Mol Gen Genet (1993) 237:177-186

© Springer-Verlag 1993

Plasmid diversity in senescent and nonsenescent strains of Neurospora

Xiao Yang and Anthony J.F. Griffiths

Department of Botany, University of British Columbia, Vancouver, B.C., Canada V6T 1Z4

Received May 28, 1992 / Accepted August 18, 1992

Summary. A sample of 171 natural isolates of Neurospora crassa and Neurospora intermedia was tested for senes- cence. Of these, 28 strains senesced within the duration of the experiment. These senescent strains, together with a selection ofnonsenescent strains, were examined for the presence of mitochondrial plasmids. This was done by digesting mitochondrial DNA preparations with protein- ase K, and running these samples on agarose gels. Most of the strains examined, both senescent and nonsenes- cent, contained plasmids, many of them new. Some new plasmids were linear, as inferred from their resistance to 5' exonuclease and sensitivity to 3' exonuclease. New circular plasmids were also found. Some strains carry several plasmids, and mixtures of circular and linear elements were common. A cross-homology study was performed on a sample of plasmid-bearing strains, and several cases of apparent relatedness were found, some between strains from distant geographical locations. Lin- ear plasmids homologous to the maranhar linear senes- cence plasmid were quite common. A new member of the LaBelle circular plasmid homology group was found. In the sample tested for homology, no strains contained elements related to the kalilo linear senescence plasmid. The relationship of the new plasmids to senescence is not known. In addition to plasmid monomers, several dif- ferent types of derivatives were found. The kalilo linear plasmid was found to occur in linear and circular forms of low mobility, presumed to be giant concatamers, and, in some strains, variant sibling structures and ladders of short derivatives were found. Circular plasmids also gave rise to extensive ladders on electrophoresis, probably representing different relaxation states and head-to-tail concatameric series. Some such forms migrated more slowly than mitochondrial DNA. One unique type of plasmid modification observed was a pair of linear ele- ments that had apparently arisen de novo which showed homology to a circular plasmid.

Correspondence. A.J.F. Griffiths

Key words: Senescence - Linear plasmids - Circular plasmids - Neurospora - Mitochondria

Introduction

The reason for the existence of plasmids is still something of a mystery. Even in prokaryotes, where plasmids were first discovered, little is known about their origin and raison d'etre, although a great deal is known about their structure and function (Broda 1979). In eukaryotes plas- raids were discovered much later, but the number of examples from different eukaryotic organisms is now considerable (Esser et al. 1986; Meinhardt et al. 1990). However, here again the origin and persistence of these elements are not understood. Furthermore, the replica- tion cycles and expression systems of such plasmids are less clear than in prokaryotes.

The current view of eukaryotic plasmids has come from assembling examples from many different organ- isms, especially fungi. No extensive survey has been per- formed on one organism in order to obtain a picture of the incidence and diversity of extragenomic elements in natural populations of that species. The present work represents the beginning of such a survey for the fungus Neurospora. This is an appropriate organism.for such research because Neurospora isolates have been collected from all over the world, and are deposited at the Fungal Genetics Stock Center.

Our interest in plasmids stems from the discovery of senescence in natural Hawaiian isolates of Neurospora intermedia (Rieck et al. 1982; Griffiths and Bertrand 1984). Most laboratory and field-isolated strains of Neurospora are immortal in that they show continuous vegetative propagation for as long as the experiment lasts. However, about 30 % of isolates from the Hawaiian island of Kauai cease growth and die in a strain-specific span of time. It has been shown that senescence is asso- ciated with the presence of a mitochondrial linear plas- mid called kalilo DNA (kalDNA, Bertrand et al. 1985;

178

Myers et al. 1989). In juvenile strains, the kalilo plasmid can replicate to high copy number with little apparent detrimental effect on the host, but ultimately the plasmid inserts into mitochondrial DNA (mtDNA), an event that seems to be the beginning of the senescence process. At death, few wild-type mtDNA molecules can be detected; most contain inserts of kalilo DNA.

After this discovery, we began to examine Neurospora intermedia and Neurospora crassa isolates from around the world, in order to document the general incidence of senescence in natural populations. A second case of senescence was soon found in a Neurospora crassa popu- lation in India. This was also caused by a mitochondrial linear plasmid, called maranhar DNA (marDNA). The maranhar plasmid also inserts into mtDNA in a manner similar to kalilo, but the two plasmids share no homol- ogy at the DNA level (Court et al. 1991). These two plasmids were the first linear plasmids to be found in Neurospora. Such elements had been found previously in other fungi and in plants, but were not associated with senescence.

