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The activity of dT kinase (EC. 2. 7. 1. 75) was estimated during the development of nucleate and anucleate cells inAcetabularia. Enzyme activity increases as the cap attains its maximum diameter (Bannwarth & Schweiger 1975). Regulation of the dT kinase activity is observed even in the absence of the cell nucleus. Twelve to fifteen days after enucleation dT kinase activity increase sharply as the cap reaches its maximum diameter and decreases 5 days later. The increase is inhibited by puromycin, chloramphenicol, rifampicin and ethidium bromide but not by cycloheximide. These findings indicate that protein synthesis on 70S organelle ribosomes is involved in the regulation of the dT kinase. Since activities of mixtures from low activity cell extracts and from high activity cell extracts are additive, it is concluded that the regulation is not mediated through an activator protein but rather throughde novosynthesis of the enzyme protein.

The activity of a ribonucleoside reductase was estimated during the life cycle of Acetabularia. During the early generative phase the enzyme activity was dramatically increased. Regulation of the ribonucleoside reductase was observed even in the absence of the nucleus. The increase in activity was inhibited by chloramphenicol but not by cycloheximide. These results indicate that the enzyme is translated on 70 S ribosomes.

The biochemical nature of the circadian regulatory system that controls many cellular activities is still unclear. Recent results obtained from the application of protein synthesis inhibitors to individual Acetabularia cells expressing circadian rhythms of photosynthesis indicate that some protein(s) must be synthesized on 80S ribosomes during a discrete part of each cycle to insure correct time-keeping. A comparative study of the effects of brief cycloheximide treatments on cells investigated at different temperature has revealed that the phase of cycloheximide sensitivity is 4-6 hr longer and occurs about 8 hr later in the cycle when cells are kept at 20 degrees rather than 25 degrees. Temperature is known to influence the function of the circadian regulatory system in Acetabularia, but the effect on frequency is small (Q10 approximately equal to 0.8) due to the existence of a temperature-compensating feature. The large effects of temperature observed here thus favor the interpretation that protein synthesis on 80S ribosomes, while providing an essential component of the circadian timing mechanism, does not itself generate the period of the photosynthesis rhythm.

Polyadenylated RNA was isolated from whole cells and from anucleate cytoplasm of Acetabularia mediterranea. It is synthesised in nucleate but not in anucleate cells. This RNA has a high molecular weight ranging from 0.5 to 3.0 × 106 daltons. In contrast to the RNA of 80 S ribosomes the synthesis of polyadenylated RNA is only slightly enhanced in nucleated cell fragments during the initial phase of regeneration. Newly synthesised polyadenylated RNA migrates from the nucleus through the stalk at about 5 mm a day. The data suggest that polyadenylated RNA migrates independently of 80 S ribosomes.

The rate of thymidine phosphorylation was estimated inAcetabularia mediterraneaby measuring thein vivoincorporation of [6-3H]thymidine into dTTP. Young cells incorporated little thymidine within 10 min. During growth of the stalk the incorporation rate increased slowly. When caps formed, no further increase was observed. But as the caps matured and the stalk became yellow-green, a steep increase in incorporation took place. This increase was also demonstrated in cells enucleated at the beginning of cap formation. Actinomycin D (3 mg/ml), when added 2 days prior to the critical stage, did not inhibit the increase, but puromycin (50 mg/l) did.

A test system allowing the estimation of the dT phosphorylating enzyme activity of one Acetabularia cell and of singular cell parts was developed. The occurrence in Acetabularia of a true dT kinase (EC. 2. 7. 1. 75) was demonstrated. This enzyme phosphorylates dT in the presence of ATP but not of AMP. dT kinase of Acetabularia is mainly bound to the particular fraction and a high percentage is localized outside the chloroplasts and mitochondria. After removal of the Triton X 100 (1%, by vol.) soluble part of the crude extract, a strong feedback inhibition by dTTP of the enzyme found in the pellet was observed. This enzyme may be solubilized with ionic detergents. The dT phosphorylating enzyme of the sediment (dT kinase) is distinct from supernatant phosphorylating enzyme in view of substrate specificity, stability, solubility, dTTP-inhibition, molecular mass, and K M value for dT.

