Decreased purine synthesis during amino acid starvation of human lymphoblasts

Normal human lymphoblasts starved for each of several essential, but not essential, amino acids had decreased DNA and RNA synthesis but no change in free intracellular purine nucleotides. The rates of purine nucleotide synthesis via the de novo and salvage pathways were measured by incorporating [14C]formate and [14C]hypoxanthine labels, respectively, into lymphoblasts starved for an amino acid or treated with a protein synthesis inhibitor. After 3 h of starvation, purine synthesis via the de novo pathway decreased 90% and via the salvage pathway decreased 60%. Cycloheximide and puromycin each reduced de novo synthesis by 96% and salvage synthesis by 72%. The decrease in purine synthesis de novo after removal of the amino acid was of first order kinetics and was fully and rapidly reversed by reconstitution with the amino acid. The synthesis of alpha-N-formylglycinamide ribonucleotide declined 97% after amino acid starvation; the synthesis of purines from 5-aminoimidazole-4-carboxamide riboside decreased 41%. The synthesis of guanylates decreased more than the synthesis of adenylates during amino acid starvation.     

Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli

Luzzati, Denise (Institut de Biologie Physico-Chimique, Paris, France). Effect of thymine starvation on messenger ribonucleic acid synthesis in Escherichia coli. J. Bacteriol. 92:1435-1446. 1966.-During the course of thymine starvation, the rate of synthesis of messenger ribonucleic acid (mRNA, the rapidly labeled fraction of the RNA which decays in the presence of dinitrophenol or which hybridizes with deoxyribonucleic acid) decreases exponentially, in parallel with the viability of the thymine-starved bacteria. The ability of cell-free extracts of starved bacteria to incorporate ribonucleoside triphosphates into RNA was determined; it was found to be inferior to that of extracts from control cells. The analysis of the properties of cell-free extracts of starved cells shows that their decreased RNA polymerase activity is the consequence of a modification of their deoxyribonucleic acid, the ability of which to serve as a template for RNA polymerase decreases during starvation.     

Purine nucleotide metabolism in phytohemagglutinin-induced human T lymphocytes

The comprehensive studies of purine nucleotide metabolism were done in nonstimulated and phytohemagglutinin (PHA)-stimulated human peripheral blood T lymphocytes. Nonstimulated lymphocytes synthesize nucleotides in two alternative pathways: via biosynthesis de novo and salvage pathways. Although synthesis of triphosphonucleosides in unstimulated lymphocytes was the predominant pathway, interconversion of monophosphonucleosides was also active. Exposure of cells to PHA affects differently various pathways of nucleotide metabolism. The most marked changes observed were rapid activation of purine salvage within minutes after exposure to PHA, and significant increase of 5-phosphoribosyl-1-pyrophosphate levels. In addition, significant increases were found in de novo purine biosynthesis, nucleotide interconversions, and RNA and DNA synthesis, whereas catabolism of nucleotides remained unchanged. These results indicate that PHA activation of T lymphocytes causes a rapid synthesis of nucleotides which may be required immediately for increases in energy metabolism and later as the precursors of nucleic acid synthesis.     

Studies of DNA bound RNA molecules isolated from nucleoids of Escherichia coli.

Methods are developed for studying RNA molecules bound directly to DNA in bacterial nucleoids. It is found that among the 1000-3000 nascent RNA chains that normally are attached to the DNA via their associated RNA polymerase molecules, 74 +/- 14 chains per nucleoid can be bound differently. These chains unlike the other nascent RNAs remained bound to the DNA after the chromosome was deproteinized and sheared. Sensitive assays using radioactive labels detected no RNA polymerase involved in the RNA-DNA linkage. The linkage was stable at low temperatures, but the RNA separated from the DNA at high temperature. The bound RNA molecules were heterodisperse (weight average length 1200 bases). Pulse-chase experiments and studies of the fate of these RNA molecules in rifampicin treated cells demonstrated that they are nascent RNAs, degraded or released from the DNA in vivo with kinetics similar to that of the total nascent RNA. Hybridization analyses showed that the chains are composed at least in part of nascent rRNA and known mRNA molecules. Some, but not more than 5% of the bound chains, contained sequences of about 300 nucleotides in length, bound to the DNA in an RNase resistant form.     

Effect of isonicotinic acid hydrazide-copper complex on Rous sarcoma virus and its genome RNA.

