strategies for high-efficiency cotransformation ofneurospora crassa

EXPERIMENTAL MYCOLOGY 14, 9-17 (1%))

Strategies for High-Efficiency Cotransformation of Neurospora crassa

BARBARA AUSTIN~ AND BRETT M. TYLER*

Research School of Biological Sciences, Australian National Universi@, P.O. Box 475, Canberra ACT 2601, Australia

Accepted for publication June 23, 1989

AUSTIN, B., AND TYLER, B. M. 1990. Strategies for high-efficiency cotransformation of Neu- rospora crassa. Experimental Mycology 14, 9-17. To facilitate the introduction of cloned genes into Neurospora crassa by transformation, we have identified strategies for carrying out high- efficiency cotransformation of Neurospora, including high-efficiency transformation with very small amounts of DNA. Using the cloned qa-2 and benomyl resistance (p-tubulin; BmlR) genes, we found that the highest rates of cotransformation (8090%) occurred when the selected gene was used at very low levels (~40 r&10’ spheroplasts) and the nonselected gene was used at high levels (1-5 t&10’ spheroplasts). As part of this study we also showed that transformation efficiencies with very small amounts of transforming DNAs are greatly enhanced (lo- to 20-fold, to 104-lo5 transformants/~g) by the presence of carrier DNA. These latter results are directly relevant to strategies for cloning genes by sib selection or by shotgun transformation. Q 1990 Academic press, hc.

INDEX DESCRIPTORS: transformation: cotransformation; Neurospora crassa; DNA; qa-2 gene; benomyl resistance.

The introduction of cloned genes into Neurospora crassa and other organisms by transformation has proven to be a powerful means of identifying cloned genes, deter- mining their functions, and testing the ef- fects of mutations on those genes. In Neu- rospora, several cloned wild-type genes including qa-2 (Case er al., 1979), am (Kinnaird et al., 1982; Kinsey and Ram- bosek, 1984), and pyr-4 (Buxton and Rad- ford, 1983) have been used as routine mark- ers for transformation in conjunction with appropriate auxotrophic mutations. More recently, a mutant p-tubulin gene confer- ring dominant benomyl resistance (Orbach et al., 1986) has been used to transform strains lacking convenient counter- selectable mutations. General purpose transformation vectors for the cloning of

’ Present address: Department of Botany, Austra- lian National University, Canberra, ACT 2601, Aus- tralia.

* Present address and address for reprints: Depart- ment of Plant Pathology, University of California, Davis, CA 95616.

Neurospora genes by expression in Neu- rospora have been constructed using both the qa-2 gene (Akins and Lambowitz, 1985) and the BmlR gene (Vollmer and Yanofsky, 1986), and these have been used to clone a large variety of genes using sib-selection strategies (Akins and Lambowitz, 1985; Vollmer and Yanofsky, 1986; Russell, 1987).

Similar vectors are also routinely used to introduce previously cloned genes into Neurospora in cases when there is no direct selection for the gene of interest. However, in this case the gene must first be ligated into the vector containing the selectable marker (e.g., qa-2 or BmlR). This process can be tedious if there are no convenient restriction sites or, for example, if it is necessary to introduce a large variety of mutant genes by transformation.

An alternative strategy for introducing nonselectable genes is cotransformation: a plasmid (or other vector) carrying the gene of interest is simply mixed with one carrying the selectable marker, and the mixture

9 0147-5975/90 $3.00 Copyright 0 1990 by Academic Press, Inc. AU rights of reproduction in any form reserved.

10 AUSTIN AND TYLER

is used for transformation. In a variety of other organisms including Escherichia coli (Kretschmer et al., 1975), yeast (Hicks et al., 1978), animal cells (Wigler et al., 1979), and Aspergillus niduluns (Timberlake et al., 1985; Wemars et al., 1987), cotransformation frequencies of 2&100% have been observed; i.e., 20-100% of transformants containing the selected marker also con- tained the nonselected marker.

