Subunit change in cytochrome c oxidase: identification of the oxygen switch in Dictyostelium
1997; Springer Nature; Volume: 16; Issue: 4 Linguagem: Inglês
10.1093/emboj/16.4.739
ISSN1460-2075
Autores Tópico(s)Photosynthetic Processes and Mechanisms
ResumoArticle15 February 1997free access Subunit change in cytochrome c oxidase: identification of the oxygen switch in Dictyostelium Roberto Bisson Corresponding Author Roberto Bisson Centro CNR Biomembrane and Dipartimento di Scienze Biomediche Sperimentali, via G. Columbo 3, 35121 Padova, Italy Search for more papers by this author Silvia Vettore Silvia Vettore Centro CNR Biomembrane and Dipartimento di Scienze Biomediche Sperimentali, via G. Columbo 3, 35121 Padova, Italy Search for more papers by this author Elisabetta Aratri Elisabetta Aratri Centro CNR Biomembrane and Dipartimento di Scienze Biomediche Sperimentali, via G. Columbo 3, 35121 Padova, Italy Search for more papers by this author Dorianna Sandonà Dorianna Sandonà Centro CNR Biomembrane and Dipartimento di Scienze Biomediche Sperimentali, via G. Columbo 3, 35121 Padova, Italy Search for more papers by this author Roberto Bisson Corresponding Author Roberto Bisson Centro CNR Biomembrane and Dipartimento di Scienze Biomediche Sperimentali, via G. Columbo 3, 35121 Padova, Italy Search for more papers by this author Silvia Vettore Silvia Vettore Centro CNR Biomembrane and Dipartimento di Scienze Biomediche Sperimentali, via G. Columbo 3, 35121 Padova, Italy Search for more papers by this author Elisabetta Aratri Elisabetta Aratri Centro CNR Biomembrane and Dipartimento di Scienze Biomediche Sperimentali, via G. Columbo 3, 35121 Padova, Italy Search for more papers by this author Dorianna Sandonà Dorianna Sandonà Centro CNR Biomembrane and Dipartimento di Scienze Biomediche Sperimentali, via G. Columbo 3, 35121 Padova, Italy Search for more papers by this author Author Information Roberto Bisson 1, Silvia Vettore1, Elisabetta Aratri1 and Dorianna Sandonà1 1Centro CNR Biomembrane and Dipartimento di Scienze Biomediche Sperimentali, via G. Columbo 3, 35121 Padova, Italy The EMBO Journal (1997)16:739-749https://doi.org/10.1093/emboj/16.4.739 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cytochrome c oxidase (COX) has a complex modular structure in eukaryotes. Depending on growth conditions, interchangeable isoforms of selected subunits are synthesized and combined to the evolutionarily conserved catalytic core of the enzyme. In Dictyostelium this structural make-up is regulated by oxygen and involves two forms of the smallest subunit, termed VIIe and VIIs. Here we show that, in spite of a considerable sequence divergency, they are encoded by adjacent genes, linked 'tail to head' by only 800 bp. Deletion analyses reveal the presence of a short intergenic segment acting as an oxygen transcriptional switch. This structural organization and the different stability of the two subunit isoforms offer a molecular explanation for the extraordinary sensitivity to oxygen of the switching mechanism. Introduction Essentially all living organisms have evolved mechanisms to adapt their metabolism to environmental changes. A striking example is offered by the energy-generating system of many prokaryotes where entirely different protein complexes, synthesized in response to fluctuations in the supply of specific substrates, constitute the flexible respiratory network that allows survival in fast-changing environments (Van Spanning et al., 1995). This ability has been drastically reduced in eukaryotes and predominantly restricted to the terminal part of the respiratory chain. Moreover, though in some microorganisms and plant tissues two alternative oxygen-reducing enzymes can still be found (McIntosh, 1994), a different adaptive strategy, based on limited subunit changes around an invariant catalytic core, was evolved and eventually prevailed in animal cells (Kadenbach et al., 1987). This modular complex is cytochrome c oxidase (COX), the integral membrane protein that in mitochondria and many aerobic bacteria catalyses electron transfer from cytochrome c to molecular oxygen coupled to proton translocation (Capaldi, 1990; Babcok and Wikstrom, 1992). Recently, its crystal structure from Paracoccus denitrificans (Iwata et al., 1995) and from bovine heart (Tsukihara et al., 1996) at 2.8 Å resolution has been published. The enzyme catalytic core is constituted by the two largest subunits that contain three redox centres (cytochrome a, CuA, and the binuclear cytochrome a3-CuB site). With a third subunit, they represent the typical structure of the bacterial complex that has been conserved throughout evolution (Castresana et al., 1994). In eukaryotes, they are encoded by the mitochondrial genome and are assembled with 4–10 different smaller subunits coded for in the nucleus (Capaldi, 1990). As mentioned, some of these additional components have alternative forms that are tissue specific or developmentally regulated