Elicitin‐like proteins Oli‐D1 and Oli‐D2 from Pythium oligandrum trigger hypersensitive response in Nicotiana benthamiana and induce resistance against Botrytis cinerea in tomato

The biocontrol agent Pythium oligandrum and its elicitin‐like proteins oligandrins have been shown to induce disease resistance in a range of plants. In the present study, the ability of two oligandrins, Oli‐D1 and Oli‐D2, to induce an immune response and the possible molecular mechanism regulating the defence responses in Nicotiana benthamiana and tomato were investigated. Infiltration of recombinant Oli‐D1 and Oli‐D2 proteins induced a typical immune response in N. benthamiana including the induction of a hypersensitive response (HR), accumulation of reactive oxygen species and production of autofluorescence. Agrobacterium‐mediated transient expression assays revealed that full‐length Oli‐D1 and Oli‐D2 were required for full HR‐inducing activity in N. benthamiana, and virus‐induced gene silencing‐mediated knockdown of some of the signalling regulatory genes demonstrated that NbSGT1 and NbNPR1 were required for Oli‐D1 and Oli‐D2 to induce HR in N. benthamiana. Subcellular localization analyses indicated that both Oli‐D1 and Oli‐D2 were targeted to the plasma membrane of N. benthamiana. When infiltrated or transiently expressed in leaves, Oli‐D1 and Oli‐D2 induced resistance against Botrytis cinerea in tomato and activated the expression of a set of genes involved in the jasmonic acid/ethylene (JA/ET)‐mediated signalling pathway. Our results demonstrate that Oli‐D1 and Oli‐D2 are effective elicitors capable of inducing immune responses in plants, probably through the JA/ET‐mediated signalling pathway, and that both Oli‐D1 and Oli‐D2 have potential for the development of bioactive formulae for crop disease control in practice.     

Identification and cloning of nodulation genes from the stem-nodulating bacterium ORS571

Summary After random Tn5 mutagenesis of the stem-nodulating Sesbania rostrata symbiont strain ORS571, Nif^-, Fix^- and Nod^- mutants were isolated. The Nif^- mutants had lost both free-living and symbiotic N₂ fixation capacity. The Fix^- mutants normally fixed N₂ in the free-living state but induced ineffective nodules on S. rostrata. They were defective in functions exclusively required for symbiotic N₂ fixation. A further analysis of the Nod^- mutants allowed the identification of two nod loci. A Tn5 insertion in nod locus 1 completely abolished both root and stem nodulation capacity. Root hair curling, which is an initial event in S. rostrata root nodulation, was no longer observed. A 400 bp region showing weak homology to the nodC gene of Rhizobium meliloti was located 1.5 kb away from this nod Tn5 insertion. A Tn5 insertion in nod locus 2 caused the loss of stem and root nodulation capacity but root hair curling still occurred. The physical maps of a 20.5 kb DNA region of nod locus 1 and of a 40 kb DNA region of nod locus 2 showed no overlaps. The two nod loci are not closely linked to nif locus 1, containing the structural genes for the nitrogenase complex (Elmerich et al. 1982).     

Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti

Summary R. meliloti strain 41 (Rm41) was shown to harbour two indigenous plasmids with molecular weights of 140 Mdal (pRmc41a) and more than 300 Mdal (pRme41b), respectively. Using a heat-treatment procedure, derivatives of Rm41 defective in nodulation (Nod^-) or nitrogen fixation (Fix^-) have been readily obtained. In some Nod^- mutants the deletion of a segment of plasmid pRme41b was found.Based on the demonstrated homology between the nitrogen fixation (nif) genes of Klebsiella pneumoniae and of R. meliloti the Rhizobium nif region has been cloned into the cosmid vector pHC79, then recloned into pBR322 and the restriction map of the nif region has been determined. ³²P-labelled nick-translated probe prepared from the cloned nif DNA fragment hybridized to pRme41b of Rm41 but for most Nod^- mutants this hybridization was not detected. Hybridization of a cosmid containing Rm41 DNA to total DNA digests from the wild-type bacterium and from a series of Nod^- mutants revealed that at least a 24 kb DNA fragment including the nif structural genes was missing from most of the Nod^- mutants. These results, together with the genetic analyses of these symbiotic mutations suggest that some nod and fix genes are located on pRme41b.     