Several natural isolates of Neurospora have also been shown to contain circular mitochondrial plasmids (Col- lins et al. 1981; Stohl et al. 1982; Natvig et al. 1984; Taylor et al. 1985). Some of these elements show homol- ogy at the DNA level and this has been used to suggest evolutionary groupings (Natvig et al. 1984; Taylor et al. 1985). Generally these circular plasmids are not per- ceived as determinants of senescence, but abnormal growth has been observed in rare cases where circular plasmids have recombined with mtDNA (Akins et al. 1986, 1989). In the present report we describe the results of a larger survey spanning the globe, and show that there is a profusion of plasmids in Neurospora natural populations. In fact, strains often contain an assortment of different plasmids, some linear and some circular. These plasmids show a variety of interesting distribution patterns. All these plasmids are mitochondrial in their location. In addition, we have discovered that strains sometimes contain derivative forms of basic plasmid types found in the same cultures. Some plasmid-contain- ing strains are senescent and others show no symptoms of senescence within the time span of the experiments.

Materials and methods

Strains. The natural isolates that form the basis of this study were obtained from the Fungal Genetics Stock Center (F.G.S.C., Department of Microbiology, Univer- sity of Kansas Medical School, Kansas City, Kan.) or from Dr. David Perkins (Biological Sciences, Stanford University, Stanford, Calif.). Other strains were deriva- tives obtained in previous studies, and these will be re- ferenced in the Results section.

Culturing. In general, standard Neurospora protocols were used (Davis and DeSerres 1970). Procedures specific to the study of senescence were those described by Rieck et al. (1982), and by Griffiths and Bertrand (1984). The best technique for allowing senescence determinants to

express themselves is serial subculturing (Griffiths et al. 1986). Strains were subjected to 20 serial subcultures before being provisionally classified as nonsenescent.

The sequence of experiments was as follows. A total of 51 strains of N. crassa, and 120 strains of N. intermedia was obtained, representing collections from North and South America, Africa, Asia, and Australia. These were all were serially subcultured. Several strains were found to be senescent. (The inheritance patterns of these senes- cent strains will be reported elsewhere.) The senescent strains, and a random selection of nonsenescent strains, were then analyzed for plasmid content.

DNA analysis. DNA isolation protocols have been de- scribed by Myers et al. (1989). The radioactive labelling procedures were those used by Griffiths et al. (1992). Exonuclease treatments were based on those of Vierula et al. (1990). The kalilo-specific probes were various restriction fragments cloned in either pUC18 or 19. These clones are described by Vickery (1991). All other ele- ments used as probes were isolated from low melting point agarose gels. General DNA protocols were stan- dard, and based on those in Maniatis et al. (1982). The l kb ladder used was from BRL (Gaithersburg, Md.).

Results

New plasmids from nature

A total of 28 strains were found to be senescent, out of the total of 171 surveyed. Two of the strains were from Hawaiian islands other than Kauai (the source of the original kalilo strains). These two proved to be typical kalilo strains bearing the kalilo plasmid, showing for the first time that the kalilo element is present on other Hawaiian islands. Three strains were from Aarey, India, the population from which the original maranhar strain was identified, and these all proved to be typical maran- har strains. The other senescent strains were of more interest because they represented new locales for the occurrence of senescence.

These remaining senescent strains were all subjected to plasmid analysis together with some nonsenescent strains included for comparison. The first procedure was to electrophorese uncut mitochondrial DNA prepara- tions treated with proteinase K. Linear plasmids gener- ally show up as bright bands after staining with ethidium bromide. Virtually every characterized linear eukaryotic plasmid has a protein covalently bound at each 5 ~ ter- minus (Meinhardt 1990), so linearity of plasmids was confirmed by checking for 5' exonuclease resistance and 3' exonuclease sensitivity. Circular plasmids do not al- ways show up in uncut ethidium bromide stained preparations. Classification as a circular plasmid was based on extensive restriction analysis, on hybridization analysis, and on resistance to both 3' and 5' exonucleases.

The results are summarized in Table 1. In this Table uncharacterized linear plasmids are listed according to molecular weight estimated from a 1 kb calibration lad- der. The two characterized linear plasmids kalilo and

179

Table 1. Plasmid content of a sample of senescent and nonsenescent natural isolates of Neurospora

Strain No. Plasmids a N/S b Origin

Neurospora

P10 P27 P277

P361 P362 P370 P901 P3785 P3789 1809 1826 1827 1830 1833

1881 1940 2557 2562 3336

3976 3977 3978 3979 3980 3981 3983

3984 3985 3986 3991 3992 4853

5014

Neurospora

74 ORA 74 ORa 2499 4717 4719 4720

intermedia

2.8, Har-1 N 8.6", 4.0 Har-1 N

S

Har-1 S 9.0, 3.9, Har-1, Har-2 S

S S

8:8, Har-1, Har-2 S Har-1 S Han-2, Har-2, Var, VS S 9.0, 3.9, 2.0, Har-1 S 9.0, 3.9, Har-1 S 4.0 S 4.0 S

9.0, 3.9, Har-1 S 7.0", Lab S 7.2, C S C c S 7.0", Har-1 S

4.0, Har-1 N 9.0, 4.0, Har-l S 9.0, Har-1 N Har-1 N 9.0, 4.0, Har-1 S Har- 1 N 9.0*, 8.0*, 7.0*, 4.0, S 1.4, Har-1, Har-2 9.0, Har-1 Har-1 7.4, 7.2*, 7.0*, Har-1

