Addendum: Properties of an in vitro system for studying temperature-sensitive DNA synthesis in ts A1S9 mouse L-cells

1978 ◽  
Vol 56 (10) ◽  
pp. 998-998
Author(s):  
Jerome Humbert ◽  
Rose Sheinin
Keyword(s):  
Dna Synthesis ◽  
Temperature Sensitive ◽  
In Vitro System ◽  
L Cells

Properties of an in vitro system for studying temperature-sensitive DNA synthesis in ts A1S9 mouse L-cells

1978 ◽  
Vol 56 (6) ◽  
pp. 444-451 ◽  
Author(s):  
Jerome Humbert ◽  
Rose Sheinin
Keyword(s):  
Dna Synthesis ◽  
Buoyant Density ◽  
Velocity Sedimentation ◽  
Synthetic Activity ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Activity Restriction ◽  
L Cells

The in vitro DNA synthesis has been observed in whole cell lysates and in cytosol and nuclear fractions of wild-type (WT-4) mouse L-cells and ts A1S9 cells which exhibit temperature-sensitive (ts) DNA replication in vivo. The product, labelled with substrate 3H-labelled TTP, is resistant to alkali and has the buoyant density (1.709 g/cm3) expected for normal mouse DNA. Pulse-chase studies, in which newly made, single-stranded DNA was analyzed by velocity sedimentation in alkaline sucrose density gradients, revealed that in vitro DNA synthesis proceeds by a discontinuous mechanism. Approximately half of the DNA made in a 30-s pulse sedimented at 3–8S; the rest was very heterogeneous with S values between [Formula: see text] and 30S. After incubation for up to 300 s, a majority of the newly made DNA (>85%) sedimented as the larger, heterogeneous material, with some cosedimenting with chromosomal size DNA.The ts DNA synthesis phenotype of ts A1S9 cells is expressed in vitro. Thus, the activity of extracts of ts cells incubated at the nonpermissive (38.5 °C) temperature was commensurate with the in vivo activity. Restriction of the ts phenotype to DNA synthesis is evident in vitro since the RNA synthetic activity of lysates of temperature-inactivated ts A1S9 cells was equivalent to that of extracts obtained from cells grown at the permissive temperature (33.5 °C). The DNA synthetic activity of nuclei from WT-4 or ts A1S9 cells grown at 33.5 °C plus homologous cytosol is equivalent to that of the whole lysate. In contrast, such cytosol preparations give little, if any, enhancement of the activity of nuclei from ts A1S9 cells incubated at 38.5 °C for 16 h. The cytosol of such temperature-inactivated cells, which are almost fully effective with nuclei of control cells, produce little or no enhancement of DNA synthesis by homologous nuclei.

Damage by Dichlorvos of Human Lymphocyte DNA

1980 ◽  
Vol 66 (4) ◽  
pp. 425-430 ◽  
Author(s):  
Paolo Perocco ◽  
Angela Fini
Keyword(s):  
Dna Synthesis ◽  
Human Lymphocyte ◽  
Tritiated Thymidine ◽  
Human Lymphocytes ◽  
Ultraviolet Rays ◽  
Thymidine Uptake ◽  
Short Term ◽  
In Vitro System ◽  
Tritiated Thymidine Uptake

The action of dichlorvos (2.2-dichlorovinyldimethyl phosphate) was studied with a short-term in vitro system which utilizes human lymphocytes. The parameters studied were the action exerted by the pesticide on scheduled (semiconservative) and unscheduled (reparative) DNA synthesis measured as tritiated thymidine uptake. The results obtained show that dichlorvos affects semiconservative DNA synthesis, damages human lymphocyte DNA inducing low reparative synthesis, and interferes with DNA repair processes after damage exerted by ultraviolet rays.

scholarly journals The inhibition of nucleic acid synthesis by mycophenolic acid

1969 ◽  
Vol 113 (3) ◽  
pp. 515-524 ◽  
Author(s):  
T J Franklin ◽  
Jennifer M. Cook
Keyword(s):  
Dna Synthesis ◽  
Mycophenolic Acid ◽  
Early Stage ◽  
Acid Synthesis ◽  
Purine Nucleotides ◽  
Free System ◽  
L Cells ◽  
A Cell ◽  
Inhibition Of Dna Synthesis

