Archaeal elongation factor 1α from <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> interacts with the eubacterial antibiotic GE2270A

The thiazolyl-peptide antibiotic GE2270A, an inhibitor of the elongation factor Tu from Escherichia coli (EcEF-Tu), was used to study the effects produced in the biochemical properties of the archaeal functional analogue elongation factor 1 from Sulfolobus solfataricus (SsEF-1). GE2270A did not substantially affect the poly(U)-directed-poly(Phe) incorporation catalyzed by SsEF-1 and the formation of the ternary complex SsEF-1·GTP·Phe-tRNA^Phe. On the other hand, the antibiotic was able to increase the GDP/GTP exchange rate of SsEF-1; nevertheless, this improvement was not associated with an increase in the catalytic activity of the enzyme. In fact, GE2270A inhibited both the intrinsic GTPase of SsEF-1 (GTPase^Na) and that stimulated by ribosomes. Interestingly, GTPase^Na of both intact and C-terminal-deleted SsEF-1 resulted in a greater sensitivity to the antibiotic with respect to SsEF-1 lacking both the M- and C-terminal domains. This result suggested that, similar to what is found for EcEF-Tu, the M domain of SsEF-1 is the region of the enzyme most responsible for the interaction with GE2270A. The different behavior observed in the inhibition of protein synthesis with respect to EcEF-Tu can be ascribed to the different adaptive structural changes that have occurred in SsEF-1 during evolution.     

Genetic analysis of brown planthopper resistance in twenty varieties of rice,Oryza saliva L.

Summary The inheritance of resistance to brown planthopper, Nilaparvata lugens (Stol.), of 20 rice cultivars was studied. Single dominant genes that are allelic to Bph 3 condition the resistance in cultivars Ptb 19, Gangala (Acc. 7733), Gangala (Acc. 15207), Horana Mawee, Kuruhondarwala, Mudu Kiriyal and Muthumanikam. Single recessive genes that are allelic to bph 4 govern the resistance in cultivars Gambada Samba, Heenhoranamawee, Hotel Samba, Kahata Samba, Kalukuruwee, Lekam Samba, Senawee, Sulai, Thirissa and Vellai Illankali. The resistance in Ptb 33, Sudu Hondarwala, and Sinna Sivappu is governed by one dominant and one recessive gene which segregate independently of each other. The dominant resistance genes in these cultivars appear allelic to either Bph 1 or Bph 3. Similarly, the recessive genes in these cultivars seem allelic to either bph 2 or bph 4. Further investigations are needed to conclusively determine the allelic relationships of resistance genes in Ptb 33, Sudu Hondarwala and Sinna Sivappu.     

Ongoing ultradian activity rhythms in the common vole,Microtus arvalis,during deprivations of food,water and rest

Summary The timing mechanism underlying ultradian (2–3 h) activity patterns in the common vole, Microtus arvalis, was studied using behavioural deprivation experiments. These were aimed at distinguishing between a homeostatic control mechanism, in which the rhythmic behaviour itself is part of the causal loop, and a clock mechanism, independent of the behaviour.In 175 experiments, deprivation of food during 3 ultradian cycles in (subjective) daytime did not result in significant changes in the ultradian periodicity of attempts to obtain the food, compared with ad lib. access to food and water. A minor, but significant increase in ultradian activity time () occurred in the course of the deprivation, but this was compensated by a shorter ultradian rest (). These results were obtained both in intact animals (n = 24), which showed ultradian and circadian rhythmicity in behaviour, and in animals (n = 21) with electrolytic lesions aimed at the suprachiasmatic nuclei (SCN), which lacked the circadian modulation of behaviour. Simultaneous deprivation of water and food in 8 voles without circadian rhythmicity during 40 experiments also did not lead to any change in the ultradian periodicity of feeding attempts.Rest deprivation was studied in 5 SCN lesioned voles, by forcing running wheel activity to continue following spontaneous running. Thus, the experimental activity bout was artificially lengthened to 2–9 h in 67 experiments. The onset of the subsequent rest episodes occurred independent of the duration of the preceding . The duration of was dependent on the preceding, experimental in a periodic fashion. The interval experimental (=lengthened +following ) was equal to one, two or three times the control (obtained on nonexperimental days). This result fits the prediction of a clock model and is in conflict with a monotonicincrease of with , as expected in a homeostatic, restorative process.It is concluded that the ultradian timing of activity in the common vole can be explained neither by homeostatic hunger or thirst mechanisms nor by homeostatic rest/activity regulation. The results strongly suggest an independent clock system generating ultradian feeding rhythms in the common vole.Abbreviations DD continuous darkness - LD light-dark regime - LL continuous light - RCA retrochiasmatic area - ARC arcuate nucleus - SCN suprachiasmatic nuclei - ultradian period - ultradian activity time - ultradian rest time     

