Construction and Characterization of aMagnaporthe griseaBacterial Artificial Chromosome Library

Fungal Genetics and Biology 20, 280–288 (1996) Article No. 0042

Construction and Characterization of a Magnaporthe grisea Bacterial Artificial Chromosome Library

Silvia V. Diaz-Perez,1 V. Wayne Crouch, and Marc J. Orbach Department of Plant Pathology, University of Arizona, Tucson, Arizona 85721

Accepted for publication October 18, 1996

Diaz-Perez, S. V., Crouch, V. W., and Orbach, M. J. 1996. Construction and characterization of a Magnaporthe grisea bacterial artificial chromosome library. Fungal Genet. Biol. 20, 280–288. A bacterial artificial chromosome (BAC) library of Magnaporthe grisea containing 4128 clones with an average insert size of 66-kb has been constructed. This library represents seven genome equivalents of M. grisea and has been demonstrated to be representative of the genome by screening for the presence of several single-copy genes and DNA markers. The utility of the library for use in map-based cloning projects was shown by the spanning of a nine-cosmid, 207-kb DNA contig with only 3 BAC clones. In addition, using a lys1-3 auxotroph, we have shown that BAC clones at least 113 kb can be transformed into M. grisea to screen for complementation of mutations. Thus, BACs isolated in chromosome walks can be rapidly screened for the presence of the sought after gene. The ease of construction of BAC libraries and of isolation and manipulation of BAC clones makes the BAC system an ideal one for physical analyses of fungal genomes. r 1996 Academic Press

Index Descriptors: Magnaporthe grisea; rice blast; bacterial artificial chromosome (BAC); map-based cloning.

1

Current address: Department of Microbiology and Molecular Genetics, UCLA College of Letters and Science, 1602 Molecular Sciences Building, Los Angeles, CA 90024-1489.

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Recent advances in the genetics of Magnaporthe grisea, the rice blast fungus, have led to the identification of several genes important to the pathogenicity of this fungus. In an effort to isolate these genes using map-based methods, detailed restriction fragment length polymorphism (RFLP) maps have been constructed (Romao and Hamer, 1992; Skinner et al., 1993; Sweigard et al., 1993) allowing the identification of linked DNA markers. Two genes have been cloned based on their map position, the host-specificity gene PWL2 (Sweigard et al., 1995) and the cultivar-specificity gene AVR2-YAMO (M. J. Orbach, L. Farrall, and B. Valent, unpublished). Chromosome walks to clone these genes were performed using cosmid libraries with an average insert size of around 40 kb. A major improvement in chromosome-walking technology has been the development of vector systems able to accommodate larger DNA inserts such as yeast artificial chromosomes (YACs), bacteriophage P1 vectors, and bacterial artificial chromosomes (BACs). YAC vectors are able to accomodate extremely large inserts, with some libraries selected for clones containing megabase genomic fragments. They are the vector of choice if the maximization of insert size is of primary concern. BAC clones have been reported with genomic DNA segments up to 300 kb, although the average insert size in most libraries is usually between 75 and 150 kb. One of the great advantages of working with BAC libraries is the ease of isolation and manipulation of BAC DNA, using standard plasmid preparation methods. BAC clones have also been reported to be very stable during growth in Escherichia coli (Shizuya et al., 1992), and the frequency of chimeric clones has been reported to be much lower in BAC libraries than in YAC libraries (Shizuya et al., 1992; Woo et al., 1994). 1087-1845/96 $18.00 Copyright r 1996 by Academic Press All rights of reproduction in any form reserved.

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Magnaporthe grisea BAC Library

Here we report the preparation and evaluation of a BAC library of M. grisea strain 4224-7-8, a rice-pathogenic laboratory strain that has been used extensively for mapping, gene cloning, and karyotype analysis. The library contains 4128 clones with an average insert size of 66 kb. Based on a haploid genome size estimated at 38 Mb (Hamer et al., 1989), individual sequences should be present in the library at greater than 99% probability. Representation of the library was evaluated using several single-copy DNA probes. We also demonstrate that M. grisea can be transformed with BACs ranging between 82 and 113 kb and that transformants can be identified by direct selection.

