Immunofluorescent study of the basic chromosomal protein set of green algae and Euglena]

Using antisera to fractions H1, H2a, H3 and H4 of the calf thymus histones, a comparative immunofluorescent investigation of these proteins in the nuclei of Chlamydomonas reinhardii, Haematococcus pluvialis, Dunaliella salina and Euglena gracilis was carried out. It has been shown that according to the immunofluorescent test, the nuclei of these algae contain proteins close to fractions H2a, H3 and H4 of the calf thymus histones. H1 fraction in these algae is either absent or can be considered as a protein immunochemically non-related to H1 fraction of the calf thymus histone. For quantitative evaluation (in units of the immunological distance) of the difference between histones of the algae and of the calf thymus in situ by indirect immunofluorescence, it was suggested to use the ultimate dilutions of antisera to histones. It was shown that the ultimate dilutions were correlated with titres of antisera in the reaction of microcomplement fixation. Such an approach and the data obtained are of interest for studying into the evolution of nucleosome histones in unicellular and multicellular eukaryotes.     

DNA methylation in wheat. Purification and properties of DNA methyltransferase

The origin and function of the large amount of 5-methylcytosine in plant DNA is not well understood. As a tool for in vitro studies of methylcytosine formation in plants we have isolated and characterized the DNA methyltransferase present in germinating wheat embryo. An enzyme fraction enriched 300-fold over the tissue homogenate was obtained by salt extraction of nuclei, chromatography on DEAE-cellulose, Sephadex G-75, blue Sepharose and on DNA immobilized on cellulose. It catalyzes the methylation of cytosine residues in double-stranded DNAs isolated from wheat, maize, calf thymus or bacteria using S-adenosylmethionine as methyl donor. The efficient methylation of both an unmethylated plasmid DNA and its hemimethylated derivative indicate that the wheat DNA methylase can function de novo and in maintenance methylation. A relative molecular mass of 50,000-55,000 was estimated by gel permeation chromatography and sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis showed the presence of a protein of Mr = 50,000 and one other component (Mr = 35,000). The preference for endogenous, double-stranded DNA as substrate and the lower molecular mass distinguish wheat DNA methyltransferase from the DNA methylases obtained from mammalian sources. The properties of the wheat enzyme resemble, however, those of the DNA methylase isolated from the alga Chlamydomonas reinhardii, suggesting that plant cells possess their own type of DNA methyltransferase for the biosynthesis of their high methylcytosine content in DNA.     

The nitrate-reducing enzyme system of Chlamydomonas reinhardii

In Chlamydomonas reinhardii the reduction of nitrate to ammonia occurs in two independent enzymatic steps: 1. the two-electrons reduction of nitrate to nitrite catalyzed by NADH-nitrate reductase, and, 2. the six-electrons reduction of nitrite to ammonia catalyzed by ferredoxin-nitrite reductase. Both enzymes have been purified and characterized, and some of their properties have been studied.     

Food quality for Eudiaptomus gracilis: the importance of particular highly unsaturated fatty acids

1. Development times and survival of nauplii and copepodites of the freshwater calanoid Eudiaptomus gracilis were measured on comparably sized Cryptomonas sp. and two strains of Chlamydomonas reinhardii under food saturated conditions (1 mg C L−1) to investigate the nutritional quality of these algae.
2.  Cryptomonas sp. supported complete ontogenesis of nauplii to adults, whereas both strains of C. reinhardii were inadequate for the development of nauplii and copepodites and resulted in high mortality. The nutritional deficiency of both strains of C. reinhardii was compensated for by Cryptomonas sp. when the latter constituted ≥50% of dietary carbon. Pulses of Cryptomonas sp. had a similar compensatory effect.
3. In comparison to Cryptomonas sp., both strains of C. reinhardii were deficient in the three highly unsaturated fatty acids (HUFAs) stearidonic, eicosapentaenoic and docosahexaenoic acid. We manipulated the fatty acid content of C. reinhardii by externally adding these three HUFAs to the alga so that the fatty acid profile resembled that of Cryptomonas sp. This supplementation did not improve food quality, however, indicating that the nutritional deficiency of both strains of C. reinhardii for the ontogenesis of E. gracilis is not due to a lack of these three HUFAs.     

Analysis of kinetic parameters of gas phase bioreactors

The dependence of toluene elimination capacity on its load was obtained in five small-scale reactors filled with glass beads carrying biocatalyst cells. With increase in operation time the calculated maximal elimination capacity was shown to increase along with biomass density in the biocatalyst bed. Fivefold increase in trickling intensity did not affect the reactor performance. A simplified mathematical model for evaluation of minimal required biocatalyst bed volume at certain loading was developed based on experimental dependence of elimination capacity vs. loading.     