Genetic analysis of the non-H-2-linked Ir genes controlling the cytotoxic T-cell response to H-Y in H-2 d mice

The T-cell mediated immune responses to the male specific minor histocompatibility antigen H-Y in mice have been studied extensively as a model for immune responses to other weak antigens like tumor antigens or autoantigens. In a recent analysis of the strain distribution of the cytotoxic T-cell (Tc-cell) responsiveness to H-Y, it has been found that genes both within and outside the H-2 complex exert an interactive control. Whereas the H-2 ^b strains all are high responders, independent of their non-H-2 background, other H-2 haplotypes (d, k, and s) only allow for a response if they are combined with certain non-H-2 genes. The H-2-linked immune response genes (Ir-genes) have been previously mapped to the I and K or D region of the H-2 complex, but the mapping of the non-H-2 genes has not yet been established. In this study evidence is presented, using recombinant inbred strains and immunoglobulin heavy chain (Igh) congenic strains of mice, to show that there is more than one non-H-2 Ir-gene involved, that the main controlling genes are not linked to the Igh complex, and that at least one non-H-2 Ir-gene is linked to the H-3 region on chromosome 2. This region includes genes for beta-2-microglobulin (2m), the Ly-mllalloantigen a polymorphic cell surface glycoprotein (Pgp-1), a B-cell specific antigen Ly-4, a transplantation antigen H-3, and genes (Ir-2) controlling the immune response to Ea-1 and H-13.     

Isolation and characterization of Tn5-induced NADPH-glutamate synthase (GOGAT-) mutants of Azorhizobium sesbaniae ORS571 and cloning of the corresponding glt locus

Summary Random Tn5 mutagenesis was used to isolate two independent Azorhizobium sesbaniae ORS571 mutants disturbed in ammonium assimilation (Asm^-). Both Asm^- mutant strains were shown to lack NADPH-glutamate synthase (NADPH-GOGAT) activity and to carry Tn5 insertions ca. 1.5 kb apart in the ORS571 chromosome. The Tn5-containing region of one of the GOGAT^- mutant strains was cloned in pACYC184 and used to identify the wild-type glt (GOGAT) locus in a phage clone bank of ORS571. The cloned region was shown to have DNA homology with the Escherichia coli glt locus and to complement the Asm^- phenotype of E. coli and ORS571 GOGAT^- strains. The ORS571 GOGAT^- mutations were found to interfere with free-living as well as symbiotic nitrogen fixation. Expression of ORS571 NADPH-GOGAT activity was shown to be independent of the nitrogen regulation (ntr) system.     

Radioactive contamination of the marine environment: Uptake and distribution of3H inDunaliella bioculata

The marine flagellateDunaliella bioculata, which is easily cultivated under laboratory conditions, is a suitable organism for assessing the importance of the radioactive contamination by³H bound to organic molecules. We have studied the uptake of the following tritiated precursors: thymidine-methyl-³H, adenine-2-³H, uridine-5-³H, l-leucine-4-³H, glycine-2-³H, l-arginine-3.4-³H, 1-aspartic acid-2. 3-³H, 1-phenylalanine-2.3-³H, D-glucose-2-³H and D-glucose-6-³H. Under the experimental conditions (2000 lux; incubation time 30 min), all tritiated molecules are taken up byD. bioculata. Their intracellular concentration may reach that of the external medium. However, leucine and adenine accumulate in the algae: their respective concentrations are 10 and 100 times higher than in the culture medium. The molecular distribution of³H has been studied by various biochemical techniques and by sieve chromatography on sepharose 4B. It has been found that more l-leucine-4-³H is incorporated into acid and acetone soluble substances than into proteins. Adenine-2-³H is mainly incorporated into macromolecules of biological significance (RNA, DNA). CsCl gradient centrifugation has shown that the total DNA ofDunaliella is constituted by a major (ϖ=1.707 g/cm³) and by a minor (ϖ=1.693 g/cm³) component. Fellow of the Commission of the European Communities     

