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Fungal mitochondrial plasmids
EXPERIMENTAL MYCOLOGY9, 285-293 (1985)
TOPICAL Fungal
REVIEW
Mitochondrial
Plasmids
FRANK E. NARGANG Department
of Genetics, Univevsidy of Alberta, Edmonton, Alberta T6G ZE9, Camada Accepted for publication August 23, 1985
INDEX DESCRIPTORS:mitochondrial
plasmids; fungi; review.
Mitochondrial plasmids have been found in many fungi and also in higher plants (Esser et al., 1983; Sederoff, 1984). Fungal mitochondrial plasmids fall into two major groups. The first consists of those that are in fact defective versions of the normal mitochondrial genome of the host organism and there is some question as to whether or not these are properly referred to as plasmids. They are derived wholly from some portion of the parent mtDNA’ molecule, have the capacity to replicate independently, and are generally suppressive toward the parental mtDNA. Examples include the DNAs found in the mitochondria of the ragged mutant of Aspergillus (Lazarus et al., 1980), stopper mutants of Neuvospora (Bertrand et al., 1980), petite mutants of yeast (Locker et nl., 1979), and senescing strains of Podospora (JametVierny et ul., 1980; Wright et al., 1982). In each of these examples the defective version of the mtDNA adversely affects the host by a disruption of mitochondrial function The second group of fungal mitochondrial plasmids consists of what have been referred to as “true” mitochondrial plasmids (Tudzynski, 1982) and will be the topic of this review. The plasmids of this group have little or no homology to the standard ’ Abbreviations used: mtDNA, mitochondrial DNA: kb, kilobase pairs; bp, base pairs; ORF, open reading frame; FGSC, Fungal Genetics Stock Centre.
mitochondrial genome of the host organism. Members of this group were first discovered in Neurospora during searches for mtDNA polymorphisms in strains recently isolated from nature (Collins et al., 1981). They have now also been form Claviceps purpwea (Tudzynski et ai., 1983). Many of the plasmid-co~t~~~~~g Neurospora strains have been examined with respect to growth rate, rni~ocbon~~~a~ cytochrome content, mitochondrial ribosome profiles, and possible production of inhibitors, but to da.te, no trait has been. associated with the presence of a plasmid in any strain (Collins et al., 1981; St&l et al., 1982). The intramitochondrial location of these true plasmids has been examined with varying degrees of rigor in different studies. Though the analyses in some cases are not totally definitive, all the data are consistent with a purely i~tramitochondr~ai location of the plasmids with the exception of the LaBelle plasmid of N. intermedia, which may also exist in the cytosol (Stohl et al., 1982). The intramitochondrial location of the plasmids suggests that they should be maternally inherited and this has been demonstrated for several of the NeLrrospora plasmids (Collins et al., 1981; Stohl et a/., 1982). A mitochondrial plasmid recently discovered in Cochir’obolus hete~os~~~~~~~~s (Garber el ai., 1984) has properties
285 0147-5975185 $3.00 Copyright ‘B 1985 by Academic Press. inc. Ali rights of reproduction in any form revxved.
286
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E. NARGANG
common to both groups of mitochondrial plasmid. It has no immediately obvious effect on the phenotype of the strain but is totally homologous to a certain region of the standard mtDNA of the parent strain as well as to the mtDNA of non-plasmid-containing strains. The problem in categorizing this plasmid arises from the question of whether or not it is suppressive. It does appear to coexist with the standard mtDNA; however, it has been noted that most aged cultures of the plasmid-containing strain have an altered growth phenotype. As yet, it has not been-conclusively shown if the plasmid is directly responsible for this effect. If the latter can be demonstrated, then this plasmid could more properly be placed in the category of defective plasmid-like DNAs rather than the true plasmid category. Examination of the mode of inheritance of the CochZiobo2us plasmid has yielded unusual results. No ascospore progeny were found to contain the free form of the mitochondrial plasmid even when the plasmid strain served as the female parent. Explanations cited for this result include the possibility that the plasmid is present only at a certain developmental stage of the organism or that not all the cells of a plasmid strain culture actually contain the plasmid. Thus, the ascospores collected may have been derived from the fertilization of non-plasmid-containing cells. Alternatively, the presence of the plasmid may cause low fertility, since no complete tetrads were found (Garber et al., 1984). It is also conceivable that the presence of several independently segregating nuclear genes, occurring only in the plasmid-containing strain, are required to maintain the plasmid in its free form. The fungal mitochondrial plasmids are of interest in that they constitute a new category of genetic elements. Their functions, if any, are still completely enigmatic and approaches to disderning their role are not obvious. In fact, it is entirely possible that they have no real role, but are simply genetic elements capable of replication. Light
may be shed on this question by elucidation of their origin (see below). Most of the information currently available on the true mitochondrial plasmids has come from studies on Neurospora. In this genus their distribution in strains isolated recently from the wild is remarkably ubiquitous and the evolution, distribution, and transmission of the plasmids is of some interest and may even be useful in elucidating taxonomic relationships (Natvig et al., 1984). The efficacy of mitochondrial plasmids as cloning vectors has also received some attention (discussed below). PROPERTIES MITOCHONDRIAL
OF
FUNGAL PLASMIDS
All of the Neurospora mitochondrial plasmids examined to date exist as a series of circular molecules containing one or more monomer units joined in a head-to-tail arrangement. More than six monomer units in a single molecule have been visualized by electron microscopy. Similarly, in Cochlioboks, 17 or more monomer units have been found in the largest of its head-to-tail multimers. In Neurospqra, estimates of the ratio of the total number of monomeric plasmid units present, relative to the number of mitochondrial DNA molecules present, vary from plasmid to plasmid; but the ratio always appears to be greater than one and may be as high as 3:l in some strains (Collins et al., 1981; Stohl et al., 1982; Natvig et al., 1984). For the Cochliobolus plasmid the ratio is about 30 (Garber et al., 1984). Eight different mitochondrial plasmids have been described in Neurospora but only one species of plasmid has ever been found in any one strain. Based on hybridization data Natvig et al. (1984) have placed the Neurospora plasmids into three homology groups that cross species lines (see Table 1). The Mauriceville and Varkud plasmids differ from the others in that they give rise to readily detectable transcripts (Collins et al., 1981; Akins, Stohl, Grant, Nargang, and Lambowitz, in preparation). Al-
FUNGAL
MITOCHONDRIAL
TABLE 1 True Fungal Mitochondrial
Species
287
PLASMIDS
Plasmids
Strain and/or isolation point (FGSC number)
Structure
Size (kb)
Neurospora plasmid homology group”
Neurospora
tetrasperma
Lihue, Hawaii (2509) Hanalei, Hawaii (2510) Waimea Falls, Hawaii (3296) Surinam (1270)
Circle Circle Circle Circle
5.0 5.0 5.0 5.0
P 1 ! 1
Neurospora
intermedia
Fiji (435) LaBelle, Florida (1940) Varkud, India (1823)
Circle Circle Circle
5.2 4.1
1 3
3.8
2
Mauriceville,
Circle
3.6
2
Neurospora Claviceps Cochliobolus
crassa purpurea heterostrophusb
Texas (2225)
K T40
Linear Circle
1.1, 5.3, 6.6, 10
-
1.9
-
a From Natvig et al. (1984). b Further analysis may reveal that this strain does not carry a true mitochondrial defective plasmid-like DNA.
