2 Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405, USA. Received October 18, 1991/February 3, 1992.
Current Genetics
Curr Genet (1992)22:129-134
9 Springer-Verlag 1992
Mitochondrial D N A of Sch ,ophyllum commune: restriction map, genetic map, and mode of inheritance Charles A. Specht 1, ,, Charles P. Novotny 2, and Robert C. Ullrieh 1
1 Department of Botany, University of Vermont, Burlington,VT 05405, USA 2 Department of Microbiologyand Molecular Genetics, University of Vermont, Burlington,VT 05405, USA Received October 18, 1991/February 3, 1992
Summary. Mitochondrial DNA (mtDNA) found in the basidiomycete Schizophyllum commune (strain 4 40) is a
circular molecule 49.75 kbp in length. A physical map containing 61 restriction sites revealed no repeat structures. Cloned genes from Neurospora crassa, Aspergillus nidulans, and Saccharomyces cerevisiae were used in Southern hybridizations to locate nine mitochondrial genes, including a possible pseudogene of ATPase 9, on the restriction map. A probe from a functional ATPase 9 gene identified homologous fragments only in the nuclear genome of S. commune. Restriction fragment length polymorphisms (RFLPs) between mtDNA isolated from different strains of S. commune were used to show that mitochondria do not migrate with nuclei during dikaryosis. Key words: Mitochondrial DNA - Fungi - Basidiomycete - Schizophyllum
Introduction
Mitochondrial genomes in fungi vary ten-fold in size from 17.6 kb in Schizosaccharomyces pombe (Zimmer et al. 1987) to 176 kb in Agaricus bitorquis (Hintz et al. 1985). Related taxa exhibit less variability. For example, mtDNA from species of the genus Suillus have been found to vary three-fold in size (36-121 kb, Bruns et al. 1988); and strains of the species S. pombe vary 1.4-fold (17.624.6 kb, Zimmer et al. 1987). Length heterogeneity in related taxa is generally due to optional introns, duplications, and insertions and deletions between transcribed regions. The gene order is usually not altered by these mutations. Common to all fungi are a set of genes encoded by the mtDNA. These genes code for: two ribosomal RNAs, a * Present address." Center for Cancer Research, Massachusetts In-
stitute of Technology,Cambridge, MA 02139, USA Correspondence to." R. C. Ullrich
set of transfer RNAs, and proteins that comprise part of ATPase, coenzyme QHz-cytochrome c reductase, and the cytochrome oxidase complexes found in the inner mitochondrial membrane (reviewed in Grossman and Hudspeth 1985; Chomyn and Attardi 1987). Some genes are found in the mitochondrial genome of one species and in the nuclear genome of another. These include subunits of the NADH dehydrogenase complex, ATPase subunit 9, maturases, transposition factors, and mitochondrial ribosomal proteins. For example, the ATPase 9 gene is encoded by mtDNA in S. cerevisiae, and nuclear DNA in N. crassa (Sebald et al. 1979) and A. nidulans (Ward and Turner 1986). ORFs that have homology to the S. cervisiae ATPase 9 gene are also present in the mtDNA of N. crassa (van den Boogaart et al. 1982) and A. nidulans (Brown et al. 1984). Even though mitochondrial genomes are heterogeneous with regard to length and gene content, the DNA sequences coding for RNAs and proteins have been succesfully used to identify homologous sequences in unrelated taxa. Little is known about the mitochondrial genomes in basidiomycetes. Partial genetic maps have been constructed for Agaricus brunnescens (Hintz et al. 1988a), two species of Coprinus (Weber et al. 1986), and five species of Suillus (Bruns et al. 1988). One mitochondrial gene, the CO III gene from Schizophyllum commune, has been sequenced (Phelps et al. 1988). In order to characterize more fully the mitochondrial genome of S. commune we located nine mitochondrial genes on a restriction map of the mtDNA from one strain. The mtDNAs isolated from different strains of S. commune exhibit RFLP's (Specht et al. 1983). We have used these as genetic markers to examine the mode of inheritance of mitochondria in S. commune. Materials and methods
Strains. S. commune homokaryoticstrain UVM 4-40 and dikaryotic strains derived from mating UVM 4-40 with homokaryoticstrains UVM 1-50, UVM 1-54, and UVM 1-106were used in this study.E. coli strain HB101 was the host for plasmid DNA.
