Effect of 5-fluorouracil post-treatment on UV-resistance ofEscherichia coli cells

The effect of 5-fluorouracil (5-FU) and uracil starvation on the UV-sensitivity ofEscherichia coli 15 was followed in a 60 minutes period after the irradiation. The presence of 5-FU caused a significant increase of radioresistance when compared with the control, on the other hand under conditions of uracil starvation the cells were slightly more sensitive than in the control sample. The DNA synthesis was highly inhibited under conditions of uracil starvation, but the same effect was observed in the presence of 5-FU. Maximum inhibition of protein synthesis was observed also when uracil was omitted from the cultivation medium and approximately 50% of inhibition was noticed in the sample with 5-FU. The increase of resistance caused by the presence of 5-FU in the 60 minutes period after UV-irradiation cannot be explained by prolonging the sensitive postirradiation phase. It seems probable that in the presence of 5-FU the conditions are favourable for a better course of the repair processes.     

Effect of some pre-treatments on UV-sensitivity and on the course of macromolecular synthesis inEscherichia coli 15 T−, U−, his−

The UV-sensitivity ofEscherichia coli 15 T⁻, U⁻, his⁻ cells after a 45 minutes glucose, thymine uracil, or histidine pre-irradiation starvation, as well as the course of DNA, RNA, and protein synthesis during starvation and during a 60 minute post-treatment in a complete medium were investigated. An increased radioresistance was observed when starvation for some compounds resulted in a consequent inhibition of protein synthesis, as it was observed in the case of glucose, histidine, or uracil starvation. During thymine starvation, which led to a decreased resistance, no inhibition of protein synthesis was recorded. The postirradiation time-course of DNA synthesis did not show any correlation with the increased rate of resistance. The DNA synthesis after U⁻ pre-treatment was greatly delayed, however, after glucose pre-treatment no retardation was observed although both factors increased the rate of surviving cells approximately to the same extent. We assume that the factors which increase the radio-resistance could act by a similar mechanism which would take part in the inhibition of protein synthesis. This mechanism could consist in a decrease of the m-RNA turnover.     

Stability of beta-galactosidase messenger ribonucleic acid in Escherichia coli

Ben-Hamida, Fakher (Washington University School of Medicine, St. Louis, Mo.), and David Schlessinger. Stability of beta-galactosidase messenger ribonucleic acid in Escherichia coli. J. Bacteriol. 90:1611-1616. 1965.-Synthesis of beta-galactosidase stops within several minutes when preinduced, permeaseless cultures are diluted into medium containing 40 mug/ml of 5-fluorouracil (5-FU) but no inducer. However, if inducer (isopropylthiogalactoside) is left in the medium, enzyme formation in the presence of 5-FU continues for at least 11 min. Thus, inducer may increase the differential metabolic stability of the corresponding messenger ribonucleic acid (RNA; defined as the capacity to produce measurable enzyme) in inducible strains. However, such an interpretation requires that 5-FU rapidly arrest the further synthesis of messenger RNA competent to form active enzyme. C(14)-5-FU, like uracil, does appear to enter cells without measurable lag, saturating the pool of uracil nucleotides, and thereby the messenger RNA being formed, within several minutes. That 5-FU acts very quickly is also supported by the similar continuation of enzyme synthesis in the presence of inducer and antibiotics (actinomycin D and proflavine) which shut off all RNA synthesis, as well as by the response to 5-FU of enzyme synthesis in various constitutive mutants.     

The control of ribonucleic acid synthesis in bacteria. Steady-state content of messenger ribonucleic acid in Escherichia coli M.R.E. 600

1. The technique of DNA-RNA hybridization was used to follow changes in the amount and average lifetime of unstable messenger RNA in Escherichia coli M.R.E. 600 over a wide range of different growth conditions. The method of analysis was based on the kinetics of incorporation of exogenous labelled nucleic acid bases into the RNA of steadily growing cultures, as described by Bolton & McCarthy (1962). 2. The ratio of the average lifetime of messenger RNA to the mean generation time of E. coli cultures was constant over the temperature range 25-45 degrees C in a given medium, but the constant varied with the nature of the growth medium. For cultures growing in sodium lactate-salts or glucose-salts media the ratio was 0.046+/-0.005 and in enriched broth it was 0.087+/-0.009. Measurements of the amounts of transfer RNA, ribosomal RNA and messenger RNA were also made. The results confirmed earlier reports that the ratio of the amount of messenger RNA to the amount of ribosomes in the cells is virtually constant. On the other hand, the ratio of the amount of transfer RNA to the amount of ribosomal RNA decreased with increasing growth rate at a given temperature. 3. In cultures at temperatures higher than necessary for optimum rates of growth the average lifetime of messenger RNA lengthened in harmony with the increased time required for cell division. It seems that suboptimum growth rates at higher temperatures cannot be explained simply as a combination of increased rates of synthesis and breakdown of messenger RNA with a grossly decreased efficiency of translation. The absolute rate of messenger RNA synthesis was lowered, and its amount in the cells was typical of all other cultures grown at lower temperatures in the same medium. 4. The rate of entry of exogenous labelled uracil into unstable messenger RNA and stable ribosomal RNA was constant in all media at all temperatures in the approximate ratio 1:2. In media supporting a lower rate of growth, e.g. lactate-salts or glucose-salts media, the messenger RNA fraction constituted 2.2+/-0.3% of the total cellular RNA. In enriched broth 3.6+/-0.3% of the total RNA was messenger.     

