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Current Genetics (1981) 4:167-171
© Springer-Verlag 1981
Review Article
Mitochondrial Genes at Cold Spring Harbor L. A. Grivell Section for Molecular Biology, Laboratory of Biochemistry, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands
The flowering dogwood trees and green lawns of Cold Spring Harbor provided the setting for a meeting devoted to Mitochondrial Genes from May 13-17th, 1981. Dedicated to the memory of Boris Ephrussi, who pioneered mitochondrial genetics at a time when the only kinds of genetics were nuclear or unclear, the meeting showed that the study of mtDNA has had impact on many areas of molecular biology including the genetic code and decoding, tRNA function, mechanisms of splicing and molecular evolution. Curiously, as Herschel Roman pointed out in his opening address, Ephrussi took great pains to avoid any mention of mitochondrial DNA in connection with his observations on cytoplasmic inheritance, preferring instead to refer to 'cytoplasmic particles, endowed with genetic continuity' (Ephrussi 1953). This reticence was not shared by participants at the meeting, as the following, brief report will show.
1. Mammalian Mitochondrial Genes and Transcripts
The first session opened with concise summaries of gene organization in the human, beef and mouse mitochondrial genomes, as deduced from DNA sequence analysis (S. Anderson, MRC Cambridge; D. Clayton, CalTech) and of the transcript analysis carried out for the human organelle by G. Attardi's group at CalTech. The recent publication of the human sequence and the corresponding transcript analysis (Anderson et al. 1981; Ojala et al. 1981; Montoya et al. 1981) meant that there were few major surprises still to report. All three genomes show a similar, compact organization, with few if any non-coding nucleotides between genes for the two mitochondrial rRNAs, 22 tRNAs, five proteins of known function and eight long open reading frames (URFs). Nevertheless, detailed comparisons reveal a high degree of sequence divergence, even for rRNA and tRNA genes, which in
other organisms tend to be highly conserved. Why this should be is not clear. Possibly, the comparative simplicity of the mitochondrial genetic system, together with the limited spectrum of gene products, permits the fixation of mutations which would be lethal in more sophisticated genetic systems. No import of tRNA has been detected in mammalian mitochondria and thus the 22 tRNAs, defined bysequence analysis, must be sufficient to read all codons. This is achieved by having only a single tRNA for each codon family in the genetic code, with tRNAs for two-codon families having G:U wobble anticodons and those for four-codon families having U in the first position of the anticodon so that U:N wobble is possible. How twocodon tRNAs with U in the wobble position are restricted to the recognition of codons ending in A or G is still not clear, however. In Neurospora mitochondria the wobble U residue in such tRNAs is modified (Heckman et al. 1980) and in the course of the meeting R. P. Martin (CNRS, Strasbourg) reported that a similar mechanism operates in yeast mitochondria. In contrast, direct sequence analysis of bovine mitochondrial tRNAs (B. A. Roe, Kent State University, Ohio) has not revealed any modification of U residues at the wobble position.
2. Yeast Mitochondrial Genes and Transcripts In human mtDNA, transcription is initiated at a single site on each strand and the genome is completely and symmetrically transcribed. Differential control of gene expression is then achieved by a combination of transcription-attenuation, control of the cleavages required to generate transcripts of individual genes and by the stability of the final products. In yeast, more scope for transcriptional controls must exist, since use of the guanylyl transferase capping assay (D. Levens, Univ. of 0172-8083/81/0004/0167/$01.00
168
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Fig. 1. The gene for apocytoehrome b in Saccharomyces cerevisiae. Dependent on strain, the gene for apocytochrome b can exist in 'long' or 'short' versions, as a result of the presence or absence of 'optional' introns. The figure shows the structures of the most commonly occurring long and short forms, together with the positions of a number of mutational clusters - the box loci - which affect apocytochrome b, or its synthesis. Exons are indicated by solid black bars (E1_6), while sequences coding for untranslated regions of the mRNA for cytochrome b are stippled. Cross-hatched areas in intron-2 and -4 represent open reading frames in phase with upstream exons. Adapted from Borst and Grivell (1981a) and Jacq et al. (1980). See text for discussion
Chicago) to pick out RNAs still containing 5'-diphosphate or triphosphate groups, has detected independent, primary transcripts from at least five regions on the mitochondrial genome, including those encoding the two ribosomal RNAs. Since the latter genes are separated by more than 28,000 bp in the mtDNAs of most laboratory yeast strains, the further analysis of their transcription initiation sites should provide useful information on how the synthesis of the ribosomal RNAs is coordinated. As in mammalian mitochondria, RNA processing also plays an important part in yeast mitochondrial gene expression and this was reflected in the number of reports dealing with the topic. Of the split genes in this DNA, that for cytochrome b continues to puzzle. This gene occurs in long or short forms, dependent on strain, and in most laboratory strains it contains either two or five introns (see Fig. 1). Lazowska et al. (1980) have recently shown that a protein (the box 3 maturase) encoded in part by intron-2 of the 'long gene' is involved in the splicing of this intron from precursors of the mtDNA for cytochrome b, thus opening up the possibility that URFs located in other introns of both this and other mitochondrial genes (Nobrega and Tzagoloff 1980; Bonitz et al. 1980) might specify proteins capable of acting in a similar fashion. Of these, intron-4 of the cytochrome b gene is at first sight such an intron. Like intron-2 it contains a long URF (1,152 nucleotides) in phase with the upstream exon, mutations within it impair splicing and some of these (box 7, see Fig. 1) are complementable in trans, thus defining a diffusible, intron-encoded product necessary for splicing, while others (box 9 and box 2-2) located on either side of box 7 are cis-dominant. An interesting difference between the two introns concerns the specificity of the trans-acting products they specify,
