Purification and characterization of variant alcohol dehydrogenase isozymes from durum wheat

Three alcohol dehydrogenase (ADH) isozymes from embryos of the durum wheat cultivar Bijaga Yellow having the variantAdh-Alb allele were purified using (NH₄)₂SO₄ precipitation, gel filtration, and ion-exchange chromatography. ADH is a dimeric enzyme. The variant isozyme ADH-1-1, which is a homodimer composed of b monomers, was compared with ADH-1-5 (homodimer composed of a monomers), the product ofAdh-B1, and the ADH-1-3 isozyme (ba heterodimer) on a number of parameters includingK _m, substrate specificities, and molecular weights. No appreciable differences among the three isozymes were found, except for the faster electrophoretic mobility of bb dimers (ADH-1-1). The results indicate that the variant isozyme is the result of a mutation altering only the charge of the isozyme.     

Comparative chemical composition of the Barents Sea brown algae

Comparative study of phytochemical compositions of the most widespread brown algae species (one laminarian and four fucoid algae) from Barents Sea has been performed. A modified technique for mannitol determination in brown algae is proposed. It was revealed that fucus algae (fam. Fucaceae) contain 3% (of total dry weight) less mannitol than laminaria (Laminaria saccharina). The contents of alginic acid and laminaran in the Barents Sea fucoids are more than 10% less compared to laminaria. The alga L. saccharina contains almost two times more iodine than the species of fam. Fucaceae. The amounts of fucoidan and sum lipids in the Barents Sea fucoid algae is higher than in Laminaria saccharina (4–7% and 1–3%, respectively). In terms of contents of main biologically active compounds, fucus and laminarian algae from Barents Sea are inferior to none of the Far-Eastern species. The Barents Sea algae may become an important source of biologically active compounds.     

Degradation of Fucoidan by the Marine Proteobacterium Pseudoalteromonas citrea

It was found that Pseudoalteromonas citrea strains KMM 3296 and KMM 3298 isolated from the brown algae Fucus evanescens and Chorda filum, respectively, and strain 3297 isolated from the sea cucumber Apostichopus japonicus are able to degrade fucoidans. The fucoidanases of these strains efficiently degraded the fucoidan of brown algae at pH 6.5–7.0 and remained active at 40–50°C. The endo-type hydrolysis of fucoidan resulted in the formation of sulfated -L-fucooligosaccharides. The other nine strains of P. citrea studied (including the type strain of this species), which were isolated from other habitats, were not able to degrade fucoidan.     

A unique surface membrane anchored purine-salvage enzyme is conserved among a group of primitive eukaryotic human pathogens

Previously, we isolated and characterized the gene encoding the 3-Nucleotidase/Nuclease (Ld3NT/NU) from the human pathogen, Leishmania donovani. This unique cell surface enzyme has been shown to be involved in the salvage of host-derived purines, which are essential for the survival of this important protozoan parasite. In this report, we assessed whether the 3-Nucleotidase/Nuclease was conserved amongst other pathogenic Leishmania and related trypanosomatid parasites. Results of pulsed field gel electrophoresis and Southern blotting showed that a Ld3NT/NU gene homolog was present in each of the visceral and cutaneous Leishmania species tested (i.e. isolates of L. donovani, L. infantum, L. tropica, L. major and L. mexicana, respectively). Further, results of colorimetric assays using 3-adenosine monophosphate as substrate demonstrated that each of these organisms also expressed significant levels of 3-nucleotidase enzyme activity. In addition, we showed that a Ld3NT/NU gene homolog was expressed in each of these Leishmania species as a > 40 kDa 3-nucleotidase enzyme activity. A Ld3NT/NU gene homolog was also identified in two Crithidia species (C. fasciculata and C. luciliae) and Leptomonas seymouri but was only marginally detectable in Trypanosoma brucei, Trypanosoma cruzi and Phytomonas serpens. Cumulatively, results of this study showed that an Ld3NT/NU homolog was conserved amongst pathogenic Leishmania sp. which suggests that this enzyme must play an critical role in purine salvage for all members of this group of human pathogens.     