Laboratory culture studies of Trichodesmium isolated from the Great Barrier Reef Lagoon, Australia

Cultures of Trichodesmium from the Northern and Southern Great Barrier Reef Lagoon (GBRL) have been established in enriched seawater and artificial seawater media. Some cultures have been maintained with active growth for over 6years. Actively growing cultures in an artificial seawater medium containing organic phosphorus (glycerophosphate) as the principal source of phosphorus have also been established. Key factors that contributed to the successful establishment of cultures were firstly, the seed samples were collected from depth, secondly, samples were thoroughly washed and thirdly, incubations were conducted under relatively low light intensities (PAR 40–50molquantam^–2s^–1). N₂ fixation rates of the cultured Trichodesmium were found to be similar to those measured in the GBRL. Specific growth rates of the cultures during the exponential growth phase in all enriched media were in the range 0.2–0.3day^–1 and growth during this phase was characterised by individual trichomes (filaments) or small aggregations of two to three trichomes. Characteristic bundle formation tended to occur following the exponential growth phase, which suggests that the bundle formation was induced by a lack of a necessary nutrient e.g. Fe. Results from some exploratory studies showed that filament-dominated cultures of Trichodesmium grew over a range of relatively low irradiances (PAR 5–120molquantam^–2s^–1) with the maximum growth occurring at 40–50molquantam^–2s^–1. These results suggest that filaments of the tested strain are well adapted for growth at depth in marine waters. Other studies showed that growth yields were dependent on salinity, with maximum growth occurring between 30 and 37psu. Also the cell yields decreased by an order of magnitude with the reduction of Fe additions from 450 to 45nM. No active growth was observed with the 4.5nM Fe addition.     

Susceptibility of clinical isolates of yeasts to anti-fungal agents

The antimicrobial activity of amphotericin B, 5-fluorocytosine, nystatin, clotrimazole and miconazole were compared in vitro against 244 strains of yeasts that had been isolated from clinical specimens. The yeasts used in this study included 20 species of Candida, Cryptococcus, Saccharomyces Geotrichum, Rhodotorula, Torulopsis and Trichosporon. The majority of the strains (78%) had an MIC of 0.5 g/ml for amphotericin B, 81% an MIC of 1 g/ml for 5-fluorocytosine, 99% 8 g/ml for nystatin, 91%, 8.0 g/ml for clotrimazole and 98% had an MIC of 4.0 for miconazole. Of the anti-fungal agents tested, 5-fluorocytosine and nystatin were found to have the greatest antifungal activity.     

Expression of α and β tropomyosin subunits during early myogenesis in somites and limb buds of chick embryos

Summary The appearance of and subunits of skeletal tropomyosin in early myogenesis was studied histochemically using monoclonal antibody to tropomyosin and affinity-purified polyclonal antibody to tropomyosin. In muscle cells, in both somites and limb buds, the and subunits are simultaneously expressed and first appear in the somites at the 30–36 somites. The relatively greater amount of than tropomyosin found in early myogenesis is thus likely to result from a higher rate of tropomyosin synthesis.     