H ar - 1 7.0*

kal, Han-2

crassa

mar*, C Har-1

Har-1

Unzen, Japan Manila, Philipines Singapore, Malaysia Bogor, Java Bogor, Java Tjisarua, Java Bogor, Java Djoue, Congo Djoue, Congo Kurubara, India Besakih, Indonesia Besakih, Indonesia Cairns Australia Townsville, Australia Jakarta, Indonesia LaBelle, USA Jakarta, Indonesia Babakan, Indonesia Monte Alegre, Brazil Beijing, China Beijing, China Hangzhou, China Changdu, China Hefei, China Jinan, China Harbin, China

N Taiyan, China N Guiyang, China N Shengyang, China N Baoding, China N Changdu, China S Wan, Papua

New Guinea S Hanalei, Hawaii

N Oak Ridge N Oak Ridge S Aarey, India N Madurai, India N Mallilinathan, India N Vallancheri, India

" New linear plasmids are listed by size in kb; previously charac- terized linear plasmids are kal (kalilo) and mar (maranhar); aster- isks show maranhar homology. New mapped circular plasmids are Har-1, Har-2 (Harbin), and Han-2 (Hanalei); C is unmapped; previously characterized circular plasmids are Lab (LaBelle), Var and VS (Varkud) b N, nonsenescent; S, senescent c Homologous to, but different from, Harbin-1

maranhar are designated kal and mar, respectively. As- terisks denote hybridization with a probe consisting of the entire maranhar plasmid. Uncharacterized circular plasmids are indicated by the letter C, and characterized circular plasmids are listed as Han-2 (from Hanalei), Har-1, Har-2, (from Harbin), Lab (from LaBelle) and

Var and VS (from Varkud). The supporting data for designations will be shown later. Several points are note- worthy. First, most strains, both senescent and nonsenes- cent, contain plasmids. Second, both senescent and non- senescent strains can contain linear and circular plas- mids. Third, many strains contain several different plas- raids. Such assemblages of plasmids have not been re- ported previously. Fourth, some senescent strains have no detectable plasmids.

Electrophoresis of uncut DNAs from a sample of 19 strains is shown in Fig. 1A, together with a 1 kb ladder for size calibration. The positions of m t D N A (d), ka lDNA (k), m a r D N A (m), and an unusual new linear plasmid to be discussed later (1) are labeled. Other new bands are not labeled. Some strains, such as those in lanes 19 and 20, have no visible bands, but it is clear that most strains have one or more linear plasmids visible as strong bands, and that most show indications of circular plasmids, visible as faint bands. Most strains show bands that migrate more slowly than the m t D N A and these are investigated in experiments described below.

Cross homology to k a l D N A has been tested by hy- bridizing Southern transfers of the gel with a mixture of cloned kalilo fragments E, G, and X3, which span most of the plasmid. Cross homology to other plasmids was tested using gel-extracted plasmids as probes. The results are also shown in Fig. 1, panels B-F. The hybridization of the ka lDNA probe is shown in Fig. lB. Of all the Neurospora strains tested, only the single kalilo control strain showed hybridization, and this was to the k a l D N A band itself, as expected. Interestingly, several kalilo-sized 9 kb bands proved not to be related to kalilo. Therefore, the kalilo element is apparently not geographically wide- spread.

The maranhar probe showed several cases of cross- homology to a range of linear plasmids with molecular weights similar to, and different from, the original mar- anhar plasmid (Fig. 1C). The maranhar plasmid was originally found in Neurospora crassa, but the new hy- bridizations all involve Neurospora intermedia, f rom New Guinea (lane 4), the USA (lane 9), Brazil (lane 10), and China (lanes 14 and 16). The USA strain in lane 9 is the LaBelle, Louisiana, strain f rom which the well-charac- terized circular LaBelle plasmid originates (Stohl et al. 1982; Schulte and Lambowitz 1991; Nargang et al. 1992); however these previous studies did not detect the linear plasmid homologous to maranhar . Evidently the maranhar element or parts of it are found in both these

Fig. IA-F. The plasmids of 19 senescent and nonsenescent Neuro- spora strains. A Ethidium bromide-stained gel of uncut DNA from mitochondria. Lane 1, Pl0; 2, 1830; 3, P362; 4, 4853; 5, P3785; 6, 1809; 7, 1827; 8, 1881; 9, 1940; 10, 3336; 11, 1 kb marker ladder; 12, 3977; 13, 3980; 14, 3983; 15, 3983 (variant subculture); 16, 3986; 17, 5014; 18, 2499; 19, 4717; 20, 4720. Arrowheads: d, mtDNA; k, kalilo DNA; m, maranhar DNA; 1, Harbin-L plasmid. B Blot of A probed with a mixture of cloned kalDNA restriction fragments. C Blot of A probed with marDNA. D Blot of A probed with Harbin-1 circular plasmid. E Blot of A probed with LaBelle circular plasmid. F Blot of A probed with Hanalei-2 circular plas- mid. See Table 1 for details of strains used

1 2 3 4 5 6 7 8 91011121314151617181920 1 2

D

kl~

ml~

II,

3 4 5 6 7 8 91011121314151617181920 A

dl, ,d

,k 4m

,41

E

d=,

k=.

