Current Genetics
Curr Genet (1992)21-169-172
9 Springer-Verlag 1992
Linear, non-mitochondrial plasmids of Alternaria alternata Hurley S. Shepherd United States Department of Agriculture, ARS Southern Regional Research Center, P.O. Box 19687, New Orleans, LA 70179, USA Received August 6, 1990/August 6, 1991
Summary. Three plasmids, with sizes o f 7.0 kbp, 6.8 kbp, and 5.0 k b p a n d designated pAal-1, pAal-2 and pAal-3 respectively, have been f o u n d in a t e n t o x i n - p r o d u c i n g isolate o f Alternaria alternata. Exonuclease digestions show these plasmids to be linear with blocked 5' ends. Plasmid pAal-1 does n o t hybridize to nuclear D N A , m i t o c h o n d r i a l D N A , or d o u b l e - s t r a n d e d R N A f r o m a m y c o v i r u s f o u n d in the isolate, b u t does hybridize weakly to a series o f linear D N A s which are n o t visible on gels a n d m a y include pAal-2 and pAal-3. Cellular fractionation shows that, unlike other linear fungal plasmids, these plasmids are n o t localized in the m i t o c h o n d r i a . Plasmids have n o t been f o u n d in other tentoxin-producing isolates a n d there is n o evidence that these plasmids have any effect on the p r o d u c t i o n o f tentoxin.
Key words: mids
Alternaria
-
Linear plasmids - Fungal plas-
Introduction Plasmids have been f o u n d in a n u m b e r o f filamentous fungi (see B t c k e l m a n n et al. 1986; S a m a c and L e o n g 1989). All o f those for which the location and structure have been determined are m i t o c h o n d r i a l a n d m o s t are linear. The m a j o r exceptions are the circular m i t o c h o n drial ptasmids o f Neurospora species (Natvig et al. 1984). F u n c t i o n s relating to fungal p h e n o t y p e are n o t apparent, a n d plasmidless-isolates generally are indistinguishable m o r p h o l o g i c a l l y f r o m those with plasmids (Samac and L e o n g 1989). Some isolates o f the imperfect fungus Alternaria alternata (Fr.) Keissler p r o d u c e tentoxin, a peptide which causes chlorosis in some plant species (Templeton et al. 1967). The synthesis o f high levels o f tentoxin is associated with the presence o f virus-like particles (VLPs; Shepherd 1988). I n the process o f purifying VLPs, three plasmids, which are n o t localized to the m i t o c h o n d r i a , were f o u n d in one isolate o f the fungus. N o n - m i t o c h o n d r i a l
plasmids have n o t been previously reported in filamentous fungi.
Materials and methods Culture conditions. A. alternata isolate number 8 and other isolates were derived from surface-sterilized cotton seeds germinated on agar. Cultures were grown on Fries Medium (Tuite 1969) at 28 ~ in continuous light without shaking. A 1 1 culture yielded a fungal mat of approximately 50 g. Plasmid &olation. Fungal mats were frozen in liquid nitrogen and ground in 1 ml/g 30 mM sodium phosphate, pH 7.6, using a mortar and pestle. After removing cellular debris by centrifugation at 27000 g in a Sorvall SS-34 rotor, the supernatant was adjusted to 40% CsC1 (w/v) and spun at 163 000 gm,xin a Sorvall TH-641 rotor for between 64 and 70 h. One ml fractions were collected from the tube and the pellet was suspended in 1 ml TE buffer (10 mM Tris, 1 mM EDTA, pH 7.6). Samples from each fraction and the suspended pellet were diluted 1 : 2 with distilled water, then precipitated by the addition of one vol. of 7.5 M ammonium acetate followed