Rapid micropropagation of <Emphasis Type="Italic">Curcuma longa</Emphasis> using bud explants pre-cultured in thidiazuron-supplemented liquid medium

Multiple shoots of Curcuma longa were induced by culture of bud explants for 1week in Murashige and Skoog (MS) liquid medium supplemented with 72.64M thidiazuron (TDZ) prior to culture on MS gelled medium without growth regulator for 8weeks. The regeneration rate was up to 11.4±1.7shoots/explant. Rooting was spontaneous and the regenerated plants were successfully transferred to soil. This protocol can be an alternative for rapid micropropagation of C. longa used for phytomedicine raw material production.     

Immunological comparison of enzymes of the β-ketoadipate pathway

-Carboxy-cis,cis-muconate lactonizing enzyme and -carboxymuconolactone decarboxylase catalyze sequential reactions in the -ketoadipate pathway, the subunit sizes of the enzymes from Pseudomonas putida, biotype A, are 40000 and 13000, respectively. The cross reaction of antisera prepared against the enzymes was tested with the isofunctional enzymes formed by representatives of other bacterial species. Despite the differences in the subunit sizes of the enzymes, the antisera revealed the same general pattern: cross reaction was observed with the corresponding enzymes formed by other strains in the fluorescent Pseudomonas RNA homology group I and generally was not observed with enzymes from other Pseudomonas species or from other bacterial genera. Exceptions were provided by representatives of Pseudomonas cepacia. Members of this species are classified outside the fluorescent Pseudomonas RNA homology group. Nevertheless, the -carboxymuconolactone decarboxylases from these organisms formed precipitin bands with antisera prepared against the corresponding enzyme from P. putida, biotype A; the lactonizing enzymes from the two species did not appear to cross react. Immunodiffusion experiments with -carboxymuconolactone decarboxylase indicated that a common set of antigenic determinants for the enzyme is conserved among strains that have been classified together by other criteria; the relative immunological distances of the decarboxylases of each taxon from the reference P. putida, biotype A, enzyme were indicated by spurring patterns on Ouchterlony plates. These results suggested that the interspecific transfer of the structural gene for the enzyme is not a common event in Pseudomonas.Non-Standard Abbreviations CMLE -carboxy-cis,cis-muconate lactonizing enzyme (EC 5.5.1.2) - CMD -carboxymuconolactone decarboxylase (EC 4.1.1.44) - MLE cis,cis-muconate lactonizing enzyme (EC 5.5.1.1) - MI muconolactone isomerase (EC 5.3.3.4) Dedicated with affection and admiration to Professor R. Y. Stanier on his 60th birthday     

Distance-constraint approach to protein folding. I. Statistical analysis of protein conformations in terms of distances between residues