Cotransformation has also been reported for N. crassu [unpublished observation quoted in Vollmer and Yanofsky (1986)]. To improve the utility of cotransformation in Neurosporu, we have developed strategies for optimizing the frequency of cotransformation in Neurosporu. As part of this study, we have also identified proce- dures for achieving high-efficiency transformation when the selectable plasmid con- stitutes only a small fraction of the DNA used for transformation, which are relevant to cloning strategies involving sib selection and shotgun transformation.

MATERIALS AND METHODS

Strains and Plusmids

Plasmid pQa2H/Xh, bearing the qu-2 gene, carried a 2.7-kb HindIII-XhoI fragment derived from pMSK335 (Schweizer et al., 1981) inserted into the Hind111 and &rfI sites of pBR322. The benomyl resistance plasmid pBT6 carried a mutant B-tubulin gene on a 3.1-kb Hind111 fragment inserted into the Hind111 site of pUC12 (Orbach et al., 1986) and was a gift of Marc Orbach and Charles Yanofsky. The N. crussu strain used for transformation was 246-89601-2A (A qu-2 uro-9 inl) (Case et al., 1977) and was a gift of Mary Case.

Transformation

Transformation was carried out using the Novozyme procedure of Vollmer and Yanofsky (1986), including storage of spheroplasts at -80°C. In a “standard”

transformation, 1 pg circular plasmid DNA was incubated with lo7 spheroplasts on ice for 20 min in 110 pl of 1 M sorbitol, 50 mM CaC&, 50 mM Tris, pH 7.4,8% PEG 3350,3 1% DMSO, 50 l&ml heparin (Vollmer and Yanofsky, 1986); diluted to 600 ~1 with 40% PEG 3350,50 mM CaCl,, 50 mM Tris-HCl, pH 7.4 (Vollmer and Yanofsky, 1986); and incubated at room temperature for 20 min or longer. With this protocol, transformation rates with either a qu-2 or a BmlR plasmid ranged between lo3 and lo4 transformants per microgram of DNA, depending on the preparation of spheroplasts. In other experiments, the amounts of DNA or spheroplasts were varied as described. qu-2+ transformants were selected by plating l- to 500-~1 aliquots of the final transformation mixture in 15 ml SFG medium (Vogel’s minimal medium containing 2% sorbose, 0.05% glucose, 0.05% fructose) plus 50 pg/ml inositol, 2.5% agar, and 1 M sorbitol, onto 9-cm Petri plates containing SFG medium plus inositol and 1.5% agar. Benomyl-resistant transformants were selected by plating in SFG medium plus 50 pg/ml inositol, 2.5% agar, 80 pg/ml phenyl- alanine, 80 p&ml tyrosine, 80 t&ml tryp- tophan, 10 kg/ml p-aminobenzoic acid, onto Petri plates containing similar medium (only 1.5% agar and no sorbitol) plus 1 Kg/ml benomyl. Transformant colonies were grown 3-5 days at 30°C.

Counting Transformants

To determine the transformation rate in a particular experiment, several different di- lutions of the transformation mix were plated to obtain at least one set of plates containing between 20 and 100 colonies-a number which could be accurately counted and which yielded a low statistical error.

3 Abbreviations used: PEG, polyethylene glycol; DMSO, dimethyl sulfoxide; SFG medium, Vogel’s medium containing 2% sorbose, 0.05% glucose, and 0.05% fructose.

COTRANSFORMATION OF Neurospora crassa 11

When necessary to reduce crowding, transformants were plated on U-cm Petri plates instead of 9-cm plates. Only strongly growing colonies were counted as true transformants, i.e., those colonies larger than 5 mm in diameter and producing aerial hyphae after 5 days at 30°C. When tested, 80-100% of such transformants proceeded to grow and conidiate when transferred to selective Vogel’s/sucrose medium. More weakly growing colonies were considered abortive transformants and were not included in the calculation of transformation rates. When tested, only lO-20% of this class of transformants continued to grow and conidiate when transferred to selective Vogel’s/ sucrose medium. In a typical experiment, the weak “abortive” transformants out- numbered the strong “true” transformants by one- to twofold. The number of regen- erable spheroplasts in the transformation mix was determined by plating a suitable dilution of the mix on nonselective SFG/agar medium.