by two vol. of ethanol. The pellet was resuspended in TE and electrophoresed on a 1% agarose gel in TBE buffer (Maniatis et al. 1982) or else subjected to 1 Ixg/ml RNase A digestion in TE buffer prior to electrophoresis. Some samples were electrophoresed in a CHEF-DRII apparatus (Bio-Rad, Richmond, CA, USA) at 90 V with a 4 sec switch at 14~ for 16 h. Bands from the gel were eluted with an Elutrap (Schleicher and Schuell, Keene, N.H.) and labeled using a Tropix Chemiluminescent Labeling and Detection System (Bios Corp., New Haven, Conn.). Hybridizations were to blots on Tropilon-45 Nylon membrane (Tropix, Bedford, Mass.) according to the manufacturer's instructions. Exonuclease and restriction enzyme digestions. Exonuclease III and lambda exonuclease were purchased from BRL (Gaithersburg, Md.) while restriction enzymes were purchased from IBI (New Haven, Conn.) and US Biochemical Corp. (Cleveland, Ohio). All enzymes were used according to the supplier's instructions. Ethanolprecipitated DNAs were dissolved in TE buffer, dialyzed against TE buffer overnight, ethanol precipitated, and then redissolved in TE before digestion. HindIII-digested lambda DNA (1.5 gg) was added to some exonuclease digestions to affirm exonuclease activity. Cellularfractionation. Cells were broken with a chilled mortar and pestle in 10 mM tricine buffer pH 7.5, containing 0.2 mM EDTA and 15% sucrose. Components were then fractionated using a mito-
170
Fig. 2. Exonuclease digestions of dialyzed CsC1fraction 10. Lane 1, lambda HindlII fragments; lane 2, untreated fraction 10 with added lambda HindlII fragments; lane 3, untreated fraction 10; lane 4, fraction 10 treated with lambda exonuclease (5' specific); lane 5, fraction 10 treated with exonuclease III (3' specific);lane 6, same as lane 2, treated with lambda exonuclease; lane 7, same as lane 2, treated with exonuclease III
Fig. 1A, B. Fractions 5-10 and the pellet from a CsC1 gradient, either untreated (A) or treated with RNase A (B), separated on a 1% agarose gel. M, lambda HindlII fragments; P, pellet from gradient tube. Fraction 5 is in the middle of the gradient, fraction 10 is at the bottom. Bands infractions 7, 8 and 9 are dsRNA from a VLP. DNA bands are in fraction 10 and the pellet
chondrial isolation procedure (Lambowitz 1979), with the 1000 g pellet designated as nuclear and a 27000 g pellet designated as mitochondrial. Pellets were disrupted with UNSET buffer (8.0 M urea, 2% SDS, 150 mM NaC1, 1 mM EDTA, 100 mM Tris-HC1, pH 7.5; Garriga et al. 1984) or with a mitochondrial lysis buffer (0.5 M NaC1, 10 mM EDTA, 2% sarcosyl, 0.5% SDS) with or without 150 ~tg/ml proteinase K for 1 h at 37 ~
Results and discussion Plasmids were initially isolated from 40% CsC1 gradients used for the purification of VLPs. VLPs band at about 1.40 g/cm 3, while the plasmid bands were seen in the bottom fraction (fraction 10) of the tube or in the pellet (Fig. 1 a). When the nucleic acids from fraction 10 or the pellet were treated with RNase A before electrophoresis, dsRNAs from the VLPs were digested, leaving three D N A bands with approximate sizes 7.0 kbp, 6.8 kbp, and 5.0 kbp (Fig. 1 b). These DNAs were seen more clearly in later samples which did not contain high levels of CsC1, and were designated as plasmids pAal-1, pAal-2, and pAal-3 in descending size order; pAal-1 is always present in the highest amount, with pAal-2 and pAal-3 in lower amounts.
