Efficient use of DNA molecular markers to construct industrial yeast strains.

Saccharomyces cerevisiae yeast strains exhibit a huge genotypic and phenotypic diversity. Breeding strategies taking advantage of these characteristics would contribute greatly to improving industrial yeasts. Here we mapped and introgressed chromosomal regions controlling industrial yeast properties, such as hydrogen sulphide production, phenolic off-flavor and a kinetic trait (lag phase duration). Two parent strains derived from industrial isolates used in winemaking and which exhibited significant quantitative differences in these traits were crossed and their progeny (50-170 clones) was analyzed for the segregation of these traits. Forty-eight segregants were genotyped at 2212 marker positions using DNA microarrays and one significant locus was mapped for each trait. To exploit these loci, an introgression approach was supervised by molecular markers monitoring using PCR/RFLP. Five successive backcrosses between an elite strain and appropriate segregants were sufficient to improve three trait values. Microarray-based genotyping confirmed that over 95% of the elite strain genome was recovered by this methodology. Moreover, karyotype patterns, mtDNA and tetrad analysis showed some genomic rearrangements during the introgression procedure

Single QTL mapping and nucleotide-level resolution of a physiologic trait in wine Saccharomyces cerevisiae strains.

International audienceNatural Saccharomyces cerevisiae yeast strains exhibit very large genotypic and phenotypic diversity. However, the link between phenotype variation and genetic determinism is still difficult to identify, especially in wild populations. Using genome hybridization on DNA microarrays, it is now possible to identify single-feature polymorphisms among divergent yeast strains. This tool offers the possibility of applying quantitative genetics to wild yeast strains. In this instance, we studied the genetic basis for variations in acetic acid production using progeny derived from two strains from grape must isolates. The trait was quantified during alcoholic fermentation of the two strains and 108 segregants derived from their crossing. A genetic map of 2212 markers was generated using oligonucleotide microarrays, and a major quantitative trait locus (QTL) was mapped with high significance. Further investigations showed that this QTL was due to a nonsynonymous single-nucleotide polymorphism that targeted the catalytic core of asparaginase type I (ASP1) and abolished its activity. This QTL was only effective when asparagine was used as a major nitrogen source. Our results link nitrogen assimilation and CO(2) production rate to acetic acid production, as well as, on a broader scale, illustrating the specific problem of quantitative genetics when working with nonlaboratory microorganisms

Utilisation d'un filtre presse pour la clarification des bourbes de moûts blancs après décantation statique. Comparaison avec la centrifugation

Les auteurs étudient la clarification des bourbes de moûts blancs avec un filtre presse équipé de plateaux à membrane. L'addition d'un adjuvant sous forme de perlite (BECOLITE 5000, perméabilité 2,100 Darcy) à la dose de 1.500 g par hectolitre permet d'atteindre des débits moyens suffisants, de l'ordre de 120 à 150 litres par heure et par m2 pour des cycles de filtration de 90 minutes. La qualité de la clarification est satisfaisante. A cet égard, le filtre presse se montre plus performant que la centrifugation. Les jus bourbeux ainsi clarifiés peuvent être réincorporés dans le moût débourbé statiquement. La dégustation ne permet pas de distinguer les vins issus de moût additionné ou non de leurs dépôts bourbeux clarifiés (par filtration ou centrifugation)

Genetic determination of <em>Saccharomyces cerevisiae</em> et <em>Saccharomyces bayanus</em> species by PCR/RFLP analysis of the <em>MET2</em> gene

Several yeast strains of the species S. cerevisiae, S. bayanus and S. paradoxus, first identified by hybridization experiments and DNA/DNA hybridization were characterized by using Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (PCR/RFLP) of the MET2 gene. The concordance between this tool and classical genetic analyses did not reveal any exception for all the strains analysed, so PCR/RFLP of the MET2 gene proves to be a reliable and fast tool for delimiting S. cerevisiae and S. bayanus. OEnological strains race bayanus, chevalieri, capensis gave S. cerevisiae restriction patterns, whereas most of strains race S. uvarum belong to S. bayanus and displayed a specific chromosomal band patterns different from band patterns of S. cerevisiae strains. To avoid confusion in oenological terminology, oenologists should no longer use the name of bayanus to designate industrial or wild S. cerevisiae Gal- strains, and should consider S. bayanus as a distinct species

Relación entre los sistemas respiratorios del oxígeno y el nitrato en vesículas de membrana de Escherichia coli K-12 efecto del 2-N-helptil-4-hidroxyquinolina-N-oxido y de la luz ultravioleta.

