Chapter 1 Synthesis of Macromolecules and Morphogenesis in Acetabularia

CHAPTER 1

SYNTHESIS OF MACROMOLECULES AND MORPHOGENESIS IN ACETABULARIA

1. Brachet LABORATOIRE DE MORPHOLOGIE ANIMALE,

FACULTB DES

SCIENCES,

UNIVERSITC

LIBRE DE BRUXELLES,

BRUSSELS, BELGIUM; AND LABORATORIO INTERNAZIONALE DI CENETICA E BIOFISICA, NAPLES, ITALY

I. Introduction .................................... 11. Morphogenetic Substances and mRNA’s ............ 111. Protein Synthesis in Nucleate and Anucleate Fragments of Acetabuluria .................................. IV. RNA Synthesis in Nucleate and Anucleate Fragments of Acetabuloria. Importance of the Chloroplasts ...... A. Net RNA Synthesis in the Absence of the Nucleus B. RNA Synthesis in Subcellular Particles Isolated from Nucleate and Anucleate Fragments ........ C. Base Composition of the RNA’s Localized in the Various Regions of the Alga .................. V. Other Functions of the Chloroplasts: DNA Replication, Photosynthesis ................................... A. Generalities ................................. B. Cytoplasmic DNA in Acetabularia .............. C. Rhythm in Photosynthetic Capacity ............ VI. Concluding Remarks ............................. References .....................................

1.

1 4 11

15 15

17 23 25 25 25 31 33 35

Introduction

The unicellular alga Acetabularia owes its fame to its giant size (several centimeters) and to its remarkable capacity to regenerate in the 1

2

J. BRACHET

absence of the nucleus ( Hammerling, 1934). It can very easily be cut in two so as to separate the nucleate from the anucleate half; the latter not only is able to survive for several months in the absence of the nucleus, but can even regenerate a very complex structure, the “cap,” which is normally the reproductive organ of the alga. A very important fact, also discovered by Hammerling ( 1934), is that the caps formed by anucleate fragments are typical of the species to which the nucleus belonged. Furthermore, interspecific grafts (for instance, the grafting of a nucleate fragment of Acetabularia mediterrunea on the anucleate stalk of Acetabularia crenulata) have shown that the nucleus of the alga produces species-specific morphogenetic substances ( Hammerling, 1953). In interspecific grafts, there is a kind of competition between the morphogenetic substances produced by the grafted nucleus of one species (med. in this instance) and preexisting morphogenetic substances accumulated in the anucleate stalk of the other species (cren. in this case). The result is often the production of a “hybrid cap, which can degenerate and be replaced by the type of cap typical of the nucleus. Furthermore, elegant experiments of Hammerling (1934) have shown that, in a normal alga, the morphogenetic substances formed by the nucleus migrate toward the tip of the stalk and are distributed according to a decreasing apicobasal gradient: anucleate apical fragments regenerate very well, whereas basal anucleate fragments (although they are very close to the nucleus) are unable to form a cap. The life cycle, the results of experiments made on algae which have been cut into fragments and on interspecific grafts have been the subject of many reviews (Hammerling, 1953; Brachet, 1957, 1961; Brachet and Lang, 1965; Puiseux-Dao, 1963; Gibor, 1966; Werz, 1965, etc. ) . Figures 1-3 will suffice to illustrate what has just been summarized, since the emphasis, in the present review article, will be placed on biochemical events. Figure 1 represents the life cycle, as described by Hammerling (1953); some of its aspects have been criticized by Puiseux-Dao (1963), but these criticisms are of minor importance for the biochemical work to be discussed here, Figure 2 is the most important of the three: it represents the results of experiments in which algae have been cut at different levels and depicts the distribution of the morphogenetic substances in the organism. Figure 3 gives a summary of the interspecies grafting experiments of Hammerling and his school. In the following, we shall place the emphasis on very recent work, including experiments made in our laboratory and still unpublished at the time of the writing of the present chapter. The main questions to be dis-

1.

SYNTHESIS AND MORPHOGENESIS IN

Acetabularia

FIG. 1. Life cycle of Acetabularia mediterranea. N, nucleus; rh, rhizoid; tid; st, stigma.

FIG. 2. Cap formation in the absence of the nucleus.

3

plas-

4

J. BRACHET

cussed are the following: Are the morphogenetic substances stable iiiessenger RNA (mRNA) molecules? Is the synthesis of enzymes possible in the absence of the nucleus, and, if so, how is it regulated? Are RNA and DNA syntheses possible in the absence of the nucleus? Are chloroplasts largely independent from the nucleus for multiplication and macromolecule synthesis?

-..- - - - - - -.... .

-

-. ....

-.

Fie. 3. Distribution of inorphogenetic substances according to Hliniincrling’s cxperiments.

II. Morphogenetic Substances and mRNA‘s

It is unquestionable that the synthesis of the “morphogenetic substances,” whatever their chemical nature might be, must ultimately bc controlled by the genetic material present in the nucleus-that is, by the chromosomal DNA. The same is also true for another-easier to analyze -property of anucleate fragments of Acetahulatla, the capacity to synthesize proteins (Brachet et al., 1955): as will be discussed in more detail in Section 111, a %fold increase in protein content can be observed during the 3 weeks following the removal of the nucleus. Among these proteins are many enzymes; the information required for the synthesis of such specific proteins must reside in DNA and be transferred, through mRNA molecules, to the protein-synthesizing machinery of the cell (the ribosomes), This hypothesis was presented by the author of the present review in 1957 and, in a more precise form, in 1961, at a time when the term “messenger” RNA had not yet been coined. But a number of facts, which were later discussed in detail by Brachet (1965)

1.

SYNTHESIS AND MORPHOGENESIS IN

Acetabularia

5

and by Werz (1965), already made clear that some kind of RNA, which is synthesized in the nucleus, moves into the cytoplasm and accumulates at the tip of the alga; there, the complex information needed for cap formation is stored, in a stable form, for several weeks. This stability is demonstrated by the fact that anucleate fragments (which do not regenerate in the dark) can still form caps when they are kept in the absence of light for 2-3 weeks, and then illuminated (Brachet, 1957; Hammerling, 1963). The main evidence for the identity of morphogenetic substances and mRNA molecules (obviously, a very large amount of information is needed in order to form such a complex structure as the “cap” and many different species of mRNA must be synthesized) is of two different kinds: indirect experiments, which will be summarized here, and comparative studies on RNA synthesis in nucleate and anucleate fragments of the alga, which will be discussed in Section IV. First of all, autoradiography clearly shows that, as in all other cells, nuclear (and particularly nucleolar) RNA is labeled first after a short pulse with uridine; by transferring the alga, after a few hours, to unlabeled seawater, it can be demonstrated that the previously labeled nuclear RNA moves out of the nucleus and migrates toward the apex of the stalk (Olszewska and Brachet, 1961; Olszewska et al., 1961; de Vitry, 1965a). However, this behavior is not specific for RNA:autoradiography experiments by Olszewska et aZ. (1961) and de Vitry (1965a) have demonstrated that certain proteins, which are also synthesized first in the nucleus, move into the cytoplasm and accumulate at the tip of the alga ( experiments using labeled methionine and lysine) . Cytochemical observations carried out independently by Werz ( 1961) have also demonstrated that high-molecular RNA and certain proteins accumulate at the apex of the stalk. Indirect experiments strongly suggest that the integrity of the RNA molecules is needed in order to get normal morphogenesis in anucleate fragments of Acetabularia: for instance, the effects of ultraviolet irradiatwn on regeneration have been studied by several workers, a11 of whom concluded that RNA is one of the factors controlling morphogenesis. Among the various papers dealing with the subject, let us mention that Olszewska et al. (1961), reported that UV radiation at 2540 A inhibits both the synthesis of RNA in the nucleus and its accumulation a t the tip of the alga (experiments on local irradiation of the various regions of the alga ) . Very interesting also are the experiments of Werz and H8mmerling ( 1961) who implanted UV-irradiated nuclei from Acetabularia

6

J. BRACHET

mediterranea into anucleate stalks of Acetabularia crenulata and found that the caps that formed were mainly of the crenulata type. These experiments show that nucleic acids are responsible for the species-specificity of the morphogenetic substances, but they do not of course allow a discrimination between the respective roles of DNA and RNA. More direct evidence for a role of RNA in the morphogenesis of Acetabularia comes from experiments in which fragments of the alga have been treated in vivo with ribonuclease. As shown by Stich and Plaut

(0)

(b)

(C)

FIG. 4. Distribution of mRNA’s. ( a ) Normal algae; ( b ) algae treated with ribonuclease; ( c ) algae treated with actinomycin.

