Eunice H. Froeliger 1, Alfredo M. Mufioz-Rivas 2' *, Charles A. Specht 2, Robert C. Ullrieh 2, and ...... Johnstone IL, Hughes SG, Clutterbuck AJ (1985) EMBO J.
CurrentGenetics
Curr Genet (1987)12:547-554
© Springer-Vedag 1987
The isolation of specific genes from the basidiomycete SchizophyUum commune Eunice H. Froeliger 1, Alfredo M. Mufioz-Rivas 2' *, Charles A. Specht 2, Robert C. Ullrieh 2, and Charles P. Novotny 1 Departments of IMicrobiologyand 2Botany, Universityof Vermont, Burlington,VT 05405, USA
Summary. We have developed a routine way to isolate genes directly from the basidiomycete fungus, Schizophyllum commune. Plasmid DNA from a genomic gene library was used to isolate five specific genes by complementation of Schizophyllum mutations via transformation. The mutant strains were deficient in the ability to synthesize either adenine (ade2 and ade5), uracil (ural, encoding orotidine-5'-phosphate decarboxylase; OMPdecase), tryptophan (trpl, encoding indole-3-glycerol phosphate synthetase; IGPS) or para aminobenzoic acid (pabl). In each case, Southern analysis revealed that transformation to prototrophy was concomitant with the integration of vector sequence into the genome of the S. commune mutant. Total DNA from transformants was restricted, religated, and used to transform E. coil Ampicillin resistant plasmids were recovered from E. coli and tested for their ability to transform the corresponding mutant of S. commune. Plasmids complementing the ade2, ade5, pabl, trpl, and ural mutations were recovered. Key words: Schizophyllum commune -Transformation - Gene isolation - Basidiomycetes - Recombinant DNA
Introduction
The developmental events in the sexual cycle of Schizophyllum commune are regulated by a tetrapolar system consisting of two unlinked mating-type genes, A and B. * Present address: Department of Plant Science,MacDonaldCollege of McGiUUniversity, Ste. Anne de Bellevue,Quebec, Canada H9X 1CO Offprint requests to: E. H. Froeliger
Comprehensive cytological, physiological, biochemical and mutational analyses have been employed to study the series of developmental changes that occur after fusion of two monokaryotic hyphae (haploid uninucleate cells) of compatible mating-types (for reviews see Raper 1966; Raper 1983). These changes, regulated by the mating-type genes, involve the establishment of dikaryotic hyphae (one nucleus from each compatible parent per cell) and the induction of fruit body formation. Within specialized cells of the fruiting bodies (basidium), nuclear fusion, meiosis and sexual spore production take place. A remarkable feature of the developmental system in S. commune is that the mating-type genes can exist as any one of a number of different alleles. Thus many combinations of different alleles of each mating-type gene can interact to produce the developmental changes controlled by that gene. Molecular studies of mating-type and gene expression during differentiation have been hampered by the lack of a DNA mediated transformation system in this organism and by the absence of methods for isolating specific genes. The recent demonstration of transformation in SchizophyUum (Mufioz-Rivas et al. 1986a) indicated that the Schizophyllum TRP1 gene (initially identified by complementation of trpC in E. coli) could be used to transform the corresponding Schizophyllum trpl mutant to prototrophy. A major disadvantage of this method of gene cloning is that only genes that produce an observable phenotypic change in mutants ofE. coli can be isolated. In order to isolate Schizophyllum genes that are not functional in E. coli (e.g. mating-type and developmental genes), it is necessary to use a protocol that 1) allows selection of the gene of interest directly in SchizophyUum mutants and 2) permits rescue of the gene from the mutant. In this report we assess the feasibility of directly recovering Schizophyllum genes by transformation of
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E.H. Froeliger et al.: The isolation of specific genes from the basidiomycete Schizophyllum commune
