Mitochondrial dysfunction in yeast expressing the cytoplasmic male sterility T-urf13 gene from maize: analysis at the population and individual cell level

Summary The urf13TW gene, which is derived from the mitochondrial T-urf13 gene responsible for Texas cytoplasmic male sterility in maize, was expressed in Saccharomyces cerevisiae by targeting its translation product into mitochondria. Analysis by oxygraphy at the population level revealed that in the presence of methomyl the oxygen uptake of intact yeast cells carrying the targeted protein is strongly stimulated only with ethanol as respiratory substrate and not with glycerol, lactate, pyruvate, or acetate. When malate is the substrate oxidized by isolated mitochondria, interaction between the targeted protein and methomyl results in significant inhibition of oxygen uptake. This inhibition is eliminated and oxygen uptake is stimulated by subsequent addition of NAD⁺. Using 3,3-dihexyloxacarbocyanine iodide [DiOC₆(3)] as probe, interactive laser scanning and flow cytometry, which permit analysis at the individual cell level, demonstrated that specific staining of the mitochondrial compartment is obtained and that DiOC₆(3) fluorescence serves as a measure of the membrane potential. Finally, it was shown that, as in T cytoplasm maize mitochondria, HmT toxin and methomyl dissipate the membrane potential of yeast mitochondria that carry the foreign protein. Furthermore, the results suggest that the HmT toxin and methomyl response is related to the plasmid copy number per cell and that the deleterious effect induced by HmT toxin is stronger than that of methomyl.     

Cloning and expression of a putative cytochrome P450 gene that influences the colour of Phalaenopsis flowers

Anthocyanins are responsible for reds through blues in flowers. Blue and violet flowers generally contain derivatives of delphinidin, whereas red and pink flowers contain derivatives of cyanidin or pelargonidin. Differences in hydroxylation patterns of these three major classes of anthocyanidins are controlled by the cytochrome P450 enzymes. Flavonoid-3',5'-hydroxylase, a member of the cytochrome P450 family, is the key enzyme in the synthesis of 3',5'-hydroxylated anthocyanins, generally required for blue or purple flowers. Here we report on the isolation of a cDNA clone of a putative flavonoid-3',5'-hydroxylase gene from Phalaenopsis that was then cloned into a plant expression vector. Transient transformation was achieved by particle bombardment of Phalaenopsis petals. The transgenic petals changed from pink to magenta, indicating that the product of the putative flavonoid-3',5'-hydroxylase gene influences anthocyanin pigment synthesis.     

Isolation of trace amounts of 3′,4′-dehydro-4′-deoxydothistromin from peanut plants naturally infected with Cercospora personata

Trace amounts of the anthraquinonoid toxic metabolite, 3,4-dehydro-4-deoxydothistromin have been identified for the first time, from peanut tissues naturally infected by Cercospora personata. Characteristic uv spectral and Chromatographic properties of the metabolite and its tetraacetate as well as the mass spectrum of the latter have established its identity.     

Characterization and localization of fusicoccin-binding sites in leaf tissues of Vicia faba L. probed with a novel radioligand

Tritiated 9-nor-fusicoccin-8-alcohol provides a highly bioactive radioligand of high specific activity which is easily prepared by oxidation of fusicoccin and subsequent reduction with tritiated sodium borohydride. Using this radioligand, we have identified and characterized a selective binding site for fusicoccin (K_a for [³H]-9-nor-fusicoccin-8-alcohol=0.20·10⁹ M^-1; K_a, apparent for fusicoccin=0.21·10⁹ M^-1) located at the plasmalemma of Vicia faba leaf tissue. The site is thermolabile, readily degraded by trypsin and located at the apoplastic face of the plasmalemma based on results obtained using right-side-out plasmalemma vesicles prepared by aqueous two-phase partitioning and macromolecular fusicoccin-derivatives. The binding-protein is present in guard cells of Vicia faba, as shown by the use of purified guard-cell protoplasts.Abbreviations BSA bovine serum albumin - DTT dithiothreitol - EDTA ethylenediaminetetraacetate - FC fusicoccin - FCol 9-nor-fusicoccin-8-alcohol - Mes 2(N-morpholino)ethanesulfonic acid - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