(1958), whose observations have been confirmed by de Vitry (1962) in our laboratory and extended to another unicellular alga ( Batophora) by Puiseux-Dao ( 1958), ribonuclease inhibits completely regeneration of nucleate or anucleate halves as long as it is present in the medium; after transfer of the treated fragments into normal seawater, the inhibition becomes reversible in the case of the nucleate halves but remains irreversible in the case of the anucleate ones. The simplest explanation for these findings is depicted schematically in Fig. 4b: in this scheme, preexisting mRNA, required for morphogenesis, is destroyed by the ribonuclease added to the medium; the loss is irreversible in the anucleate half, but RNA could be reformed by the nucleus. That this simple explanation is not necessarily the correct one is indicated by the rather paradoxical results that we obtained more recently (Rrachet and Six, 1966) : we observed an increase (80%) in the total RNA content of the two kinds of fragments or whole algae after in vivo treatment (4-9 days) with ribonuclease ( 1 mg/ml). Such treatments produced a parallel increase in the incorporation of phenylalanine into the proteins. But, as we shall see in Sections IV and V, the problem of RNA synthesis in Acetabularia is greatly complicated by the presence of considerable amounts of

1.

SYNTHESIS AND MOFWHOGENESIS IN

Acetabularia

7

RNA in the chloroplasts. Our results (Brachet and Six, 1966) may reflect a stimulatory effect of ribonuclease on “chloroplastic” RNA synthesis; they do not necessarily exclude the simple possibility (Fig. 4b) that the enzyme destroys an informational RNA which is present in small amounts only and is particularly ribonuclease-sensitive. Other useful chemical tools for the analysis of morphogenesis in Acetabularia are the various inhibitors of synthesis of nucleic acids ( fluorodeoxyuridine, hydroxyurea, actinomycin ) and proteins ( puromycin, cycloheximide, p-fluorophenylalanine) , Fluorodeoxyuridine inhibits DNA synthesis by blocking the enzyme thymidylate synthetase; its effects on Acetabularia have been studied in detail by de Vitry (1965b), who found that morphogenesis is stopped in the two types of fragments. Autoradiography analysis shows that fluorodeoxyuridine inhibits the transfer from the nucleus to the cytoplasm of RNA synthesized in the dark; this suggests an inhibition of DNA-dependent synthesis of mRNA. Hydroxyurea is a powerful inhibitor of DNA synthesis; it acts primarily on the reduction of ribonucleotides into d-ribonucleotides (one of the initial steps in DNA synthesis); at high concentrations, it might modify the DNA molecule itself in such a way as to make it unsuitable for either transcription or replication. We found (Brachet, 1967) that hydroxyurea (100 pg/ml) inhibits growth and morphogenesis in both nucleate and anucleate fragments; the inhibition remains reversible by simple transfer into seawater after a l-week treatment. Preliminary experiments by S. Limbosch and V. Heilporn indicate that the inhibition can be partially lifted by the addition to hydroxyurea of either thymidine or the coenzyme NADPH. They also found that hydroxyurea quickly inhibits the incorporation of thymidine and uridine into the nucleic acids of Acetabularia; on the other hand, it has no visible effect on the buoyant density of the DNA extracted from the alga (see Section V). Taken together, these observations indicate that hydroxyurea, in Acetabularia as in other cells, acts upon the reduction of ribose to d-ribose and that this step of DNA synthesis is required for cap formation, even in anucleate fragments of the alga. The effects of actinomycin (the now classical inhibitor of RNA synthesis) have been extensively studied in Acetabuhria by Brachet et al. (1964) and by Zetsche (1964, 1966a). If morphogenetic substances and mRNA molecules are identical, one would predict that actinomycin, which inhibits RNA synthesis without destroying preexisting RNA molecules, will not inhibit regeneration in anucleate halves; on the other

8

J. BRACHET

hand, regeneration of the nucleate fragments should be prevented as long as actinomycin is present in the culture medium; the inhibition should be reversible after transfer to normal medium. As shown in Fig. 4c in a diagrammatic way and in Fig. 5a, this is indeed the result obtained. However, a comparison between Figs. 2 (control anucleate fragments ) and 5a ( actinomycin-treated anucleate fragments) clearly shows that the caps formed in the presence of actinomycin are smaller than those in the controls and that often they are abnormal. In other words, actinomycin has no effect on the initiation of cap formation, but it inhibits the subsequent growth of the caps. We (Brachet et al., 1964) have suggested that this secondary effect of actinomycin could be due to the combination of actinomycin with DNA present in the chloroplasts (see Section V ) . In favor of this interpretation is the fact that when labeled actinomycin-14C is added to the medium, its binding to the chloroplasts can be demonstrated by autoradiography ( d e Vitry, 1964, 1 9 6 5 ~ )It . has also been shown that actinomycin produces alterations of the ultrastructure of the nucleolus and of the chloroplasts ( Boloukhhre-Presbourg, 1965); such a finding fits in well with the idea that this antibiotic combines with a component of the chloroplasts, probably DNA. Before leaving actinomycin, mention should be made of a recent analysis made by Zetsche ( 1 9 6 6 ~ )of the effects exerted by this substance on the formation, respectively, of the stalk and the cap: he comes to the interesting conclusion that the genes responsible for the formation of these two components of the alga must be active simultaneously for mRNA synthesis. The genetic information for stalk formation would express itself immediately in the cytoplasm, while that for cap formation would be stored and remain inactive. The idea that the mRNA's responsible for cap formation can be stored in a stable form in the cytoplasm is essentially the one we had presented in 1957 and 1961. But Zetsche ( 1 9 6 6 ~ ) has added two new ideas: the passage from one information (Zetsche, 1966c) to the other would occur at the level of the translation, and the nucleus would exert an inhibitory influence upon the expression, in the cytoplasm, of the genetic information specific for the cap. The effects of puromycin on regeneration in Acetabularia fragments have also been studied by Brachet et al. (1964) and by Zetsche (1966b). f i e main results are as follows: regeneration of the anucleate fragments is blocked in an almost irreversible manner; there is no regeneration in nucleate halves as long as they are kept in the presence of puromycin. After retransfer of fragments to normal seawater, inhibition is usually reversible in the case of these nucleate fragments; but regeneration in

1. SYNTHESIS AND MORPHOCENESIS IN

Acetabularia 9

10

J. BRACHET

such cases is often abnormal, resulting for instance in bifid or trifid stalks (Fig. 5 b ) . We have recently obtained similar results with another powerful inhibitor of protein synthesis at the polysomal level, c!yclolaeximide (Brachet, unpublished). These experiments clearly show that an ordered synthesis of proteins is required for the formation of a cap (Brachet et al., 1964). The analysis of the effects of puromycin on fragments of Acetabularia has been carried one step further by Zetschc (1966b); he actually measured protein synthesis and found that it is completely inhibited by puromycin in the two kinds of fragments. The inhibition is almost irreversible in the anucleate halves. It is reversible in the nucleate fragments, where there is, during the puromycin treatment, an accumulation of substances which stimulate protein synthesis when the nucleus is removed at the end of the treatment with puromycin. Experiments in which puromycin and actinomycin were combined suggest that these substances are probably mRNA's produced by the nucleus. These biochemical results of Zetsche (196613) are, of course, in excellent agreement with the excessive regeneration (bifid or trifid stalks depicted in Fig. 5b) that we observed under similar experimental conditions. The evidence obtained from electron microscopy ( BoloukhkrePresbourg, 1965) agrees with Zetsche's ( 1966b) biochemical expcriments: the emission in the cytoplasm of RNA-containing material (by the nuclear membrane) is not inhibited under conditions where protein synthesis is 60-7070 inhibited. On the other hand, puromycin modifies the structure of the chloroplasts and the mitochondria, a finding which might explain why the effects of puromycin are irreversible in the case of anucleate fragments. Finally, the effects on regeneration of various metabolites and m i i metabolites have been studied by various authors (Rrachet, 1958, 1959; de Vitry, 1962; Zetsche 1 9 6 6 ~ )It. would take us too far to discuss a11 the results which have been described. One should, however, mention the interesting effects of certain amino acid analogs: for instance, ethionitic inhibits growth and promotes the formation of caps (usually abnormal) in anucleate parts; it has no effect in nucleate parts (Brachet, 1958, 1959). More recently, Zetsche ( 1966c) discovered that fluorophenylalanine inhibits, in a rather specific way, thc growth of the caps without affecting their initiation or the growth of the stalks. This effect, which is similar to that of actinomycin, can be reversed by treatments with phenylalanine or tyrosine; it suggests that cap formation might be associated with the synthesis of specific proteins. In conclusion, the indirect experiments described in the present sec-

1.

