Enzymes Associated with Defence against Toxic Oxygen Species in PCNB-Tolerant and Sensitive Soil Fungi

The specific activities of enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPOX) and glutathione reductase (GR), which are involved in protection against toxic species of oxygen, were determined in mycelia extracts of pentachloronitrobenzene (PCNB)-tolerant and susceptible soil fungi. The organisms assayed were the highly PCNB-sensitive Rhizoctonia solani and Rhizopus arrhizus; Sclerotium rolfsii and Trichoderma harzianum, which are moderately susceptible to PCNB, and the fungicide-tolerant Fusarium oxysporum f. sp. melonis and Pythium aphanidermatum. No GPOX activity was detected in the six examined fungi. Significant differences in the specific activities of the other enzyme systems among the fungi were evident. Remarkably low levels of CAT activities were measured in R. solani. Except for T. harzianum, no meaningful differences regarding SOD, CAT and GR activities with age of the fungi cultures were observed. The electrophoretic patterns of SOD and CAT displayed dissimilarities among the fungi under study. P. aphanidermatum is more polymorphic with respect to both SOD and CAT enzyme systems as compared to the other fungi. The SOD of F. oxysporum f. sp. melonis, R. arrhizus and T. harzianum is a cuprozinc enzyme, while the mangano-SOD species was detected in S. rolfsii, R. solani and T. harzianum.     

Variation of plastid and mitochondrial DNAs in the genus Hedysarum

Summary Plastid and mitochondrial DNAs from Hedysarum species of the western Mediterranean basin, H. spinosissimum ssp eu-spinosissimum, H. spinosissimum ssp capitatum, H. carnosum, H. coronarium and H. flexuosum, were compared by restriction endonuclease fragment analysis. ctDNA fragment patterns for ssp eu-spinosissimum and ssp capitatum were indistinguishable in different enzyme digests. An identical ctDNA variation was found in Hpa II digests with two Sardinian populations of ssp capitatum. Each of the two subspecies was characterized by specific mt DNA patterns with Pst I, Bam HI, Sma I and EcoRI. No variation was detected in populations of different geographical origins for a given subspecies. H. carnosum, H. coronarium and H. flexuosum generated specific ct and mt DNA patterns. Comparison of mitochondrial fragments indicated: — a strong homology between the two subspecies, — a closer homology among the three other diploids, each being closer to the other two than to H. spinosissimum subspecies — as was also the case for the plastid genomes.     

Evolution of patterns of gene expression in Hawaiian picture-wingedDrosophila

Summary The tissue and stage specificity of expression of five enzymes was examined by electrophoretic analysis of relative enzyme levels in extracts of 13 larval and adult tissues in 27 species of Hawaiian picture-wingedDrosophila. The developmentally regulated patterns of enzyme expression thus characterized were compared to a modal standard phenotype. About 30% of the pattern features analyzed differed significantly from the standard in one or more species. Many of these regulatory differences are essentially qualitative, with tissue specific differences in enzyme activity in excess of 100 fold for some species pairs. The adaptive significance of these pattern differences is unknown, but the results provide strong direct evidence for rapid evolution of new patterns of gene regulation in this group of organisms.     

Genetic diversity in the top onion,Allium ×proliferum (Alliaceae), analysed by isozymes

Polymorphisms for six enzyme systems (GPI, IDH, PDG, PGM, SKD, and TPI) were analysed in the top onion,Allium ×proliferum. Five multilocus isozyme genotypes were found. The banding patterns of top onions were compared with those ofA. ×wakegi, A. cepa, A. fistulosum, A. altaicum, and artificial hybrids between these three species. One top onion type and one artificial hybrid had identical banding patterns. Shallots andA. altaicum, the wild progenitor ofA. fistulosum, cannot be distinguished from the common onion andA. fistulosum, respectively; these species are also potential contributors to the top onion's gene pool.     