1. Mycophenolic acid, an antibiotic of some antiquity that more recently has been found to have marked activity against a range of tumours in mice and rats, strongly inhibits DNA synthesis in the L strain of fibroblasts in vitro. 2. The extent of the inhibition of DNA synthesis is markedly increased by preincubation of the cells with mycophenolic acid before the addition of [14C]thymidine. 3. The inhibition of DNA synthesis by mycophenolic acid in L cells in vitro is reversed by guanine in a non-competitive manner, but not by hypoxanthine, xanthine or adenine. 4. The reversal of inhibition by guanine can be suppressed by hypoxanthine, 6-mercaptopurine and adenine. 5. Mycophenolic acid does not inhibit the incorporation of [14C]thymidine into DNA in suspensions of Landschütz and Yoshida ascites cells in vitro. 6. Mycophenolic acid inhibits the conversion of [14C]hypoxanthine into cold-acid-soluble and -insoluble guanine nucleotides in Landschütz and Yoshida ascites cells and also in L cells in vitro. There is some increase in the radioactivity of the adenine fraction in the presence of the antibiotic. 7. Mycophenolic acid inhibits the conversion of [14C]hypoxanthine into xanthine and guanine fractions in a cell-free system from Landschütz cells capable of converting hypoxanthine into IMP, XMP and GMP. 8. Preparations of IMP dehydrogenase from Landschütz ascites cells, calf thymus and LS cells are strongly inhibited by mycophenolic acid. The inhibition showed mixed type kinetics with Ki values of between 3·03×10−8 and 4·5×10−8m. 9. Evidence was also obtained for a partial, possibly indirect, inhibition by mycophenolic acid of an early stage of biosynthesis of purine nucleotides as indicated by a decrease in the accumulation of formylglycine amide ribonucleotide induced by the antibiotic azaserine in suspensions of Landschütz and Yoshida ascites cells and L cells in vitro.

scholarly journals Structure of interphase nuclei in relation to the cell cycle. Chromatin organization in mouse L cells temperature-sensitive for DNA replication

1978 ◽  
Vol 77 (1) ◽  
pp. 246-263 ◽  
Author(s):  
G Setterfield ◽  
R Sheinin ◽  
I Dardick ◽  
G Kiss ◽  
M Dubsky
Keyword(s):  
Dna Synthesis ◽  
Marked Change ◽  
Cell Types ◽  
Condensed Chromatin ◽  
Heat Inactivation ◽  
Temperature Sensitive ◽  
Primary Defect ◽  
L Cells ◽  
Mutant Lines ◽  
Temperature Upshift

Mutant lines of mouse L cells, TS A1S9, and TS C1, show temperature-sensitive (TS) DNA synthesis and cell division when shifted from 34 degrees to 38.5 degrees C. With TS A1S9 the decline in DNA synthesis begins after 6-8 h at 38.5 degrees C and is most marked at about 24 h. Most cells in S, G2, or M at temperature upshift complete one mitosis and accumulate in the subsequent interphase at G1 or early S as a result of expression of a primary defect, failure of elongation of newly made small DNA fragments. Heat inactivation of TS C1 cells is more rapid; they fail to complete the interphase in progress at temperature upshift and accumulate at late S or G2. Inhibition of both cell types is reversible on return to 34 degrees C. Cell and nuclear growth continues during inhibition of replication. Expression of both TS mutations leads to a marked change in gross organization of chromatin as revealed by electron microscopy. Nuclei of wild-type cells at 34 degrees and 38.5 degrees C and mutant cells at 34 degrees C show a range of aggregation of condensed chromatin from small dispersed bodies to large discrete clumps, with the majority in an intermediate state. In TS cells at 38.5 degrees C, condensed chromatin bodies in the central nuclear region become disaggregated into small clumps dispersed through the nucleus. Morphometric estimation of volume of condensed chromatin indicates that this process is not due to complete decondensation of chromatin fibrils, but rather involves dispersal of large condensed chromatin bodies into finer aggregates and loosening of fibrils within the aggregates. The dispersed condition is reversed in nuclei which resume DNA synthesis when TS cells are downshifted from 38.5 degrees to 34 degrees C. The morphological observations are consistent with the hypothesis that condensed chromatin normally undergoes an ordered cycle of transient, localized disaggregation and reaggregation associated with replication. In temperature-inactivated mutants, normal progressive disaggregation presumably occurs, but subsequent lack of chromatin replication prevents reaggregation.