Es wurden der anorganische, säurelabile, säurelösliche und alkalilösliche sowie der Lipoid-Phosphor in kernhaltigen und kernlosen Acetabularien bestimmt. Auch mit Hilfe der Kohleadsorption gelang es nicht, die Polyphosphate vollständig abzutrennen. Mit Ausnahme einer Fraktion, die vor allem aus RNS-Nucleotiden bestand, wurden keine wesentlichen qualitativen Unterschiede zwischen kernhaltigen und kernlosen Pflanzen gefunden. Die einzelnen Fraktionen lagen in den kernhaltigen Zellen in höheren Konzentrationen vor als in den kernlosen. Die Zunahme in den verschiedenen Fraktionen dauert wahrscheinlich länger als das Wachstum.

The incorporation of 3H-uridine into 80S cytosol and into 70S chloroplast ribosomes has been studied in Acetabularia in order to find out whether or not the nuclei undergo changes in the capability of rRNA formation during differentiation. To reinforce the differentiation process regenerating Acetabularia cells were chosen. Regeneration was induced by amputation of the apical part of the cell. The incorporation into cytosol ribosomes increased as early as 24 h after amputation. The stimulation reaches a maximum three to four days after amputation and does not depend on light at least for three days. It can be prevented, however, by addition of cycloheximide to the culture medium. The stimulation pattern of the uridine incorporation into chloroplast ribosomes is similar to the cytosol ribosomes with the exception that it is delayed by two days. This process is dependent on light and is not inhibited by cycloheximide. The response to amputation seems to be independent of chloroplast energy production as small and large fragments are stimulated to the same degree.

In four different species of Dasycladaceae (Acetabularia) the isozyme pattern of malic dehydrogenase was found to be species specific. Interspecific nuclear transfers changed the isozyme patterns of the recipient species to that of the nuclear donor species.

A method is described for the electrochemical determination of the oxygen balance of aquatic organisms. With this method it was possible to demonstrate that individual Acetabularia cells kept under continuous illumination showed a diurnal rhythm of photosynthesis for a period of several weeks. The rhythm could be observed even 40 days after removal of the nucleus. The role of the nucleus in this periodicity is discussed.

Isolated Acetabularia crenulata nuclei were injected with Mengo virus RNA solution and then implanted into anucleate posterior Acetabularia mediterranea cell fragments or fused with Acetabularia ryukyuensis cytoplasts. The injected animal virus RNA was actively translated in the plant cell cytoplasm. Mengo virus proteins were detected and localized in Acetabularia cytoplasts by use of an immunofluorescence method on the first to fifth day after injection.

In contrast to prokaryotes the eukaryotic cell is distinguished by a nuclear membrane separating the cell nucleus where replication and transcription occurs and the cytoplasm where the mRNAs originating from the nucleus are translated. Why eukaryotic cells developed this compartmentation cannot as yet be definitely answered. One possible function for the nuclear membrane may be a controlled transport from the nucleus into the cytoplasm or vice versa and therefore may play a role in gene expression.

Unicellular systems, and particularly individual cells, are advantageous in investigating the molecular mechanism of the circadian rhythm. The greeen alga Acetabularia has proven to be a uniquely suitable organism. Experiments are described which indicate that the circadian rhythm is due to a sequence of events which are casually interrelated. A model is presented which is based on a two-step mechanism, the first step of which is the synthesis of "essential polypeptides" on 80 S ribosomes. In a second step these polypeptides are consumed. The function of the "essential polypeptides" is to change a membrane component of the circadian clock. The membrane participates in a feed-back mechanism which produces a periodic alteration of the rate of synthesis of "essential polypeptides" and of photosynthesis. It is possible to draw parallels between this circadian rhythm model and other repetitive and non-repetitive types of temporal organization within the cell, e.g., the cell cycle; however, it must be stressed that in the case of the circadian rhythm, much stronger and more efficient mechanisms exist to keep the period constant. This is true particularly for temperature compensation. Such a conservatism is understandable if one recalls that during evolution, the circadian rhythm represents an adaptive advantage which permits the organism to adjust it activities in advance to periodic processes resulting from the earth's rotation.

