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14 recordsLinked to original sources
PubMed1976

Enrichment for auxotrophic and heat-sensitive mutants of Neurospora crassa by tritium suicide.

Tritium suicide is shown to be an effective technique for mutant enrichment in Neurospora crassa. When mutagenized conidia were labelled to a high specific radioactivity either with a tritiated amino acid mixture or with [5-3H]uridine at a non-permissive temperature and stored at 4 degrees C to accumulate decays, there was a 13-15 fold enrichment for temperature-sensitive mutants relative to the original mutagenized cultures. For a wild type culture of Neurospora crassa labelled with [5-3H]uridine at 35 degrees C the probability of cell killing per tritium decay was calculated to be 3.64 X 10(-5).

Dose-Response Relationship, Drug↗
PubMed1977

Tritium suicide selection of mammalian cell mutants defective in the transport of neutral amino acids.

Mouse lymphocytic cells of the established line GF-14 were allowed to accumulate intracellular 3H-labeled aminoisobutyric acid (AIB), frozen, and stored over liquid N2. After internal radiation had reduced survival to 1 in 10(4), survivors were plated and tested for their ability to transport AIB. Out of 200 clones tested, two (designated GF-17 and GF-18) were found to have reductions to 13-35% of the parent in the rate of transport of AIB, L-alanine, L-proline, and L-serine; GF-18 also showed significant reductions in the rate of transport of L-glutamate and DL-cysteine. Little or no change was observed for 10 other amino acids or for thymidine. Kinetic analyses revealed that the mutants were not altered in Km for AIB uptake, but had Vmax values approximately 20% the value of the parent strain, GF-14, suggesting that either the number of AIB transport sites or the turnover rate of the sites has been reduced in the two mutants.

Amino Acids↗
PubMed1978

Molecular suicide studies of 125I and 3H disintegration in the DNA of Chinese hamster cells.

The shape of the survival curve for cells inactivated by tritium decay in DNA is modified by the presence of halogenated pyrimidines in the DNA in a manner analogous to their effect on X-ray induced reproductive death. The large shoulder found for tritium suicide is removed completely by coincorporation of 10(-6) M IUdR. The oxygen enhancement ratio for 125I and tritium disintegrations in unsynchronized Chinese hamster cells was determined for cells permitted to accumulate damage from these events at 4 degrees C. The oxygen enhancement ratio for 125I induced damage is 1.4. This is much smaller than the OER found for tritium decay which is similar or more than that found for X-ray exposure under the same conditions. These results suggest that the nature of the lesions produced by 125I decay in DNA are analogous to those produced by high LET radiation while those lesions produced by tritium are similar to lesions produced by roentgen rays. In synchronous V79 cells the effects of 125I induced damage in different regions of the mammalian cell DNA was examined taking advantage of the fact that DNA replication in hamster nuclei follows a time-dependent three dimensional pattern. The experiments indicate that 125I decays accumulated in the G2-period of the cell cycle have different efficiences for the induction of reproductive death depending on the region of the DNA which is labeled. The efficiency for the induction of reproductive death appears to be a maximum in DNA that replicates in V79 cells near the end of the DNA replication cycle. Electron capture events are dramatically efficient in the production of lethal chromosome aberrations. In CHO cells synchronized in the G1-stage of the cell cycle stored in the frozen state the efficiency for the induction of dicentric and ring chromosomes is 0.03. The dose response curve for the induction of these aberrations is linear in contrast to the curvilinear response found for roentgen ray exposure under the same conditions. Data on this kind suggest that there may exist "critical" regions within mammalian cell nuclei where chromatin fibers from two different chromosomes are in close proximity to each other and both are damaged non-repairably by a single electron capture event.

Animals↗
PubMed1979

A cold-sensitive mutant of Saccharomyces cerevisiae defective in ribosome processing.

