©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression of Cytochrome c Oxidase during Growth and Development of Dictyostelium(*)

(Received for publication, October 18, 1994; and in revised form, December 16, 1994)

Dorianna Sandonà Stefano Gastaldello Rosario Rizzuto Roberto Bisson (§)

From the Consiglio Nazionale delle Ricerche Unit for the Study of Physiology of Mitochondria and Laboratory of Molecular Biology and Pathology, Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, I-35121 Padova, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the slime mold Dictyostelium discoideum, the subunit composition of cytochrome c oxidase depends on oxygen that inversely regulates the concentrations of two alternative isoforms of the smallest enzyme subunit (Schiavo, G., and Bisson, R.(1989) J. Biol. Chem. 264, 7129-7134). In order to investigate their role in the Dictyostelium life cycle, the expression of the oxidase subunits was monitored during cell growth and development. The results obtained demonstrate that exponentially growing amoebae respond rapidly and precisely to hypoxia by switching the expression of the two isoforms and also by increasing the levels of the mRNAs of the different oxidase subunits in a highly coordinated process. During normal development the ``hypoxic'' subunit is not synthesized, but its level of expression appears to parallel the sensitivity to oxygen of development, rising steeply below 10% oxygen when the differentiation program is virtually blocked. Under these conditions, the expression of the alternative subunit isoform is essentially oxygen-insensitive. These findings suggest that the physiological relevance of the subunit switching concerns primarily the vegetative phase of growth, possibly as part of a more general mechanism evolved in order to evade conditions that do not allow development. Taken together, the data obtained offer an intriguing example of the fine control exerted on the expression of a key respiratory enzyme in a strictly aerobic organism.


INTRODUCTION

In many aerobic prokaryotes the enzyme composition of the terminal part of the respiratory chain depends on growing conditions, which can influence the expression of different operons encoding alternative cytochrome oxidases(1) . This feature allows the cell to cope with rapid and drastic modifications in the environment(2, 3) . Though structural and functional similarities suggest that many of these enzymes are part of the same large superfamily(4, 5) , differences in subunit number, prosthetic groups, and utilized substrates are frequently found. These radical changes do not involve the mitochondrial respiratory chain, where a single member of the superfamily, cytochrome c oxidase, is constantly present. Nevertheless, limited changes of the enzyme polypeptide composition have been described both in lower eukaryotes and in higher organisms. These structural modifications do not involve the catalytic core of the protein, constituted by the two largest and highly conserved mtDNA encoded subunits, but some of the additional polypeptides that are assembled in the complex as products of nuclear genes(6) . Not only does the number of these nuclear subunits appear to increase with the degree of evolution of the organism, but also some of the subunits are present with alternative isoforms that are tissue-specific in multicellular organisms and environmentally controlled in lower eukaryotes(7, 8, 9) .

In the slime mold Dictyostelium discoideum, two cytochrome c oxidase isozymes have been found. They differ only in the smallest nuclear encoded component, subunit VII, which is assembled with three other polypeptides to the catalytic core of the complex(10) . The relative concentrations of the two isozymes depend on oxygen. The smallest subunit present at normal oxygen tension, termed VIIe, is in fact replaced by a larger polypeptide, termed VIIs, under hypoxia(9) . Structural analyses have shown that the presence of the two alternative polypeptides is the consequence of an early gene duplication event(11) . More recently, evidence for a functional role of subunit VII both in the assembly and in the modulation of the enzyme activity was also provided(12) . Nevertheless, the selective advantages conferred to the organism by the two subunit isoforms remain unclear. This aspect is particularly relevant because the existence of isoforms for some nuclear encoded subunits of cytochrome c oxidase is, as mentioned above, an intriguing, typical feature of the enzyme in mammalian tissues(6, 14, 15, 16, 17) . In this context, Dictyostelium appears as a model system, because it is a strictly aerobic microorganism with the structurally simplest cytochrome c oxidase so far isolated from eukaryotes(18, 19) . The presence in its life cycle of a developmental stage, which includes the formation of multicellular bodies and an elementary differentiation program(20) , is an additional interesting feature that raises the possibility that the subunit switching might be linked to this process(10) .

