transformation ofschizophyllum commune: an analysis of specific properties

EXPE~MENTAL MYCOLOGY IS, 326-335 (191)

Transformation of Sc~~zo~~y//~~ commute; An Analysis of Specific Properties

CHARLES A. SPECHT, *J ALFREDO Mw~~oz-RIVAS, *72 CHARLES P. NOVOTNY,? AND ROBERTC.ULLRICH*

D~~~~~~~~i~ o~~Bota~y, and ~M~crobioiogy and Molecular Genetics, Universi@ of Vermont* Burlington, Vermont 05405

Accepted for publication July 27, 1991

SPECHT, C. A, MUFJOZ-RIVAS, A., NOVOTNY, C. P., AND ULLRICH, R. C. 1991. Transformation of Schizophyllum commune: An analysis of specific properties. Experimental Mycology 15, 326- 33.5. Several properties of transformation in the basidiomycete, SchizophylIum commune, were examined. The ~ansformation efficiency of protoplasts made from germ~ating basi~os~res is dependent upon the length of time that the spores are incubated under conditions that promote germination. Protoplasts prepared from ungerminated spores transform at least 10 times more efficiently than protoplasts prepared from germlings (25 km in length) or from mycelium. Trans- formation fr.equencies of 1000 transformantslpg of control plasmid DNA and 10’ protoplasts are sufficient for obtaining transformants with 2 x 107 protoplasts and 10 pg of bar&DNA from a genomic plasmid library. The probability of cotransfo~ing with two plasmids is dependent on the DNA concen~~ons of each; ~oncen~ations can be adjusted to yield nearly 100% cotransformants. The -presence of a nonselected plasmid in the reaction mix improves the transformation frequency of a selected marker carried on another plasmid; this is not true if linear fragments of Schizophyllum genomic DNA are used as the nonselected DNA. Transformation of a Schizophyl- lum protoplast does not require its fusion to another protoplast. o 1991 Academic press, I~C.

INDEX DESCRIPTORS: Schizophyllum commune; fungi; protoplast; protoplast fusion; transformation; t~nsfo~ation frequency; transformation efficiency; genomic library; cot~sfo~ation; car- rier DNA.

INTRODUCTION

Genetic transformation is a versatile tool that can be used for recovering genes, studying gene expression, and introducing new genetic traits. Although transformation is being used to study genetics in a rapidly increasing number of filamentous fungi, transformation and the mechanisms for uptake of DNA by cells and recombination into the chromosomal DNA are understood in only the most rudimentary terms (for re- view see Fincham, 1989; Timberlake and Marshall, 1989).

’ Present address: Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139.

2 Present address: Campina 62, Col. Pastores, Nauchlpas, Edo. Mex., Mexico.

Previously we defined conditions (e.g., concentrations of DNA, protoplasts, CaCl,, and PEG3) used to attain transformation frequencies of l-2000 transformants/~g of plasmid DNA when using 10’ protoplasts of Schizophyllum commzme Fr. (Specht et al., 1988). This paper examines related features and uses of that transformation protocol. For example, because our previous study ident~ed substanti~ variation in the transformation efficiency of protoplasts prepared from different lots of spores, transformation efficiency is examined here as a function of protoplasts pre-

3 Abbreviations used: PEG, polyethylene glycol; IGPS, indole-3-glycerol phosphate synthetase; CYM, complex yeast medium; CYMT, CYM plus tryptophan; MM, minimal medium; OM, magnesium osmoticum; SM, sorbitol osmoticum.

326 0147-5975191 $3.00 Copyrisht 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

TRANSFORMATION OF S. commune 327

pared from mycelium or from spores in different stages of germination.

