Tritium Suicide Research Articles

Tritium Suicide Selection Identifies Proteins Involved in the Uptake and Intracellular Transport of Sterols in Saccharomyces cerevisiae

Sterol transport between the plasma membrane (PM) and the endoplasmic reticulum (ER) occurs by a nonvesicular mechanism that is poorly understood. To identify proteins required for this process, we isolated Saccharomyces cerevisiae mutants with defects in sterol transport. We used Upc2-1 cells that have the ability to take up sterols under aerobic conditions and exploited the observation that intracellular accumulation of exogenously supplied [(3)H]cholesterol in the form of [(3)H]cholesteryl ester requires an intact PM-ER sterol transport pathway. Upc2-1 cells were mutagenized using a transposon library, incubated with [(3)H]cholesterol, and subjected to tritium suicide selection to isolate mutants with a decreased ability to accumulate [(3)H]cholesterol. Many of the mutants had defects in the expression and trafficking of Aus1 and Pdr11, PM-localized ABC transporters that are required for sterol uptake. Through characterization of one of the mutants, a new role was uncovered for the transcription factor Mot3 in controlling expression of Aus1 and Pdr11. A number of mutants had transposon insertions in the uncharacterized Ydr051c gene, which we now refer to as DET1 (decreased ergosterol transport). These mutants expressed Aus1 and Pdr11 normally but were severely defective in the ability to accumulate exogenously supplied cholesterol. The transport of newly synthesized sterols from the ER to the PM was also defective in det1Delta cells. These data indicate that the cytoplasmic protein encoded by DET1 is involved in intracellular sterol transport.

Isolation and characterization of beta-ketoacyl-acyl carrier protein reductase (fabG) mutants of Escherichia coli and Salmonella enterica serovar Typhimurium.

FabG, beta-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of beta-ketoacyl-ACP substrates to beta-hydroxyacyl-ACP products, the first reductive step in the elongation cycle of fatty acid biosynthesis. We report the first documented fabG mutants and their characterization. By chemical mutagenesis followed by a tritium suicide procedure, we obtained three conditionally lethal temperature-sensitive fabG mutants. The Escherichia coli [fabG (Ts)] mutant contains two point mutations: A154T and E233K. The beta-ketoacyl-ACP reductase activity of this mutant was extremely thermolabile, and the rate of fatty acid synthesis measured in vivo was inhibited upon shift to the nonpermissive temperature. Moreover, synthesis of the acyl-ACP intermediates of the pathway was inhibited upon shift of mutant cultures to the nonpermissive temperature, indicating blockage of the synthetic cycle. Similar results were observed for in vitro fatty acid synthesis. Complementation analysis revealed that only the E233K mutation was required to give the temperature-sensitive growth phenotype. In the two Salmonella enterica serovar Typhimurium fabG(Ts) mutants one strain had a single point mutation, S224F, whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the twofold axes of symmetry at the dimer interfaces in this homotetrameric protein, suggesting that the quaternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature.

Biochemical evidence against protein-mediated uptake of myristic acid in the bioluminescent marine bacterium Vibrio harveyi.

The bioluminescent marine bacterium, Vibrio harveyi, can utilize exogenous myristic acid (14:0) for beta-oxidation, phospholipid and lipid A synthesis, and as an source of myristyl aldehyde for light emission in the V. harveyi dark mutant M17. A variety of genetic and biochemical strategies were employed in an attempt to isolate V. harveyi mutants defective in myristate uptake and to characterize proteins involved in this process. Although [3H]myristate uptake in a tritium suicide experiment decreased the survival of nitrosoguanidine-treated M17 cells by a factor of 10(5), none of the surviving cells characterized were defective in either incorporation of exogenous myristate into phospholipid or stimulation of light emission. These parameters were also unaffected when intact M17 cells were treated with proteases. Moreover, M17 double mutants selected on the basis of diminished luminescence response to myristate all incorporated [3H]myristate into lipids normally. Finally, no resistant colonies were obtained using the bacteriocidal fatty acid analogue, 11-bromoundecanoate, and experiments with decanoate (10:0) indicated that the V. harveyi cell envelope is very sensitive to physical disruption by fatty acids. Taken together, these results support an unfacilitated uptake of myristic acid in V. harveyi, in contrast with the regulated vectorial transport and activation of long chain fatty acids in Escherichia coli.