To investigate this hypothesis, the expression of the nuclear encoded subunits of cytochrome c oxidase has been analyzed during both growth and development in normal and hypoxic environments. Our data show that the efficiency of the coordinated mechanism that controls both structure and concentration of the mitochondrial enzyme in vegetatively growing amoebae is largely reduced after cell aggregation. Nevertheless, a similar pattern is found between the level of expression of the hypoxic subunit isoform and the inhibition of cell differentiation induced by oxygen. Though this polypeptide is not synthesized under normal development, these data are interpreted to suggest a link between the oxidase subunit switching and differentiation that, with a sophisticated oxygen-sensing mechanism, could have been evolved to prevent conditions that do not allow sporulation.


EXPERIMENTAL PROCEDURES

D. discoideum Growth and Development

D. discoideum amoebae (strain AX3) were grown axenically at 22 °C in suspension as described previously(10) . Conditions of limited oxygen supply were created either by fluxing the culture flasks with suitable oxygen/nitrogen mixtures or by limiting their opening as detailed in the legend to Fig. 2. For development, cells (3 times 10^6 cells/ml) were harvested, washed in 1.4 mM KH(2)PO(4), 2.0 mM Na(2)HPO(4), pH 6.1, and layered on 2% agar, prepared in the same buffer, at approximately 3 times 10^6 cells cm. Development at low oxygen tension was induced in a incubator chamber in the presence of a light source.


Figure 2: Expression of the nuclear encoded subunits of cytochrome c oxidase in amoebae growing exponentially under a variable oxygen tension. The experiment is schematically represented (top,a-e). Cells from a preculture (a) were inoculated in 6-liter culture flasks containing 1.8 liters of medium (b) with an opening limited to a diameter of 1 cm in order to induce a progressive decrease of the oxygen concentration (shown by open squares in the diagram) as the cell density increased (from b to c). The hypoxic environment was then suddenly released by diluting the cell in culture flasks with a normal (6-cm diameter) opening (d). In order to control the rate of decrease of the oxygen tension, suitable cell concentrations (indicated as cells/ml by the number reported in the scheme) were considered. The relative concentrations of the transcripts of the different enzyme subunits (indicated by roman numerals) are shown by the autoradiograms in the lower part of the figure. Northern analyses were performed with total cellular RNA extracted from amoebae harvested at different times (indicated by the vertical segments) during the 48-h experiment. The shaded area indicates the time lapse required to completely reverse the expression of the two subunit isoforms VIIe and VIIs from hypoxia to normoxia. For only these two polypeptides, the corresponding concentrations in the mitochondrial membrane, analyzed by Western blotting, are reported in the graph (circle, subunit VIIe; bullet, subunit VIIs). No significant changes of the growth rate were noticed during the course of the experiment.



Polymerase Chain Reaction

Two oligonucleotide pools corresponding to the extreme regions (MTHALPKVV and FFKYGV) of the known amino terminus of cytochrome c oxidase subunit VIIs (11) with degeneracies of 8192 and 256, respectively, were synthesized and used as primers to amplify the first half of the gene. The polymerase chain reaction (21) was performed in a Perkin-Elmer thermal cycler. 1 µg of total Dictyostelium genomic DNA and 100 pmol of each oligonucleotide were mixed with 200 nmol of each dNTP and 2 units of Taq polymerase (Boehringer Mannheim) in a 100-µl total volume and subjected to 25 cycles of denaturation at 94 °C for 90 s, annealing at 37 °C for 2 min, and polymerization at 72 °C for 90 s. The reaction products were separated on 1.5% low melt agarose gel. The major band, which exhibited the expected 100-bp (^1)size, was isolated for further characterization.