Cotransformation of Escherichia coli (Kretschmer et al., 1975) and yeast (Hicks et al., 1979) first demonstrated that a com- petent cell may readily take up and express more than one DNA molecule. Cotransfor- mation has become a powerful feature of transformation systems because it allows recovery of screenable, but nonselectable, genes by coupling transformation of the nonselectable gene to that of a selectable marker. Cotransformation is studied here as a function of the concentrations of the two transforming DNAs. During the course of the cotransformation study, we observed that a second plasmid included in the transformation mix promotes the transformation efficiency of the first. Therefore, the influence of additional DNA in the transformation mix and its effect upon transformation frequency (transformants/p,g DNA) are re- corded.

Previous studies have documented that auxotrophic mutations of Schizophyllum can be complemented by transformation with DNA from a Schizophyllum gene library (Froeliger et al., 1987). We repeated these transformations with DNA from a genomic library, selecting for adef or trp’ transformants. Each preparation of protoplasts was also transformed with a control plasmid containing either the TRPl or the ADES gene. The transformation frequencies with DNA from the gene library and the control plasmids were compared. These data provide a means to relate the frequency of transformation with a control plasmid to that necessary to obtain transformation with DNA from genomic plasmid libraries.

The fusion of yeast protoplasts is reported to occur during transformation

s et al., 1979); some yeast transformants become polyploid during this pro- cess,. Fusion has been suggested as a required event in the general mechanism for yeast transformation (Harashima et al.,

1984; Brzobohaty and Kovac 19 have examined whether transfo~a~t~ of Schizophyllum are derived from proto fusion events.

MATERIALS AND MOTHERS

Transforming Protoplasts Prepare from Spores

Procedures for obtaining spores, prepar- ing protoplasts, and performing transforms- tions are as previously reported (Specks et al., 1988). The dikaryotic strains of $. commune that were used are: UVM 8-87 (frpB) X UVM 8-89 (trpl), UVM 8-90 ( x UVM 8-91 (trpl, ade5), and (trpl STY, ad&) X UVM 8-92 ade5). The strain numbers refer to the homokaryotic strains that were mate is a mutation for indole-3-glycerol phate synthetase (IGPS), ade.5 blocks bio- synthesis of adenine by a mutation of the

phosphoribosyl-aminoimidazole sy~t~~tase gene (Alit et al., 1990), and sty is a reces- sive, morphological mutation that seduces colonial size.

Making Protoplasts from ~yce~i~~

Mycelium of homokaryotic st 8-87 (l-2 cm2 of a mycelial mat) to 50 ml of complex yeast medium (CY Raper and Hoffman, 1974) containin plementary (4 mM) tryptophan (C and blended for I min at 20,000 Eberbach semimicro co blended cultures were shak for 24 h at 30°C. Blending was r the cultures were incubated wi for 20 h. Mycelium was harvest trifugation and transferred to 0.5 50 m&Y maleic acid, pH 5.9 and Wessels, 1972). Gne milli cells was washed twice with (collecting the cells by centrifu~atio~ at 2509 each time) and resuspended in of OM; 0.5 ml Novozym 234 stoc

328 SPECHT ET AL.

added (100 mg of batch No. 1199/ml of OM), and the cells were incubated at 30°C for 4 h. Protoplasts generated from mycelium were purified and transformed as described for those from spores.

Light Microscopy

A hemocytometer was used for counting spores and protoplasts. Spore lengths were measured with an ocular micrometer cali- brated in micrometers with a stage micrometer.

Plating

Aliquots of regenerated protoplasts were mixed with 4 ml of CYM medium (42°C) containing 1% (w/v) Sea Plaque Agarose (FMC Corp.). This was overlayered in plates containing either CYM or aspar- agine-glucose-salts minimal, semisolid media (MM; Schwalb and Miles, 1967). The MM was supplemented with 0.1 m&I adenine when selecting for TRPl transformants and with 4 mM tryptophan when selecting for ADE.5 transformants. ADES and TRPl double transformants were selected on MM.

