Effect of Purine Analogs and Their Interactions with Benzyladenine on Bud Burst and Shoot Growth in Apple Bud Explants

The purine analogs 8-azaadenine, 8-azaguanine, 4-aminopyrazolo [3,4-d] pyrimidine, 2-fluoroadenine and 6-methylpurine inhibited bud burst in non-dormant vegetative axillary bud explants obtained from apple (Malus domestica Borkh.) seedlings. The reversal of inhibition was attempted by incorporating increasing concentrations of benzyladenine (BA) in the culture medium. The inhibition caused by 8-azaadenine and 8-azaguanine was reduced especially when BA was added to the media at equimolar ratios along with the inhibitors. The inhibition due to 4-aminopyrazolo [3,4-d] pyridine was reduced only with equimolar BA, BA was ineffective or only minimally effective in reducing inhibition due to 2-fluoroadenine and 6-methypurine. Short pretreatment with 8-azaadenine and 8-azaguinine failed to cause any inhibition when explants were subsequently transferred to BA -containing medium. The effectiveness of 2-fluoroadenine and 6-methylpurine was maintained or only slightly reduced, indicating their long-term inhibitory action.     

ACTION OF PURINE AND PYRIMIDINE ANALOGS ON THE GROWTH AND DIFFERENTIATION OF THE GAMETOPHYTES OF THE FERN ASPLENIUM NIDUS

The purine analogs, 8-azaadenine, 8-azaguanine, 8-azaxanthine and 8-azahypoxanthine, and the pyrimidine analogs, 2-thiocytosine, 5-fluorouracil, 2-thiouracil and 6-azauracil, inhibited the induction of 2-dimensional growth in the gametophytes of the fern Asplenium nidus L. In contrast, thymine analogs such as 5-fluorodeoxyuridine, 2-thiothymine, 6-azathymine and 5-bromouracil caused non-specific growth inhibitions without suppressing 2-dimensional growth. Subinhibitory concentrations of 8-azaxanthine, 8-azahypoxanthine, and 2-thiouracil promoted both 1-dimensional and 2-dimensional phases of growth of the gametophytes. Inhibitory effects of the analogs were observed on treatment of the spores or of gametophytes of different ages. Gametophytes growing in the analogs for different periods of time recovered from inhibition on transfer to the basal medium.     

Eremothecium ashbyii mutants resistant to 8-azaguanine. II. Mutants with different degrees of resistance to 8-azaguanine]

Guanine, unlike adenine and hypoxanthine, can not eliminate the inhibitory effect of adenine analogues on the growth and flavinogenesis of Eremothecium ashbyii. Guanine does not restore riboflavin synthesis inhibited with 5-10(-3) M 8-azaguanine. Low adenine concentrations (10(-4)-3-10(-4) M), which do not influence the inhibitory effect of 5.-10(-3) M 8-azaguanine, restore the riboflavin synthesis in combination with guanine. On the basis of the data obtained as well as the data of biochemical analysis it is concluded that the riboflavin producer studied lacks guanosinemonophosphate reductase. The mutants resistant to various concentrations of 8-azaguanine have been obtained. In all mutants resistant to 8-azaguanine the efficiency of the incorporation of 14C-guanine and 14C-adenine into mycelium is decreased as compared with the susceptible strain. The mutant Azg-R 10 resistant to high (3-10(-3) M) concentrations of 8-azaguanine, 8-azaadenine and 2,6-diaminopurine secretes inosine-like compounds when grown in a synthetic medium. The stepwise increase of the mutant resistance to 8-azaguanine from 10(-4) M TO 3-10(-3) M did not result in further enhancement of riboflavin synthesis.     

Ascaridia galli: effects of immunosuppressive drugs or bursectomy in chickens

Immunosuppressive drugs significantly increasing numbers of A. galli and incidences of infection were: cortisone, cortisol, 9-α-fluorohydrocortisone, 2-methyl-9-α-fluorohydrocortisone, prednisone, prednisolone, 6-mercaptopurine, 2, 6-diaminopurine, 6-thioguanine, 5-bromodeoxyridine, 5-fluorouracil, methotrexate, chlorambucil, and actinomycin D. These drugs and/or worm burdens significantly suppressed weight gains of hosts, and neither altered the male:female ratio of worms nor their growth. The following drugs neither altered worm burdens nor increased incidences of infection: corticosterone, 2-azaadenine, 8-azaguanine, azathioprine, 6-azauridine, busulfan, thio-TEPA, triethylenemelamine, vincristine, acriflavine, reserpine, and l-phenylalanine.Worm burdens and incidences of infection were increased significantly in chickens surgically bursectomized when 3 or 14 but not 35 days old. Chicks bursectomized in ovo with testerosterone propionate on Day 5 or 14 of incubation and infected on Day 14 after hatching developed significantly increased worm burdens and incidences of infection.Applying the Kolmogorov-Smirnov test for goodness of fit to data on increased worm burdens showed that the immunosuppressive drugs or bursectomy had a normalization effect on the statistical distribution.     