Seed esterases,leucine aminopeptidases and catalases of species of the genus Gossypium

Summary Polyacrylamide and starch gel electrophoresis were used to analyze the isozyme makeup of three enzyme systems (esterases, leucine aminopeptidases and catalases) from the dormant seeds of twenty-nine species within the genus Gossypium.Isozyme variation was observed for all three enzymes between the species of the different genome groups. The within species polymorphism noted for the esterases was not observed for the leucine aminopeptidase and catalase patterns. In general, only minor qualitative banding pattern differences distinguished the A and B genome species, whereas, band variations were greatest between the more distantly related species in the C, D and E genomes. Gossypium longicalyx (F genome) showed an overall banding pattern unique to itself. The species of the genomes (C, D, E and F) removed from the postulated area of genetic origin (Southern Africa) also exhibited greater isozyme variability than that of the wild species of the A and B genomes, both located in Southern Africa.Synthetic mixtures of seed extracts from parent species of recently formed synthetic allopolyploids produced additive isozyme patterns for esterase, leucine aminopeptidase and catalase that were closely comparable to the zymograms produced by their hybrids. In contrast all three enzyme systems showed significant qualitative isozyme variations between the three natural allotetraploids, G. tomentosum, G. barbadense and G. hirsutum when compared to the zymograms of the synthetic mixtures of their alleged parental forms.This paper is part of a dissertation by the first author for the degree of Doctor of Philosophy in Genetics. Journal paper 1763 of the Arizona Agricultural Experiment Station.National Research Council Postdoctoral Research Associate.     

Characterization of Two Sibling Species of the Genus Stylonychia (Ciliata,Hypotricha): S. mytilus Ehrenberg, 1838 and S. lemnae n. sp. II. Biochemical Characterization1

The two closely related hypotrichous ciliate species, Stylonychia lemnae and S. mytilus, have been compared with respect to their isoenzyme patterns, their macronuclear DNA banding patterns, and micronuclear DNA banding patterns after restriction enzyme digestion. Since macronuclear DNA contains mainly protein-coding sequences and since the micronuclear DNA patterns represent mainly the repetitive fraction of the genome, these results, together with the isoenzyme patterns, reflect differences on three different levels of molecular evolution between morphologically very similar species. Each of the three methods allows an unequivocal identification of each of the two species. Intraspecific variation seems to be greatest among the repetitive sequences of the micronuclear genomes. By using the isoenzyme data and the formula of Nei (19) the genetic distance between the two species is calculated and compared with the results from other protozoa and different Drosophila species. Despite their morphological similarity, the two species show a considerable amount of evolutionary divergence on the three molecular levels which have been investigated.     

Allozyme diversity and the evolution ofSymplocos (Symplocaceae) on the Bonin (Ogasawara) Islands

To study the origin and speciation of plants in oceanic islands, electrophoretic analyses have been done on three endemic species ofSymplocos in the Bonin Islands as well as on three other species;S. kuroki, S. nakaharae andS. tanakae which are considered to be closely related to the Bonin endemics. There occur three species:S. kawakamii, S. pergracilis andS. boninensis in Bonin. The genusSymplocos is one which is considered to be diversified in the Bonin Islands. Seven enzyme systems presumed to be encoded by 18 loci were examined. The genetic diversity was low in the island species, as reported in some oceanic island plants of Hawaii and the Bonin Islands. The three endemics share high genetic identities and they clustered together in the tree drawn by the UPGMA method, suggesting that they are a monophyletic group, that is, they result from a single introduction.     