Isolation of Animal Cell Mutants Defective in Long-chain Fatty Aldehyde Dehydrogenase : SENSITIVITY TO FATTY ALDEHYDES AND SCHIFF'S BASE MODIFICATION OF PHOSPHOLIPIDS: IMPLICATIONS FOR SJÖGREN-LARSSON SYNDROME

Using tritium suicide, we have isolated a variant of the Chinese hamster ovary cell line, CHO-K1, that is deficient in long-chain fatty alcohol:NAD+ oxidoreductase (FAO; EC 1.1.1.192). Specifically, it was the fatty aldehyde dehydrogenase component that was affected. The enzymatic deficiency found in this mutant strain, designated FAA. K1A, was similar to that displayed by fibroblasts from patients with Sjögren-Larsson syndrome (SLS), an inheritable neurocutaneous disorder. Complementation analyses suggested that the deficiency in fatty alcohol oxidation in the FAA.K1A cells and the SLS fibroblasts is a result of lesions in homologous genes. The FAA.K1A cells were unable to convert long chain fatty aldehydes to the corresponding fatty acids. This resulted in a hypersensitivity of the FAA.K1A cells to the cytotoxic effects of long chain fatty aldehydes. The difference between the mutant and wild-type cells was most obvious when using fatty aldehydes between 14 and 20 carbons, with the greatest difference between wild-type and mutant cells found when using octadecanal. Fibroblasts from a patient with SLS also displayed the hypersensitivity phenotype when compared with FAldDH+ human fibroblasts. In both CHO and human FAldDH- cell lines, addition of long chain fatty aldehydes to the medium caused a dramatic increase in aldehyde-modified phosphatidylethanolamine, presumably through Schiff's base addition to the primary amine of the ethanolamine head group. When 25 microM hexadecanal was added to the growth medium, approximately 10% of the phosphatidylethanolamine was found in the fatty aldehyde-modified form in FAA.K1A, although this was not observed in wild-type cells. Modified phosphatidylethanolamine could be detected in FAldDH- cells even when exogenous fatty aldehydes were not added to the medium. We propose a possible role for fatty aldehydes, or other aldehydic species, in mediating some of the symptoms associated with Sjögren-Larsson syndrome.

Selection of Chinese hamster ovary cells (CHO-K1) with reduced glutamate and aspartate uptake.

Transport of L-[3H]glutamate into Chinese hamster ovary cells (CHO-K1) was characterized and the results used to design a tritium suicide selection for cells with transport defects. Replicas of surviving colonies on polyester cloth disks were screened by autofluorography for reduced uptake and two mutant clones, Ed-A1 and Ed-B8, were obtained. Uptake of glutamate through a sodium-dependent system in both mutants was characterized by significant reductions in Vmax and increases in Km compared to parental cells, but their response to removal of extracellular sodium differed, suggesting distinct mutations in the two lines. The Vmax of aspartate uptake through this system was reduced in both mutants, to one-ninth in the case of Ed-B8. Glutamate uptake through a sodium-independent system was not altered in either mutant. Surprisingly, acid-soluble intracellular pools of several amino acids were higher in both mutants.

Single-step selection of mammalian cell mutants deficient in CTP synthetase.

A single-step selection of Chinese hamster V79 cells deficient in CTP synthetase (CTPS-) is presented. The underlying principle of the direct selection is the differential and efficient killing of synchronized wild-type cells through incorporation of [3H]uridine and [3H]thymidine. The CTPS- mutant cells were recovered by virtue of their not engaging in DNA synthesis, because (1) CTPS- cells are deficient in CTP synthetase and thus are unable to convert [3H]UTP into [3H]CTP, which eventually is converted into [3H]dCTP and incorporated into DNA; (2) the growth of CTPS- mutant cells was arrested as a result of cytidine deprivation, thus escaping the killing by the incorporation of [3H]thymidine. The isolated mutant clones are auxotrophic for cytidine and are stable in phenotype with a reversion frequency of less than 1 x 10(-7). The mutant cells have no or very low CTP synthetase activity when tested by in vitro CTP synthetase assay or by whole-cell [3H]uridine labeling assay. This modified "tritium suicide" method combined with the S-phase cell synchronization could provide a powerful means for the recovery from the cell population of nondividing mutant cells that are auxotrophic for some special nutrient requirement.

Cell cycle progression during chronic hyperthermia in S phase CHO cells

S phase CHO cells chronically exposed to 41.5 degrees C exhibited an apparent resistance to further cell killing after 4-6 h. During the treatment interval, cells were observed to progress out of S phase and into G2, mitosis, and the next cell cycle. Progression through S was delayed by about 2 h and an equivalent mitotic delay was observed. After entry into mitosis, heated cultures showed an altered nuclear morphology, presumably as the result of abnormal division occurring during the treatment. Tritium suicide experiments at this temperature showed that clonogenic, as well as nonclonogenic, cells progressed during this period. When S phase cultures were chronically heated at 42 degrees C, however, the delay in transit through S phase was 8-10 h, and an accumulation in G2 phase was observed. Therefore, our results show that, in S phase cultures heated at 42 degrees C, cell killing continues while cells progress through S, suggesting that chronic thermotolerance cannot be expressed in S phase. Furthermore, at 41.5 degrees C, cells which progress out of S during the treatment express resistance to heat killing in subsequent cell cycle phases. In summary, our results indicate that, although chronic tolerance is not expressed during heating in S phase, it is expressed after the cells progress out of S phase.