DNA for Transformation

Three plasmids were used in transformations. Two plasmids containing TRPl, pAM1 and pSc1, have been previously described (Munoz-Rivas et al., 1986a; Specht et al., 1988). Plasmid pAM7 complements the S. commune ade5 mutation (Froeliger et al., 1987).

The plasmid gene library used to isolate the PAM plasmids was constructed in the vector pRK9 (Schechtman and Yanofsky, 1983) and consists of 135,000 clones of which 77% contain plasmids with inserts averaging 5.6 kb (Munoz-Rivas et al., 1986b). The library was divided into three subsets consisting of 73,000, 34,000, and 28,000 clones. The 28,000 clone subset was

further divided into six subsubsets of 4700 clones each.

Plasmid DNA was prepared as previously described (Specht et al., 1988). Schizophyllum DNA was prepared by our methods (Specht et al., 1982).

RESULTS AND DISCUSSION

Effect of Germination Time on Transformation

The rapid physical and physiological changes that attend spore germination may influence the properties of the protoplasts derived. We speculated that these changes may be responsible for the variation in transformation efficiencies previously reported for protoplasts derived from different batches of spores (Specht et al., 1988). Consequently, we studied transformation efficiency as a function of the length of time that the spore has been incubated under conditions that support germination (or its corollary, length of the germinating spore). The results of this experiment are presented in Fig. 1. The data show a correlation between the duration of incubation for spores in CYMT and spore (germling) length and an inverse correlation with transformation efficiency. Ungerminated spores are nearly uniform in length. As spores germinate germ tubes are produced; these lengthen, but not uniformly, as evi- denced by the increase in standard deviations with incubation time. Each batch of spores varied slightly in the rate of elonga- tion between 8 and 16 h with faster rates associated with lower spore concentrations .

Protoplasts prepared from each batch of spores showed a decrease in transformation efficiency as germination progressed. The rate of decline was slightly different for each batch; however, the greatest rate of decline always occurred between 0 and 2 h, a period when spores have changed little in size. This suggests that a physiological


SPORE LENGTH NSFORMANTS

0 2 4 6 6 ” 12 16

GERMINATION TIME (hr)

FIG. 1. Effect of duration of incubation on elonga- tion of germ tubes and efficiency of transformation of protoplasts produced from germinating basidiospores. Three batches of spores were collected from fruiting bodies 4-6 days after sporulation had begun and then stored 7-10 days in water on ice. The spores were incubated with shaking in CYMT at 30°C for the times shown. Spore concentrations were 1.3, 1.4, and 1.6 x IO’ spores/ml. For each sample, spore (and germling) lengths were measured microscopically with an ocular micrometer (n = 30) and averaged. For transformations, protoplasts in 100 ~1 SM, 50 miW CaCl, were mixed with 1 p,g of plasmid pSc1 in 60 ~1 TE, 50 m&f CaCl,. Samples were mixed with 160 )~l 50% (w/v) PEG (M, = 3350, 9. T. Baker); Protoplasts were regenerated in CYM, 0.5 MMgSO,. Transformants were selected on CYM agar. When other than 5 x lo6 protoplasts were used in a transformation mix, the number of transformants was corrected by the factor (5 x lo61 X)liZ; X is the number of experimental protoplasts. In the heading # TRPl TRANSFORMANTS*, the (*) indicates that the data have been normalized for a standard transformation mix containing 5 x lo6 protoplasts and 1 pg of DNA. The data presented are averages for three batches of spores, spore lengths with standard deviations, and average number of transformants. Standard deviations for transformation data were on the average 35% of the mean (not shown).

change, in addition to cell size, is important in determining protoplast competency for transformation. These observations may explain variability in transformation efficiency for protoplasts prepared from different batches of spores. Unless special cau- tion is used to isolate batches of spores in the same germination state from sporulat- ing cultures, variation in transformation efficiency may prevail.