Cold-sensitive mutants of Saccharomyces cerevisiae isolated by tritium suicide were screened for defects in ribosome biosynthesis. The biochemical defects of mutant dip-1 (defective in processing) were characterized; it is defective in ribosome biosynthesis at the level of production of the primary 35S transcript. At restrictive conditions mutant dip-1 accumulates abnormal rRNA in addition to wild-type rRNA. In the mutant the first observable transcription product was a 14SRNA species which had sequence homologies to 18S rDNA and was the major rRNA component of the 40S ribosomal subunit. In addition, the ribonucleoprotein particles of dip-1 harbored RNA molecules with homologies to yeast rDNA which comprises the spacer region between 18S and 25S rDNA cistrons. Possible causes for the defective production of rRNA and its assembly into subunits are discussed.

Cold Temperature↗
PubMed2025

Approaches to Study Proteins Encoded by Essential Genes.

Although the phenotypes and functions of nonessential proteins can be studied by deletion of their coding sequences (both gene copies in diploid organisms), essential genes cannot be deleted unless loss of the encoded protein can be bypassed. Bypass is often achieved by supplementation with the product of the enzyme. However, supplementation cannot bypass loss of essential genes such as those encoding enzymes of DNA or RNA synthesis. To study proteins encoded by essential genes that cannot be bypassed, the mutations must be conditional in nature. The mutant cells must be able to grow under a permissive condition, but fail to grow under a different condition, the nonpermissive condition. Several methods have been developed to obtain conditional mutations in essential genes. Mutations that result in proteins abnormally sensitive to high temperatures are called temperature-sensitive (Ts) mutants and are a widely used type of conditional mutation. An alternative to Ts mutants is the "degron" system to target proteins for destruction by cellular proteases. Approaches to conditionally control the functions of proteins encoded by essential genes, plus the advantages and disadvantages of these and other approaches, will be considered.

Genes, Essential↗
PubMed1978

Apparent suicidal inactivation of DNA polymerase by adenosine 2',3'-riboepoxide 5'-triphosphate.

Adenosine 2',3'-riboepoxide 5'-triphosphate (epoxyATP) has been found to be a suicidal inactivator of DNA polymerase I from Escherichia coli by the following criteria. Inactivation is complete, is first order in enzyme activity, and shows saturation kinetics with an apparent KD of 30 +/- 10 micron for epoxy ATP. This KD is comparable to the KM of the substrate dATP. The t1/2 for inactivation is 1.3 min. Inactivation requires Mg2+ and the complementary template. The enzyme is protected by dATP but not by an excess of template. Gel filtration of the reaction mixture after inactivation with [3H]epoxy ATP results in the comigration of E. coli DNA polymerase I, the tritium-labeled inactivator, and the DNA template. The stoichiometry of binding approaches 1 mol of [3H]epoxy nucleotide per mol of inactivated enzyme. These results are consistent with the hypothesis that epoxy ATP initially serves as a substrate for the polymerase reaction, elongating the DNA chain by a nucleotidyl unit, and subsequently alkylates an essential base at the primer terminus binding site of the enzyme. Epoxy ATP also inactivates human and viral DNA polymerases but not E. coli RNA polymerase or rabbit muscle pyruvate kinase. Hence epoxy ATP may be a specific suicide reagent for DNA polymerases.

Adenosine Triphosphate↗
PubMed1978

Suiciding of lymphocytic precursor cells by tritiated nucleosides, in vitro.

Differences in suiciding by various tritiated nucleosides were observed between two functional assays for in vitro lymphocytic precursor cell development, the hemolysin plaque-forming cell (PFC) assay and the B lymphocytic colony-forming cell (CFC-L) assay, using BDF1 mouse spleen cells. PFC growth was markedly reduced by an early (days 0-1) pulse of tritiated deoxyadenosine ([3H]dAdo), but relatively unaffected by a pulse of tritiated thymidine ([3H]dThd) during the same interval. In contrast, CFC-L formation significantly dropped after an early (day 0) [3H]dThd pulse, as well as after pulses of [3H]dAdo and the corresponding tritiated ribosides, uridine and adenosine. This implied a cycling state in an early lymphocytic precursor cell, as opposed to the PFC insensitivity to an early [3H]dThd pulse. The response pattern of colonies and clusters to [3H]dThd supported our notion of a delayed suiciding of CFC contributing to the increase in cluster numbers.