Cloning and Sequencing of the Polymerase Chain Reaction Fragment

The isolated 110-bp DNA fragment was phosphorylated with T4 DNA kinase and ligated to HincII-digested and dephosphorylated pBS+ vector (Stratagene). Plasmid DNA containing the insert, obtained from transformed JM109, was isolated and purified. Both strands of the insert were sequenced by Sanger's dideoxy chain termination method (22) using the M13 forward and reverse sequencing primers.

Isolation of Mitochondria and Western Blot Analyses

Mitochondria were purified from exponentially growing cells as reported elsewhere(18) . Western blotting was performed as described previously by using purified polyclonal antibodies and densitometric scanning for quantitation(9, 12) . Concentration of cytochrome c oxidase was determined spectrophotometrically by using a Perkin-Elmer Lambda 5 UV-visible spectrophotometer(12) .

RNA Extraction and Northern Blot Analyses

RNA was isolated from growing and developing cells according to Nellen et al.(23) . Quantitation was performed as previously reported (12) and by using the Packard InstantImager electronic autoradiography system. Other recombinant DNA techniques were according to standard procedures(24) .


RESULTS

Cloning of a Subunit VIIs Gene Fragment

As isolated, Dictyostelium cytochrome c oxidase is constituted by two large mitochondrial subunits, indicated by the roman numerals I and II, and four smaller polypeptides encoded by nuclear genes, termed subunits IV, V, VI, and VII(18) . The cloned cDNAs of the subunit isoform present at normal oxygen tension, subunit VIIe, and of the remaining three nuclear polypeptides were used to follow gene expression in the different conditions tested. A suitable probe for the enzyme subunit isoform present at low oxygen, subunit VIIs, was obtained as shown by Fig. 1. On the basis of the available sequence of the polypeptide NH(2)-terminus(11) , two degenerated oligonucleotides were designed and used as primers for a polymerase chain reaction with genomic DNA to amplify the corresponding coding region of the gene, as described under ``Experimental Procedures.'' The electrophoretic analysis of the reaction mixture (data not shown) exhibited a major band at 110 bp, as expected by the relative position of the primers in the DNA sequence deduced from protein (Fig. 1). Fig. 1reports the sequence of the 110-bp amplified and cloned DNA segment. Translation to protein demonstrates the exact correspondence to the NH(2)-terminus of subunit VIIs. Because the apparent molecular mass of the polypeptide is 6.6 kDa (approximately 60 residues), the target sequence represents the first half of the coding region of the gene. Fig. 1also shows the identities of this DNA segment with the homologous gene encoding the isologue subunit VIIe. The relatively low degree of similarity of the two genes (60%) appears to be comparable with the values found in yeast and mammals between isoforms of the same subunit(13, 14, 15, 25) .


Figure 1: Cloning of the 5` coding region of the subunit VIIs gene. The available sequence at the amino-terminal end of the polypeptide (shown in the figure as a ribbon with a length proportional to the size of the protein) was used to derive the nucleotide sequences for two pools of degenerate primers employed in polymerase chain reaction with total Dictyostelium genomic DNA. A 110-bp amplified fragment was purified from the reaction mixture, cloned into a pBS+ vector, and sequenced. This confirmed the isolation of the truncated subunit VIIs gene encoding residues 1-34. The figure reports the homology of the cloned fragment with the corresponding DNA of the alternative subunit VIIe gene(40) . The alignment of the corresponding protein regions (11) is also shown for comparison. The primer sequences are given using standard abbreviations to represent ambiguity (R, G or A; Y, C or T; N, A or C or G or T). The corresponding peptides are highlighted in boldface. See ``Experimental Procedures'' for details.



