Electrophoretic variation in glutamate dehydrogenase and other isozymes in wild carrot cells cultured in the presence and absence of 2,4-dichlorophenoxyacetic acid

Summary Electrophoretic analysis of isozymal differences was performed with extracts of wild carrot (Daucus carota L.) cells, grown in the presence and absence of 2,4-dichlorophenoxyacetic acid (2,4-D). There were no differences in the patterns of malate dehydrogenase, acid phosphatase, aspartate aminotransferase, and γ-glutamyl transferase. Quantitative differences in peroxidase isozymes were detected, the plus 2,4-D cultures having lower activities. Esterase patterns were similar, but there were differences in individual isozyme activities and an additional form present in the minus 2,4-D cells. the greatest differences were in patterns of glutamate dehydrogenase with the minus 2,4-D cultures containing only the slowly migrating isozymes. The changes in glutamate dehydrogenase, as revealed by isozyme changes, together with the requirement for ammonia in embryogenesis, suggests that this enzyme may be associated with differentiation in wild carrot cells.     

Genetic Diversity Among Iranian Isolates of Rhizoctonia solani Kühn Anastomosis Group1 Subgroups Based on Isozyme Analysis and Total Soluble Protein Pattern

Rhizoctonia solani is a destructive fungal pathogen with a wide host range. The R. solani complex species includes several divergent groups delimited by affinities for hyphal anastomosis. In this study, genetic variation among 20 isolates of R. solani anastomosis group 1 (AG1) subgroups (AG1‐IA and AG1‐IB) collected from Mâzandaran province, Iran, and standard isolates of these subgroups, was determined by isozyme analysis and total soluble protein profile. Mycelial protein pattern and isozyme analysis were studied using denaturing and non‐denaturing polyacrylamide gel electrophoresis, respectively. A total of 15 enzyme systems were tested, among which six enzymes including esterase, alkaline phosphatase, superoxide dismutase, octanol dehydrogenase, lactate dehydrogenase and mannitol dehydrogenase generated distinct and reproducible results. The soluble protein patterns were similar among the R. solani isolates examined; however, minor differences in banding pattern were observed between the two subgroups. In isozyme analysis, a total of 64 electrophoretic phenotypes were detected for all six enzymes used. Based on cluster analysis and similarity matrix, the fungal isolates were divided into two genetically distinct groups of I and II consistent with the previously reported AG1‐IA and AG1‐IB subgroups in AG1. Group I represented all isolates belonging to AG1‐IA subgroup, whereas group II represented all isolates belonging to AG1‐IB subgroup. Results from isozyme analysis suggest that the subgrouping concept within AGs is genetically based.     

Aluminum stress in the roots of naked barley

Phytotoxicity of aluminum (Al) is the major limiting factor for the crops grown in acid soils rapidly inhibiting root elongation. In this study, changes in root growth, total activity and isozyme patterns of antioxidant enzymes such as peroxidase, ascorbate peroxidase, catalase and glutathione reductase by Al stress were investigated in the roots of naked barley (Hordeum vulgare L. cv. Kwangwhalssalbori). As Al concentration increased up to 500 M, the rooting rate and root elongation substantially decreased. Growth results suggested that this cultivar is an Al-sensitive species. Total activities of antioxidant enzymes generally increased at lower Al concentrations and then gradually decreased at higher Al concentrations. They also increased when the exposure time to Al was extended up to 48 hr. Changes in the isozyme patterns of antioxidant enzymes were investigated byin situ enzyme activity staining on a non-denaturing PAGE gel. They generally coincided with the changes in the total activity in parallel. Changes in the total activity of antioxidant enzymes also coincided with the changes of the root growth. Since growth reduction in the roots by Al stress could be related with the changes in the activities of antioxidant enzymes, these results suggested that Al might cause the oxidative stress in the roots of this cultivar of naked barley.     