Animals↗
PubMed1977

Reduction of fatal graft-versus-host disease by 3H-thymidine suicide of donor cells cultured with host cells.

The effect of the tritiated thymidine (3H-TdR) suicide technique on the ability of donor cells to induce fatal graft-versus-host disease (GVHD) was studied. C57BL/6 (H-2b) spleen cells were stimulated in vitro with irradiated BALB/c (H-2d) Moloney lymphoma cells in mixed culture and 3H-TdR of high-specific activity added to eliminate proliferating cells. The ability of such cells to induce fatal GVHD was assayed by injecting them i.v. into adult BALB/c mice immunosuppressed with cyclophosphamide (180 mg/kg). These cells induced fatal GVHD in fewer mice (52 per cent) than did C57BL/6 cells cultures with BALB/C lymphoma cells but without 3H-TdR (87%) and C57BL/L cells cultured with irradiated C57BL/6 cells with (95 per cent) or without 3H-TdR (86 per cent). Thus, the 3H-TdR suicide technique greatly diminished the ability of cells to induce lethal GVHD.

Animals↗
PubMed1979

Suicide inactivation of bacterial cystathionine gamma-synthase and methionine gamma-lyase during processing of L-propargylglycine.

L-Propargylglycine, a naturally occurring gamma, delta-acetylenic alpha-amino acid, induces mechanism-based inactivation of two pyridoxal phosphate dependent enzymes of methionine metabolism: (1) cystathionine gamma-synthease, which catalyzes a gamma-replacement reaction in methionine biosynthesis, and (2) methionine gamma-lyase, which catalyzes a gamma-elimination reaction in methionine breakdown. Biphasic pseudo-first-order inactivation kinetics were observed for both enzymes. Complete inactivation is achieved with a minimum molar ratio ([propargylglycine]/[enzyme monomer]) of 4:1 for cystathionine gamma-synthase and of 8:1 for methionine gamma-lyase, consistent with a small number of turnovers per inactivation event. Partitioning ratios were determined directly from observed primary kinetic isotope effects. [alpha-2H]Propargylglycine displays kH/kD values of about 3 on inactivation half-times. [alpha-3H]-Propargylglycine gives release of tritium to solvent nominally stoichiometric with inactivation but, on correction for the calculated tritium isotope discrimination, partition ratios of four and six turnovers per monomer inactivated are indicated for cystathionine gamma-synthase and methionine gamma-lyase, respectively. The inactivation stoichiometry, using [alpha-14C]-propargylglycine, is four labels per tetramer of cystathionine gamma-synthase but usually only two labels per tetramer of methionine gamma-lyase (half-of-the-sites reactivity). Two-dimensional urea isoelectrofocusing/NaDodSO4 electrophoresis suggests (1) that both native enzymes are alpha 2 beta 2 tetramers where the subunits are distinguishable by charge but not by size and (2) that, while each subunit of a cystathionine gamma-synthase tetramer becomes modified by propargylglycine, only one alpha and one beta subunit may be labeled in an inactive alpha 2 beta 2 tetramer of methionine gamma-lyase. Steady-state spectroscopic analyses during inactivation indicated that modified cystathionine gamma-synthase may reprotonate C2 of the enzyme--inactivator adduct, so that the cofactor is still in the pyridoxaldimine oxidation state. Fully inactivated methionine gamma-lyase has lambda max values at 460 and 495 nm, which may represent conjugated pyridoximine paraquinoid that does not reprotonate at C2 of the bound adduct. Either species could arise from Michael-type addition of an enzymic nucleophile to an electrophilic 3,4-allenic paraquinoid intermediate, generated initially by propargylic rearrangement upon a 4,5-acetylenic pyridoximine structure, as originally proposed for propargylglycine inactivation of gamma-cystathionase [Abeles, R., & Walsh, C. (1973) J. Am. Chem. Soc. 95, 6124]. It is reasonable that cystathionine gamma-synthase is the major in vivo target for this natural acetylenic toxin, the growth-inhibitory effects of which are reversed by methionine.