SchizophyUum mutants with DNA from a Schizophyll u m p l a s m i d gene l i b r a r y f o l l o w e d b y gene rescue in E. coli. We a s k e d i f five d i f f e r e n t genes o f Schizophyllum commune c o u l d b e selected b y c o m p l e m e n t a t i o n o f S c h i z o p h y U u m m u t a n t s a n d i f i t was possible t o recover the integrated transforming sequences from chrom o s o m a l D N A as a m p R p l a s m i d s in E. coli. Similar m e t h o d s h a v e b e e n u s e d t o isolate genes f r o m t h e A s c o m y c e t e s Saccharomyces cerevisiae (Hicks et al. 1 9 7 9 ) a n d Aspergillus nidulans ( B a l a n c e a n d T u r n e r 1 9 8 5 , 1 9 8 6 ) , a n d f r o m t h e Z y g o m y c e t e Mucor circinelloides ( v a n H e e s w i j c k a n d R o n c e r o 1 9 8 4 ) . T h e five S c h i z o p h y l l u m genes t a r g e t e d f o r r e c o v e r y w e r e : A D E 2 and A D E 5 ( s y n t h e s i s o f a d e n i n e ) , PAB1 ( s y n t h e s i s o f p a r a a m i n o b e n z o i c acid), TRP1 ( e n c o d i n g indole-3-glycerol p h o s p h a t e s y n t h e t a s e ) , a n d URA1 ( e n c o d i n g orot i d i n e - 5 ' - p h o s p h a t e d e c a r b o x y l a s e ) . T r a n s f o r m a n t s were o b t a i n e d f o r m u t a n t s o f e a c h o f t h e s e genes a n d t h e c o m p l e m e n t i n g s e q u e n c e s were r e c o v e r e d f o r e a c h o f t h e five. This m e t h o d a p p e a r s t o b e a n e f f i c i e n t w a y t o isolate d i r e c t l y S c h i z o p h y l l u m genes a n d s h o u l d m a k e it p o s s i b l e t o isolate S c h i z o p h y U u m m a t i n g - t y p e a n d develo p m e n t a l genes f o r w h i c h m u t a n t s are available.
Materials and methods S. commune strains and culture. S. commune strains UVM12-43 (A42B42, ural), UVM12-44 (AxBx, ural), UVM16-13 (AxBx, ade2), UVM16-15 (A42Bx, ade2), UVM8-12 (A41B41, pabl), UVM16-35 (AxBx, ade5, pabl), UVM12-18 (A43B42, ade5), UVM8-89 (AxBx, trpl), UVM8-87 (A91B91, trpl) were used for transformation. Strain UVM4-40 (A43B43) was the source of DNA used as a control in Southern analyses. A and B refer to the mating-type genes; the numbers identify specific matingtype alleles. The symbol " x " denotes that mating-type has not been determined. Schizophyllum strains were cultured as previously described (Mufioz-Rivas et al. 1986a).
E. coli strains and culture. E. coli K-12 strain MC1066 [A lac (IPOZYA)X74, galU, galK, strA, hsdR-, trpC9830, leuB6, pyrF74::Tn5(KmR); Casadaban et al. 1983] and strain JA228 (argH, str, hsdR-, hsdM+; obtained from Dr. John Carbon) were used for the rescue of transforming plasmids from S. commune and for plasmid propagation. Strain JA228 was also the host for the plasmid gene library. Strain NM539 (Promega Biotec, Madison, WI) was used to screen the gene library in bacteriophage k EMBL4. E. coli cultures for transformation were grown in yeast extract tryptone broth (Z medium) as described by Mun~z-Rivas et al. (1986b). E. coli cells for h phage infection were grown in NZYM medium with maltose as described (Maniatis et al. 1982).
Construction of the plasmid gene library. Construction of the S. commune gene library in the plasmid vector pRK9, a derivative of pBR322, was previously described (MunSz-Rivas et al. 1986b). Plasmid DNA for transformation was prepared from a culture of cells containing clones from the entire gene library.
Isolation of DNA. Plasmid DNA was prepared by CsC1-EtBr centrifugation (Clewell 1972). Small quantities of plasmid DNA were prepared by a rapid boil method (Holmes and Quigley 1981). DNA from S. commune transformants was isolated from mycelia grown in liquid minimal medium (MM) as previously described (Specht et al. 1982). In cases where transformants produced excessive polysaccharides in the growth medium, 0.25% xylose was substituted for 2% glucose in the growth medium. Lambda DNA was prepared by infecting E. coli strain NM539 in liquid culture essentially as described (Maniatis et al. 1982).
