Transmission of wheat striate virus by the eggs of the vectorCalligypona pellucida Fabr

Персистентная природа вируса стерилйности и карликовостн овса (ВСКО) и вируса полосатости пшеницы (ВПП) изучалась также в опыте с личинками вектораCalligypona pellucida F. происходящими нз яиц, в условиях исключающих возможность их контаминации сосанием на зараженных вирусом растениях. Яйца происходили от самок из популяции, у которой в преодшествующем теете установелено 14,4% неинфекционных особей, 61,2% зараженных ВСКО, 1,9% зараженных ВПП и 22,5% зараженных одновременно ВСКО и ВПП. При индивидуальном несколько месяцев длившемся изучении инфекционности контрольных личинок, которые никогда не имели возможности сосать на зараженных вирусом растениях, ни одна личинка не перенесла ВСКО, несмотря на то, что им их родители в высокой степени (83,7%) заражены. Наоборот, 14,9% насекомых перенесло ВПП, которым их родители заражены в меньшей степени (24,4%). Опытные личинки, следовательно, приобрели ВПП от родительских самок трансовариально и в высокой степени. Это замечательное снойство позволило отличить ВПП от ВСКО, которые в Чехословакии выступают часто вместе. Личинки, которые прошли приобретающим сосанием на овсе вараженном ВПП, проявляли по сравнению с контрольными повышсние инфективиости на 61,6% носле происшествия срока, отвечающего 26–34-хдневной циркуляции вируса в теле нереносчика и на 98,2% после циркуляционного срока в 40–51 дней. В теле вектора, новиюимому, не имеет места интерферренция между ВСКО и ВПП, так как заражение цикады одним вирусом, повидиному, не являестя препятствием се заражению друтим. Перекрестной защиты между ВСКО и ВПП в растениях—хозяевах или не существует или она незначительна, так как растения могут страдать от обенх вирусов одновременно. Признаки одного вируса, однако, могут в разной стопени, перекрываться симптомами вируса другого.     

Purification of Leuconostoc mesenteroides Citrate Lyase and Cloning and Characterization of the citCDEFG Gene Cluster