斯氏并殖吸虫乳酸脱氢酶,苹果酸脱氢酶及酯酶同工酶的研究

本文采用Disc-PAGE电泳,首次对我国独有的斯氏并殖吸虫(Paragonimus skrjabini Chen,1959)成虫、童虫、囊蚴的乳酸脱氢酶(以下简称LDH)、苹果酸脱氢酶(以下简称MDH)和酯酶(以下简称EST)同工酶进行了研究。 在成虫、童虫、囊蚴间,LDH、MDH、EST同工酶在酶带数、排列型式、Rf值、相对活性和优势酶带的位置都存在差异。 根据虫体和宿主组织同工酶谱的不同,可以认为是本虫本身所具有。 同工酶作为其分类指标时,不仅要比较不同虫种成虫稳定的同工酶谱,也要比较同工酶在个体发育型式间的差异。     

Variation of isozyme patterns among Arachis species

The genus Arachis contains a large number of species and undescribed taxa with patterns of genetic variation that are little understood. The objectives of this investigation were to estimate genetic diversity among species of Arachis by utilizing electrophoretic techniques and to establish the potential for use of isozymes as markers for germplasm introgression. One-hundred-and-thirteen accessions representing six of the seven sections of the genus were analyzed for isozyme variation of 17 enzymes. Section Rhizomatosae species were not included because they produce very few seeds. Seeds were macerated and the crude extract was used for starch-gel electrophoretic analyses. Although the cultivated species has few polymorphic isozymes, the diploid species are highly variable and two-to-six bands were observed for each isozyme among accessions. Because of the large number of isozyme differences between A. hypogaea and A. batizocoi (the presumed donor of the B genome), this species can no longer be considered as a progenitor of the cultivated peanut. Seed-to-seed polymorphisms within many accessions were also observed which indicate that germplasm should be maintained as bulk seed lots, representative of many individuals, or as lines from individual plants from original field collections. The area of greatest interspecific genetic diversity was in Mato Grosso, Brazil; however, the probability of finding unique alleles from those observed in A. hypogaea was greatest in north, north-central, south and southeast Brazil. The large number of polymorphic loci should be useful as genetic markers for interspecific hybridization studies.     

Developmental genetics of teleosts: a biochemical analysis of lake chubsucker ontogeny

Developing embryos of the lake chubsucker, Erimyzon sucetta, were analyzed with regard to both gross morphological changes and specific enzymatic changes from the unfertilized egg stage until some 3 weeks posthatching. Total activities of three enzymes—lactate dehydrogenase, glucose-6-phosphate dehydrogenase, and isocitrate dehydrogenase—were determined throughout the course of development. Each of these different enzymes exhibited a different pattern of change during ontogeny. Electrophoretic analysis of qualitative changes in isozyme patterns was accomplished for these three enzymes and for α-amylase, glucosephosphate isomerase, mannosephosphate isomerase, creatine kinase, esterase, glutamate dehydrogenase, alkaline phosphatase, aspartate aminotransferase, malate dehydrogenase, hexose diphosphatase, phosphoglucomutase, and phosphogluconate dehydrogenase. Many of the enzyme systems investigated exhibited rich patterns of ontogenetic change, while a few remained relatively unchanged throughout the interval studied. Several of the enzymes in particular metabolic pathways exhibited coincident changes suggestive of coordinate control. The appearance of several rather “tissue-specific” isozymes was closely correlated with the morphological and functional differentiation of these particular tissues or organs.     

Patterns of genetic variability within and among populations of wild radish,Raphanus raphanistrum (Brassicaceae)

The genetic structure of populations is an important determinant of the evolutionary potential of a species. Colonizing plants tend to be characterized by low within- and high among-population variability. Genetic differentiation of both floral traits and isozymes was studied in six populations of wild radish (Raphanus raphanistrum). Evidence for differentiation in both sets of traits was found, but patterns of differentiation of floral traits did not coincide with isozyme differentiation. Contrary to most colonizing species, wild radish showed high within- and only moderate among-population variability at isozyme loci. In addition, levels of differentiation did not correspond to geographic distance between the populations. These results are likely due at least in part to the self-incompatibility system of this species, long-distance movement of large numbers of wild radish seeds by humans, and introgression from cultivated radish (R. sativus).     