however. Whereas the box 3 maturase is apparently specific for the processing of the intron which encodes it, the box 7 product has been implicated in the processing of the transcripts of both the gene for cytochrome b and that for subunit I of cytochrome c oxidase (OXI3), since box 7 mutants or deletion mutants lacking intron-4 show altered patterns of OXI3 transcripts and fail to produce this protein in addition to cytochrome b (Dhawale et al. 1981). Consistent with the maturase model, box 7 mutants contain mutations in the open reading frame (C. Jacq, CNRS, Gif-sur-Yvette) and at least one of these (H. Mahler, Indiana Univ.) produces a novel polypeptide (p26) which might represent a defective maturase. Synthesis of this and of the corresponding wildtype polypeptide (p27) is prevented by premature chain termination in upstream exons, by lack of splicing in upstream introns and by deletion of intron-4, thus confirming that ribosome readthrough from upstream exons into the intron is necessary for its production. Surprisingly, however, both the size of the polypeptide and its lack of cross-reactivity With antisera directed against cytochrome b imply that exon sequences are absent. The protein must thus arise either by post-translational cleavage of a larger, fusion protein or by re-initiation of protein synthesis within the intron. To explain all results, the latter process should occur by a mechanism which is dependent on prior translation of upstream sequences. Not so easy to fit into a simple maturase model are the cis-dominant box 9 and box 2-2 mutations. These lie within the intron, in the open reading frame, and in the untranslatable region immediately following it and both classes produce subunit I of cytochrome c oxidase normally. Quite how the mutations prevent splicing in COB without affecting that in OXI3 is unclear. Jacq suggests that the effect on COB may be mediated by alterations in RNA secondary structure, such that progress of a 'splicing complex' (perhaps similar to that proposed by Sharp (1981)) is hampered. Clearly, this explanation accounts for only part of the phenomenon and further work will be required to resolved it fully. Another gene that may require maturases to process its transcripts is that coding for subunit I of cytochrome c oxidase. Undeterred by the complexities of a gene which contains, dependent on strain, anything up to eight introns and at least four long, unassigned open reading frames, L. A. M. Hensgens (Univ. of Amsterdam)briefly reviewed evidence for such an idea in the case of the first two introns in strains with the 'long' version of the gene. So far, however, the protein products corresponding to the reading frames either in these or other introns in OXI3 have eluded detection. Evidence that maturases are not the only mitochondrially-coded components required for RNA processing was provided by N. C. Martin (Univ. of Texas). In wildtype cells and in cytoplasmic petite mutants able to make
L. A. Grivell: MitochondrialGenes tRNA, Northern blot experiments show that 4S RNA is the most prominent transcript of tRNA genes. In other petites, 4S RNA is absent and high-molecular-weight tRNA gene transcripts are found. The two classes of petites retain different segments of the mitochondrial genome, suggesting that a mitochondriai function is required for tRNA processing. Now, the existence of a locus encoding such a function has been demonstrated directly by showing that the capacity to process tRNA gene transcripts can be restored to petites lacking this activity by petites retaining it in a type of zygotic complementation assay. Restoration is detectable as early as three hours after mating and is thus presumably mediated by a trans-acting element. Its expression in petite mutants (which lack mitochondrial protein synthesis) suggests that its product is an RNA rather than a protein, perhaps as part of an RNase P-type enzyme imported from the cytoplasm. Deletion mapping of the tRNA locus shows it to be sited in the proximity of the gene for tRNAmet in a relatively 'empty' region of the genome between the genes for subunit III of cytochrome c oxidase (OXI2) and 15S rRNA. So far, no yeast mitochondrial tRNA precursors have been characterized, but to judge from the available sequence data on the regions flanking many tRNA genes (Bonitz and Tzagoloff 1980), the sequences and structures of the various tRNA precursors will be many and varied. While the secondary structure of the final tRNA is likely to be an important factor in directing correct processing, it seems likely that the tRNA locus product and its associated imported enzyme will have to exercise a certain amount of versatility in its mode of action. Its isolation and further characterization is thus eagerly awaited. The finding of the tRNA locus emphasizes that even with the extensive DNA sequence data currently available for yeast mtDNA, new genes may still await discovery. This should provide a modicum of comfort for the group of R. A. Butow (Univ. of Texas, Dallas), who is still unable to locate the structural gene for the varl polypeptide, a protein associated with the small subunit of the mitochondrial ribosome. Some years ago, the situation regarding this protein seemed cut and dried (Strausberg et al. 1978): the apparent size of the protein varies in different strains and the size differences can be correlated with the presence or absence of small insertions in a restriction fragment, which petite deletion mapping identifies as carrying the varl genetic determinant. The first setback came with the results of DNA sequence analysis (Tzagoloff et al. 1980), which showed not only that the varl region - like many other regions in yeast mtDNA - was extremely AT rich, but that it also lacked any open reading frames longer than a hundred or so nucleotides. While a series of splicing events would have taken care of this problem, the second setback came with the characterization of transcripts derived from the varl
169 region. Of these, a 16S RNA (about 1,700 nucleotides) is a good candidate for the varl mRNA: it shows straindependent length variation consistent with the length of the protein produced. So far, however, only some 250 nucleotides of the varl genetic determinant region hybridize to this RNA and the RNA itself fails tohybridize either with flanking regions, with regions elsewhere on the mitochondrial genome, or even with nuclear DNA! Although some controls to role out trivial causes still have to be carded out, there seems little doubt that an interesting explanation will underlie this paradoxical result and hopefully, Butow and his colleagues will not abandon the search for the var 1 structural gene just yet. Comparison of mammalian and yeast mtDNA sequences shows that, while a number of the products of these DNAs have been conserved in evolution, the organization and mode of expression of the genes specifying them have not. The mammalian genes lack intervening sequences and are compressed into a mere 16,569 bp. In yeast (Saccharomyces)three genes are split and roughly the same information is scattered over some five times this amount of DNA, with no obvious logic to the order and with vast expanses of virtually pure AT interspersed between coding sequences. Furthermore, comparison of different yeasts (G. D. Clark-Walker, Australian National Univ., Canberra) shows that there are few constraints either on gene order, or overall size. A possible explanation for this was put forward by G. Bemardi (Univ. of Pads), based on the observation that there are multiple replication origins in yeast mtDNA (also made by H. Blanc, Stanford Univ.) and that sequences resembling these occur frequently in intergenic regions. Duplication of these origins (selfish DNA?) and their translocation throughout the genome would not only provide the molecules containing them with a selective replicative advantage, but would also open up the possibility for sequence rearrangements v/a non-homologous cross-overs.