A citrate lyase (EC 4.1.3.6) was purified 25-fold from Leuconostoc mesenteroides and was shown to contain three subunits. The first 42 amino acids of the β subunit were identified, as well as an internal peptide sequence spanning some 20 amino acids into the α subunit. Using degenerated primers from these sequences, we amplified a 1.2-kb DNA fragment by PCR from Leuconostoc mesenteroides subsp. cremoris. This fragment was used as a probe for screening a Leuconostoc genomic bank to identify the structural genes. The 2.7-kb gene cluster encoding citrate lyase of L. mesenteroides is organized in three open reading frames, citD, citE, and citF, encoding, respectively, the three citrate lyase subunits γ (acyl carrier protein [ACP]), β (citryl-S-ACP lyase; EC 4.1.3.34), and α (citrate:acetyl-ACP transferase; EC 2.8.3.10). The gene (citC) encoding the citrate lyase ligase (EC 6.2.1.22) was localized in the region upstream of citD. Protein comparisons show similarities with the citrate lyase ligase and citrate lyase of Klebsiella pneumoniae and Haemophilus influenzae. Downstream of the citrate lyase cluster, a 1.4-kb open reading frame encoding a 52-kDa protein was found. The deduced protein is similar to CitG of the other bacteria, and its function remains unknown. Expression of the citCDEFG gene cluster in Escherichia coli led to the detection of a citrate lyase activity only in the presence of acetyl coenzyme A, which is a structural analog of the prosthetic group. This shows that the acetyl-ACP group of the citrate lyase form in E. coli is not complete or not linked to the protein.Lactic acid bacteria of the genus Leuconostoc play important roles in the dairy industry because of their ability to produce carbon dioxide and C₄ aroma compounds through lactose heterofermentation and citrate utilization. The carbon dioxide produced is responsible for eye formation in certain types of cheese. Citrate utilization by these bacteria leads to the production of diacetyl, which is considered a main flavor compound of a range of fermented dairy products such as cultured butter, buttermilk, and cottage cheese.The citrate utilization by lactic acid bacteria requires specifically three enzymes involved in the conversion of citrate to pyruvate: a citrate permease, a citrate lyase, and an oxaloacetate decarboxylase. The energetic role of citrate metabolism in Leuconostoc mesenteroides has been recently described (24, 25). The citrate permease catalyzes an electrogenic exchange of divalent anionic citrate and monovalent lactate, resulting in the generation of a membrane potential (Fig. ​(Fig.1,1, reaction 1) (24, 25). The intracellular citrate is cleaved by a citrate lyase (EC 4.1.3.6), yielding acetate and oxaloacetate (Fig. ​(Fig.1,1, reactions 2 and 3). The oxaloacetate is decarboxylated into carbon dioxide and pyruvate in a reaction catalyzed by the enzyme oxaloacetate decarboxylase (Fig. ​(Fig.1,1, reaction 4). Open in a separate windowFIG. 1Citrate fermentation pathway in L. mesenteroides and role of the different subunits in the reaction catalyzed by citrate lyase (EC 4.1.3.6). The proteins involved are citrate permease (1), citrate lyase α subunit citrate:acetyl-ACP transferase (EC 2.8.3.10) (2), citrate lyase β subunit citryl–S-ACP lyase (EC 4.1.3.34) (3) oxaloacetate decarboxylase (4), acetate:SH-CL ligase (EC 6.2.1.22) (5), and lactate dehydrogenase (6). ACP, γ subunit of ACP; R, prosthetic group. Acetic anhydride is used for chemical specific acetylation of the prosthetic group. Acetic anhydride is an analog of the mixed anhydride of citric and acetic acids which corresponds probably to an intermediate analog in the acyl-exchange reaction (7a, 14a).Understanding of the molecular genetics of these lactic acid bacteria is not far advanced, and the genes encoding the enzymes citrate lyase and oxaloacetate decarboxylase are unknown.On the basis of previous studies (22, 33), the citrate lyase of Lactococcus lactis subsp. lactis biovar diacetylactis and Leuconostoc can be considered a functional complex (M_r, 585,000) composed of three proteins: α, β, and γ subunits in a stoichiometric relationship of 6:6:6. The structure and the mechanism of action are similar to those of the citrate lyase of Klebsiella pneumoniae, which has been extensively studied (1, 15, 16, 34, 36). The citrate lyase is active only if the thioester residue of the prosthetic group linked to its acyl carrier protein (ACP) (γ subunit) is acetylated. This activation is catalyzed by an acetate:SH-citrate lyase ligase (CL ligase) (EC 6.2.1.22), which converts HS-ACP with ATP and acetate into the acetyl-S-ACP (Fig. ​(Fig.1,1, reaction 5) (32). The α subunit replaces the acyl group with a citryl group to form the citryl-S-ACP (Fig. ​(Fig.1,1, reaction 2) (16). At last, the β subunit cleaves citryl-S-ACP into oxaloacetate and regenerates the acyl-S-ACP (Fig. ​(Fig.1,1, reaction 3) (16).Different mechanisms of regulation of citrate lyase have been reported, such as configurational changes, reversible covalent modification by acetylation-deacetylation, and phosphorylation-dephosphorylation (1, 2). In microorganisms like Klebsiella, in which the reactions of the tricarboxylic acid cycle are operative and therefore contain citrate synthase, a strict regulation of citrate lyase activity is necessary to avoid a futile cycle between citrate fermentation and the l-glutamate biosynthetic pathway. After citrate depletion from the growth medium or upon transfer from an anaerobic citrate medium to an aerobic glucose medium, the synthesis of l-glutamate from oxaloacetate and acetyl coenzyme A (CoA) via citrate can be ensured only if the citrate fermentation pathway is turned off. The intracellular l-glutamate concentration controls these pathways by modulating the activity of the citrate lyase complex (1, 2).An induction of citrate lyase activity has been observed in Leuconostoc but never in all Lactococcus strains tested (21, 26). In L. mesenteroides, the citrate lyase activity is induced by citrate and rapidly repressed after the citrate consumption in the medium. However, the regulation mechanisms remain unknown. In this paper, we report the purification of L. mesenteroides citrate lyase and an approach based on reverse genetics that yielded the full-length sequence of CL ligase and citrate lyase genes encoding the α, β, and γ subunits. The citrate lyase and CL ligase genes were sequenced and expressed in Escherichia coli.     

Forcing expression of a soybean root glutamine synthetase gene in tobacco leaves induces a native gene encoding cytosolic enzyme

Glutamine synthetase (GS; EC 6.3.1.2) is present in different subcellular compartments in plants. It is located in the cytoplasm in root and root nodules while generally present in the chloroplasts in leaves. The expression of GS gene(s) is enhanced in root nodules and in soybean roots treated with ammonia. We have isolated four genes encoding subunits of cytosolic GS from soybean (Glycine max L. cv. Prize). Promoter analysis of one of these genes (GS15) showed that it is expressed in a root-specific manner in transgenic tobacco and Lotus corniculatus, but is induced by ammonia only in the legume background. Making the GS15 gene expression constitutive by fusion with the CaMV-35S promoter led to the expression of GS in the leaves of transgenic tobacco plants. The soybean GS was functional and was located in the cytoplasm in tobacco leaves where this enzyme is not normally present. Forcing this change in the location of GS caused concomitant induction of the mRNA for a native cytosolic GS in the leaves of transgenic tobacco. Shifting the subcellular location of GS in transgenic plants apparently altered the nitrogen metabolism and forced the induction in leaves of a native GS gene encoding a cytosolic enzyme. The latter is normally expressed only in the root tissue of tobacco. This phenomenon may suggest a hitherto uncharacterized metabolic control on the expression of certain genes in plants.     