Counting Cotransformants

To test for cotransformation, transformant colonies were first retested on Vogel’s/sucrose medium selective for the appropriate gene. Colonies which grew and conidiated were then tested on Vogel’s/ sucrose medium selective for the second gene. For example if qa-2 and BmlR plasmids were used for cotransformation, qa-2 + transformant colonies would be transferred to medium lacking aromatic amino acids and p-aminobenzoic acid (i.e., selective for qa-2+), then retested on medium containing aromatic amino acids and p-aminobenzoic acid plus 1 t&ml benomyl (i.e., nonselective for qa-2, selective for BmfR), to identify BmlR cotransformants. The reverse procedure would be used for identifying qa-2+ cotransformants among BmZR transformants. Colonies were counted as cotransformants if they showed moderate to strong growth on the second

medium and they conidiated. They were not counted if they produced only diffuse mycelia and did not conidiate after 2 weeks at 25°C. Typically, the transformants ex- pressing the cotransformed character strongly (and which were counted as cotransformants) remained fully stable for this character. Transformant colonies which grew poorly and did not conidiate on retesting were also routinely tested for cotransformation, but these only rarely showed evidence of cotransformation.

RESULTS AND DISCUSSION

Preliminary experiments showed that simply mixing the qa-2 and BmlR plasmids at the levels we routinely used for single- gene transformation (1 pg DNA per lo7 spheroplasts) yielded a cotransformation rate of 2&50%; i.e., 20-50% of qa-2 + transformants were also BmlR, and vice versa. To determine optimal conditions for cotransformation, we focused on the rela- tive proportions of spheroplasts, selective DNA, and nonselective DNA.

Optimizing Single-Gene Transformation

Reasoning that the highest efficiencies of cotransformation might be found under conditions where the efficiency of single- gene transformation was highest, we first carried out experiments to optimize the efficiency of single-gene transformation, varying the ratio of DNA to spheroplasts. Transformation efficiency was measured in two ways-as transformants per lo7 spheroplasts and as transformants per microgram of DNA, since either index of transformation could have been important to optimizing cotransformation, depending on the mechanism of cotransformation.

Figure 1 shows the results of three experiments measuring the rate of transformation with the qa-2 plasmid, pQa2HKh. Figure 1A presents the results as transformants per lo7 spheroplasts, while Fig. 1B presents the results as transformants per microgram

12 AUSTIN AND TYLER

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~9 DNA / 10’spheroplasts ~9 DNA i IO’spheroplasts

FIG. 1. Dependency of transformation rates on DNA-to-spheroplast ratios. (A) Transformation rates expressed as transformants per unit of spheroplasts. (B) Transformation rates expressed as transformants per unit of DNA. In three independent experiments, aliquots of 10’ 246-89601-2A spheroplasts were transformed with amounts of pQa2H/Xh plasmid DNA ranging from 2 ng to 1 p,g (Expt Ia, open circles), from 40 ng to 5 pg (Expt II, open triangles), or from 1 kg to 30 pg (Expt III,

open squares). In Experiment III, the volume of the transformation reaction was varied to maintain a constant DNA concentration equivalent to that of a standard reaction. In Experiment Ib, carrier DNA, pBR322 (solid circles), or pBT6 (solid squares) was added to the transformation reactions to maintain a constant total DNA concentration equal to that in the standard reaction. In the three diierent experiments, the transformation rate with 1 ng DNA per 10’ spheroplasts varied threefold. Therefore, to directly compare the results from the three experiments, the results of Experiments II and III were normalized to those of Experiment I using the formula (adjusted yield of transformants per 10’ spheroplasts) = (actual yield of transformants per 10’ spheroplasts) X (transformation rate at 1 l&O’ spheroplasts in Expt I) + (transformation rate at 1 l&O’ spheroplasts in Expt II or III). In A, least-squares analysis was used to lit straight lines to the transformation data in the presence (solid line) or absence (stippled line) of carrier DNA over the range 2 ng to 0.2 pg DNA/lo’ spheroplasts. These lines yielded the following expressions for extrapolating transformation rates from the data, as discussed in the text: in the presence of carrier, transformation rate = 2350 x (DNAkpheroplast ratio)‘.‘*‘; and in the absence of carrier, transformation rate = 3890 x (DNAkpheroplast ratio)‘.35.