Novel Carbocyclic Nucleosides Containing A Cyclobutyl Ring. Guanosine and Adenosine Analogues

Abstract

(1R,cis)-2-(3-Amino-2,2-dimethylcyclobutyl)ethanol (4) was used as a precursor in the synthesis of cyclobutyl nucleoside analogues containing guanine, 8-azaguanine, adenine or 8-azaadenine. All the compounds were evaluated as antiviral agents in a variety of assay systems. Some activity was noted for compound 13, 17, 19 and 20 against vaccinia virus and for compounds 11, 12, 13, 17, 19 and 20 against herpes simplex virus, at concentrations that were up to 10-fold below the cytotoxic concentrations for the host cells.     

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.     

Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis

A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of ¹³C-labeled diC8PC ((methyl-¹³C)₃-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-¹³C)₃-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.     

Mutants of Pichia guilliermondii yeasts with multiple sensitivity to antibiotics and antimetabolites. I. The selection and properties of the mutants

Riboflavin deficient mutant Pichia guilliermondii MS1 which requires approximately 1000-fold lower concentration of exogenous vitamin B2 for growth when compared with a non-adapted riboflavin deficient mutants of this species was isolated by means of of UV-irradiation. The growth of the mutant was strongly inhibited by actinomycin D and L-canavanine. The revertant MS8 and MS14 which synthesized riboflavin were selected from the strain MS1. These revertants posses a multiple sensitivity to actinomycin D, rifamycin, euflavine, mitomycin C, antimycin A, 8-azaadenine, 8-azaguanine, L-canavanine and 7-methyl-8-trifluoromethyl-10-(1'-D-ribityl)isoalloxazine. The ability to utilized glycerol and ethanol as a sole carbon source for growth was impaired in these mutants. The mutants which can utilize glycerol were isolated from the strain MS14. Such mutants were resistant to actonomycin D. Mutation (s) which determines a multiple sensitivity and inability to utilized glycerol was recessive.     

Excision of the oxidatively formed 5-hydroxyhydantoin and 5-hydroxy-5-methylhydantoin pyrimidine lesions by Escherichia coli and Saccharomyces cerevisiae DNA N-glycosylases

Background

(5R?) and (5S?) diastereomers of 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2′-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes.

Methods

Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements.

Results

In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the k_cat/K_m ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase.

Conclusions

The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates.

General significance

The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.     

Genetics of resistance to 4-aminopyrazolo-(3,4-d)-pyrimidine in Saccharomyces cerevisiae

Summary Thirty six mutants resistant to the purine analogue 4-aminopyrazolo(3,4-d)pyrimidine were isolated from a prototrophic strain of yeast carrying su-pur, a suppressor of purine excretion. The mutants were allocated to seven genes app1 to app7. Linkage was found between app3, app5, app6 and app7 but not close enough to suggest functional grouping. Some of the alleles of app1, app3 and app4 are dominant. None of the mutants excreted purine when out-crossed to remove su-pur, nor did they show allelism or linkage to any of the pur (purine excretion) genes. Two mutants, app6-30 and app7-31, are recessive in diploids homo-/or heterozygous for su-pur but are dominant in diploids homozygous for su-pur ⁺. The mutants exhibit gene specific, and in one case allele specific, patterns of cross-resistance to other analogues of adenine, hypoxathine and guanine. In the presence of su-pur all seven genes confer resistance to the adenine analogue 8-azaadenine; in addition mutants of app1, app3 (dominant alleles only) and app4 are resistant to 6-methylpurine, 6-mercaptopurine, 8-azaguanine and guanine. Combination with su-pur ⁺ confers resistance to all analogues tested except in the cases of app7, which remains sensitive to 8-azaguanine and app2 which is rendered sensitive to the five purine analogues. Exogenous adenine or hypoxanthine increases the growth rate of wild type (su-pur), app2, app6 and app7 but not app1, app3, app4 and app5. These effects of purine supplementation on strains of the genotype ade2 app sup-pur suggest that mutants of app1, app3, app4 and app5 are defective in the utilisation of exogenous purines. It is suggested that the other three genes may have defects in the control of de novo purine synthesis.Supported by a Medical Research Council Research Training Scholarship Awarded to W.R.P.     