Examination by the molecular orbital method of the intrinsic conformation preferences of nucleic acid sequence]

This paper reports a systematic PCILO study of the conformation of the nucleic acid backbone. The authors principally studied the ω′ and ω phosphodiester torsion angles of the disugar triphosphate model as a simultaneous function of (1) the sugar nature, ribose or deoxyribose, (2) the different combinations of the sugar ring puckers C(2′)-endo-C(2′)-endo, C(3′)-endo-C(3′)-endo, C(3′)-endo-C(2′)-endo, and C(2′)-endo-C(3′)-endo, and (3) the different conformations around the ψ(C4′–C5′) exocyclic bond. The dependence of the (ω′,ω) conformational energy maps upon these different factors, is discussed. The results are in very good agreement with the observed structures of ribonucleic (RNA10, RNA11, A′-RNA12, tRNA^Phe) and deoxyribonucleic acids (D-DNA, C-DNA 9.3, B-DNA 10, A-DNA 11). Thus the validity of this model, the disugar triphosphate unit, is ensured. The main conclusions that can be drawn from this systematic study are the following:

• 1 The torsion around P-05′ (angle ω) is, as a general rule, more flexible than the torsion around P-03′ (angle ω′).
• 2 There is no notable difference between the ribose–triphosphate units and the deoxyribose–triphosphate units for the C(3′)-endo–C(3′)-endo and C(3′)-endo–C(2′)-endo sugar puckers.
• 3 The deoxyribose–triphosphate units with C(2′)-endo–C(2′)-endo and C(2′)-endo–C(3′)-endo sugar puckers show much more ω′ flexibility than the ribose–triphosphate units with the same sugar puckers and cis position for the 2′hydroxyl group.
• 4 The preferred values of ω′ are independent of the sugar nature (ribose or deoxyribose) and of ψ values; they are correlated with the sugar pucker of the first sugar-phosphate unit:
  • C(3′)-endo-C(3′)-endo and C(3′)-endo-C(2′)-endo puckers ? ω′ ? 240° (g^? region)
  • C(2′)-endo-C(2′)-endo and C(2′)-endo-C(3′)-endo puckers ? ω′ 180° (t region)
• 5 The preferred values of ω are independent of the nature and the puckering of the sugars; they are correlated with the rotational state of the torsion angle ψ(C4′–C5′): ψ ? 60° (gg) ? ω ? 300° (g^?), ψ ? 180° (gt) or 300° (tg) ? ω ? 60° (g⁺)

     

Triterpene saponins from Salsola imbricata

Continuing our investigations on medicinal plants of the Egyptian desert, two new triterpene glycoside derivatives, along with three known compounds have been isolated from the roots of Salsola imbricata, a shrub widely growing in Egypt. Their structures have been established as 3-O-β-d-xylopyranosyl-(1 → 2)-O-β-d-glucuronopyranosyl-akebonic acid 28-O-β-d-glucopyranoside and 3-O-β-d-xylopyranosyl-(1 → 2)-O-β-d-glucuronopyranosyl-29-hydroxyoleanolic acid 28-O-β-d-glucopyranoside on the basis of spectroscopic methods including 1D- (¹H, ¹³C) and 2D-NMR (DQF-COSY, HSQC, HMBC) experiments as well as mass spectrometry analysis.     

