INTERACTION OF LIGHT QUALITY AND NUCLEASES IN THE GROWTH OF THE GAMETOPHYTES OF ASPLENIUM NIDUS

Appropriate concentrations of ribonuclease A and B selectively inhibited initiation of two-dimensional morphology in the gametophytes of Asplenium nidus, grown under a photoperiod of 5½ hr white light. Filamentous growth was promoted in such sporelings, the individual cells of which were significantly longer than corresponding cells of the control. Higher concentrations of enzymes were required to inhibit two-dimensional growth in the gametophytes grown in blue light. Concentrations of ribonuclease A or B which inhibited two-dimensional growth in white light promoted growth in length of the protonema in red light. Growth modifications in the sporelings induced by deoxyribonuclease in different light conditions were similar to those induced by the ribonucleases. The results lend further support to the postulated role of RNA in the regulation of two-dimensional growth in fern gametophytes.     

Ribonucleic Acid and Protein Changes in the Subcellular Components of the Gametophytes of Pteridium aquilinum during Growth in Red and Blue Light

The distribution of RNA and protein in the gametophytes of bracken fern, Pteridium aquilinum is affected by the quality of light in which they are grown. Two-dimensional gametophytes growing in blue light have a greater amount of RNA and protein than one-dimensional protonema growing in red light. Fractions rich in nuclei, chloroplasts, mitochondria, ribosomes and soluble supernatants obtained from blue-light grown gametophytes by differential centrifugation contain greater amounts of RNA and protein than corresponding fractions of red-light grown plants. Differences in RNA and protein content are detected in some of the fractions within 24 hours after start of the treatment.     

Eine Analyse der durch Blaulicht bewirkten Steigerung der Proteinsynthese bei Farnvorkeimen auf der Ebene der Aminosäuren

Zusammenfassung Bei der quantitativen Analyse der Protein-Aminosäuren von Farnvorkeimen (Dryopteris filix-mas) ergab sich, daß die Steigerung des Proteingehalts durch Blaulicht mit einer Zunahme aller untersuchten Aminosäuren einhergeht. Dabei bleibt der prozentuale Anteil der einzelnen Aminosäuren am Gesamtprotein immer annähernd gleich, abgesehen vom Prolin. — Offenbar kommt es unter dem Einfluß von Blaulicht hauptsächlich zu einer quantitativen Vermehrung und nur im geringen Maße zu einer qualitativen Veränderung der Proteinfraktion. — Da die pools der erfaßbaren freien Aminosäuren im Hellrot stets größer sind als im Blaulicht, ist der Schluß berechtigt, daß das Blaulicht seine Wirkung im Zusammenhang mit der Polypeptidsynthese und nicht über eine Steigerung der Aminosäuresynthese ausübt.

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An analysis of the blue light mediated increase of protein synthesis in fern gametophytes on the level of amino acids

Summary Morphogenesis and metabolism of the sporelings (= young gametophytes) of the common male fern Dryopteris filix-max are controlled by visible radiation. Short wavelengths visible radiation (= blue light) leads to an increase in protein synthesis and makes possible the formation of normal two-dimensional prothallia. Under long wavelengths visible radiation (= red light) the sporelings grow as cellular filaments, the protein contents of which are markedly lower than under blue light even under conditions of equal rate of dry matter accumulation in red and blue light (Fig. 2). — Quantitative amino acid analysis of the total protein of blue and red grown sporelings did not reveal any striking difference. The contents of all those amino acids which can be measured quantitatively after protein hydrolysis show about the same increase under the influence of blue light (Figs. 3, 4). Only in the case of proline are the differences between red grown and blue grown sporelings indicative of a qualitative change in the nature of the protein fraction (Fig. 3/h). —The pools of the free amino acids are always smaller in blue grown sporelings than in red grown ones (Figs. 6, 7). — The facts reported in this paper indicate that blue light leads mainly to a quantitative increase in the rate of protein synthesis. The influence of blue light on the qualitative nature of the protein fraction seems to be slight. On the other hand, the data on the pool sizes of the free amino acids support the conclusion that blue light controls the rate of protein synthesis at the stage of polypeptide synthesis and not through amino acid synthesis.

