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(Received for publication, October 18, 1994; and in revised form, December 16, 1994) From the
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.
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.
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 (
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.
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.
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 (
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%). 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.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
Z30963[GenBank].
Volume 270,
Number 10,
Issue of March 10, 1995 pp. 5587-5593
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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
10
cells/ml) were harvested, washed in 1.4 mM KH
PO
, 2.0 mM NaHPO, pH 6.1, and layered on 2% agar,
prepared in the same buffer, at approximately 3 10 cells cm
. Development at low oxygen tension
was induced in a incubator chamber in the presence of a light source.
, subunit VIIe;
, 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 (
)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) .
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-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-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) .
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).
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) .
, 21%; &cjs3581;, 15%;
, 10%; 
, 5%; 
, 3%). The data are simultaneously
compared with the level of pDd63 mRNA, which measures the
progress of differentiation. See the text for
details.
)
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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