Transformation of S. commune. Protoplasts were prepared essentially as described by Mufioz-Rivas et al. (1986a). The protoplast suspension was centrifuged at 250 x g and room temperature (RT) for 5 rain. to pellet spores and debris while protoplasts remained in the supernatant. Protoplasts were recovered in the supernatant and mixed with approximately 10 ml 1 M sorbitol, 20 mM MES, pH 6.3 (SM). Protoplasts were pelleted in conical tubes by centrifugation at 250 x g and RT for 10 rain. After washing with SM protoplasts were suspended in SM (~1 x 107 per 100 #1 SM), adjusted to 40 mM CaC12 and kept on ice for 20 rain. Six to thirty ~tg of DNA from the plasmid gene library in 60/~1 10 mM Tris, pH 7.6, 1 mM Na2EDTA (TE) was adjusted to 40 mM CaC12. The DNA was then mixed gently with 100 /~1 of protoplasts and kept on ice for 15 rain. As a control protoplasts were also treated with 60 #1 of TE adjusted to 40 mM CaC12. An equal volume of polyethylene glycol (PEG; 160~tl of 44% w/v, Mr = 4,000; BDH, Poole, Eng.) was layered into the tubes below the protoplasts. Protoplasts were then centrifuged into the PEG at 250 x g at RT for 5 min. Protoplasts and PEG were resuspended in 5 ml CYM or CYM + tryptophan (trpl strains) containing 0.5 M MgSO 4 and dispensed to 10 cm plastic Petri dishes. Filamentous cells were regenerated from protoplasts by incubation at RT for 1 0 - 1 5 h. Cells were recovered by centrifugation as previously described (Mufioz-Rivas et al. 1986a). To select for ade +, pab +, or ura + transformants, the cells were plated in MM agarose overlays (4 ml each) on four to ten MM agar plates. The selective medium for trp+ transformants was CYM. Transformants were macroscopically visible after 4 8 - 6 0 h at 30 °C. Southern analysis of DNA from transformants. Total DNA ( 2 - 3 /~g) from transformants was restricted, electrophoresed in 0.85% agarose gels and transferred to nitrocellulose (Southern 1975). Nick translation of plasmid pBR322 DNA and DNADNA hybridizations were by standard procedures (Maniatis et al. 1982). Enzymes were from Bethesda Research Laboratories and were used according to the manufacturer's directions.
Recovery of transforming sequences from S. commune transformants as plasmids in E. coli. Total DNA from transformants (10 #g) was partially or totally restricted with one or more of the following restriction enzymes: EcoRI, XbaI, SalI, BamHI, or HindIII. Restriction enzymes were removed and DNA was ligated (5/zg/ml) by the method~ of Maniatis et al. (1982). The ligated DNA was extracted, precipitated (Maniatis et al. 1982) and suspended in TE at a concentration of 50/~g/ml. Portions of the religated fragments (2.5 #g) were mixed on ice with 0.5 ml of E. coli cells made competent as previously described (Dagert and Ehrlich 1979). After 15 min at 0 °C, the mixture was heated to 37 °C for 5 rain and then added to 100 ml of Z broth. The cells recovered for 60 min at 37 °C with shaking. Ampicillin
E. H. Froellger et al.: The isolation of specific genes from the basidiomycete Schizophyllum commune resistant (amp R) cells were selected by adding 100 t~g of ampicillin/ml to the liquid cultures and continuing incubation overnight. At that time some cultures showed no growth; others had grown to about 2 x 109 cells per ml. To isolate plasmid DNA, approximately one ml of the amp R cultures were inoculated into 250 ml Z broth containing 100 #g ampicillin/ml. Plasmid DNA from the amp R cultures was prepared as previously described. These mixed pools of plasmid DNA were used to transform protoplasts from either ade2, ade5, pabl, trpl, or ural strains of S. commune. If the auxotrophs were transformed to prototrophy by the pool of plasmids, portions of the E. coli cultures were spread on Z-broth agar plates containing 100 t~g ampicillin and incubated overnight. Individual colonies were selected and used to prepare plasmid DNA. These plasmid preparations were used to transform the appropriate S. commune auxotroph.