Antimikrobielle Inhaltsstoffe in Algen

Zusammenfassung 1. Acrylsäure ist innerhalb der einfachen ungesättigten Carbonsäuren ein relativ spezifischer Hemmstoff fürEscherichia coli, Staphylococcus aureus und andere Bakterien.2. Acrylsäure hat nur in sehr hohen Konzentrationen bei sehr langen Inkubationszeiten schwach bakterizide Eigenschaften.3. Sie hemmt besonders bei älterenE. coli-Zellen die Atmung sehr viel stärker als das Wachstum. Bei jungenE. coli-Kulturen und beiSt. aureus sind Wachstums- und Atmungshemmung etwa gleich stark.4. Die antibakteriallen Eigenschaften der Acrylsäure gegenE. coli werden durch SH-haltige Verbindungen aufgehoben. 2,4-Dinitrophenol wirkt dagegen stark synergistisch. BeiSt. aureus sind diese Wirkungen nur schwach ausgeprägt.5. Der Einbau von Leucin in Protein wird durch Acrylsäure beiE. coli weniger als das Wachstum, der Einbau von Thymidin in DNS fast ebenso stark wie das Wachstum und der Einbau von Uracil in RNS von Anfang an sehr viel stärker gestört. DNP steigert die Hemmung des Uracil-Einbaues durch Acrylsäure.6. Die Synthese der r-RNS und s-RNS wird in etwa gleichem Umfang gestört; die Synthese der m-RNS scheint nicht beeinflußt zu werden.

---

Antimicrobial components in algae4th contribution. Effect of acrylic acid on respiration and macromolecular synthesis inStaphyloccus aureus andEscherichia coli

Among the simple unsaturated carbonic acids, acrylic acid is a comparatively specific inhibitor forStaphylococcus aureus, Escherichia coli and other bacteria. Insignificant bactericidal action can only be observed at high concentrations and long incubation time of acrylic acid. In olderE. coli cells, acrylic acid inhibits respiration more strongly than growth. On the other hand, in youngerE. coli cells and inSt. aureus, inhibition of growth is more pronounced than that of respiration. Antibacterial activities of acrylic acid againstE. coli can be reduced by compounds with thiol groups; they can be increased by 2.4-dinitrophenol (DNP). The effect of acrylic acid and DNP is synergistic; such effects are very small inSt. aureus. Incorporation of leucine into the protein ofE. coli cells is less inhibited by acrylic acid than their growth rate; incorporation of thymidine into DNA is inhibited to the same extent as growth; incorporation of uracil into RNA is inhibited to a much higher extent right from the beginning of the experiment. DNP is capable of increasing the degree of inhibition of uracil incorporation caused by acrylic acid. Syntheses of r-RNA and s-RNA are inhibited to nearly the same extent; there seems to be no influence on the synthesis of m-RNA.

---

---

Teil der Dissertation vonR. Heyser, Bonn, 1970 (D5).     

Arabidopsis Uracil DNA Glycosylase (UNG) Is Required for Base Excision Repair of Uracil and Increases Plant Sensitivity to 5-Fluorouracil

Uracil in DNA arises by misincorporation of dUMP during replication and by hydrolytic deamination of cytosine. This common lesion is actively removed through a base excision repair (BER) pathway initiated by a uracil DNA glycosylase (UDG) activity that excises the damage as a free base. UDGs are classified into different families differentially distributed across eubacteria, archaea, yeast, and animals, but remain to be unambiguously identified in plants. We report here the molecular characterization of AtUNG (Arabidopsis thaliana uracil DNA glycosylase), a plant member of the Family-1 of UDGs typified by Escherichia coli Ung. AtUNG exhibits the narrow substrate specificity and single-stranded DNA preference that are characteristic of Ung homologues. Cell extracts from atung^−/− mutants are devoid of UDG activity, and lack the capacity to initiate BER on uracil residues. AtUNG-deficient plants do not display any apparent phenotype, but show increased resistance to 5-fluorouracil (5-FU), a cytostatic drug that favors dUMP misincorporation into DNA. The resistance of atung^−/− mutants to 5-FU is accompanied by the accumulation of uracil residues in DNA. These results suggest that AtUNG excises uracil in vivo but generates toxic AP sites when processing abundant U:A pairs in dTTP-depleted cells. Altogether, our findings point to AtUNG as the major UDG activity in Arabidopsis.     

