymidylate synthetase from Streptomyces aureofaciens has been purified to homogenity by a two-step chromatographic procedure including anion-exchange chromatography and affinity chromatography on methotrexate-Sepharose 4B. The enzyme was purified 1025-fold with a 34% yield. Basic characteristics of the enzyme were determined: molecular weight of the enzyme subunit (28,000), pH and temperature optimum, effect of cations, dependency on reducing agents, Km values for dUMP, mTHF, and NADPH (3.78, 21.1, and 38.9 microM, respectively), and inhibition effect of 5-FdUMP. Binding studies revealed the enzyme mechanism to be ordered sequential: dUMP bound before mTHF. S. aureofaciens thymidylate synthetase exhibits an absolute requirement for NADPH for the enzyme activity--a unique feature not displayed by any of the thymidylate synthetases isolated so far. NADPH is not consumed during enzyme reaction, indicating its regulatory role. The properties of S. aureofaciens thymidylate synthetase show that it is a monofunctional bacterial enzyme. 相似文献
20.
Stable mutants of Chinese hamster V79 cells deficient in thymidylate synthetase (TS; E.C. 2.1.1.45) have been selected from cultures grown in medium supplemented with folinic acid, aminopterin, and thymidine (FAT). After chemical mutagenesis, the frequency of colonies resistant to the "FAT" medium increased more than 100-fold over the spontaneous frequency. The optimal expression time of the mutant phenotype was 5-7 days after mutagen treatment. The recovery of FAT-resistant colonies in the selective medium was not affected by the presence of wild-type cells at a density below 9,000 cells per cm2. All 21 mutants tested exhibited thymidine auxotrophy; neither folinic acid nor deoxyuridine could support mutant cell growth. There was no detectable TS activity in all 11 mutants so far examined and only about 50% of wild-type activity in three prototrophic revertants, as measured by whole-cell and cell-free enzyme assays. The apparent Michaelis-Menten constant (Km) for deoxyuridine-5'-monophosphate and inhibition constant (Ki) for 5-fluoro-deoxyuridine-5'-monophosphate, measured by whole-cell enzyme assay, appear to be similar for the wild-type and revertant cell lines. Using 5-fluoro-[6-3H]-2'-deoxyuridine 5'-monophosphate as active site titrant, the relative amounts of TS in crude cell extract from the parental, revertant, and mutant cells were shown to exist in a 1:0.5:0 ratio. Furthermore, the enzymes from two revertants were more heat labile than that of V79 cells. These properties, taken together, suggest that the FAT-resistant, thymidine auxotrophic phenotype may be the result of a structural gene mutation at the TS locus. The availability of such a mutant facilitates studies on thymidylate stress in relation to DNA metabolism, cell growth, and mutagenesis. 相似文献