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1.
Differential expression of two linked selection genes (HSVI-tk and Eco.gpt) in transformed teratocarcinoma and in L cells 总被引:1,自引:0,他引:1
C Lasserre J Thillet C Besnard D Bucchini R Pictet J Jami 《Journal of cellular physiology》1985,124(1):37-42
Upon transfection of (TK-)F9 teratocarcinoma stem-cells and (TK-)L fibroblasts with a plasmid carrying two selection genes, Eco.gpt and HSVI-tk, selection for gpt gene yielded ten times fewer colonies than selection for tk. Only the transformed clones selected for gpt had measurable xanthine guanine phosphoribosyltransferase (XGPRT) activity (Jami et al., 1983). Eco.gpt coding for XGPRT was under the control of simian virus 40 (SV40) early genes' regulating sequences (SV-gpt). In the present study, it was verified that the low efficiency of gpt selection in mouse cells was not due to the eucaryotic controlling sequences added to the bacterial gene. The transformed clones selected for tk that had no XGPRT activity possessed at least one uninterrupted copy of the composite SV-gpt gene and as many copies of the transforming plasmid as the cells selected for gpt expression. In a further test, the gpt gene was placed under the control of tk-regulating sequences and inserted with the tk gene in the same vector. Under these conditions, expression of XGPRT in the transformed clones selected for tk was improved, even though relative selection for gpt remained low. 相似文献
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K. Michalova J. Jami J. -P. Rousset E. Ritz D. Bucchini 《Experimental cell research》1978,114(2):293-300
Metaphases collected from cultures grown for three cell cycles in 5-bromodeoxyuridine (BUdR) and then for one or two further cell cycles without BUdR show persistence of differentially FPG-stained chromatids. The cell cycle length is not altered by the presence of BUdR. After removal of BUdR, the cells synthesize DNA and incorporate mainly thymine, as demonstrated by density gradient analysis of DNA. Our observations suggest that chromatids with T-B DNA stain lightly after removal of BUdR, in contrast with their dark staining when cultures are maintained in BUdR. Thus, in any experimental condition, there is a correspondence between the nature (T-DNA or B-DNA) of the “old” DNA strands and the FPG-staining (dark or light) of the chromatids. 相似文献
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Fimognari C Sestili P Lenzi M Bucchini A Cantelli-Forti G Hrelia P 《Mutation research》2008,648(1-2):15-22
In contrast to damage of genomic DNA and despite its potential to affect cell physiology, RNA damage is a poorly examined field in biomedical research. Potential triggers of RNA damage as well as its pathophysiological implications remain largely unknown. While less lethal than mutations in genome, such non-acutely lethal insults to cells have been recently associated with underlying mechanisms of several human chronic diseases. We investigated whether RNA damage could be related to the exposure of particular xenobiotics by testing the RNA-damaging activity of a series of chemicals with different mechanisms of action. Cultured human T-lymphoblastoid cells were treated with ethyl methanesulfonate (EMS), H(2)O(2), doxorubicin, spermine, or S-nitroso-N-acetylpenicillamine (SNAP). Furthermore, we studied the potential protective activity of a pomegranate extract against RNA damage induced by different chemicals. Special attention has been paid to the protective mechanisms of the extract. The protective effect of pomegranate can be mediated by alterations of the rates of toxic agent absorption and uptake, by trapping of electrophiles as well as free radicals, and protection of nucleophilic sites in RNA. We used two different treatment protocols (pre- and co-treatment) for understanding the mechanism of the inhibitory activity of pomegranate. We demonstrated that total RNA is susceptible to chemical attack. A degradation of total RNA could be accomplished with doxorubicin, H(2)O(2), spermine and SNAP. However, EMS, a well-known DNA-damaging agent, was devoid of RNA-damaging properties, while spermine and SNAP, although lacking of DNA-damaging properties, were able to damage RNA. Pomegranate reduced the RNA-damaging effect of doxorubicin, H(2)O(2), and spermine. Its inhibitory activity could be related with its ability to forms complexes with doxorubicin and H(2)O(2), or interacts with the intracellular formation of reactive species mediating their toxicity. For spermine, an alteration of the rates of spermine absorption and uptake can also be involved. 相似文献
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Targeted disruption of the insulin receptor gene in the mouse results in neonatal lethality. 总被引:19,自引:0,他引:19
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R L Joshi B Lamothe N Cordonnier K Mesbah E Monthioux J Jami D Bucchini 《The EMBO journal》1996,15(7):1542-1547