in higher organisms and environmentally controlled in lower eukaryotes (Kadenbach et al., 1987; Hodge et al., 1989; Schiavo and Bisson, 1989; Parson et al., 1996). The nuclear subunits could therefore act as regulators of the enzyme activity either directly or via binding of allosteric effectors. Recent findings add support to both possibilities. In yeast, the switching between two subunit isoforms affects the binuclear reaction centre and alters the kinetics of interaction with cytochrome c (Allen et al., 1995). In mammals, under physiological concentration, ATP allosterically influences the enzyme kinetics by differential binding to the liver and heart isoforms of the same nuclear subunit (Anthony et al., 1993; Frank and Kadenbach, 1996). The in vivo physiological meaning of these observations is a matter of debate not only for the general understanding of the structure–function relationship of the key enzyme of the aerobic metabolism, but also for the implications concerning an emerging group of mitochondrial diseases involving oxidative phosphorylation defects (Wallace, 1993; Brown and Wallace, 1994; Kadenbach et al., 1995; Tiranti et al., 1995). In this respect, the selection of suitable model systems is an important aspect of these studies. We are investigating these issues in the slime mould Dictyostelium discoideum because of some interesting features that characterize its lifestyle and the enzyme structure. This strictly aerobic organism lives in the forest soil as single cell amoeba, feeding on bacteria and other nutrients from decaying leaves. Upon starvation, cells can aggregate and differentiate in two major cell types that, eventually, after a 24 h developmental stage, form a cellulose stalk holding on top a balloon-like structure filled with spores (Loomis, 1982). The process shows an extraordinary sensitivity to oxygen that also affects the relatively simple subunit composition of COX (Sandonà et al., 1995). A 20–30% decrease of the oxygen tension is in fact sufficient to trigger the expression of an alternative form of subunit VII, the smallest polypeptide of the enzyme, which exclusively prevails under hypoxia (Schiavo and Bisson, 1989). In order to investigate the molecular mechanism involved in the oxygen-dependent, highly coordinated expression of the two subunit isoforms (termed VIIe and VIIs), we have cloned and characterized the genes. They are located in a short segment of genomic DNA, in a tail-to-head array. This organization, which is unique among the COX isogenes of other eukaryotic organisms, appears related to the presence of a common cis-active oxygen-responsive element(s) located in the middle of the intergenic region. Though expression is regulated at the transcriptional level, we show that the different efficiency in enzyme assembly of the two subunit isoforms, a possible consequence of their diverse stability, has considerably increased the sensitivity of the cell response under conditions of mild hypoxia. Results Characterization of the two genes encoding alternative forms of COX subunit VII As isolated, Dictyostelium COX comprises six polypeptides, usually identified by roman numerals. Subunits I and II are the largest mitochondrially encoded subunits, while subunits IV, V, VI and VII are the products of nuclear genes. A third mitochondrial component, subunit III, which is common to all eukaryotic and most bacterial COX, is lost during purification (Bisson et al., 1985). Subunit VIIe is the smallest subunit that is substituted by an alternative isoform, termed VIIs, under hypoxia. A subunit VIIe cDNA (Rizzuto et al., 1990) and a subunit VIIs gene fragment obtained by PCR (Sandonà et al., 1995) were used for restriction analysis of the genes. Southern blot hybridization of the genomic DNA, after digestion with appropriate restriction enzymes (Figure 1A), shows that they are present as a single copy. Thus, in confirmation of a previous observation (Rizzuto et al., 1993), the nuclear genes of Dictyostelium COX do not share the complexity found in mammals, characterized by the existence of dispersed multigene families with a number of pseudogenes (Suske et al., 1987; Carrero-Valenzuela et al., 1991; Taanman et al., 1991; Arnaudo et al., 1992; Seelan and Grossman, 1993; Mell et al., 1994). In this connection, an additional unprecedented feature, immediately evident from the identical position of several restriction fragments after re-hybridization of the filter with the alternative probe, is the close proximity of the two genes. As shown by the restriction map of Figure 1B, they are ∼800 bp apart and, as later confirmed by sequencing, in a 'tail-to-head' configuration. Figure 1.Structural organization of the subunit VII genes of Dictyostelium cytochrome c oxidase. (A) Southern blot analysis of subunit VIIe (left) and VIIs (right) genes. The hybridization pattern of total genomic DNA digested with different restriction enzymes is shown. The samples were run on the