though not all the plasmid strains have been examined for the presence of plasmid transcripts, it is known that at least the Fiji and LaBelle plasmids do not give rise to easily detectable transcripts. Though the latter plasmids may simply not give rise to transcripts it is also conceivable that a particular inducing condition is required for the production of transcripts from certain plasmids. The situation for the C. puvpurea plasmids is quite different. Here, four plasmids were found in one strain. The two most predominant (6.6 and 5.3 kb)’ were shown to be linear, were present in high copy number, and were homologous to each other. The two minor plasmids were not studied in detail. The Claviceps and Cochliobolus plasmids have not been examined for the production of transcripts. One feature that the plasmid-containing strains from Neurospora, Claviceps, and Cochliobolus share is that all are recent isolates from nature. The single, plasmid-contaming strain of Claviceps was found in one of two wild strains examined. Four descendants of production strains did not have pla,smids. The Cochliobolus plasmid was
plasmid but, rather, a
found in 1 of 23 field isolates examined; 2 laboratory strains did not contain plasmids. In Neurospora, 8 plasmid-containing strains have been described from 2 natural isolates; 2 laboratory strains examined did not contain plasmids (Collins and Lambowitz, 1983; Natvig et al., $984). In a separate study the presence of mitochondrial plasmids in natural isolates of N. intermedia was noted, but the numbers and characteristics of the plasmids were not reported (Bertrand et al., 1985). It could be coincidence that no laboratory strain in any species contains a plasmid, since the number of laboratory strains examined is relatively small. On the other hand, it is conceivable that the plasmids are lost under laboratory conditions. As yet, there is no evidence that repeated subculturing of plasmid-containing strains leads to plasmid loss. DNA SEQUENCE ANALYSIS OF THE MAURICEVILLE MITOCHONDRIAL PLASMID
The plasmid from the N. crassa auriceville strain has been sequenced entinely (Nargang et al., 1984). Perhaps the most intriguing feature revealed by the sequence
288
FRANK
E. NARGANG
amino acid sequence between the ORFs of Mauriceville and Varkud is strongly indicative of functional conservation. What might the function of the ORF product be? The first obvious possibility is a function that imparts a beneficial trait to the host in a particular environment. Speculation on the possible nature of such a, function should consider the fact that any expression of genetic information present on the plasmid probably occurs within the mitochondrion. The type of function that would evolve to be expressed, and one would assume utilized, in the mitochondrion is not immediately obvious. A product affecting oxidative phosphorylation either directly or indirectly, perhaps by imparting resistance to an inhibitor, seems the most appealing in this context. Another possible function of the ORF product would be one that is necessary for the efficient maintenance of the plasmid, such as replication or partitioning. Interestingly, a short block of amino acid homology between the Mauriceville ORF and the putative polymerase of Cauliflower mosaic virus (Gardner et al., 1981; Toh et et al., 1984). al., 1983) was noted in the original analysis It is conceivable that the ORF exists of the sequence (Nargang et al., 1984). This solely by chance and does not represent a homology has been extended to other short gene that encodes an actual product, but blocks of amino acid sequence common to this is unlikely for a number of reasons. The reverse transcriptases (Michel and Lang, ORF is 2133 bp long and over 30 stop co- 1985). Whether or not reverse transcripdons appear in each of the other reading tase activity exists in the Mauriceville frames along the same length of DNA se- strain is not yet known but, in any case, quence. Furthermore, sequence analysis of such an activity would not necessarily be the homologous region of DNA from the related to replication. Still, since the Mauclosely related Varkud plasmid has re- riceville piasmid does produce unit-length vealed an ORF of the same length that con- transcripts (see below), the possibility retains no insertions or deletions and only a mains open that the plasmid may replicate few amino acid differences in comparison through an RNA intermediate using reverse to the MauriceviIle ORF (Nargang, 1986). transcriptase as is postulated for the DNA Considering that the two plasmids are virus, cauliflower mosaic virus (Pfeiffer and found in different species and in isolates Hahn, 1983). from regions separated by a considerable In view of the considerable indirect evigeographical distance (see Table l), it is dence for the existence of a product from highly unlikely that the ORF is conserved the Mauriceville ORF, the obvious quesentirely by chance. The conservation of tion is: Why has no protein product been
analysis is a long ORF that could encode a hydrophihc protein of up to 710 amino acids. Varying lengths of protein product are possible depending on which of several internal AUG codons might be used as a translational start signal. Mapping of the plasmid transcripts revealed that the ORF is encoded on the appropriate DNA strand and is in the correct position to be completely expressed in the plasmid’s major transcripts. Codon usage in the plasmid ORF is quite unusual. Plasmid ORF codons end in C or G about 40% of the time whereas such codons occur in the genes and ORFs of the standard mtDNA of Neurospora only about 20% of the time. Remarkably, despite this overall difference, usage of certain codons in the plasmid ORF strongly resembles that found in the ORFs of mtDNA introns. The latter differ strikingly in their usage of codons when compared to the standard mtDNA genes that encode proteins involved in oxidative phosphorylation (e.g., Hudspeth et al., 1982). It should be noted that codon usage in the plasmid ORF does not resemble that in Neurospora nuclear genes either (Nargang
FUNGAL
MITOCHONDRIAL
definitively assigned to the Mauriceville plasmid? Possibly, the protein may be expressed only under certain conditions. Such conditions might be highly specific and might be found only in the natural environment of the strain. On the other hand, the product may be present, but only in very small amounts. This might be due to inefficient translation due to the aberrant codon usage in the ORF and/or a poor ribosome binding site on the transcript. Another feature of the Mauriceville plasmid revealed by DNA sequence analysis was the presence of the so-called PstI palindromes. These are 18-bp sequence elements that contain two PstI sites and are palindromic (.5’-CCCTGCAGTACTGCAGW-3”). These elements were initially discovered flanking various genes on the standard mtDNA of Neurospova (Yin et al., 1981). Five such elements were found clustered in a region of less than 400 bp on the plasmid. Two of these were identical to the canonical sequences found on the standard mtDNA and three were found to differ by a few nucleotides. Similar sequences have also been found on the related Varkud plasmid and in the latter case the sequences appear to have undergone less divergence from. the canonical sequences than those on the auriceville plasmid (Akins et al., in preparation). Both the Mauriceville and Varkud plasmids contain directly repeated units of about 170 bp within the region of clustered PstI palindromes. The origin and significance of the PstI clusters on the plasmid and on mtDNA are not understood but one hypothesis consistent with all the data available to date was originally put forward by Yin et al. (1981) for the standard mtDNA. The PstI palindromes may be related to mobile sequence elements that propagate or were once capable of propagating in mitochondria. Thus the sequences would be expected to insert randomly throughout the mtDNA-except in coding sequences, which would be rendered nonfunctional by their presence. This appears
289
PLASMIDS
to be the case on mtDNA and, i~teresti~~l~~ the PstI elements are not found within the plasmid ORF even though the ORF comprises almost 60% of the plasmid sequence. The Mauriceville plasmid was also foun to contain a group of sequence ele highly related to elements that a served in group I mtDNA introns ( et al., 1982; Michel and Dujon, These elements are present in the plastic transcript in the correct 5’ to 3’ order expected in the group I class of introns. On the other hand, a relationship between the Mauriceville plasmid and a family of group II mtDNA introns with ORFs that contain short stretches of amino acid sequence homology to reverse transcriptases was recently discovered (Michel and Lang, 19&Q, The relationship of the a~~ic~vil~e plasmid to this intron family thought to be quite distant since the observed homology extends over only short stretc of amino acids. IS THE
MAURICEVILLE INTRON
PLASMID
AN
PROGENITOR?