Expression of Cytochrome c Oxidase in Vegetatively Growing Cells at Different Oxygen Concentrations

The sensitivity of the cell response to environmental changes was analyzed at both mRNA and protein levels. Large oscillations of the oxygen tension in the culture medium from 240 µM, the normal value at 22 °C, to 10 µM were obtained as shown schematically in Fig. 2(top) and were monitored by an oxygen electrode. Cells were inoculated into culture flasks with a reduced opening. As their density increased, the rate of oxygen diffusion from the environment to the flask became limited and was not sufficient to compensate for the consumption in the medium. Close to anaerobiosis, the slow decline of the oxygen tension (shown by open squares in the diagram of Fig. 2) was rapidly reversed by diluting the cells in culture flasks with a normal opening. This rapid transition to normal conditions was induced essentially to evaluate the time required by the cells for adaptation in a new environment (see below). It is noteworthy that, in spite of the large drop in oxygen tension, the cells maintained normal exponential growth (data not shown). This observation indicates that the Dictyostelium amoebae did not suffer any damage during the 2-day experiment that likely simulates conditions that the slime mold is prepared to face in its physiological environment (for instance, when the amoebae grow in a nutrient-rich but poorly aerated puddle in forest soil).

As shown by the Northern blots of Fig. 2, the levels of subunit VIIe and VIIs mRNAs are dramatically and inversely affected by oxygen. The sensing mechanism that controls the process is tailored to respond to oxygen fluctuations in the medium from 200 to 10 µM. It should be noted, however, that the response is not linear but increases considerably between 100 and 10 µM oxygen.

An additional relevant aspect concerns the rate of adaptation to new conditions of growth, which can be clearly evaluated when the cells exposed to 10 µM oxygen are suddenly diluted into a fresh medium. As shown by Fig. 2, the mRNA for subunit VIIe becomes detectable on Northern blots within 30 min. The steady state is attained after 100 min when, simultaneously, in a tightly coordinated process, the level of the messenger of the alternative subunit isoform becomes negligible.

The relative concentrations of the two isoforms in the mitochondrial membranes of cells grown under the same conditions are also reported in the diagram of Fig. 2(open and closed circles for subunit VIIe and VIIs, respectively) for comparison. The data were obtained by immunoblotting using subunit-specific antibodies, as described under ``Experimental Procedures.'' It may be noticed that the subunit switching correlates with mRNA changes, though only qualitatively. This is particularly evident following the rapid transition from 10 to 240 µM oxygen. Whereas, as reported above, subunit VIIs mRNA disappears in about 2 h (shaded section of Fig. 2), the protein is still detectable after 20 h. Taking into account the 8 h of cell doubling time, these data suggest that the half-lives of mRNA and protein are approximately 1 and 40 h, respectively.

The drastic changes discussed above do not involve the remaining nuclear encoded subunits of cytochrome c oxidase. Nevertheless, a careful inspection of the Northern blots shows an apparent increase in the cell concentration of subunit V and VI mRNAs at low oxygen.

This possibility was further tested by quantitative Northern blotting of RNA samples extracted from cells grown under normal oxygen or exposed to a nitrogen atmosphere for 2 h. This latter condition allows complete conversion from subunit VIIe to subunit VIIs mRNA without any damage of the cells, which in fact can grow and develop regularly if then brought back to normoxia. As an additional control, RNA samples taken from cells re-exposed to normal oxygen for 2 h after the hypoxic treatment were also considered. Fig. 3A shows the results obtained. Hypoxia induces a remarkably similar 20-30% increase of the level of subunit IV, V, and VI mRNA (A, shaded bars). This behavior of the oxidase genes appears to be specific, because similar analyses of two control genes, guk and gip17 encoding a mitochondrial and a cytoplasmic form of the enzyme nucleoside diphosphate kinase, respectively(26, 27) , show no significant change (guk) or rather an inhibition (gip17) of their expression. The possibility that the observed changes may result in an increase of the cell enzyme concentration was also investigated. In this case, the amoebae were exposed for 30 h to 5% oxygen. Again, these are conditions that induce a complete switching between the two subunit isoform mRNAs (9) but also allow exponential growth for the time needed to approach the steady state of protein concentration in the new environment (diagram of Fig. 2). As shown by Fig. 3B, exposure to hypoxia induces a small but significant rise in the concentration of spectroscopically detectable cytochrome c oxidase, which is comparable to the increase of the level of the enzyme transcripts.