same 1% agarose gel and transferred to nitrocellulose. The filter was hybridized with a specific DNA probe for subunit VIIe and, after stripping, for VIIs (left and right panel, respectively). Molecular-size markers from HindIII-digested phage λ DNA are shown on the left margin. Restriction enzymes are indicated. (B) Map of the region containing the two subunit VII genes, as determined by restriction of genomic DNA and confirmed by sequencing of the cloned 5 kb BglII fragment. The position of the coding regions is shown by boxes. Three ORFs, present in the DNA segment, are hatched using a motif that suggests their direction of transcription. Horizontal arrows indicate direction (5′ → 3′) and extent of sequencing of fragments obtained by DNA deletion. The map of the two oxidase genes is shown in greater detail in the lower diagram, where open arrows show the direction of transcription. The nucleotide sequence of the segment defined by the two open arrowheads is reported in Figure 2. The restriction enzyme sites are as follows: Bg, BglII; Sa, Sau3A; Ni, NsiI; Ha, HaeIII; Tq, TaqI; EV, EcoRV; Ra, RsaI; Hf, HinfI. (C and D) Presence of the oxygen switch in the cloned region. (C) Southern blot analysis of EcoRI-digested genomic DNA obtained from the wild-type (wt) and from a Dictyostelium stable transformant (st) created by transfection with multiple copies of a plasmid containing the BglII fragment shown in (B). Total genomic DNA was digested either with EcoRI (Ec) or, to excise the cloned 4.9 insert, with both EcoRI and HindIII (Ec/Hd). The filter was hybridized with random primer-labelled subunit VIIe cDNA. The correspondent expression of the two isogenes under different conditions is shown by Northern blots (D). Total cellular RNA was extracted from exponentially growing amoebae harvested before (O2) and after a 2 h exposure to a nitrogen environment (N2). The blots were probed with random primer-labelled subunit VIIe or a subunit VIIs cDNA-specific probe. Download figure Download PowerPoint The 4.9 kb BglII restriction fragment (Figure 1A) was partially purified by fractionation in agarose gel, inserted into the BamHI site of a pUC19 vector and cloned as detailed in Materials and methods. A set of deleted subclones, extending either 5′ or 3′ of the DNA region (Figure 1B), were then generated by a nested deletion system based on exonuclease III/mung bean nuclease degradation of DNA and used for sequencing and analysis of gene expression. The presence in the cloned region of the cis-active sequences needed for oxygen regulation was directly tested in Dictyostelium stable transfectants obtained by electroporation (Rizzuto et al., 1993), in the presence of the plasmid containing the BglII fragment and selection. Southern blotting analysis of total DNA shows that multiple copies of the construct can be inserted in the slime mould genome. In the example reported in Figure 1C, the comparison of the hybridization signals obtained from a stable transformant (st) and the wild-type (wt) demonstrates the integration of at least 12–15 copies/cell of the cloned region. As suggested by the intense 8 kb band of the EcoRI-restricted genomic DNA and by the presence of a unique site for this enzyme in the plasmid used for transformation, insertion occurs predominantly into a single chromosomal site. The excision of the 4.9 kb genomic fragment by EcoRI/HindIII double digestion, excludes major rearrangements. The small shift to lower molecular weights of the band that contains the endogenous genes, indicates a site of recombination in this region, an event with a relatively low frequency in this type of experiments (see Figure 5B). Northern analysis of total RNA extracted from the Dictyostelium transformant grown in normal oxygen shows the expected proportional increase of the subunit VIIe mRNA, while the alternative transcript remains barely detectable (Figure 1D, O2). Hypoxia completely reversed the expression pattern (Figure 1D, N2), as it normally occurs in the parental cells (Sandonà et al., 1995). These results demonstrate that, in the cloned BglII fragment, both structural genes and the sequences required, in cis, for oxygen control are functional. It may also be noticed that under our experimental conditions the presence of the active endogenous genes does not hamper interpretation of the data. The above approach was therefore extended to the investigation of the effects of selected deletions on gene expression (see below). Figure 2 reports the sequence of the two COX isogenes and their flanking regions. At first sight, the segment shows the typical structure of the Dictyostelium DNA, with the two genes embedded in long poly(dA–dT) tracts. Nevertheless, as will be shown later (Figure 5), these 2 kb of genomic DNA contain all the sequence elements needed for oxygen regulation and, ultimately, that are responsible for the presence of COX isoenzymes in the slime mould. Figure 2.Nucleotide sequence of the subunit