The copper complex of the antituberculous drug, insonicotinic acid hydrazide (INH), inhibits the RNA-dependent DNA polymerase of Rous sarcoma virus and inactivates its ability to malignantly transform chick embryo cells. The INH-copper complex binds to the 70S genome RNA of Rous sarcoma virus (RSV), which may account for its ability to inhibit the RNA-dependent DNA polymerase. The complex binds RNA more effectively than DNA in contrast to M-IBT-copper complexes, which bind both types of nucleic acids equally. The homopolymers, poly rA and poly rU, are bound by the INH-copper complex to a greater extent than poly rC. Isonicotinic acid hydrazide alone and CuSO4 alone bind neither DNA, RNA, poly (rA), poly (rU), nor poly (rC). However, CuSO4 alone binds poly (rI); INH alone does not. In addition to viral DNA synthesis, chick-embryo cell DNA synthesis is inhibited by the INH-copper complex. The extent of inhibition of cellular DNA synthesis is greater than that of cellular RNA and protein synthesis. No selective inhibition of transformation in cells previously infected with Rous sarcoma virus is observed.     

The effect of amino acid starvation on nucleoside uptake and RNA synthesis in Tetrahymena

The uptake of nucleosides and the synthesis of RNA in Tetrahymena thermophila were examined following amino acid starvation. Omission of leucine, phenylalanine, or arginine from the medium resulted in a rapid decrease in the incorporation of [3H]uridine into the acid-soluble pool and acid-insoluble material (RNA). Amino acid starvation inhibited the uptake of all ribo- and deoxyribonucleosides tested but did not affect the uptake of amino acids or glucose. In addition, under the conditions used, the omission of an amino acid did not result in a large decrease in amino acid incorporation into total protein. Treatment of cells with cycloheximide or emetine gave results similar to the effects of amino acid starvation, but in these experiments the inhibition of protein synthesis was essentially complete. Nucleotide pool sizes were also measured following amino acid starvation. ATP and UTP levels were essentially unchanged, but the dTTP pool size was decreased by 40%. The decrease in RNA synthesis in vivo in the absence of an essential amino acid was reflected in the endogenous RNA synthetic activity of isolated nuclei. However, when solubilized RNA polymerase activity was measured with calf thymus DNA as template, no significant difference was observed between control and amino acid-starved cells.     

The effect of feeding with a tryptophan-free amino acid mixture on rat liver magnesium ion-activated deoxyribonucleic acid-dependent ribonucleic acid polymerase

1. The Widnell & Tata (1966) assay method for Mg(2+)-activated DNA-dependent RNA polymerase was used for initial-velocity determinations of rat liver nuclear RNA polymerase. One unit (U) of RNA polymerase was defined as that amount of enzyme required for 1 mmol of [(3)H]GMP incorporation/min at 37 degrees C. 2. Colony fed rats were found to have a mean RNA polymerase activity of 65.9muU/mg of DNA and 18h-starved rats had a mean activity of 53.2muU/mg of DNA. Longer periods of starvation did not significantly decrease RNA polymerase activity further. 3. Rats that had been starved for 18h were used for all feeding experiments. Complete and tryptophan-deficient amino acid mixtures were given by stomach tube and the animals were killed 15-120min later. The response of RNA polymerase to the feeding with the complete amino acid mixture was rapid and almost linear over the first hour of feeding, resulting in a doubling of activity. The activity was still elevated above the starvation value at 120min after feeding. The tryptophan-deficient amino acid mixture produced a much less vigorous response about 45min after the feeding, and the activity had returned to the starvation value by 120min after the feeding. 4. The response of RNA polymerase to the feeding with the complete amino acid mixture was shown to occur within a period of less than 5min to about 10min after the feeding. 5. Pretreatment of the animals with puromycin or cycloheximide was found to abolish the 15min RNA polymerase response to the feeding with the complete amino acid mixture, but the activity of the controls was unaffected. 6. The characteristics of the RNA polymerase from 18h-starved animals and animals fed with the complete or incomplete amino acid mixtures for 1h were examined. The effects of Mg(2+) ions, pH, actinomycin D and nucleoside triphosphate omissions were determined. The [Mg(2+)]- and pH-activity profiles of the RNA polymerase from the animal fed with the complete mixture appeared to differ from those of the enzyme from the other groups, but this difference is probably not significant. 7. [5-(3)H]Orotic acid incorporation by rat liver nuclei in vivo was shown to be affected by the amino acid mixtures in a similar manner to the RNA polymerase. 8. The tryptophan concentrations of plasma and liver were determined up to 120 min after feeding with the amino acid mixtures. Feeding with the complete mixture produced a rapid increase in free tryptophan concentrations in both plasma and liver, but feeding with the incomplete mixture did not alter the plasma concentration. The liver tryptophan concentration increased at about 45min after feeding with the tryptophan-deficient diet. 9. There was a good correlation between the liver tryptophan concentration and RNA polymerase activity in all groups of animals. 10. It was concluded that the rat liver nucleus responded to an increase in amino acid supply by increased synthesis of RNA as a result of synthesis of RNA polymerase de novo. The correlation of tryptophan concentration and RNA polymerase activity appears to reflect the general amino acid concentration required to support hepatic protein synthesis and to produce new RNA polymerase. This new polymerase appears to differ from the basal RNA polymerase by its rapid synthesis and destruction, which may be a means of regulating RNA synthesis by the amino acid concentration in the liver.     

