Synchronous Cultures Of Chlorella Research Articles

Phytohormone effects on cell division inChlorella pyrenoidosa chick (TX-7-11-05) (chlorellaceae)

Phytohormone effects on cell division of synchronous cultures ofChlorella pyrenoidosa (TX-7-11-05) were studied under different photo flux densities. The time required for initiation of incipient cell division was reduced significantly by treatment with kinetin (6-furfurylaminopurine), gibberellic acid, and indole-3-acetic acid. Data show that kinetin was more effective than gibberellic acid, which was more effective than indole-3-acetic acid. Studies with combinations of the phytohormones revealed no antagonistic, additive, or synergistic effects.

The Variability of the Photosynthetic Unit in Chlorella II. The Effect of Light Intensity and Cell Development on Photosynthesis, P-700 and Cytochrome f in Homocontinuous and Synchronous Cultures of Chlorella

The effect of light intensity of steady state photosynthesis, the contents of P-700 and cytochrome f was studied in homocontinuously grown Chlorella cells. The adaptation to higher light intensities drastically provokes the decrease of chlorophylls per dry weight, per packed cell volume and per cell. The light saturating curves show that high light grown cells produce threefold more oxygen per chlorophyll, whereas the oxygen evolution per cell is reduced by half. The rates of saturated photosynthesis per chlorophyll are directly correlated with increasing light intensities. The content of P-700 is slightly altered by light intensity. On the other hand, cytochrome f is changed in very strict accordance with the alterations of biomass productivity and photosynthetic capacity. The mathematical equation for the relationship between maximal photosynthesis and cytochrome f concentration is found to be linear with a correlation coefficient of 0.96. In addition to the results of homocontinuously grown Chlorella cells, the relationship between photosynthesis and the content of cytochrome f was analyzed in synchronous cultures. After the onset of light the photosynthetic activity rises as well as the content of cytochrome f. During the dark period, however, when the photosynthetic capacity is low, there still remains a high amount of cytochrome f. The results are discussed on the basis of the variability of the photosynthetic unit and the in-tactness of the thylakoid membrane. It is proposed to introduce the term of the «physiological photosynthetic unit» defined by the ratio of chlorophyll to cytochrome f. Its size is proved to be used as an indicator of the rate limiting step in the photosynthetic electron transport.

Poly(A) RNA populations, polypeptide synthesis and macromolecule accumulation in the cell cycle of the eukaryote Chlorella.

Synchronous cultures of Chlorella, that were obtained with minimum metabolic perturbation by centrifugal selection, reveal that progress through the cell cycle requires no change in the poly(A)+ mRNA population, although changes do occur during nutritional adaptation. Of the abundant soluble proteins, 93% are synthesized continuously through the cell cycle and those that are discontinuous show similar patterns in control cells. The synthesis of proteins is compared with parallel studies of accumulation of enzyme activity and it is shown that there is no discrepancy in their pattern of accumulation when both are studied under the same culture conditions. The eukaryote cell cycle can allow stable relative rates of synthesis of most proteins and balanced rates of accumulation of most enzyme activities. Macromolecule classes differ in their rates of accumulation throughout the cell cycle: total RNA increases linearly, poly(A)+ RNA accumulation is restricted to G1 phase, but total protein accumulation accelerates smoothly through G1, S and mitosis phases, pausing at cytokinesis. There is no evidence that the cell cycle requires an extensive programme of differential enzyme synthesis. The cycle can therefore proceed with minimum disturbance of metabolism required for growth.

Regulation of accumulation and turnover of an inducible glutamate dehydrogenase in synchronous cultures of Chlorella.