The observations discussed above have led to the hypothesis that the ~a~riceville plasmid belongs to a class of genetic elements that are or were the progenitors of mtDNA introns (Nargang et ak., 1984.). It has been pointed out that m~toc~o~drial introns may have arisen from colonization and insertion of completely indepen foreign DNA into preexisting rnit~c~~n genes, since there are codon usage differences between intron ORFs and standard mtDNA genes (Borst and Grivell, 19&l; Hudspeth et al., 1982). Furtbe~m’~re, there is reason to believe that have arisen via insertion cular intermediate into mt et al., 1983). The Mauriceville mi drial plasmid is circular and its 0 certain codon usage properties ~erni~i~ce~t of mtDNA intron ORFs. However, the codon usage is in general somewhat aberrant from that found in rnit~ch~~~dr~~ be-
290
FRANK
E. NARGANG
cause of the high proportion of G or C ending codons. It is conceivable that the unusual codon usage is characteristic of that found in an independent intron progenitor that is not closely related to mtDNA. Following integration, the codons might tend to evolve toward those preferred by the mitochondrial protein synthesizing apparatus. The contention that the plasmid is not closely related to mtDNA is also supported by the fact that it is not suppressive toward the replication of the standard mtDNA. That is, if an origin of replication on the plasmid was very similar to (or derived from) that of the standard mtDNA, it might be expected to compete with the standard mtDNA and cause loss of mitochondrial function as appears to be the case with certain of the defective plasmid-like mtDNAs. In order to more convincingly fit the role of a putative intron progenitor, the Mauriceville plasmid should also have the characteristics of mobile genetic elements. The structure of the plasmid transcripts suggests a relationship to such elements in that the major transcripts of the plasmid are approximately unit length having a single major 3’ end and several major 5’ ends. Some of the major 5’ and 3’ ends overlap with each other by a few nucleotides thus generating RNAs with short terminal repeats (Nargang et al., 1984). Therefore, even though the terminal repeats on the plasmid transcripts are probably quite short in most cases, the plasmid’s transcripts do bear some resemblance to those of mobile genetic elements such as retroviruses (Varmus, 1983) and the Ty elements of yeast (Elder et al., 1983). This putative relationship is strengthened by the observed blocks of homology between the plasmid ORF and the reverse transcriptases found in retroviruses (Michel and Lang, 1985). Interestingly, it has been shown that the Ty elements also contain regions of homology to reverse transcriptases (Clare and Farabaugh, 1985) and to transpose through an
RNA intermediate (Boeke et al., 1985). At some time in its past the Mauriceville tran; script may have represented a similar transposition intermediate. Recent work on the plasmid-like a-sen DNA of Podospora supports the idea that intron DNAs are capable of existing autonomously. Sequence analysis of this DNA has shown that it is an excised intron of the mitochondrially encoded cytochrome c oxidase subunit 1 gene (Osiewacz and Esser, 1984). This excised intron shares many features with the Mauriceville plasmid. It is transcribed (Wright and Cummings, 1983) and belongs to the family of group II introns whose ORFs bear homology to reverse transcriptases (Michel and Lang, 1985). Thus, a remote structural similarity exists between o-sen DNA and the Mauriceville plasmid. However, a-sen DNA differs from Mauriceville in two major respects. First, the codon usage in the ORF of ai-sen DNA exhibits the strong A or U third position bias characteristic of mitochondrial genes and mitochondrial intron ORFs and, second, it contains a sequence strongly resembling that of certain known mtDNA autonomously replicating sequences (Osiewacz and Esser, 1984). The latter observation fits well with the suppressive nature of a-sen DNA toward the parental mtDNA. These observations are all consistent with the hypothesis that ol-sen DNA is an independently existing excised intron while the Mauriceville plasmid may be an intron progenitor. If the Mauriceville plasmid is an intron progenitor why is it not found integrated, at least in the Mauriceville strain, in various regions of the standard mtDNA? This is difficult to answer but it is conceivable that the plasmid is very ancient and has, for some reason, lost the potential to integrate. If it is assumed that the plasmid’s ancestor was once capable of integrating by a mechanism similar to that of retroviruses, then the loss of an appropriate retroviral-like endonuclease activity could account for the
FUNGAL
MITOCHONDRIAL
present inability to integrate (see Varmus, 1985, and references therein). One might also expect that the presence of the PstI palindromes on both the plasmid and mtDNA could provide sites for the occurrence of recombination, but no homology between the Mauriceville plasmid and mtDNA has been detected in hybridization studies (Collins et al., 1981). It is possible that all fungal mitochondrial plasmids bear relationships to mitochondrial introns and all may in fact be intron progenitors. In order to determine if this is the case, DNA sequence analysis of other mitochondrial plasmids is required.
PLASMIDS
BE
gang, 1986). These two plasmids are found in isolates of different species t are geographically well separated (Table The data on observed amino acid differences between the two strongly suggest that the ORF has been conserved through its evolution in each of the two strains. Over the entire length of 2133 nucleotides of the ORF in each plasmid, only 34 nucleotide differences were observed. Most of the differences between Varkud and Mauriceville are transitions (82%) and most are silent (71%). Thus, only nine amino acid differences exist in the protein sequences predicted from the ORFs, and five of these still belong to the same chemical group of EVOLUTION amino acid. Studies on animal mt~NAs Natvig et al. (1984) have studied the ev- have revealed that within protein coding olutionary relationship between the Neugenes, the higher the ratio of transitions to rospora mitochondrial plasmids, particutransversions and the higher the ratio of silarly those from homology group 1 (Table lent to replacement substitutions, the more 1). The plasmids from the Hawaiian Island closely related are the species ( 1983). These ratios have been used to esstrains of N. tetrasperma are indistinguishable by restriction analysis and differ only timate evolutionary distances between anslightly from the N. tetrasperma Surinam imal species. While it would be unwise to compare directly Neurospora to animals, plasmid. The N. intermedia Fiji plasmid has diverged substantially from the other the high number of transitions and silent four. These data were taken as evidence differences observed between the supporting the contention that the fourville and Varkud plasmid ORFs spored isolates of Neurospora represent a reflects a fairly close relationship between the plasmids. separate taxonomic group. Two hypotheses concerning the distribution and origins of USE OF MITOCHONDRIAL PLASMIDS AS these plasmids were put forward. First, the CLONING VECTORS ancient group 1 plasmid might have been present before the divergence of N. interThere has been considerable interest in media and N. tetrasperma and has simply using the mitochondrial plasmids as general cloning vectors. This was undoubt been retained in both species over time stimulated by previous demonstrations while undergoing evolutionary divergence. portions of mtDNA were able to function The second possibility is that the plasmid might have been transferred between the as replicons in other organisms (e.g., Zalkian, 1981; Hyman et al., 1982). Though the species since their divergence. This could have occurred by interspecific crossing, hy- use of mitochondrial plasmids in this regard phal fusions, or some form of infectious has not yet been shown to be generally transmittance. fruitful, there are some reports of initial As mentioned above, a striking level of success. Early results with Neurospora suggested that a segment of DNA from the homology has been observed at the DNA sequence level between the ORFs of the LaBelle plasmid might increase transformation frequencies, but the efficacy of the Mauriceville and Varkud plasmids (Nar-
292
FRANK
E. NARGANG
LaBelle plasmid sequence in such systems was subsequently found to be questionable (Stohl et al., 1984). Success has also been reported in transforming Podospora with derivatives of the plasmid-hke ol-sen DNA (Stahl et al., 1982). The latter system has the disadvantage that successful transformants are in the process of senescence. The Cochliobolus mitochondrial plasmid has been used in a system to give transformants in yeast, though these are relatively unstable (Garber et al., 1984). A small and variable amount of DNA from the LaBelle cloning plasmids has been reported to appear in mitochondria (Stohl and Lambowitz, 1983). Potentially, such a system might be used for cloning and transformation of mtDNA. This would be of interest to those studying mitochondrial biogenesis, particularly in species where mitochondrial mutations are difficult to generate. A mitochondrial cloning system would allow the creation of specific gene alterations in vitro which could be examined for their effects following their introduction into mitochondria. FUTURE
WORK
Some of the most obvious areas for future work include determination of the role of the Cochliobolus plasmid in senesence, the search for a protein product from the Mauriceville and Varkud strains of Neurospora, discovering any relationship of other mitochondrial plasmids to introns, and the assay of reverse transcriptase activity in the mitochondria of plasmid-containing strains. Other points of interest include the distribution of mitochondrial plasmids in other fungal species and determining the reasons for their purely intramitochondrial location. REFERENCES BERTRAND,
H.,
CHAN,
B.
S-S.,
AND
GRIFFITHS,
A. J. F. 1985. Insertion of a foreign nucleotide sequence into mitochondrial DNA causes senescence in Neuvospora intermedia. Cell 41: 877-884.
L. L., A. M. 1980. Deletion mutants of Neurospora crussa mitochondrial DNA and their relationship to the “stopstart” growth phenotype. Proc. Natl. Acad. Sci.
BERTRAND, GOEWERT,
USA
H., R.
COLLINS, R., AND
R. A.,
STOHL,
LAMBOWITZ,
77: 6032-6036.