Isozymic Characterization of Three Mating Groups of the Tetrahymena pyriformis complex*

SYNOPSIS. Strains of 3 unnamed mating groups of the Tetrahymena pyriformis complex have been subjected to starch gel electrophoresis followed by staining the gels for the enzymes isocitrate dehydrogenase (NADP), tyrosine aminotransferase, and tetrazolium oxidase (superoxide dismutase). With respect to the electrophoretic mobilities of these enzyme systems, the mating groups referred to here as 5, 13 and 14 are very similar to Tetrahymena americanis (syngen 2), the most common North American species of the complex. Cultures in our collection labeled Tetrahymena cosmopolitans (formerly syngen 4) are either amicronucleate, with unique isozyme patterns, or micronucleate cells which mate with and have isozyme patterns similar to Tetrahymena canadensis (syngen 7). Immature progeny have been derived from crosses between the latter strains and T. canadensis recently collected in Colorado. The amicronucleate strains are now placed in the Tetrahymena sp. category, and we conclude that strains identifiable as T. cosmopolitanis are no longer available. The reliability of isozymes as characters in ciliate taxonomy was evaluated by comparing the present results for 3 enzymes in 15 groups of strains (syngens and phenosets) that had been compared in an earlier study. These enzyme systems gave correlation coefficients (r) of 0.75 or higher in the separate studies, and can be considered useful diagnostic traits. Other enzymes that were present at threshold levels of detectability or varied highly in concentration from species to species are too unreliable to be of diagnostic value. Some of the strains in the complex are so evolutionarily divergent at the molecular level that we have difficulty finding growth and electrophoretic conditions under which orthologous enzyme activities can be detected simultaneously for all the strains being compared.     

Studying genetics of adaptive variation in model organisms: flowering time variation in Arabidopsis lyrata

Arabidopsis thaliana has emerged as a model organism for plant developmental genetics, but it is also now being widely used for population genetic studies. Outcrossing relatives of A. thaliana are likely to provide suitable additional or alternative species for studies of evolutionary and population genetics. We have examined patterns of adaptive flowering time variation in the outcrossing, perennial A. lyrata. In addition, we examine the distribution of variation at marker genes in populations form North America and Europe. The probability of flowering in this species differs between southern and northern populations. Northern populations are much less likely to flower in short than in long days. A significant daylength by region interaction shows that the northern and southern populations respond differently to the daylength. The timing of flowering also differs between populations, and is made shorter by long days, and in some populations, by vernalization. North American and European populations show consistent genetic differentiation over microsatellite and isozyme loci and alcohol dehydrogenase sequences. Thus, the patterns of variation are quite different from those in A. thaliana, where flowering time differences show little relationship to latitude of origin and the genealogical trees of accessions vary depending on the genomic region studied. The genetic architecture of adaptation can be compared in these species with different life histories.     

ISOZYME NUMBER IN SUBTRIBE ONCIDIINAE (ORCHIDACEAE): AN EVALUATION OF POLYPLOIDY

The Oncidiinae has attracted attention because of the variation it exhibits in chromosome number, n = 5–30, which is greater than the range in the rest of the Orchidaceae. The genus Psygmorchis, with n = 5 and 7, has been a particular focus of controversy, and many authors have suggested that 5 and 7 are the base numbers for the subtribe. The other taxa in the subtribe presumably evolved through hybridization and polyploidy. Other workers have found that the lowest counts correlate with derived morphological conditions and have hypothesized that these low numbers result from aneuploid reductions, while higher numbers are associated with ancestral morphologies and are not the result of polyploidy. These two hypotheses were evaluated by determining isozyme numbers for 13 enzymes in species that span the chromosomal range known for the Oncidiinae (n = 5–30). Isozyme number has been shown to be a reliable indicator of polyploidy in angiosperms because polyploids display isozyme multiplicity relative to diploids. This analysis revealed no differences among species in isozyme number for the enzymes examined. Therefore, our data reject the hypothesis that species with higher chromosome numbers are polyploid.     

Differential expression of esterase and MDH isozymes during in vitro culture in maize (Zea mays L.)

Isozyme patterns of esterase and malate dehydrogenase were analyzed at different stages of in vitro culture of immature embryos and glumes of Zea mays L. viz. explant, callus formation, root formation and shoot formation. Significant changes in isoenzyme patterns of esterase and MDH were observed besides the appearance of specific and new isozymes. Specific fast migrating isozymes were noted in differentiating calli of embryo and glume calli which were absent at other stages suggesting a possible association of these isozyme patterns with in vitro differentiation.     