Earlier studies indicated that the gene of an ammonium-inducible glutamate dehydrogenase (GDH) was inducible throughout the cell cycle and was expressible shortly after replication early in the S-phase in synchronous Chlorella cells growing at a rate of 13% per h in the absence of inducer. In the present study, synchronous cells cultured at the same growth rate in the continuous presence of inducer accumulated this enzyme in a linear manner, with a positive rate change observed late instead of early in the S-phase. At a growth rate of 26% per h, the positive rate change appeared to be displaced to 1.5 h before the S-phase in the next cell cycle. With 2'-deoxyadenosine, an in vivo inhibitor of deoxyribonucleic acid (DNA) synthesis, the magnitude of the positive rate change was shown to be proportional to the relative increase in DNA in the previous cell cycle. Collectively, these data support the idea that expression of newly replicated genes of this enzyme can be delayed into the subsequent cell cycle in cells in the continuous presence of inducer. Studies with cycloheximide indicated that the inducible GDH and another GDH isozyme were stable in fully induced cells in the absence of protein synthesis. However, after ammonium was removed from the culture medium, the activity of the inducible GDH decreased rapidly in vivo, with a half-time of 5 to 10 min at 38.5 degrees C, whereas the rate of accumulation of the other GDH isozyme did not change. Addition of cycloheximide, at the time of inducer removal, prevented this loss in activity of the inducible GDH. The inability to rescue the activity of the inducible GDH, by readdition of ammonium during the deinduction period, indicates that this enzyme probably underwent irreversible inactivation and/or proteolytic degradation.

Selection of synchronous bacterial cultures by density sedimentation.

A procedure previously used to select synchronous cultures of Chlorella was found to produce similar results with the bacterium Lineola longa (Bacillus macroides). A midlog culture of L. longa was layered onto a 31-42% dialyzed Ficoll gradient and ceitruged at 51 000 3 g. The culture sedimented into a broad band in 30 min. Continued centrifugation failed to cause further migration. Cells taken from the top of the band and reinoculated into the broth in which they had previously grown, pH adjusted to 7.0, grew without a lag, doubled in optical density at the same rate as midlog cultures, and divided synchronously. Coulter counter sizing of these cells showed a doubling in volume just before division followed by a halving of volume after division. The major advantages of this method are the low osmolarity of Ficoll and the large volume of cells that can be separated.

The effect of light on RNA and nucleotide synthesis in synchronous cultures of chlorella

The increase of the soluble nucleotide content ceased immediately when young Chlorella cells were removed from the light. Later in the cell cycle (14 h after the beginning of the light period) the soluble nucleotide content also increased significantly upon transfer to the dark. The nucleotide triphosphate levels remained constant or even increased noticeably when the RNA synthesis ceased in the dark. It is concluded, therefore, that the regulation of RNA synthesis by light does not occur at the level of the nucleoside triphosphates. The appropriate rate of nucleotide biosynthesis is apparently regulated to a great extent by feedback control.

Dissociation of nuclear DNA replication from concomitant protein synthesis in synchronous cultures of chlorella

The interrelationship between nuclear DNA replication and cytoplasmic protein synthesis in Chlorella was studied by use of hydroxyurea and cycloheximide as specific inhibitors. 15 μM cycloheximide completely blocked the synthesis of protein and depressed DNA and RNA formation by about 90 %. Protein synthesis resumed immediately after removal of the inhibitor, while DNA synthesis was fully restored after a lag time of about 2 h. 10 mM hydroxyurea completely inhibited DNA synthesis. A decrease in protein and RNA synthesis gradually ensued after about 3 h of exposure. DNA synthesis was restored immediately after removal of the inhibitor, but the final DNA levels were markedly lower than in control cultures. During the recovery from the hydroxyurea treatment about 50 % increase of the DNA content was observed in the presence of cycloheximide. The results suggest that simultaneous synthesis of specific proteins required for DNA replication occurs during the S phase. These proteins continue to be synthesized when nuclear DNA replication is stopped by specific inhibitors, and can then be used to support subsequent DNA replication in the absence of protein synthesis.