Relationship between the nitrate and oxygen respiratory systems in membrane vesicles of Escherichia coli k-12 efect of 2-N-heptyl-4-hydroxyquinoline-n-oxide and ultraviolet light. (Crispín Sánchez, José; Dubourdieu, Michel, Chippaux, Marc y Puig, Juan) Abstract Membrane vesicles prepared from anaerobic nitrate-grown Escherichia coli cell were shown to contain both oxygen and nitrate reducing systems. A scorbate-reduced phenazine methosulfate and NADH-reduced menadione were used as artificial electron donors for the enzymatic reduction of nitrate. By using ascorbate phenazine methosulfate, it was shown that nitrate reductase is essentially not inhibited by molercular oxygen. Nitrate and oxygen compete for available reducing equivalents from NADH or formate. Electrons proceeding from oxidation of NADH and formate pass through common electron carries. This favors the idea that electron transfer chains in E. coli are of the ramified type. 2-N-hepty1-4-Hydroxyquinoline-N-oxide (HQNO) inhibited electron transfer to oxygen and nitrate in at least one site. Arguments are given which support the idea that these site are also targets for ultraviolet light. Surprisingly, ultraviolet light irradiation inhibited the benzyl viologen mediated reduction of nitrate in membranes, but not in purified nitrate reductase. Relación entre los sistemas respiratorios del oxígeno y el nitrato en vesículas de membrana de Escherichia coli K-12 efecto del 2-N-helptil-4-hidroxyquinolina-N-oxido y de la luz ultravioleta. (Crispín Sánchez, José; Dubourdieu, Michel, Chippaux, Marc y Puig, Juan) Resumen Fueron obtenidas vesículas de membranas, preparadas a partir de células de Escherichia coli crecidas anaerobiosis con nitrato, que contienen sistemas reductores del oxígeno y del nitrato. Estas vesículas son capaces de reducir el nitrato en presencia de dos donadores artificiales de electrones: Fenacina metasulfato reducido por ascorbato y menadiona reducido por Nicotinamida adenindinucleótido reducido (NADII). Con el primero de estos donadores, se demostró que la enzima nitrato reductasa no es inhibida por el oxígeno molecular. El NO3 y el O2 compiten por equivalentes reductores provenientes del NADH o del formiato. Los electrones producidos por la oxidación de estos donadores pasan a través de transportadores que son comunes a las cadenas respitatorias del O2 y de NO3. Estas observaciones son argumentos en favor del carácter ramificado de las cadenas respiratorias en E. coli. 2-N-helptil-4-hidroxyquinolina-N-oxido (HQNO) inhibió la transferencia de electrónes hacia el O2 y el NO3 en, al menos, un sitio de la cadena. Los argumentos presentados confirman la idea de que la luz ultravioleta actúa también sobre estos sitios. Sorpresivamente, la irradiación de las vesículas con luz ultravioleta inhibió la reducción del nitrato dependiente del benzil viológeno pero esa irradiación no afecta a la nitrato reductasa purificada. Artículo publicado en: Revista Acta Científica Venezolana. Vol. 34 Nº [email protected] monográfic

Etude comparée des tests de stabilité protéique

Différents essais de laboratoire sont utilisés depuis longtemps pour évaluer le risque d'apparition d'un trouble protéique. Les auteurs comparent ces différents tests en utilisant les méthodes récentes de mesure de la limpidité (néphelométrie) et de dosage des protéines (CLHP). Les résultats obtenus montrent que le test à la chaleur (80° C-30 mn) reste l'essai de laboratoire le plus fiable, car il est simple à mettre en oeuvre et il se rapproche le plus des conditions d'apparition des troubles protéiques dans la pratique. +++ Various laboratory stability tests have been used for a long time to check the risks of protein turbidity. The authors have been comparing those different tests with the help of more modern methods of measuring out cloudiness (nephelometry) and protein (HPLC). Six tests were examined : - The test consisting in heating for 5 minutes at 80° C in a double-boiler. - The test consisting in heating for 30 minutes at 80° C in a double-boiler. - The test consisting in heating for 10 days at 35° C in an oven. - The adding of 0.5 9 of tannin per litre. - The adding of a reagent to phosphomolybdic acid (bentotest). - The adding of trichloracetic acid. The results are as follows : - For the tests involving heat, the longer the heating at 80° C, the higher the measures of the turbidity are; the cloudiness observed for the test consisting in heating at 35° C for 10 days is quite important. If the results obtained with these heating tests are to be compared to the real protein values, the most efficient test consists in heating 30 minutes at 80° C. - For the tests consisting in adding tannins, a high variation of turbidity values must be related to the results obtained through high performance liquid chromatography. Moreover, the important cloudiness observed when the tannins were added does not correspond to the small difference observed between the chromatograms of the wine streated with the tanins and those of the original samples. - The reagent to phosphomolybdic acid (bentotest) and the trichloracetice acid test create an important turbidity. After chromatographic examination, the use of these reagents proved to be acting more thoroughly on every sort of protein than the use of heat in the heating tests. For two out of the four wine-samples examined, the bentotest was more drastic than the addition of trichloracetic acid. As a conclusion the heating test (80° C for 30 minutes) remains the most reliable laboratory test, because it is sample and easy to make; it is also very close to the conditions in which protein turbidity may appear in reality

Identification des souches de levures isolées de vins par l'analyse de leur ADN mitochondrial

