Electrophoretic Patterns Of Enzymes Research Articles

Parity among interpretation methods of MLEE patterns and disparity among clustering methods in epidemiological typing of Candida albicans

The typing of C. albicans by MLEE (multilocus enzyme electrophoresis) is dependent on the interpretation of enzyme electrophoretic patterns, and the study of the epidemiological relationships of these yeasts can be conducted by cluster analysis. Therefore, the aims of the present study were to first determine the discriminatory power of genetic interpretation (deduction of the allelic composition of diploid organisms) and numerical interpretation (mere determination of the presence and absence of bands) of MLEE patterns, and then to determine the concordance (Pearson product-moment correlation coefficient) and similarity (Jaccard similarity coefficient) of the groups of strains generated by three cluster analysis models, and the discriminatory power of such models as well [model A: genetic interpretation, genetic distance matrix of Nei ( d ij ) and UPGMA dendrogram; model B: genetic interpretation, Dice similarity matrix ( S D1 ) and UPGMA dendrogram; model C: numerical interpretation, Dice similarity matrix ( S D2 ) and UPGMA dendrogram]. MLEE was found to be a powerful and reliable tool for the typing of C. albicans due to its high discriminatory power (> 0.9). Discriminatory power indicated that numerical interpretation is a method capable of discriminating a greater number of strains (47 versus 43 subtypes), but also pointed to model B as a method capable of providing a greater number of groups, suggesting its use for the typing of C. albicans by MLEE and cluster analysis. Very good agreement was only observed between the elements of the matrices S D1 and S D2 , but a large majority of the groups generated in the three UPGMA dendrograms showed similarity S J between 4.8% and 75%, suggesting disparities in the conclusions obtained by the cluster assays.

Diversity of Enzyme Electrophoretic Patterns in the Eggplant Complex

Studies on electrophoretic patterns of five enzymes, aspartate aminotransferase (AAT), glucose-phosphate isomerase (GPI), glutamate dehydrogenase (GDH), shikimate dehydrogenase (SKDH) and triose-phosphate isomerase (TPI) resulted in the identification of 17 phenotypes in 21 accessions of Solanum melongena L, S. insanum L and S. incanum L. The results provided evidence of 10 isozyme loci represented by 20 alleles involved in the control of the above enzymes. GPI and AAT showed high number of phenotypes, while GDHpresented a single morpho The most frequent phenotypes were identical in S. melongena, S. insanum and S. incanum indicating close genetic proximity of the three taxa. S. melongena accessions showed high degree of zymogram homogeneity, while the other two species were more diverse.

The Mating Systems of Some Epiphytic Polypodiaceae

Genetic loads, estimated from sporophyte production by isolated gametophyte cultures, indicate mating systems of intragametophytic selfing in Campyloneurum angustifolium (in part), C. phyllitidis, Phlebodium aureum and Phymatosorus scolopendria, and intergametophytic mating in C. angustifolium (in part), Microgramma heterophylla and Polypodium pellucidum. Polyploidy characterizes the intragametophytic-selfing species, whereas the intergametophytic-mating taxa are diploid. The duplicated loci of polyploid taxa may mitigate the expression of recessive lethal alleles caused by intragametophytic selfing, whereas genetic load probably maintains the mating systems of the intergametophytically mating taxa. Enzyme electrophoretic patterns of fixed heterozygosity support allopolyploid origins of C. phyllitidis and P. aureum and confirm their intragametophytic mating systems. Antheridiogens, present in both groups, may promote intergametophytic mating in diploids through promotion of the early development of male plants in gametophyte populations and bisexuality in isolated gametophytes of polyploids if these gametophytes delay or do not attain insensitivity to their own antheridiogen. In the polyploids, antheridiogens may also alleviate low genetic variability through promotion of occasional outcrossing. The perennial, clone-forming habit of epiphytic Polypodiaceae increases the duration and the physical space occupied by derivatives of a single spore, thus expanding the chance of interaction with a later migrant. Genetic load, duplicated genes, and antheridiogens, together with a perennial and clone-forming gametophyte growth habit, interact to produce successful breeding strategies of these epiphytic species.