Identification of restriction fragments for probing a lambda library. To identify probes suitable for screening a lambda library it was necessary to identify from the recovered plasmids restriction fragments that contained transforming activity for the gene in question. Simple restriction maps were prepared for the recovered plasmids pADE5, pPAB1, and pURA1. Each gene was mapped using a transformation assay. Restriction digests (0.5-5/zg of plasmid DNA) were transformed into Schizophyllum protoplasts as above; if no transformants were observed, it was concluded that the restriction digest disrupted the function of the transforming sequence. When restriction digests yielded transformants, the activity could be mapped to a specific fragment of the plasmid. Restriction digests that yielded transformants were electrophoresed and DNA from each band was recovered either by electroelution or by heating gel slices of low melting temperature agarose (Maniatis et al. 1982). Individual restriction fragments were used in the transformation assay. Single restriction fragments with transforming activity were identified for ADE5 and URA1. For PAB1 a sequence containing the transforming activity was defined by a duster of restriction sites that destroyed the transforming activity. A lambda clone containing the TRP1 gene had been previously isolated (Mufioz-Rivas et al. 1986b). No attempt was made to recover the ADE2 gene from the lambda library.
Screening the lambda library. The S. commune gene library, in bacteriophage k EMBL4, has been previously described (MufiozRivas et al. 1986b). About 1.6 x 104 PFU were plated on two plates with E. coli NM539 as the indicator cells. Plaques were screened for either the ADE5, PAB1, or URA1 genes of S. commune by plaque hybridization Benton and Davis 1977; Maniatis
549
et al. 1982). Fragments to be used as probes were labeled by replacement synthesis using 32p-dCTP (New England Nuclear) and T4 polymerase as described (Maniatis et al. 1982). Intact lambda DNA was isolated from lambda clones that hybridized to the probes. This DNA was tested for its ability to transform the appropriate S. commune mutants.
Results Transformation o f S. c o m m u n e D N A f r o m the S. c o m m u n e plasmid gene library was used to transform protoplasts carrying either ade2, ade5, pabl, trpl, or ural m u t a t i o n s to p r o t o t r o p h y . A total o f 5 ADE2, 5 ADE5, 5 PAB1, 3 TRP1, and 8 URA1 putative transformants were o b t a i n e d (Table 1). Initially 30 g g o f D N A was used in each e x p e r i m e n t , b u t as the t r a n s f o r m a t i o n p r o c e d u r e was i m p r o v e d (unpublished results), D N A was decreased to 6 #g. Control transformations using buffer w i t h o u t D N A did n o t yield colonies e x c e p t w i t h the p a b l m u t a t i o n which is h y p o m o r p h i c . Cells w i t h this m u t a t i o n produce a background o f slow growing mycelia and transformants were d e t e c t e d as rapidly growing colonies. There was n o background g r o w t h on selective plates w i t h the o t h e r mutants.
Analysis o f transformants Representative transformants o f each gene were selected at r a n d o m and tested for the presence o f vector D N A (Fig. 1) and for m e i o t i c stability o f the transformed phen o t y p e (Table 2). F o r S o u t h e r n analysis, t w o transformants o f each t y p e and a n o n t r a n s f o r m e d c o n t r o l strain ( U V M 4 - 4 0 ) were g r o w n in liquid selective media. T o t a l D N A was isolated f r o m ceils, samples were restricted w i t h E c o R I , and subjected to S o u t h e r n h y b r i d i z a t i o n using p B R 3 2 2 D N A as a p r o b e (Fig. 1). All the transfor-
Table 1. Transformation experiments Mutation
Average # protoplasts per expt.
Average # regenerates per expt.
Per cent protoplast viability
Amount plasmid library DNA used per expt.