Evaluation of the Kinetic Parameters of Trickle Bed Vapor Phase Bioreactors

The dependence of toluene elimination capacity on its load was determined in five small-scale reactors filled with glass beads carrying biocatalyst cells. With an increase in the operation time, the calculated maximum elimination capacity was shown to increase in parallel with the biomass density in the biocatalyst bed. A fivefold increase in the trickling rate did not affect the reactor performance. A simplified mathematical model for evaluating the minimal required biocatalyst bed volume at a certain loading was developed based on the experimental curve of elimination capacity versus loading.     

Photodimerization of uracil in films and their use for dosimetry of genetically active ultraviolet irradiation

On the basis of the known photodimerization of uracil it is proposed to use its films obtained by sublimation in vacuum for the dosimetry of genetically active UV radiation. Experimental estimations are presented of radiation dose rate resulting in a change of uracil optical density delta D/D0 under irradiation due to photodimerization for erythemal lamp lo-30. The data obtained are used for dosimetry of lethal damage of Chlamydomonas reinhardii. Estimations are also presented of the sun radiation dose rate which induce uracil photodimerization on the earth surface and in the extraterrestrial atmosphere.     

Sequence of the chloroplast 16S rRNA gene and its surrounding regions of Chlamydomonas reinhardii.

The sequence of a 2 kb DNA fragment containing the chloroplast 16S ribosomal RNA gene from Chlamydomonas reinhardii and its flanking regions has been determined. The algal 16S rRNA sequence (1475 nucleotides) and secondary structure are highly related to those found in bacteria and in the chloroplasts of higher plants. In contrast, the flanking regions are very different. In C. reinhardii the 16S rRNA gene is surrounded by AT rich segments of about 180 bases, which are followed by a long stretch of complementary bases separated from each other by 1833 nucleotides. It is likely that these structures play an important role in the folding and processing of the precursor of 16S rRNA. The primary and secondary structures of the binding sites of two ribosomal proteins in the 16SrRNAs of E. coli and C. reinhardii are considerably related.     

Nucleotide sequence and structure of cytoplasmic 5S RNA and 5.8S RNA of Chlamydomonas reinhardii.

Three small RNAs of the cytoplasmic 8OS ribosomes of the green unicellular alga Chlamydomonas reinhardii have been sequenced. They include two species of ribosomal 5S RNA, a major and a minor one of 122 and 121 nucleotides respectively, which differ from each other by 17 bases, and also the ribosomal 5.8S RNA of 156 nucleotides. Novel structural features can be recognized in the 5S RNAs of C. reinhardii by a comparison with published 5S RNA sequences. In addition the secondary structure of these small RNA molecules has been examined using a newly developed method based on differential nuclease susceptibility.     

Intracellular calcium redistribution during mating in Chlamydomonas reinhardii

Gametes of the unicellular green alga Chlamydomonas reinhardii recognize and adhere to cells of the opposite mating type by flagellar contact. Adhesion between these specialized organelles signals a rapid series of mating events which result in gamete fusion. The sequence of morphological changes (flagellar tip activation, cell wall loss, and mating structure elongation), which occur as a consequence of the sexual signalling, have been characterized. The signalling mechanisms have, however, not been defined. Calcium is known to be involved during fertilization of animal species. Increased intracellular free calcium, which can be achieved either by calcium influx or by mobilization of ions from intracellular stores, has been observed during activation of both eggs and sperm. A recent report by Bloodgood & Levin that gametes of C. reinhardii preloaded with 45Ca showed a transient increase in Ca efflux following mating, suggests that intracellular Ca redistribution may also accompany mating in this algal species. We have used X-ray microanalysis to analyze the subcellular distribution of bound calcium during mating in Chlamydomonas reinhardii. X-ray maps reveal that calcium is sequestered in discrete granules within the gamete cell body prior to mating and that during activation and cell fusion, calcium is diffuse throughout the cell. This suggests the possibility that calcium serves as a second messenger in this species.     