J. D., GARFINKEL, D. J., STYLES. C. A.. AND G. R. 1985. Ty elements transpose through an RNA intermediate. Cell 40: 491-500. BORST, P., AWD GRLVELL, L. A. 1981. One gene’s intron is another gene’s exon. Nature (Landon) 289: 439-440. BROWN, W. M. 1983. Evolution of animal mitochondrial DNA. In Evobtion of Genes and Proteins (M. Nei and R. K. Koehn, Eds.), pp. 62-88. Sinauer Assoc. Inc., Sunderland, Mass. CLARE, J., AND FARABALJGH, P. 1985. Nucleotide sequence of a yeast Ty element: Evidence for an unusual mechanism of gene expression. Proc. Nat/. BOEKE, FINK,
Acad.
Sci.
USA 82: 2829-2833.
COLLINS, R. A., AND LAMBOWITZ, A. M. 1983. Structural variations and optional introns in the mitochondrial DMAs of Neurospora strains isolated from nature. P/amid 4: 53-70. COLLINS, R. A., STOHL. L. L., COLE, M. D., AND LAMBOWITZ, A. M. 1981. Characterization of a novel plasmid DNA found in mitoehoudria of N. crussa.
Cell 24: 443-452.
ELDER, R. T., LOH, E. W., AND DAVID, R. N. 1983. RNA from the yeast transposable element Tyl has both ends in the direct repeats, a structure similar to retrovirus RNA. Proc. Natl. Acad. Sci. USA 80: 2432-2436. ESSER, K., KIJCK, U., STAHL, U., AND TUDZYNSKI, P. 1983. Cloning vectors of mitochondrial origin for eukaryotes: A new concept in genetic engineering. Curr. GARBER,
Genet. R. C.,
I: 239-243. TURGEON,
B. G.,
AND
CODER,
0.
C.
1984. A mitochondrial plasmid from the plant pathogenic fungus Cachliobolus heterostrophus. Mol. Gen. Genet. 196: 301-310. GARDNER,
R. C., HOWARTH,
A. J., HAHN,
P., EROWN-
LUEDI, M., SHEPHERD, R. .I., AND MESSING, J. 1981. The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencing. Nucl. Acids Res. 9: 28712888. HENSGEMS, L. A. M., BONEN, L., DE HAAN, M., VAN DER HCIRST,G., AND GRIVELL, L. A. 1983. Two intron sequences in yeast mitachorvdrial COXI gene: Homology among URF-co,ntaining introns and strain dependent variations in flanking exons. Ceil 32: 379-389. HUDSPET~, M. D. S., BUTOW,
E. S., AINLEY, W. M., R. A., AND GROSSMAN,
Location and structure of the varl
gene
SHUMARD, L. I. 1982.
on yeast
FUNGAL
MITOCHONDRIAL
mitochondrial DNA: Nucleotide sequence of the 40.0 allele. Cell 30: 617-626. HYMAN. B. C., CRAMER, J. H., AND ROWND, R, H. 1982. Properties of a Saccharomycees cerevisiae mtDNA segment conferring high frequency yeast transformation. Proc. Nat/. Acad. Sci. USA 79: 1578-1582. JAMET-VIERNY, C., BEGEL, O., AND BELC~OUR, L. 1980. Senescence in Podospora anserina: Amplification of a mitochondrial DNA sequence. Cell 21: 189-194. LAZARUS. C. M., EARL, A. J., TURNER, G., AND KUNTZEL, H. 1980. Amplification ofa mitochondrial DNA sequence in the cytoplasmically inherited “ragged” mutant of Aspergillus amstelodami. Eur. J. Biochem. 106: 633-641. LOCKER, J., LEWIN, A., AND RABINOWITZ, M. 1979. The structure and organization of mitochondrial DNA from petite yeast. Plasmid 2: 155-181. MICHEL, E, AND DLJJON, B. 1983. Conservation of RNA secondary structures in two iniron families including mitochondrial-, chloroplast-, and nuclearencoded members. EMB0 J. 2: 33-38. MICIIEL, E, JACQUIER, A.; AND DUJ~N, B. 1982. Comparison of fungal mitochondrial introns reveals extensive homologies in RNA secondary structure. Biochimie 64: 867-881. MICHEL, F., AND LANG, B. F. 1985. Mitochondrial class II introns encode proteins related to the reverse transcriptases of retroviruses. Nature 316: 641-643. NARGANC-, F. E. 1986. Conservation of a long open reading frame in two Neurospora mitochondrial plasmids. IMoE. Bin/. Evd., 3, in press. NARGANG, E E., BELL, J. B., STOHL, L., AND LAMBOWITZ, A. M. 1984. The DNA sequence and genetic organization of a Neurosporu mitochoadrial plasmid suggest a relationship to introns and mobile elements. Cell 38: 441-453. NATYIG, D., MAY. G., AND TAYLOR, J. W. 1984. Distribution and evolutionary significance of mitochondrial piasmids in Neurospora spp. J. Bacterioi. 159: 288-293. OSIEWACZ. H. D., AND ESSER, K. 1984. The mitochondrial plasmid of Podospora anserina: A mobile intron of a mitochondrial gene. Cur. Genet. 8: 299305. PFEIFFER, P., AND HOHN, T. 1983. Involvement of reverse transcription in the replication of cauliflower mosaic virus: A detailed model and test of some aspects. Ceil 33: 781-789. SEDEROFF:R. 1984. Structural variation in mitochondrial DNA. In Advances in Generics, Vol. 22, Mo-
PLASMIDS
lecular Genetics of Plants (J. G. Scandalios, Ed.). pp. l-108. Academic Press, Orlando, Fla. STAHL, U., TUDZYNSKI, P., KUCK, U.. AND ESSER, K. 1982. Replication and expression of a bacterial-mitochondrial hybrid plasmid in the fungus Podospora anserina. Proc. Natl. Acad. Sci. USA 79: 36413645. STOHL, L. L.. AKIN& R. A., AND LAMBOWITZ, A. M. 1984. Characterization of deletion derivatives of an autonomously replicating Nearospora plasmid. Nucl. Acids Res. 12: 6169-6178. STOHL, L. L., COLLINS, R. A., COLE, M. D., AND LAMBOWITZ~ A. M. 1982. Characterization of two new plasmid DNAs found in mitochondiia of wiidtype Neurospora intermedia strains. Nucl. Acids Res. 10: 1439-1458. STOHL, L. L., AND L,AMBOWITZ, A. M. 1983. Construction of a shuttle vector for the filamrntous fungus Newospora crassa. Proc. Nat/. Acad. Sci. USA 80: 1058- 1062. TOH, H., HAYASHIDA, H., AND MIYATA, T. 1983. Sequence homology between retroviral reverse t.ranscriptase and putative polymerases of hepatitis B virus and cauliflower mosaic virus. Natlrre (London) 305: 827-829. TUDZYNSKI, P. 1982. DNA plasmids in eukarjotes with emphasis on mitochondria. Progu. Bat. 44: 297-307. TUDZYNSKI, P.. DUVELL, A., ANU ESSER. K. 1983. Extrachromosomal genetics of Claviceps purpurea. I. Mitochondrial DNA and mitochondrial piasmids. Curr. Genet. 7: 145- 150. VARMUS, H. 1983. Retroviruses. In Mobile Genetic Elements (J. A. Shapiro, Ed.), pp. 411-503. Academic Press, New York. VARXIUS. H. 1985. Reverse transcriptase rides again. Nature (London) 314: 583-584. WRIGHT, R. M., AND CUMMINGS, D. 9. 1983. Transcription of a mitochondrial plasmid during senescence in Podospora anserina. Can. Genet. I: 457464. WRIGHT, R. M., HORRUM, M. A., AND CUMMINGS. D. J. 1982. Are mitochondrial structural genes selectively amplified during senescence in Podnspora anserina? Cell 29: 505-515. YIN,
S.,
HECKMAN,
J.,
AND
RAJBHANDAXY,
U.
L.
1981. Highly conserved CC-rich palindromic DNA sequence.s flank tRNA genes in Neorospora cmssa mitochondria. Cc/i 26: 325-332. ZALKIAN, V. A. 1981. Origin. of replication from Xrnopits laevis mitochondrial DNA promotes high frequency transformation of yeast. Proc. Nail. Acad. Sci. USA 78: 3128-3132.