of DNA. In Experiment I, the amount of plasmid DNA added to 10’ spheroplasts was varied from 2 ng to 1 kg, either with (closed symbols) or without (open circles) the addition of carrier DNA. In Experiment II (open triangles) the amount of plasmid DNA was varied from 40 ng to 5 yg without carrier. In Experiment III (open squares), 1 p,g of DNA was added to varying amounts of spheroplasts (10’ down to 3.3 x Id). Since the transformation rate under the standard conditions (1 pg DNA + 10’ spheroplasts) varied among the three experiments (lSOO/~g in Expt I; 384O/kg in Expt II; 432O/p.g in Expt III) the transformation results in Experiments II and III

were normalized to those of Experiment I to more readily compare the results of the three experiments (as detailed in the figure legend).

The results shown in Fig. 1A and B show that with less than 1 p,g DNA (Expt I, open circles), the number of transformants per 10’ spheroplasts is roughly proportional to the amount of input DNA. Above 1 pg DNA/lo’ spheroplasts (Expts II and III, open triangles and squares) the number of transformants increases by only a further two- to threefold. This plateau suggests that spheroplasts become saturated with DNA as the DNA ratio is increased above 1 l&10’ spheroplasts. In Fig. lB, the same


experiments show the number of transfor- formants per unit of transforming DNA mants per microgram of DNA roughly con- were achieved with very low ratios of trans- stant below 1 pg DNA/lo’ spheroplasts, forming DNA to spheroplasts (~2 ng/lO’ then rapidly dropping above 1 kg/IO’ spheroplasts) in the presence of carrier spheroplasts . DNA (1 p&O’ spheroplasts).

In the second part of Experiment I, carrier DNA was used to maintain a constant ratio of total DNA to spheroplasts (1 kg/IO’) in the transformation mixture, to determine whether the transformation rate would be improved at very low levels of transforming DNA, or whether there would be competition between the DNAs for up- take into spheroplasts. Figure IB shows that the number of transformants per microgram of input DNA is strikingly increased at low levels of transforming DNA by the addition of carrier DNA (solid circles and squares). The improvement is less- ened as the amount of transforming DNA approaches 1 pg. In Experiment I, the transformation rate with 2 ng of qa-2 DNA was 600 transformants per microgram in the absence of carrier, and 13,500 transformants per microgram in the presence of 1 p,g carrier DNA. The rate with 1 p,g qa-2 DNA was 1500 transformants per microgram.

Optimizing Cotransformation

In optimizing cotransformation, it was our initial expectation that, based on simple probabilities, cotransformation would be maximal when the rate of transformation per spheroplast was maximal. This prediction was based on the assumption that all spheroplasts which could take up and ex- press exogenous DNA could do so with approximately equal efficiencies. Therefore we measured the rates of cotransformation in experiments in which increasing amounts of qa-2 and BmlR plasmid DNAs were mixed and used to transform IO7 spheroplasts. qa-2 + transformants were selected and then tested for benomyl resistance. The results of two such experiments are shown in Table 1. Experiment II in this table cor- responds to Experiment III in Fig. 1.

TABLE 1

Two types of carrier DNA were used, plasmid vector pBR322 DNA and BmlR plasmid (pBT6) DNA. The two DNAs gave identical results, suggesting that at the levels used, there was no competition for up- take into spheroplasts even when the com- peting DNA carried another Neurosporu gene (Le., BmlR) and was present in a 500- fold excess. Possible explanations for the improvement of the transformation rate in the presence of carrier DNA could be pro- tection of the transforming DNA from small amounts of nuclease or from nonproductive adsorption to spheroplasts.