Alkynes↗
PubMed1975

Cell-mediated immune responses in vitro. III. Elimination of specific cytotoxic lymphocyte responses by 3H-thymidine suicide.

The role of cellular proliferation in the development of cytotoxic lymphocyte (CL) responses in one-way mixed lymphocyte reactions was investigated by using tritiated thymidine of high specific activity to kill proliferating cells. To develop maximum CL responses, responding lymphoid cells must proliferate for approximately 72 hr; thereafter, precursors of CL appear to differentiate into active CL without further proliferation. Different alloantigen-sensitive precursor cell populations participate in the CL responses to each of two sets of stimulating alloantigens. When cells responding to one set of alloantigens were selectively destroyed after incorporating the hot thymidine, the surviving cells retained the capacity to develop a normal CL response to the second set of alloantigens.

Animals↗
PubMed1979

Buoyant density analysis of myeloid colony-forming cells in germfree and conventional mice.

Granulocyte-macrophage colony-forming cells (CFUc), in the bone marrow of germfree and conventioal CBA mice, were compared quantitatively and qualitatively. Cells were separated on the basis of their buoyant density by equilibrium centrifugation in continuous albumin density gradients. CFUc in the density subpopulations were detected by culture in agar containing three different types of colony stimulating factor (CSF). The sources of the CSF were post-endotoxin mouse serum (CSFES), mouse lung conditioned medium (CSFMLCM) and human urine (CSFHU). Mice were removed from the germfree environment and the buoyant density status of their CFUc was examined 1, 4 and 8 weeks later. No difference was found between germfree and conventional mice in the number of nucleated cells per femur or in their modal density. Neither was the number of CFUc per femur different. The cell cycle status of CFUc, as determined by the thymidine suicide technique was not significantly different. Functional heterogeneity was found among the density subpopulations for both groups of mice. This depended on the type of CSF. The density distribution of CFUc was significantly different in germfree mice. There were proportionately more low density CFUc. The mean modal density of CFUc under CSFES stimulation was less by 0.0045 g/cm3 in germfree mice. The removal of mice from the germfree environment resulted in a shift of the distribution to higher densities. The trend was towards the conventional situation. The significance of the buoyant density status of CFUc is discussed.

Animals↗
PubMed1978

Subpopulations of mouse T lymphocytes. II. Suppression of graft-vs.-host reactions by naturally proliferating splenic T cells.

The immunological role of a naturally proliferating subpopulation of splenic T cells was investigated using the graft-vs.-host (GvH) reaction on the mouse. Normal parental spleen cells, purified splenic T cells or lymph node cells were pulse-treated for one hour in vitro with tritiated thymidine of high specific activity ([3H]dThd, "thymidine suicide"). The treatment specifically and selectively kills proliferating cells which are actively synthesizing DNA, i.e. cells in S phase. Following treatment, the cells were transferred to F1 recipients and the GvH reaction measured by the splenomegaly assay. The results showed that the GvH effector cells in the donor spleen and lymph node are nonproliferating T cells. Furthermore, donor spleen cells treated with [3H]dThd consistently had enhanced GvH reactivity when compared to the controls, while the phytohemagglutinin response of these same treated cell suspensions was significantly inhibited. When purified splenic T cells were used, treatment with [3H]dThd also caused an increase in the GvH reaction, showing that a T cell population was being affected by the cycleactive agent. These results indicated that some naturally proliferating T cells have suppressor functions, and their specific inactivation allows nonproliferating effector T cells to mount a more vigourous GvH reaction.

Animals↗