Population differentiation in Collinsia verna Nuttal (Scrophulariaceae): a multifaceted approach

Three species of Collinsia (commonly called Blue-eyed Mary, Innocence, Chinese Houses) including 12 populations of C. verna were examined for genetic variability and differentiation using three investigative techniques: enzyme electrophoresis, chiasma frequencies, and morphological measurements. Analysis of isozyme data showed higher heterozygosity was maintained in the C. verna populations (previously reported to be self pollinating) than in C. heterophylla and C. tinctoria, which are outcrossers. C. heterophylla and C. tinctoria were separated by a significant genetic distance from each other and especially from C. verna. Within C. verna isozyme, morphological, and chiasma frequency analyses separated the populations into subsets showing some geographical trends, possibly influenced by water dispersal of seeds. High genetic variability was maintained and differentiation has occured in the C. verna populations. The explanation may lie in a combination of factors associated with the heterozygosity paradox including microgeographical influence and inappropriate mating system classification.     

Gene duplication in salmonid fishes: Evidence for duplicated but catalytically equivalent A4 lactate dehydrogenases

Skeletal muscle tissues from many species of salmonid fish are known to exhibit a set of three to five isozymes for A₄-type lactate dehydrogenase (l-lactate: NAD oxidoreductase, E.C. 1.1.1.27), but the genetic basis for this isozyme system has not previously been assessed. This isozyme system was purified to homogeneity from salmon (Oncorhynchus tshawytscha) and shown to be composed of two polypeptides, A and A, in binomial tetrameric combinations. Amino acid analysis revealed that A and A are closely related but genetically distinct proteins, and thus are coded for by recently duplicated structural loci. Catalytic studies on the purified intact salmon isozyme system and on the isozymically pure A₄ and A₄ homotetramers from brown trout (Salmo trutta) revealed no significant differences in catalytic properties among these enzymes, suggesting equivalent catalytic function of A and A. These results, in combination with studies on polypeptide and isozyme ratios, suggest that one of the duplicated loci in salmon may be drifting toward a nonfunctional state by accumulation of mutations in regulatory DNA rather than in the structural gene itself.This work was supported in part by research grants from the University Grants Committee, the University of Otago Research Committee, and the Medical Research Council of New Zealand. Part of this work was performed while one of us (S. T. L.) was supported by predoctoral fellowships from the Medical Research Council of New Zealand and from the Lee Foundation.     

Identification and isolation of a unique esterase from the medium of non-embryogenic cell line of cultured carrot cells

A unique esterase isozyme z with very low electrophoretic mobility on the anionic polyacrylamide gel (PAGE) was found in the medium of a non-embryogenic (Ca-4) line of cultured carrot (Daucus carota L.) cells. The protein corresponding to this esterase isozyme z was purified by electroelution from preparative PAGE and the esterase migrated as a single band with an apparent M _r of 35 000 on SDS-PAGE. The purified esterase isozyme z exhibited at least 350-fold higher specific activity than that in the total medium proteins.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Associations between isozyme phenotypes and environment in the slender wild oat (Avena barbata) in Israel

Summary Collections from 31 populations of A. barbata from diverse habitats in Israel were assayed electrophoretically for seven enzyme systems. Phenotype frequencies were scored in nine enzyme zones, probably representing 27 loci, to determine isozyme variability within and among populations. Many different isozyme phenotypes were found in all of the populations; also the array of isozyme phenotypes found in each population differed distinctly from that found in each other population. Overlays of phenotypic frequencies on map locations showed that isozyme variability is distributed in mosaic patterns not related to geographical distance. Principal-component and multiple-regression analyses revealed that temperature and moisture-related variables are significantly correlated with particular isozyme phenotypes. Further, the mosaic patterns of isozyme variation were found to correspond closely to mosaic patterns of the habitat. This structuring of the genetic variability into multilocus combinations was attributed to the combined effects of directional and diversifying selection. Comparisons of patterns and extent of genetic variation in Israel and California led to the conclusion that the evolution of ecotypes, each adapted to a specific habitat and marked by a particular set of enzyme alleles, has proceeded further in Israel, where A. barbata is endemic, than in California, where it is a recent introduction.This study was supported in part by NSF Grant BMS-01113-A01. Seed collections were supported by a United States-Israel Binational Science Foundation Grant     