The cloning and transposon Tn5 mutagenesis of the glnA region of Klebsiella pneumoniae: identification of glnR, a gene involved in the regulation of the nif and hut operons

Summary A 15 kilobase HindIII fragment of Klebsiella pneumoniae DNA containing the glnA gene was cloned into the plasmid vector pACYC184. The resulting plasmid, pFB51, complements glnA ^- mutations in Escherichia coli and K. pneumoniae. pFB51 also complements the GlnR phenotype of a Klebsiella pneumoniae gln regulatory mutant (KP5060) defined by the restoration of Hut⁺ and Nif⁺ (histidine utilization and nitrogen fixation) phenotypes to this strain. Three recombinant plasmids containing subsegments of the 15 kb HindIII fragment were derived from pFB51. Plasmid pFB514 which contains a spontaneous 4 kb delection of K. pneumoniae DNA from pFB51 is more stable than pFB51 and is still able to complement glnA ^- mutations and the GlnR^- phenotype of KP5060. Plasmids pFB53 and pFB54, which contain a 6.5 kb SalI DNA fragment from pFB51 recloned into pACYC184 in opposite orientations, complement glnA ^- mutations but not the GlnR^- phenotype of KP5060. Plasmids pFB514 and pFB53 were mutagenized by transposon Tn5 resulting in a total of 92 Tn5 insertions in the cloned fragments. Utilizing these insertion mutations, a correlated physical and genetic map was constructed by determining the physical location of each Tn5 insertion and by analyzing the ability of each Tn5 mutated plasmid to complement a glnA ^- strain of E. coli and a glnA ^- GlnR^- strain of K. pneumoniae. Two classes of Tn5 insertions with an altered Gln phenotpye were obtained. One cluster of insertions spanning a 1.3 kb region abolished complementation of the glnA ^- mutations. A second 2 kb cluster of Tn5 insertions, immediately adjacent to the first cluster, abolished the ability of pFB514 plasmid to complement the GlnR^- phenotype, while glnA ^- complementation was unaffected. We propose that the second cluster of Tn5 insertions define a DNA region coding for a positive regulatory factor for nitrogen fixation (nif) and histidine utilization (hut) genes (glnR).     

Design and Synthesis of LNA-Based Mercaptoacetamido-Linked Nucleoside Dimers

Three LNA-based mercaptoacetamido-linked nonionic nucleoside dimers T^L-S-T, T-S-T^L , and T^L-S-T^L have been synthesized by HOBT and HBTU catalyzed condensation of silyl-protected 2-S-(thymidin-5?′-yl)mercaptoacetic acid or 2-S-(2?′-O,4?′-C-methylenethymidin-5?′-yl)mercaptoacetic acid with 3?′-amino-3?′-deoxy-5?′-O-DMT-2?′-O,4?′-C-methylenethymidine or with 3?′-amino-3?′-deoxy-5?′-O-DMT-β-thymidine followed by desilylation of the protected dimers. The 3?′-O-phosphoramidite derivative of one of the nucleoside dimers was successfully prepared by condensation with [P(-Cl)(-OCH₂CH₂CN)-N(iPr)₂}] in DCM in the presence of N,N-diisopropylethylamine (DIPEA), which is a building block for the preparation of mercaptoacetamido-linked oligonucleotides of therapeutic applications.     

The Constituents of the Essential Oil from Japanese Quince Fruit,Cydonia oblonga Miller

The biosyntheses of sescandelin (1) and sescandelin B (2) were studied by feeding of [¹³C]labelled precursors to Sesquicilium candelabrum. The labelling pattern of those compounds enriched from the [1-¹³C], [2-¹³C], and [l,2-¹³C]acetates, and from the [¹³C]formate showed that both compounds were derived from a pentaketide chain and a C₁ unit. The isocoumarin skeleton of 1 and 2 is considered to have been formed by the cyclization of a pentaketide chain and a C₁ unit, and of a pentaketide, respectively.     

Synthesis of 2′-Deuterio Tubercidin and Adenosine and the Corresponding Arabino Analogs

Abstract

The 2′-deuterio arabino analogs of tubercidin and adenosine have been prepared by Swern oxidation of the 3′,5′-TPDS derivatives of tubercidin and adenosine and reduction with NaBD_4. Subsequent inversion of stereochemistry at C-2′ yielded [2′-²H]tubercidin and [2′-²H]adenosine with 98% deuterium incorporation.     

Two modes of loss of the tol function from Pseudomonas putida mt-2

Summary Some of a set of independently arising Tol^- (non toluate-utilising) derivatives of Pseudomonas putida mt-2 have lost the unique plasmid present in the parent strain. In others this plasmid has suffered a deletion of a specific region of about 27 Md.     

