Effects of Indole-3-acetic Acid and Gibberellin on Synchronous Cultures of Chlorella fusca

The applicability of synchronous cultures of Chlorella fusca as a reproducible experimental system for the study of growth regulators has been investigated using indole-3-acetic acid (IAA) and gibberellin.

• 1 None of these compounds stimulated growth or sporulation.
• 2 IAA inhibited growth and sporulation at concentrations higher than 6 × 10^?5M, the effect increasing with increasing acidity. Gibberellin had no effect.

     

Isolation and characterization of microsatellite loci in a marine fish species,the tusk (Brosme brosme)

We developed polymerase chain reaction primers for nine dinucleotide microsatellite loci in the marine deep‐sea fish, the tusk (Brosme brosme). All markers were obtained from a partial genomic DNA library, and characterized in 100 unrelated individuals from one putative population. The number of alleles ranged from two to 60 with an average of 15/locus. The observed heterozygosity ranged from 0.020 to 0.826 (average 0.438). Several of the markers amplified multiple alleles from the two other gadoid species tested.     

Uptake of Uracil by Synchronous Chlorella fusca

Uptake of radioactive uracil by light-dark synchronized Cblorella fusca Shihira and Krauss was studied. For the characterization of the uptake system autospores were used and the following results obtained. Autospores kept in the dark accumulated uracil against a concentration gradient in a process having an observed activation energy of 10 keal/mol in the 10–40°C interval. Addition of glucose to the reaction suspension did not affect the uptake, but, 100 γM dinitrophenol inhibited the process by 90%. Abrupt changes in rate were found upon changing the conditions from light to dark and vice versa, and the rates measured in light were about 2.5 times larger than those found in the dark. Initial rates measured in the dark followed saturation kineties with half maximal rate found at 0.25 γM uracil, and with an apparent maximal rate of 1.7.10^-10 mol/10 min . 10⁷ cells. The effect of 14 pyrimidines on uptake was tested, and it was found that uracils which were substituted in the 5′ or 5′+ 6′ positions were strongly inhibitory. Of these, thymine and dihydrouracil were tested and shown to inhibit uracil uptake competitively. Initial uptake rates, measured in the dark with 1.0 γM uracil, were recorded at intervals during the 24 h synchronous cycle. The uptake rate per ml culture was constant during the first 9 h, thereafter increasing to reach a peak value at 14 h. This peak was followed by a strong increase from 18 h onwards, this increase being concomitant with the sporulation process, and closely followed its time course.     

Uptake of l-Phenylalanine in Synchronous Chlorella fusca. Characterization of the Uptake System

Phenylalanine uptake in Chlorella fusca was measured, using the membrane filter technique. The cells were synchronized, and harvested at specific points of the life cycle. Experiments with autospores showed that the uptake followed saturation kinetics, with a K_m= 5 μM. V_max, was 0.1 nmol/min × 10⁷ cells. The optimum temperature for the uptake was 40°C, and the activation energy was 1700 J/mol. The uptake showed a high specificity towards l -phenylalanine; presence of the unlabelled stereoisomer did not inhibit the uptake. Uptake of l -phenylalanine was inhibited in the presence of other analogues or other amino acids, but only if they were present in concentrations considerably higher than that of L-phenylalanine. Variations in the ratio of Na4⁺ to K⁺ in the external solution during uptake experiments did not have any influence upon the uptake rate of l -phenylalanine. The cells were able to take up the amino acid against a concentration gradient. At pool maximum the ratio between internal and external amino acid concentration was 1000/1. 2,4-Dinitro-phenol inhibited the uptake completely. Exchange between internal and external l -phenylalanine could not be demonstrated. The K_m value did not change during the life cycle of the cells. The uptake rate reached a maximum at the end of the light period, and fell to a minimum just before sporulation started. It is concluded that Chlorella fusca cells have a highly specific, active uptake system for l -phenylalanine. The system is constitutive, independent on the K or Na concentration, and the mechanism of uptake does not change during the life cycle of the cells.     