Characterization of active barley alpha-amylase 1 expressed and secreted by Saccharomyces cerevisiae

Recombinant barley -amylase 1 isozyme was constitutively secreted by Saccharomyces cerevisiae. The enzyme was purified to homogeneity by ultrafiltration and affinity chromatography. The protein had a correct N-terminal sequence of His-Gln-Val-Leu-Phe-Gln-Gly-Phe-Asn-Trp, indicating that the signal peptide was efficiently processed. The purified -amylase had an enzyme activity of 1.9 mmol maltose/mg protein/min, equivalent to that observed for the native seed enzyme. The k _cat/K _m was 2.7 × 10² mM^–1.s^–1, consistent with those of -amylases from plants and other sources.     

Nucleotide sequence and phylogenetic implication of the ATPase subunits β and ε encoded in the chloroplast genome of the brown alga Dictyota dichotoma

We have cloned and sequenced the genes atpB and atpE, coding for CF₁ subunits and , respectively, of the chloroplast genome of the brown alga Dictyota dichotoma. Although the coding site of atpE cannot be demonstrated by heterologous Southern hybridizations, a 417 bp reading frame 3 to atpB was identified as the gene atpE by sequence similarities with atpE genes from other sources. A maximum sequence identity of 30% is found between the predicted amino acid sequence of the Dictyota subunit and the corresponding cyanobacterial subunits. Including conserved amino acid replacements, the Dictyota subunit exhibits about 70% sequence similarity with the cyanobacterial and land plant subunits. As in cyanobacteria, the atpE gene does not overlap the preceding gene atpB. The deduced amino acid sequence of atpB is 74–79% identical to the corresponding cyanobacterial and chloroplast subunits. Entirely conserved are regions referred to as the catalytic and/or regulatory sites of ATP formation, including interacting regions between subunits and . A phylogram predicted from F₁/CF₁- subunits of eleven different organisms suggests a common evolutionary origin of plastids from chlorophytes and brown algae.     

Effect of the Meristem on Sporification of Laminaria cichorioides

Isolated disks from vegetative thalli of Laminaria cichorioides Miyabe collected in April in Amursky Bay, Sea of Japan, cultivated separately from meristematic sites, developed sporangial sori after 4–6 weeks of cultivation, i.e., 4 months earlier than in natural conditions. The development of reproductive organs in intact plants, on meristematic sites, and also on vegetative disks, cultivated together with meristematic sites, was not observed. The effect of the meristem on L. cichorioides reproduction is supposed to be conditioned by inhibitors of sporification produced by this tissue. We offer a mechanism of sporification regulation for laminarian algae in natural conditions.     

Identification of the protein products of the rrnC, ilv, rho region of the Escherichia coli K-12 chromosome

Summary Two methods have been used to identify the protein products of the Escherichia coli K-12 ilv region at 84 min and the flanking rrnC (counterclockwise) and rho (clockwise) loci. First, a set of dilv specialized transducing phages, including some phages that carry rho and others that carry part of rrnC, was used to infect UV irradiated cells. The proteins produced by the infecting dilv phage were selectively labelled with radioactive amino acids and identified by SDS gel electrophoresis and autoradiography. Second, restriction enzyme fragments were cloned from the dilv phage into pBR322 and the plasmid specific gene products produced in maxicells were similarly identified by SDS gel electrophoresis and autoradiography. The proteins produced were correlated with specific genes and restriction enzyme fragments present in the dilv phage and the pBR322 derivatives. Several ilv gene products that have previously been refractory to protein purification attempts have been identified for the first time by this technique. The presence of mutations at the ilvO site is shown to activate the cryptic ilvG gene and to result in the production of a 62,000 dalton protein. A 15,000 dalton protein of unknown function is synthesized from a DNA segment between ilv and rrnC. The rho gene was cloned from dilv phage into pBR322 and shown to be dominant to a rho mutation on the host cromosome. The rho gene product and four additional proteins coded by genes near or between rho and ilv have been detected.     