Figure 3: Influence of oxygen on cell concentration of cytochrome c oxidase. A shows the increase of the mRNA concentration of different subunits of cytochrome c oxidase when exponentially growing cells are shifted from a normal (open bar) to a nitrogen environment for 2 h (dotted bar). The process is fully reversed when the hypoxic amoebae are re-exposed for 2 h to a normal atmosphere (striped bar). guk and gip17 are the two Dictyostelium genes used as controls, encoding a mitochondrial and a cytoplasmic form of the enzyme nucleoside diphosphate kinase, respectively. As demonstrated by the results of B, if the cells are grown in a suitable, hypoxic environment (5% oxygen) for a time sufficient to complete the enzyme subunit switching (at least 30 h), the increased mRNA expression results in a parallel increase of the enzyme (COX) concentration (dotted bar). The data reported in the figure averaged different measurements performed on 12 and 4 independent experiments in the cases of mRNA and protein, respectively. The same filters were rehybridized with the different oligonucleotide probes to minimize the effect of any possible unequal loading of the gels on the comparison of the data.



Expression in Developing Cells

As mentioned above, an interesting phase of the Dictyostelium life cycle, normally induced by starvation, is development. In this stage, amoebae aggregate in multicellular bodies termed pseudoplasmodia and initiate a differentiation program. Two major cell types, termed prespore and prestalk cells, are formed that, after approximately 24 h, lead to formation of the fruiting bodies constituted by a cellulose stalk holding a balloon-like structure filled with spores(20) .

Northern blotting was again used to monitor the expression of the cytochrome c oxidase subunits. Progress of development was evaluated by the time course of the different morphological stages, while the synchrony of the process was followed with two specific cDNA probes termed gip17 and pDd63, respectively. As already mentioned, gip17 encodes the cytoplasmic form of the enzyme nucleotide diphosphokinase, whose synthesis is strongly reduced within a few hours after cell aggregation and again resumed during terminal differentiation(28) . On the contrary, the pDd63 cDNA recognizes a prestalkspecific mRNA, which appears only in the late stages of development(29) .

Fig. 4shows the result obtained during normal development. As expected, the gip17 gene is strongly down-regulated at the time of cell aggregation(28) . Only a few hours later, when the center of the aggregates forms a small tip and rises into the air as an elongated cylinder (the ``first finger'' morphological state), the pDd63 gene is activated(29) . In contrast with the behavior of these two genes, those encoding the cytochrome c oxidase subunits, represented in the Northern blots of Fig. 4by the mRNA of the largest (subunit IV) and the smallest (subunit VIIe) nuclear polypeptides, appear to evenly decrease their expression following a remarkably similar pattern. The down-regulation begins after the formation of tight aggregates leading to a 50% decrease of the mRNA concentration at culmination. As shown by Fig. 4, the mRNA of the alternative subunit isoform, subunit VIIs, remains undetectable in the different stages of normal development.


Figure 4: Time course of mRNA synthesis during normal development. Cells were grown and set for development as described under ``Experimental Procedures.'' Time 0 corresponds to the removal of nutrients from vegetative cells, which then aggregate during the the next 8-10 h. The dominant morphological stages, present at different times from aggregates to culminants, are schematically indicated at the top. The autoradiograms obtained from filters hybridized with probes for the largest and the two alternative isoforms of the smaller nuclear subunits of cytochrome c oxidase are reported. gip17, a cDNA encoding a protein whose expression is sharply reduced at aggregation, and the prestalk-specific marker pDd63 were used to probe the degree of synchrony of development.