     

Die Aufnahme von 14CO2 und die Verteilung des 14C auf freie Aminosäuren und auf Proteinaminosäuren im Hellrot und im Blaulicht. [Objekt: Farnvorkeime von Dryopteris filix-mas (L.) Schott]

Summary Morphogenesis and metabolism of the early gametophytes (= sporelings) of the common male fern are controlled by light. The normal two-dimensional development of the gametophytes in white or blue light is correlated with an increase in protein content; inred light alone, on the other hand, the sporelings remain filamentous, and the protein content is markedly lower (cf. Mohr, 1965). The problem has been whether blue light increases the rate of protein synthesis or decreases the rate of protein degradation. This problem was solved in the present paper by the use of ¹⁴CO₂. Blue light promotes specifically the rate of protein synthesis as indicated by the increase of ¹⁴C incorporation into protein-bound amino acids under blue light as compared with red light.Using ¹⁴CO₂ we have analyzed the kinetics of free amino acid synthesis (Fig. 4) and protein synthesis (Fig. 5) under steady state conditions of photosynthetic CO₂ incorporation in blue or red light (Fig. 3). Under our conditions the rate of photosynthesis is about 1.5 times higher under blue light than under red light (Fig. 3, Table 1).The facts that the total pool sizes of the free amino acids are smaller in blue than in red light (v. Deimling and Mohr, 1967; Table 2) and that, on the other hand, the ¹⁴C-contents of the thoroughly labelled amino acid pools are virtually identical in blue and red (Table 3) indicate (a) that the pool sizes of these labelled amino acids may be equal in both light qualities and (b) that there is a compartmentation of free amino acid pools in the fern sporeling. This problem will be dealt with more in detail in a forthcoming paper on the behaviour of alanine in the fern sporeling (Payer, 1969).Protein synthesis is obviously much stronger under blue light than under red light. The detailed kinetics (Fig. 5b) indicate the involvement of two sorts of proteins: a relatively small part with high turnover which is rapidly labelled with a small but significant difference in red and blue, and a larger part with a slower turnover, the synthesis of which is strongly favored by blue light. — The first sort could be enzyme protein; the latter sort might be structural protein of the chloroplasts. These organelles increase dramaticly in size under the influence of blue light (Bergfeld, 1963). The amino acid composition of the protein, however, does not show any qualitative difference in gametophytes grown in blue or red light (v. Deimling and Mohr, 1967, Table 4).

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Die Aufnahme von ¹⁴CO₂ und die Verteilung des ¹⁴C auf freie Aminosäuren und auf Proteinaminosäuren im Hellrot und im Blaulicht. [Objekt: Farnvorkeime von Dryopteris filix-mas (L.) Schott]

     

Chloroplastenausbildung und Morphogenese der Gametophyten von Dryopteris filix-mas (L.) Schott nach Applikation von Chloramphenicol und Actidion (Cycloheximid)

Summary Morphogenesis and differentiation of the young gametophytes (=sporelings) of Dryopteris filix-mas are controlled by light. Blue light leads to the formation of normal two-dimensional prothallia; under red light, however, the gametophytes grow as cellular filaments. Morphogenesis in blue light is connected with an increase in protein synthesis; in red light the protein content of the sporelings is markedly lower. The size of the chloroplasts is correlated with the protein content of the sporelings.In the present paper the diverse effect of chloramphenicol (CAP) and actidione (cycloheximide, ACT) was studied in connection with the formation of chloroplasts. ACT blocks the growth of the gametophytes, while the chloroplasts remain functional. On the other hand, CAP does not influence morphogenesis of the gametophytes. In particular the activity of the dividing apical cells remains untouched. Even when the light quality is changed during the development the corresponding specific effect of the light quality on morphogenesis is normal. The chloroplasts, however, become smaller, probably by inhibition of synthesis of structural proteins. But their synthetic activity is not completely suppressed. The specific blue or red light dependent morphogenesis is not changed, when protein synthesis in the chloroplasts is inhibited.     