Polyphasic characterization of rhizobia that nodulate Phaseolus vulgaris in West Africa (Senegal and Gambia).

Fifty-eight new isolates were obtained from root nodules of common bean (Phaseolus vulgaris) cultivated in soils originating from different agroecological areas in Senegal and Gambia (West Africa). A polyphasic approach including both phenotypic and genotypic techniques was used to study the diversity of the 58 Rhizobium isolates and to determine their taxonomic relationships with reference strains. All the techniques performed, analysis of multilocus enzyme electrophoretic patterns, SDS-PAGE profiles of total cell proteins, PCR-RFLP analysis of the genes encoding 16S rRNA and of the 16S-23S RNA intergenic spacer region (ITS-PCR-RFLP), auxanographic tests using API galleries and nodulation tests lead to the consensus conclusion that the new rhizobial isolates formed two main distinct groups, I and II, belonging to Rhizobium tropici type B and Rhizobium etli, respectively. By MLEE R. etli and group II strains showed several related electrophoretic types, evidencing some extent of internal heterogeneity among them. This heterogeneity was confirmed by other techniques (ITS-PCR-RFLP, SDS-PAGE and host-plant-specificity) with the same nine distinct strains of group II showing some differences from the core of group II (54 strains).

Genetic variation amongTrypanosoma cruzi populations

The analysis of enzyme electrophoretic patterns in 141 extracts of Trypanosoma cruzi isolated from humans and domesticated and wild animals from the endemic zone of Chagas' disease in Argentina allowed their characterization into 12 different "isozymic strains" or zymodemes (Z1 to Z12). In humans, the parasites most frequently found belong to Z1 and Z12. These zymodemes differ significantly in pathogenic activity, Z1 having fewer deleterious effects than Z12. Because of the good correlation existing between zymodemes and the groups of parasites characterized by the study of kinetoplast DNA (kDNA), it is possible to identify zymodemes by analyzing kDNA. A 270-base-pair probe of the highly variable region of minicircles (HVRm) of kDNA, purified from Z1 and Z12 reference stocks, hybridized specifically with PCR-amplified HVRm from T. cruzi isolates, allowing identification of the "major natural clones." It is suggested that this technique of zymodeme identification could be useful for the prognosis of the probable evolution of the disease.

Use of electrophoretic enzyme patterns for streptomycete systematics

The relative electrophoretic mobilities of various enzymes from 24 different streptomycetes were determined on polyacrylamide gels in order to examine the relatedness of species and strains of the genus Streptomyces. Of 11 different enzymes tested in this study, hexokinase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, malate dehydrogenase and isocitrate dehydrogenase showed a limited number of constant and reproducible polymorphic enzyme patterns, by comparing which the inter-specific relationships could be examined. In contrast, glucose dehydrogenase, alcohol dehydrogenase, 3-hydroxybutyrate dehydrogenase, phosphoglucose isomerase, peroxidase and esterase exhibited either weak non-reproducible or highly heterogeneous band patterns which were suitable for dissecting the strains within a species and a cluster group.

Use of electrophoretic enzyme patterns for streptomycete systematics

The relative electrophoretic mobilities of various enzymes from 24 different streptomycetes were determined on polyacrylamide gels in order to examine the relatedness of species and strains of the genus Streptomyces. Of 11 different enzymes tested in this study, hexokinase, glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, malate dehydrogenase and isocitrate dehydrogenase showed a limited number of constant and reproducible polymorphic enzyme patterns, by comparing which the inter-specific relationships could be examined. In contrast, glucose dehydrogenase, alcohol dehydrogenase, 3-hydroxybutyrate dehydrogenase, phosphoglucose isomerase, peroxidase and esterase exhibited either weak non-reproducible or highly heterogeneous band patterns which were suitable for dissecting the strains within a species and a cluster group.

Candida sojae, a new species of yeast isolated from an extraction process of water-soluble substances of defatted soybean flakes.