VII genes and their flanking regions. The coding regions are indicated by the corresponding amino acids (shown in single-letter code below the sequence). The ORFs with initiation codon at nucleotide 501 and 1521 encode subunit VIIe and subunit VIIs, respectively. Highlighted are restriction sites (dotted underlined), and the nucleotide sequence previously characterized in a subunit VIIe cDNA clone (underlined; Rizzuto et al., 1990). A segment of the intergenic region that, on the basis of the data of this work, plays a key role in oxygen regulation is shown in boldface. This sequence is available from the DDBJ/EMBL/GenBank Data Library under the accession number X99344. Download figure Download PowerPoint Subunit structure and interactions The existence of contiguous COX genes in nuclear genomes has never been previously described, but it could be explained by a recent duplication event. This possibility, however, is not supported by the relatively low degree of similarity of the encoded proteins (Figure 3A). Indeed, with 23 invariant residues, subunits VIIe and VIIs are only 44% similar, a value that is the lowest found among COX subunit isoforms, even when the isogenes are dispersed in different chromosomes (Arnaudo et al., 1992). Half of these residues are in the single hydrophobic stretch that, folded in α-helix, spans the membrane (Rizzuto et al., 1991; Tsukihara et al., 1996). As shown in Figure 3B, they cluster on one face of the helix. Since the conservation of the hydrophobic character is the only requirement for a protein surface interacting with lipids, this distribution implicitly indicates their involvement in protein–protein interaction (Figure 3C), suggesting that the two polypeptides compete for the same site in the protein complex. The latter observation may appear obvious, but only if one does not consider the substantial differences that frequently characterize the primary structure of COX subunit isoforms in different organisms (Capaldi, 1990). In this regard, the relatively large sequence divergency of the two Dictyostelium polypeptides and the presence of a clearly identifiable element of secondary structure (the transmembrane helix) are a clarifying coincidence. The recruitment of 50% of the conserved residues for protein interaction within the enzyme hydrophobic sector, may also explain early observations concerning the sensitivity of the subunit to perturbation by detergents of the lipid–protein boundary (Bisson et al., 1985). Figure 3.Sequence alignment and membrane organization of the two oxidase subunit isoforms. (A) Comparison of the deduced amino acid sequences of the two subunit isoforms. The dash indicates a gap inserted by the computer program (Myers and Miller, 1988) to maintain alignment. Bullets (●) denote identical residues. As shown by the hydropathy plot (Klein et al., 1985), half are localized in the hydrophobic stretch present in the middle of the polypeptide chain. The solid and the dashed lines refer to subunit VIIe and VIIs, respectively. A window of nine amino acids across the length of the protein was employed for calculation. The dotted line divides hydrophobic regions (above) from hydrophilic regions (below). (B and C) Membrane topology. When the hydrophobic segment, that is part of the enzyme membranous sector (see Figure 4), is folded in a α-helix, the invariant residues (darkened area) are clustered on one face of the helix (B), probably in contact with the rest of the protein complex, as indicated schematically in (C). Download figure Download PowerPoint Dictyostelium subunit VII is the only nuclear-encoded subunit of COX that has been characterized in yeast, plant and animal cells. As shown in Figure 4A, in spite of the strictly conserved location of the hydrophobic stretch (horizontal bar), the degree of similarity among the cognate polypeptides is low. It is also evident, from the dendrogram of Figure 4B, that the differences between the two Dictyostelium isoforms are larger than those found for the same subunit in mammals and even comparable with those existing among different organisms. These data provide evidence for the extent of the dramatic change that occurred in the ancestral gene during evolution, to the point that the sequence of the encoded proteins might now be insufficient, without additional information (in this case, the presence and position of the hydrophobic segment), in order to establish homology. These observations suggest that the nuclear-encoded subunits of COX are rapidly evolving, probably to satisfy different function in different organisms, thus increasing the plasticity of this key mitochondrial enzyme. Of particular note is that the existence of isoforms of subunit VII remains a unique feature of the slime mould enzyme, since the presence of multigene families in higher organisms, reported also for this subunit, has been ascribed to processed pseudogenes (Suske et al., 1988). Figure 4.Evolution of a nuclear-encoded