MATERIALS AND METHODS Preparation of High-Molecular-Weight DNA M. grisea strain 4224-7-8 (Sweigard et al., 1995), one of the parental strains in our M. grisea RFLP map (Sweigard et al., 1993), was used to isolate high-molecular-weight (HMW) DNA for construction of the BAC library. The DNA was prepared from protoplasts embedded in agarose by a modification of our previously published protocol (Orbach et al., 1996). Mycelium from a 2-week-old culture grown on oatmeal agar (Valent et al., 1991) was macerated in a blender with 100 ml of Iwasaki medium (per liter: 20 g glucose, 0.5 g KH2PO4, 0.5 g K2HPO4, 0.01 g CaCl2, 5 g yeast extract) (Chida and Sisler, 1987). This was used as inoculum for 1 liter of Iwasaki medium in a 3-liter Fernbach flask. The culture was grown with constant agitation (225 rpm) for 40 h at room temperature. Harvesting and preparation of protoplasts were as described previously (Orbach et al., 1996) except that incubation of the mycelium with Novozym 234 to release protoplasts was done at 35°C instead of room temperature. Protoplasts were embedded in low melting point (LMP) agarose (Life Technologies) at a concentration of 1.0 3 109 protoplasts/ml as described previously (Orbach et al., 1996). Lysis of the protoplasts was modified to include two treatments with NDS buffer (0.4 M EDTA, 10 mM Tris–Cl, pH 9.5, 1% N-lauroylsarcosine) containing proteinase K at 2 mg/ml for 18 to 24 h at 50°C, prior to storage in 50 mM EDTA (Orbach et al., 1988). This step increased the purity of the DNA as measured by the rate of its digestion with HindIII in the agarose plug/DNA mixture.

Partial Digestion and Size Fractionation of the BAC insert DNA The DNA embedded in agarose was dialyzed against TE, pH 8.0, for 30 min at 50°C and then overnight at 4°C. Partial digestion of the DNA was done in agarose using the limiting Mg21 method (Albertsen et al., 1989; Birren and Lai, 1993), where HindIII is diffused into the DNA/ agarose plugs that are equilibrated with restriction enzyme buffer in the absence of Mg21. We used 2.5 units HindIII (Gibco BRL, Life Technologies) per 50 mg of DNA/ agarose plug mixture to achieve the desired partial digestion (see Results). The enzyme reaction was initiated by addition of MgCl2 to 10 mM and terminated by addition of EDTA to 50 mM. The digested DNA was fractionated on CHEF gels (Chu et al., 1986) using two different methods for the purpose of removing small DNA fragments that could be ligated into the BAC vector and be overrepresented in the library. In the first method, a double size-selection approach was used where the DNA was separated on a CHEF gel, a size-selected fraction of DNA (see Results) removed from the gel as an agarose slice, and rerun on a second gel. Both gels were made of 1% LMP agarose in 0.53 TBE and were run at 11°C for 18 h with a 90-s switching interval at 6 V/cm. The basis of this approach is to allow any small fragments trapped with the larger DNA in the first gel to segregate away from the desired fragments in the second gel. We termed the second approach ‘‘selective small-fragment removal,’’ where the small fragments were electrophoresed out of the fractionated DNA/agarose plugs prior to using them for library preparation. In this method, following fractionation of the sized fragments as in the first LMP gel above, the DNA-containing LMP agarose slices were placed in the wells of a second (regular agarose) CHEF gel and subjected to a brief pulsed-field electrophoresis regime allowing the small fragments to enter the gel while the large DNAs remained in the LMP agarose slices. Conditions for this second gel were 1% agarose gel in 0.53 TBE, run at 11°C for 70 to 85 min with a 15-s switch interval at 7 V/cm. The DNA slices were then removed from the gel well and used for ligation (see below).