I t

¢

17

B

4d

4k

41

4

dr

mD.

9 10 14 16 18 17

F

, d

4 m

Fig. 1A-F. (for legend, see page 179)

E5 K E5

P

K

X

Fig. 2. Restriction maps of the new circular plasmids found in this study

181

H E1 ES B p

Neurospora species and in diverse geographical loca- tions. In all five cross-hybridizations there is one band of a size roughly comparable to maranhar DNA itself. It is not known whether this is the same basic structure or if this size equivalence is a coincidence. One strain from Harbin, China, showed three linear plasmids with ho- mology to maranhar. In a different subculture of this same strain (lane 15) all these plasmids were no longer detectable. The weak background radioactivity in panel C results from contamination of the maranhar plasmid during excision of the gel-isolated probe. In this connec- tion it should be noted that the faint signal at the position of kalilo DNA in lane 17 is an artifact not found in other maranhar-to-kalilo hybridizations (for example Fig. 1 B); in fact no sequence homology exists between kalilo and maranhar DNAs (Court et al. 1992).

We have analysed a circular plasmid that we originally isolated from strain 3983 from Harbin, China, hence- forth referred to as the Harbin-1 (Har-1) plasmid. The restriction map of this plasmid is shown in Fig. 2. After linearization by restriction enzyme cleavage, this plasmid was used as probe in a Southern blot of the gel in Fig. 1A. The hybridization in Fig. 1D reveals some interesting findings. First, this circular plasmid exists in a multitude of different forms. We have not specifically tested the nature of these forms, but the ladders probably represent different degrees of DNA relaxation. The different lad- ders could represent concatamers of the basic unit. Plas- mid ladders have been reported before in Neurospora (Collins et al. 1981; Taylor et al. 1985; Collins and Saville 1990), but to the best of our knowledge these are the most extensive published arrays derived from a single plasmid. Treatment of the array with a restriction enzyme having one target site in the basic unit reduces the entire array to one intense band (data not shown).

The second important finding from this gel is that Har-1 sequences are clearly widespread in natural pop- ulations ofN. intermedia and N. crassa (see also Table 1). They were found in 12 out of the 19 strains shown on the gel in Fig. 1A and in 24 strains overall. The strains in lanes 19 and 20 showed no evidence of plasmids on the ethidium bromide-stained gel (Fig. 1A) but hybridization shows that they do contain circular plasmids homolo-

gous to Har-1. Restriction analysis (not shown) has dem- onstrated that all sequences hybridizing to Har-1 give identical fragments to Har-1 and are therefore derived from equivalent structures. Note that the Har-1 plasmid hybridizes to circular plasmid ladders in the LaBelle strain (lane 9). A third important point that emerges from the data in Fig. 1D is that the Har-1 plasmid probe hybridizes to two linear plasmids in the Harbin strain subculture that had lost the three prominent maranhar- related bands and the Har-1 ladders (lane 15). These elements will be discussed below in the section on plas- mid derivatives. (Only the more prominent one is marked (1) in Fig. 1D.)

The LaBelle circular plasmid, extensively studied by others (Stohl et al. 1982; Schulte and Lambowitz 1991; Nargang et al. 1992), was also used as a probe. The hybridization patterns produced were virtually identical to those of the Har-1 plasmid (Fig. 1E). Therefore Har- bin-1 and Labelle are clearly closely related. However, it should be noted that there are differences (compare Fig. 2 with Stohl et al. 1982); for example Labelle has an EcoR 1 site whereas Har-1 has none, and Labelle has one Pstl site whereas Har-1 has two. Furthermore, we have not detected any homology of the Har-1 plasmid with mtDNA, as has been found for Labelle (Nargang et al. 1992). (Notice that the hybridization at the d position is not to mtDNA, as demonstrated by the unlabelled lanes.)

The final hybridization in Fig. 1 uses as probe a circular plasmid originally identified in a Neurospora intermedia kalilo strain 5014 (P561) from Hanalei, Kauai (Bertrand et al. 1985). This will be referred to as the Hanalei-2 plasmid, because it is not the first plasmid reported from that location (Taylor et al. 1985). The hybridization pattern is shown in Fig. 1F. Like the other circular plasmids, an extensive array of presumptive con- catamers and relaxation states is seen. Only one other strain contains a homologous plasmid, Neurospora inter- media strain 1809 from India, which shows an identical hybridization pattern (lane 6). Restriction analysis showed an identical structure in each. The restriction map of Hanalei-2 is shown in Fig. 2. The Hanalei-2 plasmid is approximately the same size as the Hanalei plasmid originally isolated from Neurospora tetrasperma

182

(Taylor et al. 1985), but there is only a limited match of restriction sites. We have not performed cross-hybridiza- tions between the two.