130
Isolation ofdikaryotic cells. Inocula from strains to be mated were placed 1 cm apart on complete yeast extract agar medium (CYM, Raper and Hoffman 1974). Cells were incubated at 30~ until fruiting body primordia were visible (4-5 days). Pieces of dikaryotic mycelium (2 mm 3) were removed from both sides of the mating at points furthest from the line of mycelial fusion and cultured for D N A extraction.
gelling. Agarose was not removed for digestion with restriction enzymes or for Southern hybridizations. Incorporation of 3zp ranged from 0.4 to 14.0x106 cpm/fragment. Schizophyllum m t D N A and cloned ribosomal D N A (Specht et al. 1984) isolated from strain UVM 4-40 were each labelled with e.32p dCTP by nick translation (Mackey et al. 1977). The specific activity of the labelled D N A was approximately 10 s cpm/Ixg.
Growth of cells for DNA extraction. Schizophyllum mycelia were grown in liquid CYM and E. coli cells in yeast broth as previously described (Mufioz-Rivas et al. 1986).
Electrophoretic analysis of restriction digests. Restriction digests of Sehizophyllum m t D N A were analyzed by standard methods to determine the size of fragments to be mapped. Electrophoresis was in 1% or 2% (w/v) agarose gels buffered with TAE. Samples o f a 1 kb ladder (Bethesda Research Labs, Gaithersburg, MD, USA) were used as size standards. When restriction digests of labelled m t D N A were analyzed the standard was added to each sample. Standard bands were marked after staining; gels were dried and exposed to X-ray film.
Isolation of DNA. Whole cell D N A from S. commune was purified and used for the isolation of m t D N A by two rounds of centrifugation on CsCl-bisbenzimide gradients (Specht et al. 1983). Minipreparations of plasmid D N A were purified from E. coli by a rapid boil method (Holmes and Quigley 1981). Large preparations of plasmid D N A were purified on CsCl-ethidium bromide gradients, essentially as described by Clewell (1972). They were used for restriction mapping and preparing hybridization probes. Cloning Schizophyllum mtDNA. EeoRI and HindlII digests o f m t D NA from strain UVM 4-40 were ligated to the plasmid pYIP5 (Botstein et al. 1979). BglII and XbaI m t D N A digests were ligated to a derivative of pYIP5 that has unique BgIII and XbaI restriction sites. Cloning procedures were as described by Maniatis et al. (1982). Labelling DNA. Restriction fragments from directly isolated or cloned Schizophyllum m t D N A were labelled with c~-32PdCTP by T4 polymerase (Maniatis et al. 1982). Labelled fragments were separated by electrophoresis in SeaPlaque agarose (FMC Corp. Rockland, ME, USA) and Tris-Acetate EDTA buffer (TAE, Maniatis et al. 1982). Gels were stained with ethidium bromide in TAE and illuminated with 366 nm of UV light. Bands of D N A were excised and suspended in 10 m M Tris-HC1, 1 mM EDTA, pH 8.0 (TE), 50 m M NaC1 to a final volume of 0.5 ml. Samples were heated at 70 ~ to melt the agarose and maintained at 37~ to keep the agarose from
Southern hybridizations. Schizophyllum m t D N A (0.5 gg) was restricted with either of five enzymes: AvaII, BglII, EeoRI, HindIII, and XbaI. Restricted D N A was electrophoresed and transferred to Zeta Probe membranes (BioRad, Richmond, CA, USA) according to Southern (1975). Restriction fragments of Schizophyllum m t D N A were labelled and individually hybridized to the Southern blots. Hybridizations were at 65~ in 6x SSC containing 5x Denhardt's, 0.1% (w/v) SDS, l x TE abbreviated as HB. Rinses were at 65~ in 2x SSC containing 0.1% SDS, 0.1% disodium pyrophosphate, 0.5x TE abbreviated as RB. Labelled restriction fragments of m t D N A from other fungi (Table 1) were used as probes in Southern hybridizations. Whole cell D N A (1 Isg) or m t D N A (0.5 gg) from Schizophyllum was digested with AvaII, BglII, EcoRI, HindIII, or XbaI, electrophoresed and blotted as above. Hybridization conditions were 6x or 10x SSC HB, 60~ Filters were washed successively in 2x, 1 x, 0.5x, and 0.25 x SSC RB at 60 ~ with exposure to X-ray film after each rinse. Schizophyllum m t D N A and ribosomal D N A labelled by nick translation were used in Southern hybridizations to probe whole cell D N A (1.0 p.g) prepared from dikaryotic mycelia. Hybridizations were in 6 x SSC HB, 65~ and rinse conditions were 2x SSC RB, 65~