Influence of the Volume Fraction,Size and Surface Coating of Hard Spheres on the Microstructure and Rheological Properties of Model Mozzarella Cheese

This study investigated the influence of model filler particles (glass beads) on the microstructure and rheological properties of Mozzarella cheese. Model Mozzarella cheese composites with increasing volume fractions of glass beads of various sizes and surface properties were processed in a Rapid Visco Analyser (RVA). Confocal laser scanning microscope images showed that all the hard spheres were dispersed in the protein phase, rather than in the fat phase. Dynamic oscillatory rheology revealed that the volume fraction of the glass beads had a major influence on the complex modulus (G*) of the cheese composites, whereas the size and the coating of the glass beads had no influence. However, the zero shear viscosity (η ₀), measured using the creep-compliance test, was affected by both the size and the volume fraction of the glass beads. This indicated that there were some interactions between the glass beads and the cheese matrix. Filler–matrix interactions played a major role in the fracture properties of the cheese composites. The fracture stress (σ _f ) was highly dependent on the coating and the size of the glass beads. Simple equations for filled gels from the literature fitted well with the experimental results and could be successfully applied for future predictions. According to this study, the transfer of knowledge from filled polymer composites to model cheese appears relevant. This can provide a good basis for designing new dairy product structures.     

Isolation,Purification, and Characterization of Mitochondria from Chlamydomonas reinhardtii

Mitochondria were isolated from autotrophically grown Chlamydomonas reinhardtii cell-wall-less mutant CW 92. The cells were broken by vortexing with glass beads, and the mitochondria were collected by differential centrifugation and purified on a Percoll gradient. The isolated mitochondria oxidized malate, pyruvate, succinate, NADH, and [alpha]-ketoglutarate. Respiratory control was obtained with malate (2.0) and pyruvate (2.2) but not with the other substrates. From experiments with KCN and salicylhydroxamic acid, it was estimated that the capacity of the cytochrome pathway was at least 100 nmol O2 mg-1 protein min-1 and the capacity of the alternative oxidase was at least 50 nmol O2 mg-1 protein min-1. A low sensitivity to oligomycin indicates some difference in the properties of the mitochondrial ATPase from Chlamydomonas as compared to higher plants.     

Immunochemical properties of erythrocyte-specific histone H5 in salmonid fishes

Comparative studies have been made on the immunochemical properties of histone H5 in Salmonidae species. High degree of homology between these histones and H5 histone from Oncorhynchus tschawytscha was demonstrated by microcomplement fixation. Properties of H5 histones in fish and birds are discussed.     

Unusual 5 S ribosomal RNAs. An analysis of individual segments can reveal phylogenetic relatedness

Sequence comparisons of 5 S and other ribosomal RNAs by segments can be useful in understanding anomalous primary and secondary structures and in assessing phylogenetic relationships. In a segmented analysis, the 5'-half of the Chlamydomonas reinhardii chloroplast 5 S ribosomal RNA is found to have a very close sequence homology to the green plant chloroplast and cyanobacterial 5 S RNAs; however, the 3'-half has a highly unusual sequence. Further comparisons of homologies between regions of the 5 S RNAs from C. reinhardii and the green plant chloroplasts suggest that genetic rearrangements within the 5 S DNA may have produced the unusual sequence at the 3'-half. Segmented analyses of the C. reinhardii and green plant chloroplast 5 S RNAs suggest a close relationship which is not revealed by overall sequence comparisons.     

Construction and characterization of autonomously replicating plasmids in the green unicellular alga Chlamydomonas reinhardii

Plasmids that replicate autonomously in Chlamydomonas reinhardii were constructed by inserting random DNA fragments from this alga into a plasmid containing the yeast ARG4 locus. Arginine prototrophy was used as a selective marker. The presence of free plasmids in the DNA of the transformants was demonstrated by hybridization with a specific plasmid probe and by recovering these plasmids in E. coli after transformation. Four of them were characterized. Their inserts of 415, 257, 153, and 102 by all hybridize to chloroplast DNA and were localized on the physical map of the chloroplast genome. One of these plasmids also promotes autonomous replication in yeast. Sequence analysis of the inserts of the plasmids reveals several short direct and inverted repeats and two semiconserved AT-rich elements of 19 and 12 by that may play a role in promoting autonomous replication in C. reinhardii.     

Lipid composition and (Na+ + K+)-ATPase activity in rat lens during triparanol-induced cataract formation