Isolation and characterization of a glycine transport mutant in an established mammalian cell line, CHO(PEOT/1)

Glycine uptake in CHO(PEOT/1) cells is mediated by at least three systems, of which two have been identified and partially characterized in this study: (1) a low affinity "A" system that transports a number of small neutral amino acids including glycine and methylaminoisobutyric acid (MeAIB), and (2) a high-affinity system, specific for glycine and sarcosine. By a combination of tritium suicide and replica plating, we have isolated a mutant (CHY-3) with a 47% decrease in glycine transport at the standard test concentration of 2.5 microM. Uptake studies with radioactive glycine, MeAIB, and sarcosine revealed that the mutant lacks the glycine-sarcosine system, but has undergone a compensatory 30-50% increase in the A system. Thus, there appears to be a regulatory interaction between these two systems for glycine uptake by CHO cells.

11] Use of tritium-labeled precursors to select mutants

A killing selection is certainly a method of choice for rapid mutant isolation. However, it is very often difficult to meet the “comfortable” situation of having, for selection, a toxic compound analogous to an intermediate in the pathway of interest. This chapter illustrates with four examples that the use of tritiated-labeled precursors overcomes this difficulty and provides a powerful and general method of selection for mutants defective in membrane transport and in metabolic pathways. With these four examples of tritium suicide selection, the chapter shows that it is possible to devise a selection procedure allowing mutant isolation in a reasonable time period with either metabolizable or nonmetabolizable precursors. The tritium suicide has several advantages and is called “radiosuicide,” and was first used with bacterial genetics. This method is highly selective as the substrate itself of the pathway to “mutate” is the killing agent. The principle is based on the killing effects generated by the radiolytic decay of the incorporated labeled precursor. A killing selection is certainly a method of choice for rapid mutant isolation. However it is very often difficult to meet the “comfortable” situation of having, for selection, a toxic compound analogous to an intermediate in the pathway of interest.

Characterization of the pathways for phosphatidylethanolamine biosynthesis in Chinese hamster ovary mutant and parental cell lines.

A tritium suicide procedure was devised to facilitate the isolation of Chinese hamster ovary cell mutants defective in phosphatidylethanolamine biosynthesis. One mutant with a 20-50% reduction in [3H]ethanolamine incorporation was chosen for further analysis and was shown to have reduced activity of CTP: phosphoethanolamine cytidylyltransferase. Levels of phosphatidylethanolamine and rates of its biosynthesis were compared in the mutant and parent cell lines. Despite the reduced activity of the CDP-ethanolamine pathway in the mutant, levels of phosphatidylethanolamine were the same in mutant and parent cells. Rates of phosphatidylethanolamine synthesis de novo, as measured by incorporation of 32PO4 into phosphatidylethanolamine, were also the same in mutant and parent cells, as was the rate of incorporation of [3H]serine into both phosphatidylserine and phosphatidylethanolamine. After a long term labeling with [3H]serine, the specific radioactivity of phosphatidylserine was the same as that of phosphatidylethanolamine, and there was no difference in the specific radioactivities of the two lipids between mutant and parent cells. These results implicate decarboxylation of phosphatidylserine as the sole route for synthesis of phosphatidylethanolamine under normal culture conditions.

Isolation by tritium suicide of uridine-cytidine kinase-defective mutants in Chinese hamster V79 cells

Tritium suicide was shown to be a highly effective method for isolating mutants defective in uridine-cytidine kinase in the Chinese hamster lung cell line V79. The tritium suicide procedure consisted of three kill cycles. Survivors of one kill cycle were used for the next kill cycle. The kill cycles involved incorporation of [ 3H]uridine for 10 min, followed by storage of 3H-labelled cells at −70 °C for 4–7 days. Nine clones that survived the third kill cycle were tested for incorporation of [ 3H]uridine and for uridine kinase activity in extracts. Eight of these clones were defective in whole-cell uridine incorporation and in uridine kinase activity. A kinetic study was made on the uridine-cytidine kinase activity of three of the mutants. The apparent V max of the mutants was reduced approx. 10-fold when either uridine or cytidine was used as substrate. In contrast, the apparent K m of uridine was reduced approx. 12-fold in the mutants with only a 2-fold (probably insignificant) reduction in K m's for cytidine or for ATP.

Isolation of a spontaneous CHO amino acid transport mutant by a combination of tritium suicide and replica plating.