subunit of COX. (A) Sequence comparison of the two Dictyostelium COX isoforms with their homologous counterparts in a yeast and a mammal. Conserved amino acids are boxed. Conservative replacements (period) are marked only for the two alternative Dictyostelium subunits. The position of the common hydrophobic stretch is highlighted (horizontal bar). Numbers refer to the subunit VIIs sequence. (B) Hypothetical phylogenetic tree. The dendrogram, inferred by the method of Higgins and Sharp (1989), condenses and extends the above observations to the different organisms where the same oxidase subunit was characterized (Dd, D.discoideum; Sc, Saccharomyces cerevisiae; Ib, Ipomea batatas; Hs, Homo sapiens; Bt, Bos taurus; Rn, Rattus norvegicus). Subunit nomenclature is according to authors that first identified the polypeptide. The asterisk in one of the two rat genes is to indicate that the protein sequence was deduced from the open reading frame of a putative pseudogene (Suske et al., 1988). Download figure Download PowerPoint Figure 5.Deletion analysis. (A) Effect on oxygen-regulated gene expression of progressive upstream and downstream deletions of the genomic DNA fragment shown in Figure 2, monitored by Northern analysis (right). The schematic to the left illustrates the relative length and position of each deletion. The coordinates of the deletion endpoints are given in base pairs 5′ of the ATG starting codon of both subunit VIIe (upper number) and VIIs (lower number) genes. For a prompt evaluation of the relative extent of each deletion, a ruler is reported at the top of the figure. Total RNA was extracted from stable transformants containing multiple copies of the deleted region, after vegetative growth either in the presence (+) or in the absence (−) of oxygen. In the latter case, as previously described (Sandonà et al., 1995), cells were exposed to a nitrogen atmosphere for 2 h before harvesting. Under these conditions, the expression of the endogenous genes is negligible. For this reason, in the parental cells (wt) their presence as a single copy is highlighted by a dashed scheme. The comparison of the signals obtained after hybridization of the different sample with either a VIIe- or a VIIs-specific cDNA probe shows that a key cis-active sequence element for oxygen regulation is located in the middle of the intergenic region (shaded area). As reported in Figure 1C and D, the relative intensities of the bands in the overexpressing transformants reflect the integrated gene copy number. (B and C) Deletion of the promoter region 5′ to the upstream gene activates backwards transcription from the downstream hypoxic promoter. Southern blot analysis (B) of the subunit VIIs gene in the wild-type and two transformants previously shown in rows 3 and 4 of (A). Genomic DNA was digested as reported in Figure 1C, either with EcoRI (Ec) or with both EcoRI and HindIII (Ec/Hd) to allow the precise excision of the cloned insert. Autoradiographs of the Northern blot (C) obtained from the same cells after hybridization with a random primer-labelled subunit VIIe cDNA probe (upper panel) and, after stripping, with a single-stranded, antisense-specific DNA probe (lower panel). The position of the subunit VIIe mRNA is shown by the open arrowhead. The size of the transcripts (kb) is shown on the left margin. Download figure Download PowerPoint A short intergenic sequence inversely regulates both genes As an alternative to a recent gene duplication event, functional reasons could have determined the present structural organization. To investigate this possibility and, more generally, to understand the molecular mechanism of oxygen regulation of gene expression, Dictyostelium stable transformants containing multiple copies of different 5′ and 3′ deleted versions of the sequenced DNA segment (Figure 2) were created and analysed under normal and hypoxic conditions. As shown in Figure 5A, upstream deletions up to 300 bp from the ATG start codon of the subunit VIIe gene have no effect on the regulated synthesis of both VIIe and VIIs transcripts (rows 1 and 2). The loss of the promoter obviously abolishes transcription of the correspondent mRNA, but still has no influence on the activity of the hypoxic VIIs isogene (row 3). An unexpected result, however, is the expression under hypoxia of high molecular weight transcripts, with a predominant size of 3 kb, that hybridize with the subunit VIIe probe. Traces of a similar product are also present in stable transformants transfected with the construct shown in row 4 that, in spite of the deletion of the subunit VIIe coding region, still includes 200 bp corresponding to the untranslated trailer region of the mRNA (Figure 2 and Rizzuto et al., 1990). Figure 5B and C analyses the origin of these products. As shown by the Southern blot (Figure 5B), the two transformants contain ∼30–40 copies of the selected inserts that, upon excision, exhibit the expected molecular weights. Northern