A new anamorphic yeast species, Candida sojae Nakase et al., is proposed for two strains isolated from an extraction process of water-soluble substances of defatted soybean flakes. Candida sojae resembles Candida tropicalis and Candida albicans, but differs from these two species by mol% G+C, DNA-DNA relatedness, electrophoretic enzyme patterns, and the proton magnetic resonance spectra of cell wall mannans. Practically, C. sojae is distinguished from C. tropicalis and C. albicans by its inability to ferment maltose and low maximum growth temperature below 40°C. It is also distinguishable from C. tropicalis and C. albicans serotype A by the lack of agglutination with mouse monoclonal antibody CA4-2.

Genetic structure of Trypanosoma cruzi populations from Argentina estimated from enzyme polymorphism

Isolates of Trypanosoma cruzi from human patients, domestic and sylvatic animals and vector insects were obtained in different areas of Argentina. Electrophoretic patterns of enzymes from extracts of 95 isolates were analysed. On the basis of zymograms providing information on 10 loci, 12 zymodemes are described according to their genotypes. Data presented show fixed heterozygosity, absence of segregation of genotypes, significant departures from Hardy-Weinberg equilibrium, and over-represented genotypes. This evidence supports the hypothesis that sexual reproduction is very restricted or absent in this parasite. The proportion of polymorphic loci is 80%. The expected mean heterozygosity per locus (He) is 0.43, while the observed value (Ho) is 0.24. Differences between these values may be explained by accepting a basically clonal structure for T. cruzi. The data matrix of 12 zymodemes using 28 characters was analysed using a Wagner parsimony algorithm. Two equally most parsimonious unrooted trees were generated; both have 39 steps. The results show clusters clearly separated according to the geographical origin of the stocks. There are some indications of some correlations between genetic composition of the parasite and the clinical picture of the infection in human patients.

Utilization of Carbon Substrates, Electrophoretic Enzyme Patterns, and Symbiotic Performance of Plasmid-Cured Clover Rhizobia

Plasmids in Rhizobium spp. are relatively large, numerous, and difficult to cure. Except for the symbiotic plasmid, little is known about their functions. The primary objective of our investigation was to obtain plasmid-cured derivatives of Rhizobium leguminosarum bv. trifolii by using a direct selection system and to determine changes in the phenotype of the cured strains. Three strains of rhizobia were utilized that contained three, four, and five plasmids. Phenotypic effects observed after curing of plasmids indicated that the plasmids were involved in the utilization of adonitol, arabinose, catechol, glycerol, inositol, lactose, malate, rhamnose, and sorbitol and also influenced motility, lipopolysaccharide production, and utilization of nitrate. Specific staining of 26 enzymes electrophoretically separated on starch gels indicated that superoxide dismutase, hexokinase, and carbamate kinase activities were affected by curing of plasmids. Curing of cryptic plasmids also influenced nodulation and growth of plants on nitrogen-deficient media. The alteration in the ability to utilize various substrates after curing of plasmids suggests that the plasmids may encode genes that contribute significantly to the saprophytic competence of rhizobia in soil.

Electrophoretic comparison of enzymes from 22 single-spore cultures obtained from Frankia strain ORS 140102

Individual spores of Frankia strain ORS 140102 were allowed to germinate on Qmod medium. Germination percentages were approximately 30%. Colonies from germinated spores were individually picked off and subcultured on two media giving 22 single-spore cultures. Although colony morphology and pigmentation of the single-spore cultures differed, the study of the electrophoretic patterns of enzymes showed that malate dehydrogenase and esterase profiles of the 22 strains and the mother-strain were very similar.

Candida fragi, a new species of anamorphic yeast isolated from fermenting strawberry.