Table 1. Hybridization probes for gene mapping Gene
Organism
Plasmid
CO I
N. crassa
pUCB3
CO II
N. crassa
pUCB4
Probe a P
Pv
P
P
P
P
~
Reference Pv P
I
,i
P
Burger et al. 1982 Macino and Morelli 1983 Browning and RajBhandary 1982
CO III
N. crassa
pBP582
Cyt b
N. crassa
pJBP5b
ATPase 6
N. crassa
pUCB7b
ATPase 6
S. c e r e v i s i a e
pSC40ME6
ATPase 8
S. c e r e v i s i a e
pSC40ME62
E
x
Novitski et al. 1984
ATPase 9
N. crassa
pUCB4
~
J
van den Boogaart et al. 1982
25s r R N A
N. crassa
pHRB1
Heckman and RajBhandary 1979
17s r R N A
N. crassa
pMB3
Heckman and RajBhandary 1979; Taylor and Smolich 1985
ATPase 9 (nuclear)
A. nidulans
pMWll
t~
Burke et al. 1984 Morelli and Macino 1984
~=ff
Novitski et al. 1984
x~s
Ward et al. 1986
Each D N A fragment is shown 5' to 3' with shaded boxes indicating coding regions and open boxes, introns. Probes were made using restriction fragments generated by the indicated restriction enzymes: P, PstI; Pv, PvulI; H, HindlII; X, XbaI; E, EcoRI; B, BamHI; S, ScaI
a
131
Results
EcoRI, HindIII, KpnI, and XbaI) were used for mapping; five, which gave 10-15 restriction sites each, are shown in Fig. 1. Cloned and uncloned restriction fragments were used to establish a restriction map (Fig. 2). Attempts to clone each EcoRI and HindIII fragment did not succeed (data not shown). Cloned versions of EcoRI fragment 1 (El) and EcoRI fragment 2 (E2) were always smaller than their uncloned counterparts and plasmids with HindIII fragment 3 (H3) were not recovered. This result prompted the cloning of BglII and XbaI fragment to more completely represent the mitochondrial genome. Eighty-one percent of the mitochondrial genome has been isolated as cloned fragments: BgIII fragment 4a (Bg4a) and Bg6-9; E4-12; H10, H7, H6a, H4, H2; XbaI fragment 10 (XI0), X8, X7, X5. Each cloned fragment was examined for the inclusion of restriction sites for the other enzymes of the set (i.e., AvaII, BglII, EcoRI, HindIII, and XbaI). Because AvaII produced many restriction fragments from the vector alone, when Avail was used as the second enzyme, vector D N A was digested with both enzymes and electrophoresed in an adjacent lane in order to aid in identifying m t D N A fragment(s). Each plasmid was also labelled and hybridized to Southern blots of restriction fragments of m t D N A . Ninety-four percent of the genome was also mapped for restriction sites using uncloned restriction fragments. AvaII fragments 4 - 1 4 (A4-14), B g 6 - 9 , E 4 - 1 2 , H7 - 10, and X2 11 (Fig. 1) were labelled and isolated. Aliquots of each sample were restricted and electrophoresed to map restriction sites within each fragment. Incomplete digestion, which often occurred, aided the ordering of fragments. Labelled, uncloned fragments were also used as probes for Southern hybridizations. Data from the analysis of cloned and uncloned mtDN A provided sufficient information to establish and confirm the order of restriction fragments. The XbaI digest was chosen as the standard because there were no doublets and the largest fragment was about 10 kb (Fig. 1). A first approximation for the order of fragments was based on hybridization data. The precise order was established from restriction mapping of cloned and uncloned fragments. Two unanticipated problems arose. First, gaps in the map made it evident that small AvaII and XbaI fragments (i.e., A I 2 - 1 4 and X11) were being missed. These fragments are not visible in Fig. 1. Each fragment, how-