To determine whether low oxygen could still stimulate the expression of this polypeptide during differentiation, the influence of an atmosphere containing variable oxygen concentrations, ranging from 21 to 3%, was investigated. As expected from previous studies on cell differentiation under submerged conditions (30) and as was immediately evident from the morphology of the aggregates reported in Fig. 5, Dictyostelium late development is dramatically affected by low oxygen. Though no significant influence on the formation of tight aggregates could be found (Fig. 5, photographs at 12 h), a slowing down of the differentiation process was apparent after only a few percent reduction of the oxygen tension. As shown by Fig. 5, even after 28 h, the fruiting bodies are present only in the sample exposed to a normal (21% oxygen) atmosphere. Below 10% oxygen most of the aggregates exhibit an elongated shape that resembles the first finger morphological stage but are unable to further differentiate.


Figure 5: Sensitivity to oxygen of development. The figure shows the morphology of the aggregates obtained at 12 and 28 h after plating of the amoebae for development under a normal and an hypoxic (10 and 3% oxygen, respectively) atmosphere.



A quantitative analysis of this aspect is shown by the data of Fig. 6, where the expression of the prespore gene pDd63, again used as a marker of differentiation, is compared with the behavior of the two oxidase isogenes. In 15% oxygen there is a perceptible lengthening of the time needed to reach culmination, shown by the broadening of the pDd63 mRNA peak. This mild hypoxia is already sufficient to trigger the expression of the subunit VIIs gene. The concentration of the transcript, however, is low and becomes almost undetectable at the first finger morphological stage. A considerable change occurs below 10% oxygen. Under these conditions, which basically prevent the formation of fruiting bodies, the level of the subunit VIIs mRNA rises steeply, apparently following the increasing inhibition of the pDd63 gene expression. It is noteworthy that the block of the differentiation program can be removed by shifting the aggregates to normal oxygen, but the efficiency of culmination is negatively affected by the amount of time spent in the hypoxic environment (not shown).


Figure 6: Hypoxia and development. The figure shows the expression of the two cytochrome c oxidase alternative subunits analyzed by quantitative Northern blotting during development performed at different oxygen tensions (bullet, 21%; &cjs3581;, 15%; , 10%; box, 5%; , 3%). The data are simultaneously compared with the level of pDd63 mRNA, which measures the progress of differentiation. See the text for details.



The behavior of subunit VIIe, the isoform normally present in cytochrome c oxidase, is clearly oxygen-dependent only in the first 4 h from the beginning of starvation, when the amoebae are still present as individual cells. As shown in Fig. 6, after aggregation the expression pattern of this polypeptide is virtually the same in a wide range of oxygen concentration (from 15 to 3%).


DISCUSSION

Much evidence has recently been provided suggesting a regulative role for the nuclear encoded subunits of cytochrome c oxidase and their isoforms(31, 32, 33, 34) . Nevertheless, the in vivo function of the different isozymes, tissue-specific in mammals and environmentally controlled in some lower eukaryotes, remains speculative(6) . The possibility of studying this complex problem in simple systems is attractive especially when, as is the case of Dictyostelium, the life cycle includes features that are typical of multicellular organisms(20) . In this study, the expressions of cytochrome c oxidase and in particular of its two alternative, oxygen-regulated subunit isoforms have been investigated in detail in both exponentially growing and developing cells. As shown by the data obtained, the fine control exerted by oxygen on the mitochondrial enzyme well represents the extraordinary sensitivity of Dictyostelium to this environmental factor. In this context, the similarities between the pattern of increase of the subunit VIIs mRNA concentration and that of inhibition of differentiation, as monitored by the pDd63 prespore gene, are remarkable. This observation favors the idea that the polypeptide exerts an advantageous function also in the multicellular stage of the organism, increasing the chance of survival in case of a prolonged exposure to low oxygen. Ultimately, this would preserve the capability of the aggregates to resume differentiation as soon as normal conditions are restored.

The release of oxygen control on the subunit VIIe isoform after cell aggregation is provocative, because it raises the possibility that the two polypeptides might work independently and therefore exert different functions during development. The tight coordination of the expression in vegetatively growing cells, however, suggests that the sensing mechanism was primarily evolved to operate in advance, before development, possibly to prevent individual amoebae from entering a hostile environment that would not allow sporulation.