SYNTHESIS AND MORPHOGENESIS IN

Acetabularia

11

tion clearly show that the transcription of nuclear DNA into stable, longlived mRNA molecules is a process of fundamental importance for morphogenesis in Acetabularia. On the other hand, growth and cap formation are very complex processes, which must require the synthesis of many ditferent kinds of mRNA's and of proteins (structural proteins and enzymes). Control of morphogenesis must occur also at the level of the translation of the genetic information encoded in these stable mRNA molecules: as we have seen, the expression of the genetic message (cap formation) is suppressed when protein synthesis is blocked. Let us now examine what is actually known about the control of protein synthesis (enzymes, in particular) in nucleate and anucleate fragments of Acetabularia. 111. Protein Synthesis in Nucleate and Anucleate Fragments of Acetabularia

As we showed in 1955 (Brachet et al.), net synthesis of proteins occurs, under conditions of good illumination, in anucleate fragments of the alga. In both halves, the total protein content can increase to 3-fold during the 3 weeks that follow the removal of the nucleus. After that time, net protein synthesis can no longer be demonstrated in the anucleate halves; but the use of radioactive precursors ( 14C02-labeled amino acids) shows that turnover of the proteins still remains quite active at that time. According to Clauss (1958), both chloroplastic and cytoplasmic proteins increase in the absence of the nucleus (respectively, 170 and 265%) . Among the proteins synthesized by anucleate fragments are a number of enzymes: invertase (Keck and Clauss, 1958) , aldolase ( Baltus, 1959), phosphorylase ( Clauss, 1959), UDPG-4-epimerase (Zetsche, 1966d). Enzymes not involved in carbohydrate metabolism are also synthesized in the absence of the nucleus; this is the case, for instance, for two soluble ribonucleases, having different pH optima, studied by Schweiger ( 1966). Their activity increases in the anucleate as well as in the nucleate fragments; however, their synthesis is more important in the latter. Special attention has been concentrated on acid phos)3hatase, which is present in several isozyme forms. This is the reason why, in their first experiments, Keck and Clauss (1958) failed to find any synthesis of acid phosphatase in anucleate fragments. But further analysis by Spencer and Harris (1964) and by Triplett et al. (1965) showed that acid phosphatase in Acetabulariu is a complex of many isozymes, which be-

12

J. BRACE-IET

have differently in the absence of the nucleus. For instance, it was found in our laboratory (Triplett et al., 1965) that five distinct isozymes can be detected in extracts of the alga; thcy may increase in quantity in the absence of the nucleus; but the activity of each of these enzymes seems to be regulated independently of the others, even in anucleate cytoplasm. Two of these enzymes, which are associated with the chloroplast fraction, greatly increase in activity when caps are formed by anucleate fragments. The activity of the third major isozyme, which is not found in the chloroplasts, as time goes on diminishes progressively in the absence of the nucleus. Very different experimental conditions were adopted by Spencer and Harris (1964), who also studied alkaline phosphatase. But their main conclusion is the same: there is an independent regulation of the synthesis of the various enzymes, even in the absence of the nucleus. Comparable results have been obtained more recently by Zetschc ( 1966d) for UDPG-Cepimerase: the enzymatic activity sharply increases at the time of cap formation, even in anucleate halves. The logical conclusion which emerges from all these observations is that enzyme synthesis, in anucleate fragments of Acetabulariu, must be controlled at the level of translation. But the presence of chloroplasts in Acetabularia and the fact that they are capable of autonomous protein synthesis (see Section V) make this conclusion less certain. Very recently, we (Brachet and Lievens, 1968) tried to find out whether the level of phosphatase activity could be modified in nucleate and anucleate fragments of Acetabularia by changing the phosphate content of the medium. If, as in bacteria, enzyme synthesis is regulated by the presence or absence of small molecules (substrate, reaction product), one would expect that the culture of the algae in a phosphatedeficient medium would lead to a derepression of the mechanisms for enzyme synthesis (in other words, to an increase in phosphatase activity). It was of obvious interest to see whether the well-known model of Jacob and Monod ( 1961), which is the result of extensive studies on the bacterial chromosome, is valid in the case of anucleate fragments of an alga. Figure 6 gives a summary of our main results. In a first series of experiments, total acid phosphatase activity was compared in whole algae, nucleate and anucleate fragments cultivated either in seawater enriched in phosphate (normal medium) or in phosphate-deficient medium. In all cases, transfer in the phosphate-deficient medium was followed by a &crease (instead of the expected increase) in phosphatase activity: but

I

0

I

10

10

Days

1

20

20

Days

.

1

30

30

0

medium,

-. Deficient medium, -.-.-.-

FIG.6. Regulation of acid phosphatase in algae, nucleate, and anucleate fragments. ( a ) Anucleate fragments. ( b ) Nucleate fragments. ( c ) Whole algae. Complete

(C)

(b)

x

c I

2,

e

In

.-e

14

J. BRACIIET

this is a transient phcnomcnon in whole algac :md nucleutc fragincnts where, after a few days, the initial level of activity is reached again. On the other hand, in anucleate fragments, phosphatase activity r c m i n s loto all the time: in this respect, therc is a marked difference betwcen thc nucleate and anucleate systems. In a second step, the three main isozymes of acid phosphatasc havc: been separated by agar gel electrophoresis and followed quantitatively. It was found that the initial decrease in total acid phosphatase activity is due to decreased activity of only one of the isozymes (isozyme 111, which is localized in the chloroplasts). The secondary increase in total acid phosphatasc activity, which occurs only whcn the nucleus is present. is due to increased activity of another cliloroplastic isosyntc (isozyme I ) . This isozyme I behaves in the way one would expect for a repressiblc cnzyme, except for the puzzling fact that its synthesis requires a time 1;ig (several days) before it starts. One of the reasons for this anomalous behavior is the presence of large amounts of polyphosphates in Acctabularia: we have studied them, using thin-layer chromatography as a method, and found that culture of the whole algae in phosphate-poor medium induces the breakdown of high-molecular ( n > 6 ) polyphosphates. In the case of the fragments, the “surgical shock” produced by the section is sufficient to induce an almost complete disappcarance of these high-molecular polyphosphatcs : this occurs both in nucleatc and anuclcate fragments, in normal as well as in phosphate-deficient medium. The presence of these polyphosphates and their breakdown makcs conclusions difficult before other more favorable inducible enzymes havc. been studied. It remains, however, clear that the increase in the actiuit!/ of i s o z y e I , despite the fact that it has a clrloro)h.stic localization, is under nuclear control.* The different behavior of this enzyme, in nucleatc and anucleatc fragments, cannot be explained on the basis of diff ereiiccs in the polyphosphate content. The conclusion that the synthesis of chloroplastic proteins might be partly under nuclear control [an opinion which is shared by Zetsche (1966a) and for which we will find additional arguments in Section V] reinforces the idea that the control of protciii synthesis in anucleate fragments of Agetabularia occurs mainly at thc translational level of protein synthesis. One of the main problems, which will require close study, concerns the molccular incchanisms for the formation of the caps: we have already seen that the activities of certain enzymes markedly increase when caps are produced, even in the absence of the nucleus. Among thcse enzymes,

* Elegant grafting experiments of H. G . Schweiger et nl. (1967) have clearly shown that the isozyme pattern for malic dehydrogenase also is under nuclear control.

1.

SYNTHESIS A N D MORPEIOGENESIS IN

Acetabularia

15

special attention should perhaps be given to the UDPG-4-epimerase studied by Zetsche (1966d), because of the possibility that enzymes involved in the synthesis of the polysaccharides forming the cell wall are as important for morphogenesis in Acetabularia as they are in slime molds (see the review by M. Sussman, 1966, in Volume 1 of this serial). Interesting biochemical and biophysical changes certainly must occur at the tip of the stalk when the cap begins to form: for instance, Zetsche (1966d) recently found that the cell wall of the cap is richer in galactose than that of the stalk. Werz (1965,1966a,b) has shown that cellular differentiation during cap formation is initiated by lytic processes within the already existent cell wall. The tip of the stalk, especially prior to cap formation, gives a metachromatic (red) staining when the algae are placed in seawater containing toluidine blue (J. Brachet, unpublished) : such a metachromasia might indicate a localized accumulation of mucopolysaccharides in the regions where growth and morphogenesis occur. Very suggestive also is the fact that Zetsche (1966d) discovered in Acetabularia three different enzymes taking part in the synthesis of UDP-galactose at the expense of fructose 6-phosphate. The polysaccharide synthesized by the chloroplasts in Acetabulurk is not starch (as was usually thought), but inulin: Vanden Driessche and Bonotto (1967) have clearly shown that the only hydrolysis product of this polysaccharide is fructose. One can therefore imagine that the inulin which is stored in the chloroplasts gives rise to fructose phosphate, which would be converted to UDP-galactose; the latter would finally be incorporated into cell wall mucopolysaccharides. The continuation of work along these lines might yield results very important in the future for our understanding of the realization (expression) of the genetic information stored in stable mRNA molecules: after all, a cap is something more complex than a mixture of mRNA's! IV. RNA Synthesis in Nucleate and Anucleate Fragments of Acetabularia. Importance of the Chloroplasts

Is RNA synthesis possible in the absence of the nucleus in Acetabulurk? If so, which kinds of RNA molecules are made by nucleate and anucleate fragments? What is the base composition of the RNA's present in the various parts of the alga? These are the main questions to be discussed now. IN THE ABSENCEOF THE NUCLEUS A. NET RNA SYNTHESIS This problem has been the subject of long discussions, because contradictory results were obtained in different laboratories: while Brachet