Mapping restriction sites M t D N A from S. commune strain 4-40 was chosen because of molecular and genetic studies previously conducted with this strain and the ease with which D N A could be isolated. For every 500 gg of whole-cell D N A prepared by our methods 15-25 gg o f m t D N A was readily purified by centrifugation in CsCl-bisbenzimide gradients. Seven restriction enzymes (AvaII, BamHI, BglII,
Ava
II
Bgl
II
E c o RI
Hind
III
Xba
I
--10180
-5090 --4072 -3054
-2036
- 1018
-511
-210 ]
-148 - 75
Fig. 1. Restriction digests of S. commune strain 4-40 mtDNA. Each lane contains 0.5 p~g of mtDNA electrophoresed into 1.0% (w/v) agarose and stained with ethidium bromide. Each fragment is identified by a letter/number code including fragments not visible under these conditions. Bars at right indicate standards with lengths in bp
11a
1
Aya.
,
BamHI Bgllls EcoR/
H,n~,.. Kpn, xba,[
4a a
I H
5
4b
,os
/8
I.I
2
I.I
2 4
, 4
10
I ol51
i
]
13
3 6
181 , I
3
3
I
'
1 2
5
I ~ J~,o i61
I-~
I-----1
CO-Ig
I.rRNA
11t
If ~
5
7 i
2
' ' I 2 I'1 ~ It''~
' []
9tl716
1
1
~
s-rRNA ATP.6 (kbp)
[]
12t
I
3
; 1'
I
]
~
CO-[ ATP.8 ATP.9 CO.]I
9 3
'
~
I r--] Cyt-b
Fig. 2. Restriction map and gene map of S. commune strain 4-40 mtDNA. Restriction sites are indicated by vertical bars. The box above each gene shows the smallest region hybridized with each probe. The arrow in the CO III box indicates the direction of transcription of the CO III gene (from Phelps et al. 1988)
132 ever, was visible when electrophoresing 2 l.tg of restricted m t D N A in 2.0% (w/v) agarose gels (data not shown). Second, two AvalI sites were cut by the enzyme using uncloned m t D N A , but not recognized within the cloned fragments X8 and H2; the inability to restrict these sites m a y be due to methylation by the dcm methlyase in E. coli strain HB101. The order of restriction sites is given in Table 2 and depicted in Fig. 2. There was no evidence of duplicated D N A . The mitochondrial genome of strain 4-40 m t D N A is circular and 49.75 kb in size.
Mapping genes The restriction m a p made possible the approximate positioning of mitochondrial genes. Genes cloned from other fungi were used to identify homologous sequences, as shown in Table 3. The a m o u n t of homology between each probe and Schizophyllum D N A was estimated by repeatedly rinsing each filter at 60 ~ in sequentially lower concentrations of SSC RB until the probe was removed. Probes melted from Schizophyllum D N A within concentrations of 0 . 2 5 - 1 . 0 x SSC RB. O f the probes tested only the ATPase 6 gene probe from N. crassa did not hybridize (10x SSC HB, 55 ~ to SchizophylIum m t D N A . However, the yeast probe encoding ATPase 6 did (10x SSC HB, 60 ~ Each of the other probes hybridized to a unique set of restriction fragments from the m t D N A of Schizophyllum and the order of genes was resolved, as shown in Fig. 2. N. crassa and A. nidulans each have two ATPase 9 genes. The gene that encodes a functional protein is found in the nuclear genome of each organism and the pseudogene version in the mitochondrial genomes. The Neurospora pseudogene hybridized only to Schizophyllum m t D N A and the Aspergillus nuclear gene hybridized only to Schizophyllum nuclear D N A . Probes for the Neurospora functional gene and Aspergillus pseudogene were not available to us. The Sehizophyllum CO III gene was m a p p e d using a Neurospora probe, and subsequently it was sequenced (Phelps et al. 1988). The direction of transcription for the CO I I I gene (see Fig. 2) is known from the D N A sequence. Two overlapping restriction fragments containing portions of the Neurospora CO I gene were used as probes; one contained the entire gene and the other only the 3' end. The different AvaII fragments that hybridized (see Table 3 and Fig. 2) indicate that transcription of CO I is also in the same direction as the CO III gene.