DNA synthesis and multinucleation in embryonic stem cell-derived cardiomyocytes

1995 ◽  
Vol 269 (6) ◽  
pp. H1913-H1921 ◽  
Author(s):  
M. G. Klug ◽  
M. H. Soonpaa ◽  
L. J. Field
Keyword(s):  
Dna Synthesis ◽  
Thymidine Incorporation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Isolated Cells ◽  
In Vitro System ◽  
Multinucleated Cells

The proliferative capacity of embryonic stem (ES) cell-derived cardiomyocytes was assessed. Enriched preparations of cardiomyocytes were isolated by microdissection of the cardiogenic regions of cultured embryoid bodies. The identity of the isolated cells was established by immunocytology, and mitotic activity was monitored by [3H]thymidine incorporation and pulse-chase experiments. ES-derived cardiomyocytes were mitotically active and predominantly mononucleated at 11 days after cardiogenic induction. By 21 days postinduction, cardiomyocyte DNA synthesis was markedly decreased, with a concomitant increase in the percentage of multinucleated cells. Interestingly, the duration of active cardiomyocyte reduplication in the ES system appeared to be roughly similar to that observed during normal murine cardiogenesis. Given these observations, as well as the genetic tractability of ES cells, ES-derived cardiogenesis might provide a useful in vitro system with which to assess the molecular regulation of the cardiomyocyte cell cycle.

scholarly journals The Size and Transit Time of Nondividing Subpool of Precursor Cells in Acute Leukemia

Blood ◽  
1967 ◽  
Vol 29 (1) ◽  
pp. 63-76 ◽  
Author(s):  
MAX SHIUMING LIN ◽  
BERTHA A. BOURONCLE
Keyword(s):  
Acute Myeloid Leukemia ◽  
Acute Leukemia ◽  
Dna Synthesis ◽  
Transit Time ◽  
Myeloid Leukemia ◽  
Precursor Cells ◽  
Synthesis Time ◽  
In Vitro System ◽  
Acute Myeloid

Abstract 1. In order to elucidate the postulation and to quantitate the size and transit time of the nondividing subpool of precursor cells in acute leukemia, mathematical formulas based on a prolonged-labeling model were derived for the following kinetic parameters: (1) DNA synthesis time and generation time of dividing precursor cells in normal hemopoiesis and in acute leukemia. (2) size and transit time of the nondividing subpool of precursor cells in acute leukemia. The validity of the assumptions made in the model was discussed. 2. In vitro experimental estimation of the above kinetic parameters was done for the myeloid precursor pool in four cases: two with normal hemopoiesis; one, acute myeloid leukemia; one, subacute myeloid leukemia. 3. Variable decrease with time in availability of tritiated thymidine given initially in single dose to the in vitro system was observed and discussed. 4. DNA synthesis time and generation time of the dividing myeloid precursor cells in the two leukemic patients were not significantly different from, and were no shorter than, the corresponding values for normal myeloid precursor cells in the two nonleukemic patients. Thus it allows the prediction that as much as 86 per cent of the myeloid precursor cells in the marrow may be nondividing in acute myeloid leukemia. 5. In a case of acute myeloid leukemia about 87 per cent of the myeloid precursor cells in the marrow were estimated as nondividing with nondividing subpool transit time of about 32 days in closed in vitro system. The probable meanings of these in vitro estimates in vivo were discussed. 6. The result and analytical review of literature support the postulation of heterogeneous acute leukemia precursor cells with the nondividing fraction consisting of end cells being maintained by the dividing fraction. It is also suggested that a small fraction of nondividing cells may be capable of DNA synthesis and thus be initially-labeling. More definitive approaches to the problems await future investigation.