Transforming Protoplasts from Homokaryotic Mycelium

Because the mycelium is the the fully germinated spore we to examine the efficiency of with protoplasts generated from myceIi~m. It requires about 4 h incubation w zyme to enable recovery of 2-5 x 1 toplasts from 1 ml of pelleted these experiments, myceli homokaryotic strain UVM 8-87 was incubated with Novozym 234, and release was monitored microsco significant increase in the numb plasts was seen after 4 h (data not show

Five preparations of mycelia were u to make protoplasts. The averag of protoplasts purified was 2.9 x ml of packed cells. From each bat lo6 protoplasts were trans of plasmid pSc1 in a 160+1 mix. An average of 40 tr DNA (range 20-70 transforman obtained when DNA and proto~last concentrations were normalized for a transformation mix containing 5 X 106 and 1 pg of DNA. This is similar to transformation efficiencies observed for protoplasts made from the germlings of s germinated for 16 h (Fig. 1). resent the low end of trans ciencies that was being approac mination progressed. Part of the decline ; attributable to a reduction in the protoplasts that regenerate, der to undefined physiological factors. toplasts from homokaryot germlings 25 urn (16 h) in le 10 times fewer transformants t plasts from ungerminated many regenerates (data no

Eflect of Nonselected DNA ~rz Transformation

In the course of our cotransformatio~ experiments (discussed below) it became

330 SPECHT ET AL.

parent that the presence of one plasmid had effects on the transformation efftciency of a second plasmid containing the selected marker. Therefore, we pursued the following set of experiments. When increasing amounts of nonselected plasmid, e.g., pBR322, were added to 5 kg of pAM1 DNA, and used to transform protoplasts to trp+, the number of trp+ transformants increased (Table 1). Approximately 1.6 times more transformants were obtained when 5 pg of pAM1 was mixed with 5 pg of plasmid lacking the selected marker. This result may be specific for plasmid DNA because restricted (SadA) Schizophyllum genomic DNA did not enhance transformation. The result may also derive from homology between the vector of the transforming DNA and the nonselected DNA. In either case it is likely that transformation using plasmid library DNA may also enhance transformation rates because the bulk of the DNA should behave as though it were nonselected plasmid DNA with homology to the vector carrying the selected trait.

Cotransformation

Cotransformation makes it feasible to re- cover transformants for a nonselectable marker by simultaneously transforming

TABLE 1 Effect of Nonselected DNA on

Transformation Frequencies

Relative IJS I-G I% number

pAM1 pBR322 Schizophyllum of Trp+ DNA DNA DNA transformants

5 0 0 1.00 5 0.5 0 1.13 5 2.5 0 1.38 5 5.0 0 1.60 5 0 0.5 0.63 5 0 2.5 0.88 5 0 5.0 0.84

Note. Data using pBR322 are averages of two experiments and data using Schizophyllum DNA cut with Sau3A averages of three experiments.

with a DNA molecule containing a selectable marker. The number of cotransformants should be dependent on the concentration of each DNA, with increasing concentrations approaching a theoretical limit defined by transformation with single DNA molecules. We have used a double auxotrophic mutant and plasmids encoding comple- menting wild-type genes to test this hypoth- esis in a quantitative way.

A series of experiments was performed to examine cotransformation as a function of the absolute and relative concentrations of the two transforming DNAs (Table 2). The number of ade+ transformants in- creases with increasing amounts of plasmid pSc1 (the TRPI-containing plasmid) even though the amount of ADES-containing plasmid (pAM7) is held constant (Table 2A). This result is consistent with the effect of nonselected plasmid DNA enhancing transformation (discussed above). Also the number of cotransformants (ade+ and trp’ double transformants) increased as the amount of pSc1 DNA was increased (Table 2A). Cotransformation was frequent; even when the ratio of pScl:pAM7 was 1:8 (0.125 pg: 1 pg, respectively). Fifty-two percent of the ade+ transformants and es- sentially all of the trp’ transformants were also transformed with the other gene (TRPl and ADES, respectively). When equal amounts of pSc1 and pAM7 were used, the number of ade+ transformants and the number of trp+ transformants were about equal, indicating both plasmids transform with comparable frequencies (Table 2B). However, when equal, but increasing, amounts of pSc1 and pAM7 were used in a series of transformations (Table 2B), the ratio of cotransformants to single transformants increased. The data suggest that at saturating DNA concentrations this ratio could approach 1 .OO.