3. Mitochondrial Genes of the Filamentous Fungi, Plants and Trypanosomes The fact that the sequences of many mitochondrial gene products are evolutionarily conserved has been exploited to examine gene organization in the mtDNAs of a wide range of organisms from filamentous fungi to higher plants with, in some cases, unexpected results. In Neurospora, the use of yeast probes to pick up homologous sequences has unearthed what appears to be a mitochondrial pseudo gene for ATPase subunit 9 (P. Van den Boogaart, State Univ. Groningen). This is surprising, since Sebald et al. (1977) showed quite convincingly that in Neurospora, this protein is specified by a nuclear gene on chromosome VII, and synthesized on cell-sap ribosomes. The newly-discovered copy contains a continuous sequence with homol-
170 ogy to the yeast mitochondrial gene, followed by an amber stop and a short stretch of non-homology. Beyond this, and a small gap in the sequence, homology with the yeast gene is again found. Van den Boogaart suggests that the pseudogene is the remains of an ancestral gene that also gave rise to the present day, active version in the nucleus, by a process of duplication and translocation. If this is so, it is somewhat paradoxical, first that the product of the silent gene has as much homology with the yeast mitochondrially-coded protein, as with its functional nuclear-coded counterpart and second, that the gene has accumulated so few deleterious mutations. These findings imply, on the one hand, that the gene duplication was a relatively ancient event and, on the other, that the mitochondrial copy has only recently lost its ability to function. Although it is in principle possible that the two genes have co-existed for some time in functional state, it may be useful at this stage to consider an alternative explanation for this finding, namely that the nuclear copy may have had an independent origin, representing a recent acquisition via some kind ofinterspecies DNA transfer. Either way, the final solution is sure to be interesting. In Aspergillus, sequence comparisons with the yeast and mammalian mitochondrial genomes (R. Wayne-Davis, University of Essex) have brought out two points of interest. The first is that the gene for cytochrome b in this mtDNA contains only a single intron, which corresponds both in position and sequence to intron-3 of the X cerevisiae gene (presented by-J. Lazowska, CNRS, Gif-sur-Yvette). Like intron-2, which specifies the box 3 maturase, the Aspergillus intron contains an open reading frame in phase with the preceding exon, implying that in this organism too, mitochondrial maturases may mediate splicing. Perhaps of even greater interest, however, is the finding that Aspergillus mtDNA contains two open reading frames homologous to UFRs 1 and 4 of the human genome. This finding should convince even the most hardy of sceptics that the URFs must fulfil important roles in both organisms. It has been suggested (Borst and GfiveU, 1981b) that the human URFs may specify mitoribosomal proteins, or RNA processing enzymes. Their occurrence in Aspergillus means that these speculations can be tested with the help of genetic analysis. Going further afield, yeast mitochondrial gene segments have also been used to probe sequence organization in the kinetoplast DNA of trypanosomes and the mtDNA of higher plants. L. Simpson (Univ. of Calif.) reported that under hybridization conditions of extremely low stringency (Tin -45 ° !), the maxi-circle DNA of Leishmania tarentolae contains sequences corresponding to the yeast genes for cytochrome b and subunits I-III of cytochrome c oxidase. Specific transcripts of these sequences, which map in close proximity to each other, have been detected and their further characterization
L.A. Grivell: MitochondrialGenes will no doubt soon be forthcoming. In maize, a similar approach has culminated in the determination of the complete DNA sequence of the gene for subunit II of cytochrome c oxidase (T. D. Fox, Biocenter Basel and C. Leaver, Univ. of Edingburgh) and examination of the sequence has justified the effort involved on two counts. The first is that maize mitochondria introduce yet a further variation on the once universal genetic code. Unlike the fungal and mammalian mitochondrial genomes, which use UGA to specify tryptophan instead of stop, maize apparently uses CGG in addition to the usual UGG to specify this amino acid, since at three positions in the sequence, this codon lines up with tryptophan residues in the yeast and beef proteins. The maize gene, unlike its yeast counterpart, contains a 794-bp long intron, thus raising yet again the question why some mitochondrial genes should be split, while others are not. To afficionados of mitochondrial maturases, the intron itself must be a disappointment, however. The two longest reading frames contain 118 and 110 codons, respectively; neither of them is in phase with either exon and no homology with either yeast or mammalian URFs is detectable. Several speakers presented data on the complexity of plant mtDNAs with estimates ranging from around 1.5 x l0 s bp for wheat mtDNA up to some 2.4 x 106 bp for musk melon mtDNA (A. J. Bendich, Univ. of Washington). While some scepticism was expressed as to the reality of the values at the upper end of the scale, it is clear that the coding potential of higher plant mtDNAs far exceeds that of all other organism (cf. Kolodner and Tewari 1972) and that this, coupled with the curious heterogeneity displayed by these DNAs, represents a biological problem of the first order. One indication of what a small part of the extra coding potential does was given by C. Leaver, who showed that different forms of cytoplasmically-inherited male sterility in maize can be correlated with changes in the pattern of mitochondrial translation products, and in one case, also with the accumulation of plasmid-like molecules in the mitochondria.