The base-alteration spectrum of spontaneous and ultraviolet radiation-induced forward mutations in the URA3 locus of Saccharomyces cerevisiae

Summary A forward mutation system has been developed to obtain rapidly clonable mutants at the URA3 locus in yeast by means of selection for 5-fluoroorotic acid resistance. We have used this system to determine base changes in 35 spontaneous and 34 ultraviolet radiation-induced ura3 base substitution mutants. Other mutants (frameshift, deletion, duplication, replacement) were detected as well. Evidence is reported which suggests cyclobutane dimers are the principal mutagenic lesions induced by UV radiation in stationary phase cells of the yeast Saccharomyces cerevisiae. Since most of the induced lesions are at 5-TT-3 sites, the results suggest that the A-rule, preferential insertion of adenine residues opposite poorly pairing sites in DNA, does not apply for yeast cells irradiated in stationary phase, whereas the spontaneous mutation data indicate that the A-rule applies for cells in logarithmic phase. Most of the spontaneous mutations are transversions. UV-induced transitions and transversions occur at approximately equal frequencies.     

Genetic control of chalcone-flavanone isomerase activity in Callistephus chinensis

A mutant blocked in anthocyanin synthesis leads to an accumulation of 4,2,4,6-tetrahydroxy-chalcone-2-glucoside (isosalipurposide) in blossoms of Callistephus chinesis (L.) Nees, whereas in geno-types with the wild-type allele, higher oxidized flavonoids and anthocyanins are synthesized. Measurements of chalcone-flavanone isomerase activity of 18 lines of Callistephus chinensis showed a clear correlation between accumulation of chalcone in the recessive genotypes (ch ch) and deficiency of this enzyme activity. Both the chemogenetic and the enzymologic evidence lead to the following conclusions: 1. The first product of the synthesis of the flavonoid skeleton should be tetrahydroxychalcone.-2. The chalcone-flavanone isomerase catalyzes the formation of flavanone from chalcone in a stereospecific way and there-with furnishes the substrate for the further reactions in the flavonoid biosynthesis.Abbreviations EGME ethylene glycol monomethyl ether - HOAc acetic acid - MeOH methanol - PVP polyvinylpyrrolidone - TBA tert. butanol-acetic acid-water, 3:1:1 - TLC thin-layer chromatography     

Degradation products of myelin proteins in a light CNS subcellular fraction

The fraction floating on 0.32 M sucrose when normal mammalian spinal cord homogenate is submitted to discontinuous density gradient centrifugation is highly enriched in Marchi-positive material. In situ this material is located along paranodal myelin sheath segments. We here show by immunoblotting that degradation products of the myelin-associated glycoprotein (MAG) and of the enzyme 2,3-cyclic nucleotide 3-phosphodiesterase (CNP) is present in the Marchi-positive floating fraction but is not found in the myelin fraction. Since previous biochemical analyses of the floating fraction show a gross composition closely resembling myelin and since metabolic studies show the specific activity of incorporated amino acids to proceed with time from beavier to lighter myelin subfractions the results strongly suggest that normally occurring Marchi-positive bodies represents an intermediate stage in myelin catabolism.     

Normal coordinate analysis of 2′-deoxythymidine and 2′-deoxyadenosine

The proposed valence force field allows us to reproduce the vibration modes of 2-deoxythymidine and 2-deoxyadenosine. The present calculations are based on the Wilson GF-method and a non-redundant set of symmetrical coordinates. The calculated wavenumbers have been compared to the available Raman and infrared peak positions observed in solid, amorphous or aqueous samples. Moreover, the results obtained with the present force field allow us to assign some of the characteristic vibration modes for the thymidine and adenosine residues involved in DNA double-helical chains.     