There is the possibility that two individuals each transformed for the opposite marker could fuse by virtue of a compatible mating or form a heterokaryon driven by

TRANSFORMATION OF S. cmmnune

TABLE 2A &otransformatiou

Plasmid used Transformed phenotype Ratio of cotr~s~ormants (PEd (number of transformants) to single tr~~~~rrnants

PSCl pAM7 ade+ and Single transformant

TR?! Am3 trp+ de+ trp’ ade+ trp+

0 I 0 530 0 Q.QQ - 0.125 1 310 660 34 0.52 1.10 0.25 1 490 620 420 0.68 0.86 Q.5 1 690 750 520 0.69 0.75 1 1 820 780 590 0.76 0.71 2.5 1 1220 820 660 0.80 0.54 5 I 1560 904 760 0.84 0.49

nutritional selection. The products of these reactions would mimic cotransformants. Therefore, we included the following control expe~ment. One aliquot of protoplasts was transformed with one plasmid; a second aliquot was transformed with the other plasmid. After regeneration overnight the cells were mixed and plated on MM. No cells prototrophic for both markers were recovered when scored during the usual as- say period. This control experiment dem- onstrates that cotransformants are the result of two (or more) transformation events per cell and not the result of interactions between two transfo~ed individuals.

~otransformation confirms that more than one molecule of DNA is being taken

up and stably incorporated by eorn~~~e~~ protoplasts. Data in Table 2 suggest two al- ternative ways to ensure a high ~r~que~~~ of cotransfo~ation even if tra~sfo~~a~t~ for the second DNA cannot rectly. One is to make the ratio of se DNA to nonselectable DNA 1 1: lo), the other is to keep the two equal concentration, but high saturate the proto~lasts being

Transforming ~S~hizophyl6um with from Plasmid Gene Libraries

This section provides data that s useful to researchers deve~op~~~ trans~~~~ mation systems in fungi. The data relate the

TABLE 2B Cotransformation

Plasmid used (WI

pSC1 pAM7 TRPl ME5

Transformed phenotype Ratio of cot~ansform~ts (number of transformants) to single tra~sform~~t~

-- ade+ and

w’ ade+ trp+ Average

0.1 0.1 60 80 20 0.29 0.25 0.25 210 270 I40 0.58 0.5 0.5 ‘MO 480 330 0.72 1 1 890 710 680 0.85

Note. For transformation, 5 x lo6 protoplasts derived from the following dikaryon (UVM g-90 a&5 w~I x UVM 8-91 a&5, trpI) were mixed with plasmid DNA as indicated in a 160+1 transformation mix. Protoplasts were regenerated overnight in MM, 0.5 &f MgSO, and then plated on selective media. Trp+ tr~sfo~~a~~s were selected on MM containing 0.1 n&4 adenine; ade + tr~sfo~~ts were selected on MM conta~iu~ 4 octopi; and de + trp’ cotransformants were selected on MM.