The Varkud and VS plasmids had previously been documented in strain 1809 (Collins and Saville 1990). We have detected and confirmed the identity of the Varkud plasmid by restriction mapping (not shown), but we have not detected the VS element. In hybridizations to the panel of strains in Fig. 1, the Varkud plasmid binds only to itself (not shown). Strain 1809 contains a total of four plasmids, two of which were previously undetected. One of the new plasmids in this strain, Harbin-2, was initially detected in the Harbin strain 3983. The plasmid in strain 3983 was mapped (Fig. 2) and used as a probe. It is a 4.9 kb element that did not hybridize to the Harbin-l, Hanalei-2 or Varkud plasmids, but did detect homolo- gous elements in three other strains (Table 1 ; hybridiza- tions not shown). Subsequent analysis showed the same restriction fragments in all four.

PIasmid derivatives

The ladders of presumptive concatamers and relaxation states of the circular plasmids represent one type of derivative. However, several previously undescribed additional derivative types were detected and these were investigated because of their possible importance in plas- mid evolution.

Kalilo strains. Short derivatives of kalDNA were first observed in a subculture of N. crassa kalilo strain BC3-3. This strain was derived from an ascus that was segregat- ing for a senescence-suppressing nuclear allele (Griffiths et al. 1992) and the strain itself contained the suppressor. The ethidium bromide-stained gel in Fig. 3 (lanes 5 and 6) shows a conspicuous set of shorter linear elements in this strain ranging in size from 6.5 to 0.5 kb. This gel was accidentally destroyed, but a blot of a similar gel prepared from these strains and probed with a cloned restriction fragment X3 from the terminal repeat from kalDNA, showed the homology of the novel bands to the kalilo element (lanes 8 and 9). These same bands do not hybridize to mtDNA, or to Hanalei-2, a circular plasmid present in this strain (hybridizations not shown). The sister ascospore BC3-2, which bore the same nuclear genotype and equivalent amounts of kalDNA, did not show the array of short derivatives (Fig. 3, lanes 1 and 2), so some random event must be involved in their synthesis.

The short fragments are resistant to 5' nuclease attack (lanes 6 and 9), but are degraded by 3' exonuclease (lanes 7 and 10). This suggests that these smaller bands rep- resent linear plasmid derivatives with occluded 5' ends (Vierula et al. 1990 showed that proteinase K does not remove all amino acids from the blocked 5' ends). No positive control for the action of the 5' exonuclease is included in this figure. However the action of this sample of nuclease is demonstrated in a later figure. The short derivatives have been investigated further by probing with cloned restriction fragments representing the entire

length of the kalilo plasmid, and these results are shown in Fig. 4. The probes containing terminal kalilo restric- tion fragments hybridize to more small plasmids than probes containing internal fragments. In particular the very low molecular weight elements hybridize only to the terminal probes.

The action of the senescence suppressor allele ulti- mately reduces kalilo elements to very low levels, so these extra bands were ephemeral. Furthermore, new subcul- tures from the same ascospore culture sometimes showed a different pattern of smaller bands suggesting a state of flux in this strain. Small derivatives have also been ob- served in other strains not containing suppressors, but never at such high levels as in this strain.

Different subcultures of the same suppressor strains illustrate another type of variant of the kalilo plasmid, which will be referred to as sibling plasmids. These are apparently higher molecular weight variants that have slightly slower electrophoretic mobilities. Examples are shown in lanes 1 and 2 of Fig. 5. Notice that the siblings can apparently replace the regular kalilo band.

Figs. 3 and 5 show that there are several prominent kalilo-related bands that have migrated to the region of the gel between the kalilo plasmid and the well. Figure 3, lanes 11 to 13 are overexposed to demonstrate these bands more clearly. The exonuclease digestions in Fig. 3, lanes 9, 10, 12 and 13, show that three of these elements are most likely linear, and one is circular (labelled L and C). These apparently giant plasmids are regularly ob- served in kalilo strains, generally in low copy number, and are generally detectable only by Southern hybridiza- tion. They most probably do not represent hybrids of kalDNA and mtDNA. The evidence for this is as follows. Firstly, they are not visualized by probes consisting of whole labelled mtDNA from non-kalilo strains. Second- ly, they can be found in kalilo strains in which there is no detectable insertion of kalDNA into mtDNA, and, thirdly, they are not altered by PstI (data not shown), an enzyme that does not cut within kalDNA (see restriction map in Fig. 4). We have found that when giant kalilo elements are digested with enzymes that cut at positions within kalDNA, and probed with the kalilo terminal repeat, autoradiograms show bands that most probably represent rearranged fragments derived from the slowly migrating forms. An example of such an analysis is shown in Fig. 6, which uses a different kalilo strain from those in Figs. 3-5 that contains no apparent inserts in the mtDNA. Lanes 1 and 8 show the undigested kalilo sam- ple; no giant bands are visible as they are prominent only when larger amounts of DNA are loaded (compare with Fig. 3). Lanes 3 to 6 and 10 to 13 show fragments produced by different enzymes. Several lanes show unex- pected bands that cannot be derived from head-to-tail concatamers. For example digestion with HindIII (lanes 4 and 11), which cleaves kalDNA once, produces two intense bands of approximately 7.0 and 1.6 kb corre- sponding to the fragments of the free plasmid, but in addition two faint bands appear above the 1 kb ladder and these are too large to be kalilo-sized pieces liberated from a tandem array by a single cleavage. In another example, BgllI digestion (lanes 5 and 12) reveals the two