A statistical analysis of protein conformations in terms of the distance between residues, represented by their C atoms, is presented. We consider four factors that contribute to the determination of the distanced _i,i+k between a given pair ofith and(i+k)th residues in the native conformation of a globular protein: (1) the distancek along the chain, (2) the size of the protein, (3) the conformational states of theith to(i+k)th residues, and (4) the amino acid types of the and(i+k)th residues. In order to account for the dependence on the distancek along the chain, the statistics are taken for three ranges, viz., short, medium, and long ranges (k8; 9k20; andk21; respectively). In the statistics of short-range distances, a mean distanceD _k and its standard deviationS _k are calculated for each value ofk, with and without taking into account the conformational states of all residues fromi toi+k (factors 1 and 3). As an Appendix, the relations for converting from the distances between residues into other conformational parameters are discussed. In the statistics of long-range distances, a reduced distanced* _ij (the actual distance divided by the radius of gyration) is used to scale the data so that they become independent of protein size, and then a mean reduced distanceD _l (a, a) and its standard deviation _l (a, a) are calculated for each amino acid pair (a, a) (factors 2 and 4). The effect of the neighboring residues along the chain on the value of the distanced* _ij is explored by a linear regression analysis between the actual reduced distanced* _ij and the mean value over theD _l for all possible pairs of residues in the two segments of the (i–2)th to the (i+2)th and the (j–2)th to the (j+2)th residues. The effect is assessed in terms of the tangentA _l (a, a) of the calculated regression line for each amino acid pair (a, a). In the statistics of medium-range distances, only factors 1 and 4 are considered, to simplify the analysis. The scaled distanced _i,i+k =(d _i,i+k -D _k )/S _k is used to eliminate the dependence onk, the distance along the chain. The propertiesD _m (a, a), _m (a, a) andA _m (a, a) corresponding toD _l (a, a), _l (a, a), andA _l (a, a), and also calculated for each amino acid pair (a, a). The results are interpreted as follows: the smaller values ofD _l (a, a) andD _m (a, a) indicate a preference of the pair (a, a) for a contact (e.g., pairs between hydrophobic amino acids, and pairs of Cys with aromatic amino acids), and the larger values of these quantities indicate a preference for distant mutual location (e.g., pairs between strong hydrophilic amino acids); the smaller values of _l (a, a) and _m (a, a) indicate a strong preference for either contact or noncontact (e.g., pairs between hydrophobic amino acids, and pairs between strong hydrophobic and hydrophilic amino acids, respectively), and the larger values of these quantities indicate the ambivalent/neutral nature of the preference for contact and noncontact (e.g., pairs containing Ser or Thr); the smaller values ofA _l (a, a) andA _m (a, a) indicate that the distance of an (a, a) pair is determined independently of the amino acid character of the neighboring residues along the chain (e.g., some pairs of Cys or Met with other amino acids) and the larger values of these quantities indicare that such amino acid character contributes strongly to the determination of the distance (e.g., pairs containing Ser or Thr, and pairs between amino acids with small side chains). The difference between the statistics for the long- and medium-range distances is also discussed; the former reflect the difference between the hydrophobic and hydrophilic character of the residues, but the latter cannot be easily interpretable only in terms of hydrophobicity and hydrophilicity. The data analyzed here are used in the optimization of an object function to compute protein conformation in a subsequent paper.     

Expression of a novel yeast gene that detoxifies the proline analog azetidine-2-carboxylate confers resistance during tobacco seed germination,callus and shoot formation

A novel acetyltransferase (Mpr1) found in Saccharomyces cerevisiae (strain 1278b) has been shown to specifically detoxify a proline analog, l-azetidine-2-carboxylic acid (A2C) in yeast cells [M. Shichiri et al. (2001) J Biol Chem 276: 41998–42002]. We investigated whether the yeast MPR1 gene would function similarly in a plant system and if its expression could confer resistance to proline analogs. The MPR1 gene coding sequence driven by two different constitutive promoters, with or without the 5- and 3-noncoding sequence from the MPR1 gene adjacent to the conventional NOS terminator, was transformed into tobacco (Nicotiana tabacum L. cv. Xanthi) plants via Agrobacterium tumefaciens infection. The presence of the yeast 5- and 3-noncoding sequences appeared to increase the likelihood of MPR1 gene expression in the transgenic plants. The kanamycin-selected transgenic plants with a high level of Mpr1 activity grew normally, and their progeny expressed acetyltransferase activity that could utilize A2C, azetidine-3-carboxylic acid and 4-hydroxy-l-proline as substrates. Resistance to A2C, but not to the other two analogs, was exhibited during leaf tissue culture and seed germination. The A2C toxicity to the wild-type plants was reversed by the addition of proline, suggesting that A2C acts as a proline analog. Our studies confirm that MPR1 can function in a similar fashion in tobacco as in yeast to detoxify the toxic proline analog A2C, so it could potentially be used as a new selectable marker for plant transformation. However, our attempts to utilize MPR1 as an efficient selectable marker gene for the A. tumefaciens-mediated transformation of tobacco were unsuccessful.Abbreviations A2C: l-Azetidine-2-carboxylic acid - A3C: Azetidine-3-carboxylic acid - Hyp: 4-Hydroxy-l-proline - hpt: Hygromycin phosphotransferase II - NPTII: Neomycin phosphotransferase II Communicated by H. Wang     

