Characterization of the arginine repressor from Salmonella typhimurium and its interactions with the carAB operator.

Primer extension experiments showed that the argR gene, encoding the arginine repressor in Salmonella typhimurium, is transcribed from a single promoter that is negatively regulated by arginine. A repressor overproducing strain was constructed and the repressor was purified to homogeneity. Gel filtration, sedimentation and cross-linking studies established that the native repressor is a hexamer of identical 17,000 Mr subunits. Gel retardation experiments indicate that the apparent dissociation constant for repressor/carAB operator is 6 x 10(-12) M. These experiments showed that arginine is essential for binding of the repressor to the DNA and that pyrimidine nucleotides have no significant effect on this binding. These results indicate that the effect of pyrimidines on expression of the arginine sensitive "downstream" carAB promoter is not directly mediated by the arginine repressor. These experiments also suggest that a single hexamer binds to the carAB operator, which carries two previously defined "ARG box" sequences that characterize operators for arg genes. Gel retardation experiments with DNA fragments carrying the individual ARG boxes showed that both boxes are required for effective binding of the hexameric repressor to the operator, indicating that the ARG boxes comprise a single binding site for the repressor. Analysis of the potential secondary structure of the arginine repressor does not reveal any of the recognizable structural motifs common to a number of DNA-binding proteins. A combination of DNase I, premethylation interference, depurination and hydroxyl radical footprinting techniques were employed to characterize the interactions of the repressor with the carAB operator, with the results suggesting that the repressor predominantly interacts with A.T residues in this region. Comparative DNA sequence analysis of the known arginine operators of enteric bacteria further indicates that the specificity of interaction may be based more on the precise distance between two defined A.T-rich regions rather than on the specific nucleotide sequence.     

Amino acid catalyzed condensation of purines and pyrimidines with 2-deoxyribose.

Mild heating of aqueous mixtures containing 2-deoxyribose, amino compounds, and purines or pyrimidines produces derivatives of the purines and pyrimidines in high yield. Among the major products formed are 2,3-dideoxy-3-(1'-pyrimidino)pentose and 2,3-dideoxy-3-(9'-purino)pentose. The mechanism of the reaction includes amine-catalzyed dehydration of the alpha, beta positions of the sugar followed by addition of the purine or pyrimidine to the double bond. Rapid addition of purines and pyrimidines to alpha, beta-unsaturated carbonyl compounds (such as acrolin) is a general phenomenon which does not require an amine catalyst. While multiple derivatization of the purines will take place, the N-9 derivative is formed first.     

Nucleotide sequence analysis of genes purH and purD involved in the de novo purine nucleotide biosynthesis of Escherichia coli

5'-Phosphoribosylglycinamide synthetase (EC 6.3.4.13) and 5'-phosphoribosyl 5-aminoimidazole-4-carboxamide transformylase (EC 2.1.2.3) are enzymes involved in the de novo purine nucleotide synthesis and are encoded by purD and purH genes of Escherichia coli, respectively. A 3535-nucleotide sequence containing the purHD locus and the upstream region of the rrnE gene was determined. This sequence specifies two open reading frames, ORF-1 and ORF-2, encoding proteins with the expected Mr of 57,329 and 46,140, respectively. The plasmids carrying ORF-1 complemented not only the mutant cells defective in purH of E. coli but also the cells of Salmonella typhimurium lacking the activity of IMP cyclohydrolase (EC 3.5.4.10) which catalyzes the conversion of 5'-phosphoribosyl 5-formylaminoimidazole-4-carboxamide to IMP. The E. coli purH gene, therefore, specifies bifunctional 5'-phosphoribosyl 5-aminoimidazole-4-carboxamide transformylase-IMP cyclohydrolase. The plasmids carrying ORF-2 were able to complement the mutant cells defective in purD. Both purH and purD genes constitute a single operon and are coregulated in expression by purines as other purine genes are. A highly conserved 16-nucleotide sequence termed the PUR box (Watanabe, W., Sampei, G., Aiba, A., and Mizobuchi, K. (1989) J. Bacteriol. 171, 198-204; Tiedeman, A.A., Keyhani, J., Kamholz, J., Daum, H. A., III, Gots, J.S., and Smith, J.M. (1989) J. Bacteriol. 171, 205-212) was found in the control region of the purHD operon and compared with the sequences of the control regions of other purine operons.     