16

J. BRACIIET

et al. (1955) found a net increase in the RNA content of anucleate fragments of Acetabularia, Richter (1959) and Naora et al. (1959) could not confirm this finding. That the discrepancy was probably due to diffcrences in the culture conditions (in particular, the amount of light given to the algae) was suggested by the work of Naora et aZ. (1960) : they found that, in anucleate fragments, RNA increases in the chloroplastic fraction and decreases in the cytoplasmic ones (supernatant after removal of the chloroplasts by low speed centrifugation). That the amount of light given to the algae is indeed an essential factor in these experiments has been conclusively demonstrated by Schweiger and Bremer (1960, 1961): they showed that the RNA content of the two kinds of fragments markedly decreases (30-40c;rO) if the fragments are kept in darkness for 10 days; but, if the anucleate fragments are reilluminated, significant RNA synthesis takes place. In particular, if nucleate fragments kept in the dark for 10 days are amputated from their nucleus and if the resulting anucleate fragments are illuminated, their RNA content almost doubles within 10 days. It is thus well established now that net R N A synthesis occurs in anucleate fragments of Acetabuluria if they are given adequate illumination. Under the experimental conditions adopted at that time in our laboratory, Brachet and Six ( 1966) observed a 50% increase (in 4-9 days) in the RNA content of the anucleate fragments in a series of 6 experiments (initial value: 0.38 pg of RNA per fragment; final value: 0.57 pg of RNA per fragment). At the present time, a 2-fold increase in the RNA content of anucleate fragments, within 5-7 days, is often observed in our laboratory. We know that RNA synthesis always takes place on a DNA template and that it is blocked when actinomycin reacts with this DNA. Is RNA synthesis sensitive to actinomycin in anucleate fragments of Acetabularia also? The answer to this question is a positive one: it has been found by Brachet and Six ( 1966) that prolonged actinomycin treatment (30 pg/ml) (7-15 days) results in a decrease in the amount of RNA in anucleate fragments as well as in whole algae. Curiously enough, the basal anucleate fragments of the stalk (in contrast to the apical ones) resist the actinomycin treatment. We know (see Fig. 3) that these basal fragments are extremely limited in their morphogenetic capacities, and it is possible that there exists some relation between the two phenomena (insensitivity to nctinomycin and restricted morphogenetic potentialities). More recent, still unpublished, experiments by Janowski and Bonotto

1.

SYNTHESIS AND MORPIIOGENESIS IN

Acetabularia

17

have confirmed, with different methods, that RNA synthesis in anucleate halves is actinomycin sensitive: the inhibition of ~ r i d i n e - ~incorporaH tion, after a 42-hour treatment with concentrated actinomycin (100 pg/ ml) is even greater in the anucleate than in the nucleate fragments (respectively, 70 and 30%). Inhibition of RNA synthesis (followed by uridine incorporation and autoradiography ) by actinomycin has also been found to occur in both kinds of fragments by Shephard (196%). In conclusion, there is no doubt that net synthesis of RNA is possible in anucleate fragments of Acetabularia and that this RNA synthesis is sensitive to actinomycin. It must, therefore, presumably take place on a cytoplasmic DNA template. Since, as we shall see in detail in Section V, Acetabularia chloroplasts contain DNA, one would conclude that, as suggested by Naora et al. in 1960, the chloroplasts are the main (or even the only) site for RNA synthesis in the absence of the nucleus. We shall now see that the situation is more complicated than that and, in fact, remains obscure. IN SUBCELLULAR PAiwrcLEs IsoLxrm B. RNA SYNTHESIS NUCLEATE AND ANUCLEATEFRAGMENTS

FROM

There are many difficulties in thc study of the RNA’s prcsent in the various fractions which can be collected by differential centrifugation from a homogenate of Acetabularia: for instance, it has not yet been possible to isolate clean cytoplasmic ribosomes in sufficient amounts to make a base analysis; the algnc contain very active enzymes for the degradation of RNA, and these enzymes differ from the classical ribonucleases in their response to inhibitors; the presence of chloroplasts complicates the interpretation of the data, since it is known that they contain ribosomes that are different in size and composition from those present in the cytoplasm. For this reason, the work done in our laboratory (which is still largely unpublished) must be considered too incomplete to allow the drawing of definite conclusions. The following should therefore be taken as a progress report. The important role played by the chloroplasts in the synthesis of RNA has been well demonstrated by Janowski (1965), who fractionated the 52P-labeledRNA of the algae by centrifugation and column chromatography. He was able to demonstrate the presence of three different kinds of RNA labeled in the absence of the nucleus: a small molecular weight RNA (tRNA or degradation products of larger RNA molecules) which is ubiquitous, a ribosomal type of RNA associated with the Chloroplasts, and a chloroplastic RNA that is closely associated with the

18

J. BRACHET

chloroplastic DNA, possibly in the form of a molecular hybrid. The presence of rapidly labeled (with 32P)RNA in both nucleate and anucleate fragments has been confirmed by Richter (1966). More recent experiments of Schweiger et al. (1967) have clearly shown that a &day incorporation of uracil-14C results in the labeling of the RNA’s present in the chloroplastic mitochondria1 and soluble fractions of both nucleate and anucleate fragments. The sucrose density gradient centrifugation profile of radioactivity is similar to that of isolated Escherichia coli ribosomes. Moreover, Berger ( 1967) has shown that isolated chloroplasts from Acetabularia retain their ability to synthesize 23, 16, 9, and 4 S RNA. Incorporation of RNA precursors is inhibited by darkness, actinomycin, and deoxyribonuclease. Recently, Janowski (1966, 1967; see also Baltus et al., 1968) worked out methods for the detection of ribosomes and polysomes in whole algae and in their fragments. For these experiments, algae or fragments were labeled with uridine-aH or with a mixture of 14C-labeled amino acids; the sedimentation profile, in a sucrose gradient, was compared beforc and after treatment with dilute ribonuclease (which destroys the fiber of mRNA which links together the ribosomes into a polysome). The results, which are shown in Fig. 7, can be summarized as follows: in whole plants, three peaks of uridine-incorporating particles can be seen (Fig. 7a); they have sedimentation coefficients of 82, 65, and 48 S. Ribonuclease converts heavier radioactive components to 82 S particles; they probably are polysomes made of 82 S monomeres. When the Mg++ concentration in the homogenate is increased (Fig, %), the only change that can be seen is a decrease in the yield of polysomes. It seems therefore unlikely that the 65 and 48 S components consist of subunits of dissociated monosomes. Anucleate fragments, 2 days after the removal of the nucleus, are still able to incorporate uridine into their polysomes, as well as into their 82, 65, and 48 S particles (Fig. 7c). The incorporation transferred from the polysomal region to the 82 S ribosomes, after ribonuclease treatment, is, however, very small. But, if the anuclcntc fragments are previously kept in the dark [as in Schweiger and Bremcr’s ( 1961) experiments], an important increase of radioactivity is observed in the 82, 65, and 48 S peaks and, especially, in the polysomes ( Fig. 7d ) . It has been demonstrated that the 65 S particles contain 23 S uridinelabeled RNA, while the 48 S particles contain 16 S RNA. The fact that anucleate fragments can still incorporate uridinc into the polysomes is quite unexpected and deserves careful verification; however, the strong effect of light on the formation of polysomes in anucleate

(a)

Fraction number

2oo

--

Fraction number

(b) 400r--1:

_-

I

1

10 20 30 (dl Fraction number (C) Fraction number FIG.7. Sedimentation profile of cytoplasmic ribosomes of Acetabularia labeled with uridine-8H. The plants were incubated for 2 hours in their growth medium containing 100 pC of uridine-3H per milliliter. Centrifugation was performed in a 15-3070 sucrose gradient (4.8 ml) for 2 hours at 37,500 rpm in the SW 39 rotor of a Spinco Model L 50 centrifuge. 0-0-0,Untreated sample; 0-0-0, RNase-treated sample. ( a ) Whole plants; final Mg" concentration in the homogenate: 5 x lO-3M. ( b ) Whole plants; final Mg" concentration in the homogenate: 8 x 103M. (c) Twoclay anucleate fragments that were allowed to regenerate in the light. ( d ) Two-day anucleate fragments that were allowed to regenerate in the dark.

20

J. BRACHET

fragments which had been previously cultivated in darkness suggests, as a hypothcsis, that mRNA molecules synthesized on a c h b r o p h t i c DNA templnte might diffuse out of the chloroplasts and combine with the cytoplasmic ribosomes to form active polysomes. A few recent, still unpublished, observations of Janowski and Bonotto deserve mention, although they might still need confirmation. For instance, these workers found that it is possible to isolate, from the chloroplasts, three different kinds of ribosomes or ribosomal subparticles corresponding approximately to the above-mentioned sedimentation constants for radioactive materials (i.e., 82, 65, and 48 S ) . It was impossible, with the same methods (involving the use of strong detergents) to isolate appreciable amounts of cytoplasmic ribosomes. It was also observed that, 4 weeks after the removal of the nucleus, only the chloroplasts are still the site of ribosome synthesis (in agreement with the conclusions of Naora et al., 1960). Finally, it was found that chloramphenicol inhibits the incorporation of uridine into the RNA of the polysomes and the S2 S ribosomes; the effects of this antibiotic on the 65 S and 48 S particles are different, a fact which confirms the idea that these particles are synthesized in an independent way: these particles do not derive one from the other; nor are they breakdown products of the 82 S ribosomes. Nothing more is known about the nature, origin, and possible role of these 65 S and 48 S particles; but their very existence shows the complexity of RNA metabolism in Acetabularia Other attempts have been made in our laboratory, in order to isolate, in undegraded form, various RNA fractions. In particular, since it is so difficult to isolate ribosomes from the algae, Baltus and Quertier (1966) have tried to characterizc the RNA present in these particles without their previous isolation. The classical 16 S and 25 S ribosomal RNA's could be recovered from normal algae; the two peaks are of equal size, whereas in most cases the 25 S peak is more prominent than the 16 S one. In anucleate fragments (Baltus et al., 1968), a complete disappearance of the two ribosomal peaks was sometimes observed; in other cases, only one peak had disappeared. Specially designed experiments (Baltus and Quertier, 1966; Baltus et al., 1968; M. Janowski and S. Bonotto, unpublished) have shown that the disappearance of one or of both ribosomal RNA (rRNA) peaks is not due to degradation during the isolation procedure; it has been demonstrated also that, in the ribosomes of normal algae, the content of 16 S rRNA is higher than of 25 S RNA, and that the latter does not undergo degradation into the former. Removal of the nucleus thus exerts strong effects on the rRNA's; but it is unlikely that the ribosomes themselves disappear in anucleate frag-

1.