1 2 3 4 5 6 7 8 9 10 11 12 13

4L

B3 B4 E X2b G X3 C

k~ S~ S~

2 3

183

4

• 3.5D

Fig. 3. Demonstration of short and giant kalilo derivatives Arrow- heads. d, mtDNA; k, kalDNA; L, linear giant derivatives; C, circular giant derivative; dots indicate examples of short linear derivatives. Lanes 1-7, ethidium bromide-stained gel of uncut DNA from mitochondria; lanes 8 13, blot of a similar gel probed with cloned kalilo fragment X3 (lanes 11-13 are overexposed to show certain bands more clearly). Lanes 5-13 are from the strain BC3-3, and lanes 1-3 are from its ascus sister spore BC3-2. Lanes 1, 5, 8, 11, undigested DNA; lanes 2, 6, 9, 12, digestion with 5' exonuclease; lanes 3, 7, 10, 13 digestion with 3' exonuclease

Fig. 4. Investigation of the structure of short kalilo derivatives. Cloned kalilo restriction segments (indicated in the maps in the

X3 Xl X2b J X2a J XbaI C G E J C EcoRl

E1 I E3 E2 EcoRV B2 [ B/, I B3 I a l Bg[]I

kl J k2 Hindlll PstI

~ l kb ~

lower panel) were used to probe the same blot of short derivatives. Each pair of lanes shows a short and a long exposure to optimize band visualization. The lower arrowhead indicates a 3.5 kb band referred to in the text. The arrows in the restriction map of kalDNA shown below indicate the terminal repeats

Fig. 5. Sibling plasmids of kalilo DNA. Uncut preparations were hybridized with a probe consisting of a cloned kalDNA inverted repeat (fragment X3, see Fig. 4). Lanes 3 and 4 show the more common pattern, with kalDNA (k) and giant forms visible, but lanes 1 and 2 are different subcultures of the same strain showing sibling structures labelled S

normal terminal fragments of the monomer (2.4 and 3.3 kb), but does not produce the single 5.7 kb junction fragment expected of a head-to-tail array; instead, two novel large bands are seen. PstI , which does not cut within kalilo, does not generate any novel fragments (lane 13), so the rearrangement that produces the giant bands generates no new PstI sites. The absence of a mtDNA-flanked kalilo fragment in the PstI digest (Grif- fiths et al. 1992) confirms that this strain has no detect- able ka lDNA inserts into mtDNA.

Harbin strains. The plasmid content of the original senes- cent Harbin strain 3983 and its deviant subculture (lanes 14 and 15 of Fig. 1) is complex. The inheritance of such elements and their connection with senescence will be described elsewhere. In these strains there are three pro-

minent bands of 9.0, 8.0 and 7.0 kb, which are linear elements with homology to maranhar (Fig. 1C, lane 14).

We have also discovered giant elements derived from the circular Har-1 plasmid originally found in this strain. Examples are shown in Fig. 1 D and E. Since these ele- ments are resistant to 5' and 3' digestion, a circular form is indicated. Such circularity has not been demonstrated previously. The elements show characteristics of head-to- tail concatamers of the basic circular plasmid monomer. For example, we know that the enzyme E c o R V cuts once within the Har-1 plasmid (Fig. 2). When treated with EcoRV, the giant forms disappear, and one Har-1 plas- mid band remains (data not shown).

One subculture of strain 3983 was unusual in that it appeared to have lost the three linear maranhar-related elements and the circular plasmid Har-1, but had gained

184

1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 910111213 14 151617181920

k ,

t

Fig. 6. Investigation of the structure of giant kalilo derivatives in a subculture of ascospore 4 (Myers et al. 1989) in which there was no detectable insertion into mtDNA. Lanes 1-7, ethidium bromide- stained gel of uncut (lane 1) and enzyme-digested preparations from a kalilo strain; lanes 8-14, blot of the same gel hybridized with cloned restriction fragment X3 from the kalDNA terminal inverted repeat (see map in Fig. 4). Lanes 1 and 8, uncut DNA, 2 and 9, treatment with 3' exonuclease, lanes 3 and 10, EcoR1, lanes 4 and 11, HindIII, lanes 5 and 12, Bg/II, lanes 6 and 13, PstI, lanes 7 and 14, 1 kb ladder (hybridization is to pBR322-derived sequences com- mon to the vector)

Fig. 7. Investigation of the structure of the linear plasmids, called Harbin L and L', with homology to the Harbin-1 circular plasmid,

L;.

L~.