4. Nucleocytoplasmic Interactions The contribution of mtDNA to mitochondrial biogenesis, though essential, is a small one. Only some 10% ofmitochondfial protein is specified by this DNA, while the remainder is encoded by nuclear genes, synthesized on cell-sap ribosomes and imported into the mitochondria. That this had not been entirely forgotten was indicated by the fact that a small but significant number of reports on nucleocytoplasmic interactions had crept into a meeting ostensibly devoted exclusively to mitochondrial genes. Of especial interest were reports from A. M. Lam-
L. A. Grivell: Mitochondrial Genes
bowitz (St. Louis Univ.), on the characterization of nuclear mutants impaired in the splicing of the mitochondrial large rRNA in Neurospora and from C. Dieckmann (Columbia Univ.), who briefly described a nuclear mutant that is apparently specifically blocked in its ability to produce transcripts of the cytochrome b gene in yeast. Although it is perhaps surprising that a nuclear factor should be required for the transcription of a particular mitochondrial gene, the nature of the lesion in this mutant should soon be known, since plasmids taken from a nuclear clone bank of a wild-type strain complement the mutation in transformation tests and the further characterization of these is underway.
5. Evolutionary Aspects Finally, an example of what Piotr Slonimski may term 'extrovert' applications of our knowlege ofmitochondrial genes was given by the use of sequence comparison of mtDNAs to determine evoJutionary relationships. In higher eukaryotes, the combination of maternal inheritance, with a lack or extremely low rate of recombination permits lineages to be followed withoutthe 'scrambling' effects of meiosis, while the high fixation rate of mutations in mtDNA ( 5 - 1 0 times higher than singlecopy nuclear DNA) makes it an extremely sensitive indicator of relationships over short evolutionary distances. Of course, this approach is only feasible if all mtDNA molecules in a given individual are identical and the fact that this indeed is the case, notwithstanding the high mutation rate, is something of a paradox, as pointed out by R. A. Lansman (Univ. of Georgia). One solution to this may be that only a small number ofmtDNA molecules in a female's germ-line cells determines the sequence of the mtDNA molecules observed in the somatic tissues of her progeny. Whatever the reason, the utility of the approach was demonstrated in several instances and, applied to our own origins by R. L. Cann (Univ. of Calif.) it indicates that Homo sapiens may have arisen as
171
recently as 130,000 years ago, from a small group of individuals. Furthermore, the presence of extremely divergent mtDNA lineages within the human species suggests that non-sapiens mitochondrial genomes still persist in some human populations.
References Anderson S, Bankier AT, Barrell BG, de Bruijn MHL, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Smith AJH, Staden R, Young IG (1981) Nature 290:457 465 Bonitz SG, Tzagoloff A (1980) J Biol Chem 255:9075-9081 Bonitz SG, Coruzzi G, Thalenfeld BE, Tzagoloff A, Macino G (1980) J Biol Chem 255:11927-11941 Borst P, Grivell LA (1981a) Nature 289:439-440 Borst P, Grivell LA (1981b) Nature 290:443-444 Dhawale S, Hanson DK, Alexander N J, Perlman PS, Mahler HR (1981) Proc Natl Acad Sci USA 78:1778-1782 Ephrussi B (1953) Nucleo-Cytoplasmic Relations in MicroOrganisms. Clarendon Press, Oxford Heckman JE, Sarnoff J, Alzner De Weerd B, Yyn S, RajBhandary UL (1980) Proc Natl Acad Sci USA 77:3159-3163 Jacq C, Lazowska J, Slonimski PP (1980) In: Kroon AM, Saccone C (eds) The Organization and Expression of the Mitochondrial Genomes. North-Holland, Amsterdam, pp 139-152 Kolodner R, Tewari KK (1972) Proc Natl Acad Sci USA 69: 1830-1834 Lazowska J, Jacq C, Slonimski PP (1980) Cell 22:333-348 Montoya J, Ojala D, Attardi G (1981) Nature 290:465-470 Nobrega FG, Tzagoloff A (1980) J Biol Chem 255:9828-9837 Ojala D, Montoya J, Attardi G (1981) Nature 290:470-474 Sebald W, Sebald-Althaus M, Wachter E (1977) In: Bandlow W, Schweyen R J, Wolf K, Kaudewitz F (eds) Mitochondria 1977, Genetics and Biogenesis of Mitochondria. De Gruyter, Berlin, pp 433-440 Sharp PA (1981) Cell 23:643-646 Strausberg RL, Vincent RD, Perlman PS, Butow RA (1978) Nature 276:577-583 Tzagoloff A, Nobrega M, Akai A, Macino G (1980) Curr Genet 2:149-157
Communicated by F. Kandewitz Received August 31, 1981
Current Genetics (1981) 4:167-171
© Springer-Verlag 1981
Review Article
Mitochondrial Genes at Cold Spring Harbor L. A. Grivell Section for Molecular Biology, Laboratory of Biochemistry, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands
The flowering dogwood trees and green lawns of Cold Spring Harbor provided the setting for a meeting devoted to Mitochondrial Genes from May 13-17th, 1981. Dedicated to the memory of Boris Ephrussi, who pioneered mitochondrial genetics at a time when the only kinds of genetics were nuclear or unclear, the meeting showed that the study of mtDNA has had impact on many areas of molecular biology including the genetic code and decoding, tRNA function, mechanisms of splicing and molecular evolution. Curiously, as Herschel Roman pointed out in his opening address, Ephrussi took great pains to avoid any mention of mitochondrial DNA in connection with his observations on cytoplasmic inheritance, preferring instead to refer to 'cytoplasmic particles, endowed with genetic continuity' (Ephrussi 1953). This reticence was not shared by participants at the meeting, as the following, brief report will show.