BAC Library Construction pBeloBAC11, a gift from Dr. H. Shizuya, was grown as described by Woo et al. (1994) in 5 liters of LB media containing chloramphenicol (Cm) (12.5 µg/ml) at room temperature for 24 h with constant agitation. The vector

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was isolated by the alkaline–lysis method and further purified by treatment with RNase A (50 µg/ml) prior to two rounds of separation by cesium chloride–ethidium bromide equilibrium centrifugation at 100,000g for 17 h (Sambrook et al., 1989). The vector was treated with 10 units of HindIII per microgram of DNA and dephosphorylated with calf intestinal alkaline phosphatase (Boehringer Mannheim Biochemicals). The completeness of the HindIII digestion and dephosphorylation was functionally tested by ligation of the dephosphorylated vector with and without specific small HindIII fragments. These ligations, and vector DNA that was digested with HindIII but not ligated, were transformed into the BAC library recipient strain, HS996, by electroporation (Sheng et al., 1995). Strain HS996 is a derivative of DH10B (Life Technologies) resistant to phage T1 (H. Shizuya, personal communication). Vector preparation was considered acceptable when, using blue/white screening, less than 4% of the colonies were white from ligations lacking insert DNA while greater than 80% were white when insert DNA was added to the ligation. Size-fractionated DNA from strain 4224-7-8 was released from LMP agarose by treatment with b-agarase (New England Biolabs). Agarose gel slices were equilibrated overnight at 4°C in b-agarase buffer (10 mM Bis–Tris–HCl, pH 6.5, 1 mM EDTA, 50 mM NaCl) prior to melting at 65°C for 10 min. The sample was cooled to 42°C before addition of b-agarase (2 units/50 mg of agarose slice) and then incubated at 42°C for 2 to 3 h. This DNA solution was used directly for ligations following the b-agarase treatment. The concentration of the insert DNA was estimated by comparison to known amounts of uncut l DNA on 1% agarose gels. Ligations were done in a final volume of 35 µl using 10 ng of vector prepared as described above and 20 ng of insert DNA for a predicted vector to insert ratio of 10:1. Prior to the addition of ligase, the reaction mixture was heated at 65°C for 5 min to release any annealed HindIII ends and then cooled to room temperature. Ligation reactions were incubated overnight at 15°C with 0.25 units of T4 DNA ligase (Promega). Prior to electroporation, the ligations were drop-dialyzed against 25 ml of 0.53 TE on Type VS 0.025-µm pore size filters (Millipore VSWP 02500) for 1.5 h. In some cases ligations were dropdialyzed against 0.53 TE containing 0.75 mM spermine and 0.30 mM spermidine (Sheng et al., 1995). Electrocompetent HS996 cells were prepared as described by Sheng et al. (1995). Electroporations were carried out using a BTX Electro Cell Manipulator 600

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Diaz-Perez, Crouch, and Orbach

electroporator. Thirty microliters of thawed HS996 cells was mixed with 2 µl of dialyzed ligation mixture and placed in 0.2-cm cuvettes. The electroporation conditions were 1.8 kV, 100 W, and 25 mF (Sheng et al., 1995). Following electroporation, cells were incubated with agitation (225 rpm) in 1 ml of SOC medium for 45 min at 37°C and then plated on LB plates containing 12.5 µg/ml Cm, 50 µg/ml X-gal, and 25 µg/ml IPTG. After a 24-h incubation at 37°C, the plates were transferred to 4°C to allow color development in order to identify recombinant clones. HS996 transformants containing recombinant BACs were picked into microtiter-dish wells containing LB plus 12.5 µg/ml Cm and 10% glycerol. After 24 h of growth at 37°C, the microtiter dishes were replicated and stored at 280°C.

Preparation and Analysis of BAC Clones DNA from individual BAC clones was isolated by alkaline lysis from 5-ml cultures grown in LB with 12.5 µg/ml Cm for 24 h at room temperature. To further purify the BAC DNAs, after the initial precipitation and resuspension of nucleic acids, LiCl was added to a final concentration of 2.5 M and the mixtures were incubated for 10 min on ice. The precipitate was removed by centrifugation. The BAC DNAs in the supernatant were then precipitated by addition of 0.1 vol of 3 M sodium acetate, pH 6.0, and 2 vol of ethanol. The DNAs were resuspended in 40 µl of TE and 10 µl was used for restriction digestions. The sizes of individual BAC clones were determined by analyzing a combination of HindIII digestions run on 0.7% agarose gels in 13 TBE and NotI digestions electrophoresed on CHEF gels. The CHEF gel conditions were 1% agarose gels in 0.53 TBE run at 6 V/cm with a 1.5-s switching interval for 14 h at 11°C.