TOPICAL Fungal
REVIEW
Mitochondrial
Plasmids
FRANK E. NARGANG Department
of Genetics, Univevsidy of Alberta, Edmonton, Alberta T6G ZE9, Camada Accepted for publication August 23, 1985
INDEX DESCRIPTORS:mitochondrial
plasmids; fungi; review.
Mitochondrial plasmids have been found in many fungi and also in higher plants (Esser et al., 1983; Sederoff, 1984). Fungal mitochondrial plasmids fall into two major groups. The first consists of those that are in fact defective versions of the normal mitochondrial genome of the host organism and there is some question as to whether or not these are properly referred to as plasmids. They are derived wholly from some portion of the parent mtDNA’ molecule, have the capacity to replicate independently, and are generally suppressive toward the parental mtDNA. Examples include the DNAs found in the mitochondria of the ragged mutant of Aspergillus (Lazarus et al., 1980), stopper mutants of Neuvospora (Bertrand et al., 1980), petite mutants of yeast (Locker et nl., 1979), and senescing strains of Podospora (JametVierny et ul., 1980; Wright et al., 1982). In each of these examples the defective version of the mtDNA adversely affects the host by a disruption of mitochondrial function The second group of fungal mitochondrial plasmids consists of what have been referred to as “true” mitochondrial plasmids (Tudzynski, 1982) and will be the topic of this review. The plasmids of this group have little or no homology to the standard ’ Abbreviations used: mtDNA, mitochondrial DNA: kb, kilobase pairs; bp, base pairs; ORF, open reading frame; FGSC, Fungal Genetics Stock Centre.
mitochondrial genome of the host organism. Members of this group were first discovered in Neurospora during searches for mtDNA polymorphisms in strains recently isolated from nature (Collins et al., 1981). They have now also been form Claviceps purpwea (Tudzynski et ai., 1983). Many of the plasmid-co~t~~~~~g Neurospora strains have been examined with respect to growth rate, rni~ocbon~~~a~ cytochrome content, mitochondrial ribosome profiles, and possible production of inhibitors, but to da.te, no trait has been. associated with the presence of a plasmid in any strain (Collins et al., 1981; St&l et al., 1982). The intramitochondrial location of these true plasmids has been examined with varying degrees of rigor in different studies. Though the analyses in some cases are not totally definitive, all the data are consistent with a purely i~tramitochondr~ai location of the plasmids with the exception of the LaBelle plasmid of N. intermedia, which may also exist in the cytosol (Stohl et al., 1982). The intramitochondrial location of the plasmids suggests that they should be maternally inherited and this has been demonstrated for several of the NeLrrospora plasmids (Collins et al., 1981; Stohl et a/., 1982). A mitochondrial plasmid recently discovered in Cochir’obolus hete~os~~~~~~~~s (Garber el ai., 1984) has properties
285 0147-5975185 $3.00 Copyright ‘B 1985 by Academic Press. inc. Ali rights of reproduction in any form revxved.
286
FRANK
E. NARGANG
common to both groups of mitochondrial plasmid. It has no immediately obvious effect on the phenotype of the strain but is totally homologous to a certain region of the standard mtDNA of the parent strain as well as to the mtDNA of non-plasmid-containing strains. The problem in categorizing this plasmid arises from the question of whether or not it is suppressive. It does appear to coexist with the standard mtDNA; however, it has been noted that most aged cultures of the plasmid-containing strain have an altered growth phenotype. As yet, it has not been-conclusively shown if the plasmid is directly responsible for this effect. If the latter can be demonstrated, then this plasmid could more properly be placed in the category of defective plasmid-like DNAs rather than the true plasmid category. Examination of the mode of inheritance of the CochZiobo2us plasmid has yielded unusual results. No ascospore progeny were found to contain the free form of the mitochondrial plasmid even when the plasmid strain served as the female parent. Explanations cited for this result include the possibility that the plasmid is present only at a certain developmental stage of the organism or that not all the cells of a plasmid strain culture actually contain the plasmid. Thus, the ascospores collected may have been derived from the fertilization of non-plasmid-containing cells. Alternatively, the presence of the plasmid may cause low fertility, since no complete tetrads were found (Garber et al., 1984). It is also conceivable that the presence of several independently segregating nuclear genes, occurring only in the plasmid-containing strain, are required to maintain the plasmid in its free form. The fungal mitochondrial plasmids are of interest in that they constitute a new category of genetic elements. Their functions, if any, are still completely enigmatic and approaches to disderning their role are not obvious. In fact, it is entirely possible that they have no real role, but are simply genetic elements capable of replication. Light
may be shed on this question by elucidation of their origin (see below). Most of the information currently available on the true mitochondrial plasmids has come from studies on Neurospora. In this genus their distribution in strains isolated recently from the wild is remarkably ubiquitous and the evolution, distribution, and transmission of the plasmids is of some interest and may even be useful in elucidating taxonomic relationships (Natvig et al., 1984). The efficacy of mitochondrial plasmids as cloning vectors has also received some attention (discussed below). PROPERTIES MITOCHONDRIAL
OF
FUNGAL PLASMIDS
All of the Neurospora mitochondrial plasmids examined to date exist as a series of circular molecules containing one or more monomer units joined in a head-to-tail arrangement. More than six monomer units in a single molecule have been visualized by electron microscopy. Similarly, in Cochlioboks, 17 or more monomer units have been found in the largest of its head-to-tail multimers. In Neurospqra, estimates of the ratio of the total number of monomeric plasmid units present, relative to the number of mitochondrial DNA molecules present, vary from plasmid to plasmid; but the ratio always appears to be greater than one and may be as high as 3:l in some strains (Collins et al., 1981; Stohl et al., 1982; Natvig et al., 1984). For the Cochliobolus plasmid the ratio is about 30 (Garber et al., 1984). Eight different mitochondrial plasmids have been described in Neurospora but only one species of plasmid has ever been found in any one strain. Based on hybridization data Natvig et al. (1984) have placed the Neurospora plasmids into three homology groups that cross species lines (see Table 1). The Mauriceville and Varkud plasmids differ from the others in that they give rise to readily detectable transcripts (Collins et al., 1981; Akins, Stohl, Grant, Nargang, and Lambowitz, in preparation). Al-
FUNGAL
MITOCHONDRIAL
TABLE 1 True Fungal Mitochondrial
Species
287
PLASMIDS
Plasmids
Strain and/or isolation point (FGSC number)
Structure
Size (kb)
Neurospora plasmid homology group”
Neurospora
tetrasperma
Lihue, Hawaii (2509) Hanalei, Hawaii (2510) Waimea Falls, Hawaii (3296) Surinam (1270)
Circle Circle Circle Circle
5.0 5.0 5.0 5.0
P 1 ! 1
Neurospora
intermedia
Fiji (435) LaBelle, Florida (1940) Varkud, India (1823)
Circle Circle Circle
5.2 4.1
1 3
3.8
2
Mauriceville,
Circle
3.6
2
Neurospora Claviceps Cochliobolus
crassa purpurea heterostrophusb
Texas (2225)
K T40
Linear Circle
1.1, 5.3, 6.6, 10
-
1.9
-
a From Natvig et al. (1984). b Further analysis may reveal that this strain does not carry a true mitochondrial defective plasmid-like DNA.