Studies on isozymes of sorbitol dehydrogenase in some vertebrate species

Summary In swine, the enzyme sorbitol dehydrogenase exhibits a genetically determined polymorphism as identified by multiple electrophoretic bands suggesting a tetrameric structure (Op't Hof, 1969). In order to obtain further information on the genetics of this polymorphism, a number of other vertebrate species were examined. Multiple alleles at the SDH gene locus were found in the Cyprinid fishes Leuciscus and Rutilus, and in the Salmonid fish Salmo trutta. The electrophoretic patterns found in these species are compatible with the model of a tetrameric structure of the enzyme. This model was further verified by dissociation-reassociation studies with the homomeric enzymes of swine and Leuciscus. Since in the heterozygote an intensity gradient of the isozymes was seen, the three phenotypes observed in swine were further characterized by means of activity measurements and application of different substrates. The results point to a differential activity of the two different homomeres.Supported by the Deutsche Forschungsgemeinschaft.     

Nitrogen preferences and plant-soil feedbacks as influenced by neighbors in the alpine tundra

Plant resource partitioning of chemical forms of nitrogen (N) may be an important factor promoting species coexistence in N-limited ecosystems. Since the microbial community regulates N-form transformations, plant partitioning of N may be related to plant–soil feedbacks. We conducted a ¹⁵N tracer addition experiment to study the ability of two alpine plant species, Acomastylis rossii and Deschampsia caespitosa, to partition organic and inorganic forms of N. The species are codominant and associated with strong plant–soil feedbacks that affect N cycling. We manipulated interspecific interactions by removing Acomastylis or Deschampsia from areas where the species were codominant to test if N uptake patterns varied in the presence of the other species. We found that Deschampsia acquired organic and inorganic N more rapidly than Acomastylis, regardless of neighbor treatment. Plant N uptake—specifically ammonium uptake—increased with plant density and the presence of an interspecific neighbor. Interestingly, this change in N uptake was not in the expected direction to reduce niche overlap and instead suggested facilitation of ammonium use. To test if N acquisition patterns were consistent with plant–soil feedbacks, we also compared microbial rhizosphere extracellular enzyme activity in patches dominated by one or the other species and in areas where they grew together. The presence of both species was generally associated with increased rhizosphere extracellular enzyme activity (five of ten enzymes) and a trend towards increased foliar N concentrations. Taken together, these results suggest that feedbacks through the microbial community, either in response to increased plant density or specific plant neighbors, could facilitate coexistence. However, coexistence is promoted via enhanced resource uptake rather than reduced niche overlap. The importance of resource partitioning to reduce the intensity of competitive interactions might vary across systems, particularly as a function of plant-soil feedbacks.     

Studies on Pectic Enzymes of Microorganisms

To pick out potent strains which specifically produce one of several pectic enzymes, endo- and exo-polygalacturonase, pectin esterase, macerating, and apple juice clarifying activities were examined with regard to 344 strains of mold (containing 71 strains of phytopathogenic mold) grown on a bran culture medium and 56 strains of shakingly cultured yeast. As the result of screening, Asper gillus saitoi and Penicillium islandicum were isolated as potent specific producers of endo-polygalacturonase. And the composition of pectic enzymes of mold was found to be rather genus or species specific. So far as examined in crude enzyme systems, there was no parallelism between anyone of pectic enzyme activities and apple juice clarifying or macerating activities.     

Patterns of distribution and abundance of chitons of the genus Ischnochiton in intertidal boulder fields