Inheritance of mitochondrial DNA We indirectly examined the migration of mitochondria during dikaryosis to determine the type of inheritance that could be expected o f m t D N A . Strains known to have R F L P s in both nuclear and mitochondrial D N A were mated. Three different pairwise combinations of strains were made and dikaryotic cells were subsequently sampied from the mycelium which grew at each side of each mating. Nuclear D N A from each strain was identified by
Table 2. Mapping restriction sites of
S. commune strain 4 40
mitochrondrial DNA
Avail
BamHI BgllI
12.400 a 9 . 7 8 0 1 . 7 0 0 14.675 18.830 6 . 1 0 0 15.615 6.710 17.520 10.290 17.760 14.750 17.900 19.385 18.385 28.400 18.870 47.205 19.760 47.730 19.845 49.215 24.245 25.705 34.575 35.500 43.635
EcoRI
HindIII
KpnI
XbaI
6.210 7.490 9.165 11.940 14.425 30.150 31.125 33.650 35.940 46.020 47.060 48.565
1 . 0 2 5 18.680 0.000b 12.650 41.220 5.475 14.875 9.115 19.360 17.090 23.520 19.030 24.330 20.020 28.500 30.050 30.950 31.725 36.820 35.005 42.970 35.135 49.600 40.270 49.750
" Units are kilobase pairs b 0.000 was arbitrarily chosen to begin at one end of fragment X4
Table 3. Mapping genes to
S. commune strain 4-40 mitochondrial
DNA Hybridization probe
CO CO CO CO
I (3.0 PstI) I (1.85 PvuII) II III
Restriction fragments of Schizophyllum mitochondrial DNA that hybridized a A2; A2, A3; A1;
Bgl; E7; H4; X7 A9; Bgl; E7; H4; X7 Bgl; E2; H3; X2 Bg4b; E4; HI; X3
Cyt b
AI; Bg7, Bg9; E9; H2; X2
ATPase 6 (N. crassa) ATPase 6 (S. cerevisiae)
No hybridization A2; Bg2; El; H6b; XI
ATPase 8 ATPase 9 (mitochondrial, N. crassa) 25s rRNA 17s rRNA ATPase 9 (nuclear, A. nidulans)
A3; Bgl; E2; H3; X5 A3; Bgl; E2; H3; X2, X5 A6, Alia, b; Bg3; El; H5; X8 A7; Bg2; El; HI0; X1 No hybridization to mtDNA b
" Fragments are labelled as in Figs. 1 and 2 b ATPase 9 (nuclear) hybridized to total Schizophyllum DNA. The fragment sizes that were detected were: AvaII, 0.75 kb, 0.55 kb; BgflI, 9.1 kb; EcoRI, 23 kb; HindIII, 9.4 kb, 7.8 kb; XbaI, 11.3 kb
characteristic R F L P s in ribosomal D N A . The ribosomal D N A and m t D N A restriction patterns obtained are shown in Fig. 3. Each dikaryon produced a ribosomal D N A pattern that is a composite representing the polymorphisms of the two haploid mates (Specht et al. 1984). This indicates that nuclear migration had occurred. The m t D N A patterns, however, showed no composites; each pattern is that of the respective h o m o k a r y o n prior to dikaryosis (Specht et al. 1983). We conclude that mitochondria do not migrate with their respective h o m o k a r y -
133 Discussion
Fig. 3A, B. Southern blot hybridizationsto determine inheritance of nuclear, ribosomal DNA and mtDNA. Strain numbers identify which side of each of three matings (indicatedby an "x") was the source of genomicDNA in eachlane. Nuclear, ribosomalDNA was detectedin EeoRI restrictiondigests (A) with labelled nuclear, ribosomal DNA. Arrows in A indicate the positions of ribosomal DNA fragments found in respectivehomokaryoticstrains before mating. Mitochondrial DNA was detectedin HindIII restriction digests (B) with labelled mtDNA. Arrows in B indicate the positions of mtDNA fragmentsfound for strains 1-50, 1-54, and 1-106, respectively, prior to mating.The positionof fragmentsfor strain 4-40 before and after matingis indenticalto the HindIII digestin Fig. 1. HindIII fragmentsof lambdaDNA are indicatedby triangleswithrespective sizes in kbp
otic nuclei during dikaryosis and, therefore, S. commune will exhibit uniparental inheritance of its mtDNA. DNA prepared from spores derived from the dikaryotic mycelium have confirmed this (data not shown).