Regulation of purine biosynthetic genes expression inSalmonella typhimurium IV Oc mutation site ofpurG and its function analysis

Salmonella typhimurium 5 phosphoribosylformylglycinamide (FGAR) amidotransferase encoded bypurG gene catalyzes the conversion of FGAR to formylglycinamide ribonucleotide (FGAM) in the presence of glu- tamine and ATP for thede novo purine nucleotide biosynthesis.purG gene is negatively regulated by a repressor-operator system. The O⁺ purG and O^c purG were cloned respectivelyin vivo. Restriction enzymes analysis of preliminary clones pLBG-1 (O⁺) and pLBG-2 (O^c) were carried out. The hybrid plasmids pLB1933 (O⁺) and pLB1927 (O^c) containing 5′ control region ofpurG were constructed and the DNA sequences were determined respectively, DNA sequences data showed that O^c mutation ofpurG occurred at the 3rd position of 16 bp PUR box in the 5′ control region (G→A). Gel retardation experiment indicated that the repressor bound well with O⁺ PUR box, but not with O^c PUR box. The result strongly supported the idea that PUR box is the binding region of repressor protein and the 3rd position base G of PUR box is essential for the binding function with repressor protein.     

Regulation of purine biosynthetic genes expression in Salmonella typhimurium Ⅳ Oc mutation site of purG and its function analysis

Salmonella typhimurium 5 phosphoribosylformylglycinamide (FGAR) amidotransferase encoded bypurG gene catalyzes the conversion of FGAR to formylglycinamide ribonucleotide (FGAM) in the presence of glu- tamine and ATP for thede novo purine nucleotide biosynthesis.purG gene is negatively regulated by a repressor-operator system. The O⁺ purG and O^c purG were cloned respectivelyin vivo. Restriction enzymes analysis of preliminary clones pLBG-1 (O⁺) and pLBG-2 (O^c) were carried out. The hybrid plasmids pLB1933 (O⁺) and pLB1927 (O^c) containing 5′ control region ofpurG were constructed and the DNA sequences were determined respectively, DNA sequences data showed that O^c mutation ofpurG occurred at the 3rd position of 16 bp PUR box in the 5′ control region (G→A). Gel retardation experiment indicated that the repressor bound well with O⁺ PUR box, but not with O^c PUR box. The result strongly supported the idea that PUR box is the binding region of repressor protein and the 3rd position base G of PUR box is essential for the binding function with repressor protein. Project supported by the National Natural Science Foundation of China.     

On the conformational dependence of the proton chemical shifts in nucleosides and nucleotides. II. Proton shifts in the ribose ring of purine nucleosides as a function of the torsion angle about the glycosyl bond

The variations of the ring current, the local diamagnetic susceptibility anisotropy and the polarization contributions to the chemical shift of the non exchangeable protons of the ribose ring of purine nucleosides are computed as a function of the torsion angle about the glycosyl bond, χ_CN. The results show that the ring current effect is relatively more important in the purines than in the pyrimidines. In addition, N₃ of purines has a local magnetic anisotropy effect similar to the one of the carbonyl group C₂O₂ of pyrimidine nucleosides. The experimental differences between the chemical shift of the ribose protons of purine nucleosides and of 8 substituted derivatives are discussed in relation to the theoretical variations.     