L'analyse des profils de restriction de l'ADN mitochondrial des levures permet une caractérisation fine des souches de Saccharomyces cerevisiae. Cette analyse a été appliquée à deux souches de levures sèches actives et à une vingtaine de souches indigènes, isolées de différents moûts lors de la fermentation spontanée. Les profils de restriction de l'ADN mt des souches étudiées présentent une grande diversité. La méthode mise en oeuvre est décrite de façon détaillée et les applications pratiques discutées. +++ The analysis of restriction patterns from yeast's mitochondrial DNA leads to a fine characterization of different strains of Saccharomyces cerevisiae. This analysis has been applied to two commercial strains and to twenty wild yeasts, isolated from different musts in case of natural fermentations. The DNA restriction patterns of the strains studied present a wide diversity. The method we used is minutely described and the practical applications are discussed

The expression and intracellular distribution of phosphoglycerate kinase isoenzymes in Trypanosoma cruzi

In this paper, we report the subcellular distribution of phosphoglycerate kinase (PGK) in epimastigotes of Trypanosoma cruzi. Approximately 80% of the PGK activity was found in the cytosol, 20% in the glycosomes. Western blot analysis suggested that two isoenzymes of 56 and 48 kDa, respectively, are responsible for the glycosomal PGK activity, whereas the cytosolic activity should be attributed to a single PGK of 48 kDa. In analogy to the situation previously reported for PGK in Trypanosoma brucei, these isoenzymes were called PGKA, C and B, respectively. However, in T. cruzi, PGKA seems not to be a minor enzyme like its counterpart in T. brucei. Whereas PGKC behaved as a soluble glycosomal matrix protein, PGKA appeared to be present at the inner surface of the organelle's membrane. After alkaline carbonate treatment, the enzyme remained associated with the particulate fraction of the organelles. Upon solubilization of glycosomes with Triton X-114, PGKA was recovered from the detergent phase, indicating its (partial) hydrophobic character and therefore, a possible hydrophobic interaction with the membrane. The PGKA gene was cloned and sequenced, but the predicted amino-acid sequence did not reveal an obvious clue as to the mechanism by which the enzyme is attached to the glycosomal membrane, (C) 2001 Elsevier Science B.V. All rights reserved

Incidence of a vine protection using a commercial formula of Bordeaux mixture on the Sauvignon grapes maturity and the wines varietal aroma (results of a 3-year study)

The incidence of cupric sprayings using a commercial formula of Bordeaux mixture (containing 20% of copper sulphate or an organic fungicide), on Sauvignon grapes composition and wines varietal aroma, has been studied in three areas of the Bordeaux region (Entre-deux-Mers, Graves, Médoc). Copper content has been noticed to increase in the clarified juices in all cases after these treatments, values ranging from 0,3 mg/l to 14,5 mg/l, whereas natural copper content did not exceed 0,4 mg/l. The value of residual copper by treatment was found to be significantly different from one year to the other (2 to 3,5 mg/l in 1993, 0,3 to 0,9 mg/l in 1994 and 0,7 to 0,9 mg/l in 1995) mainly due to the grapes composition and the different climatic conditions, whereas for the same year in the three different areas, this value was almost the same. The operation known as «skin contact» during which juices were left with their skins under CO2 at 10°C, during 18 hours before pressing, was found to decrease copper content in the clarified juices from 60 p. cent to 80 p. cent, whereas immediate pressing of grapes led to a decrease of only 20 p. cent ; the combination of copper during skin contact with glutathione, which is present in Sauvignon grapes, is a possible explanation. Under the ripening conditions of Bordeaux's oceanic temperate climate, we have noticed that even one single spray with a commercial formula of Bordeaux mixture at 3 000 g/ha, between the stage of closing of the cluster and the post-veraison, is sufficient to significantly reduce the content in 4-mercapto-4-methylpentan-2-one (4-MMP), a main component of the characteristic Sauvignon wines aroma. Increase of the introduced copper quantities in the must as well as of the number of applications, results in a greater reduction of the content in this thiol, as 4-MMP reacts with copper residues during alcoholic fermentation while its precursor is revealed into an aroma. Nevertheless, the experiments with a polymer that eliminates copper from the juice prior to alcoholic fermentation, showing in some cases no recovery in the wines 4-MMP content, lead us to the hypothesis that copper might as well intervene during ripening of the grapes, causing thus a limitation of the aroma's precursor synthesis. By the sensory analysis, Sauvignon wines produced from copper treated vines showed much less characteristic aroma than those produced from not treated vines (having nevertheless been protected by an organic fungicide) and were lower graded. While the average grade for the last ones was 7,9/10, this grade was lowered to 2,9/10 and to 2,8/10 for the samples produced after one and two or three vine's copper treatments respectively. Grapes sugar concentration can also be affected by the vine's cupric treatments. Throughout the 3-year study, differences in sugar concentration up to 40 g/l have been observed, especially concerning the treatments made between stages of closing of the cluster and veraison. Nevertheless, sugar content seems highly dependent on the number of cupric applications, the period of their application as well as climatic conditions during vine sprayings