When dialyzed samples from the CsC1 gradient were treated with exonuclease III, which digests D N A from the 3' end, all the pAal-1 plasmid band disappeared (Fig. 2, lane 5). Plasmid pAal-2 also disappeared, but it was difficult to determine the fate o f pAal-3 due to the small amount initially present, and interference from the VLP d s R N A which is still present. Later blots suggested that pAal-3 is also linear (see Fig. 5). Digestion with lambda exonuclease, which digests progressively from 5' ends, had no effect on the plasmids (Fig. 2, lane 4). Nuclear D N A and lambda D N A H i n d l I I fragments (lanes 2, 6, and 7) were digested in both reactions, verifying enzyme activity. The differential susceptibility of the plasmids to the exonucleases indicates that these plasmids are linear with blocked 5' ends. This type o f blockage is commonly found on linear D N A species and is thought to depend on a protein involved in replication (see references in Samac and Leong 1989). We found, however, that the addition of protease (proteinase K or pronase) to isolation protocols has no effect on the isolation of the plasmids reported here. This could mean that the blockage involves something other than protein, such as the hairpin loops found in plasmids of R h i z o c t o n i a solani (Miyashita et al. 1990). When fractions from CsC1 gradients were digested with restriction enzymes, plasmid pAal-1 gave distinct bands, with fragment sizes adding to about 7.0 kbp (Fig. 3). The possibility that the pAal-1 band is an unresolved doublet is unlikely since all digestions give an unambiguous size of about 7.0 kbp. Plasmids pAal-2 and pAal-3 were not present in high enough amounts to be visible in this preparation. To determine the cellular location of the plasmids, mitochondria and nuclei were isolated. Restriction en-
171
Fig. 3. Digestion of dialyzed CsCI fraction 10 with restriction enzymes. Lane 1, uncut; lane 2, BamHI; lane 3, EcoRI; lane 4, PstI; lane 5, HaelI
Fig. 4. A nucleic acids from gradient-purified mitochondria run on a CHEF-DRII. 1, uncut; 2, cut with PstI. The band at around 6 kbp is dsRNA from the VLP. Large circular DNAs, such as those of mitochondria, form a diffuse band above 23 kbp (Skelly and Maleszka 1989). B nucleic acids from the supernatant (cytoplasmic) fraction of isolate number 8 (which contains the plasmids) and two other tentoxin-producing isolates digested with PstI. 1, Isolate 8; 2, isolate 64; 3, isolate 202. Plasmid fragments are indicated by aYl'OWS
zyme digestion verified the presence of mitochondrial D N A at the 44%/55% sucrose interface of flotation gradients while to plasmid D N A was seen (Fig. 4 A). This was the case whether mitochondrial preparations were treated with proteinase K or not. Plasmid D N A was also not detectable in the mitochondrial pellets probed with
Fig. 5. A nucleic acids found in the supernatant after cell fractionation. Lane I was treated with exonuclease III and RNase A; lane 2 was treated only with RNase A. B blot of lanes in A hybridized to the excised pAal-1 band. C same as B, exposed six times longer isolated plasmid (data not shown). Plasmid D N A was found exclusively in the supernatant while no mitochondrial D N A was seen in the supernatant (Fig. 4 B, lane 1), indicating a non-mitochondrial location for the plasmids. Since a large amount of nuclear D N A is found in the cytoplasmic fraction, it is not certain whether the plasmids are nuclear or cytoplasmic. Small amounts ofpAalI and pAal-3 were sometimes seen in the nuclear and mitochondrial fractions, which could result from plasmid adhering to the outside of the organelles, as observed by Ligon et al. (1989). The plasmids were not seen when the pellets were treated with DNase I. In cultures older than 10 days, increasing amounts of the plasmids were found in the initial mitochondrial pellet, but not in mitochondria from the flotation gradient interface. A similar finding has been made for VLPs which apparently become enclosed in vesicles as the mycelia age (Shepherd 1990). Plasmid pAal-1 was electroeluted from agarose gels, labeled, and hybridized to a blot o f mitochondrial and supernatant fractions, pAal-1 hybridized strongly to itself and to two other bands not visible on the original gel (Fig. 5 B, lane 2), one of which was near to the location of pAal-3. Some hybridization was noted to a large number of bands when the blot was exposed for longer periods of time (Fig. 5 C). The relation of these bands to pAal-1 is not known but they are all linear since they are digested by exonuclease III (Fig. 5 C, lane 1). Increasing the stringency of the hybridization, by increasing temperature and decreasing salt concentration, did not affect the number of bands. If these bands are related to pAal-1, this could explain the apparent increase in the amount of plasmid sometimes seen following restriction digestion. Plasmids found previously in filamentous fungi have been localized only to mitochondria, although the locations of many have not yet been determined (Samac and Leong 1989). Linear plasmids of yeasts, however, have been found in the cytoplasm (Stam et al. 1986; Shepherd et al. 1987; Ligon et al. 1989). The plasmids reported here show no homology with nuclear or mitochondrial DNA, or with viral dsRNAs present in the same isolate. All