A unique pattern of intrastrand anomalies in base composition of the DNA in hypotrichs

The 50 non-coding bases immediately internal to the telomeric repeats in the two 5′ ends of macronuclear DNA molecules of a group of hypotrichous ciliates are anomalous in composition, consisting of 61% purines and 39% pyrimidines, A>T (ratio of 44:32), and G>C (ratio of 17:7). These ratio imbalances violate parity rule 2, according to which A should equal T and G should equal C within a DNA strand and therefore pyrimidines should equal purines. The purine-rich and base ratio imbalances are in marked contrast to the rest of the non-coding parts of the molecules, which have the theoretically expected purine content of 50%, with A = T and G = C. The ORFs contain an average of 52% purines as a result of bias in codon usage. The 50 bases that flank the 5′ ends of macronuclear sequences in micronuclear DNA (12 cases) consist of ~50% purines. Thus, the 50 bases in the 5′ ends of macronuclear sequences in micronuclear DNA are islands of purine richness in which A>T and G>C. These islands may serve as signals for the excision of macronuclear molecules during macronuclear development. We have found no published reports of coding or non-coding native DNA with such anomalous base composition.     

A Role for Purines and Pyrimidines of Ribonucleic Acid in the Induction of Two-dimensional Growth in the Gametophytes of the Fern, Asplenium nidus

The inhibition of two-dimensional growth in the gametophytesof Asplenium nidus induced by purine and pyrimidine analoguesand the reversal of inhibition by natural purine and pyrimidinebases and their derivatives have been studied. Adenine and guanineand their ribosides and ribotides were more effective than cytosine,uracil, thymine, and their derivatives in preventing the inhibitiondue to 8-azaadenine and 8-azaguanine. Likewise, the inhibitoryeffects of 2-thiocytosine, 2-thiouracil,6-azauracil, and 5-fluorouracilwere overcome by the pyrimidines and their derivatives, butnot usually by the purines.Combinations of two purine analoguesor two pyrimidine analogues or one purine analogue and one pyrimidineanalogue inhibited growth more effectively than single compounds.The combined inhibitions were maximally reversed when both naturalbases or their derivatives were added to the medium. It is concludedthat there is a requirement for both purines and pyrimidinesof ribonucleic acid in the induction of two-dimensional growthin the gametophytes of Asplenium nidus.     

Origin of ultraviolet damage in DNA

A novel ultraviolet (u.v.) footprinting technique has been used to analyze the formation of u.v. photoproducts at 250 bases of a 5 S rRNA gene under conditions where the gene is either double or single-stranded. Because many more types of u.v. damage can be detected by the u.v. footprinting technique than has been previously possible, we have been able to examine in detail why certain bases in DNA are damaged by u.v. light while others are not. Our measurements demonstrate that the ability of u.v. light to damage a given base in DNA is determined by two factors, the sequence of the DNA in the immediate vicinity of the photoproduct, and the flexibility of the DNA at the site of the photoproduct. For pyrimidines, the predominant photoreaction in double-stranded DNA involves covalent dimerization between adjacent pyrimidine residues. Dimerization is much easier in melted DNA because the geometrical changes required for adjacent pyrimidine residues to dimerize are easier in single-stranded DNA. The absorption of a u.v. photon cannot simultaneously induce the geometrical changes required for adjacent pyrimidines or other bases to dimerize with one another. Rather, upon the absorption of a u.v. photon, only those thermally excited bases that are in a geometry capable of easily forming a photodimer during excitation, can photoreact. In contrast to adjacent pyrimidines, non-adjacent pyrimidines (pyrimidines flanked on either side by a purine) do not readily form u.v. photoproducts in double-stranded DNA. Because photoreactions at non-adjacent pyrimidine residues are greatly enhanced in single-stranded DNA, their failure to form in double-helical DNA is attributed to torsional constraints imposed by the double helix which make it difficult for non-adjacent pyrimidines to adopt a geometry necessary for photoreaction. Although purines are believed to be resistant to u.v. damage, our measurements demonstrate that at moderate u.v. dosages purines which are flanked on their 5' side by two or more contiguous pyrimidines readily form u.v. photoproducts in double-stranded DNA. Flanking pyrimidines appear to activate purine photoreactions by transferring triplet excitation energy to the purine. Melting of the DNA helix greatly inhibits the ability of flanking pyrimidines to activate purine photoreactions, presumably by disrupting intimate orbital overlap required for triplet transfer.     