Total # experiments
Total # transformants obtained
ural trpl ade2 ade5 pabl
2x 2x 8x 3x 4x
2x 6x 9x 8x 1x
10.0 3.0 1.0 3.0 3.0
30 ttg 30 ~tg 30/~g 6 ~tg 6 gg
20 5 5 5 5
8 3 5 5 5
107 107 106 107 107
106 105 104 105 106
Number of protoplasts determined by direct haemocytometer count. The number of regenerates is estimated by plating an aliquot of protoplasts on nonselective media
E . H . Froeliger et al.: The isolation of specific genes f r o m the basidiomycete Schizophyllum commune
550
Fig. 1. Southern analysis of DNA from ten S. commune transformants and a n o n t r a n s f o r m e d (control) strain. Analyses of two transf o r m a n t s are s h o w n for each m u t a t i o n used in transformation experiments. Genetic symbols are described in Materials and methods; for transformants, the second digit refers to a particular isolate. Samples o f total DNA from transformants and the n o n t r a n s f o r m e d strain, (HVD4-40) were digested with EcoRI and fractionated by electrophoresis in 0.85% agarose gels. Blots were hybridized with radiolabeled p B R 3 2 2 DNA. Plasmid pBR322, restricted with EcoRI, was added to one sample of HVD4-40 DNA as a positive control for hybridization. Bars to the left of each lane indicate the positions of molecular weight standards which are, from top to b o t t o m , 23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kilobases
Table 2. Marker distribution of h o m o k a r y o t i c segregants from transformants Expt.
Transformant
# Spores
+: - : nonviable
x2 1 : 1
X2 1 : 1 : 2
1 2 3 4 5 6 7 8 9 10 11
ade2-4 + ade5-2 + ade5-3 + pabl-1 + pabl-3 + pabl-4 + trpl-1 + ural-1 + ural-2 + ural-7 + ural-8 +
94 66 63 59 63 68 98 64 66 91 96
27:19:48 21:45 24:39 29:30 38:30 40:23 48:50 36:28 30:36 41:40 21:29:46
8.0 3.1 0 0.72 4.1 0.01 0.77 0.38 0 -
1.07 1.15
(D) (D) (D) (D) (D) (D) (D) (H) (D) (D) (D)
P (1 : 1)*
P (1 : 1 : 2)*
0.05-0.1 > 0.99 0.3-0.4 0.04-0.05 0.9-0.95 0.3-0.4 0.5-0.6 > 0.99 -
0.5-0.6 0.001-0.005 0.5-0.6
P r o t o t r o p h y is designated by +; a u x o t r o p h y by - . (D) refers to transformants recovered as dikaryons; (H) to those recovered as h o m o karyons. * Significant at P = 0.05
mants
contained
DNA.
The
pBR322
sequences complementary
size o f
the
fragments
ranged from about 2 to approximately
DNA from the nontransformed hybridize
to pBR322
that hybridized
to the probe
UVM4-40
to
23 kb.
strain did not
(negative control).
A positive
control (UVM4-40
DNA + 2 ng pBR322
that
was possible if pBR322
hybridization
DNA) shows sequences
were present. With unrestricted DNA from transformants (data not shown), there was no evidence of rapidly migrating
DNA
indicative
of
autonomously
replicating
E. H. Froeliger et al.: The isolation of specific genes from the basidiomycete Schizophyllum commune plasmids and hybridization with pBR322 occurred at the same position as the high molecular weight chromosomal DNA ( > 80 kb). This suggests that vector sequences are integrated into the genome of the transformants and that unintegrated copies of the vector are not present in the transformants. Eleven transformants were tested for meiotic stability (Table 2) by fruiting the dikaryotic (i.e. fertile state) mycelium of transformants. When plating protoplasts derived from basidiospores at high density, dikaryons were usually established on selective media during transformation. This occurs when a homokaryotic (haploid) transformant mates with a compatible homokaryotic cell (not transformed for the selective marker) shortly after plating on selective media. In one case (Table 2, Expt. 8), where the transformant was recovered as a homokaryon, the transformant was mated with a compatible homokaryotic strain carrying the same auxotrophic mutation. Spores were germinated on nonselective agar medium and portions of the mycelia were transferred to selective and nonselective media. Seven of the eleven transformants (Expts. 3, 4, 5, 7, 8, 9, 10) gave a 1 : 1 ratio for prototrophy versus auxotrophy indicating that the transforming DNA segregates stably as a single gene in meiosis. For Experiments 1 and 11, approximately half of the segregants were not viable on nonselective media. Those viable gave a 1:1 ratio for prototrophy versus auxotrophy. It is not clear why half of the segregants were not viable on nonselective media. One possibility is that more than one integration event has occurred. The selectable marker segregates stably as a single gene in meiosis, but a second transforming molecule may have integrated into another linkage group causing a recessive lethal gene disruption. This is likely since cotransformation with more than one molecule of plasmid DNA has been observed (unpublished results). One pab + dikaryon (Expt. 6) and one ade+ dikaryon (Expt. 2) gave ratios for prototrophy versus auxotrophy that do not fit a 1 : 1 ratio at the level of significance of P = 0.05. We do not understand the basis for these segregation ratios at this time.