1. Mammalian Mitochondrial Genes and Transcripts
The first session opened with concise summaries of gene organization in the human, beef and mouse mitochondrial genomes, as deduced from DNA sequence analysis (S. Anderson, MRC Cambridge; D. Clayton, CalTech) and of the transcript analysis carried out for the human organelle by G. Attardi's group at CalTech. The recent publication of the human sequence and the corresponding transcript analysis (Anderson et al. 1981; Ojala et al. 1981; Montoya et al. 1981) meant that there were few major surprises still to report. All three genomes show a similar, compact organization, with few if any non-coding nucleotides between genes for the two mitochondrial rRNAs, 22 tRNAs, five proteins of known function and eight long open reading frames (URFs). Nevertheless, detailed comparisons reveal a high degree of sequence divergence, even for rRNA and tRNA genes, which in
other organisms tend to be highly conserved. Why this should be is not clear. Possibly, the comparative simplicity of the mitochondrial genetic system, together with the limited spectrum of gene products, permits the fixation of mutations which would be lethal in more sophisticated genetic systems. No import of tRNA has been detected in mammalian mitochondria and thus the 22 tRNAs, defined bysequence analysis, must be sufficient to read all codons. This is achieved by having only a single tRNA for each codon family in the genetic code, with tRNAs for two-codon families having G:U wobble anticodons and those for four-codon families having U in the first position of the anticodon so that U:N wobble is possible. How twocodon tRNAs with U in the wobble position are restricted to the recognition of codons ending in A or G is still not clear, however. In Neurospora mitochondria the wobble U residue in such tRNAs is modified (Heckman et al. 1980) and in the course of the meeting R. P. Martin (CNRS, Strasbourg) reported that a similar mechanism operates in yeast mitochondria. In contrast, direct sequence analysis of bovine mitochondrial tRNAs (B. A. Roe, Kent State University, Ohio) has not revealed any modification of U residues at the wobble position.
2. Yeast Mitochondrial Genes and Transcripts In human mtDNA, transcription is initiated at a single site on each strand and the genome is completely and symmetrically transcribed. Differential control of gene expression is then achieved by a combination of transcription-attenuation, control of the cleavages required to generate transcripts of individual genes and by the stability of the final products. In yeast, more scope for transcriptional controls must exist, since use of the guanylyl transferase capping assay (D. Levens, Univ. of 0172-8083/81/0004/0167/$01.00
168
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Fig. 1. The gene for apocytoehrome b in Saccharomyces cerevisiae. Dependent on strain, the gene for apocytochrome b can exist in 'long' or 'short' versions, as a result of the presence or absence of 'optional' introns. The figure shows the structures of the most commonly occurring long and short forms, together with the positions of a number of mutational clusters - the box loci - which affect apocytochrome b, or its synthesis. Exons are indicated by solid black bars (E1_6), while sequences coding for untranslated regions of the mRNA for cytochrome b are stippled. Cross-hatched areas in intron-2 and -4 represent open reading frames in phase with upstream exons. Adapted from Borst and Grivell (1981a) and Jacq et al. (1980). See text for discussion
Chicago) to pick out RNAs still containing 5'-diphosphate or triphosphate groups, has detected independent, primary transcripts from at least five regions on the mitochondrial genome, including those encoding the two ribosomal RNAs. Since the latter genes are separated by more than 28,000 bp in the mtDNAs of most laboratory yeast strains, the further analysis of their transcription initiation sites should provide useful information on how the synthesis of the ribosomal RNAs is coordinated. As in mammalian mitochondria, RNA processing also plays an important part in yeast mitochondrial gene expression and this was reflected in the number of reports dealing with the topic. Of the split genes in this DNA, that for cytochrome b continues to puzzle. This gene occurs in long or short forms, dependent on strain, and in most laboratory strains it contains either two or five introns (see Fig. 1). Lazowska et al. (1980) have recently shown that a protein (the box 3 maturase) encoded in part by intron-2 of the 'long gene' is involved in the splicing of this intron from precursors of the mtDNA for cytochrome b, thus opening up the possibility that URFs located in other introns of both this and other mitochondrial genes (Nobrega and Tzagoloff 1980; Bonitz et al. 1980) might specify proteins capable of acting in a similar fashion. Of these, intron-4 of the cytochrome b gene is at first sight such an intron. Like intron-2 it contains a long URF (1,152 nucleotides) in phase with the upstream exon, mutations within it impair splicing and some of these (box 7, see Fig. 1) are complementable in trans, thus defining a diffusible, intron-encoded product necessary for splicing, while others (box 9 and box 2-2) located on either side of box 7 are cis-dominant. An interesting difference between the two introns concerns the specificity of the trans-acting products they specify,
however. Whereas the box 3 maturase is apparently specific for the processing of the intron which encodes it, the box 7 product has been implicated in the processing of the transcripts of both the gene for cytochrome b and that for subunit I of cytochrome c oxidase (OXI3), since box 7 mutants or deletion mutants lacking intron-4 show altered patterns of OXI3 transcripts and fail to produce this protein in addition to cytochrome b (Dhawale et al. 1981). Consistent with the maturase model, box 7 mutants contain mutations in the open reading frame (C. Jacq, CNRS, Gif-sur-Yvette) and at least one of these (H. Mahler, Indiana Univ.) produces a novel polypeptide (p26) which might represent a defective maturase. Synthesis of this and of the corresponding wildtype polypeptide (p27) is prevented by premature chain termination in upstream exons, by lack of splicing in upstream introns and by deletion of intron-4, thus confirming that ribosome readthrough from upstream exons into the intron is necessary for its production. Surprisingly, however, both the size of the polypeptide and its lack of cross-reactivity With antisera directed against cytochrome b imply that exon sequences are absent. The protein must thus arise either by post-translational cleavage of a larger, fusion protein