BAC Library Screening For hybridization analyses, the BAC library was replicated from microtiter dishes onto MagnaCharge filters (MSI) that were placed on LB agar containing 12.5 µg/ml Cm and grown as described by Woo et al. (1994). The colony hybridization conditions were as described by Sambrook et al. (1989). Radiolabeled probes were prepared by the random-hexamer priming method of Feinberg and Vogelstein (1983). In some cases, colony hybridizations were done using a method to suppress vector hybridization (Kim et al., 1994) by preannealing 32Plabeled probes with unlabeled vector DNA (either

Magnaporthe grisea BAC Library

pMOcosX for cosmid probes or pBluescript KS(2)). Single-copy probes used for screening the library were b-tubulin (TUB1, carried on plasmid pCB635), acetolactate synthase (ILV1, on pCB634), a cosmid (CP866E1) containing the lysine biosynthetic gene LYS1, and the RFLP marker cosmid 12B5 (Sweigard et al., 1993). A complex probe consisting of seven cosmids that represent a 207-kb genomic region was also used to probe the BAC library. This probe represented a nine-step walk initiated from cos72 (Sweigard et al., 1993) in a pMOcosX (Orbach, 1994) cosmid library of M. grisea strain 4392-1-6 (U. Gunawardena, S. V. Diaz-Perez, and M. J. Orbach, unpublished). The cosmids used in the complex probe were cos72, from a cosmid library in the vector pArc of strain 4091-5-8 (F. G. Chumley, unpublished), and cosmids 14G6, 19A12, 25G5, 34B1, 35G11, and 40D10 from the 4392-1-6 library. Two other 4392-1-6 cosmids from the walk, 3H6 and 29D2, were not used in the complex probe because they contained repetitive sequences. The cosmid DNAs of the complex probe were mixed in equal masses, and 100 ng of the mixture was used to make a radiolabeled probe. To determine the placement of each BAC clone in the 207-kb cosmid contig, the BAC DNAs were subjected to Southern analysis (Sambrook et al., 1989) using individual cosmids as probes.

Transformation of M. grisea lys1-3 with BACs Strain 4389-R-2, a lysine auxotroph (lys1-3), was transformed as described by Sweigard et al. (1995). Cosmid CP866E1, and BAC clone DNAs isolated by alkaline lysis mini-preps, were used to transform 4389-R-2 protoplasts either alone or by cotransformation with the hygromycin resistance vector pMP6 (Orbach, 1994). Prototrophic colonies were selected on modified Vogel’s minimal medium (Vogel, 1964) containing or lacking hygromycin (100 µg/ml; CalBiochem). In modified Vogel’s medium, NH4NO3 is replaced with urea as the nitrogen source.

RESULTS Construction of the M. grisea BAC Library For preparation of HMW DNA from strain 4224-7-8 for making insert DNA, we modified published methods for the generation of protoplasts. Standard M. grisea liquid culture methods often resulted in the production of

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melanized mycelia, which are resistant to digestion with Novozym 234. To reduce the melanin level in mycelial cultures, we used young oatmeal agar plate cultures for initiation of liquid cultures which were grown in modified Iwasaki medium (Chida and Sisler, 1987). Additionally, the protoplasts from 4224-7-8 mycelia were generated at 35°C instead of 24°C. Use of these conditions increased the efficiency of protoplasting over 10-fold, yielding 8 3 108 protoplasts per gram of mycelia. The DNA in agarose was subjected to two 18- to 24-h treatments in NDS buffer with 2 mg/ml proteinase K in order to improve the efficiency of digestion with HindIII. DNA prepared in this manner was subjected to partial digestion with varying amounts of HindIII to determine the optimal amount for generation of maximal amounts of DNA in the 100- to 350-kb range (data not shown). For construction of our library, we used 2.5 units of HindIII per 50 mg of agarose plug. A library of 4128 clones was prepared and stored as individual clones. An initial 1248 clones were prepared using a double size-selection method that is often used to remove small contaminating DNA fragments from the partially digested HMW DNA. This method involved resolving partially digested genomic DNA on a preparative CHEF gel and cutting out the 150- to 300-kb region of the gel. This fraction was rerun on a second preparative CHEF gel to allow small fragments whose migration may have been retarded in the initial gel to be resolved from the 150to 300-kb fragments to be used for ligation. To test whether this method was effective, 25 random BAC clones were analyzed for size (Fig. 1). These clones had an average insert size of 38 kb and ranged between 15 and 92 kb. The observation that the average insert size was much lower than the size of the fractionated DNA has been seen by others (Woo et al., 1994); however, the production of clones with very small inserts was unacceptable. To increase average insert size and eliminate the generation of small BAC clones, two modifications were employed. First, the range of partially digested DNA used for ligation was increased to 280 to 450 kb. This increased the average insert size of the clones, but did not eliminate small clones. To selectively remove small DNAs from the size-fractionated DNA we subjected the DNA-containing agarose slices from the first size-selection gel to a brief pulsed-field gel electrophoresis regime. These slices were then removed from the gel wells and used for ligations. This method successfully removed small fragments from the size-selected DNA. Insert DNA prepared in this manner was used to generate 2880 BAC clones to com-