Amino acid substitutions at conserved tyrosine 52 alter fidelity and bypass efficiency of human DNA polymerase eta

DNA polymerase eta (Pol eta) is a member of a new class of DNA polymerases that is able to copy DNA containing damaged nucleotides. These polymerases are highly error-prone during copying of unaltered DNA templates. We analyzed the relationship between bypass efficiency and fidelity of DNA synthesis by introducing substitutions for Tyr-52, a highly conserved amino acid, within the human DNA polymerase eta (hPol eta) finger domain. Most substitutions for Tyr-52 caused reduction in bypass of UV-associated damage, measured by the ability to rescue the viability of UV-sensitive yeast cells at a high UV dose. For most mutants, the reduction in bypass ability paralleled the reduction in polymerization activity. Interestingly, the hPol eta Y52E mutant exhibited a greater reduction in bypass efficiency than polymerization activity. The reduction in bypass efficiency was accompanied by an up to 11-fold increase in the incorporation of complementary nucleotides relative to non-complementary nucleotides. The fidelity of DNA synthesis, measured by copying a gapped M13 DNA template in vitro, was also enhanced as much as 15-fold; the enhancement resulted from a decrease in transitions, which were relatively frequent, and a large decrease in transversions. Our demonstration that an amino acid substitution within the active site enhances the fidelity of DNA synthesis by hPol eta, one of the most inaccurate of DNA polymerases, supports the hypothesis that even error-prone DNA polymerases function in base selection.     

Avian myeloblastosis virus DNA polymerase. Kinetic studies on the incorporation of noncomplementary nucleotides.

The high error rate characteristic of DNA polymerases from RNA tumor viruses has permitted measurements on the simultaneous incorporation of complementary and noncomplementary nucleotides during DNA synthesis. For example, avian myeloblastosis virus DNA polymerase incorporates 1 molecule of dCMP for approximately 500 molecules of dTMP polymerized using polyriboadenylic acid as a template. The parallel incorporation of complementary and noncomplementary nucleotides afer gel filtration of avian myeloblastosis virus DNA polymerase indicates that the observed fidelity is catalyzed by the polymerase itself. Nearest neighbor analysis of the product indicates that noncomplementary nucleotides are incorporated as single base substitutions. The incorporation of the noncomplementary dCMP is not reduced by a 20-fold greater amount of the complementary nucleotide, dTTP. Conversely, the concentration of the noncomplementary nucleotides does not effect the rate of incorporation of the complementary nucleotide. A similar lack of competition between complementary dGTP and noncomplementary dATP is exhibited using poly(rC)-oligo(dG) as a template-primer. Furthermore, there was no detectable competition between the different noncomplementary nucleotides. Possible explanations for this lack of competition are considered.     

Intrasubunit nucleotide binding in ribonucleic acid polymerase.

1. Periodate oxidation of the ribose ring was used to synthesize derivatives of nucleoside triphosphates. 2. These oxidized nucleoside triphosphates. 2. These oxidized nucleoside triphosphates are competitive inhibitors of RNA polymerase. 3. On incubation, together with NaBH4, these oxidized labelled nucleotides are covalently bound to Escherichia coli RNA polymerase. 4. Nucleoside triphosphate substrates decrease the extent of labelling. 5. A lysine residue in an alpha-subunit is labelled. 6. The significance of these results in relation to the location of the nucleotide-binding site is discussed.     