Recovery of transforming DNA from S. commune as plasmids in E. coli Because the transforming DNA and plasmid vector sequences frequently integrate together into the genome of Schizophyllum transformants, we attempted to recover the transforming DNA as a plasmid in E. coli (Hicks et al. 1979). This entailed restricting total genomic DNA from transformants and religating the fragments to form circular molecules. We wanted to use conditions that maximized the possibility of generating a viable plasmid that would transform E. coli to ampicillin resistance;
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several factors were considered. First, was the restriction map of the pRK9 vector DNA. Restriction enzymes that cut the plasmid sequences frequently or disrupt either the ampicillin resistance gene or the origin of replication were eliminated as choices. Restriction enzymes that cut plasmid sequences only once (EcoRI, SalI, BamHI) preferably at or near the insert, or not at all (HindlII, XbaI) were considered as possible choices. Because restriction enzymes that preserve critical vector sequences may also disrupt the function of the putative genes, both partial and complete digests of transformant DNA were done. The choice of transformant was also deemed important. Transformants to be used for gene recovery were selected on the basis of Southern analyses of genomic DNA using plasmid pBR322 as the probe. Transformants that yielded fragments hybridizing to pBR322 that were greater than the size of the pRK9 vector (4 kb) were chosen. It was likely that these fragments contained sufficient vector sequences to make plasmid recovery possible. Three ADE2, three PAB1, two ADE5, two TRP1, and three URA1 transformants were selected and grown in liquid MM and total DNA was prepared. The DNA was restricted, religated at a concentration of 5 gg per ml and used to transform E. coli as described. Ampicillin resistant E. coli cultures were obtained with DNA from all of the above mentioned S. commune transformants. Plasmid DNA was prepared from the mixed cultures of ampicillin resistant E. coli cells and used to retransform the appropriate S. commune mutant to prototrophy. The pool of plasmid DNA generated from one PAB1 transformant (partially restricted with EcoRI), one ADE5 transformant (completely restricted with EcoRI), one ADE2 (partially restricted with EcoRI), one TRP1 (completely restricted with EcoRI), and from two different URA1 transformants (partially restricted with EcoRI) were able to transform the appropriate auxotrophic mutants to prototrophy at frequencies of 10 or more transformants//~g of mixed plasmid DNA. Plasmid DNA was then extracted from individual E. coli clones and for each gene one plasmid with sequences able to transform the appropriate S. commune mutant was recovered. Plasmids of the following sizes were obtained: pADE2 (14.0 kb), pADE5 (12.8 kb), pPAB1 (14.5 kb),pTRP1 (4.6 kb), and pURA1 (18.5 kb). Transformation frequencies using these plasmids as DNA ranged from 100-500 Schizophyllum prototrophic transformants per pg DNA. Genes were not recovered from every transformant used. Among the transformants tested, one of three ADE2, one of two ADE5, one of three PAB1, one of two TRP1, and two of three URA1 transformants yielded amp R plasmids that were also able to transform the appropriate S. commune mutant to prototrophy. Loss or rearrangements of essential vector sequences during
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E. H. Froeliger et al.: The isolation of specificgenes from the basidiomyceteSchizophyllum commune
integration, excision, or both may be responsible for the failure to recover specific genes from some transformants.