In vitro complementation of the soluble assimilatory NAD(P)H-nitrate reductase (NAD(P)H:nitrate oxidoreductase, EC 1.6.6.2) was attained by mixing cell-free preparations of Chlamydomonas reinhardii mutant 104, uniquely possessing nitrate-inducible NAD(P)H-cytochrome c reductase, and mutant 305 which possesses solely the nitrate-inducible FMNH2- and reduced benzyl viologen-nitrate reductase activities. Full activity and integrity of NAD(P)H-cytochrome c reductase from mutant 104 and reduced benzyl viologen-nitrate reductase from mutant 305 are needed for the complementation to take place. A constitutive and heat-labile molybdenum-containing cofactor, that reconstitutes the NAD(P)H-nitrate reductase activity of nit-1 Neurospora crassa but is incapable of complementing with 104 from C. reinhardii, is present in the wild type and 305 algal strains. The complemented NAD(P)H-nitrate reductase has been purified 100-fold and was found to be similar to the wild enzyme in sucrose density sedimentation, molecular size, pH optimum, kinetic parameters, substrate affinity and sensitivity to inhibitors and temperature. From previous data and data presented in this article on 104 and 305 mutant activities, it is concluded that C. reinhardii NAD(P)H-nitrate reductase is a heteromultimeric complex consisting of, at least, two types of subunits separately responsible for the NAD(P)H-cytochrome c reductase and the reduced benzyl viologen-nitrate reductase activities.     

Constancy of wheat histones during development

Summary Histones were extracted from leaves of winter- and spring-wheat seedlings, flowering shoots of spring wheat, shoots of vernalized and control winter wheat, and roots and shoots of winter wheat, and were compared by polyacrylamide-gel electrophoresis. No differences were found either in the electrophoretic mobility or relative quantity of the various fractions. Wheat histones contained fractions of the exact electrophoretic mobility as F2a1 and F3 of calf thymus and pea histones. Other major fractions of the wheat histones had electrophoretic mobilities similar to those of F1, F2b and F2a2 of peas.     

The gene for the large subunit of ribulose-1,5-bisphosphate carboxylase in Euglena gracilis chloroplast DNA: location, polarity, cloning, and evidence for an intervening sequence

The gene for the large subunit (LS) of ribulose-1,5,-bisphosphate carboxylase of Euglena gracilis Z chloroplast DNA has been mapped by heterologous hybridization with DNA restriction fragments containing internal sequences from the Zea mays and Chlamydomonas reinhardii LS genes. The Euglena LS gene which has the same polarity as the Euglena rRNA genes has been located with respect to Pst I, Pvu I, and HindIII sites within the Eco RI fragment Eco A. The region of Euglena chloroplast DNA complementary to an 887 bp internal fragment from the Chlamydomonas chloroplast LS gene is interrupted by a 0.5-1.1 kbp non-complementary sequence. This is the first chloroplast protein gene located on the Euglena genome, and the first evidence for an intervening sequence within any chloroplast protein gene.     

Influence of concanavalin a on autolysis of gametes from Chlamydomonas reinhardii.

The phytohemagglutinin concanavalin A inhibited zygote formation of Chlamydomonas reinhardii. 15--50 mug lectin/ml not only interfered with the mating reaction, but also with cell wall lysis of gametes and zoospores in a crude autolysin preparation gained from copulating gametes. Further, the structure of cell walls shed into the medium after autolysis in the course of the mating reaction and after lysis "from without" in the crude autolysin preparation was stabilized by Con A. Therefore, it must be assumed that the lectin inhibited zygote formation of C. reinhardii by interfering with autolysis of the cell walls of the gametes. Though Con A inhibited the lytic processes of C. reinhardii, an activation of the autolytic system in theta gametes by the lectin was found to compete with its inhibitory reaction. Con A induced autolysis of theta gametes was dependent on adherence of the cells by their flagella to the surface of the culture vessel or the liquid medium and did not occur in cultures stirred by rotation. The interferences of Con A with the autolytic serum of C. rienhardii were inhibited by methyl-alpha-D-mannopyrano-side and to a lesser degree by glucose, indicating that the carbohydrate binding sites of the lectin were involved in its reactions with the cells.     

Urate oxidase of Chlamydomonas reinhardii

Urate oxidase (EC 1.7.3.3) of Chlamydomonas reinhardii cells grown on purines and purine derivatives has been partially characterized. Crude enzyme preparations have a pH optimum of 9.0, require O₂ for activity, have an apparent K_m of 12 μ M for urate, and are inhibited by high concentrations of this substrate. Enzyme activity was particularly sensitive to metal ion chelating agents like cyanide, cupferron, diethyldithiocarbamate and o -phenanthroline, and to structural analogues of urate like hypoxanthine and xanthine. Chlamydomonas cells grow phototrophically on adenine, guanine, hypoxanthine, xanthine, urate, allantoin or allantoate as sole nitrogen source, indicating that in this alga the standard pathway of aerobic degradation of purines of higher plants, animals and many microorganisms operates. As deduced from experiments in vivo , urate oxidase from Chlamydomonas is repressed in the presence of ammonia or nitrate.