scholarly journals Maintenance of Human Germinal Center B Cells In Vitro

Blood ◽  
1997 ◽  
Vol 89 (3) ◽  
pp. 919-928 ◽  
Author(s):  
John D. Pound ◽  
John Gordon
Keyword(s):  
B Cells ◽  
Dna Synthesis ◽  
B Cell ◽  
Germinal Center ◽  
Culture System ◽  
Cell Phenotype ◽  
Common Component ◽  
Cell Numbers ◽  
L Cells

Abstract The ability to maintain germinal center (GC) B cells in culture should facilitate studies on the molecular and cellular events which accompany affinity maturation and the generation of memory in T-dependent responses. We have investigated the ability of cytokines to maintain human tonsillar GC B cells (IgD−/CD39−/CD38+/CD77+) in the “CD40 culture system.” In the absence of added cytokines, CD40 monoclonal antibody held on CD32-transfected L cells effectively sustained DNA synthesis in GC B cells for a maximum 3 to 4 days. Of the following cytokines (interleukin-1β [IL-1β], IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, and stem cell factor), only IL-2 and IL-4 provided a significant enhancement to DNA synthesis in the CD40 culture system; this was modest and shortterm. Following a study on the cooperative activity between pairs of cytokines, triple combinations were identified that could maintain high levels of GC B-cell stimulation for at least 10 days. IL-10 was a common component of these synergistic cytokine cocktails, which were IL-10 + IL-4 + IL-7; IL-10 + IL-3 + IL-7; IL-10 + IL-1β + IL-2; IL-10 + IL-1β + IL-3, and IL-10 + IL-3 + IL-6. Culture of GC B cells with these cytokine combinations resulted in a net increase in viable cell numbers of 50% to 100% whereas total cell numbers increased up to fourfold. Cells recovered from these cultures retained a GC B-cell phenotype with a significant proportion being CD38+/CD44−, features characteristic of centroblasts. Studies with metabolically inactive CD32-L cells supported a role for stromal cell-derived soluble factors in maintaining GC B cells in vitro.

scholarly journals ISOLATION AND CHARACTERIZATION OF dnaX AND dnaY TEMPERATURE-SENSITIVE MUTANTS OF ESCHERICHIA COLI

Genetics ◽  
1979 ◽  
Vol 92 (4) ◽  
pp. 1041-1059
Author(s):  
Joan M Henson ◽  
Herman Chu ◽  
Carleen A Irwin ◽  
James R Walker
Keyword(s):  
Escherichia Coli ◽  
Dna Synthesis ◽  
Fold Increase ◽  
Temperature Sensitive ◽  
E Coli ◽  
Temperature Sensitive Mutants ◽  
Isolation And Characterization ◽  
Ts Mutations

ABSTRACT Escherichia coli mutants with temperature-sensitive (ts) mutations in dnaX and dnaY genes have been isolated. Based on transduction by phage PI, dnaX and Y have been mapped at minutes 10.4-10.5 and 12.1, respectively, in the sequence dnaX purE dnaY. Both dnaXts36 and YtslO are recessive to wild-type alleles present on episomes. F13 carries both dnaX  + and Y  +; the shorter F210 carries dnaY  +, but not X  +. Lambda transducing phages that carry dnaX  + or Y  + have been isolated, and hybrid plasmids of Col E1 and E. coli DNA from the CLARKE and CARBON (1976) collection also carry portions of the dnaX purE dnaY region. Results obtained with the λ transducing phages and the hybrid plasmids suggest that dnaX is a different gene from the previously characterized dnaZ gene, which is also near minute 10.5.—The dnaXts36 mutant, after a shift to 42°, stopped DNA synthesis gradually, and the total amount of DNA increased two-fold. When this mutant was shifted to M°, the rate of DNA synthesis dropped immediately and the final increment of DNA was only 10% of the initial amount. Replicative DNA synthesis in toluene-treated cells was completely inhibited at 42° and was partially in-hibited even at 30°.—When the dnaYtslO mutant was shifted to 42°, DNA synthesis gradually stopped, and the amount of DNA increased 3.6-fold. At 44°, residual DNA synthesis amounted to a two-fold increase. Replicative DNA synthesis in vitro in toluene-treated cells was inactivated after 20 minutes at 42° or by "preincubation" of cells at 42° before toluene treatment.— The dnaX and dnaY products probably function in polymerization of DNA, although participation also in initiation cannot be excluded.

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