Effect of temperature in the regulation of DNA synthesis in synchronous cultures of Chlorella

The influence of temperature on DNA replication of Chlorella pyrenoidosa grown at 30 °C was studied in synchronous cultures. Final DNA content and cell counts/ml were 30 to 40% less than controls after a 4 h exposure to 15 °C, but reached normal levels after being kept temporarily at 10 °C. The rates of DNA synthesis at these two temperatures were, respectively, one-sixth and one-tenth of the rate at 30 °C, but during the 15 °C treatment the cells lost the ability to enter a further replication cycle more rapidly than at 10 °C. This loss of capacity to initiate further replication cycles was prevented by inhibiting protein synthesis with cycloheximide. The results suggest that depletion, in the dark, of specific substances required for initiation of DNA replication is a process coupled with protein synthesis.

Periodic change of deoxyribonuclease activity in synchronous cultures of chlorella

A deoxyribonuclease, highly specific for denatured DNA, has been demonstrated in Chlorella. Maximal velocity of DNA degradation was observed in Tris-HCl buffer at pH 8.5 in the presence of 7.5 mM MgCl 2. In synchronously growing cultures high enzyme activity was found only during the period of DNA synthesis. After this time the deoxyribonuclease activity was strongly depressed by a heat-stable nondialysable inhibitor. A possible involvement of this deoxyribonuclease in the regulation of DNA synthesis in vivo is suggested.

Induction of cell division in synchronous cultures of Chlorella

In synchronen Chlorella-Kulturen wird wahrend der 2. Halfte der 16stundigen Lichtzeit eine starke Erhohung der durchschnittlichen Autosporenzahl stimuliert. Wurde die Belichtung wahrend dieser Induktionsphase abgebrochen, dann konnte die Sporenzahl durch eine 4stundige Temperatursenkung von 30 auf 18°C erheblich reduziert werden, sofern diese innerhalb der ersten 2–3 Std nach dem Dunkelbeginn einsetzte. Der Erniedrigung der Sporenzahl entsprach eine im Vergleich zur Proteinsynthese stark verminderte DNS-Syntheserate bei der niedrigen Temperatur. Wurden andererseits, beginnend innerhalb desselben Zeitraums einige Stunden Zusatzlicht gegeben, dann konnte noch eine betrachtliche Erhohung der Sporenzahl beobachtet werden. Danach erwies sich die Sporenzahl als irreversibel fixiert, d.h. sie konnte weder durch eine vorubergehende Kalteperiode noch durch Zusatzlicht beeinflust werden.

Continuous Synchronous Culture of Photosynthetic Micro-organisms

A series of stirred tanks, some illuminated and some in the dark, have been used to provide synchronous cultures of Chlorella, the largest overall cell size being found in the illuminated tanks.

The effect of light on DNA synthesis and related processes in synchronous cultures of chlorella.

In autotrophic cultures of Chlorella synchronised by alternating light and dark periods of 16:8 hours the DNA content duplicated normally 4 times successively during the S mphase, i. e. between the 10th and 18th hour after the beginning of the light period. This finding together with electron microscopical observations revealed that one duplication of the DNA and of the nuclei per cell proceeds every 110 minutes. All nuclei of a cell seem to undergo successive DNA syntheses and nuclear divisions synchronously. The rate of DNA synthesis was independent from illumination. On appropriate reduction of the light period the last duplication cycle fell out and the average final spore number per cell was accordingly lower. If a culture was transferred to darkness or low light intensity 3 hours before the normal end of the light period the release of spores was promoted by approximately 1 1/2 hours, provided a strong decrease of metabolically accessible carbohydrates was prevented by either an additional short illumination during the dark period or by continuing the weak light. A possible explanation for the shortening of the cell development is that, by passing over one DNA duplication and one protoplast division, the cell can enter sooner the respective subsequent developmental stages.

A New Type of “Light Inhibition”, Occurring in Synchronous Cultures of Chlorella

A New Type of “Light Inhibition”, Occurring in Synchronous Cultures of Chlorella