172
References
Garriga G, Bertrand H, Lambowitz A (1984) Cell 36:623-634 Lambowitz A (1979) Methods Enzymol 59:421-433 Ligon J, Bolen P, Hill D, Bothast R, Kurtzman C (1989) Plasmid 21:185-194 Maniatis T, Fritsch E, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Miyashita S, Hirochika H, Ikeda J, Hashiba T (1990) Mol Gen Genet 220:165-171 Natvig D, May G, Taylor J (1984) J Bacteriol 159:288-293 Samac D, Leong S (1989) Molec Plant-Microbe Interac 2:155-159 Shepherd H (1988) J Virol 62:3888-3891 Shepherd H (1990) Exp Mycol 14:294-298 Shepherd H, Ligon J, Bolen P, Kurtzman C (1987) Curt Genet 12:297-304 Skelly P, Maleszka R (1989) Nucleic Acids Res 17:7537 Stam J, Kwakman J, Meijer M, Stuitje A (1986) Nucleic Acids Res 14:6871-6884 Templeton G, Grable G, Fulton N, Bollenbacher K (1967) Phytopathology 57:516-518 Tuite J (1969) Plant pathological methods: fungi and bacteria. Burgess Publishing Co., Minneapolis
B6ckelmann B, Osiewacz HD, Schmidt FR, Schulte E (1986) Extrachromosomal DNA in fungi. In: Buck KW (ed) Fungal virology. CRC Press, Boca Raton, pp 237-283
C o m m u n i c a t e d by H. Bertrand
three plasmid bands, as well as the VLP d s R N A , were found in four single-spore isolates o f the culture (data not shown); thus, their occurrence together is not the result of a mixed culture. N o plasmids have been found in ten other isolates of A. alternata, either with or without VLPs or tentoxin production (see Fig. 4 B, lanes 2 and 3). N o other phenotypic differences are apparent between isolate 8 and other isolates of the fungus in which no plasmids have been found. Thus, the plasmids do not seem to play a role in tentoxin production or in the normal life cycle of the fungus, but m a y still be useful as vectors for studying the molecular biology of development and tentoxin production.
Acknowledgements. The able technical assistance of David Ambrogio is gratefully acknowledged.
Curr Genet (1992)21-169-172
9 Springer-Verlag 1992
Linear, non-mitochondrial plasmids of Alternaria alternata Hurley S. Shepherd United States Department of Agriculture, ARS Southern Regional Research Center, P.O. Box 19687, New Orleans, LA 70179, USA Received August 6, 1990/August 6, 1991
Summary. Three plasmids, with sizes o f 7.0 kbp, 6.8 kbp, and 5.0 k b p a n d designated pAal-1, pAal-2 and pAal-3 respectively, have been f o u n d in a t e n t o x i n - p r o d u c i n g isolate o f Alternaria alternata. Exonuclease digestions show these plasmids to be linear with blocked 5' ends. Plasmid pAal-1 does n o t hybridize to nuclear D N A , m i t o c h o n d r i a l D N A , or d o u b l e - s t r a n d e d R N A f r o m a m y c o v i r u s f o u n d in the isolate, b u t does hybridize weakly to a series o f linear D N A s which are n o t visible on gels a n d m a y include pAal-2 and pAal-3. Cellular fractionation shows that, unlike other linear fungal plasmids, these plasmids are n o t localized in the m i t o c h o n d r i a . Plasmids have n o t been f o u n d in other tentoxin-producing isolates a n d there is n o evidence that these plasmids have any effect on the p r o d u c t i o n o f tentoxin.
Key words: mids
Alternaria
-
Linear plasmids - Fungal plas-
Introduction Plasmids have been f o u n d in a n u m b e r o f filamentous fungi (see B t c k e l m a n n et al. 1986; S a m a c and L e o n g 1989). All o f those for which the location and structure have been determined are m i t o c h o n d r i a l a n d m o s t are linear. The m a j o r exceptions are the circular m i t o c h o n drial ptasmids o f Neurospora species (Natvig et al. 1984). F u n c t i o n s relating to fungal p h e n o t y p e are n o t apparent, a n d plasmidless-isolates generally are indistinguishable m o r p h o l o g i c a l l y f r o m those with plasmids (Samac and L e o n g 1989). Some isolates o f the imperfect fungus Alternaria alternata (Fr.) Keissler p r o d u c e tentoxin, a peptide which causes chlorosis in some plant species (Templeton et al. 1967). The synthesis o f high levels o f tentoxin is associated with the presence o f virus-like particles (VLPs; Shepherd 1988). I n the process o f purifying VLPs, three plasmids, which are n o t localized to the m i t o c h o n d r i a , were f o u n d in one isolate o f the fungus. N o n - m i t o c h o n d r i a l
plasmids have n o t been previously reported in filamentous fungi.