Die Regulation der RNS-Synthese in Farngametophyten Durch Licht

Zusammenfassung In einer früheren Arbeit wurde gezeigt (Ohlenroth und Mohr, 1963), daß sich die Vorkeime von Dryopteris filix-mas im Blaulicht zu normalen zweidimensionalen Prothallien mit einem relativ hohen Proteingehalt entwickeln. Im Hellrot hingegen bilden sich Zellfäden (= Protonemen) aus, deren Proteingehalt bei gleicher Photosyntheseleistung wesentlich geringer ist.In der vorliegenden Arbeit wird gezeigt, daß auch der RNS-Gehalt im Blaulicht stets größer als im Hellrot ist. Die blaulichtabhängige RNS-Zunahme setzt zeitlich früher ein als die Proteinzunahme.In Umsetzexperimenten von Hellrot nach Blau manifestiert sich die morphogenetische Umsteuerung der Protonemen zeitlich eher als die blaulichtabhängige Steigerung des Proteingehalts. Kasemir und Mohr (1965) konnten zeigen, daß es sich bei dem Blaulichtprotein in erster Linie um eine Vermehrung des Strukturproteins der Chloroplasten hanhandelt. Die blaulichtabhängige RNS-Zunahme dagegen ist spätestens zum Zeitpunkt der morphologischen Umsteuerung faßbar. Dieses Ergebnis wird dadurch gestützt, daß Blaulicht im Vergleich zu Hellrot rasch einen gesteigerten Einbau von ¹⁴C, als ¹⁴C-Uridin (U) geboten, in die RNS-Fraktion verursacht. Das Blaulicht scheint in den Farnvorkeimen zwei verschiedene Vorgänge zu verursachen. 1. Steigerung einer autonomen Proteinsynthese in den Chloroplasten. 2. Auslösung oder Steigerung einer spezifischen Enzymsynthese im Cytoplasma. Die blaulichtabhängige Steigerung der RNS-Synthese scheint damit in Zusammenhang zu stehen. Die Daten der vorliegenden Arbeit werden als Indizien dafür angesehen, daß das Blaulicht seine morphogenetische Wirkung über eine differentielle Genaktivierung ausübt.

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The regulation of RNA synthesis in fern gametophytes by light

Summary Morphogenesis and metabolism of the gametophytes (= sporelings) of the common male fern Dryopteris filix-mas are controlled by visible radiation. Short wavelengths visible radiation (= blue light) leads to an increase in protein synthesis and makes possible the formation of normal two-dimensional prothallia. Under long wavelengths visible radiation (= red light) the sporelings grow as cellular filaments the protein contents of which are markedly lower than under blue irradiation even under conditions of equal rate of dry matter accumulation in red and blue (Ohlenroth and Mohr, 1963). — It is shown in the present paper that the RNA content of sporelings of the same age is always higher in blue light than in red light (Figs. 1, 3). The blue-dependent increase of RNA occurs faster than the blue-dependent increase of protein (Fig. 2). Furthermore the increase of protein per sporeling is much larger than the increase of RNA (Fig. 4). These facts are in agreement with the hypothesis that in some way or another blue light initiates differential gene activation.The blue light-dependent morphological changes which occur if we put red grown filamentous sporelings under blue light can be measured much faster than the blue light-dependent increase of the bulk protein (Figs. 5, 6). We have to conclude as we did in a previous paper (Kasemir and Mohr, 1965) that the blue light-dependent increase in the protein content of the sporelings might be mainly due to an increase of chloroplast protein. — The blue light-dependent increase of the RNA content occurs at least as fast as the morphological changes (Figs. 5, 6). This finding is supplemented by the observation (Fig. 8) that blue light markedly and rapidly stimulates the incorporation of ¹⁴C into RNA. The ¹⁴C was applied as ¹⁴C-uridine (U). — It seems that blue light causes an increase of protein synthesis in the chloroplasts as well as in the cytoplasm. Blue light-dependent RNA synthesis seems to be involved in this response. These data support the view that blue light might exert its morphogenetic control through differential gene activation.

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Die Arbeit wurde durch Sachbeihilfen der Deutschen Forschunggemeinschaft und der Stiftung Volkswagenwerk gefördert.     

Untersuchungen zur Kompartimentierung der freien Aminosäure Alanin in den Farnvorkeimen von Dryopteris filix-mas (L.) Schott im Rotlicht und im Blaulicht