though not all the plasmid strains have been examined for the presence of plasmid transcripts, it is known that at least the Fiji and LaBelle plasmids do not give rise to easily detectable transcripts. Though the latter plasmids may simply not give rise to transcripts it is also conceivable that a particular inducing condition is required for the production of transcripts from certain plasmids. The situation for the C. puvpurea plasmids is quite different. Here, four plasmids were found in one strain. The two most predominant (6.6 and 5.3 kb)’ were shown to be linear, were present in high copy number, and were homologous to each other. The two minor plasmids were not studied in detail. The Claviceps and Cochliobolus plasmids have not been examined for the production of transcripts. One feature that the plasmid-containing strains from Neurospora, Claviceps, and Cochliobolus share is that all are recent isolates from nature. The single, plasmid-contaming strain of Claviceps was found in one of two wild strains examined. Four descendants of production strains did not have pla,smids. The Cochliobolus plasmid was
plasmid but, rather, a
found in 1 of 23 field isolates examined; 2 laboratory strains did not contain plasmids. In Neurospora, 8 plasmid-containing strains have been described from 2 natural isolates; 2 laboratory strains examined did not contain plasmids (Collins and Lambowitz, 1983; Natvig et al., $984). In a separate study the presence of mitochondrial plasmids in natural isolates of N. intermedia was noted, but the numbers and characteristics of the plasmids were not reported (Bertrand et al., 1985). It could be coincidence that no laboratory strain in any species contains a plasmid, since the number of laboratory strains examined is relatively small. On the other hand, it is conceivable that the plasmids are lost under laboratory conditions. As yet, there is no evidence that repeated subculturing of plasmid-containing strains leads to plasmid loss. DNA SEQUENCE ANALYSIS OF THE MAURICEVILLE MITOCHONDRIAL PLASMID
The plasmid from the N. crassa auriceville strain has been sequenced entinely (Nargang et al., 1984). Perhaps the most intriguing feature revealed by the sequence
288
FRANK
E. NARGANG
amino acid sequence between the ORFs of Mauriceville and Varkud is strongly indicative of functional conservation. What might the function of the ORF product be? The first obvious possibility is a function that imparts a beneficial trait to the host in a particular environment. Speculation on the possible nature of such a, function should consider the fact that any expression of genetic information present on the plasmid probably occurs within the mitochondrion. The type of function that would evolve to be expressed, and one would assume utilized, in the mitochondrion is not immediately obvious. A product affecting oxidative phosphorylation either directly or indirectly, perhaps by imparting resistance to an inhibitor, seems the most appealing in this context. Another possible function of the ORF product would be one that is necessary for the efficient maintenance of the plasmid, such as replication or partitioning. Interestingly, a short block of amino acid homology between the Mauriceville ORF and the putative polymerase of Cauliflower mosaic virus (Gardner et al., 1981; Toh et et al., 1984). al., 1983) was noted in the original analysis It is conceivable that the ORF exists of the sequence (Nargang et al., 1984). This solely by chance and does not represent a homology has been extended to other short gene that encodes an actual product, but blocks of amino acid sequence common to this is unlikely for a number of reasons. The reverse transcriptases (Michel and Lang, ORF is 2133 bp long and over 30 stop co- 1985). Whether or not reverse transcripdons appear in each of the other reading tase activity exists in the Mauriceville frames along the same length of DNA se- strain is not yet known but, in any case, quence. Furthermore, sequence analysis of such an activity would not necessarily be the homologous region of DNA from the related to replication. Still, since the Mauclosely related Varkud plasmid has re- riceville piasmid does produce unit-length vealed an ORF of the same length that con- transcripts (see below), the possibility retains no insertions or deletions and only a mains open that the plasmid may replicate few amino acid differences in comparison through an RNA intermediate using reverse to the MauriceviIle ORF (Nargang, 1986). transcriptase as is postulated for the DNA Considering that the two plasmids are virus, cauliflower mosaic virus (Pfeiffer and found in different species and in isolates Hahn, 1983). from regions separated by a considerable In view of the considerable indirect evigeographical distance (see Table l), it is dence for the existence of a product from highly unlikely that the ORF is conserved the Mauriceville ORF, the obvious quesentirely by chance. The conservation of tion is: Why has no protein product been
analysis is a long ORF that could encode a hydrophihc protein of up to 710 amino acids. Varying lengths of protein product are possible depending on which of several internal AUG codons might be used as a translational start signal. Mapping of the plasmid transcripts revealed that the ORF is encoded on the appropriate DNA strand and is in the correct position to be completely expressed in the plasmid’s major transcripts. Codon usage in the plasmid ORF is quite unusual. Plasmid ORF codons end in C or G about 40% of the time whereas such codons occur in the genes and ORFs of the standard mtDNA of Neurospora only about 20% of the time. Remarkably, despite this overall difference, usage of certain codons in the plasmid ORF strongly resembles that found in the ORFs of mtDNA introns. The latter differ strikingly in their usage of codons when compared to the standard mtDNA genes that encode proteins involved in oxidative phosphorylation (e.g., Hudspeth et al., 1982). It should be noted that codon usage in the plasmid ORF does not resemble that in Neurospora nuclear genes either (Nargang
FUNGAL
MITOCHONDRIAL
definitively assigned to the Mauriceville plasmid? Possibly, the protein may be expressed only under certain conditions. Such conditions might be highly specific and might be found only in the natural environment of the strain. On the other hand, the product may be present, but only in very small amounts. This might be due to inefficient translation due to the aberrant codon usage in the ORF and/or a poor ribosome binding site on the transcript. Another feature of the Mauriceville plasmid revealed by DNA sequence analysis was the presence of the so-called PstI palindromes. These are 18-bp sequence elements that contain two PstI sites and are palindromic (.5’-CCCTGCAGTACTGCAGW-3”). These elements were initially discovered flanking various genes on the standard mtDNA of Neurospova (Yin et al., 1981). Five such elements were found clustered in a region of less than 400 bp on the plasmid. Two of these were identical to the canonical sequences found on the standard mtDNA and three were found to differ by a few nucleotides. Similar sequences have also been found on the related Varkud plasmid and in the latter case the sequences appear to have undergone less divergence from. the canonical sequences than those on the auriceville plasmid (Akins et al., in preparation). Both the Mauriceville and Varkud plasmids contain directly repeated units of about 170 bp within the region of clustered PstI palindromes. The origin and significance of the PstI clusters on the plasmid and on mtDNA are not understood but one hypothesis consistent with all the data available to date was originally put forward by Yin et al. (1981) for the standard mtDNA. The PstI palindromes may be related to mobile sequence elements that propagate or were once capable of propagating in mitochondria. Thus the sequences would be expected to insert randomly throughout the mtDNA-except in coding sequences, which would be rendered nonfunctional by their presence. This appears
289
PLASMIDS
to be the case on mtDNA and, i~teresti~~l~~ the PstI elements are not found within the plasmid ORF even though the ORF comprises almost 60% of the plasmid sequence. The Mauriceville plasmid was also foun to contain a group of sequence ele highly related to elements that a served in group I mtDNA introns ( et al., 1982; Michel and Dujon, These elements are present in the plastic transcript in the correct 5’ to 3’ order expected in the group I class of introns. On the other hand, a relationship between the Mauriceville plasmid and a family of group II mtDNA introns with ORFs that contain short stretches of amino acid sequence homology to reverse transcriptases was recently discovered (Michel and Lang, 19&Q, The relationship of the a~~ic~vil~e plasmid to this intron family thought to be quite distant since the observed homology extends over only short stretc of amino acids. IS THE
MAURICEVILLE INTRON
PLASMID
AN
PROGENITOR?
