The retinoblastoma gene product (the RB protein) is phosphorylated In a cell cycle-dependent manner and this modification is believed to be important for cells to progress through the cell cycle. We found that purified cdk2 (cyclin-dependent kinase/cell division kinase 2) can phosphorylate the RB protein in vitro at the sites phosphorylated in the cell. The timing ofactivation ofcdk2 in the cell cycle was similar (3)(4)(5). The RB protein also interacts specifically with several cellular proteins, including transcription factor E2F and the protooncogene product Myc (6-10). Thus, the normal function of the RB protein may involve formation of complexes with cellular proteins. Recently, microinjection of the RB protein into cells early in the G1 phase of the cell cycle was found to inhibit progression to the S phase, suggesting that the RB protein regulates cell proliferation by restricting cell cycle progression at a specific point in the G1 phase (11). The function of the RB protein is thought to be regulated mainly by phosphorylation. The RB protein has been shown to be phosphorylated in a cell cycle-dependent manner, being underphosphorylated in Go/Gj and highly phosphorylated during the G1 to S transition (12-16). Interestingly, only underphosphorylated RB protein can form a complex with large T antigen (17) and transcription factor E2F (7). The underphosphorylated RB protein is tightly associated with the nucleus, whereas the hyperphosphorylated form is easily extracted under the same conditions, suggesting that the underphosphorylated RB protein interacts tightly with nuclear proteins (18,19). These findings suggest that underphosphorylated RB protein inhibits transition ofcells from G, to S and that phosphorylation is a regulatory event leading to inactivation of the growth-suppressing activity of the RB protein.The RB protein possesses a number of consensus sequences for phosphorylation by cdc2 kinase. In fact, cdc2 kinase phosphorylates the RB protein in vitro at the same sites as those phosphorylated in living cells, and the amino acid sequences of the five sites phosphorylated in the cells were recently shown to correspond closely to the consensus sequence for phosphorylation by cdc2 kinase (20-23). However, cdc2 kinase is known to be active in the late G2 and M phases (24), whereas the RB protein becomes highly phosphorylated at about the G1 to S transition. On the other hand, although in yeast a single cdc2 kinase regulates both the G, to S and G2 to M transitions (24)

Euglena gracilis strain (Z) cells were synchronized under photoautotrophic conditions using a 14 hour light:10 hour dark regimen. The cells grew during the light period (growth phase) and divided during the following 10 hour period either in the dark or in the light (division phase). Changes in morphology of the pyrenoid and in the distribution of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) within the chloroplasts were followed by immunoelectron microscopy during the growth and division phases of Euglena cells. Epon-embedded sections were labeled with an antibody to the holoenzyme followed by protein A-gold. The immunoreactive proteins were concentrated in the pyrenoid, and less densely distributed in the stroma during the growth phase. During the division phase, the pyrenoid could not be detected and the gold particles were dispersed throughout the stroma. Toward the end of the division phase, the pyrenoid began to form in the center of a chloroplast, and the immunoreactive proteins started to concentrate over that rudimentary pyrenoid. During the growth phase, small areas rich in gold particles, called ;satellite pyrenoid,' were observed, in addition to the main pyrenoid. From a comparison of photosynthetic CO(2)-fixation with the total carboxylase activity of Rubisco extracted from Euglena cells in the growth phase, it is suggested that the carboxylase in the pyrenoid functions in CO(2)-fixation in photosynthesis.

Summary Euglena grown to stationary phase in the dark without aeration accumulated lipids. When these high lipid cells are transferred to an inorganic medium and aerated, lipids were rapidly metabolized and the respiratory rate declined concomitant with the decline in cellular lipid content. Prolamellar bodies, propyrenoids and prothylakoids developed within the proplastid of dark aerated cells and the cells developed an increased capacity for chlorophyll synthesis manifested upon subsequent exposure to light. Lipid content did not decline in cells exposed to nitrogen and chlorophyll synthesis ability did not increase. The addition of an organic carbon source to cells at the start of aeration did not prevent lipid degradation. Organic carbon source addition and inhibitors of RNA and protein synthesis did however inhibit the development of an increased capacity for chlorophyll synthesis. These results suggest that oxygen triggers light independent proplastid development with the oxidative metabolism of lipids providing the carbon and energy for the synthesis of nucleic acids and proteins required for proplastid development in the dark. Growth of microorganisms requires carbon and energy for the synthesis of cellular components. Autotrophs utilize sunlight or the energy released by oxidization of inorganic compounds for growth while the breakdown of organic compounds provides the energy needed for the growth of heterotrophs. Most pathogenic bacteria, protozoa, and fungi are heterotrophs. The objective of this study was to analyze the mechanism of conversion from heterotrophy to autotrophy using the unicellular alga, Euglena gracilis. Key wordsEuglena gracilis grows heterotrophically on a variety of carbon sources in the dark showing a typical animal-type metabolism and when it is placed in the light on an inorganic medium it grows autotrophically exhibiting plant-type metabolism. Euglena is the only organism that can reversibly switch from animal-like heterotrophic metabolism to plant-like autotrophic metabolism making its taxonomic classification as a plant or animal problematic (Johnson 1968). Schiff's group (Osafune et al. 1990, Schiff andSchwartzbach 1982) has studied the transformation from a heterotroph to a phototroph, the greening of Euglena gracilis var. bacillaris, by transferring dark grown non-dividing cells, resting cells, into the light to induce chloroplast development. They found that the breakdown of the storage carbohydrate paramylum provided carbon and energy for the light induced synthesis of proteins, nucleic acids and lipids needed for chloroplast development (Schwartzbach et al. 1975, Rosenberg et al. 1964 found that lipids, mainly wax esters, accumulated in the cytoplasm of dark grown stationary Euglena and their breakdown was induced by light exposure providing an additional source of carbon and energy for light induced chloroplast development.

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Changes in the morphology of pyrenoid and the distribution of RuBisCO in the chloroplast of Euglena gracilis were followed by immunoelectron microscopy during the cell cycle in a light (14 h)- dark (10 h) synchronized culture under photoautotrophic conditions. The imrnunoreactive proteins wereconcentrated in the pyrenoid, and less densely distributed in the stroma during the light period (growth phase, Fig. 1-2), but the pyrenoid disappeared during the dark period (division phase), and RuBisCO was dispersed throughout the stroma. Toward the end of the division phase, the pyrenoid began to form in the center of the stroma, and RuBisCO is again concentrated in that pyrenoid region. From a comparison of photosynthetic CO2-fixation with the total carboxylase activity of RuBisCO extracted from Euglena cells in the growth phase, it is suggested that the carboxylase in the pyrenoid functions in CO2-fixation in photosynthesis.