Convergent evolution masks extensive biodiversity among marine coccoid picoplankton

Coccoid picoplankters are minute unicellular algae that, when viewed with a light microscope, appear as little balls. They comprise an important component of open-ocean phytoplankton, and, except for colour differences (i.e. red, green, brown), many eukaryotic picoplankters are morphologically similar. To evaluate the biological diversity of the little brown ball subpopulation of little balls, we randomly selected nine undescribed algal strains and compared the nucleotide sequences of their nuclear 18S ribosomal RNA genes. The results indicate that little brown balls have evolved independently in three distinct eukaryotic lineages (heterokont algae, haptophyte algae, and green algae), and at least four taxonomic classes, and that, even within the four classes, considerable genetic diversity exists. These findings suggest that a tiny coccoid morphology confers some adaptive advantage in the open ocean, that repeated convergent evolution has occurred, and that molecular data may be necessary for taxonomic distinction of closely related coccoid picoplankters.     

Chromosome number evolution in the tribeBrassiceae (Brassicaceae): Evidence from isozyme number

Variation in isozyme number was used to assess the evolution of haploid chromosome numbers (n=6–75) and systematic relationships in the tribeBrassiceae, which is believed to be one of the few monophyletic tribes in theBrassicaceae. Ten enzyme systems were surveyed among 108 species in 35 genera of tribeBrassiceae and for 11 species from seven other tribes. The data indicated that taxa with n=7–13 and n=14–18 were similar in isozyme number, suggesting that genera with n=14–18 did not arise from polyploidy (i.e. entire duplication) of the n=7–13 genomes. These results suggest that aneuploidy and/or chromosome fusion/splitting have played a more significant role than polyploidy in the evolution of higher base chromosome numbers in the tribe. The detection of widespread isozyme duplication in the tribe is consistent with reports of extensive gene duplication in theBrassica crop species, and suggests that the common ancestor of the tribe already had undergone a polyploid event, i.e. complete genome duplication, prior to aneuploid divergence. Inheritance studies conducted onSinapis arvensis showed that segregation ratios at seven loci (Fbp-2,Gpi-2,Idh-2,Pgm-2,Pgm-2,Tpi-1,Tpi-1) conformed to those expected under Mendelian inheritance. Isozyme duplications were phylogenetically informative at various taxonomic levels in the tribe. In particular, duplications for cytosolic phosphoglucomutase (Pgm-2,Pgm-2) and plastid triosephosphate isomerase (Tpi-1,Tpi-1) were evident in 33 of the 35 genera examined, supporting the monophyletic status of theBrassiceae with the inclusion ofOrychophragmus and the exclusion of controversial membersCalepina andConringia.     

Genetic mapping of a quantitative trait locus (QTL) that enhances the shoot differentiation rate in Hordeum vulgare L.

A quantitative trait locus (QTL) controlling shoot differentiation from immature embryo callus was identified by linkage analysis with morphological and isozyme markers in barley, Hordeum vulgare L. Immature embryos were isolated from cvs Azumamugi (difficult to differentiate), Kanto Nakate Gold (easy to differentiate), their hybrids (F₁) and a backcross population derived from a cross Azumamugi x F₁. The embryos were cultured in vitro for callus initiation and subsequent shoot differentiation. The shoot differentiation rate was closely associated with ear type (v locus), isocitrate dehydrogenase isozyme (Idh-2), and esterase isozyme (Est-11). These markers were found to reside in a chromosome segment of approximately 30cM on chromosome 2. Recombination frequency was 9.9% between v and a proposed QTL named Shd1 (shoot differentiation), 11.5% between Idh-2 and Shd1, and 21.3% between Est-11 and Shd1. All data showed the Idh-2, v, Shdl and Est-11 loci to be arranged in this order from proximal to distal on the long arm of chromosome 2.     

α-Amylase structural genes in rye

Summary Rye -Amy1, -Amy2, and -Amy3 genes were studied in the cross between inbred lines using wheat -amylase cDNA probes. The -Amy1 and -Amy2 probes uncovered considerable restriction fragment length polymorphism, whereas the -Amy3 region was much more conserved. The numbers of restriction fragments found and the F₂ segregation data suggest that there are three -Amy1 genes, two or three -Amy2 genes, and three -Amy3 genes in rye. These conclusions were supported by a simultaneous study of -amylase isozyme polymorphism. The F₂ data showed the three individual -Amy1 genes to span a distance of 3cM at the locus on chromosome 6RL. The genes were mapped relative to other RFLP markers on 6RL. On chromosome 7RL two -Amy2 genes were shown to be separated by 5 cM. Linkage data within -Amy3 on 5RL were not obtained since RFLP could be detected at only one of the genes.     