I

4L

43.8 ,L'

42.4

41.6 41.4

41.2

harbin- L

3.9 I 1.3 EcoRV

4.9 10.3 Psi I

5.0 I XbaI /,.9 EcoRI

3.8 I 1,~ KpnI 1.2 ! 2.4 ] 1.6 Bg[]f

HindllI

that arose spontaneously during culture (see lane 15 in Fig. 1). Lanes 1-3, a gel showing uncut DNA (lane 1), 3' exonuclease treatment (lane 2), and 5' exonuclease treatment (lane 3). Lanes 4-6 show a blot of the same gel probed with the linear plasmid (L). The lanes marked with triangles were loaded with 1 ug of the 1 kb DNA ladder. Lanes 7 13 are from another gel of the same DNA prepara- tion with undigested and restricted samples probed with plasmid L. Lanes 14-20 are a blot of the same gel probed with a 1.4 kb KpnI terminal fragment. Lanes 7 and 14, uncut DNA; lanes 8 and 15, EcoRV; lanes 9 and 16, PstI; lanes 10 and 17, XbaI; lanes 11 and 18, BglII; lanes 12 and 19, EcoRI; lanes 13 and 20, KpnI. The bands labelled are Bg/II and KpnI fragments discussed in the text. Restric- tion maps of Harbin-L are shown below

two novel elements tha t hybridized to the circular Harb in plasmid probe (Fig. 1D, lane 15). The linear nature o f these derivatives and the presence o f prote in b o u n d to the 5' ends is demons t ra ted in Fig. 7. Lanes 1 and 4 show undigested samples. A l though the L ' element is generally less abundan t than L, the Southern transfer (lane 4) seems to have been inefficient. L ' generally transfers better as shown in lanes 7 and 14. The 5' digestions are shown in lanes 3 and 6, and the 3' digestions in lanes 2 and 5. Lanes 2, 3, 5, and 6 (marked) also conta in 1 gg o f the 1 kb ladder and this has been fully digested.

Since this is the first recorded case o f linear plasmids with h o m o l o g y to a circular element, we carried out a prel iminary investigation o f their structure. The larger o f

the two (L) is more abundan t and we focussed u p o n it. A restriction m a p was constructed, also shown in Fig. 7. Compar i son with the restriction map o f the circular Har- bin-1 plasmid shows that there is no ma tch o f restriction sites. To determine whether the element has a terminal repeat (characteristic o f all linear plasmids) the 1.4 kb KpnI f ragment was used as a probe. Hybridizat ions o f this p robe to undigested and restricted prepara t ions are shown in lanes 14 to 20, and for compr i son the full-length plasmid was used as a p robe in lanes 7 to 13. The 1.4 kb f ragment hybridizes to all f ragments that include a ter- minus, but no t to the internal 2.4 kb BglII f ragment (lane 18). The 3.8 kb Kpn! f ragment binds to all f ragments (not shown). These results are compat ible with the existence

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of a terminal repeat. (The figure does show a few anoma- lies. The hybridization of the 1.4 kb probe to the 1.2 kb BgIII fragment in lane 18 is weaker than expected, al- though there is a signal; an undetected terminal BgIII site might account for the decrease in signal intensity. In the same lane the signal from the 2.4 kb fragment probably results from the previous hybridization shown in lane 11. The figure is also complicated by cross hybridizations with the second plasmid, L'; for example in lane 11, the strong 1.6 kb signal is caused by a comigrating fragment of L'. Finally, KpnI digestion (lanes 13 and 20) did not remove all of band L, even though band L' was fully digested.)

Discussion

We have studied a large sample of Neurospora strains from around the world. Of this sample, 28 strains were found to be senescent. These senescent strains and some nonsenescent strains and laboratory strains were studied for plasmid content. This study revealed a plethora of DNA types that proved to be plasmids or plasmid deriva- tives. In any one strain, the number of plasmids ranged from one to seven. Plasmids were categorized as either circular or linear, depending on sensitivity to 5' and 3' exonucleases. Both types of plasmids are common, and several strains contain both types. A number of strains (such as LaBelle) have been examined previously and did not show some of the plasmids reported here. (Probably the two key elements of our procedure are treatment with proteinase K and the avoidance of the use of CsC1 den- sity gradient centrifugation for DNA isolation.) This rich diversity of plasmids was unexpected. Furthermore, it is surprising that a strain can sometimes carry several linear and circular plasmids, and concatamers and derivatives of both, with little apparent detrimental effect upon growth and general vigour.

The general relationship of plasmids to senescence remains unclear. Although other studies have established that the kalilo and maranhar plasmids are causally con- nected with senescence, this relationship has not been established for any of the new plasmids reported in senes- cent strains in this study. Furthermore, the potential of plasmids to cause senescence in more extensive trials is a subject of some interest. We have terminated subcul- ture series at 20 subcultures, but this is an arbitrary cut-off point. Nevertheless, there might be two classes of linear plasmids, one capable of causing senescence and one benign. The kalilo and maranhar plasmids are the only Neurospora linear plasmids for which there is sequence data (Chan et al. 1991; Court et al. 1991), so it is not yet possible to pinpoint any senescence-determin- ing properties. However, any presumptive special senes- cence-determining features must be relatively subtle, be- cause the overall architecture of these two plasmids is similar to that of all linear plasmids in other organisms, none of which cause senescence.