or by re-initiation of protein synthesis within the intron. To explain all results, the latter process should occur by a mechanism which is dependent on prior translation of upstream sequences. Not so easy to fit into a simple maturase model are the cis-dominant box 9 and box 2-2 mutations. These lie within the intron, in the open reading frame, and in the untranslatable region immediately following it and both classes produce subunit I of cytochrome c oxidase normally. Quite how the mutations prevent splicing in COB without affecting that in OXI3 is unclear. Jacq suggests that the effect on COB may be mediated by alterations in RNA secondary structure, such that progress of a 'splicing complex' (perhaps similar to that proposed by Sharp (1981)) is hampered. Clearly, this explanation accounts for only part of the phenomenon and further work will be required to resolved it fully. Another gene that may require maturases to process its transcripts is that coding for subunit I of cytochrome c oxidase. Undeterred by the complexities of a gene which contains, dependent on strain, anything up to eight introns and at least four long, unassigned open reading frames, L. A. M. Hensgens (Univ. of Amsterdam)briefly reviewed evidence for such an idea in the case of the first two introns in strains with the 'long' version of the gene. So far, however, the protein products corresponding to the reading frames either in these or other introns in OXI3 have eluded detection. Evidence that maturases are not the only mitochondrially-coded components required for RNA processing was provided by N. C. Martin (Univ. of Texas). In wildtype cells and in cytoplasmic petite mutants able to make
L. A. Grivell: MitochondrialGenes tRNA, Northern blot experiments show that 4S RNA is the most prominent transcript of tRNA genes. In other petites, 4S RNA is absent and high-molecular-weight tRNA gene transcripts are found. The two classes of petites retain different segments of the mitochondrial genome, suggesting that a mitochondriai function is required for tRNA processing. Now, the existence of a locus encoding such a function has been demonstrated directly by showing that the capacity to process tRNA gene transcripts can be restored to petites lacking this activity by petites retaining it in a type of zygotic complementation assay. Restoration is detectable as early as three hours after mating and is thus presumably mediated by a trans-acting element. Its expression in petite mutants (which lack mitochondrial protein synthesis) suggests that its product is an RNA rather than a protein, perhaps as part of an RNase P-type enzyme imported from the cytoplasm. Deletion mapping of the tRNA locus shows it to be sited in the proximity of the gene for tRNAmet in a relatively 'empty' region of the genome between the genes for subunit III of cytochrome c oxidase (OXI2) and 15S rRNA. So far, no yeast mitochondrial tRNA precursors have been characterized, but to judge from the available sequence data on the regions flanking many tRNA genes (Bonitz and Tzagoloff 1980), the sequences and structures of the various tRNA precursors will be many and varied. While the secondary structure of the final tRNA is likely to be an important factor in directing correct processing, it seems likely that the tRNA locus product and its associated imported enzyme will have to exercise a certain amount of versatility in its mode of action. Its isolation and further characterization is thus eagerly awaited. The finding of the tRNA locus emphasizes that even with the extensive DNA sequence data currently available for yeast mtDNA, new genes may still await discovery. This should provide a modicum of comfort for the group of R. A. Butow (Univ. of Texas, Dallas), who is still unable to locate the structural gene for the varl polypeptide, a protein associated with the small subunit of the mitochondrial ribosome. Some years ago, the situation regarding this protein seemed cut and dried (Strausberg et al. 1978): the apparent size of the protein varies in different strains and the size differences can be correlated with the presence or absence of small insertions in a restriction fragment, which petite deletion mapping identifies as carrying the varl genetic determinant. The first setback came with the results of DNA sequence analysis (Tzagoloff et al. 1980), which showed not only that the varl region - like many other regions in yeast mtDNA - was extremely AT rich, but that it also lacked any open reading frames longer than a hundred or so nucleotides. While a series of splicing events would have taken care of this problem, the second setback came with the characterization of transcripts derived from the varl
169 region. Of these, a 16S RNA (about 1,700 nucleotides) is a good candidate for the varl mRNA: it shows straindependent length variation consistent with the length of the protein produced. So far, however, only some 250 nucleotides of the varl genetic determinant region hybridize to this RNA and the RNA itself fails tohybridize either with flanking regions, with regions elsewhere on the mitochondrial genome, or even with nuclear DNA! Although some controls to role out trivial causes still have to be carded out, there seems little doubt that an interesting explanation will underlie this paradoxical result and hopefully, Butow and his colleagues will not abandon the search for the var 1 structural gene just yet. Comparison of mammalian and yeast mtDNA sequences shows that, while a number of the products of these DNAs have been conserved in evolution, the organization and mode of expression of the genes specifying them have not. The mammalian genes lack intervening sequences and are compressed into a mere 16,569 bp. In yeast (Saccharomyces)three genes are split and roughly the same information is scattered over some five times this amount of DNA, with no obvious logic to the order and with vast expanses of virtually pure AT interspersed between coding sequences. Furthermore, comparison of different yeasts (G. D. Clark-Walker, Australian National Univ., Canberra) shows that there are few constraints either on gene order, or overall size. A possible explanation for this was put forward by G. Bemardi (Univ. of Pads), based on the observation that there are multiple replication origins in yeast mtDNA (also made by H. Blanc, Stanford Univ.) and that sequences resembling these occur frequently in intergenic regions. Duplication of these origins (selfish DNA?) and their translocation throughout the genome would not only provide the molecules containing them with a selective replicative advantage, but would also open up the possibility for sequence rearrangements v/a non-homologous cross-overs.