SYNTHESIS AND MORPHOGENESIS IN

Acetabularia

21

ments: electron micrographs taken by M. Boloukhbre (Baltus et d., 1968) show that ribosomelike particles remain unchanged in their aspect and concentration in anucleate fragments. It might well be-but it remains to be proved-that removal of the nucleus is followed by degradation or loss of ribosomal RNA without disappearance of the

, 10

.-.-.,

20 Fraction number FIG. 8. Sedimentation profile of total RNA extracted from Acetabularia. Centrifugation was carried out in a 5-200/0 siicrose gradient (4.8 ml) for 5 hours at 87,500 rpm in the SW 39 rotor of a Spinco Modrl L 50 centrifuge. Optical density at 260 mpc.

ribosomal proteins. Such a conclusion would be in keeping with the often expressed idea that the rRNA's that have been synthesized in the nucleolus (on the DNA of the nucleolar organizer as template) combine, in the cytoplasm, with preexisting ribosomal proteins. Another approach i s being followed by F. Farber (unpublished), who precipitates the nucleic acids with 2 A1 LiCI, and then extracts them with phenol. This method yields only high-molecular, undegraded RNA's (tRNA molecules are too small to be precipitated by LiC1). Three peaks can be obtained from whole algae, in a reproducible way (Fig. 8), but their identity is not yet certain. At 2-7 days after the operation, there is no appreciable change in the pattern obtained from anucleate fragments; in the nucleate ones, peak I1 decreases momentarily. In whole algae submitted to a pulse with label uridine, peak I11 is quickly labeled ( Fig. 9),and looses its radioactivity in 3 days.

22

J. BRACHET

Finally, peak I disappears in both nucleate and anucleate fragrncnts after 4 weeks: this change might be correlated with cap formation. As already pointed out, it is difficult to draw conclusions from these experiments at their present stage. Regarding mRNA's and their possible

0.4

a3

z

d

0

V

a2

0.I

10 20 Fraction number

FIG. 9. Sedimentation profile of Acetabularia RNA exposed to a 10-minute puke of uridine-3H. Plants were incubated in their growth medium containing 12.5 &/ml of uridine-SH and centrifuged according to the procedure defined in Fig. 8. Optical density at 260 mp; 0-0-0, radioactivity.

- -

,

role in morphogenesis, two things are puzzling: ( 1 ) the existence of the hypothetical stabb mRNA molecules is certainly not easy to demonstrate with the methods used (so far, none of the fractions isolated by Farber have had any template activity in a system incorporating amino acids in uitro) "; ( 2 ) on the other hand, the synthesis of mRNA molecules in anucleate fragments is certainly surprising. New experiments are ccrtainly needed before we have to revise completely our conceptions about mRNA's and their role in Acetabularia: it might be that the stable mRNA is present in small amounts only or is easily degraded during its isolation; synthesis of mRNA in the absence of the nucleus could occur, as already seen, on a chloroplastic DNA template. Since this review was written, it was found by F. Farber et al. (in press) that bulk RNA isolated with the LiCl method has template activity, which decreases when caps are formed. This suggests that informational RNA is destroyed during cap formation.

1.

c.

23

Acetuliulariu

SYNTHESIS AND MORPHOGENESIS IN

BASECOMPOSITION OF THE RNA’S LOCALIZED IN THE REGIONSOF THE ALGA

VARIOUS

The work that will now be presented has been done in collaboration with Professor E. J. Edstrom (whose ultramicromethod for base analysis has been used in many cases) and has now been published (Baltus et al., 1968). The results are summarized in Tables I and 11. TABLE I BASE COMPOSITION OF RNA’s EXTRACTED FROM NUCLEOLUS, NUCLEAHSAP, CHLOROPLASTS, TIP, AND POSTERIOR PART OF TKE ALGAE.@ ADENINE CONTENT OF GAMETICDNA ( MICROELECTROPHORESIS ) MEASUREMENTS Originof thesample

n

A

u

G

RNA

Nucleolus Nucleoplasm Apex of the stalk Basal part of the stalk Whole chloroplasts

7 4 4 5 8

29.2 25.2 25.5 25.7 27.9

1.5 2.4 0.82 2.3 1.4

19.7 17.1 24.8 26.4 23.3

DNA

Gametes

9 29.0

1.7

-

Sample

C

Q

U

rs

1.3 1.4 1.1 2.2 0.90

20.6 19.8 23.0 23.7 18.8

2.2 1.4 1.1 0.66 1.4

30.0 33.2 26.7 24.3 29.8

2.0 3.3 2.5 1.4 2.1

-

-

-

(J

- -

n: Number of experiments; u: standard deviation; A, G, C, U: adenylic, guanylic, cytidylic, and uridylic acids. a

TABLE I1 BASECOMPOSITION OF RNA’s FROM CHLOROPLASTIC AND “TRUE” CYTOPLASMIC Rmosoms OF Acetabulariaa OriginoftheRNA

n

A

u

G

Q

C

“True” cytoplasmic ribosomes “rRNA” extracted for whole chloroplasts Chloroplastic ribosomes

3

21.0

1.7

22.5

0.17

2

24.0

0.92

26.0

2

27.0

-

30.0

n: Number of experiments;

cytidylic, and uridylic acids.

0:

0

U

18.8

0.11

38.0

1.0

0.78

28.5

1.7

21.5

0.10

-

21.1

21.9

-

-

rs

standard deviation; A, G, C, U: adenylic, guanylic,

The following comments can be made, although many of the results still remain difficult to interpret. 1. The RNA of the nucleolus is definitely a DNA-like RNA: the amounts of A and U, and those of G and C correspond very closely. Furthermore, the A content of the nucleolar RNA and the nuclear (gametic) D N A are the same. This situation is different from that found in the nucleoli of amphibian (Edstrom and Gall, 1963) and starfish (Edstrom,

24

J. BnACllET

1965) oocytes: thcir RNAs are, likc ribosotnal RNA, rich in guaninc. However, the nucleolus of Chironomus salivary gland cells has a basc composition similar to that of Acetabularia (Edstrom and Beermann, 1962). It would be tempting to speculate, in view of the probable necessity of large amounts of informational RNA’s for the morphogenesis of Acetabularia, and of the scarcity of cytoplasmic ribosomes in this alga, that the nucleolar RNA is an accumulation of mRNA (DNA-like composition, fast labeling). But it would be dangerous to take this speculation too seriously: according to a personal communication from Professor Edstrom, the nucleolar RNA in Chironomus and Drosophila, despite its DNA-like composition, is entirely preribosomal. 2. The base composition of the n u c b o p h m RNA is entirely different from that of the nucleolar RNA: it is very rich in U (38.2%), like the nuclear RNA’s which have been found recently in bird erythrocytes and HeLa cells by Attardi and his colleagues (1986) and by Houssais and Attardi (1966). Their exact role remains unknown: there is no simple relation between these U-rich nuclear RNA’s and cytoplasmic mRNA; they may play a role in the control of genetic activity, by acting as mRNA’s for the synthesis of nuclear proteins. In Acetabularia, nucleoplasm RNA may have something to do with the fact that the nucleus exerts an inhibitory influence on cap formation. 3. The chloroplastic ribosomes are, like the cytoplasmic ribosomes isolated from many other cells, of the GC type. The rRNA extracted from whole chloroplasts has a somewhat higher G C content ( 54.5% ) than that extracted from isolated chloroplastic ribosomes (51.1%) , This small difference has probably little-if any-significance; but significant differences are found when the composition of the total RNA isolated from whole chloroplasts ( G C = 42’;h ) is compared with that of chloroplastic rRNA. This large difference confirms that the chloroplasts of Acetabularia contain other RNA species than rRNA (see above, Section IV). It is worth noting that chloroplastic DNA has a G C content (45%) not very different from that of the RNA present in whole chloroplasts (see Section V ) . 4. No significant difference in base composition between the total RNA’s present in the apex and the basal part of the stalk could be detected, despite the fact that their content in “morphogenetic substances” must be very different (Fig. 3). But cyclosis is very active in Acetabulark and certainly makes the distribution of the quantitatively major species of RNA’s (the chloroplastic ones, in particular) fairly homogeneous.