Methotrexate induced differentiation in colon cancer cells is primarily due to purine deprivation

The folate antagonist methotrexate (MTX) inhibits synthesis of tetrahydrofolate (THF), pyrimidines and purines, and induces differentiation in several cell types. At 1 microM, MTX reduced proliferation and induced differentiation in HT29 colon cancer cells; the latter effect was augmented (P < 0.001) by thymidine (100 microM) but was reversed (P < 0.001) by the purines, hypoxanthine (Hx; 100 microM) and adenosine (100 microM). In contrast 5-fluoro-uracil (5-FU), a specific thymidylate synthase (TS) inhibitor, had no effect on differentiation, suggesting that MTX-induced differentiation is not due to a reduction in thymidine but to the inhibition of purine biosynthesis. Inhibition of cyclic AMP (cAMP) by RpcAMP (25 microM) further enhanced (P < 0.001) MTX induced differentiation, whereas the cAMP activator forskolin (10 microM) reversed (P < 0.001) MTX induced differentiation. These observations implicate a central role of adenosine and cAMP in MTX induced differentiation. By combining Western blot analysis with liquid chromatography-mass spectrometry (LC-MS)and HPLC analyses we also reveal both the expression and activity of key enzymes (i.e. methionine synthase (MS), s-adenosylhomocysteinase, cystathionine beta-synthase and ornithine decarboxylase) regulating methyl cycle, transsulfuration and polyamine pathways in HT29 colon cancer cells. At 1 microM, MTX induced differentiation was associated with a marked reduction in the intracellular concentrations of adenosine and, consequently, S-adenosylmethionine (SAM), S-adenosylhomocysteine, polyamines and glutathione (GSH). Importantly, the marked reduction in methionine that accompanied MS inhibition following MTX treatment was non-limiting with respect to SAM synthesis. Collectively, these findings indicate that the effects of MTX on cellular differentiation and single carbon metabolism are primarily due to the intracellular depletion of purines.     

Purine and pyrimidine salvage in a clonal Drosophila cell line

A new culture medium, ZH1%, for Drosophila cell lines is described in which treatment of cells with the folate antagonist methotrexate (MTX) leads to overt double auxotrophy for both purines and pyrimidines. Using this medium the response of a clonal Drosophila cell line, KcAlo, to various purines and pyrimidines was measured. Guanosine was found to balance the toxicity of adenosine (AR), and vice versa. In addition AR toxicity was shown to be strongly dependent on the pH of the culture medium. Using ZH1% supplemented with MTX + thymidine (TdR) or with MTX + inosine (HR) several purines or pyrimidines, respectively, were tested for their capacity to support cell proliferation. This provided evidence for pathways for the salvage of thymine, TdR, thymidylic acid, adenine (A), AR, and HR. Bromodeoxyuridine and fluorodeoxyuridine are very active and specific TdR analogues inhibiting Drosophila cell proliferation. Of the many purine analogues tested the only potent growth inhibitor with demonstrable pathway specificity (antagonizable by A only) was methylpurine.     

Genetical and biochemical evidence that a novel dinucleoside polyphosphate coordinates salvage and de novo nucleotide biosynthetic pathways in mammalian cells

Studies with ‘wild type’ Chinese hamster ovary cells and mutant derivatives defective in purine salvage and $\text{de novo}$ nucleotide biosynthesis pathways have brought to light the possibility that an unusual dinucleoside polyphosphate, HS-3 (see appendix) is a crucial regulator of these two pathways. Three antitumor drugs, methotrexate, 5-fluorouracil and azaserine as well as L-glutamine, purines and pyrimidines were used to define the loci of HS-3 metabolism. Wild type and salvage pathways mutants accumulated HS-3 in the absence of glutamine. $\text{De novo}$ pathways mutant accumulated HS-3 only when purine was absent. Depletion of HS-3 was induced in wild type and $\text{de novo}$ mutant cell lines by purine compounds. Salvage pathways mutants did not cause depletion of HS-3 when supplied with purines or pyrimidines, except 5-fluorouracil. Data indicate that HS-3 is probably synthesised when an early step in purine nucleotide synthesis is blocked and depleted when the salvage pathways are operative. HS-3 may be an important factor in certain diseases involving nucleotide metabolism.