A restriction map was determined for Schizophyllum strain UVM 4-40. Based on these data we conclude that the mitochondrial genome of S. commune is moderate in size when compared to other fungi. Its mtDNA is circular and 49.75 kbp in length. No regions of repeated DNA were detected during the process of ordering restriction fragments by Southern hybridizations. Nine genes commonly found in the mtDNA of fungi were positioned on this restriction map. The use of DNA probes from other fungi to map genes to Schizophyllum mtDNA was supported by results with the CO III gene. The Neurospora CO III probe identified a unique EcoRIBglII fragment that was subsequently sequenced (Phelps et al. 1988). One ORF translates into a protein with 51% homology to the Neurospora CO III protein; 72 amino acids are also identical with CO III genes from other organisms. The DNA homology between Schizophyllum and Neurospora in the CO III translated region is 59% including 97 bp 5' of ATG, 807 bp of translated DNA, and 4 bp 3' of TAA. The Neurospora CO III probe was removed from membrane-bound Schizophyllum DNA following Southern hybridizations between 0.5-1.0x SSC RB, 60 ~ Each of the other probes that were used for gene mapping hybridized to a unique set of restriction fragments under the same conditions of stringency as the Neurospora CO III DNA. Therefore, it is likely that each heterologous probe identifies its Schizophyllum homologue. Results from Southern hybridizations indicate that S. commune, like A. nidulans and N. crassa, has a nuclear and mitochondrial homologue to ATPase 9. Qualitatively, (based on conditions of stringency used for rinses) both were equally homologous to their respective DNA probes and neither probe cross-hybridized to its nuclear or mitochondrial counterpart. Further analysis is needed to identify which of these regions encodes a functional gene. The CO I and the CO III genes are transcribed from the same DNA strand. This suggests that S. commune is similar to other fungi and uses principally one strand for transcription. The inheritance of mtDNA has been shown to be uniparental or biparental in various fungal species, and the mode of inheritance is unresolved when recombination occurs (reviewed in Taylor 1986). S. commune is like many other basidiomycetes, in that, when compatible homokaryotic mycelia are paired, their nuclei are reciprocaly exchanged in order to form sexually competent dikaryotic mycelia. Mitochondria do not co-migrate with nuclei in either Coprinus cinereus (Casselton and Condit 1972; May and Taylor 1988) or A. bitorquis (Hintz et al. 1988b). S. commune showed no evidence for mitochondria to migrate with nuclei if dikaryotic mycelium is sampled 3 4 cm distant from the zone of fusion. Each side of a mating contains mitochondria from one parent and nuclei from both parents. Our results place in perspective an earlier report (Watrud and Ellingboe 1973) that showed cobalt-stained mitochondria migrated short distances, but not long distances (e.g., 3 - 4 cm), into recipient royce-
134 lia following h y p h a l a n a s t o m o s i s o f c o m p a t i b l e h o m o k a r y o n s o f S. commune. Acknowledgements. We are grateful to Drs. J. M. Burke, J. W. Taylor, M. Ward, I. F. Connerton and C. Breitenberger for providing cloned mtDNA from other fungi and Dr. J. M. Burke for assistance with Southern blot hybridizations. This work was supported by the Vermont Agricultural Experiment Station, University of Vermont, Burlington, Vt., and grant DCB-8402107 of the National Science Foundation to RCU and CPN.
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