332 SPECHT ET AL.

TABLE 3A Transformation Frequencies Using Subsets of Plasmid Bank DNA

DNA

Number of Transformation frequency transformants transformants/ug

Expt 1 Expt 1

TRP ADE TRP ADE

10 subset 1 (73,000 pg clones) 99.9% completea 6 0 0.6 0.0

10 subset 2 (34,000 pg clones) 99.0% complete” 4 5 0.4 0.5

10 subset 3 (28,000 ug clones) 99.0% complete” 4 0 0.4 0.0

2 PSCL I% 1400 700 2 pAM7 w 1850 925

a The method of Clarke and Carbon (1976) was used to estimate the probability that each subset includes every sequence of the Schizophyllum genome at least once.

number of transformants obtained with by Specht et al. (1988). The results are re- plasmid DNA extracted from various num- ported in Table 3. TRPZ transformants bers of clones (e.g., genomic library, sub- were recovered using 10 t.r,g of plasmid sets, and subsubsets thereof) to those ob- DNA from each of the three subsets of the tained with the DNA of single control plas- gene library and from four of the six sub- mids (i.e., pSC1 containing TRPI or pAM7 subsets. ADE.5 transformants were recov- containing ADES). DNA from a plasmid ered from one subset; no subsubsets gene library was used to transform trpl or yielded ADES transformants (data not ade5 auxotrophs to prototrophy using the shown). Verification that putative transfor- optimized transformation protocol reported mants were true transformants and that

TABLE 3B Transformation Frequencies Using Subset 3 DNA

DNA

Number of TRP transformants

Expt 2 Expt 3 (5 l-4 (10 IJd

Transformation frequency transformants/p,g

Expt 2 Expt 3 (5 IKid (10 l-4

Subsubset 3-l (4700 clones) Subsubset 3-2 (4700 clones) Subsubset 3-3 (4700 clones) Subsubset 34 (4700 clones) Subsubset 3-5 (4700 clones) Subsubset 3-6 (4700 clones)

1 kg plasmid pSc1 2 pg plasmid pSc1

0 2 0.0 0.2 2 3 0.4 0.3 0 4 0.0 0.4 2 6 0.4 0.6 0 0 0.0 0.0 0 0 0.0 0.0

710 710 2130 1065

Note. For transformation Expt’s 1 and 3, 10 pg of bank DNA or 2 ug of control plasmid DNA was added to 2 x lo7 protoplasts (Expt 1: UVM 8-90 X UVM 8-91; Expt 3: UVM 8-87 X UVM 8-89) in a 320~u.1 transformation mix. For transformation Expt 2, 5 pg of bank DNA or 1 pg of control plasmid DNA was added to 4 x lo6 protoplasts (UVM 8-87 X UVM 8-89) in a 160~pl transformation mix. Each subsubset is calculated (Clarke and Carbon, 1976) to have a 45% likelihood that each Schizophyllum sequence is represented at least once.


transformation for other Schizophyllum mutations is possible has been reported elsewhere (Froeliger et al., 1987). The sta- tistical likelihood that each gene is present in each set is included in Table 3A; however, the completeness of extracted library DNA may not be equivalent to the theoretical completeness of the library because various plasmids and cells may not repli- cate equally well during amplification. The results demonstrate there is no a priori way to anticipate which DNA preparations will yield transformants. On the other hand, the transformation frequencies obtained with control plasmids (pSc1 and pAM7, Table 3) can be used to estimate the transformation frequency anticipated of bank DNA. This is instructive in the development of a transformation system because it suggests the likelihood of transforming for a marker from a gene library. For example, if protoplasts yield 1000 transformants/pg of control plasmid, transformants for unique sequence genes should be obtained with 10 p,g of bank DNA. This guide from Schizophyl- lum may be especially useful to those es- tablishing transformation in other species and hopeful of recovering genes from genomic libraries.