The effect of anaerobiosis on the induced synthesis of β-galactosidase in non-growingEscherichia coli

Depending on conditions of aeration maltose and glucose were found to exhibit different effects on the inducible synthesis of β-galactosidase in aerobically grown cells ofEscherichia coli starving for an exogenous source of nitrogen; both saccharides repressed the synthesis of the enzyme under aerobic conditions, while the above-mentioned saccharides were essential for the enzyme synthesis under anaerobic conditions. The presence of maltose in the medium resulted in the repression of the enzyme synthesis in anaerobically grown cells starving for an exogenous nitrogen source under anaerobic conditions. The synthesis of β-galactosidase-specific messenger RNA was completely blocked and the synthesis of the enzyme proper considerably inhibited in aerobically grown cells incubated anaerobically in a medium without nitrogen and carbon sources.     

ALT: A new factor involved in the synthesis of RNA by Escherichia coli

Summary We have defined a new gene, alt, which affects RNA synthesis in Escherichia coli. Mutants for alt arise among revertants of strains lacking the CRP-cAMP system necessary for full expression of catabolite-sensitive operons. Studies on a temperature-sensitive alt mutant indicate that the alt gene product is necessary for the synthesis of an important class of messenger RNA molecules.     

Non-coordinate regulation of RNA synthesis in escherichia coli exposed to 0°C

A non-coordinate mode of regulation of RNA synthesis is observed inEscherichia coli cells during exposure to 0°C. The stable RNA synthesis is preferentially inhibited with simultaneous accumulation of messenger RNA. The species of RNA synthesized at 0°C was determined by several criteria such as sedimentation value in sucrose gradients, DNA-RNA hybridization, half life measurements, protein synthesizing capacity and its functional rate of decay. The mode of regulation of RNA synthesis at 0°C is unique and is distinct from the non-coordinate regulation observed during amino acid starvation under stringent control.     

Diphosphate concentration does not correlate with the level of inorganic diphosphatase inEscherichia coli

Partial inhibition of DNA synthesis stimulates the production of inorganic diphosphatase inEscherichia coli but the changes in diphosphate (PP _i) level observed did not correlate with the enzyme activity. An accumulation ofPP _I was observed in the presence of inhibitors of RNA synthesis or nucleotide synthesis. In the former case the level of the enzyme did not change but in the latter case it increased. Thus the amount of inorganic diphosphatase alone does not determine the concentration ofPP ₁ inE. coli.     

Functional characterization of a gene encoding a dual domain for uridine kinase and uracil phosphoribosyltransferase in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Uridine kinase (UK) and uracil phosphoribosyltransferase (UPRT) are enzymes catalyzing the formation of uridine 5′-monophosphate (UMP) from uridine and adenine 5′-triphosphate (ATP) and from uracil and phosphoribosyl-α-1-pyrophosphate (PRPP), respectively, in the pyrimidine salvage pathway. Here, we report the characterization and functional analysis of a gene AtUK/UPRT1 from Arabidopsis thaliana. Sequencing of an expressed sequence tag clone of this gene revealed that it contains a full-length open reading frame of 1461 nucleotides and encodes a protein with a molecular mass of approximately 53 kDa. The sequence analysis revealed that the N-terminal region of AtUK/UPRT1 contains a UK domain and the C-terminal region consists of a UPRT domain. Expression of AtUK/UPRT1 in upp and upp-udk mutants of Escherichia coli supplied with 5-fluorouracil (5-FU) and 5-fluorouridine (5-FD) led to growth inhibition. Identical results were obtained with 5-FD and 5-FU treatments when the UK and UPRT domains were separated by the introduction of translation initiation and stop codons prior to complementation into the upp-udk and upp mutants. These results suggest that the AtUK/UPRT1 product can use uracil and uridine as substrates for the production of UMP. We also investigated the function of AtUK/UPRT1 in an Arabidopsis mutant. The wild-type Arabidopsis plants showed drastic growth retardation when they were treated with 5-FU and 5-FD while the growth of atuk/uprt1 mutant plants was not significantly affected. These findings confirm that AtUK/UPRT1 has a dual role in coding for both uridine kinase and uracil phosphoribosyltransferase that form UMP through the pyrimidine salvage pathway in Arabidopsis.     