The observations discussed above have led to the hypothesis that the ~a~riceville plasmid belongs to a class of genetic elements that are or were the progenitors of mtDNA introns (Nargang et ak., 1984.). It has been pointed out that m~toc~o~drial introns may have arisen from colonization and insertion of completely indepen foreign DNA into preexisting rnit~c~~n genes, since there are codon usage differences between intron ORFs and standard mtDNA genes (Borst and Grivell, 19&l; Hudspeth et al., 1982). Furtbe~m’~re, there is reason to believe that have arisen via insertion cular intermediate into mt et al., 1983). The Mauriceville mi drial plasmid is circular and its 0 certain codon usage properties ~erni~i~ce~t of mtDNA intron ORFs. However, the codon usage is in general somewhat aberrant from that found in rnit~ch~~~dr~~ be-
290
FRANK
E. NARGANG
cause of the high proportion of G or C ending codons. It is conceivable that the unusual codon usage is characteristic of that found in an independent intron progenitor that is not closely related to mtDNA. Following integration, the codons might tend to evolve toward those preferred by the mitochondrial protein synthesizing apparatus. The contention that the plasmid is not closely related to mtDNA is also supported by the fact that it is not suppressive toward the replication of the standard mtDNA. That is, if an origin of replication on the plasmid was very similar to (or derived from) that of the standard mtDNA, it might be expected to compete with the standard mtDNA and cause loss of mitochondrial function as appears to be the case with certain of the defective plasmid-like mtDNAs. In order to more convincingly fit the role of a putative intron progenitor, the Mauriceville plasmid should also have the characteristics of mobile genetic elements. The structure of the plasmid transcripts suggests a relationship to such elements in that the major transcripts of the plasmid are approximately unit length having a single major 3’ end and several major 5’ ends. Some of the major 5’ and 3’ ends overlap with each other by a few nucleotides thus generating RNAs with short terminal repeats (Nargang et al., 1984). Therefore, even though the terminal repeats on the plasmid transcripts are probably quite short in most cases, the plasmid’s transcripts do bear some resemblance to those of mobile genetic elements such as retroviruses (Varmus, 1983) and the Ty elements of yeast (Elder et al., 1983). This putative relationship is strengthened by the observed blocks of homology between the plasmid ORF and the reverse transcriptases found in retroviruses (Michel and Lang, 1985). Interestingly, it has been shown that the Ty elements also contain regions of homology to reverse transcriptases (Clare and Farabaugh, 1985) and to transpose through an
RNA intermediate (Boeke et al., 1985). At some time in its past the Mauriceville tran; script may have represented a similar transposition intermediate. Recent work on the plasmid-like a-sen DNA of Podospora supports the idea that intron DNAs are capable of existing autonomously. Sequence analysis of this DNA has shown that it is an excised intron of the mitochondrially encoded cytochrome c oxidase subunit 1 gene (Osiewacz and Esser, 1984). This excised intron shares many features with the Mauriceville plasmid. It is transcribed (Wright and Cummings, 1983) and belongs to the family of group II introns whose ORFs bear homology to reverse transcriptases (Michel and Lang, 1985). Thus, a remote structural similarity exists between o-sen DNA and the Mauriceville plasmid. However, a-sen DNA differs from Mauriceville in two major respects. First, the codon usage in the ORF of ai-sen DNA exhibits the strong A or U third position bias characteristic of mitochondrial genes and mitochondrial intron ORFs and, second, it contains a sequence strongly resembling that of certain known mtDNA autonomously replicating sequences (Osiewacz and Esser, 1984). The latter observation fits well with the suppressive nature of a-sen DNA toward the parental mtDNA. These observations are all consistent with the hypothesis that ol-sen DNA is an independently existing excised intron while the Mauriceville plasmid may be an intron progenitor. If the Mauriceville plasmid is an intron progenitor why is it not found integrated, at least in the Mauriceville strain, in various regions of the standard mtDNA? This is difficult to answer but it is conceivable that the plasmid is very ancient and has, for some reason, lost the potential to integrate. If it is assumed that the plasmid’s ancestor was once capable of integrating by a mechanism similar to that of retroviruses, then the loss of an appropriate retroviral-like endonuclease activity could account for the
FUNGAL
MITOCHONDRIAL
present inability to integrate (see Varmus, 1985, and references therein). One might also expect that the presence of the PstI palindromes on both the plasmid and mtDNA could provide sites for the occurrence of recombination, but no homology between the Mauriceville plasmid and mtDNA has been detected in hybridization studies (Collins et al., 1981). It is possible that all fungal mitochondrial plasmids bear relationships to mitochondrial introns and all may in fact be intron progenitors. In order to determine if this is the case, DNA sequence analysis of other mitochondrial plasmids is required.
PLASMIDS
BE
gang, 1986). These two plasmids are found in isolates of different species t are geographically well separated (Table The data on observed amino acid differences between the two strongly suggest that the ORF has been conserved through its evolution in each of the two strains. Over the entire length of 2133 nucleotides of the ORF in each plasmid, only 34 nucleotide differences were observed. Most of the differences between Varkud and Mauriceville are transitions (82%) and most are silent (71%). Thus, only nine amino acid differences exist in the protein sequences predicted from the ORFs, and five of these still belong to the same chemical group of EVOLUTION amino acid. Studies on animal mt~NAs Natvig et al. (1984) have studied the ev- have revealed that within protein coding olutionary relationship between the Neugenes, the higher the ratio of transitions to rospora mitochondrial plasmids, particutransversions and the higher the ratio of silarly those from homology group 1 (Table lent to replacement substitutions, the more 1). The plasmids from the Hawaiian Island closely related are the species ( 1983). These ratios have been used to esstrains of N. tetrasperma are indistinguishable by restriction analysis and differ only timate evolutionary distances between anslightly from the N. tetrasperma Surinam imal species. While it would be unwise to compare directly Neurospora to animals, plasmid. The N. intermedia Fiji plasmid has diverged substantially from the other the high number of transitions and silent four. These data were taken as evidence differences observed between the supporting the contention that the fourville and Varkud plasmid ORFs spored isolates of Neurospora represent a reflects a fairly close relationship between the plasmids. separate taxonomic group. Two hypotheses concerning the distribution and origins of USE OF MITOCHONDRIAL PLASMIDS AS these plasmids were put forward. First, the CLONING VECTORS ancient group 1 plasmid might have been present before the divergence of N. interThere has been considerable interest in media and N. tetrasperma and has simply using the mitochondrial plasmids as general cloning vectors. This was undoubt been retained in both species over time stimulated by previous demonstrations while undergoing evolutionary divergence. portions of mtDNA were able to function The second possibility is that the plasmid might have been transferred between the as replicons in other organisms (e.g., Zalkian, 1981; Hyman et al., 1982). Though the species since their divergence. This could have occurred by interspecific crossing, hy- use of mitochondrial plasmids in this regard phal fusions, or some form of infectious has not yet been shown to be generally transmittance. fruitful, there are some reports of initial As mentioned above, a striking level of success. Early results with Neurospora suggested that a segment of DNA from the homology has been observed at the DNA sequence level between the ORFs of the LaBelle plasmid might increase transformation frequencies, but the efficacy of the Mauriceville and Varkud plasmids (Nar-
292
FRANK
E. NARGANG
LaBelle plasmid sequence in such systems was subsequently found to be questionable (Stohl et al., 1984). Success has also been reported in transforming Podospora with derivatives of the plasmid-hke ol-sen DNA (Stahl et al., 1982). The latter system has the disadvantage that successful transformants are in the process of senescence. The Cochliobolus mitochondrial plasmid has been used in a system to give transformants in yeast, though these are relatively unstable (Garber et al., 1984). A small and variable amount of DNA from the LaBelle cloning plasmids has been reported to appear in mitochondria (Stohl and Lambowitz, 1983). Potentially, such a system might be used for cloning and transformation of mtDNA. This would be of interest to those studying mitochondrial biogenesis, particularly in species where mitochondrial mutations are difficult to generate. A mitochondrial cloning system would allow the creation of specific gene alterations in vitro which could be examined for their effects following their introduction into mitochondria. FUTURE
WORK
Some of the most obvious areas for future work include determination of the role of the Cochliobolus plasmid in senesence, the search for a protein product from the Mauriceville and Varkud strains of Neurospora, discovering any relationship of other mitochondrial plasmids to introns, and the assay of reverse transcriptase activity in the mitochondria of plasmid-containing strains. Other points of interest include the distribution of mitochondrial plasmids in other fungal species and determining the reasons for their purely intramitochondrial location. REFERENCES BERTRAND,
H.,
CHAN,
B.
S-S.,
AND
GRIFFITHS,
A. J. F. 1985. Insertion of a foreign nucleotide sequence into mitochondrial DNA causes senescence in Neuvospora intermedia. Cell 41: 877-884.
L. L., A. M. 1980. Deletion mutants of Neurospora crussa mitochondrial DNA and their relationship to the “stopstart” growth phenotype. Proc. Natl. Acad. Sci.
BERTRAND, GOEWERT,
USA
H., R.
COLLINS, R., AND
R. A.,
STOHL,
LAMBOWITZ,
77: 6032-6036.