A new anamorphic yeast species, Candida fragi Suzuki, Nakase et Fukazawa, is proposed for a strain isolated from fermenting strawberry that was formerly identified as C. sake, or C. natalensis. Candida fragi resembles C. natalensis. C. oleophila, and C. sake but is clearly differentiated from these species by DNA-DNA relatedness, electrophoretic enzyme patterns, and the proton magnetic resonance spectra of cell wall mannans. Practically, C. fragi is distinguished from C. natalensis by its inability to assimilate trehalose, mannoheptulose, and DL-glyceraldehyde; from C. sake by its inability to assimilate trehalose and α-methyl-D-glucoside and its ability to assimilate 5-ketogluconic acid and xylitol; and from C. oleophila by its inability to assimilate trehalose, to ferment galactose, and to grow in 100ppm of cycloheximide.

Electrophoretic comparison of enzymes from 22 single-spore cultures obtained fromFrankiastrain ORS 140102

Individual spores of Frankia strain ORS 140102 were allowed to germinate on Qmod medium. Germination percentages were approximately 30%. Colonies from germinated spores were individually picked off and subcultured on two media giving 22 single-spore cultures. Although colony morphology and pigmentation of the single-spore cultures differed, the study of the electrophoretic patterns of enzymes showed that malate dehydrogenase and esterase profiles of the 22 strains and the mother-strain were very similar.

Correlation between ribosomal DNA polymorphism and electrophoretic enzyme polymorphism in Yersinia

Ribosomal DNA (rDNA) polymorphism was compared with electrophoretic enzyme polymorphism for the intra- and interspecies differentiation of Yersinia enterocolitica, Y. pseudotuberculosis, Y. intermedia, Y. aldovae, Y. frederiksenii and Y. kristensenii. DNA from 90 strains previously classified into six zymotypes (Y. enterocolitica and Y. frederiksenii) and into distinct enzyme electrophoretic patterns (the four other species) was digested with EcoRI or HindIII and analysed by Southern blotting. The six species were clearly differentiated from each other. In Y. enterocolitica, the subclassification of biotype 1 into zymotypes 1A and 1B was also reflected in the rDNA and the four other bio-zymotypes gave four different classes of restriction pattern. In Y. frederiksenii, both EcoRI and HindIII gave five distinct riboclasses which correlated with the zymotypes. In the four other species, the phenotype polymorphism appeared to be better correlated with the restriction fragment length polymorphism data in some enzymes than others. The data demonstrate that the inter- and intraspecies classification by rDNA polymorphism using two restriction enzymes is similar to that based on electrophoretic enzyme polymorphism. The analysis could be refined for taxonomic and epidemiological purposes by using other restriction enzymes.

Bullera miyagina, a new species of ballistospore-forming yeast isolated from Abies firma collected in Japan.

A new species of ballistospore-forming yeast, Bullera miyagiana Nakase, Itoh, Takematsu et Bandoni is described. This yeast was isolated from Abies firma collected at Sendai, a northeast region in Japan. It is characterized by Q-10 as the major ubiquinone, xylose in the cells and high G+C content in DNA, 61.4mol% (calculated from Tm) or 58.9mol% (analyzed by high performance liquid chromatography). These characteristics resemble those of Bullera oryzae, but there is a clear difference between B. miyagiana and B. oryzae in the electrophoretic patterns of enzymes. Practically, B. miyagiana is differentiated from B. oryzae by its assimilation of nitrate, ethanol and ribitol, and its lack of assimilation of ethylamine or L-lysine.

Significance of the coenzyme Q system in the classification of yeasts and yeast-like organisms. An electrophoretic comparison of enzymes in strains of species in the genera Lipomyces Lodder et Kreger-van Rij and Waltomyces Yamada et Nakase (lipomycetaceae).

A taxonomic study below the generic or at the specific level was performed as to the electrophoretic patterns of seven enzymes in twenty-six strains of Lipomyces and Waltomyces species. The seven enzymes were glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase, malate dehydrogenase, hexokinase, phosphoglucomutase and fumarase. The five species, L. anomalus, L.kononenkoae, L. starkeyi, L. tetrasporus and W. lipofer constituted separate clusters from each other, the similarity values being all 0%. The two strains of L. kononenkoae were quite dissimilar in their electrophoretic enzyme patterns (s = 0%). The unidentified Lipomyces strain, CBS 8113, was very similar to the type strain of L. kononenkoae (s = 100%). The five strains of L. starkeyi were divided into two groups with a similarity value of 0%. The unidentified Lipomyces strain, CBS 8064, was a member of the second L. starkeyi group (s = 43%). The eleven strains of L. tetrasporus were divided into three separ...