Formation of nucleoside 5′-phosphoramidates under potentially prebiological conditions

Summary Adenosine 5-phosphoramidates form when solutions containing adenosine 5-polyphosphates p_nA (n 3) or P₁, P₂-diadenosine 5-diphosphate and amines are allowed to dry out. Mg ions catalyze these reactions. We have studied systems containing ammonia, imidazole, glycine, ethylenediamine and histamine. The yields of adenosine 5-phosphoramidates range from 10–50 % based on the nucleotide. The prebiotic significance of the reactions is discussed.Abbreviations Im imidazole - hist histamine - gly glycine - en ethylenediamine - CDI 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride - EDTA ethylenediaminetetraacetic acid - A adenosine - P_n (n = 1, 2 ) linear polyphosphate containing n phosphate residues - p_nA adenosine 5-polyphosphate containing n phosphate residues - ADP adenosine 5-diphosphate - ATP adenosine 5-triphosphate - AppA P₁, P₂-diadenosine 5-diphosphate - gly-pA adenylyl-(5N)-glycine - ImpA adenosine 5-phosphorimidazolide - NH₂-pA adenosine 5-phosphoramidate - en-pA adenylyl-(5N)-ethylenediamine - hist (NH) - pA adenosine 5-phospho-[2-(4-imidazolyl)-ethylamide] - hist(Im)-pA adenosine 5-phospho-[4-(2-aminoethyl)-imidazolide] - enP_1,2 phosphoramidates of ethylenediamine derived from H₃PO₄ and H₄P₂O₇     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).     

Gel mobility shift scanning of the acetate-inducible promoters fromNeurospora crassa reveals a common co-inducible DNA-binding protein

The promoter regions of four acetate-inducible genes ofNeurospora crassa, acu-3, acu-5, acu-8 andacu-9, have been sequenced. Using a scanning gel mobility shift assay particular DNA regions in each promoter have been shown specifically to bind partially purified protein extracted from acetate-induced mycelia. The protein-binding regions so defined have common sequence motifs, elements of which are similar to those required for acetate induction inAspergillus nidulans.     

Microbial degradation of dehydrodiconiferyl alcohol,a lignin substructure model

Bacteria, yeasts, and molds which grew in a medium containing a synthetic lignin — a dehydrogenation polymer (DHP) of coniferyl alcohol — as a sole carbon source, were isolated from soil. One fungus, Fusarium solani M-13-1, was found to degrade the DHP most vigorously among the isolated organisms. It was shake-cultured in a medium containing dehydrodiconiferyl alcohol (DHCA) (I), an important lignin model compound, and the following six metabolic products were isolated and identified: 1) Phenylcoumaran--aldehydic (II) and -carboxylic compounds, 2) phenylcoumaran--aldehydic compound (IV), formed by release of a 2-carbon fragment from the phenylcoumaran--carboxylic compound, 3) 5-acetylvanillyl alcohol (V), formed by cleavage of the coumaran ring and reduction of the -aldehyde group, 4) 5-carboxyvanillyl alcohol (VI), formed by subsequent oxidation of the acetyl group, and 5) the -ether of DHCA (VII), considered to be a by-product. A degradation pathway for DHCA was proposed on the basis of these metabolic products.Non-Standard Abbreviations DHP dehydrogenation polymer - DHCA dehydrodiconiferyl alcohol - DDQ dichlorodicyano-p-benzoquinone - DDHQ dichlorodicyano-p-hydroquinone - Ar aromatic - TLC thin layer chromatography - GC-MS gas chromatography-mass spectrometry     

An efficient synthesis of 3′-amino-3′-deoxythymidine derivatives

An efficient method of reduction of 3-azido-3-deoxythymidine and its 5-protected derivatives to 3-aminothymidine derivatives on a palladium catalyst using ammonium formate as a source of hydrogen was suggested.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 147–150.Original Russian Text Copyright © 2005 by Seregin, Chudinov, Yurkevich, Shvets.     