The recruitment of cytochrome c oxidase among the protein involved in the organization of the cell response to environmental changes is not surprising considering the analogy with similar situations in bacteria and the central role of the enzyme in the energy metabolism. More impressive, and perhaps difficult to understand here, is the reason for the complexity of a mechanism that can influence both the structure and the concentration of cytochrome c oxidase. For example, the 20% increase of the protein following a large drop of oxygen tension is unlikely to be an absolute requirement for the survival of the organism. Indeed, antisense mutants with only 60% of the normal enzyme concentration do not show any visible growth or developmental phenotype(12) . Nevertheless, the elaborate evolutionary process that has optimized the expression of different, scattered subunit genes to produce a synchronous, slight increase of their activity eventually succeeded. We are therefore forced to conclude that the adaptive response, which presumably introduces only minor modifications in the cell metabolism, may represent a visible selective advantage only in a large time scale and/or in the native ecological environment.

Cytochrome c oxidase is also under developmental control, because in normal conditions, the level of the transcripts is significantly reduced between aggregation and the beginning of culmination. These data fit with earlier observations indicating a progressive decrease of the oxygen consumption as Dictyostelium amoebae progress into development(35) . Indeed, the breakdown of protein and RNA and the subsequent oxidation of the components become the major sources of energy for differentiation(36) . In this context, the down-regulation of cytochrome c oxidase and the increased sensitivity to oxygen of late development are intriguing. The change of the oxidative metabolism, however, implies the activation of different enzyme pools that include some oxygenases and that are responsible for a massive loss of protein and RNA that, at culmination, halves the values normally found in vegetative cells.

Oxygen is also a substrate of different oxidases, hydroxylases, and other oxygenases involved in the synthesis of important components of the cell(37) , which in part could be developmentally regulated. Under hypoxia, the low affinity for oxygen of only one of these enzymes could result in reduced activity with the consequent inhibition of differentiation. It is noteworthy that oxygen has been suggested to play a fundamental role in prestalk-prespore formation (38) and possibly in size determination of the aggregates, a crucial aspect of efficient spore dispersal(39) .

These observations may account for the evolution of a highly sensitive oxygen-detecting mechanism in Dictyostelium and the relevant differences found in comparison with another lower eukaryote, the yeast Saccharomyces cerevisiae. In this unicellular facultative aerobe, the lack of oxygen can also induce switching between the two isoforms of a different cytochrome c oxidase subunit(25) . However, the activation of this process requires conditions that are close to anaerobiosis, and at least 6 h are needed to approach the steady state of mRNA synthesis(8) . At the same time the concentration of cytochrome c oxidase, a dispensable enzyme in yeast, decreases dramatically. In the end, from these observations and other available data(41) , it appears that the subunit-switching mechanism of the obligate aerobe D. discoideum is at least 400 times more sensitive to oxygen than the one active in S. cerevisiae, in addition to being under different controls during growth and development. Thus, although we do not understand the precise function of this regulation, a connection with the lifestyle of the organism is clearly emerging.

The isolation of the two isogenes and the creation of suitable Dictyostelium mutants may therefore offer not only new information on the role of the enzyme nuclear encoded subunits but also a picklock to investigate at a molecular level the relation between oxygen and development in a simple multicellular system.


FOOTNOTES

*
This work was partially supported by the Consiglio Nazionale delle Ricerche (Progetto Finalizzato Ingegneria Genetica and Grant 92.02153.CT14) and by grants from the Ministero dell'Università e della Ricerca Scientifica e Tecnologica (40%) and Telethon-Italia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z30963[GenBank].

§
To whom correspondence should be addressed: Dipartimento di Scienze Biomediche Sperimentali, via Trieste 75, 35121 Padova, Italy. Fax: 39-49-828-6576.

(^1)
The abbreviation used is: bp, base pair.


ACKNOWLEDGEMENTS

We thank Dr. M. Brini for help at the beginning of this work. We are indebted to Dr. R. Mutzel for the guk and gip17 probes and to Dr. W. J. Williams for the pDd63 cDNA clone.


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