3. Mitochondrial Genes of the Filamentous Fungi, Plants and Trypanosomes The fact that the sequences of many mitochondrial gene products are evolutionarily conserved has been exploited to examine gene organization in the mtDNAs of a wide range of organisms from filamentous fungi to higher plants with, in some cases, unexpected results. In Neurospora, the use of yeast probes to pick up homologous sequences has unearthed what appears to be a mitochondrial pseudo gene for ATPase subunit 9 (P. Van den Boogaart, State Univ. Groningen). This is surprising, since Sebald et al. (1977) showed quite convincingly that in Neurospora, this protein is specified by a nuclear gene on chromosome VII, and synthesized on cell-sap ribosomes. The newly-discovered copy contains a continuous sequence with homol-
170 ogy to the yeast mitochondrial gene, followed by an amber stop and a short stretch of non-homology. Beyond this, and a small gap in the sequence, homology with the yeast gene is again found. Van den Boogaart suggests that the pseudogene is the remains of an ancestral gene that also gave rise to the present day, active version in the nucleus, by a process of duplication and translocation. If this is so, it is somewhat paradoxical, first that the product of the silent gene has as much homology with the yeast mitochondrially-coded protein, as with its functional nuclear-coded counterpart and second, that the gene has accumulated so few deleterious mutations. These findings imply, on the one hand, that the gene duplication was a relatively ancient event and, on the other, that the mitochondrial copy has only recently lost its ability to function. Although it is in principle possible that the two genes have co-existed for some time in functional state, it may be useful at this stage to consider an alternative explanation for this finding, namely that the nuclear copy may have had an independent origin, representing a recent acquisition via some kind ofinterspecies DNA transfer. Either way, the final solution is sure to be interesting. In Aspergillus, sequence comparisons with the yeast and mammalian mitochondrial genomes (R. Wayne-Davis, University of Essex) have brought out two points of interest. The first is that the gene for cytochrome b in this mtDNA contains only a single intron, which corresponds both in position and sequence to intron-3 of the X cerevisiae gene (presented by-J. Lazowska, CNRS, Gif-sur-Yvette). Like intron-2, which specifies the box 3 maturase, the Aspergillus intron contains an open reading frame in phase with the preceding exon, implying that in this organism too, mitochondrial maturases may mediate splicing. Perhaps of even greater interest, however, is the finding that Aspergillus mtDNA contains two open reading frames homologous to UFRs 1 and 4 of the human genome. This finding should convince even the most hardy of sceptics that the URFs must fulfil important roles in both organisms. It has been suggested (Borst and GfiveU, 1981b) that the human URFs may specify mitoribosomal proteins, or RNA processing enzymes. Their occurrence in Aspergillus means that these speculations can be tested with the help of genetic analysis. Going further afield, yeast mitochondrial gene segments have also been used to probe sequence organization in the kinetoplast DNA of trypanosomes and the mtDNA of higher plants. L. Simpson (Univ. of Calif.) reported that under hybridization conditions of extremely low stringency (Tin -45 ° !), the maxi-circle DNA of Leishmania tarentolae contains sequences corresponding to the yeast genes for cytochrome b and subunits I-III of cytochrome c oxidase. Specific transcripts of these sequences, which map in close proximity to each other, have been detected and their further characterization
L.A. Grivell: MitochondrialGenes will no doubt soon be forthcoming. In maize, a similar approach has culminated in the determination of the complete DNA sequence of the gene for subunit II of cytochrome c oxidase (T. D. Fox, Biocenter Basel and C. Leaver, Univ. of Edingburgh) and examination of the sequence has justified the effort involved on two counts. The first is that maize mitochondria introduce yet a further variation on the once universal genetic code. Unlike the fungal and mammalian mitochondrial genomes, which use UGA to specify tryptophan instead of stop, maize apparently uses CGG in addition to the usual UGG to specify this amino acid, since at three positions in the sequence, this codon lines up with tryptophan residues in the yeast and beef proteins. The maize gene, unlike its yeast counterpart, contains a 794-bp long intron, thus raising yet again the question why some mitochondrial genes should be split, while others are not. To afficionados of mitochondrial maturases, the intron itself must be a disappointment, however. The two longest reading frames contain 118 and 110 codons, respectively; neither of them is in phase with either exon and no homology with either yeast or mammalian URFs is detectable. Several speakers presented data on the complexity of plant mtDNAs with estimates ranging from around 1.5 x l0 s bp for wheat mtDNA up to some 2.4 x 106 bp for musk melon mtDNA (A. J. Bendich, Univ. of Washington). While some scepticism was expressed as to the reality of the values at the upper end of the scale, it is clear that the coding potential of higher plant mtDNAs far exceeds that of all other organism (cf. Kolodner and Tewari 1972) and that this, coupled with the curious heterogeneity displayed by these DNAs, represents a biological problem of the first order. One indication of what a small part of the extra coding potential does was given by C. Leaver, who showed that different forms of cytoplasmically-inherited male sterility in maize can be correlated with changes in the pattern of mitochondrial translation products, and in one case, also with the accumulation of plasmid-like molecules in the mitochondria.