Abstract Chitons of the genus Ischnochiton are found predominantly on the undersurfaces of boulders, compared with other intertidal or subtidal habitats. They therefore appear to be habitat‐specialists at this scale. This, combined with the fact that boulder fields are relatively sparse compared with other intertidal habitats, makes these animals vulnerable to natural and anthropogenic disturbances. In addition, many species of Ischnochiton are relatively rare and appear to have very patchy abundances, making them likely to have very specific requirements for habitat. We need to understand the habitat requirements in order to manage, conserve and restore disturbed habitats. The present study was carried out at three intertidal boulder fields separated along approximately 200 km of the coast of New South Wales, Australia, centred around Sydney. The boulder fields were representative of those found in this region. The boulders were made of different materials: shale in the north, sandstone in Sydney and quartzitic sandstone in the south. Some boulders in each boulder field were covered by up to 0.4 m of water during low tide. The study showed that the seven species examined were overdispersed among boulders in each of these three intertidal boulder fields. Most boulders did not have associated chitons, but there were very large abundances on a very small number of boulders. Chitons were also overdispersed among boulders that they occupied. These patterns were consistent among shores and among species, even though patterns of abundance were extremely different among different species. These species appear therefore to show specific requirements for habitat at a small spatial scale, using only a small proportion of potential patches of habitat (i.e. boulders) in any place. Extremely patchy patterns of dispersion can be caused by variation in patterns of recruitment, mortality or behavioural responses to habitat or other species. Before performing experiments to investigate such processes, it is useful to test hypotheses of association with habitat using mensurative experiments to identify environmental correlates that might explain the observed patterns. In the present study, sizes of boulders and the associated sessile and mobile assemblages were proposed as mechanisms that could affect dispersion of chitons among boulders. None of these factors, however, showed strong associations with abundances of chitons. The lack of support of these models rules out some features of habitat to which species of Ischnochiton might respond, thereby precluding manipulative experiments involving these features, which are unlikely to be involved in the very patchy patterns of dispersion of species of Ischnochiton.     

Chloramphenicol acetyltransferase fromPseudomonas aeruginosa—a new variant of the enzyme

Pseudomonas aeruginosa isolates are highly resistant to chloramphenicol (minimal inhibitory concentration, 100–1,000 g/ml). Most of the strains tested produce the enzyme chloramphenicol acetyltransferase (CAT) while 15% do not produce the enzyme, although they are highly resistant to the drug. In an attempt to understand the resistance mechanism ofP. aeruginosa to chloramphenicol, CAT produced by this microorganism was examined and compared with other known variants of the enzyme which are usually associated with high-level resistance in other species. CAT ofP. aeruginosa was found to be synthesized constitutively and to have a molecular weight of 22,500 per protomer. The specific activity of the enzyme is 30-fold lower than that ofEscherichia coli type II CAT. The same specific activity was obtained for CAT produced by two isolates ofP. aeruginosa, one with high and one with low resistance. The elution patterns from affinity and hydrophobic chromatography,K _m 's for chloramphenicol, the high affinity of the enzyme for acetyl-CoA (2- to 6-fold greater than that of any other variant) and insensitivity to sulfhydryl reagents indicate that the properties of this enzyme are not identical with those of any of the known variants.     

Musculoskeletal determinants of pelvic sucker function in hawaiian stream gobiid fishes: Interspecific comparisons and allometric scaling

Gobiid fishes possess a distinctive ventral sucker, formed from fusion of the pelvic fins. This sucker is used to adhere to a wide range of substrates including, in some species, the vertical cliffs of waterfalls that are climbed during upstream migrations. Previous studies of waterfall‐climbing goby species have found that pressure differentials and adhesive forces generated by the sucker increase with positive allometry as fish grow in size, despite isometry or negative allometry of sucker area. To produce such scaling patterns for pressure differential and adhesive force, waterfall‐climbing gobies might exhibit allometry for other muscular or skeletal components of the pelvic sucker that contribute to its adhesive function. In this study, we used anatomical dissections and modeling to evaluate the potential for allometric growth in the cross‐sectional area, effective mechanical advantage (EMA), and force generating capacity of major protractor and retractor muscles of the pelvic sucker (m. protractor ischii and m. retractor ischii) that help to expand the sealed volume of the sucker to produce pressure differentials and adhesive force. We compared patterns for three Hawaiian gobiid species: a nonclimber (Stenogobius hawaiiensis), an ontogenetically limited climber (Awaous guamensis), and a proficient climber (Sicyopterus stimpsoni). Scaling patterns were relatively similar for all three species, typically exhibiting isometric or negatively allometric scaling for the muscles and lever systems examined. Although these scaling patterns do not help to explain the positive allometry of pressure differentials and adhesive force as climbing gobies grow, the best climber among the species we compared, S. stimpsoni, does exhibit the highest calculated estimates of EMA, muscular input force, and output force for pelvic sucker retraction at any body size, potentially facilitating its adhesive ability. J. Morphol. 2013. © 2013 Wiley Periodicals, Inc.     