An electrophoretic comparison of enzymes in strains of the Q9-equipped species in the genus Sporobolomyces Kluyver et van Niel(Sporobolomycetaceae).

A taxonomic study below the generic or at the specific level was made of electrophoretic patterns of seven enzymes in 18 strains of the Q9-equipped Sporobolomyces and Bensingtonia species. The seven enzymes were glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase, malate dehydrogenase, hexokinase, phosphoglucomutase, and fumarase. Seven species, S. intermedius, S. miscanthi, S. subroseus, S. yamatoanus, S. naganoensis, S. yuccicola, and S. phylladus, constituted clusters separate from each other (s=0 or 14%). The two strains of S. miscanthi showing identical electrophoretic enzyme patterns (s=100%) were linked to Sporobolomyces species NZ-3 with a similarity value of 29%. Within the five strains of S. yamatoanus examined, the type strain had a similarity value of 86% with one other strain. The remaining three strains whose similarity values were calculated as 100% and 57% were linked to the two strains including the type strain with a similarity value of 43%. The three strains of S. phylladus had identical electrophoretic enzyme patterns (s=100%). Sporobolomyces species NZ-24 had a separate cluster (s=0%). Sporobolomyces weijmanii was synonymous with S. intermedius (s=100%). Bensingtonia ciliata constituted its own cluster (s=0%). These data are discussed from the taxonomic point of view.

Cutaneous leishmaniasis in Guatemala: isoenzyme characterization of isolates from humans.

Leishmania organisms cultivated from cutaneous lesions of humans in Guatemala were characterized by cellulose acetate electrophoresis. Six isolates had electrophoretic enzyme patterns identical to World Health Organization reference strains of Leishmania braziliensis braziliensis, and 5 had patterns identical to reference strains of Leishmania mexicana mexicana.

Glucose-6-Phosphate Dehydrogenase and Malate Dehydrogenase Enzyme Electrophoretic Patterns Amongst Strains of Bacteroides fragilis

Fifty-two strains of Bacteroides fragilis were examined for their enzyme electrophoretic patterns of glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH). All strains tested possessed high levels of both enzymes but the G6PDH reduced NADP whereas MDH was NAD-dependent. Twenty-seven strains produced single bands of both G6PDH and MDH. In all cases G6PDH migrated faster than MDH. Strains clustered by a single linkage algorithm were recovered in eight clusters at the 77% similarity level. The remaining 25 strains produced multiple bands of one or both enzymes. These were recovered in six clusters at the 72% similarity level using the same algorithm. The results of this study revealed considerable heterogeneity of enzyme patterns within B. fragilis.

An electrophoretic comparison of enzymes in strains of species in the fission yeast genera Schizosaccharomyces, Octosporomyces, and Hasegawaea.

A taxonomic study below the generic or at the specific level was made of the electrophoretic patterns of five enzymes in fourteen strains ofSchizosaccharomyces, Octosporomyces, and Hasegawaea species. The five enzymes were glucose-6-phosphate dehydrogenase, 6-phosphogluconatedehydrogenase, hexokinase, phosphoglucomutase, and fumarase. All of the six strains of Schizosaccharomyces pombe examined were linked to each other with a similarity value of 40% or more. The type strain of Schizosaccharomyces malidevorans was closely related to that of S. pombe with a similarity value of 60%. The similarity values of three strains of Octosporomyces octosporus were 80% or more. All of the three strains ofHasegawaea japonica var. japonica examined had a uniform electrophoretic enzyme pattern. The similarity value between the strains of H. japonica var. japonica and H. japonica var. versatilis was 60%. The three species, S. pombe, O. octosporus, and H. japonica had quite different electrophoretic enzyme patterns. Their similarity values were all 0%. The Co-Q systems of the strains were reinvestigated. These data are discussed from the taxonomic point of view.