Most Transformed Cells Do Not Arise from Fused Protoplasts

The following experiment was to determine if most transformant rived from protoplasts that have been fu due to the action of PEG. Spores wer lected from the dikaryon resulting mating compatible homokaryotic &rams UVM S-90 (trpl STY, ade.5) and UV (TRPl sty, ad&). Sty is a recessiv phological mutation causing c nial growth. It was recovered in transformant in which the tram DNA integrated adjacent to or w trpl gene. Sty has not been obse vert and maps 0.15-0.7 units Our experiment (Table 4) use derived from these spores and forming plasmid, pAM7, contain If transformants result preponderant protoplast fusion events more) of the ADES trans have wild-type morphology

The data in column A (Table 4) confirm the 1: 1 segregation of the two genotypes colonial morphology anticipated of t cross. Therefore, spores from t were used to make protoplasts for transfor-

TABLE 4 Protoplasts Fused versus Protoplasts Transformed

Percentage of colonies (with specified phenotype) derived from

(W (Cl (E) Anticipated percentage Protoplasts Protoplasts of ADES transformants with

(A) treated treated with (D) the specified phenotypes if Colonial Untreated with CaCl, CaCl,, DNA ADES transformants are derived

morphology spores and DNA and PEG transformants from fused protoplasts”

Wild type 46.0 50.0 54.0 56.0 75.0 QY 54.0 50.0 46.0 44.0 25.0

Note. Spores were from the cross: UVM 8-90 ade5, trpl STY x UVM 8-92 ade5, TRPI sty. (B) No transformation or fusion because PEG was omitted. (C) Fusion and transformation, but the population has not been selected forADE5 transformants. (D) Same population as (C), but ADES transformants were selected. Ceils were grown on CYMT; cells transformed for ADES were selected on MM plus 4 mM tryptophan. Data in each column are based on total colony counts of about 200.

a Assuming pairwise fusions of protoplasts.

334 SPECHT ET AL.

mation with the pAM7 plasmid. Cells were regenerated from protoplasts and plated after each of several steps in the transformation protocol (Table 4, columns B-D). Pro- toplasts mixed with DNA and CaCl, (column B) gave the same percentages of each phenotype as untreated spores. However, the addition of PEG increased the percentage of cells with wild-type morphology (column C), presumably due to the fusion of some protoplasts. Column D contains the results for ADES transformants selected on MM plus 4 mill tryptophan. With respect to the frequency of cells with each colonial morphology, the population of transformants (column D) is no different than the population from which transformants were selected (column C). These frequencies differ substantially from those anticipated if transformants are derived directly from fused protoplasts (column E). Seventy-five percent or more of the ADES transformants should have wild-type morphology if they are derived from fused protoplasts. The results demonstrate that fusions of protoplasts are promoted by the addition of PEG, but that the population of transformants is not coincident with the population of fused cells. The addition of PEG mediates the uptake of DNA by individual protoplasts; uptake does not require fusion of these protoplasts to others. The mechanism may be analogous to transformation of intact yeast cells in which PEG is required (Ito et al., 1983; Klebe et al., 1983), but for which there is little likelihood of cell to cell fusion because of the presence of the cell wall. More likely PEG mediates the uptake of DNA by a different mechanism, perhaps related to its hydrophobic charac- ter. Addition of an equal volume of 50% PEG to the reaction mix must compartmen- talize hydrophobic and hydrophilic entities within the reaction mix. This may drive the DNA in proximity to protoplasts in ele- vated concentrations, favoring DNA uptake.

ACKNOWLEDGMENTS

This research was supported by the Vermont Agri- cultural Experiment Station, University of Vermont, Burlington; National Science Foundation Grant No. PCM-8402107; and National Institutes of Health Grant No. GM34023. Novozym 234 was a gift from Novo Labs.

REFERENCES

ALIC, M., CLARK, E. K., KORNEGAY, J. R., AND GOLD, M. H. 1990. Transformation of Phanerocha- ete chrysosporium and Neurospora crassa with adenine biosynthetic genes from Schizophyllum commune. Curr. Genet. 17: 305-311.

BRZOBOHATY, B., AND KOVAC, L. 1985. Interaction of plasmid DNA with yeast protoplasts and a mechanism of genetic transformation. FEBS Lett. 183: 211-214.