Materials and methods Culture conditions. A. alternata isolate number 8 and other isolates were derived from surface-sterilized cotton seeds germinated on agar. Cultures were grown on Fries Medium (Tuite 1969) at 28 ~ in continuous light without shaking. A 1 1 culture yielded a fungal mat of approximately 50 g. Plasmid &olation. Fungal mats were frozen in liquid nitrogen and ground in 1 ml/g 30 mM sodium phosphate, pH 7.6, using a mortar and pestle. After removing cellular debris by centrifugation at 27000 g in a Sorvall SS-34 rotor, the supernatant was adjusted to 40% CsC1 (w/v) and spun at 163 000 gm,xin a Sorvall TH-641 rotor for between 64 and 70 h. One ml fractions were collected from the tube and the pellet was suspended in 1 ml TE buffer (10 mM Tris, 1 mM EDTA, pH 7.6). Samples from each fraction and the suspended pellet were diluted 1 : 2 with distilled water, then precipitated by the addition of one vol. of 7.5 M ammonium acetate followed by two vol. of ethanol. The pellet was resuspended in TE and electrophoresed on a 1% agarose gel in TBE buffer (Maniatis et al. 1982) or else subjected to 1 Ixg/ml RNase A digestion in TE buffer prior to electrophoresis. Some samples were electrophoresed in a CHEF-DRII apparatus (Bio-Rad, Richmond, CA, USA) at 90 V with a 4 sec switch at 14~ for 16 h. Bands from the gel were eluted with an Elutrap (Schleicher and Schuell, Keene, N.H.) and labeled using a Tropix Chemiluminescent Labeling and Detection System (Bios Corp., New Haven, Conn.). Hybridizations were to blots on Tropilon-45 Nylon membrane (Tropix, Bedford, Mass.) according to the manufacturer's instructions. Exonuclease and restriction enzyme digestions. Exonuclease III and lambda exonuclease were purchased from BRL (Gaithersburg, Md.) while restriction enzymes were purchased from IBI (New Haven, Conn.) and US Biochemical Corp. (Cleveland, Ohio). All enzymes were used according to the supplier's instructions. Ethanolprecipitated DNAs were dissolved in TE buffer, dialyzed against TE buffer overnight, ethanol precipitated, and then redissolved in TE before digestion. HindIII-digested lambda DNA (1.5 gg) was added to some exonuclease digestions to affirm exonuclease activity. Cellularfractionation. Cells were broken with a chilled mortar and pestle in 10 mM tricine buffer pH 7.5, containing 0.2 mM EDTA and 15% sucrose. Components were then fractionated using a mito-
170
Fig. 2. Exonuclease digestions of dialyzed CsC1fraction 10. Lane 1, lambda HindlII fragments; lane 2, untreated fraction 10 with added lambda HindlII fragments; lane 3, untreated fraction 10; lane 4, fraction 10 treated with lambda exonuclease (5' specific); lane 5, fraction 10 treated with exonuclease III (3' specific);lane 6, same as lane 2, treated with lambda exonuclease; lane 7, same as lane 2, treated with exonuclease III
Fig. 1A, B. Fractions 5-10 and the pellet from a CsC1 gradient, either untreated (A) or treated with RNase A (B), separated on a 1% agarose gel. M, lambda HindlII fragments; P, pellet from gradient tube. Fraction 5 is in the middle of the gradient, fraction 10 is at the bottom. Bands infractions 7, 8 and 9 are dsRNA from a VLP. DNA bands are in fraction 10 and the pellet
chondrial isolation procedure (Lambowitz 1979), with the 1000 g pellet designated as nuclear and a 27000 g pellet designated as mitochondrial. Pellets were disrupted with UNSET buffer (8.0 M urea, 2% SDS, 150 mM NaC1, 1 mM EDTA, 100 mM Tris-HC1, pH 7.5; Garriga et al. 1984) or with a mitochondrial lysis buffer (0.5 M NaC1, 10 mM EDTA, 2% sarcosyl, 0.5% SDS) with or without 150 ~tg/ml proteinase K for 1 h at 37 ~
Results and discussion Plasmids were initially isolated from 40% CsC1 gradients used for the purification of VLPs. VLPs band at about 1.40 g/cm 3, while the plasmid bands were seen in the bottom fraction (fraction 10) of the tube or in the pellet (Fig. 1 a). When the nucleic acids from fraction 10 or the pellet were treated with RNase A before electrophoresis, dsRNAs from the VLPs were digested, leaving three D N A bands with approximate sizes 7.0 kbp, 6.8 kbp, and 5.0 kbp (Fig. 1 b). These DNAs were seen more clearly in later samples which did not contain high levels of CsC1, and were designated as plasmids pAal-1, pAal-2, and pAal-3 in descending size order; pAal-1 is always present in the highest amount, with pAal-2 and pAal-3 in lower amounts.