A spontaneous transport mutant of Chinese hamster ovary cells, CHY-1, was isolated by a combination of [3H]proline suicide and replica plating. The mutant took up less tritium than the parent, resulting in a lower killing rate during storage. Transport by four separate amino acid transport systems (A, ASC, L, Ly+) was examined. The CHY-1 mutant exhibited normal uptake via the ASC, L, and Ly+ systems. By contrast, uptake of the most specific substrate of the A system, 2-(methylamino)-isobutyric acid, was significantly reduced at low, but not high, concentrations, due to a 3.5-fold increase in Km and a 1.5-fold increase in Vmax. Taken together, these data suggest that the CHY-1 mutation may be in the structural gene coding for the A transport protein. The tritium suicide procedure is discussed, and general equations are derived to predict the maximum storage time for the survival of one mutant cell and the optimum size of the cell population for maximum mutant enrichment.

Isolation of hypoxanthine phosphoribosyltransferase-defective mutants in chinese hamster V79 cells by tritium suicide

Tritium suicide was shown to be highly efficient method for isolating mutants defective in hypoxanthine incorporation in the Chinese hamster lung cell line V79. The tritium suicide procedure consisted of 3 kill cycles. Survivors of one kill cycle were used for the next kill cycle. The kill cycles involved incorporation of [ 3H]hypoxanthine for 5 or 10 min, followed by storage of 3H-labelled cells at −70°C for 4–10 days. 12 clones that survived the 3rd kill cycle were tested for incorporation of [ 3H]hypoxanthine and all were found to be defective. At least 6 of the clones have defective hypoxanthine phosphoribosyltransferase (HPRT) activity. One mutant, H19, chosen for further characterization, had HPRT with a 13-fold elevation in apparent K m for phosphoribosylpyrophosphate (PRPP). Thin-layer chromatography of cell extracts showed that this mutant was incapable of converting intracellular hypoxanthine to IMP or to other purine metabolites. In addition, H19 was resistant to 6-thioguanine.

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.

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.

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° 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° C the probability of cell killing per tritium decay was calculated to be 3.64 × 10 −5.

Enrichment for cold-sensitive mutants by tritium suicide

Enrichment for cold-sensitive mutants by tritium suicide

Chapter 15 Methods for Selecting Auxotrophic and Temperature-Sensitive Mutants in Yeasts

Publisher Summary This chapter describes the methods for selecting auxotrophic and temperature-sensitive mutants in yeasts. Mutant selection is defined as those processes that enrich a culture for desired mutants by decreasing the number of viable nonmutant cells. Considerable pressure toward increasing the percentage of specific mutants can be exerted at each of the four basic steps in mutant isolation: (1) choice of parental strain, (2) mutagenesis, (3) enrichment of the culture for auxotrophic or temperature-sensitive ( ts ) mutants, and (4) identification of the desired mutants in the remaining cell population. Auxotrophic and ts mutations are usually recessive and therefore should be induced in a haploid strain. It is advisable to use a standard strain carrying at least one easily identified marker mutation. The chapter describes general enrichment procedures applicable to the selection of a wide variety of auxotrophic and ts mutants in yeasts. Although used primarily for Saccharomyces cerevisiae , these techniques appear to be usable with most species of yeast. Generalized procedures for selecting auxotrophs with nystatin and for selecting ts mutants by tritium suicide are given. These are followed by specific protocols used for S. cerevisiae.

Enrichment for temperature-sensitive and auxotrophic mutants in Saccharomyces cerevisiae by tritium suicide

Tritium suicide is shown to be an efficient technique for mutant enrichment in Saccharomyces cerevisiae. Decays from incorporated [5- 3H]uridine and tritiated amino acids proved equally effective in inducing suicide; in cultures labeled to a specific activity of 50 dpm/cell, the viability fell to 2% after 12 days' storage at 4°. Mutagenized cultures were labeled with either [5- 3H]uridine or a mixture of tritiated amino acids under conditions where auxotrophic mutants and temperature-sensitive mutants in RNA or protein synthesis would not incorporate a significant amount of the tritiated percursor. When survival fell to 2%, the percentages of both auxotrophic and temperature-sensitive mutants were 10-fold higher among these survivors than in the original mutagenized culture, regardless of the radioactive precursor used.

Specific enrichment of mutants of Escherichia coli with an altered acyl CoA synthetase by tritium suicide.

In a rich growth medium, tritium-labelled oleate is incorporated preferentially into the lipid fraction of E. coli K12. The incorporation of the radioactive oleate after a temperature shift kills the majority of the cells during subsequent storage. Most of the surviving cells can no longer grow with oleate as the sole carbon source at 40°C or 30°C, though some exhibit a thermosensitive behaviour, and are able to grow at 30°C but not at 40°C. It will be shown that mutants of the first group lack the enzyme acyl CoA synthetase, and that this enzyme is thermolabile in mutants of the second group. In addition one further mutant was isolated which possessed a normal acyl CoA synthetase but which was unable to degrade or incorporate oleic acid.