Targeted disruption of the insulin receptor gene (Insr) in the mouse was achieved using the homologous recombination approach. Insr+/- mice were normal as shown by glucose tolerance tests. Normal Insr-/- pups were born at expected rates, indicating that Insr can be dispensable for intrauterine development, growth and metabolism. However, they rapidly developed diabetic ketoacidosis accompanied by a marked post-natal growth retardation (up to 30-40% of littermate size), skeletal muscle hypotrophy and fatty infiltration of the liver and they died within 7 days after birth. Total absence of the insulin receptor (IR), demonstrated in the homozygous mutant mice, also resulted in other metabolic disorders: plasma triglyceride level could increase 6-fold and hepatic glycogen content could be five times less as compared with normal littermates. The very pronounced hyperglycemia in Insr-/- mice could result in an increased plasma insulin level of up to approximately 300 microU/ml, as compared with approximately 25 microU/ml for normal littermates. However, this plasma level was still unexpectedly low when compared with human infants with leprechaunism, who lack IR but who could have extremely high insulinemia (up to > 4000 microU/ml). The pathogenesis resulting from a null mutation in Insr is discussed. 相似文献
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The state of intracellular viral DNA in cells infected with channel catfish virus has been studied by the Hirt selective extraction procedure and by restriction endonuclease digestion. The sedimentation properties and restriction patterns of viral DNA in the Hirt supernatant fraction indicate that the majority, if not all, of the DNA is in the form of linear unit-length (Mr approximately equal to 85 x 10(6)) molecules. However, restriction digests of viral DNA in the pellet fraction lacked two fragments corresponding to the molecular ends of unit-length DNA. In addition, there appeared in HpaI digests of pellet DNA a new restriction fragment interpretable as the product of fusion between the ends of unit-length molecules. The size of the new fragment requires that fusion occur in such a way that one copy of the terminally repeated sequences (Mr approximately equal to 12.3 x 10(6)) of the unit-length DNA is lost in the process. In pulse-chase experiments, radioactivity flowed from the pellet fraction to the supernatant fraction, suggesting a precursor-product relationship for these DNA species. The results are easily understood if unit-length virion DNA is generated by excision from concatemeric structures. 相似文献
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Ivana Stolić Katarina Mišković Anahi Magdaleno Ariel Mariano Silber Ivo Piantanida Miroslav Bajić Ljubica Glavaš-Obrovac 《Bioorganic & medicinal chemistry》2009,17(6):2544-2554
Novel bisbenzimidazoles (4–6), characterized by 3,4-ethylenedioxy-extension of thiophene core, revealed pronounced affinity and strong thermal stabilization effect toward ds-DNA. They interact within ds-DNA grooves as dimmers or even oligomers and agglomerate along ds-RNA. Compounds 4–6 have shown moderate to strong antiproliferative effect toward panel of eight carcinoma cell lines. Compound 5 displayed the best inhibitory potential and in equitoxic concentration (IC50 = 1 × 10?6 M) induced accumulation of cells in G2/M phase after 48 h of incubation. Fluorescence microscopy showed that 5 entered into live HeLa cells within 30 min, but did not accumulate in nuclei even after 2.5 h. Compound 5 inhibited the growth of Trypanosome cruzi epimastigotes (IC50 = 4.3 × 10?6 M). 相似文献
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Mehrshahi P Gonzalez-Jorge S Akhtar TA Ward JL Santoyo-Castelazo A Marcus SE Lara-Núñez A Ravanel S Hawkins ND Beale MH Barrett DA Knox JP Gregory JF Hanson AD Bennett MJ Dellapenna D 《The Plant journal : for cell and molecular biology》2010,64(2):267-279
Cellular folates function as co-enzymes in one-carbon metabolism and are predominantly decorated with a polyglutamate tail that enhances co-enzyme affinity, subcellular compartmentation and stability. Polyglutamylation is catalysed by folylpolyglutamate synthetases (FPGSs) that are specified by three genes in Arabidopsis, FPGS1, 2 and 3, which reportedly encode plastidic, mitochondrial and cytosolic isoforms, respectively. A mutational approach was used to probe the functional importance of folate polyglutamylation in one-carbon metabolism and development. Biochemical analysis of single FPGS loss-of-function mutants established that folate polyglutamylation is essential for organellar and whole-plant folate homeostasis. However, polyglutamylated folates were still detectable, albeit at lower levels, in organelles isolated from the corresponding isozyme knockout lines, e.g. in plastids and mitochondria of the fpgs1 (plastidial) and fpgs2 (mitochondrial) mutants. This result is surprising given the purported single-compartment targeting of each FPGS isozyme. These results indicate redundancy in compartmentalised FPGS activity, which in turn explains the lack of anticipated phenotypic defects for the single FPGS mutants. In agreement with this hypothesis, fpgs1 fpgs2 double mutants were embryo-lethal, fpgs2 fpgs3 mutants exhibited seedling lethality, and fpgs1 fpgs3 mutants were dwarfed with reduced fertility. These phenotypic, metabolic and genetic observations are consistent with targeting of one or more FPGS isozymes to multiple organelles. These data confirm the importance of polyglutamylation in folate compartmentation, folate homeostasis and folate-dependent metabolic processes, including photorespiration, methionine and pantothenate biosynthesis. 相似文献