Induction of Isocitrate Lyase in Synchronous Cultures of Chlorella fusca

Isocitrate lyase (ICL) of Chlorella was induced with acetate, and induction kinetics followed in autospores and 6 h old cells of a synchronous culture. The enzyme could not be induced in illuminated cells. With both cell types 1.2 mM acetate was the optimal inducer concentration. Freeze-thawed cells and acetone powders were used for measurement of activity. With the former the time course of increase in activity was different at the two cell ages. With 6 h old cells the activity fluctuated: There was first a period of increase, then one with decrease and again one of increase. No such variation was found with freee-thawed autospores or with acetone powders of both cell stages. Darkening 6 h old cells for different periods of time before induction reduced the peak of activity, leaving the rate of the third phase unchanged. Illumination of darkened cells before induction increased the peak. Increasing the duration of both treatments increased their respective effects. Acetone extracts taken at different times after start of induction inhibited the ICL activity of a test preparation. The inhibition decreased concurrently with the variation in the ICL activity-found-when freee-thawed cells were used in the enzyme assay. The inducibility, taken as the rate of the third phase, was measured at different times during the 24 h synchronous cycle. Using three different acetate concentrations and both methods of cell preparation, we found that the inducibility was constant for 17 h whereafter it increased rapidly to a final level.     

Incorporation of Uracil by Synchronous Chlorella

Light-dark synchronized Chlorella fusca was used to study the incorporation of radioactive uracil into the trichloroacetic acid insoluble fraction. In autospores the incorporation measured in darkened cells usually started immediately after uraci addition, and the time course showed three distinct linear phases with abrupt transitions between them. Chromatographic analyses of the radioactive pool components showed that the total amounts of monophosphate, diphosphate and triphosphate nucleotides of uracil did not limit the incorporation and thus did not cause the abrupt rate shifts. In autospores, 5, 9 and 14 h old cells, the initial incorporation rate increased with increasing initial uracil concentration to reach a constant value above 0.5 μ.M. Addition of glucose to the cells increased the incorporation rate over the whole uracil concentration range tested with autospores, but did not influence the rates of the other cells. The incorporation rate varied during the synchronous cycle in a manner which closely resembled the pattern for uptake and degradation rates, with the most pronounced similarity from 9 h onwards.     

Synchronous Cultures of Chlamydomonas reinhardti: Properties and Regulation of Repressible Phosphatases

De novo synthesis of phosphatase (derepression) in orthophosphate deprived synchronously growing Chlamydomonas reinhardti has been demonstrated by using a double labelling isotope technique coupled with cellulose acetate electrophoresis. Repressed and derepressed phosphatase exhibited different enzymatic properties as pH optimum, electrophoretic pattern, K_m and K_i values. Especially the acid phosphatase was located near the cell surface. Inorganic, cold TCA-extractable ³²P, decreased during the first 1–2 h after phosphate deprivation when there was little or no net synthesis of phosphatase. Results of experiments with additions of orthophosphate and cycloheximide to derepressed cells, indicated that the derepressible enzyme was relatively unstable, while its m-RNA was relatively stable.     

Characterization of Sulphate Uptake in Anacystis nidulans

Sulphate uptake in the blue-green alga Anacystis nidulans appears based upon an active mechanism with a Km of 0.75 μM and V_max of 0.7 pmol/min × 10⁶ cells. Sulphate uptake is competitively inhibited by thiosulphate and sulphite. The sulphate uptake has a pH optimum at 8 and a temperature optimum at 40°C. By increasing the extracellular sulphate concentration from 0.1 to 10 μM the sulphate pool in Anacystis was altered from 8.3, 10^?5M to 5.9, 10^?4M.     

Pool Control of Uracil Uptake by Synchronous Chlorella

Experiments with autospores of Chlorella fusca showed that the uptake rate of uracil was reduced as much as 40 % as the result of formation of soluble pools of uracil and its metabolites from exogenous uracil. Using synchronous cultures similar results were obtained with cells at other developmental stages.     

Bathymetric barriers promoting genetic structure in the deepwater demersal fish tusk (Brosme brosme)

Population structuring in the North Atlantic deepwater demersal fish tusk ( Brosme brosme ) was studied with microsatellite DNA analyses. Screening eight samples from across the range of the species for seven loci revealed low but significant genetic heterogeneity ( F _ST = 0.0014). Spatial genetic variability was only weakly related to geographical (Euclidean) distance between study sites or separation of study sites along the path of major ocean currents. Instead, we found a significant effect of habitat, indicated by significant differentiation between relatively closely spaced sites: Rockall, which is surrounded by very deep water (>1000 m), and the Mid-Atlantic Ridge, which is separated from the European slope by a deep ocean basin, were differentiated from relatively homogeneous sites across the Nordic Seas. Limited adult migration across bathymetric barriers in combination with limited intersite exchange of pelagic eggs and larvae due to site-specific circulatory retention or poor survival during drift phases across deep basins may be reducing gene flow. We regard these limitations to gene flow as the most likely mechanisms for the observed population structure in this demersal species. The results underscore the importance of habitat boundaries in marine species.