Summary In fern gametophytes (= sporelings) there is a strong correlation between the degree of blue light mediated photomorphogenesis and the protein content of the organism (cf. Mohr, 1963). In a previous paper (Payer et al., 1969) we have shown that blue light specifically increases the rate of protein synthesis in the fern sporelings over the rate which is maintained under red light. — In the present paper blue light mediated protein synthesis has been dealt with further using one representative amino acid, alanine, which was labelled with ¹⁴C from ¹⁴CO₂ under steady state conditions of photosynthetic ¹⁴C incorporation under blue or red light.Synthesis of free alanine is proportional to the rate of photosynthesis (Table 1). For a number of reasons we conclude that alanine is derived directly from primary photosynthetic products. Since the pool size of the thoroughly ¹⁴C-labelled pool of free alanine is much less than the actual, pool size of this amino acid, (Table 1), and since the specific activity of the isolated ¹⁴C-alanine is much below the value we can expect on the basis of the specific activity of the ¹⁴CO₂ applied we conclude that there are separate pools of free alanine; active (with respect to protein synthesis) and inactive pools which do not mingle. Taking into account this possibility of compartmentation of pools of free amino acids we have calculated in the case of ¹⁴C-alanine the rate of protein synthesis for two extreme instances (Table 2). A comparison of the theoretical values with the actual data indicates that indeed protein synthesis is fed from active pools of amino acids while the inactive pools are possibly located in the vacuoles. The total pool of alanine is much larger in red grown than in blue grown sporelings while the active pools seem to have the same size under both conditions. The cells of the red grown sporelings have much larger vacuoles than the cells of the blue grown sporelings.The rate of protein synthesis is under our conditions 1.8 times higher in blue light than in red light. The rate of turnover of the total protein is 0.29% per hour in the blue and 0.23% in the red light. The absolute turnover of protein is 1.5 times higher in blue light than in red light. It is concluded that the blue light mediated increase of protein synthesis is very real. Blue light must act specifically at the level of polypeptide synthesis.     

Die regulation der DNS-Synthese in Farngametophyten durch Licht

Zusammenfassung Bei Farnvorkeimen (Dryopteris filix-mas) steigert Blaulicht spezifisch DNS-Replikation und mitotische Aktivität, so daß sich die im Hellrot bzw. Blaulicht herangewachsenen Vorkeime, die gleichviel Trockensubstanz besitzen, bezüglich ihres DNS-Gehalts und ihrer Zellzahl wesentlich unterscheiden — Die Zelle scheint als Bezugssystem für RNS, Protein und entsprechende biochemische Größen ungeeignet zu sein. Der Gesamtkeimling ist das angemessene Bezugssystem.

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The regulation of DNA synthesis in fern gametophytes by light

Summary In young gametophytes (= sporelings) of the common male fern (Dryopteris filix-mas) short wavelength light (= blue light) specifically increases DNA replication and mitotic activity even if the sporelings have the same growth rate under blue and red light, as determined by dry matter increase. Cell number and DNA content of sporelings of the same age and the same dry matter content are much higher under blue than under red light (Figs. 1, 2). In the second part of the paper it is pointed out that the cell (or unit of DNA) may not be used as a system of reference for biochemical data such as protein or RNA content in the case of the fern sporelings (and possibly not in many other organisms either). The appropriate system of reference seems to be the entire multicellular system if precaution is taken that the systems grow with the same growth rate under the different experimental conditions (e.g. under blue and red light in the case of the fern sporelings).

     

Ein spezifischer Einfluß von Blaulicht auf den Einhau von photosynthetisch assimiliertem 14C in das Protein von Farnvorkeimen [Dryopteris filix-mas (L.) Schott]

Summary Morphogenesis and metabolism of the early gametophytes (=sporelings) of the common male fern are controlled by light. The normal two-dimensional development of the gametophytes takes place only in white or blue light; in red light alone, on the other hand, the sporelings remain filamentous even under conditions of equal photosynthetic rate.The problem has been whether blue light exerts its morphogenic influence through differential gene activation. In other words: does blue light mediate the synthesis of morphogenic enzymes which are required for normal morphogenesis. In an earlier paper (Drumm and Mohr, 1967) we have shown that blue light increases the rate of RNA synthesis within an hour whereas the first indication of a morphogenic change due to blue light is only discernible about 3 hours after the onset of blue light (Figs. 1,2). Furthermore we have shown (Mohr, 1965) that Actinomycin D specifically inhibits the blue light mediated morphogenic alterations, and Bergfeld (1967) has shown that blue light will rapidly lead to changes in nuclei and nucleoli in the fern sporelings. In the present paper it has been shown that blue light does increase the rate of protein synthesis about an hour after the transfer of the sporelings from the red into the blue light of equal quantum flux density (350 pE·cm^-2·s^-1).The rate of protein synthesis was measured in shortterm experiments (40min) using ¹⁴CO₂. The photosynthetic rate was the same in red and blue; it was not influenced by the transfer(Fig. 3). Likewise the rate of ¹⁴C incorporation into the pool of free amino acids was not significantly different in red and blue light (Fig.4). On the other hand, the rate of incorporation of ¹⁴C into the protein increased rapidly after the transfer of the sporelings from the red into the blue light (Fig. 5). The same phenomenon (no influence of blue light on the specific activity of the free amino acid; a strong promotive influence on the specific activity of the protein-bound amino acid) was observed in the case of alanine which was investigated in detail (Figs. 6, 7). Since the increase of the protein content of the sporelings is not significant during the first six hours after transfer to blue light (Fig. 8) the protein induced by blue light and directly related to morphogenesis can only be a very small fraction of the total protein of the sporeling.The data strongly support the hypothesis (Ohlenroth and Mohr, 1964), that the morphogenic effect of blue light on the fern sporelings is due to the induction of morphogenic enzymes by blue light.     