Cyclic blockade of initiation sites by streptomycin-damaged ribosomes in Escherichia coli: an explanation for dominance of sensitivity

In bacterial extracts streptomycin is known not only to inhibit ribosomal activity but also to cause gradual release of ribosomes from polysomes. Nevertheless, we now find that after streptomycin has virtually halted protein synthesis in cells of Escherichia coli K12 a substantial (though reduced) level of polysomes persists. These polysomes are evidently maintained by turnover rather than by static blockade, for in streptomycin-treated cells [³H]uracil pulses are rapidly incorporated in the polysomal messenger RNA; moreover, if the synthesis of RNA or the formylation of methionyl-transfer RNA is blocked the polysome level decreases rapidly. Streptomycin thus appears to cause a cycle of ribosomal initiation, blockage of chain extension, gradual release, and reinitiation.The resulting cyclic blockade of initiation sites can account for the dominance of streptomycin sensitivity over resistance in $\text{str}{}^{\mathrm{s}}\text{str}{}^{\mathrm{r}}$² heterozygotes. In confirmation of this model, the inactive resistant ribosomes in treated heterozygotes were found to resume activity if the cells were lysed and excess messenger was provided. These findings further suggest that in sensitive cells damage to only a fraction of the ribosomal population by streptomycin may be sufficient to block protein synthesis.     

Cold shock proteins improve E. coli cell-free synthesis in terms of soluble yields of aggregation-prone proteins

Protein folding is usually slowed-down at low temperatures, and thus low-temperature expression is an effective strategy to improve the soluble yield of aggregation-prone proteins. In this study, we investigated the effects of a variety of cold shock proteins and domains (Csps) on an Escherichia coli cell extract-based cell-free protein synthesis system (CF). Most of the 12 Csps that were successfully prepared dramatically improved the protein yields, by factors of more than 5 at 16°C and 2 at 23°C, to levels comparable to those obtained at 30°C. Their stimulatory effects were complementary to each other, while CspD and CspH were inhibitory. The Csps’ effects correlated well with their Pfam CSD family scores (PF00313.22). All of the investigated Csps, except CspH, similarly possessed RNA binding and chaperon activities and increased the messenger RNA amount irrespective of their effect, suggesting that the proper balance between these activities was required for the enhancement. Unexpectedly, the 5′-untranslated region of cspA was less effective as the leader sequence. Our results demonstrated that the use of the Csps presented in this study will provide a simple and highly effective strategy for the CF, to improve the soluble yields of aggregation-prone proteins.     

Further observations on the effect of ethionine on the synthesis of β-galactosidase inEscherichia coli: Formation of an immunologically cross-reacting protein

It was found that ethionine partially inhibits the transport of the inducer (TMG) of β-galactosidase into the cells ofEscherichia coli ML-30. The synthesis of β-galactosidase-specific messenger RNA is not inhibited. Ethionine appears to be incorporated into proteins synthesized by the strains used. The incorporation of ethionine into the molecule of β-galactosidase results in the synthesis of an enzymically inactive, immunologically cross-reacting protein.     

Pyrimidine,purine and nitrogen control of cytosine deaminase synthesis in Escherichia coli K12. Involvement of the glnLG and purR genes in the regulation of codA expression

Cytosine deaminase, encoded by the codA gene in Escherichia coli catalyzes the deamination of cytosine to uracil and ammonia. Regulation of codA expression was studied by determining the level of cytosine deaminase in E. coli K12 grown in various defined media. Addition of either pyrimidine or purine nucleobases to the growth medium caused repressed enzyme levels, whereas growth on a poor nitrogen source such as proline resulted in derepression of cytosine deaminase synthesis. Derepression of codA expression was induced by starvation for either uracil or cytosine nucleotides. Nitrogen control was found to be mediated by the glnLG gene products, and purine repression required a functional purR gene product. Studies with strains harbouring multiple mutations affecting both pyrimidine, purine and nitrogen control revealed that the overall regulation of cytosine deaminase synthesis by the different metabolites is cumulative.This paper is dedicated to Professor John Ingraham, Department of Bacteriology, University of California, Davis, on the occasion of his retirement, in recognition of his many contributions in the field of bacterial growth and metabolism     

Mechanism of Inhibition of DNA Synthesis by 1-β-D-Arabinofuranosyl-E-5-(2-Bromovinyl)uracil

The mechanism of the inhibitory action of 1-β-D -arabinofuranosyl-E-5-(2-bromovinyl) uracil triphosphate (BV-araUTP) on DNA synthesis by Escherichia coli DNA polymerase I Klenow fragment was studied. Acting as a chain terminator, BV-araUTP inhibited DNA synthesis by Klenow fragment more effectively than 2′, 3′-dideoxythymidine triphosphate (ddTTP). However, the incorporation sites of BV-araU monophosphate were restricted at consecutive dTMP sequence whereas ddTMP was incorporated at every dTMP site.