Utilization of 5-fluorouracil byMyobacterium avium

Myobacterium avium LM1 was exposed to concentrations of 5-fluorouracil (5FU) that ranged from 0 to 100 g/ml. Growth inhibition was inversely proportional to the concentration of the drug. DNA was extracted from cells grown in medium that contained [¹⁴C]5FU, but no carrier. The [¹⁴C]DNA was enzymatically hydrolyzed to deoxyribonucleotides, which were separated and fractionated by high-performance liquid chromatography (HPLC). The isotope was located in 2-deoxycytidine 5-monophosphate (dCMP) and 2-deoxythymidine 5-monophosphate (dTMP), with dCMP containing the majority. There was no radioactivity at the elution times for 5-fluoro-2-deoxyuridine 5-monophosphate or 2-deoxyuridine 5-monophosphate. These results suggested that 5FU was dehalogenated and the uracil moiety ultimately converted into cytosine and thymine deoxyribonucleotides. Cells were grown in [³H]uracil, and [³H]DNA was extracted and analyzed by HPLC. The isotope was found only in the pyrimidine deoxyribonucleotides, with dCMP containing 4.1 times that in dTMP. Thus, it was demonstrated that uracil and dehalogenated 5FU were not directly incorporated into DNA, but rather converted to cytosine and thymine and then incorporated into DNA by a salvage pathway.     

Specific adhesion of primary hepatocytes to a novel glucose-carrying polymer

A new amphiphilic glycopolymer, poly-[N-p-vinylbenzyl-d-glucuronamide] (PV6Gna), was synthesized and characterized. Glucose moieties in the polymer were confirmed to be exposed into outer surface of polystyrene (PS) by direct lectin-enzyme assay. Hepatocytes were specifically interacted with PV6Gna substituted at C-6 of glucose but not poly-[N-p-vinylbenzyl-O--d-glucopyranosyl-[14]-d-gluconamide] (PVMA) and poly-[3-O-p-vinylbenzyl-d-glucose (PVG) substituted at C-1 and C-3 of glucose, respectively, although the glycopolymers were interacted with Con A as lectin for -d-glucose and -d-mannose. The adhesion of hepatocytes was dependent on Ca²⁺ and independent on temperature for the PV6Gna surface unlike integrin-dependent adhesion. Morphologies of hepatocytes on the PV6Gna surface were significantly different from ones on collagen type-I and affected by the coating concentration of PV6Gna onto PS dish and epidermal growth factor (EGF).     

Monoclonal antibody specific for α2→8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing 28-linked polysialic acid (8Neu5Gc2) _n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (8KDN2) _n 6(KDN23Gal13GlNAc13) GalNAc1 residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues. The mAb.kdn8kdn was shown specifically to recognize the 28-linked oligo/polyKDN sequences, (8KDN2) _n , and to be able to distinguish specifically (8KDN2) _n chains from (8Neu5Ac2) _n and (8Neu5Gc2) _n chains. The antibody was used successfully for the immunohistochemical detection of reactive KDN epitopes in sections of paraffin embedded rat pancreas. Several controls verified the specificity of the immunohistochemical staining, thus providing the first demonstration of (8KDN2) _n sequences in a mammalian tissue. The mAb.kdn8kdn can now be used to search further for glycoconjugates containing (8KDN2) _n chains and will facilitate studies on their biosynthesis, intracellular localization and function.     

Geometry of the dry-state oligomerization of 2′,3′-cyclic phosphates

Summary Evaporation of a solution of thymidine plus either theexo or theendo diastereomer of uridine cyclic 2,3-O, O-phosphorothioate (U > p(S) in 1,2-diaminoethane hydrochloride buffer gave the 2,5 and 3,5 isomers of (P-thio) uridylylthymidine (Up(S)dT) in a ratio of 1:2 with a combined yield of about 20%. These isomers were re-converted to U > p(S) and dT by a reaction that is known to proceed by an in-line mechanism. Both the 2,5 and 3,5 isomers gave as product the same diastereomer of U > p(S) that had been used originally in their formation. These dry-state prebiotic reactions (Verlander, Lohrmann, and Orgel 1973) are thus shown to be stereospecific, and both the 2,5 and 3,5 internucleotide bonds are formed by an in-line mechanism.Abbreviations DAE 1,2-diaminoethane - HPLC high pressure liquid chromatography - RNase bovine pancreatic ribonuclease A, EC 3.1.4.22 - TEAB triethylammonium bicarbonate - tris tris(hydroxymethyl)aminomethane - UMP(S) uridine monophosphorothioate - U > p uridine cyclic 2,3-phosphate - U > p(S) uridine cyclic 2,3-O, O-phosphorothioate - Up(S)dT (P-thio)uridylylthymidine - U2p(R_p-S)5dT (P-thio)uridylylthymidine with theR configuration at phosphorous, and a 2,5 internucleotide linkage     