Regulation of Protein Synthesis during Photomorphogenesis of Gametophytes of the Fern Onoclea sensibilis

Gametophytes of the fern Onoclea sensibilis grow as filaments in the dark and in red light and become planar in blue light. Pulse-labeling 4-day-old gametophytes with [³⁵S]methionine at different times after transfer to dark, red, and blue light environments revealed higher rates of amino acid uptake and protein synthesis in blue light than in red light or in the dark. Characterization of the extant and newly synthesized soluble proteins by one- and two-dimensional gel electrophoresis showed that the patterns of protein accumulation and synthesis in gametophytes exposed to short periods of red or blue light were qualitatively indistinguishable from those of gametophytes maintained in the dark. However, some striking increases and decreases in the levels of certain polypeptides were noted and these changes were accentuated during continued growth of gametophytes in the different environments. The results show that photomorphogenesis of gametophytes of O. sensibilis is associated with quantitative rather than qualitative changes in the population of mRNAs available for translation.     

The effects of light on sex determination in gametophytes of the fern <Emphasis Type="Italic">Ceratopteris richardii</Emphasis>

The sexuality of homosporous fern gametophytes is usually determined by antheridiogen, a pheromone that promotes maleness. In this work the effect of photomorphogenically active light on antheridiogen-induced male development was examined for gametophytes of Ceratopteris richardii. Although blue light did not affect sensitivity to Ceratopteris antheridiogen (A_Ce) in wild-type gametophytes, it was found that the gametophytes of the her1 mutant, which are insensitive to A_Ce, developed into males when grown under blue light in the presence of A_Ce. Thus, we conclude that another A_Ce-signal transduction pathway activated by blue light exists latently in the gametophytes of C. richardii. Red light, on the other hand, suppressed male development. Because simultaneous red and blue light-irradiation did not promote male development in the her1 gametophytes, the action of red light seems to dominate that of blue light. The results of experiments with a photomorphogenic mutant also suggested that phytochrome may be involved in the action of red light.     

Growth Promotion of Vegetative Gametophytes of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> by Blue Light

Through an acclimation period of 10 days, compared to white light, the maximal net photosynthetic rates were significantly higher for gametophytes of Undaria pinnatifida cultivated under blue light (400–500 nm), and were lower under red light (600–700 nm). Chlorophyll c and the carotenoid content of gametophytes were similar under blue light and red light but were much lower under white light. The growth rate of female gametophytes under blue light was higher than that under other lights, and the growth rate of male gametophytes showed little variation with respect to blue and white light. Male and female gametophytes were mixed together to form sporophytes under white, blue and red light. After approximately 5 days, 50% gametophytes became fertile under blue and white light, but remained vegetative under red light after 10 days.     