Cotransformation Rates with High Ratios of DNA to Spheroplasts

From the experiments shown in Fig. 1, it was concluded that the highest numbers of transformants per unit of spheroplasts were achieved with high ratios of DNA to spheroplasts (>I kg DNA/lo’ spheroplasts), while the highest numbers of trans-

Ratio of DNA: spheroplasts

Cotransformation frequency

(M!tlO’l Experiment I Experiment II

qa-2 BmlR Number % Number %

1 1 10/33 36% 13132 46% 3 3 15135 43% 7140 17% 9 9 lU33 37% 13t36 36%

30 30 nt nt 406 25% 1 5 5135 14% 12l31 39%

Note. The amounts of pQa2HIXh (qa-2+) and pBT6 (BmZR) plasmid DNAs shown in the fust and second columns were used to transform 10’ spheroplasts using the standard protocol. Stable qa-2+ transformants were then selected and tested for benomyl resistance. The volume of each transformation reaction was adjusted so that the DNA concentration of pQa2H/Xh DNA remained the same in all the reactions. The cotransformants are shown as the number or percentage of stable qa-2+ transformants which were stably cotransformed to benomyl resistance. nt, not tested.

14 AUSTIN AND TYLER

In neither experiment were there significant differences among the cotransformation rates at the different ratios of DNA to spheroplasts [Expt I: x2(2) = 1.17, P > 0.5; Expt II: x2(3) = 3.73, P > 0.11. The last line of Table 1 shows further that there was no significant change in the cotransformation rate under these conditions when the ratio of qa-2 to BmlR plasmid DNAs was altered from 1:l to 1:5 [Expt I: x2(1) = 2.50, P > 0.1; Expt II: x2(1) = 1.33, P > 0.11.

Since cotransformation was not elevated under conditions in which the transformation rate per spheroplast was maximal, we measured the cotransformation rate under conditions in which the transformation rate per microgram of DNA was maximal: lo7 spheroplasts were transformed with a mixture of 40 ng each of qa-2 and BmlR plasmid DNAs in the presence of 1 p,g of carrier DNA, then qa-2+ transformants were selected and tested for the acquisition of

benomyl resistance, and vice versa. The experiment was repeated twice. As shown in Table 2, the average cotransformation rate was less under these conditions (12.S20%) than in the controls, in which 1 u.g of each plasmid was used in the cotransformation (5 l-57%).

The next set of conditions examined were those employing low levels of transforming DNA and high levels of cotransforming DNA. When cotransformation was carried out with 40 ng qa-2 plasmid plus 1 pg BmlR plasmid, 89% of the qu-2+ transformants proved to have been cotransformed with the BmlR plasmid (third line of Table 2). In the reciprocal experiment, 74% of BmlR transformants were cotransformed with the qu-2 plasmid (last line of Table 2). The difference from the control (line 2) was statistically highly significant (P < 0.005) in the first case, but less so (P < 0.1) in the reciprocal experiment, possibly because

TABLE 2 Cotransfotmation Rates with Low Ratios of Transforming DNA to Spheroplasts

Transforming plasmid DNAs (t&O’ sp)

qa-2 BmlR Expt

Number of % Cotransformation cotransformants (means of Expts I and II)

qa-2+lBmlh BmlRiqa-2 + b qa-2+lBmlRc BmlRlqa-2+d

0.04 0.04 I II

1.0 1.0 I II

1.0 0.04 I II

0.04 1.0 I II

nt 3120 2116 4116

10120 9120 10119 16123 15117 l/20 19/21 6i24 l/19 13120 1123 20124

12.5

51’

89’ (P < 0.005)

5

20

57f

15

74f (P < 0.1)