Polymerization of nucleotide analogues I: Reaction of nucleoside 5′-phosphorimidazolides with 2′-amino-2′-deoxyuridine

Summary 2-Amino-2-deoxyuridine reacts efficiently with nucleoside 5-phosphorimidazolides in aqueous solution. The dinucleoside monophosphate analogues were obtained in yields exceeding 80% under conditions in which little reaction occurs with the natural nucleosides.In a similar way, the 5-phosphorimidazolide of 2-amino-2-deoxyuridine undergoes self-condensation in aqueous solution to give a complex mixture of oligomers.The phosphoramidate bond in the dinucleoside monophosphate analogues is stable for several days at room temperature and pH 7. The mechanisms of their hydrolysis under acidic and alkaline conditions are described.Abbreviations A adenosine - C cytidine - G guanosine - U uridine - T thymidine - U_{N 3} 2-azido-2-deoxyuridine - U_{NH 2} 2-amino-2-deoxyuridine - ImpA adenosine 5-phosphorimidazolide - ImpU uridine 5-phosphorimidazolide - ImpU_{N 3} 2-azido-2-deoxyuridine 5-phosphorimidazolide - ImpU_{NH 2} 2-amino-2-deoxyuridine 5-phosphorimidazolide - pA adenosine 5-phosphate - pU uridine 5-phosphate - pU_{N 3} 2-azido-2-deoxyuridine 5-phosphate - pU_{NH 2} 2-amino-2-deoxyuridine 5-phosphate - UpA uridylyl-[35]-adenosine - UpU uridylyl-[35]-uridine - U_NpA adenylyl-[52]-2-amino-2-deoxy-uridine - U_NpU uridylyl-[52]-2-amino-2-deoxyuridine (pU_N)_n n=2,3,4 [25]-linked oligomers of pU_{NH 2} poly(A) polyadenylic acid - Im imidazole - MeIm l-methylimidazole     

In vitro and in vivo studies on the mitochondrial import of CBS1, a translational activator of cytochrome b in yeast

Summary Translation of mitochondrial cytochrome b mRNA in yeast is activated by the product of the nuclear gene CBS1. CBS1 encodes a 27 kDa precursor protein, which is cleaved to a 24 kDa mature protein during the import into isolated mitochondria. The sequences required for mitochondrial import reside in the amino-terminal end of the CBS1 precursor. Deletion of the 76 amino-terminal amino acids renders the protein incompetent for mitochondrial import in vitro and non-functional in vivo. When present on a high copy number plasmid and under the control of a strong yeast promoter, biological function can be restored by this truncated derivative. This observation indicates that the CBS1 protein devoid of mitochondrial targeting sequences can enter mitochondria in vivo, possibly due to a bypass of the mitochondrial import system.     

Red/far-red modulation in vitro of enzyme activity in a membrane fraction from Phaseolus aureus

In a membrane fraction isolated from hypocotyls of Phaseolus aureus Roxb. the activity of a number of enzymes was regulated by red and far-red irradiation in vitro, provided that the tissue received a brief red light treatment before extraction. Other enzymes showed no photoregulation. There were two types of photocontrol, neither of which could be detected in the solute fraction, nor in extracts from completely etiolated material. One (Type I) was a red/far-red reversible regulation of the rate of enzyme activity, depending on the light given (in vivo or in vitro) before the assay was begun. The second (Type II) was a promotion of enzyme activity by red or far-red light given during the assay. The action spectra for type II responses do not coincide with either the phytochrome absorption or difference spectra. However, the effectiveness of red and far-red was correlated with the P_fr/P ratio present at the beginning of the assay, such that far-red was more efficient at high P_fr/P and red at low P_fr/P ratios. All enzymes that were regulated involved ATP. In samples that showed enzyme regulation, small changes in fluorescence yield of tryptophan and the covalent probe Fluram (Roche) accompanied the photoconversion of phytochrome, but no fluorescence changes could be measured after briefly incubating the membrane fraction with ATP. The results indicate that light may affect the interaction of ATP with the membrane fraction.Abbreviations F far-red light - P_r and P_fr phytochrome in the red and far-red absorbing forms - P_tot total phytochrome - R red light - RNP ribonucleoprotein