DOES LIGHT QUALITY AFFECT THE SINKING RATES OF MARINE DIATOMS?1

Although the spectral quality of light in the ocean varies considerably with depth, the effect of light quality on different physiological processes in marine phytoplankton remains largely unknown. In cases where experiments are performed under full spectral irradiance, the meaning of these experiments in situ is thus unclear. In this study, we determined whether variations in spectral quality affected the sinking rates of marine diatoms. Semicontinuous batch cultures of Thalassiosira weissflogii (Gru.) Fryxell et Hasle and Ditylum brightwellii (t. West) Grunow in Van Huerk were grown under continuous red, white, or blue light. For T. weissflogii, sinking rates (SETCOL method) were twice as high (～0.2 m·d^?1)for cells grown under red light as for cells grown under white or blue light (～0.08 m·d^?1), but there were no significant differences in carbohydrate content (～105 fg·μm^?3) or silica content (～ 17 fg·μ^?3) to account for the difference in sinking rates. Thalassiosira weissflogii grown under blue light was significantly smaller (495 μm³) than cells grown under red light (661 μm³), which could contribute to its reduced sinking rate. However, cells grown under white light were similar in size to those grown under red light but had sinking rates not different from those of cells grown under blue light, indicating the involvement of factors other than size. There were no significant differences in sinking rate (～0.054 m·d^?1) or silica content (～20 fg·μm^?3) in D. brightwellii grown under red, white, or blue light, but cells grown under red light were significantly (20%) larger and contained significantly (20%) more carbohydrate per μm³ than cells grown under white or blue light. Spectral quality had no consistent effect on sinking rate, biochemical composition (carbohydrate or silica content), or cell volume in the two diatoms studied. The similarity in sinking rate of cells grown under white light compared to those grown under blue light supports the ecological validity of sinking rate studies done under white light.     

SPORELING COALESCENCE IN CHONDRUS CRISPUS (RHODOPHYCEAE)1

Coalescence of developing sporelings of Chondrus crispus Stackhouse was observed. Juvenile tetra-sporophytes showed a higher proportion of coalescence than developing gametophytes. Stages of complete coalescence between different sporelings are illustrated. Coalesced sporelings exhibit vertical and horizontal alignment of cells, as well as “cuticular” continuity and secondary pit connections between adjacent, coalesced sporelings. Ultimately the cells in the center of the coalesced sporelings produce upright, multiaxial fronds that grow more rapidly than fronds of non-coalesced sporelings. Other red algae, such as Gracilaria verrucosa (Hudson) Papenfuss and Gigartina stellata (Stackhouse) Batters also show a similar sequence of sporeling coalescence and enhanced growth. The ecological significance of sporeling coalescence is discussed.     

Influence of light quality on the ribosomal RNA levels inWolffia arrhiza: Comparison of phosphate and magnesium limited chemostat populations

Wolffia arrhiza (L.) Wimm. was grown axenically in the chemostat under white luminescent light (photon fluence rate 23 ujnol m^-2 s^-1) and phosphate or magnesium limitation (0.075 and 0.01 jxmol 1^-1, respectively). Aliquots (1 g fresh mass) were taken from the continuous cultures and were irradiated for 1 h with either white light (control) or monochromatic blue (453 nm) or red (654 nm) light. The amount of [5^-3H]-uridine incorporated into cytosolic and chloroplastic rRNAs during these exposures was estimated and following results were obtained: In phosphate limited plants rod light considerably reduced and blue light slightly increased label incorporation as compared with the control. Moreover, in red light, chloroplast incorporation is relatively more slowed down than that in the cytosolic compartment (34 % as compared to 59 % of the control). In blue light the enhancement is approximately equal in both compartments. In magnesium limited plants incorporation under both blue and red light is moderately slower as compared with the control. In both cases also the retardation is slightly greater in the chloroplast than in the cytoplasm. The results suggest that rRNA metabolism is controlled by light quality as well as by mineral nutrition.     

Reproduction induced by blue light in female gametophytes of Laminaria saccharina

Summary In red light at 15°C, female gametophytes of Laminaria saccharina continue to grow indefinitely without becoming fertile, but 6–12 hours' irradiation with blue light induces the production of eggs. At lower temperatures, some gametophytes become fertile in red light, but blue irradiation increases the percentage of fertile gametophytes.     

RNA SYNTHESIS IN CHINESE HAMSTER CELLS : II. Increase in Rate of RNA Synthesis during G1

Cultures of mitotic Chinese hamster cells, prepared by mechanical selection, were pulse-labeled with methionine-methyl-¹⁴C or with uridine-³H at different stages in the life cycle. The rate of ¹⁴C incorporation into 18S RNA was measured, as was the rate of uridine-³H incorporation into total RNA for both monolayer and suspension cultures. The rate of incorporation increased continuously throughout interphase in a fashion inconsistent with a gene-dosage effect upon RNA synthesis.