Evidence for a composite phylogenetic origin of the plastid genome of the brown alga Pylaiella littoralis (L.) Kjellm

The nucleotide sequence and the 5 flanking region of the rbcL gene coding for the large subunit of ribulose bisphosphate-1,5-carboxylase/oxygenase of Pylaiella littoralis, a brown alga, has been determined and the deduced amino-acid sequence has been compared to those of various photosynthetic and chemoautotrophic Eubacteria, of a red alga and of green plastids (Euglena gracilis, green algae and higher plants). Unlike the rbcL genes of green plastids which are more closely related to those of cyanobacteria the P. littoralis rbcL gene is more closely related to that of a -purple bacterium, as was found for the rbcS gene of another chromophytic alga [Boczar et al., Proc Natl Acad Sci USA 86: 4996–4999, 1989]. Matrix data of homology between the rbcL gene of P. littoralis and the same gene of other organisms are presented. Based on our previous report, the gene coding for the 16S rRNA from P. littoralis is closely related to that of E. gracilis (Markowicz et al., Curr Genet 14: 599–608, 1988). We suggest that the large plastid DNA molecule of P. littoralis is a phylogenetically composite genome which probably resulted from mixed endosymbiosis events, or from a horizontal transfer of DNA.     

Digestion of seaweeds by the marine amoeba Trichosphaerium

A crude enzyme preparation from the marine amoeba Trichosphaerium was used to produce protoplasts from Sargassum muticum, Macrocystis pyrifera Porphyra perforata, and other red and brown marcroalgae. Cortical and medullary protoplasts of Sargassum, which were impossible to obtain using mixtures of previously available enzymes have now been prepared. Intact inner cortical and medullary protoplasts of Macrocystis, which were not observed in past isolations were obtained. Improved protoplast yields of as much as 500 fold resulted from feeding the amoebae on specific seaweeds. Cuticles of live Sargassum and Macrocystis were digested easily by the amoebae. However cuticles of autoclaved Macrocystis and those of Porphyra (fresh or autoclaved) were eaten last. In spite of the absence of identifiable extracellular enzymatic activity in the medium the amoebae were able to cut and consume live fronds and blocks of gelled agars carrageenans, and alginates.     

Biochemical composition of three algal species proposed as food for captive freshwater mussels

To identify potential diets for rearing captive freshwater mussels, the protein, carbohydrate (CHO), and lipid contents of two green algae, Neochloris oleoabundans, Bracteacoccus grandis, and one diatom, Phaeodactylum tricornutum, were compared at different growth stages. The fatty acid and sterol composition were also identified. Protein was greatest (55–70%) for all species at late log growth stage (LL), and declined in late stationary (LS) growth. CHO was greatest at LS stage for all species (33.9–56.4% dry wt). No significant change in lipid levels occurred with growth stage, but tended to increase in N. oleoabundans. Mean lipid content differed significantly in the order: N. oleoabundans > P. tricornutum > B. grandis. Total fatty acids (TFA) were higher at LS stage compared to other stages in the two green algae, and stationary stage in the diatom. Mean unsaturated fatty acids (UFA) as %TFA was significantly higher in N. oleoabundans than the other species. The green algae contained high percentages of C-18 polyunsaturated fatty acids (PUFAs), while the diatom was abundant in C-16 saturated and mono-unsaturated fatty acids and C-20 PUFA fatty acids. Growth stage had no effect on sterol concentration of any species. B. grandis showed significantly higher sterol levels than the other species except P. tricornutum at S stage. B. grandis was characterized by predominantly ⁵, C-29 sterols, while N. oleoabundans synthesized ^5,7, ^5,7,22 , and ⁷, C-28 sterols. P. tricornutum produced primarily a ^5,22, C-28 sterol, and a small amount of a ^7,22, C-28 sterol.