When dialyzed samples from the CsC1 gradient were treated with exonuclease III, which digests D N A from the 3' end, all the pAal-1 plasmid band disappeared (Fig. 2, lane 5). Plasmid pAal-2 also disappeared, but it was difficult to determine the fate o f pAal-3 due to the small amount initially present, and interference from the VLP d s R N A which is still present. Later blots suggested that pAal-3 is also linear (see Fig. 5). Digestion with lambda exonuclease, which digests progressively from 5' ends, had no effect on the plasmids (Fig. 2, lane 4). Nuclear D N A and lambda D N A H i n d l I I fragments (lanes 2, 6, and 7) were digested in both reactions, verifying enzyme activity. The differential susceptibility of the plasmids to the exonucleases indicates that these plasmids are linear with blocked 5' ends. This type o f blockage is commonly found on linear D N A species and is thought to depend on a protein involved in replication (see references in Samac and Leong 1989). We found, however, that the addition of protease (proteinase K or pronase) to isolation protocols has no effect on the isolation of the plasmids reported here. This could mean that the blockage involves something other than protein, such as the hairpin loops found in plasmids of R h i z o c t o n i a solani (Miyashita et al. 1990). When fractions from CsC1 gradients were digested with restriction enzymes, plasmid pAal-1 gave distinct bands, with fragment sizes adding to about 7.0 kbp (Fig. 3). The possibility that the pAal-1 band is an unresolved doublet is unlikely since all digestions give an unambiguous size of about 7.0 kbp. Plasmids pAal-2 and pAal-3 were not present in high enough amounts to be visible in this preparation. To determine the cellular location of the plasmids, mitochondria and nuclei were isolated. Restriction en-
171
Fig. 3. Digestion of dialyzed CsCI fraction 10 with restriction enzymes. Lane 1, uncut; lane 2, BamHI; lane 3, EcoRI; lane 4, PstI; lane 5, HaelI
Fig. 4. A nucleic acids from gradient-purified mitochondria run on a CHEF-DRII. 1, uncut; 2, cut with PstI. The band at around 6 kbp is dsRNA from the VLP. Large circular DNAs, such as those of mitochondria, form a diffuse band above 23 kbp (Skelly and Maleszka 1989). B nucleic acids from the supernatant (cytoplasmic) fraction of isolate number 8 (which contains the plasmids) and two other tentoxin-producing isolates digested with PstI. 1, Isolate 8; 2, isolate 64; 3, isolate 202. Plasmid fragments are indicated by aYl'OWS
zyme digestion verified the presence of mitochondrial D N A at the 44%/55% sucrose interface of flotation gradients while to plasmid D N A was seen (Fig. 4 A). This was the case whether mitochondrial preparations were treated with proteinase K or not. Plasmid D N A was also not detectable in the mitochondrial pellets probed with
Fig. 5. A nucleic acids found in the supernatant after cell fractionation. Lane I was treated with exonuclease III and RNase A; lane 2 was treated only with RNase A. B blot of lanes in A hybridized to the excised pAal-1 band. C same as B, exposed six times longer isolated plasmid (data not shown). Plasmid D N A was found exclusively in the supernatant while no mitochondrial D N A was seen in the supernatant (Fig. 4 B, lane 1), indicating a non-mitochondrial location for the plasmids. Since a large amount of nuclear D N A is found in the cytoplasmic fraction, it is not certain whether the plasmids are nuclear or cytoplasmic. Small amounts ofpAalI and pAal-3 were sometimes seen in the nuclear and mitochondrial fractions, which could result from plasmid adhering to the outside