+

+

+

1.

SYNTHESIS

AND MORPHOGENESIS IN

Acetabularia

25

V. Other Functions of the Chloroplasts: DNA Replication, Photosynthesis

A. GENERALITIES Growth and morphogenesis in Acetabularia absolutely require light (Beth, 1955; Richter, 1962; Clauss, 1963; Terborgh and Thimann, 1965; Terborgh, 1965), which produces the energy needed for the various syntheses through photophosphorylation: there is no photosynthesis in red light, and, under these conditions, the algae stop growing (Clauss, 1963; Terborgh, 1965). Removal of the nucleus exerts no measurable effect on the photosynthetic capacity, even after several weeks (Brachet et al., 1955). As shown by Werz (1966a), although the algae do not grow in the absence of light, young chloroplasts and mitochondria of nucleate and anucleate fragments can differentiate in the dark: they can form “grana” and “cristae,” respectively, under these conditions. These findings suggest that the chloroplasts and the mitochondria of Acetabularia can differentiate in an autonomous way; this autonomy is probably linked to the presence of D N A in these cell organelles, a point to which we shall return soon. But one important question should first be answered: Is the autonomy of the chloroplasts in relation to the nucleus so great that they can multiply in anucleate fragments? This question has been carefully studied by our former colleague Shephard (1965a), who gave a positive answer: the number of chloroplasts increases in the anucleate fragments; they are capable of selfreplication in the absence of the nucleus and must be largely autonomous relative to the latter. Division of the chloroplasts occurs only in the light, whether or not the nucleus is present. When nucleate and anucleate fragments are compared, it can be seen that division of the chloroplasts is less frequent in the absence of the nucleus; many large dumbbellshaped chloroplasts are found in the anucleate fragments. Some factor of nuclear origin is probably required to ensure the normal rhythm of chloroplast multiplication. This conclusion, as we have seen in Section 111, has also been drawn for the synthesis of chloroplastic enzymes (Brachet and Lievens, 1968; Zetsche, 1966b).

B. CYTOPLASMIC DNA

Acetabularia The idea that chloroplasts contain DNA and can replicate their own IN

DNA comes from our old autoradiography observations ( Brachet, 1959) :

26

J. BRACIlET

chloroplasts are capable of thymidine incorporation into acid insoluble material, even in anucleate fragments. Later work in our and other laboratories (Shephard, 1965b; de Vitry, 1965a; Chapman et al., 1966) involving control experiments ( including digestions with deoxyribonuclease ) has conclusively shown that this material is really chloroplastic DNA. Another cytochemical approach has been used by de Vitry (1964, 1965c), who showed that 14C-labeledactinomycin is incorporated in both nucleate and anucleate fragments of Acetabuluriu and combines with a material which can probably be identiiied as chloroplastic DNA. More direct evidence for the presence of DNA in Acetabulariu chloroplasts has been obtained by chemical estimation of the DNA content of chloroplasts isolated from anucleate fragments (in order to exclude any possibility of contamination by nuclear DNA, although the latter is present in such small amounts that the Feulgen reaction is negative in the nucleus). It was found (Baltus and Brachet, 1962; Gibor and Izawa, 1963) that the chloroplasts isolated from Acetabularia ( which might, as we shall see, be slightly contaminated with mitochondria) really contain small amounts of DNA (of the order of 1 to 3 x gm per chloroplast); they should be sufficient, in principle, for the coding of several hundred proteins having a molecular weight of 20,000. More recently, we have undertaken a study of the physical properties (buoyant density measurements in a CsCl gradient) of the cytoplasmic DNA's of Acetabularia (Green et al., 1967). The results are summarized in Fig. 10. Figure 11 shows electron micrographs of fractions obtained by differential centrifugation. As one can see in Fig. 10a, two peaks of densities 1.704 and 1.714 gm/ml can be obtained from a homogenate of anucleate fragments; they correspond, respectively, to a G C content of 45 and 55%. A purified fraction of large chloroplasts is strongly enriched for the lighter peak (1.704 gm/ml), but still contains some of the 1.714 gm/ml peak (Fig. lob). But, as shown in Fig. 10a, many of these large chloroplasts have mitochondria adsorbed to them or attached by cytoplasm. These experiments strongly suggest that the 1.704 gm/ml peak corresponds to the chloroplastic DNA, while the heavier peak (1.714 gm/ml) would be mitochondrial DNA. Attempts to prove this point, by working with a purified mitochondrial fraction, have not been completely successful: in a typical experiment where 5.000 anucleate fragments were used, 3 peaks of density 1.698, 1.714, and 1.722 gm/ml were found (Fig. 1Oc). But, as shown by electron micrograph (Fig. l l b ) , this fraction contains mitochondria, small chloroplasts, and contaminating niicroorganisms; the latter are probably responsible for the lighter peak (clen-

+

1.

SYNTHESIS AND MORPHOGENESIS IN

Acetabulariu

27

sity 1.698 gm/ml). No detectable amounts of DNA were found in cell walls or microsomes. Finally, in the cysts (which contain nuclei as well as chloroplasts), a rather broad peak was found (Fig. 10d); it has an average buoyant density of 1.702 gm/ml.

i

Fraction I

I1

cysts

\nucleate fragments

M L.

p = 1.704

~'1.704

7 p3Jq . :I Fraction

p= 1.698

I

LP7

p= I702

cI L

FIG. 10. ( a ) CsCl density gradient centrifugation of DNA from anucleate fragments of Acetabularia mediterranea. M.L. = Micrococcus lysodeikticus DNA reference ( p = 1.731 gm/ml). ( b ) CsCl density gradient centrifugation of fraction I DNA M.L. = M . Zysodeikticus DNA ( p = 1.731 gm/ml). ( c ) CsCl density gradient centrifugation of fraction I1 DNA with LP, phage DNA as density reference ( p = 1.741 gm/ml). (a) CsCl density gradient centrifugation of cyst DNA. LP, DNA ( p = 1.741 gm/ml).

It can be concluded from these experiments that the densities of thc chloroplasts and nuclear DNA's are similar, but probably not identical: the G C content of the nuclear DNA would be 42%, a value which corresponds exactly to the value found for the gametes by base analysis (Baltus et al., 1968) (see Table I). The G C content of cliloroplastic DNA would be a little higher (45% ) and very similar to that of the RNA isolated from whole chloroplasts of Acetabularia (42% ) . It is likely that

+

+

28

J. BRACHET

the 1.714 gm/ml peak is the mitochondria2 DNA, which could be very different from nuclear and chloroplastic DNA's in base composition. But the presence of bacterial contaminants (which could only be demonstrated by electron microscopy) makes this identification somewhat uncertain.

FIG. 11( a ) . For legend

see opposite

page.

Does the chloroplastic DNA have the same functions as the nuclear DNA? In other words, does it carry information which can be transcribed into RNA and translated into proteins? Is it capable of independent replication? The answers to these important questions are all positive ones.

1.

SYNTHESIS AND MOFWHOGENESIS IN

Acetabularia

29

Isolated chloroplasts from anucleate fragments of Acetabularia can incorporate RNA precursors (Schweiger and Berger, 1964) as well as amino acids (Goffeau and Brachet, 1965). These RNA and protein syntheses require light and are inhibited by actinomycin which, as we have

FIG. l l ( b )

FIG. 11. ( a ) Illustration of the CsCl density gradient centrifugation of Fig. 10a. Chloroplasts ( c ) with associated mitochondria ( m ) . ( b ) Illustration of the CsCl density gradient centrifugation of Fig. 1Oc. Small chloroplasts ( c ) , mitochondria ( m ) , and contaminating microorganisms ( b ) . x 25,000.

seen, combines with chloroplastic DNA. It is clear that the synthesis of chloroplastic proteins must take place by mechanisms very similar to those known to occur in whole cells (transcription, translation). Very recently, A. Goffeau (still unpublished) has analyzed in more detail the incorporation of amino acids by chloroplasts isolated from