Kalilo plasmid sequences have so far been found only on the Hawaiian islands. However, maranhar sequences are more widespread. Maranhar was first found in N. crassa in India, but we have shown homologous se-

quences in N. intermedia from other parts of the world. The maranhar-related sequences are generally on plas- raids of roughly equal sizes, but the degree of homology is not yet known. In the Chinese Harbin strain of N. in- termedia, three plasmids show homology to the maran- har plasmid. One is about the same size as marDNA, but the others are larger and the source of the extra material is unknown at present.

The Harbin-1 plasmid is a member of the Labelle family of plasmids. This being the case, it is clear that this is quite a widespread type, present in both species, and not restricted to one isolate as previously thought (Nar- gang et al. 1992). This is particularly interesting as a 1.6 kb region of Neurospora mtDNA is homologous to a portion of the LaBelle plasmid (Nargang et al. 1992). In contrast the Harbin-2 plasmid seems to represent a new plasmid group.

The ladders derived from circular plasmids have been observed before, but the ladders shown here are the most complex reported to date. Undoubtedly this is a result of differing isolation and separation procedures. Presump- tive giant aggregates of circular plasmids have also been observed before (Natvig et al. 1984). We have shown that these are circular aggregates, most likely head-to-tail concatamers. It is not known if their slow mobility on the gels is truly a result of large size or of some other struc- tural feature. If the slow mobility is due to large size, these plasmids are so large that they cannot be resolved under the electrophoresis conditions used here.

The slow-moving, presumptive giant forms of the linear plasmid kalDNA have not been reported previous- ly. Both linear and circular versions of these giant forms occur. Such elements are always present in relatively small amounts. They seem to be more complex in struc- ture than the circular concatamers.

The other plasmid derivatives found in this study are novel for fungi. One ascospore culture produced short derivatives of the kalilo plasmid. Hybridization analysis with internal and terminal kalilo fragments has shown that all short forms carry DNA from the termini, but only the larger forms carry internal regions. However it is unlikely that such structures are simple deletions or replication intermediates: note for example that a 3.5 kb element shows homology to range of probes (Fig. 4) that together cover most of the 8.7 kb plasmid. The hybridiza- tion patterns are consistent with these short derivatives being formed from the termini inwards, but whether this process involves DNA or RNA intermediates is not known. The short kalilo forms were ephemeral, so may sult from self-propagating error that occurred by chance in the nucleic acid transactions of this kalilo strain. We know that this error occurred in cells bearing a senes- cence supressor that interferes with plasmid propagation. However, no similar patterns have been found in other suppressed strains, including the identical sister spore, so the relation of the process to suppression remains to be determined. Vierula and Bertrand (1991) have reported preliminary results that indicate the presence of a single truncated form of the kalilo plasmid in one strain. Short forms of maize linear plasmids have also been reported (Koncz et al. 1981; Bedinger et al. 1986).

In a different subculture of the suppressor strain, the

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first example was found of kalilo sibling plasmids, vari- ants of approximately the same size as kalilo. The struc- ture of these variants is not known, but is the subject of ongoing research. Similar siblings have been reported for the S1 and $2 plasmids of maize (Kemble and Marts 1983), and it was suggested that these might be obligate replication intermediates.

Perhaps the most intriguing derivative is the linear element showing homology with the Harbin-1 plasmid. Our preliminary data suggest that this plasmid has a 5' terminal protein and terminal repeats, but it has not been established if these repeats are direct or inverted. A full study of the structure of this plasmid should throw light on the origin of linear elements. It is probably significant that in the subculture in which this novel form was first seen, there had been a concomitant loss of the three linear plasmids normally present, and also of the circular Har- bin-1 plasmid (Harbin-2 remains). Although it is likely that the striking loss of plasmids and the gain of the novel linear type both stem from the same event, the new linear plasmid does not contain any regions homologous to maranhar , unlike the three original elements. However, it is conceivable that their non-maranhar segments com- bined with Harbin- I in some way.

Certainly one of the predominant impressions that has emerged f rom the present work is that plasmids are the rule in Neurospora , and not the exception. The types that we have identified are undoubtedly merely the tip of an iceberg of diversity. The linear plasmids so far de- scribed in fungi and plants (Esser et al. 1986; Meinhardt et al. 1990), despite showing no nucleotide homology, have remarkably similar general structures, apparently code for the same kinds of proteins related to viral poly- merases, and are virtually all mitochondrial in location. Assuming that all linear plasmids will have such proper- ties, is it reasonable to propose that this entire gamut of plasmid types all evolved f rom one common ancestor that inhabited the original endosymbiont that gave rise to mitochondria? Alternatively, it is possible that there is only one structure that is compatible with the selfish D N A lifestyle within mitochondria, and that the mul- titude of types have converged on this form.

Acknowledgements. We thank Dr David Perkins for donating strains and Dr Daniel Vickery for donating kalilo probes. The work was financed by grant A6599 from the Natural Science and Engi- neering Research Council of Canada.

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Communicated by D.M. Lonsdale