Selective effects of purine and pyrimidine analogues and of respiratory inhibitors on perithecial development and branching in sordaria

The initiation of perithecia in the homothallic ascomycete Sordaria fimicola was completely suppressed, without seriously inhibiting vegetative growth, by growing the fungus on an agar medium containing one of the following additions: 1) 1 μm 5-fluorouracil, 2) 10 to 100 μm 6-azauracil, 8-azaguanine or 8-azaadenine, 3) 50 to 500 μm cyanide or azide, 4) 5% (w/v) casein hydrolysate. In contrast to the selective activity of the analogues of 3 RNA bases, whose inhibition could be reversed by the appropriate normal bases only, none of the analogues of thymine were active, neither were the thio-derivatives of RNA bases. Other inhibitors of RNA and protein synthesis, like actinomycin D, puromycin and cycloheximide, were also without selective activity, although the last of these inhibited perithecial maturation at 0.1 μm concentration but not initiation. Amino acid analogues were inactive, as were the metabolic inhibitors thiourea, 2,4-dinitrophenol and fluoride. The compounds which inhibited the formation of perithecia also lowered the branching frequency of leading hyphae, but not their linear growth rates. Consequently, the branch densities were diminished in their presence. Hypotheses to account for these findings are discussed in terms of inhibition of growth in general, of the synthesis of some specific messenger RNAs, and of RNA-mediated transport across membranes, the last of which seeming the most fruitful for further work.     

Interaction of Kinetin and Various Inhibitors in the Growth of Soybean Tissue

The growth of kinetin-requiring soybean callus was inhibited by the purine analogs 2,6-diaminopnrine, 8-azaguanine and 8-azaadenine. The purine analog 6-mercaptopnrine and the antibiotic puromycin were not effective as inhibitors at the concentrations employed. Reversal of the inhibition was attempted by raising the kinetin concentration. Kinetin did not reverse the inhibition due to 2,6-dianiinopurine but it did lessen to some extent the inhibition due to 8-azagnanine. It is suggested the kinetin affects RNA metabolism.     

Stimulation of Growth of an Adenine-requiring Yeast by Purine Analogues

An adenine-requiring yeast grew in an adenine-free medium after considerably long lag period (approximately 170 hr). Supplement of adenine to the medium resulted in a marked reduction of the lag period and in a diauxic growth. Although the growth rate was much faster, a similar diauxic growth was observed in the medium containing sufficient amount of adenine. In both cases, the primary growth occurred as a result of fermentative metabolism of glucose, and after exhaustion of glucose, the secondary growth started at the expense of accumulated ethanol. When cells obtained from the adenine-deficient medium were analyzed for total adenine compounds, approximately six times as much adenine was detected as that amount of adenine added to the medium. Therefore, the yeast has no block in the biochemical sequences leading to adenine biosynthesis but has a defect in the control mechanism of adenine biosynthesis. Adenine appears to initiate adenine biosynthesis under adenine deficient conditions. In order to understand the initiation mechanism by adenine, the effect of several purine analogues on growth was examined. Among the agents tested, 6-thioguanine or 8-azaadenine shortened the lag time of growth in the adenine-free medium. Furthermore, when small amount of adenine was supplied to the medium, these compounds stimulated the primary growth without affecting the secondary growth. On the contrary, 8-azaguanine or 8-azaxanthine markedly stimulated the secondary growth without affecting the primary growth. Thus, two distinct trigger mechanisms in adenine biosynthesis were proposed.     

Isolation of a novel rmn1 gene genetically linked to spnab2 with respect to mRNA export in fission yeast

In fission yeast, Schizosaccharomyces pombe, the spnab2 gene encodes an ortholog of the budding yeast nuclear abundant poly(A)⁺ RNA-binding protein 2 (Nab2) that is an essential protein required for both mRNA biogenesis and nuclear export of mRNA to the cytoplasm. We have previously isolated three mutants (SLnab1–3) that showed synthetic lethality under the repressed condition of spnab2 expression. In this study, we isolated a novel rmn1 gene as a multicopy suppressor that complemented the defects in growth and mRNA export of SLnab1 mutant cells. The rmn1 gene contained three introns and encoded a 589 amino-acid protein with the RNA recognition motif (RRM) in the central region. The Δrmn1 null mutant was viable but showed a s light mRNA export defect. However, its over-expression caused a deleterious effect on growth accompanied by intense accumulation of poly(A)⁺ RNA in the nucleus. The combination of Δrmn1 with Δspnab2 or Δspmex67 also inhibited growth. In addition, Rmn1p was associated with Rae1p in vivo. These results suggest that rmn1 is a novel gene that is functionally linked to spnab2.     

Synthesis of protein and RNA in pollen tubes stimulated with 2-thiouracil

1. 1.
   Protein synthesis in pollen tubes ofNicotiana alata Link etOtto estimated by the incorporation of leucine-¹⁴C is linear over six hours of artificial cultivation after a short lag phase. 2-Thiouracil and other growth-stimulating antimetabolites of natural pyrimidines and purines, such as 6-azauracil, 5-nitrouracil, 8-azaguanine and 8-azaadenine, stimulate the incorporation of leucine-¹⁴C into protein. The intensity of stimulation of protein synthesis is associated with the intensity of growth stimulation by antimetabolite.     
   