30

J. BRACHET

anucleate fragments of Acetabularia. He found that it is inhibited by puromycin, chloramphenicol, and tetracycline, but not by cycloheximide; these findings are similar to the observations made on mitochondria. According to Goffeau‘s present work, 40% of the incorporation occurs in a protein fraction which is closely associated with the membrane of the chloroplast; about 10% is incorporated in the “structure protein” of the chloroplasts. This structure protein, which is insoluble in water at neutral pH, has the same amino acid composition in the chloroplasts of Acetabulark and the spinach. If, as we have seen, chloroplasts contain DNA and continue to multiply in anucleate fragments of Acetabularia, such fragments should be the site of a net synthesis of DNA. That this is really the case has been demonstrated recently by Heilporn-Pohl and Brachet ( 1966), who measured the DNA content of the anucleate fragments with a fluorometric method. It was found that DNA begins to increase a few days (between 2 and 7 ) after the section; this initial lag might be a consequence of the “surgical shock.” During the second week (7, 9, 10, 13, and 15 days), there is a marked increase in the DNA content of the anucleate halves, ranging between 70 and 140%. There is no consistent increase in fragments cultivated in the dark, i.e., under conditions where there is no chloroplast multiplication. The rates of DNA and RNA synthesis in anucleate fragments are essentially parallel, a fact which suggests that the two processes are linked together as one would expect if the two syntheses reflect the multiplication of chloroplasts. The kinetics of DNA synthesis in the nucleate and anucleate fragments are now being compared by Heilporn-Pohl and Limbosch: they find that the rates are very similar during the first 2 weeks; later on, DNA synthesis becomes higher in the nucleate halves. This is exactly what has been found for RNA and protein synthesis; obviously, after a few weeks, all the activities of the anucleate fragments (regeneration, multiplication of chloroplasts, synthesis of macromolecules) become restricted. We have seen, in Section 11, that hydroxyurea, an inhibitor of DNA synthesis, stops or slows down morphogenesis in both kinds of fragments ( Brachet, 1967). Current work by Heilporn-Pohl and Limbosch clearly shows that hydroxyurea inhibits DNA synthesis in anucleate as well as nucleate fragments of Acetabularia. It has no effect on the density of chloroplastic DNA, which probably remains intact in the treated algae. As already pointed out in Section 11, it is probable that hydroxyurea, as in other cells, acts by inhibiting the reduction of ribose into deoxyribose. Similar results have been obtained, in our laboratory, by Netrawali

1.

SYNTHESIS AND MORPHOGENESIS IN

Acetabularia

31

(unpublished), who has been using X-rays ( 5000-100,000 rads) : there is an excellent parallelism between the inhibition of DNA synthesis, the retardation of morphogenesis, and the radiation dosage. In both nucleate and anucleate fragments, irradiation at 100,000 rads inhibits DNA synthesis almost completely during at least 24 days. These experiments show that chloroplastic DNA has the same degree of radiosensitivity in the presence or the absence of the nucleus, C. RHYTHM IN PHOTOSYNTHETIC CAPACITY The existence in Acetabularia of a daily rhythm in photosynthetic capacity has been demonstrated by Sweeney and Haxo ( 1961) : the peak period of photosynthesis is at midday and its magnitude is almost five times that of the value found during the night. This endogenous rhythm (it persists for a few days in plants kept in continuous light of low intensity) remains intact after removal of the nucleus. In fact, Richter (1963) showed that anucleate fragments of Acetabularia still display a rhythm in photosynthetic capacity several weeks after enucleation, at a time when net synthesis of proteins and RNA has come to a standstill. This finding of Richter (1963) has been confirmed by Schweiger et al. (1964a), who could still detect the rhythm 40 days after enucleation. These observations suggest, at first glance, that the rhythm in photosynthetic capacity is entirely cytoplasmic and reflects the independence of chloroplasts from the nucleus. But we have already seen that this independence is not absolute: the synthesis of chloroplastic enzymes and the multiplication of the chloroplasts are partly under nuclear control. The existence of such a control, in the case of the rhythm in photosynthetic capacity, has been demonstrated by elegant experiments of Schweiger et al. (1964b), who combined nucleate and anucleate fragments of algae which were at opposite phases of the cycle; they found that it is the nucleate half which “sets the clock,” imposing its own rhythm on the anucleate, chloroplast-rich, fragment. Similar observations have been made recently in our laboratory by Vanden Driessche (1967); it was found that some of our Acetabularia cultures, which had been grown for a long time in the presence of a complex mixture of antibiotics (in the hope of getting completely sterile cultures) had lost their rhythm in photosynthetic capacity. Vanden Driessche ( 1967) made grafts of nucleate fragments of algae which had retained their rhythm into anucleate fragments of algae which had lost it, and vice versa; in all cases, the presence or absence of the rhythm in the grafted algae was found to be nucleus-dependent.

32

J. URACIIET

The study of such fine interactions between the nucleus and the cytoplasm is of great interest; unfortunately, the biochemical and even the biological analyses are only beginning. For instance, according to the work of Vanden Driessche (1966a, 1967), it seems that Acetabularia displays other “circadian” rhythms than that in photosynthetic capacity: there is also a rhythm in the shape (elongated or almost spherical) of the chloroplasts and, probably, in their inulin content. Incidentally, this work has confirmed Shephard’s conclusion (1965a) that some factor of nuclear origin is needed for chloroplast division at the maximal rate. An important question, which has been recently discussed by Vanden Driessche (1967) and by Brahmachary (1967), is the importance of RNA (mRNA in particular) and protein synthesis for the establishment and maintenance of circadian rhythms. This problem has been studied, by using inhibitors of these syntheses, by Vanden Driessche (1966b, 1967) and by Sweeney et al. (1967). The work of the two groups is difEicult to compare, since the species of Acetabulariu, the culture conditions, and the concentrations of the inhibitors used were all different. According to Vanden Driessche (1966b), actinomycin inhibits the rhythm for photosynthetic capacity in whole algae and nucleate halves; this rhythm (like the regeneration of caps, as we have seen before) is remarkably insensitive to actinomycin in anucleate halves. Sensitivity of the rhythm to actinomilcin is thus linked to the presence of the nucleus, presumably to the synthesis of mRNA by the nucleus. It is interesting to note that the rhythmic changes in the shape of the chloroplasts are also sensitive to actinomycin in nucleate algae and insensitive in the anucleate fragments; however, these changes are never as marked in the latter as in the nucleate halves. Regarding the effects of various inhibitors of protein synthesis on the rhythm of photosynthetic capacity, Vanden Driessche (1967 and still unpublished results) finds that chloramphenicol abolishes the rhythm in both kinds of fragments; puromycin reduces it, but strongly decreases photosynthesis itself. Cycloheximide also strongly affects photosynthesis; like chloramphenicol and in contrast with puromycin, it completely abolishes the rhythm. In the experiments of Sweeney et al. (1967), which were made on anucleate fragments only, high concentrations of actinomycin and puromycin inhibited photosynthesis without suppressing the rhythm. They also inhibited the incorporation of leucine into proteins; curiously enough, the algae became resistant to actinomycin after 3-4 days of treatment and resumed incorporation of leucine into their proteins.

1.

SYNTHESIS AND MORPHOGENESIS IN

Acetabularia

33

It is obviously too early to draw conclusions from this work, which is still incomplete. But there is no doubt that Acetabularia will be a very favorable material for the detailed analysis of the role played by the cell nucleus, mRNA synthesis, and protein synthesis in “biological clocks.” VI. Concluding Remarks

It is clear that Acetabularia is a strange organism for molecular biologists as well as for cell biologists; the latter are impressed by the capacity

FIG.12. Nucleus of Acetabularia. ( a ) Normal nucleus. ( b ) Nucleus treated with dinithrophenol.

of regeneration in the absence of the nucleus and by the peculiarities of this nucleus itself, which contains almost undetectable amounts of DNA and an enormous, RNA-rich nucleolus. The structure and composition of this nucleolus (and of the nucleus itself) are deeply altered when energy production in the cytoplasm is decreased, as shown in Fig. 12. Such changes can be obtained, in a reversible way, by cultivating the algae in the dark and bringing them back into the light (Stich, 1951). Another very interesting effect of the cytoplasm on the nucleus should be mentioned: as shown by Hammerling (1953), cutting the stalk each time the cap is about to form prevents the breakdown of the nucleus and its entry into mitosis. The factors that determine whether the nucleus will remain in interphase or undergo mitotic division must be cytoplasmic.

34

J. BRACHET

A biochemical study of this cytoplasmic control would be extremely interesting. Molecular biologists, at first sight, might be surprised by the existence of extensive DNA and RNA net synthesis in anucleate cytoplasm; it is a consequence of the presence of the chloroplasts and of their great (but not complete) autonomy in relation to the nucleus. The chemical nature of the “morphogenetic substances” remains unknown; it is quite possible that, as is probably the case for the inducing substances of the amphibian eggs (review by Tiedemann, 1967), they are specific proteins synthesized under the control of mRNA’s produced on chromosomal DNA. That mRNA’s synthesized in the nucleus can survive for a long time in a stable form in Acetabulariu cytoplasm seems hard to deny. However, no one has so far succeeded in isolating and identifying cap-forming substances. We also know next to nothing about another important problem: the control of gene activity in the nucleus of Acetabularia. A careful study of the histones present in this alga might be rewarding in this respect. The presence of chloroplasts in Acetabularia is a complicating factor for those who are used to work with animal cells or bacteria. But it is possible, as we have done recently (Brachet, 1967), to compare Acetabulark with anucleate fragments of sea urchin or amphibian eggs. Such a comparison would take us too far if we went into detail. In summary, anucleate fragments of eggs have very restricted morphogenetic potentialities ( cleavage, usually abnormal) as compared to Acetabularia. Like this alga, they contain cytoplasmic DNA, but it is localized in mitochondria and in yolk platelets, not in chloroplasts. Slight incorporation of thymidine, reflecting perhaps the multiplication of mitochondria in the absence of the nucleus, is detectable. All kinds of RNA’s are present in the cytoplasm, including stable mRNA molecules. Under conditions where there is no RNA synthesis, protein synthesis can occur in such anucleate fragments of eggs. Evidently the similarities between the alga and the egg are greater than might appear at first glance. A close comparison between the two systems, at the molecular level, will certainly greatly help in solving important problems of morphogenesis and cell differentiation. ACKNOWLEDGMENTS

I wish to thank Dr. P. Malpoix for help in the preparation of the English text and

all my colleagues in the laboratory for interesting and useful discussion of their work

on Acetabularia. This work has been financially supported by Euratom (Contract Euratom-U.L.B. 007-Bl-ABIB) .