analysis of RNA, extracted from the cells grown under normal and hypoxic conditions, is reported in Figure 5C. Here, the results obtained after hybridization with the double-stranded, random primer-labelled subunit VIIe cDNA probe used in Figure 5A (upper autoradiograph) and, on the same stripped filter, with a single-stranded, sense DNA probe (lower autoradiograph), are compared. The selective cross-hybridization of the latter antisense-specific probe indicates that the large RNA shown in row 3 is transcribed by a polymerase moving 'backwards' from a downstream hypoxic promoter. With the same highly specific probe, the large transcript of row 4 is undetectable. The concatameric structure of the integrated DNA, suggested by the data of Figure 5B, could explain the 3 kb apparent size of most of the transcripts. This is in fact the length that, moving upstream, separates the hypoxic promoter from the first Dictyostelium terminator in the adjacent integrated plasmid. The reproducibility of the result (present in all the 12 independent clones isolated), the absence of similar effects in transformants prepared with the different constructs containing the subunit VIIe gene shown in rows 1, 2 and 7–11 (65 clones analysed in all), and the concentration of the large transcripts (even not considering degradation, 3- to 5-fold above the level of the endogenous messenger), suggest that the above observations are not the consequence of occasional rearrangements. Returning to the data of Figure 5A, a further 300 bp deletion, between 815 and 510 bp, 5′ of the subunit VIIs gene (row 5) does not have obvious effects on its oxygen-regulated expression, but transcription is abolished when the sequence upstream of the coding region is reduced to 260 bp (row 6). Additional information is obtained by the analysis of progressive 3′ deletions of the cloned DNA region (Figure 5A, rows 7–11). As shown by the data of row 7, 100 bp downstream of the hypoxic gene appears to be sufficient for correct transcription termination. Their deletion (row 8) obviously induces the synthesis of larger subunit VIIs RNAs, but the expression remains oxygen-controlled even after the loss of about half of the subunit VIIs gene coding region. A quite different behaviour is shown by the normoxic gene. Although a downstream DNA segment of ∼600 bp still allows regulated transcription (row 9), a 100 bp deletion 3′ to this region makes expression oxygen-insensitive (row 10). A further shortening does not change the result (row 11). Within the limits of experimental error, no significant differences are found between the level of the subunit VIIe mRNA extracted from transformants containing the constructs of rows 10 and 11, grown in the two extreme conditions considered here. The relative averaged values, obtained from seven independent clones, and after correction for the contribution of the functional endogenous genes, differ in fact by <5%, indicating that regulation is completely lost. Of note is the fact that the 100 bp genomic region essential for repression of the normoxic gene under low oxygen overlaps the DNA segment whose deletion abolishes expression of the hypoxic gene (Figure 5A, shaded area). Its position coincides with a sequence (highlighted in bold in Figure 2) which is significantly richer in C and G than the neighbour DNA, dominated by long poly(A) and poly(T) stretches typical of untranscribed segments of the Dictyostelium genome. The possibility that certain deletions may induce 'backwards' transcription from the normoxic promoter, as in the case described above for the hypoxic promoter, was also tested by using a DNA probe able to recognize the 1 kb region upstream of the subunit VIIe gene. The absence of cross-hybridization on Northern blots, however, rules out this possibility (data not shown). Overexpression In Dictyostelium mutants able to constitutively and selectively overexpress subunit VIIe (Figure 5A, rows 10 and 11), one would expect the predominant presence of this polypeptide, even under the hypoxic conditions that normally favour the synthesis of the alternative subunit. If this is the case, analysis of these transformants and their comparison with wild-type cells could offer new insights on the role of COX isoenzymes. This possibility was investigated first by monitoring the level of mRNA and protein in amoebae growing exponentially either in a normal or in an hypoxic (5% O2) atmosphere. In these conditions, wild-type cells maintain normal doubling time and the switching between the two COX isoenzymes is complete (Schiavo and Bisson, 1989). As shown by the Northern blots of Figure 6A, in the three Dictyostelium transformants used in this experiment (st1–st3), the level of the subunit VIIe mRNA is approximately one order of magnitude above the wild-type (wt). The first of them (st1) integrates multiple copies of the functional region (Figure 5, row 1) and it can there
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