Phenetic groupings in bees of the tribe bombini based on the enzyme α-glycerophosphate dehydrogenase

GDH isozyme patterns from thoracic muscle of 21 species of Bombus and 4 species of Psithyrus are each characterized by at least 8 bands arranged in 3 isozyme groups. Four distinctive pattern types were found, one for the species of Psithyrus and three among the Bombus species analyzed. Phenetic groupings of the 25 species are presented. Tissue specific GDH patterns are reported. No quantitative or qualitative differences in GDH patterns from thoracic extracts were associated with caste or age. The enzymes appear to be controlled by four non-allelic genes.     

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.     

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes in tissues and callus cultures ofCereus peruvianus (cactaceae)

Malate dehydrogenase (MDH; EC 1.1.1.37) isozymes were investigated in seeds and in seedlings and calli cultures ofC. peruvianus to determine if the changes in MDH isozyme banding patterns could be used as biochemical markers to identify the origin of regenerated plants from callus tissues. Four cytoplasmic MDH isozymes (sMDH), five mitochondrial MDH isozymes (mMDH), and one glyoxysomal MDH isozyme (gMDH) were detected and showed tissue- and stage-specific expression. A relationship of mMDH and gMDH isozyme patterns with callus tissues subcultured in three hormonal combinations and with the plants regenerated from these callus tissues was demonstrated. Furthermore, temperature and mechanical stress were found to be closely related to mMDH-1 activity in callus culture. Therefore, the different patterns of MDH isozymes in the various tissues ofC. peruvianus can be used as biochemical markers for the study of gene expression during development and as powerful tools in monitoring studies on callus cultures. This research was supported by the CNPq.     

Energy Metabolism in Fish Development

SYNOPSIS. During oogenesis the fish oocytes accumulate severalsubstances of which lipids and glycogen are the major energysubstrates. Oocyte maturation is accompanied by an increasein all the enzymes of carbohydrate metabolism. After fertilization,respiration and glycogenolysis are increased and the energycharge is decreased. During early embryogenesis glycogen appearsto be the only substrate of glycolysis. Glycolysis and the citricacid cycle are the main sources of energy for the biosyntheticactivities and for the maintenance of embryo morphology. Thereare two patterns of ontogeny of glycolytic enzymes in troutembryos. One group of enzymes does not undergo appreciable changeswhereas enzymes within the second group exhibit variable activities.Marked changes in enzyme activity occur during fertilizationand gastrulation. Lactate dehydrogenase (LDH) is of particularinterest. Its activity increases during gastrulation. This increasein LDH activity is followed by a change in the isozyme patternand in the adenylate charge. 1mmunochemical and histochemicallocalization of LDH revealed that its cellular distributiondepends on the position of the cells in the embryo. Moving cellshad higher levels of LDH activity. The lactate dehydrogenaseisozymes appear to play an important role in the regulationof energy metabolism during fish embryogenesis. These gene productsare useful biochemical markers of cellular differentiation andorganogenesis.     

A labile acid phosphatase isozyme associated with the surface of vegetative Dictyostelium discoideum cells

A minor acid phosphatase isozyme (acid phosphatase I) of vegetative Dictyostelium discoideum amebae has been shown to be associated exclusively with the external surface of the plasma membrane. The isozyme is not present in phagocytic vacuoles isolated with latex beads.The isozyme disappears from cells removed from nutrient medium and does not reappear during differentiation. When inhibitors of protein synthesis (e.g. cycloheximide, choral hydrate, concanavallin A) are added to cells growing in nutrient medium, acid phosphatase I is rapidly lost. It appears that the level of protein synthesis need only be moderately reduced (less than 25%) to induce loss of enzyme activity. Treatment with inhibitors of DNA and RNA synthesis for up to 2 h had no effect on isozyme activity. It is postulated that the cells are able to “sense” (through the reduction in levels of protein synthesis) when external conditions become unfavorable, and immediately respond by reducing the activity of enzymes involved in maintaining contact with the extracellular environment. The closed system thought necessary for differentiation would then be created.     