of the organelles, as observed by Ligon et al. (1989). The plasmids were not seen when the pellets were treated with DNase I. In cultures older than 10 days, increasing amounts of the plasmids were found in the initial mitochondrial pellet, but not in mitochondria from the flotation gradient interface. A similar finding has been made for VLPs which apparently become enclosed in vesicles as the mycelia age (Shepherd 1990). Plasmid pAal-1 was electroeluted from agarose gels, labeled, and hybridized to a blot o f mitochondrial and supernatant fractions, pAal-1 hybridized strongly to itself and to two other bands not visible on the original gel (Fig. 5 B, lane 2), one of which was near to the location of pAal-3. Some hybridization was noted to a large number of bands when the blot was exposed for longer periods of time (Fig. 5 C). The relation of these bands to pAal-1 is not known but they are all linear since they are digested by exonuclease III (Fig. 5 C, lane 1). Increasing the stringency of the hybridization, by increasing temperature and decreasing salt concentration, did not affect the number of bands. If these bands are related to pAal-1, this could explain the apparent increase in the amount of plasmid sometimes seen following restriction digestion. Plasmids found previously in filamentous fungi have been localized only to mitochondria, although the locations of many have not yet been determined (Samac and Leong 1989). Linear plasmids of yeasts, however, have been found in the cytoplasm (Stam et al. 1986; Shepherd et al. 1987; Ligon et al. 1989). The plasmids reported here show no homology with nuclear or mitochondrial DNA, or with viral dsRNAs present in the same isolate. All
172
References
Garriga G, Bertrand H, Lambowitz A (1984) Cell 36:623-634 Lambowitz A (1979) Methods Enzymol 59:421-433 Ligon J, Bolen P, Hill D, Bothast R, Kurtzman C (1989) Plasmid 21:185-194 Maniatis T, Fritsch E, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Miyashita S, Hirochika H, Ikeda J, Hashiba T (1990) Mol Gen Genet 220:165-171 Natvig D, May G, Taylor J (1984) J Bacteriol 159:288-293 Samac D, Leong S (1989) Molec Plant-Microbe Interac 2:155-159 Shepherd H (1988) J Virol 62:3888-3891 Shepherd H (1990) Exp Mycol 14:294-298 Shepherd H, Ligon J, Bolen P, Kurtzman C (1987) Curt Genet 12:297-304 Skelly P, Maleszka R (1989) Nucleic Acids Res 17:7537 Stam J, Kwakman J, Meijer M, Stuitje A (1986) Nucleic Acids Res 14:6871-6884 Templeton G, Grable G, Fulton N, Bollenbacher K (1967) Phytopathology 57:516-518 Tuite J (1969) Plant pathological methods: fungi and bacteria. Burgess Publishing Co., Minneapolis
B6ckelmann B, Osiewacz HD, Schmidt FR, Schulte E (1986) Extrachromosomal DNA in fungi. In: Buck KW (ed) Fungal virology. CRC Press, Boca Raton, pp 237-283
C o m m u n i c a t e d by H. Bertrand
three plasmid bands, as well as the VLP d s R N A , were found in four single-spore isolates o f the culture (data not shown); thus, their occurrence together is not the result of a mixed culture. N o plasmids have been found in ten other isolates of A. alternata, either with or without VLPs or tentoxin production (see Fig. 4 B, lanes 2 and 3). N o other phenotypic differences are apparent between isolate 8 and other isolates of the fungus in which no plasmids have been found. Thus, the plasmids do not seem to play a role in tentoxin production or in the normal life cycle of the fungus, but m a y still be useful as vectors for studying the molecular biology of development and tentoxin production.
Acknowledgements. The able technical assistance of David Ambrogio is gratefully acknowledged.