Note. The amounts of pQdH/Xh (qa-2+) and pBT6 (BmF) plasmid DNAs shown in the fmt and second columns were used to transform 10’ spheroplasts using the standard protocol. Stable qa-2+ transformants were then selected and tested for benomyl resistance. Stable BmfR transformants were also selected from the same transformation reactions and tested for their qa-2 phenotype. In the experiments utilizing 0.04 ug of both qa-2 and BmlR plasmids, 1 p,g of pBR322 plasmid DNA was included as carrier DNA. nt, not tested.

a Number of stable qa-2+ cotransformants identified among the given number of BmlR transformants. b Number of stable BmlR cotransformants identified among the given number of qa-2+ transformants. ’ Mean percentage of stable qa-2+ cotransformants identified among stable BmlR transformants in Experi-

ments I and II. d Mean percentage of stable BmlR cotransformants identified among stable qa-2+ transformants in Experi-

ments I and II. of Pairs of results compared by x2 analysis (by pooling results of Experiments I and II) to give the indicated

signiticance probabilities.


the number of transformants examined was small. When 1 pg of the transforming plasmid and 40 ng of the cotransforming plasmid were used, the cotransformation rate was very low.

These results indicate that the optimal protocol for cotransformation in N. C~USSU is to use low levels of the transforming DNA carrying the selectable marker (2- 40 ng DNA per IO7 spheroplasts depending on the number of transformants required from the experiment) mixed with high levels of the cotransforming DNA carrying the nonselectable gene (> 1 p.g DNA per IO7 spheroplasts). The low levels of transforming DNA represent conditions under which the rate of transformation per unit of DNA is high, while high levels of cotransforming DNA represent conditions under which the rate of transformation per spheroplast is high.

The observation that both the rate of transformation per microgram of DNA and the rate of cotransformation are highest at very low levels of transforming DNA (in the presence of carrier) suggests that in a typical preparation of Neurospora spheroplasts, there is a small subpopulation of spheroplasts which is highly susceptible to DNA-mediated transformation. Under this hypothesis, when very low levels of transforming DNA are used, there is a strong selection for this subpopulation of spheroplasts, since they can incorporate the very low concentrations of DNA with high efli- ciency. Consequently, in a cotransformation experiment in which a second DNA species is present at high concentration, these same spheroplasts can also incorporate the cotransforming DNA at high efficiency. Note, however, that these highly transformable spheroplasts probably do not constitute a discrete subpopulation, quali- tatively different from the bulk of the transformable spheroplasts, because if they did, one would predict that the cotransformation rate would still be high when both the transforming and cotransforming DNAs

were present at low levels. Instead, as shown in line 1 of Table 2, the cotransformation rate under these conditions is low. In reality, therefore, as suggested by the straight line in Fig. lB, it seems more likely that spheroplasts display a continuum of susceptibilities to transformation.

In animal cells, cotransformation frequencies of 80% were obtained under conditions similar to those we have described above, with very small amounts of transforming DNA and very large amounts of cotransforming DNA (Wigler et al., 1979). In Succhuromyces cerevisiue, cotransformation rates of 30% with inte- grating vectors and rates greater than 90% with autonomously replicating vectors were reported with approximately equal levels of transforming and cotransforming DNAs (Hicks et al., 1978), but no attempt to optimize conditions was reported. In As- pergillus niduluns, Wernars et al. (1987) found that cotransformation was highest when the level of cotransforming DNA was high. They also found that cotransformation was slightly higher (e.g., 65% compared to 55%) when low levels of selectable transforming DNA were used, but they did not explore the use of very low levels of transforming DNA, probably because of the low absolute transformation rate in that fungus. It may be valuable to test whether the strategies we have identified for high- efficiency cotransformation in Neurosporu would improve cotransformation frequencies in other fungal transformation systems, especially those where transformation rates are low.