The effect of the ribosomal protein S1 from Escherichia coli on the synthesis in vitro of bacterial-, DNA phage- and RNA phage proteins.

Summary Using an in vitro preparation for protein synthesis, we have studied the effect of the ribosomal protein S1 fromEscherichia coli on the synthesis of the coat protein of the RNA-containing phages Q and MS2, on that of an early and a ate enzyme encoded by the DNA containing phage T7, and on that of anthranilate synthetase, an enzyme encoded by the bacterial tryptophan operon. Our results indicated that for the synthesis of these five proteins the presence of S1 is required. From these results we conclude that S1 is an essential protein for the translation of bacterial and bacteriophage messenger RNA.     

The effect of a taurine-containing drink on performance in 10 endurance-athletes

Summary To determine the effect of a taurine-enriched drink Red Bull on performance, 10 endurance-athletes performed three trials. After 60 min. cycling at approximately 70% VO₂ max, the subjects pedalled to exhaustion on a cycle ergometer. During each exercise, the subjects received 500 ml of a test-drink after 30 min. submaximal cycling: Red Bull without taurine, without glucuronolacton (U1), Red Bull without taurine, without glucuronolacton, without caffeine (U2) and Red Bull original drink containing taurine, glucuronolacton and caffeine (U3).The heart rate level was significantly lower in U3 (p = 0,0031) 15 min. after application. The plasma catecholamines increased slightly from begin of exercise to 15 min. after application of the drinks in all trials but remained on a significantly lower level in U3 (epinephrine (p = 0,0011) and norepinephrine (p = 0,0003). Endurance time was significantly longer with Red Bull original in U3 (p = 0,015). The results of this study show a positive effect of a taurine-containing drink on hormonal responses which leads to a higher performance.     

Assimilation of γ-butyrobetaine,and d-and l-carnitine by resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida

The metabolic pattern of utilization of [1,2,3,4-¹⁴C, methyl-³H] -butyrobetaine and d-and l-[1-¹⁴C, methyl-³H]carnitine has been examined with variously grown resting cell suspensions of Acinetobacter calcoaceticus and Pseudomonas putida. Ps. putida grown on d, l-carnitine as the sole source of carbon, degraded only l-carnitine with stoichiometric accumulation of glycinebetaine. Alternatively, when grown on -butyrobetaine, Ps. putida rapidly metabolized -butyrobetaine, and to a lesser but significant extent, both d-and l-carnitine with equivalent formation of trimethylamine and degradation of the betaine carbon skeleton. Ac. calcoaceticus grown on either d,l-carnitine or -butyrobetaine, effectively utilized all three betaines at nearly the same rates. Disappearance of each of these quarternary ammonium compounds was accompanied by stoichiometric formation of trimethylamine and degradation of the carbon backbone. Utilization of the betaines and corresponding formation of trimethylamine by resting cell suspensions of appropriately grown Ac. calcoaceticus and Ps. putida, was essentially abolished under conditions of anaerobiosis and severely impaired in the presence of sodium cyanide, sodium azide, 2,4-dinitrophenol or 2,2-bipyridine. The results of the present investigations with resting cell suspensions of both Ac. calcoaceticus and Ps. putida do not support an earlier suggestion that -butyrobetaine degradation in these organisms proceeds by its prior hydroxylation to l-carnitine. Indeed, disrupted cell-free preparations of Ac. calcoaceticus and Ps. putida grown on either d,l-carnitine or -butyrobetaine showed no detectable -butyrobetaine hydroxylase activity.     