The phosphorylated pathway of serine biosynthesis links plant growth with nitrogen metabolism

Because it is the precursor for various essential cellular components, the amino acid serine is indispensable for every living organism. In plants, serine is synthesized by two major pathways: photorespiration and the phosphorylated pathway of serine biosynthesis (PPSB). However, the importance of these pathways in providing serine for plant development is not fully understood. In this study, we examine the relative contributions of photorespiration and PPSB to providing serine for growth and metabolism in the C3 model plant Arabidopsis thaliana. Our analyses of cell proliferation and elongation reveal that PPSB-derived serine is indispensable for plant growth and its loss cannot be compensated by photorespiratory serine biosynthesis. Using isotope labeling, we show that PPSB-deficiency impairs the synthesis of proteins and purine nucleotides in plants. Furthermore, deficiency in PPSB-mediated serine biosynthesis leads to a strong accumulation of metabolites related to nitrogen metabolism. This result corroborates ¹⁵N-isotope labeling in which we observed an increased enrichment in labeled amino acids in PPSB-deficient plants. Expression studies indicate that elevated ammonium uptake and higher glutamine synthetase/glutamine oxoglutarate aminotransferase (GS/GOGAT) activity causes this phenotype. Metabolic analyses further show that elevated nitrogen assimilation and reduced amino acid turnover into proteins and nucleotides are the most likely driving forces for changes in respiratory metabolism and amino acid catabolism in PPSB-deficient plants. Accordingly, we conclude that even though photorespiration generates high amounts of serine in plants, PPSB-derived serine is more important for plant growth and its deficiency triggers the induction of nitrogen assimilation, most likely as an amino acid starvation response.

The phosphorylated pathway of serine biosynthesis is required to synthesize serine for plant growth; and its deficiency triggers an amino acid starvation response by inducing nitrogen assimilation.     

Isolation of a yeast single-strand deoxyribonucleic acid binding protein that specifically stimulates yeast DNA polymerase I

We sought a protein from yeast that would bind more strongly to single-stranded DNA than to duplex DNA and would stimulate the activity of the major yeast DNA polymerase, but not polymerases from other organisms. We isolated a protein that binds about 200 times more strongly to single-stranded DNA than duplex DNA and stimulates yeast DNA polymerase I activity 4-5-fold. It inhibits synthesis catalyzed by calf thymus DNA polymerase alpha and has little effect on T4 DNA polymerase. This yeast protein, SSB-1, has a molecular weight of approximately 40 000. At apparent saturation there is one protein molecule bound per 40 nucleotides. Protein binding causes the single-stranded DNA molecule to assume a relatively extended conformation. It binds to single-stranded RNA as strongly as to DNA. SSB-1 increases the initial rate of polymerization catalyzed by yeast DNA polymerase I apparently by increasing the processivity of the enzyme. We estimate there are 7500-30 000 molecules of SSB-1 per yeast cell, enough to bind at least 400-1600 nucleotides per replication fork. Thus it is present in sufficient abundance to participate in DNA replication in vivo in the manner suggested by these in vitro experiments.     

The control of ribonucleic acid synthesis in bacteria. Fluctuations in messenger ribonucleic acid synthesis in cultures recovering from amino acid starvation

Changes in the cell content and rate of synthesis of mRNA were studied in auxotrophs of Escherichia coli recovering from a period of amino acid deprivation. Parallel studies were carried out on bacterial strains inhibited with trimethoprim, when glycine and methionine were added to relieve an amino acid deficiency. In the latter case, protein synthesis was still severely inhibited through a lack of N-formylmethionyl-tRNA(fMet) for chain initiation, so that fewer ribosomes were attached to mRNA chains. (1) In RC(str) strains recovering from amino acid starvation, there was a transient oversynthesis of mRNA, but the amounts returned to normal after about a 15-min period of recovery. RC(rel) strains did not show this effect; any extra mRNA accumulated during the previous starvation period was rapidly lost, but no oversynthesis occurred during the resumption of growth. (2) In trimethoprim-inhibited cultures supplemented with glycine and methionine, mRNA was produced at the same rate, relative to stable RNA species, as during normal growth. The evidence implied that decreased rates of ribosome attachment had no effect on the functional or chemical lifetime of the mRNA fraction. This suggests that mRNA stability does not depend on the frequency of translation by ribosomes. (3) Changes in the mRNA contents of trimethoprim-inhibited RC(str) and RC(rel) cultures were noted soon after supplementation with glycine and methionine. These closely followed those observed in cultures recovering from simple amino acid withdrawal.