4. Nucleocytoplasmic Interactions The contribution of mtDNA to mitochondrial biogenesis, though essential, is a small one. Only some 10% ofmitochondfial protein is specified by this DNA, while the remainder is encoded by nuclear genes, synthesized on cell-sap ribosomes and imported into the mitochondria. That this had not been entirely forgotten was indicated by the fact that a small but significant number of reports on nucleocytoplasmic interactions had crept into a meeting ostensibly devoted exclusively to mitochondrial genes. Of especial interest were reports from A. M. Lam-
L. A. Grivell: Mitochondrial Genes
bowitz (St. Louis Univ.), on the characterization of nuclear mutants impaired in the splicing of the mitochondrial large rRNA in Neurospora and from C. Dieckmann (Columbia Univ.), who briefly described a nuclear mutant that is apparently specifically blocked in its ability to produce transcripts of the cytochrome b gene in yeast. Although it is perhaps surprising that a nuclear factor should be required for the transcription of a particular mitochondrial gene, the nature of the lesion in this mutant should soon be known, since plasmids taken from a nuclear clone bank of a wild-type strain complement the mutation in transformation tests and the further characterization of these is underway.
5. Evolutionary Aspects Finally, an example of what Piotr Slonimski may term 'extrovert' applications of our knowlege ofmitochondrial genes was given by the use of sequence comparison of mtDNAs to determine evoJutionary relationships. In higher eukaryotes, the combination of maternal inheritance, with a lack or extremely low rate of recombination permits lineages to be followed withoutthe 'scrambling' effects of meiosis, while the high fixation rate of mutations in mtDNA ( 5 - 1 0 times higher than singlecopy nuclear DNA) makes it an extremely sensitive indicator of relationships over short evolutionary distances. Of course, this approach is only feasible if all mtDNA molecules in a given individual are identical and the fact that this indeed is the case, notwithstanding the high mutation rate, is something of a paradox, as pointed out by R. A. Lansman (Univ. of Georgia). One solution to this may be that only a small number ofmtDNA molecules in a female's germ-line cells determines the sequence of the mtDNA molecules observed in the somatic tissues of her progeny. Whatever the reason, the utility of the approach was demonstrated in several instances and, applied to our own origins by R. L. Cann (Univ. of Calif.) it indicates that Homo sapiens may have arisen as
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recently as 130,000 years ago, from a small group of individuals. Furthermore, the presence of extremely divergent mtDNA lineages within the human species suggests that non-sapiens mitochondrial genomes still persist in some human populations.
References Anderson S, Bankier AT, Barrell BG, de Bruijn MHL, Coulson AR, Drouin J, Eperon IC, Nierlich DP, Roe BA, Sanger F, Smith AJH, Staden R, Young IG (1981) Nature 290:457 465 Bonitz SG, Tzagoloff A (1980) J Biol Chem 255:9075-9081 Bonitz SG, Coruzzi G, Thalenfeld BE, Tzagoloff A, Macino G (1980) J Biol Chem 255:11927-11941 Borst P, Grivell LA (1981a) Nature 289:439-440 Borst P, Grivell LA (1981b) Nature 290:443-444 Dhawale S, Hanson DK, Alexander N J, Perlman PS, Mahler HR (1981) Proc Natl Acad Sci USA 78:1778-1782 Ephrussi B (1953) Nucleo-Cytoplasmic Relations in MicroOrganisms. Clarendon Press, Oxford Heckman JE, Sarnoff J, Alzner De Weerd B, Yyn S, RajBhandary UL (1980) Proc Natl Acad Sci USA 77:3159-3163 Jacq C, Lazowska J, Slonimski PP (1980) In: Kroon AM, Saccone C (eds) The Organization and Expression of the Mitochondrial Genomes. North-Holland, Amsterdam, pp 139-152 Kolodner R, Tewari KK (1972) Proc Natl Acad Sci USA 69: 1830-1834 Lazowska J, Jacq C, Slonimski PP (1980) Cell 22:333-348 Montoya J, Ojala D, Attardi G (1981) Nature 290:465-470 Nobrega FG, Tzagoloff A (1980) J Biol Chem 255:9828-9837 Ojala D, Montoya J, Attardi G (1981) Nature 290:470-474 Sebald W, Sebald-Althaus M, Wachter E (1977) In: Bandlow W, Schweyen R J, Wolf K, Kaudewitz F (eds) Mitochondria 1977, Genetics and Biogenesis of Mitochondria. De Gruyter, Berlin, pp 433-440 Sharp PA (1981) Cell 23:643-646 Strausberg RL, Vincent RD, Perlman PS, Butow RA (1978) Nature 276:577-583 Tzagoloff A, Nobrega M, Akai A, Macino G (1980) Curr Genet 2:149-157
Communicated by F. Kandewitz Received August 31, 1981