Benefits of caffeine ingestion on sprint performance in trained and untrained swimmers

The influence of specific training on benefits from caffeine (Caf) ingestion was examined during a sprint test in a group of highly trained swimmers (T) and compared with the response of a group of untrained occasional swimmers (UT). Seven T and seven UT subjects swam freestyle two randomly assigned 2 x 100 m distances, at maximal speed and separated by 20 min of passive recovery, once after Caf (250 mg) and once after placebo (Pla) ingestion. Anaerobic capacity was assessed by the mean velocity (meters per second) during each 100 m and blood was sampled from the fingertip just before and 1, 3, 5, 7, and 9 min after each 100 m for resting and maximal blood lactate concentration ([la-]b,max) determination. The [la-]bmax was significantly enhanced by Caf in both T and UT subjects (P less than 0.01). However, only T subjects exhibited significant improvement in their swimming velocity (P less than 0.01) after Caf or any significant impairment during the second 100 m. In light of these results, it appears that specific training is necessary to benefit from the metabolic adaptations induced by Caf during supramaximal exercise requiring a high anaerobic capacity.     

Diel periodicity in phytoplankton productivity

Daily variation in phytoplankton productivity influences the dynamics and linkages between several large scale processes in aquatic ecosystems. As part of an opening address to the 5th International workshop for the Group for Aquatic Productivity (GAP), the daily patterns of variability in photosynthesis for different algal classes was introduced and accompanied by a discussion of the sources of environmental and endogenous regulation of repeating biological oscillations that occur in phytoplankton on timescales of one day. It is suggested that one way to develop a database that serves to sort and predict phytoplankton variability over the day may be to encourage the creation of a temporal library. Such a library would be comprised of temporally fixed maps of circadian clock-controlled rhythms for individual species, as well as temporally variable maps of diel periodicities that only can be defined for a selected set of environmental conditions.     

Purification and N-terminal sequencing of style glycoproteins associated with self-incompatibility in Petunia hybrida

We report isolation and N-terminal amino acid sequencing of three style glycoproteins, which segregate with three S (self-incompatibility) alleles of Petunia hybrida. The S-glycoproteins were expressed mainly in the upper part of the pistil and showed an increasing concentration during flower development. The glycoproteins were purified by a combination of ConA-Sepharose and cation exchange fast protein liquid chromatography. The amount of S-glycoproteins recovered from style extracts varied from 0.5 to 1.6 g per style, which was 40–60% of the amount recovered by a simplified analytical method. N-terminal amino acid sequences of S₁-, S₂- and S₃-glycoprotein showed homology within the fifteen amino terminal residues. These amino acid sequences were compared with the previously published sequences of S-glycoproteins from Nicotiana alata and Lycopersicon peruvianum.     

Synthesis and secretion of hirudin by Streptomyces lividans

Summary To examine the secretory production of heterologous proteins by Streptomyces lividans, we fused the DNA encoding the signal peptide of the -amylase inhibitor tendamistat, derived from S. tendae with a synthetic gene encoding the thrombin inhibitor hirudin. The analysis of secretion by immunoblots revealed an efficient translocation of hirudin through the membrane, with no detectable immunoreaction among the cellular proteins. The secreted hirudin was stable in the shaking culture for about 6 days. A comparison of the hirudin secreted by S. lividans and recombinant reference hirudin from yeast by immunoblots and thrombin inhibition assays shows that hirudin from Streptomyces has a lower specific activity, which may be due to a different aminoterminal sequence or to inexact processing of the precursor.Offprint requests to: J. Engels     

Comparative analysis of the individual protoxin components in P1 crystals of Bacillus thuringiensis subsp. kurstaki isolates NRD-12 and HD-1.

Two commercially important strains (NRD-12 and HD-1) of the entomopathogenic bacterium Bacillus thuringiensis subsp. kurstaki each contain three genes of partially identical sequence coding for three classes of 130-135 kDa protoxins (termed the 4.5, 5.3 and 6.6 protoxins) that display toxicity towards various lepidopteran larvae. These gene products combine to form the intracellular bipyramidal P1 crystal. Each of the genes from both strains was cloned and expressed in Escherichia coli. Analysis of the cloned genes at the restriction-endonuclease level revealed no detectable differences among genes within a particular gene class. The composition of the P1 crystal from both strains was quantitatively analysed by CNBr cleavage of the purified P1 crystal, with the purified recombinant gene products as reference proteins. Independent verification of the presence of high 6.6-protoxin gene product in the P1 crystal was provided by a rapid in vitro lawn cell toxicity assay directed against a Choristoneura fumiferana (CF-1) insect cell line. The results indicate that, although all three gene products are represented within the P1 crystal of either NRD-12 or HD-1, only the contents of the 4.5 and 5.3 protoxins vary between the two crystals, whereas the 6.6 protoxin contents are similar and represent approximately one-third of the P1 crystal in either strain.