EVIDENCE FOR POST-TRANSLATIONAL MODIFICATION OF TRIOSE PHOSPHATE ISOMERASE (TPI) IN ISOËTES (ISOËTACEAE)

As part of an electrophoretic study on Isoëtes, a number of Neotropical and North American species were examined for allozyme variation in TPI. Three of these species—I. storkii, I. flaccida, and I. mexicana—exhibit three distinct zones of TPI activity. The two most anodally migrating zones are comparable to the two zones found in most angiosperms and in several other species of Isoëtes. The single or three-banded phenotypes produced at these loci correspond, respectively, to the homozygous and heterozygous patterns typical of a dimeric enzyme. The most cathodal zone (zone III) differs in producing either single or two-banded phenotypes. Analyses of these three zones indicate a nearly perfect correlation between zones II and III in putative allelic constitution and relative allelic mobility. Explanations involving TPI gene duplications and/or null alleles fail to account for the peculiar banding characteristics and origin of activity zone III. An alternative hypothesis involving a protease duplication and differential post-translational modification is postulated. This hypothesis adequately explains the zone III phenotypes and fixation of the third activity zone in the species examined. Amino acid sequencing is suggested as the most direct test of this hypothesis. The taxonomic distribution of TPI III generally supports a previous, morphologically-based, hypothesis on species relationships in Isoëtes. The presence of this zone is regarded as an independent synapomorphy for a major clade of Neotropical Isoëtes.     

STARCH GEL ELECTROPHORESIS OF ENZYMES FROM NINE SPECIES OF POLYPORUS

Taxonomic relationships among 22 isolates of nine species of the wood-rotting fungus, Polyporus, were examined by starch gel electrophoresis, coupled with histochemical staining procedures. Crude extracts of 21-day cultures grown on malt extract agar were subjected to electrophoresis for 3 hr at a constant current of 25 ma. Esterase, peroxidase, catechol oxidase, galactose dehydrogenase, acid phosphatase, and alkaline phosphatase banding patterns were analyzed. With each enzyme, the bands for each isolate were compared with those for other isolates of the same species and for isolates of other species. A closer relationship was found in the banding patterns for different isolates of a single species than among isolates of different species. This technique may prove valuable for the evaluation of taxonomic differences among these wood-rotting fungi.     

Isozyme diversity in sour,sweet, and ground cherry

Thirty-six sour (Prunus cerasus L.), sweet (P. avium L.), and ground cherry (P. fruticosa Pall.) selections were evaluated for seven enzyme systems and principal coordinate analysis was used to examine isozyme divergence among these cherry species. The enzyme systems studied were phosphoglucose isomerase (PGI), isocitrate dehydrogenase (IDH), phosphoglucomutase (PGM), 6-phosphogluconate dehydrogenase (6-PGD), leucine aminopeptidase (LAP), shikimate dehydrogenase (SKDH), and malate dehydrogenase (MDH). The first principal coordinate, which accounted for 41% of the total variation, separated the diploid sweet cherry selections from the sour, ground, and sour x ground cherry tetraploids. An additional 86 selections were evaluated for up to six of the enzyme systems to determine the polymorphisms at the enzyme loci and the level of heterozygosity between the diploid sweet cherry and the tetraploid species and interspecific hybrids. 6-PGD was the most polymorphic enzyme exhibiting 16 patterns. The tetraploid cherry species were more heterozygous than the diploid sweet cherry with an average heterozygosity of 78% compared to 19% for the diploids.     