CLARKE, L., AND CARBON, J. 1976. A colony bank containing synthetic Co1 El plasmids representative of the entire E. coli genome. Cell 9: 91-99.

DEVRIES, 0. M. H., AND WESSELS, J. G. H. 1972. Re- lease of protoplasts from Schizophyllum commune by a lytic enzyme preparation from Trichoderma vir- ide. J. Gen. Microbial. 73: 13-22.

FINCHAM, J. R. S. 1989. Transformation in Fungi. Mi- crobiol. Rev. 53: 148-170.

FROELIGER, E. H., MUAOZ-RIVAS, A. M., SPECHT, C. A., ULLRICH, R. C., AND NOVOTNY, C. P. 1987. The isolation of specific genes from the basidiomycete Schizophyllum commune. Curr. Genet. 12: 547- 554.

HARASHIMA, S., TAKAGI, A., AND OSHIMA, Y. 1984. Transformation of protoplasted yeast cells is directly associated with cell fusion. Mol. Cell. Biol. 4: 771-778.

HICKS, J. B., HINNEN, A., AND FINK, G. 1979. Prop- erties of yeast transformation. Cold Spring Harbor Symp. Quant. Biol. 43: 13051313.

ITO, H., FUKDA, Y., MURATA, K., AND KIMURA, A. 1983. Transformation of intact yeast cells treated with alkali cations. J. Bacterial. 153: 163-168.

KLEBE, R. J., HARRISS, J. V., SHARP, Z. D., AND DOUGLAS, M. G. 1983. A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene 25: 333-341.

KRETSCHMER, P. J., CHANG, A. C. Y., AND COHEN, S. N. 1975. Indirect selection of bacterial plasmids lacking identifiable phenotypic properties. J. Bacte- riol. 124: 225-23 1.

Mu~;oz-RIVAS, A. M., SPECHT, C. A., DRUMMOND, B. J., FROELIGER, E., NOVOTNY, C. P., AND ULL- RICH, R. C. 1986a. Transformation of the basidiomycete, Schizophyllum commune. Mol. Gen. Genet. 205: 103-106.


MURQZ-RIVAS, A., SPECHT, C. A., ULLRICH, R. C., AND NOVOTNY, C. P. 1986b. Isolation of the DNA sequence coding indole-3-glycerol phosphate synthetase and phospho~bosylant~anila~ isomerase of Schizaphyllam commune. Cum. Genet. 10: 90% 913.

RAPER, 3. R., AND HOFFMAN, R. M. 1974. Schiza- phyllum commune. In Handbook of Genetics (R. C. King, Ed.), pp. 597-626. Plenum, New York.

SCHECHTMAN, M, G., AND YANOFSKY, C. 1983. Structure of the trifunctional trp-1 gene from Neu- rospora crassa and its aberrant expression in Es- ~~~e~~c~ia cofi. J. Mol. A&. Genet. 2: 83-89.

SC~WALB, M. N., AND MILES, P. G. 1967. Morpho-

genesis of Schizophyllum commune. 1. ~~~~~~og- ical variation and mating behavior of the thin mata- tion. Am. J. Bot. 54: 440-446.

SPECffT, C. A., DIWSSO, c., NOYOTNY, e. P., AND IJLLRICH, R. C. 1982. A method for ex~~~~~g big& molecular-weight deox~bonucleic acid from fungi. Anal. Biochem. 119: 158-163.

SPECHT, C. A., MUNOZ-RWAS, A. M., NOVOTW, C. P., AND IJLLRICH, R. C. 1988. Tra~s~o~a~~Qn of Schizophyllum commune: An analysis ofpar~meters for improving transformation frequencies. BP. A@- col. 12: 357-366.

TIMBERLAKE, W. E., AND MA~s~A~~* . A* 1989. Genetic e~~~ee~ng of ~l~eoto~s fungi. Science 244: 13134317.

transformation ofschizophyllum commune: an analysis of specific properties

Documents