DUPLICATE GENE EXPRESSION FOR ISOCITRATE DEHYDROGENASE AND 6-PHOSPHOGLUCONATE DEHYDROGENASE IN DIPLOID SPECIES OF ELEUSINE (GRAMINEAE)

In the course of a survey of isozyme variation in the grass genus Eleusine, complex band patterns were observed for the enzymes isocitrate dehydrogenase (IDH) and 6-phosphogluconate dehydrogenase (6PGD). These patterns were interpreted as the result of duplicate expression for one of the two genes that ordinarily codes subcellularly compartmentalized forms of each of these enzymes. The interpretation of IDH phenotypes was facilitated by intraspecific allelic variation at one of the putatively duplicated genes (Idh-2), and was verified by examining phenotype ratios in progeny arrays from selfed heterozygotes of E. indica. The lack of analogous intraspecific variation for 6PGD precluded genetic tests, but the duplicate nature of expression was supported by interspecific patterns of variation. Four out of the five diploid species of Eleusine studied (E. indica, E. jaegeri, E. multiflora, E. tristachya) exhibited both duplications; E. floccifolia appeared to lack the IDH duplication, but possessed the 6PGD duplication. Both enzymes showed evidence of hyperduplication in the tetraploid species E. coracana.     

Evaluation of isozyme systems in Citrus to facilitate identification of fusion products

Summary Ten isozymes were analyzed in nucellar calli of nine Citrus species and cultivars and roots of the corresponding apomictic seedlings. The zymograms obtained can be divided into three groups: a) isozyme patterns similar in both calli and roots, b) isozyme patterns similar in calli but variable in roots, and c) isozyme patterns variable in both calli and roots. Analysis of these ten isozyme systems may facilitate identification of fusion products in Citrus.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel. No. 354-E, 1982 series     

基于过氧化物同工酶分析月季种质资源的亲缘关系及杂种真实性

为了提高种质资源利用率,加速月季育种进程,对27份月季种质及3个杂交组合的8个杂交后代,采用过氧化物酶(POD)同工酶方法分析其亲缘关系并进行杂种真实性鉴定。结果表明:月季种质采用POD同工酶分析具有一定的可行性。酶谱分析中,在相对迁移率为0.264~0.858的位点处共获得7条酶带,其中共有酶带3条,特征酶带4条,表明不同月季种质间遗传多样性丰富,但又存在一定同源性。基于酶带特征进行聚类分析,在相似系数为0.57处,可将27份供试材料分为3个大组。合柱组与月季组材料聚在一个大组中,两个组在形态上的相似性再次得到确认。金樱子与硕苞蔷薇分别聚在两个不同的组中,表明二者之间的亲缘关系较远。供试的古老月季品种被聚在两个不同的大组中,与野生种聚成的一组呈平行关系,表明古老月季在起源上的差异较大,可利用其作为杂交亲本进行广泛杂交以选育具有丰富遗传多样性的杂交后代。根据有无父本特征酶带对杂种后代真实性进行鉴定,初步确定2个杂交组合的6个杂交后代中5个为真实杂种,1个为自交种。该研究结果为进一步开展月季遗传育种奠定了基础。     

Isozyme variation in Verticillium dahliae isolates from Crete

Fifteen isolates ofVerticillium dahliae (eight of race1, seven of race2; most from the island of Crete, Greece) were examined for isozyme and molecular variation. Among the isozyme banding patterns (zymograms) of six enzymes that were “activity-stained” after electrophoresis in 9% polyacrylamide gels, differences were observed in diaphorase, α-esterase, peroxidase and superoxide dismutase; 2, 2, 3 and 5 different types of zymograms were recorded, respectively. The zymograms could not be correlated with either race1 or2. However, all six isolates originating from the Oropedio (plateau) are, of Lasithi (Crete) showed an esterase zymogram clearly distinguishable from the other isolates. No differences were observed when staining for acid phosphatase or aspartate aminotransferase (‘glutamic-oxaloacetic transaminase’). Furthermore, electrophoresis of random-amplified polymorphic DNA (RAPD) in 2% agarose gels showed that three race-2 isolates from Oropedio of Lasithi could also be distinguished by the RAPD pattern generated with primer OPA-1. The variation observed possibly represents adaptation ofV. dahliae to the Oropedio environment.