Interaction of super-repressible and dominant constitutive mutations for the synthesis of galactose pathway enzymes in Saccharomyces cerevisiae

Summary Two dominant uninducible mutant alleles in the gal80 locus were identified. The GAL80 ^s-1 and GAL80 ^s-2 mutants showed novel phenotypes in response to the newly isolated GAL81-1 mutant allele, a dominant constitutive mutation linked to the gal4 locus; the GAL80 ^s-1 GAL81-1 strain was inducible and the GAL80 ^s-2 GAL81-1 strain was uninducible. Many galactose positive revertants from the GAL80 ^s-2 GAL81-1 strain were isolated. It was proved that each revertant was due to a secondary mutation either in the gal80 or GAL81 locus, whereas revertants due to mutation at the supposed controlling site for the structural gene cluster of the galactose-pathway enzymes have not been isolated.This study was supported in part by grant no. 048164 to Y. Oshima from the Scientific Research Fund of the Ministry of Eduction, Japan     

New complexities at the Qa-1 locus

Mouse strain and tissue distribution analyses indicate that the new antiserum A anti-A-Tla ^b recognizes the cell-surface product governed by the previously serologically undetectable Qa-I ^b allele. This cell-surface product has therefore been called Qa-1.2. Three levels of anti-Qa-1.2 cytotoxicity in the presence of complement have been observed: high, intermediate, and zero lysis. In general, high levels of lysis correlate with the presence of the Qa-1 b allele, while zero levels of lysis correlate with the presence of the Qa-1 ^aallele. The A.CA strain reacts with both anti-Qa-1.1 and anti-Qa-1.2 and may possess a third allele, Qa-1 ^d. Several strains including B6-H-2 ^k react in an intermediate fashion. Recombinant strain analyses indicate that this intermediate reaction may be due to modifying genes within the H-2D region.     

Mating preference tests with the recombinant congenic strain BALB.HTG

Tests of MHC-associated mating preference were conducted with the congenic mouse strains BALE (H-2 ^d), BALB.B (H-2 ^b), and BALB.HTG (recombinant ofH-2 ^d andH-2)^b. The results conform to a hypothesis that anRi gene (Ri-1), the expression of which influences mating preference in females, is situated to the right of theS region; and that anotherRi gene (Ri-2), the expression of which influences mating preference in males, is situated elsewhere, probably to the left ofH-2D. This hypothesis is consistent with conclusions previously reached from study of the mating preferences of B6 and B6-Tla^a congenic mice.Abbreviations according to Yamazaki et al. (1978) Ri recognition of identity     

The location of the feedback-specific region with the pyrimidine-3 locus of Neurospora crassa.

Summary Thepyrimidine-3 locus ofNeurospora crassa specifies two enzyme activities, pyrimidine-specific carbamyl phosphate synthetase (CPSpyr) and aspartate transcarbamylase (ATC). ATC is translationally distal. CPSpyr, but not ATC, is subject to feedback inhibition by uridine triphosphate (UTP). To investigate the location of the feedback-specific region within the locus, inhibition of a number ofpyr-3 alleles by UTP was investigated. All CPS⁺ ATC^- polar alleles, revertants of CPS^- ATC^- polar alleles, and 5-fluorouracil-resistant mutants had normal UTP response. The location of the feedback-specific region is in or close to the CPS-specific region.Supported by Science Research Council Grant B/RG/2981     

SYNTHETIC STUDIES TO IMPROVE THE DEUTERIUM LABELLING IN NUCLEOSIDES FOR FACILITATING STRUCTURAL STUDIES OF LARGE RNAS BY HIGH-FIELD NMR SPECTROSCOPY

Synthetic studies to prepare ribonucleosides deuterated at C2′ and the application of the developed procedures for the synthesis of ² H ₅ -ribonucleosides from 1,2-O-isopropylidene-3-O-benzyl-ribofuranose-3,4,5,5′- ² H ₄ have been reported.