Identification of molecular markers linked to the mildew resistance gene <Emphasis Type="Italic">Pl-d</Emphasis> in apple

Powdery mildew poses a serious problem for apple growers, and resistance to the disease is a major objective in breeding programmes for cultivar improvement. As selective pressure allows pathogens to overcome previously reliable resistances, there is a need for the introduction of novel resistance genes into new breeding lines. This investigation is concerned with the identification of the first set of molecular markers linked to the gene for mildew resistance, Pl-d, from the accession D12. As no prior information on the map position or markers for Pl-d were available, a bulked-segregant approach was used to test 49 microsatellite primers, 176 amplified fragment length polymorphism (AFLP) primers and 80 random amplified polymorphic DNA (RAPD) primers in a progeny segregating for Pl-d resistance, Fiesta (susceptible) × A871-14 (Worcester Pearmain × D12). The segregations of the markers identified in the resistant and susceptible bulks were scored in the progeny, then the recombination fractions between Pl-d and the most tightly linked markers were calculated and a map prepared. Three AFLP, one RAPD and two microsatellite markers were identified. One AFLP was developed into a sequence-characterised amplified region marker, while the microsatellites CH03C02 and CH01D03 were flanking markers, 7 and 11 recombination units, respectively, from Pl-d. Two more distant microsatellites on the same linkage group, CH01D09 and CH01G12, confirmed the orientation of the markers on the linkage group. These microsatellites place Pl-d on the bottom of linkage group 12 in published apple maps, a region where a number of other disease resistance genes have been identified.     

Book reviews

Consider the perturbed harmonic oscillator Ty=-y+x²y+q(x)y in L²(), where the real potential q belongs to the Hilbert space H={q, xq L²()}. The spectrum of T is an increasing sequence of simple eigenvalues _n(q)=1+2n+_n, n 0, such that _n 0 as n. Let _n(x,q) be the corresponding eigenfunctions. Define the norming constants _n(q)=lim_xlog |_n (x,q)/_n (-x,q)|. We show that for some real Hilbert space and some subspace Furthermore, the mapping :q(q)=({_n(q)}₀, {_n(q)}₀) is a real analytic isomorphism between H and is the set of all strictly increasing sequences s={s_n}₀ such that The proof is based on nonlinear functional analysis combined with sharp asymptotics of spectral data in the high energy limit for complex potentials. We use ideas from the analysis of the inverse problem for the operator -ypy, p L²(0,1), with Dirichlet boundary conditions on the unit interval. There is no literature about the spaces We obtain their basic properties, using their representation as spaces of analytic functions in the disk.     

Physicochemical and immunological characterization of the type E botulinum neurotoxin binding protein purified fromClostridium botulinum

Type E botulinum neurotoxin is produced byClostridium botulinum along with a neurotoxin binding protein which helps protect the neurotoxin from adversepH, temperature, and proteolytic conditions. The neurotoxin binding protein has been purified as a 118-kDa protein. Secondary structure content of the neurotoxin binding protein as revealed by far-UV circular dichroism spectroscopy was 19% -helix, 50%-sheets, 28% random coils, and 3%-turns. This compared to 22% -helix, 44%-sheets, 34% random coils, and no-turns of the type E botulinum neurotoxin. The complex of the two proteins revealed 25%-helix, 45%-sheets, 27% random coils, and 3%-turns, suggesting a significant alteration at least in the-helical folding of the two proteins upon their interaction. Tyrosine topography is altered considerably (28%) when the neurotoxin and its binding protein are separated, indicating strong interaction between the two proteins. Gel filtration results suggested that type E neurotoxin binding protein clearly complexes with type E neurotoxin. The interaction is favored at lowpH as indicated by an initial binding rate of 8.4 min^–1 atpH 5.7 compared to 4.0 min^–1 atpH 7.5 as determined using a fiber optic-based biosensor. The neurotoxin and its binding protein apparently are of equivalent antigenicity, as both reacted equally on enzyme-linked immunosorbent assay to polyclonal antibodies raised against the toxoid of their complex.