Relationships and Hybridization among Smilax china and Its Affinities: Evidence from Allozyme Data

Smilax china L. is a widespread species in China with different ploidy levels. It is morphologically similar to S. davidiana, S. trinervula, and S. glauco-china. In this study, the chromosome number and the variation in allozyme patterns of eight enzyme systems with 25 alleles in 11 populations of S. china and three affinitive species were investigated. The allozyme data, together with morphological and cytological data, suggest that S. glauco-china is not closely related to the other taxa investigated. The diploid species S. davidiana and S. trinervula are involved as ancestor species and share great introgressions with S. china. In S. china, populations from Guilin and Guiyang are allotetraploid; their diploid progenitors probably are diploid populations of S. china and S. trinervula. The results suggest this species arose from multiple origins.     

Paulownia taiwaniana,a hybrid betweenP. fortunei andP. kawakamii (Scrophulariaceae)

Paulownia taiwaniana, the widely cultivated, commercially important tree, has been suspected of being of hybrid origin at least since its original publication in 1975. Evidence in support of this thesis, derived from a number of different investigations, is presented in this paper.—Strong evidence comes from a controlled pollination study of the two supposed parental species,P. kawakamii andP. fortunei. F₁ seedlings, derived from reciprocal crosses between the suspected parents, exhibited identical banding patterns for a number of enzymes (such as SKDH, GOT, and IDH) withP. taiwaniana, when separated by electrophoresis. Furthermore, comparative morphological studies of trichomes and wood parenchyma patterns between the purported parents andP. taiwaniana reveal that this latter qualitatively exhibits characteristics that combine features of both of the suspected parental types. Biochemically, eight enzyme systems were compared in the three species here under discussion, and, without exception, the electrophoretic banding patterns exhibited byP. taiwaniana represented a combination of the alleles of the other two species. Perhaps the most convincing evidence comes from a genetic analysis of the progeny obtained by selfingP. taiwaniana. Genotypic segregation of the offspring based on a single locus each of SKDH and PGI fit the 1:2:1 hypothesis. Genotypic segregation of the offspring based on two loci each of SP and GOT fit the ratio of 3:6:3:1:2:1. This, taken in conjunction with the other data presented, clearly suggests thatP. taiwaniana is a hybrid involvingP. kawakamii andP. fortunei.     

Molecular authentication of ginseng cultivars by comparison of internal transcribed spacer and 5.8S rDNA sequences

Molecular authentication among three Panax species and within cultivars and accessions of P. ginseng was investigated using the DNA sequence in the ribosomal ITS1–5.8S–ITS2 region. Four single-nucleotide polymorphisms were identified between P. ginseng and other Panax species. In the electrophoresis profile, obtained after digestion with the enzyme TaqI, three fingerprinting patterns were obtained from cultivars and accessions of Panax species. Consequently, this authentication procedure based upon the restriction fragment length polymorphism in the ribosomal ITS1–5.8S–ITS2 region can now be utilized to differentiate these Panax species as well as major Korean cultivars such as Gopoong and Kumpoong from other cultivars and accessions in Panax species at the DNA level. O. T. Kim and K. H. Bang contributed equally to this paper.     

Comparison of Fusarium oxysporum and Fusarium oxysporum var. redolens by Analyzing the Isozyme and Serological Patterns

Extracts from Fusarium oxysporum (F.o.) and F. oxysporum var. redolens (F.o.r.) isolates were compared by means of electrophoresis and crossed immunoelectrophoresis. The polymorphism of five isozyme systems allowed a distinction between F.o. and F.o.r. isolates. The isozyme patterns of three other isozyme systems did not allow this distinction between F.o. and F.o.r. to be made. Both fungi appeared almost identical serologically. Relative amounts of their corresponding proteins differed but the qualitative patterns of the proteins were nearly the same with the anti-F.o.r. serum, only one specific antigen was detected in the extracts from F.o.r., isolates. Although the results obtained indicate a strong similarity between F.o. and F.o.r., they are not sufficient for an unequivocal statement that the fungi belong to the same species.