J. D., GARFINKEL, D. J., STYLES. C. A.. AND G. R. 1985. Ty elements transpose through an RNA intermediate. Cell 40: 491-500. BORST, P., AWD GRLVELL, L. A. 1981. One gene’s intron is another gene’s exon. Nature (Landon) 289: 439-440. BROWN, W. M. 1983. Evolution of animal mitochondrial DNA. In Evobtion of Genes and Proteins (M. Nei and R. K. Koehn, Eds.), pp. 62-88. Sinauer Assoc. Inc., Sunderland, Mass. CLARE, J., AND FARABALJGH, P. 1985. Nucleotide sequence of a yeast Ty element: Evidence for an unusual mechanism of gene expression. Proc. Nat/. BOEKE, FINK,
Acad.
Sci.
USA 82: 2829-2833.
COLLINS, R. A., AND LAMBOWITZ, A. M. 1983. Structural variations and optional introns in the mitochondrial DMAs of Neurospora strains isolated from nature. P/amid 4: 53-70. COLLINS, R. A., STOHL. L. L., COLE, M. D., AND LAMBOWITZ, A. M. 1981. Characterization of a novel plasmid DNA found in mitoehoudria of N. crussa.
Cell 24: 443-452.
ELDER, R. T., LOH, E. W., AND DAVID, R. N. 1983. RNA from the yeast transposable element Tyl has both ends in the direct repeats, a structure similar to retrovirus RNA. Proc. Natl. Acad. Sci. USA 80: 2432-2436. ESSER, K., KIJCK, U., STAHL, U., AND TUDZYNSKI, P. 1983. Cloning vectors of mitochondrial origin for eukaryotes: A new concept in genetic engineering. Curr. GARBER,
Genet. R. C.,
I: 239-243. TURGEON,
B. G.,
AND
CODER,
0.
C.
1984. A mitochondrial plasmid from the plant pathogenic fungus Cachliobolus heterostrophus. Mol. Gen. Genet. 196: 301-310. GARDNER,
R. C., HOWARTH,
A. J., HAHN,
P., EROWN-
LUEDI, M., SHEPHERD, R. .I., AND MESSING, J. 1981. The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencing. Nucl. Acids Res. 9: 28712888. HENSGEMS, L. A. M., BONEN, L., DE HAAN, M., VAN DER HCIRST,G., AND GRIVELL, L. A. 1983. Two intron sequences in yeast mitachorvdrial COXI gene: Homology among URF-co,ntaining introns and strain dependent variations in flanking exons. Ceil 32: 379-389. HUDSPET~, M. D. S., BUTOW,
E. S., AINLEY, W. M., R. A., AND GROSSMAN,
Location and structure of the varl
gene
SHUMARD, L. I. 1982.
on yeast
FUNGAL
MITOCHONDRIAL
mitochondrial DNA: Nucleotide sequence of the 40.0 allele. Cell 30: 617-626. HYMAN. B. C., CRAMER, J. H., AND ROWND, R, H. 1982. Properties of a Saccharomycees cerevisiae mtDNA segment conferring high frequency yeast transformation. Proc. Nat/. Acad. Sci. USA 79: 1578-1582. JAMET-VIERNY, C., BEGEL, O., AND BELC~OUR, L. 1980. Senescence in Podospora anserina: Amplification of a mitochondrial DNA sequence. Cell 21: 189-194. LAZARUS. C. M., EARL, A. J., TURNER, G., AND KUNTZEL, H. 1980. Amplification ofa mitochondrial DNA sequence in the cytoplasmically inherited “ragged” mutant of Aspergillus amstelodami. Eur. J. Biochem. 106: 633-641. LOCKER, J., LEWIN, A., AND RABINOWITZ, M. 1979. The structure and organization of mitochondrial DNA from petite yeast. Plasmid 2: 155-181. MICHEL, E, AND DLJJON, B. 1983. Conservation of RNA secondary structures in two iniron families including mitochondrial-, chloroplast-, and nuclearencoded members. EMB0 J. 2: 33-38. MICIIEL, E, JACQUIER, A.; AND DUJ~N, B. 1982. Comparison of fungal mitochondrial introns reveals extensive homologies in RNA secondary structure. Biochimie 64: 867-881. MICHEL, F., AND LANG, B. F. 1985. Mitochondrial class II introns encode proteins related to the reverse transcriptases of retroviruses. Nature 316: 641-643. NARGANC-, F. E. 1986. Conservation of a long open reading frame in two Neurospora mitochondrial plasmids. IMoE. Bin/. Evd., 3, in press. NARGANG, E E., BELL, J. B., STOHL, L., AND LAMBOWITZ, A. M. 1984. The DNA sequence and genetic organization of a Neurosporu mitochoadrial plasmid suggest a relationship to introns and mobile elements. Cell 38: 441-453. NATYIG, D., MAY. G., AND TAYLOR, J. W. 1984. Distribution and evolutionary significance of mitochondrial piasmids in Neurospora spp. J. Bacterioi. 159: 288-293. OSIEWACZ. H. D., AND ESSER, K. 1984. The mitochondrial plasmid of Podospora anserina: A mobile intron of a mitochondrial gene. Cur. Genet. 8: 299305. PFEIFFER, P., AND HOHN, T. 1983. Involvement of reverse transcription in the replication of cauliflower mosaic virus: A detailed model and test of some aspects. Ceil 33: 781-789. SEDEROFF:R. 1984. Structural variation in mitochondrial DNA. In Advances in Generics, Vol. 22, Mo-
PLASMIDS
lecular Genetics of Plants (J. G. Scandalios, Ed.). pp. l-108. Academic Press, Orlando, Fla. STAHL, U., TUDZYNSKI, P., KUCK, U.. AND ESSER, K. 1982. Replication and expression of a bacterial-mitochondrial hybrid plasmid in the fungus Podospora anserina. Proc. Natl. Acad. Sci. USA 79: 36413645. STOHL, L. L.. AKIN& R. A., AND LAMBOWITZ, A. M. 1984. Characterization of deletion derivatives of an autonomously replicating Nearospora plasmid. Nucl. Acids Res. 12: 6169-6178. STOHL, L. L., COLLINS, R. A., COLE, M. D., AND LAMBOWITZ~ A. M. 1982. Characterization of two new plasmid DNAs found in mitochondiia of wiidtype Neurospora intermedia strains. Nucl. Acids Res. 10: 1439-1458. STOHL, L. L., AND L,AMBOWITZ, A. M. 1983. Construction of a shuttle vector for the filamrntous fungus Newospora crassa. Proc. Nat/. Acad. Sci. USA 80: 1058- 1062. TOH, H., HAYASHIDA, H., AND MIYATA, T. 1983. Sequence homology between retroviral reverse t.ranscriptase and putative polymerases of hepatitis B virus and cauliflower mosaic virus. Natlrre (London) 305: 827-829. TUDZYNSKI, P. 1982. DNA plasmids in eukarjotes with emphasis on mitochondria. Progu. Bat. 44: 297-307. TUDZYNSKI, P.. DUVELL, A., ANU ESSER. K. 1983. Extrachromosomal genetics of Claviceps purpurea. I. Mitochondrial DNA and mitochondrial piasmids. Curr. Genet. 7: 145- 150. VARMUS, H. 1983. Retroviruses. In Mobile Genetic Elements (J. A. Shapiro, Ed.), pp. 411-503. Academic Press, New York. VARXIUS. H. 1985. Reverse transcriptase rides again. Nature (London) 314: 583-584. WRIGHT, R. M., AND CUMMINGS, D. 9. 1983. Transcription of a mitochondrial plasmid during senescence in Podospora anserina. Can. Genet. I: 457464. WRIGHT, R. M., HORRUM, M. A., AND CUMMINGS. D. J. 1982. Are mitochondrial structural genes selectively amplified during senescence in Podnspora anserina? Cell 29: 505-515. YIN,
S.,
HECKMAN,
J.,
AND
RAJBHANDAXY,
U.
L.
1981. Highly conserved CC-rich palindromic DNA sequence.s flank tRNA genes in Neorospora cmssa mitochondria. Cc/i 26: 325-332. ZALKIAN, V. A. 1981. Origin. of replication from Xrnopits laevis mitochondrial DNA promotes high frequency transformation of yeast. Proc. Nail. Acad. Sci. USA 78: 3128-3132.