1.

SYNTHESIS AND MORPHOGENESIS IN

Acetabularia

35

REFERENCES Attardi, C., Parnas, H., Hwang, M., and Attardi, B. (1966). J. Mol. Biol. 20, 145. Baltus, E. (1959). Biochim. Biophys. Acta 33, 337. Baltus, E., and Brachet, J. (1962). Biochim. Biophys. Acta 61, 157. Baltus, E., and Quertier, J. (1966). Biochim. Biophys. Acta 119, 192. Baltus, E., Edstrdm, J. E., Janowski, M., Quertier, J., Tencer, R., and Brachet, J. (1968). Proc. Nutl. Acad. Sci. U S . 59, 406. Berger, S. (1967). P r o t o p h m a 64, 13. Beth, K. (1955). 2. Naturforsch. lob, 267. BoloukhAre-Presbourg, M . ( 196s). J. Microscop. 4, 363. Brachet, J. ( 1957). “Biochemical Cytology.” Academic Press, New York. Brachet, J. ( 1958). Erptl. Cell Res. 14, 650. Brachet, J. (1959). Exptl. Cell Res. Suppl. 6, 73. Brachet, J. (ed. ) ( 1961). “The Weizmann Lectures.” Rehovoth Institute, Rehovoth. Brachet, J. (1965). Bull. Chsse Sci. Acad. Roy. Belg. 51, 257. Brachet, J. (1967). Nature 214, 1132. Brachet, J., and Lang, A. (1965). Handb. Pflanzenphysiol. 15, 1. Brachet, J., and Lievens, A. (1968). Biokhimiya. In press. Brachet, J., and Six, N. (1966). Planta 68, 225. Brachet, J., Chantrenne, H., and Vanderhaeghe, F. ( 1955). Biochiin. Biophys. Acta 18, 544. Brachet, J., Denis, H., and de Vitry, F. (1964). Deoelop. Biol. 9, 398. Brahmachary, R. L. (1967). Intern. Rev. Cytol. 21, 65. Chapman, C. J., Nugent, N. A., and Schreiber, R. W. (1966). Plunt Physiol. 41, 589. Clauss, H. (1958). Plantu 52, 334. Clanss, H. (1959). Phnta 52, 534. Clauss, H. ( 1963). Natzirwissenchuften 50, 719. de Vitry, F. (1962). Protoplasma 55, 313. de Vitry, F. (1964). Compt. Rend. Acad. Sci. 253, 4829. de Vitry, F. (1965a). Bull. Soc. Chim. Biol. 47, 1325. de Vitry, F. (1965b). Bull. SOC. Chim. Bwl. 47, 1353. SOC. Chim. Biol. 47, 1375. de Vitry, F. ( 1 9 6 5 ~ ) Bull. . Edstroni, J. (1965). Biochem. Biophys. Res. Commrtn. 18, 595. Edstrom, J., and Beermann, W. (1962). J . Cell B i d . 14, 371. Edstroni, J., and Gall, J. C. (1963). J. Cell Biol. 19, 279. Farber, F. et al. Proc. Natl. Acud. Sci. U . S., in press. Gibor, A. (1966). Sci. Am., p. 118. Gibor, A., and Iznwa, M. ( 1963). Proc. Nutl. Acnd. Sci. U.S.50, 1164. Goffean, A., and Brachet, J. (1965). Biochim. Biophys. Acfn 95. 302. Green, B., Heilporn, V., Limlmsch, S., BoloukhBre, M., and Brachet, J. ( 1067). Proc. Nutl. Acad. Sci. U . S . 58, 1351. Hammerling, J. ( 1934). Arch. E ~ ~ t w i c k l ~ ~ ~Organ. g ~ m 131, ~ ~ h1. . Hiimmerling, J. (1953). Intern. Reo. Cytol. 2 , 475. Hammerling, J. (1963). Ann. Reu. Plant Pathol. 13, 65. Heilporn-Pohl, V., and Brachet, J. (1966). Biochim. Biophys. Actu 119, 429. Houssnis, J., and Attardi, C. (1966). Proc. Natl. Acad. Sci. U.S. 56, 616. Tacob, F., and Monod, J. (1961). J. Mol. Biol. 3, 318. Janowski, M. (1965). Biochim. Biophys. Acta 103, 399. Janowski, M. (1966). Life Sci. 5, 2113. Janowski, M. (1967). Arch. Intern. Physio2. Biochim. 75, 172.

36

J. BRACHET

Keck, K., and Claws, H. (1958).Botan. Gaz. 120,43. Naora, H.,Richter, G., and Naora, H. (1959).Erptl. Cell Res. 16, 434. Naora, H., Naora, H., and Brachet, J. (1960).J. Gen. Physiol. 43, 1083. Olszewska, M.,and Brachet, J. (1961). Exptl. Cell Res. 23, 370. Olszewska, M., de Vitry, F., nntl Brachet, J. (1961).Exptl. CeU Re.s. 24, 58. Puiseux-Dao, S. (1958).Compt. Rend. Acad. Sci. 246, 1076. Puiseux-Dao, S. (1963). Ann. Biol. 2, 99. Richter, G. (1959).Bwchim. Biophys. Acta 34, 407. Richter, G. (1962).Naturwissenchaften 49, 238. Richter, G. (1963).Z. Naturforsch. 18, 1085. Richter, G. (1968).Nature 212, 1363. Schweiger, E.,Wallraff, H. G., and Schweiger, H. G. (1964a).Z. Naturforsch. 19b, 499. Schweiger, E., Wallraff, H. G., and Schweiger, H. G. (1964b).Science 146, 658. Schweiger, H. G. (1968).Planta 68, 247. Schweiger, H. G.,and Berger, S. (1964).Biochim. Biophys. Acta 87, 533. Schweiger, H.G.,and Bremer, J. (1960).Erptl. Cell Res. 20, 617. Schweiger, H. G., and Bremer, J. (1961).Biochim. Biophys. Acta 51, 50. Scliweiger, €I. C . , Dillards, W. L., Cibor, A., and Berger, S. ( 1967a). Protoplamia 64,1. Scliweiger, H. C.. ct 01. (19671)).Nafrtre 216, 554. Shephard, D. (1965a).Expll. Cell Res. 37, 93. Shephard, D. (1965b).Biochim. Biophys. Acta 108, 635. Spencer, T., and Harris, H. (1964).Biochem. J. 91, 282. Stich, H. (1951).2. Naturforsch. Gb, 319. Stich, H., and Plaut, W. (1958).J . Biophys. Biochem, Cytol. 4, 119. Stutz, E.,and Noll, II. (1967).Proc. Natl. Acad. Sci. US.57, 774. Sussman, M. ( 1966). In “Current Topics in Developmental Biology” (A. A. Moscona and A. Monroy, eds.), Vol. 1, p. 61. Academic Press, New York. Sweeney, B. M., and Haxo, F. T. (1961).Science 134, 1361. Sweeney, B. M.,Tnffli, C. F., and Rubin, R. H. (1967).J . Gen. Plrysiol. 50, 647. Terborgh, J. ( 1965). Nature 207, 1360. Terborgh, J., and Thimann, K. V. (1965).Planta 64, 241. Tiedemann, &I. (1967).In “Current Topics in Developmental Biology” (A. A. Moscona and A. Monroy, eds.), Vol. 1, p. 85. Academic Press, New York. Triplett, E. L., Steens-Lievens, A., and Baltiis, E. (1965). Erptl. Cell Res. 38, 366. Vanden Driessche, T. (1966a).Exptl. Cell Res. 42, 18. Vanden Driessche, T. (1966b).Biochim. Biophys. Acta 126, 456. Vanden Driessche, T. (1967).Sci. Progr. 55, 293. Vanden Driessche, T., a n d Bonotto, S. ( 1987). Arch. Intern. Pliynhl. Biuchim. 75, 186. Werz, G. (1961).Z. Naturforsch. 16b, 126. Werz, G. (1965).Brookhaven Symp. Bfol. 18, 185. Werz, G . ( 1966a).Planta 68, 256. Werz, C. (196613).Phnfa 69, 53. Werz, G.,and Hiimmcrling, J. ( 1961 ). Z.Nuturforsch. ISb, 829. Zetsche, K. (1964).2. Naturforsch. 19b, 751. Zetsche, K. (1966a).Z. Naturforsch. 21b, 375. Zetsche, K. (1966b).Z. Naturforsch. 21b, 88. Zetsche, K. ( 196Bc). Planta 68, 360. Zetsche, K. (1966d).Biochim. Biophys. Acta 124, 332.