The occurrence of a dCMP deaminase in Acetabularia mediterranea has been demonstrated. The enzyme which is found in a particulate fraction is substantially stimulated by the addition of dCTP. The activity of the enzyme is increased at the beginning of the generative phase in nucleate as well as in anucleate cells. This regulation is due to de novo synthesis of the enzyme. By means of inhibitor studies, it has been shown that the enzyme is translated on 70S ribosomes of and coced for in cell organelles.

Monocellular organisms are well suited for studies on the molecular mechanisms of circadian rhythms. Thythms in different monocellular organisms are similar. This mean that it might be anticipated that the mechanisms of all these rhythms are similar. Evidence so far available has resulted in several models, some of which emphasize the role of membranes and deserve special interest. A "coupled translation membrane model" is in full agreement with those particular results which have been obtained on the green alga, Acetabularia. Attempts to verify the role of essential polypeptides which was postulated in the model, by isolating such components appear to be successful.

In Acetabularia mediterranea the transcellular electrical potential along the cell is subjected to diurnal oscillations which persist under constant conditions. The frequency may significantly differ between individual cells even in simultaneous measurements. The free running period of a distinct cell may be subjected to slow changes during long-term measurements. Such measurements have been performed over about 100 days.

Protoplasts were isolated from cells of Acetabularia cliftonii, which are presumed to be haploid. The release of the protoplasts occurred after treatment of the cells with papain or proteinase K. They are genuine protoplasts since they contain a nucleus. Fusion was initiated by mechanically pushing together two protoplasts. Under these conditions, the efficiency of fusion was more than 90% within 30 minutes at room temperature. Haploid cells from one cyst, i.e., cells which eventually would have formed gametes of the same mating type, exhibit a greater propensity for fusion as compared to haploid cells from different cysts.

A number of problems require the measurement of functional parameters in single cell systems. A major difficulty in such measurements is the low level of signal output that can be achieved from single cells. This problem can be avoided by performing measurements on populations of single cells. In cell populations, however, an interaction between cells cannot be excluded. Under these circumstances, it may be necessary to accept the low signal level of an individual cell and to develop appropriate methods for evaluation.

Transformation of animal and prokaryotic cells is well studied (Garapin and Colbere-Garapin, 1983; Harris, 1983) but comparatively little is known on plant cell transformation (Schell et al., 1984; Ohta, 1986) and particularly about that of algae (Rochaix and van Dillewijn, 1982). In plant cells vector bound DNA is preferentially used for transformation. The Tiplasmid of Agrobacterium tumefaciens (for review see Schell et al., 1984) and the Cauliflower mosaic virus DNA (Brisson et al., 1984; Fromm et al., 1986) have proved to be useful in this respect. There have been only a few studies concerned with transforming effects of naked DNA (Paszkowski et al., 1984; Hain et al., 1985; Potrykus et al., 1985; Lorz et al., 1985; Ohta, 1986).

The kinetic properties of UDPG pyrophosphorylase (glucosyl-1-phosphate uridyl transferase, EC 2.7.7.9) suggest that it may play a key role in the regulation of metabolism in Acetabularia mediterranea. The enzyme-catalyzed reaction is readily reversible in vitro, and has been assayed in both directions. The enzyme shows substrate inhibition by UDPG and UTP at substrate concentrations in excess of 2 mM. The kinetic behavior of the enzyme is consistent with the hypothesis that it catalyzes an ordered bisubstrate biproduct reaction in which G-1-P is the leading substrate, and UTP is the leading product. A plot of initial velocity vs. PPi concentration is sigmoid, indicating a cooperative homotropic effect. PGAL inhibits the reaction in the direction: UTP + G-1-P leads to UDPG + PPi It has no effect on the reverse reaction. The responses of the enzyme may serve to regulate the allocation of G-1-P between anabolic and catabolic pathways.

In Acetabularia mediterranea, Acetabularia crenulata and Polyphysa cliftonii ribonuclease activity was demonstrated. The enzyme is heat stabile and exhibits maxima at pH 7.2 and at the region of pH 5. Even centrifugation with 100000 x g for 120 minutes does not result in sedimentation of enzyme activity.Anucleate cells of Acetabularia are capable of increasing their ribonuclease activity, but to a smaller extent than nucleate cells do.The stability of the genetic information in the cytoplasm of anucleate Acetabularia cannot be explained by a lack in ribonuclease activity.