Isolation o f the ADE5, PAB1, and URA1 genes from a lambda library A 3.8 kb BamHI-KpnI fragment from pADE5 was able to transform. Schizophyllum ade5 mutants to prototrophy. Restriction analysis showed there was one Sail site in the central region of this fragment. Restriction with Sail destroyed transforming activity suggesting that the Sail site is within the ADE5 gene. The 3.8 kb BamHI-KpnI fragment from pADE5 was used as a probe to screen a Schizophyllum gene library in X EMBIA for the ADE5 gene. One lambda clone that hybridized to the probe was recovered. Restriction analysis showed the lambda clone contained the 3.8 kb BamHI-KpnI restriction fragment with the centrally located Sail site. Total DNA from the clones transformed ade5 mutants to prototrophy at a frequency of about 500 transformants//2g h DNA. From pURA1, we identified a 2.8 kb BamHI fragment that transformed Schizophyllum ural mutants to ura +. Restriction analysis showed there were two HindlII sites within this fragment. Restriction of this fragment with HindlII destroyed transforming activity suggesting that one or both of the HindlII sites are within the URA1 gene. The 2.8 kb BamHI fragment was used to probe the lambda library for the URA1 gene. Three different lambda clones that hybridized to the probe were recovered. Restriction analysis showed each lambda contained the 2.8 kb BamHI fragment; the two HindlII sites within this fragment were also present. Total DNA from each of these three lambda d6nes was able to transform ural mutants to ura + at a frequency of about 500 transformants//~g of X DNA. Restriction of the lambda clones with HindlII destroyed transforming activity. The transforming activity of pPAB1 was shown to span adjacent (0.7 and 4.0 kb) SALI restriction fragments. Restriction sites for BamHI, EcoRI, BgllI,XhoI, SstI, and SstlI were clustered around the SalI site separating both fragments. Restriction at any one of these sites destroyed transforming activity suggesting that these sites are within the PAB1 gene. The 4.0 kb SalI fragment from pBAB1 was used to probe the lambda library for the PAB1 gene. Three different lambda clones that hybridized to the probe were recovered. Restriction analysis showed each clone had the two adjacent Sail fragments (0.7 and 4.0 kb). The duster of restriction sites around the Sail site in common to both fragments was also present. Total DNA from each of these three lambda clones transformedpabl mutants to prototrophy at a frequency of about 500 transformants//~g of X DNA.
Discussion
The ability to isolate specific genes from SchizophyUum is important for the further development of genetic molecular analyses in this organism. The data presented in this paper show it is possible to complement Schizophylhim mutations with DNA from a plasmid gene library and that the transforming sequences can be recovered from chromosomal DNA as amp g plasmids in E. coli. The direct isolation of sequences capable of complementing a mutation in a specific gene requires a relatively high transformation frequency. The initial transformation frequency we reported for S. commune, using the Schizophyllum TRP1 gene isolated by complemantalion of trpC in E. coli, was up to 30 transformants//~g of plasmid DNA (Mufioz-Rivas et al. 1986a). The transformation protocol described in this report is a modification of the initial protocol (Mufioz-Rivas et al. 1986a) and yields from 500 to 2,000 transformants//2g of plasmid DNA. This frequency was sufficient to obtain complementation of all five Schizophyllum mutations selected. For several yeasts and other filamentous fungi, high frequency transformation systems have been developed to the point where genes have been routinely isolated from these organisms by complementation of mutants. For the yeasts S. cerevisiae and Schizosaccharomyces pombe transforming DNA can be introduced as a replicating plasmid that can be easily recovered from transformants (Beggs 1978; Beach and Nurse 1981). Transformation frequencies of 104-105 transformants per/2g DNA have been reported for yeast and many yeast genes have been isolated by complementation of yeast mutations (Struhl 1983; Beach et al. 1982). Transformation of the filamentous fungi Phycomyces blakesleeanus to G-418 resistance by an autonomously replicating plasmid has been reported (Revuelta and Jayaram 1986). However, attempts have been made to construct replicating plasmids for other filamentous fungi for use in transformation, but this has not been a practical strategy for gene isolation (Hynes 1986). Our data suggests that transformation in S. commune occurs by integration of complementing DNA and bacterial plasmid sequences. In other filamentous fungi such as A. nidulans and Neurospora crassa, the transforming DNA is usually cloned in a bacterial plasmid, cosmid, or bacteriophage vector and transformation usually occurs by integration (Hynes 1986). Integrative transformation also occurs in yeast (Struhl 1983). Integrative transformation frequences reported in these systems ranges from 101 to 104 transformants per/ag of DNA (Struhl 1983; Vollmer and Yanofsky 1986; Balance and Turner 1985). In systems where transformation occurs by integration the ability to recover the integrated sequences from chromosomal DNA is essential. Because the integrated transforming sequence remains linked to the bacterial