Potential Significance for Strategies Involving Trunsformution

Our findings hold significance for several experimental strategies involving Neuros- poru transformation. For example, the cloning of Neurosporu genes by sib selection (Akins and Lambowitz, 1985; Vollmer and Yanofsky, 1986) involves transforming

16 AUSTIN AND TYLER

with a large mixture of different plasmid or phage DNAs and selecting for rare transformants resulting from DNA carrying the gene of interest. This strategy is analogous to the part of Experiment Ib in Fig. 1A in which a small amount of DNA was used for transformation in the presence of a large amount of unrelated DNA. Consequently, a prediction from our results is that the rate of recovery of transformants bearing the clone of interest should be considerably higher than would be predicted from a simple linear extrapolation from the transformation rate achieved with 1 kg/lo7 spheroplasts.

For example, interpolation of our transformation data in the presence of carrier, using the solid curve (Expt lb) in Fig. lA, predicts that under the transformation conditions used for the first round of sib- selection experiments by Vollmer and Yanofsky (1986) (i.e., a mixture of 96 plasmid clones), the transformation rate per unit DNA of a particular clone will be approximately 6% of the rate at 1 )~,g/lO~ spheroplasts, or sixfold higher than predicted by a simple linear calculation [i.e., 1196 x (rate with 1 pg), making the rough assumption that DNA molecules from the clone of interest are proportionally repre- sented within the DNA of the pool]. The figures quoted by Vollmer and Yanofsky (1986) for the cloning of his-3 by sib selection are consistent with this prediction: in that experiment the transformation rate with 1 p,g DNA was quoted as 400-1000 transformants/pg, and 27 colonies were obtained from one %-member pool using 1 p,g of mixed DNA, whereas a linear calculation would predict 4-10 colonies. Similarly, in the first-round sib-selection experiments of Akins and Lambowitz (1983, in which a mixture of 1800 plasmid clones was used, extrapolation of our data from Fig. 1A predicts that around 52 transformants/pg pool DNA should be obtained for a given clone. In fact, for the cloning of the in1 gene, Akins

and Lambowitz (1985) obtained 50 colonies from the one positive pool-matching the prediction. In contrast though, they obtained only an average of 6 transformants per pool from three positive pools when cloning the nit-1 gene, suggesting that the specific figures in Fig. IA may not be ap- plicable to all transformation conditions or all clones, e.g., clones whose DNA is un- derrepresented in a pool of library DNA.

To the extent that our data, presented in Fig. lA, can be extrapolated to transformation with other clones in other laboratories, these findings suggest that in sib-selection or shotgun cloning experiments, the number of transformants that must be produced and then screened to have a satisfactory probability of obtaining the clone of interest may be as much as tenfold fewer than predicted by a simple linear calculation. This will be of particular importance in cases in which the transformants containing the clone of interest can be identified only by screening the total population of transformants one by one. It will be important to determine whether transformation rates fol- low a similar pattern in other fungi, such as plant pathogens, in which the lack of genet- ics more frequently requires screening indi- vidual shotgun transformants one by one.

Another potential implication of our findings relates to strategies for producing multicopy transformants. Multicopy transformation has been used elegantly in Aspergil- lus nidulans to identify specific DNA sequences capable of titrating out truns- acting regulatory proteins (Kelly and Hynes, 1987). Multicopy transformation is also important for the construction of over- producing strains. A prediction of our findings is that in a Neurospora crassa cotransformation experiment utilizing a low level of transforming DNA (say qa-2) and a high level of cotransforming DNA (e.g., BmlR), the very high rate of cotransformation should yield a high proportion of cotransformants (qa-2+ BmlR) which contain mul-


tiple copies of the cotransforming plasmid (BmlR). We have not tested this prediction in Neurospora or any other fungi.

ACKNOWLEDGMENTS

We thank Mary Case and Charley Yanofsky for gifts of plasmids and Neurospora strains and Howard Ju- delson and Michael Holland for criticism of the manu- script. This work was supported by a CSIRO- Australian National University Collaborative Re- search Grant and by the Research School of Biological Sciences.

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strategies for high-efficiency cotransformation ofneurospora crassa

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