   

INHIBITION OF LAMINA INCLINATION BY CYTOKININ IN EXCISED RICE LEAVES

Effects of kinetin and other purine and pyrimidine derivativeson lamina inclination in excised rice leaves have been examinedin this work. Kinetin inhibited the lamina inclination both in the presenceand in the absence of added IAA, and was effective even at 10^–7M. The decreasing order of inhibitory activities of kinetinand its analogs was as follows; 6-benzylaminopurine, kinetin,2-amino-6-furfurylaminopurine (2-aminokinetin), 2-amino-6-benzylaminopurine,kinetin-9-glucoside, 6-benzylaminopurine-9-glucoside. Otherpurine derivatives, 8-azaadenine, 2, 6-diaminopurine and 8-azaguanine,and colchicine also inhibited the lamina inclination. The inhibitiveaction of kinetin was not reversed by the addition of adenine,hypoxanthine, uracil and thymine. (Received May 20, 1965; )     

Toxicity of 6-thioguanine and 8-azaguanine to non-dividing liver cell cultures

8-azaguanine and 6-thioguanine were both toxic to non-dividing liver cells in primary cultures. In addition, these agents were toxic to an established line of liver-derived epithelial cells brought to growth arrest by serum deprivation. These observations demonstrate that the toxicity of 8-azaguanine and 6-thioguanine can occur at least in part through mechanisms that do not involve effects on DNA synthesis or incorporation of the analogs into DNA.Abbreviations AG 8-azaguanine - ARL adult rat liver epithelial cell line - HGPRT hypoxanthine-guanine phosphoribosyl transferase - WME Williams Medium E     

Effect of aza-substituted nucleotides on the starve-refeed response of rats.

Rat liver glucose 6-phosphate dehydrogenase (G6PD) and malic enzyme (ME) activities were increased by starvation-refeeding to levels above those found in rats fed ad libitum. The increases in enzyme activities above ad libitum-fed levels were prevented by 8-azaguanine and 6-azauridine, but not by 2-azauridine. Blood insulin levels were not affected at the time studied. Two aza analogs, 8-azaadenine and 5-azacytidine, proved to be too toxic in this type of studies. Since 8-azahypoxanthine, 8-azaxanthine and 5-azauracil were neither effective in preventing the enzyme overshoot, nor toxic to the animals, it was concluded that the toxiciyty to the animals of 8-azaadenine and 5-azacytidine is due to the compounds themselves rather than to the breakdown products.     

Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins

We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc₁ complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036-36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8^m). Subsequently replacing ARG8^m with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit⁻). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.     

Study of model systems to test the potential function of Artemia group 1 late embryogenesis abundant (LEA) proteins

Embryos of the brine shrimp, Artemia franciscana, are genetically programmed to develop either ovoviparously or oviparously depending on environmental conditions. Shortly upon their release from the female, oviparous embryos enter diapause during which time they undergo major metabolic rate depression while simultaneously synthesize proteins that permit them to tolerate a wide range of stressful environmental events including prolonged periods of desiccation, freezing, and anoxia. Among the known stress-related proteins that accumulate in embryos entering diapause are the late embryogenesis abundant (LEA) proteins. This large group of intrinsically disordered proteins has been proposed to act as molecular shields or chaperones of macromolecules which are otherwise intolerant to harsh conditions associated with diapause. In this research, we used two model systems to study the potential function of the group 1 LEA proteins from Artemia. Expression of the Artemia group 1 gene (AfrLEA-1) in Escherichia coli inhibited growth in proportion to the number of 20-mer amino acid motifs expressed. As well, clones of E. coli, transformed with the AfrLEA-1 gene, expressed multiple bands of LEA proteins, either intrinsically or upon induction with isopropyl-β-thiogalactoside (IPTG), in a vector-specific manner. Expression of AfrLEA-1 in E. coli did not overcome the inhibitory effects of high concentrations of NaCl and KCl but modulated growth inhibition resulting from high concentrations of sorbitol in the growth medium. In contrast, expression of the AfrLEA-1 gene in Saccharomyces cerevisiae did not alter the growth kinetics or permit yeast to tolerate high concentrations of NaCl, KCl, or sorbitol. However, expression of AfrLEA-1 in yeast improved its tolerance to drying (desiccation) and freezing. Under our experimental conditions, both E. coli and S. cerevisiae appear to be potentially suitable hosts to study the function of Artemia group 1 LEA proteins under environmentally stressful conditions.

Electronic supplementary material

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