E. H. Froeliger etal.: The isolation of specific genes from the basidiomycete Schizophyllum commune sequences in many Schizophyllum transformants, it was possible to recover them as amp R plasmids in E. coli. This procedure has not always been successful with other fungi. The rescue of integrated transforming sequences from N. crassa as plasmids has not been successful, perhaps because essential bacterial plasmid sequences have been lost (Case 1982). Consequently, a different approach for recovering integrated transforming sequences, sib selection, was developed to overcome this problem. Sib selection involves repeated transformations of N. crassa mutants using subdivisions of a genomic library until a single transforming plasmid is identified (Akins and Lambowitz 1985). For S. cerevisiae and A. nidulans integrated transforming sequences have been recovered by restricting total DNA from transformants, rehgating the fragments, and transforming E. coli (Hicks et al. 1979; Balance and Turner 1985). Also forA. nidulans, transforming sequences that were cloned in cosmid vectors have been recovered by lambda packaging of transformant genomic DNA and rescued as a plasmid in E. coli (Yelton e t a l . 1985). Many genes from yeast, Aspergillus, and Neurospora have been isolated using the above mentioned strategies (Struhl 1983; Herskowitz 1985; Hynes etal. 1983; Yelton etal. 1985; Johnstone etal. 1985; Balance and Turner 1986; Akins and Lambowitz 1985; Vollmer and Yanofsky 1986). Several lines of evidence suggest that the cloned sequences recovered from SchizophyUum code for the structural gene being sought. First, the DNA used to construct the plasmid library is from a well characterized wild-type strain in which no evidence for extragenic suppression has been observed despite concerted attempts to generate and identify suppressors for most of the genes (ade2, ade5, pabl, ural) sought in this study (Parag 1970). Second, analyses of crosses between mutants for each of the genes studied here and wild-type strains show a 1 : 1 segregation ratio for wild-type versus mutant progeny. This indicates that wild-type strains do not normally contain extragenic suppressors of auxotrophic mutations. Furthermore, we isolated from a lambda library clones that hybridize to the putative ADE5, PAB1, and URA1 sequences recovered from transformants. DNA from each of these lambda clones has a restriction map in common with the sequences contained in the respective transforming plasmid. Each of the lambda clones also transforms the appropriate S. commune mutants to prototrophy. Because these clones were isolated from a lambda library that was never subjected to nutritional selection, it is unlikely that the cloned sequences contain spontaneous suppressor mutations. Finally, it is clear that one of the genes recovered, TRP1, is the actual gone and not a suppressor of TRP1. The TRP1 sequence recovered from a transformant by plasmid rescue has a restriction map corresponding to the S. commune TRP1 gone isolated by comple-
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mentation of trpC in E. coli. Subsequent studies showed this gene to have indole-3-glycerol phosphate synthetase activity (Mufioz-Rivas etal. 1986b). The methods used in this study make it feasible to isolate directly SchizophyUum genes. This adds a new dimension to the genetic analysis of S. commune in that it should now be possible to identify essentially any SchizophyUum gene for which mutants can be obtained. Mating-type mutants for both the A and the B loci exist; these include mutants which have lost various degrees of mating-type function as well as mutants which are constitutive in mating-type function. There are many developmental genes scattered throughout the genome that are expressed only during sexual morphogengenesis and are regulated by the mating-type genes. Mutants also exist for these genes (see Raper 1983 for a review of mating-type and developmental mutants). Attempts are underway in our laboratories to use procedures described in this report to isolate several SchizophyUum genes involved in mating-type and sexual development. Acknowledgements. Thisresearch was supported by the Vermont Agricultural Experiment Station, University of Vermont, Burlington, by National Science Foundation Grant No. PCM-8402107 and by National Institutes of Health Grant No. GM34023.
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C o m m u n i c a t e d b y C. P. Hollenberg Received July 10, 1987