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project, which overlapped to cover a 207-kb region (U. Gunawardena, S. V. Diaz-Perez, and M. J. Orbach, unpublished). Eighteen BACs that hybridized to this complex probe were identified and their locations within the 207-kb contig were determined by probing with subsets of the cosmids that spanned the region (data not shown). These data showed that the region could be covered by three steps in the BAC library instead of the nine it took using the cosmid library.

Use of BACs for Complementation of M. grisea

FIG. 1. Size distribution of BAC clones obtained using two different methods for insert preparation. The sizes of 25 BAC clones obtained using the double size-selection method are indicated by striped bars (§). The sizes of 50 BAC clones obtained using the method that selectively removes small fragments from the sized-insert DNA are indicated by the dotted bars (b).

plete the library. Analyses of 50 clones for size indicated that the inserts of these BACs averaged 78 kb in length and the range observed was between 30 and 140 kb (Fig. 1).

Sampling of the BAC Library The M. grisea BAC library corresponds to 7.2 equivalents of the genome based on the estimated genome size of 38 Mb (Hamer et al., 1989). To test representation of the genome, three gene probes and one RFLP marker were hybridized to colony filter membranes of the library. Seven BACs containing the M. grisea b-tubulin gene were identified; the ILV1 gene was present in three BACs and the LYS1 cosmid, CP866E1, hybridized to eight BACs. The RFLP marker cosmid 12B5 was used to probe the BAC library in order to initiate a chromosome walk to the avirulence gene AVR1-MARA. Four BACs that mapped to this locus were identified (M. A. Mandel and M. J. Orbach, unpublished results). These results, while a limited sample, support the estimated representation of the BAC library. In addition to examining the BAC library for representation of individual loci in the M. grisea genome, we tested for completeness of the library by screening with a complex probe representing a 207-kb segment of the genome (Fig. 2). This complex probe consisted of seven cosmids, identified in nine steps of a chromosome-walking

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The M. grisea lysine auxotrophic mutant lys1-1 (Crawford et al., 1986) was previously complemented using a cosmid library by selection for prototrophic growth (Parsons, 1988). One cosmid from a complemented strain, designated CP866E1, was recovered and retransformed into lys1-1 to demonstrate that it contained the complementing sequences. As mentioned above, the cosmid CP866E1 hybridized to eight BACs in our library (Fig. 3B). Five of these clones were transformed into M. grisea either alone or by cotransformation with the hygromycin resistance vector pMP6 (a gift from M. Plamann). Four of them, ranging from 82 to 113 kb, restored prototrophic growth ability to the lys1-3 mutant strain 4389-R-2 (Fig. 3A). Prototrophic strains were detected either by direct selection on minimal media or by coselection on minimal media with hygromycin. The fifth BAC, which did not complement the mutant, showed only limited hybridization to CP866E1 (Fig. 3B), suggesting that it only contains sequences corresponding to an end of the cosmid, and thus may not contain the LYS1 gene. Cosmid CP866E1 yielded about 39 transformants/µg, while the four BACs averaged 16 transformants/µg (data not shown).

DISCUSSION The application of BAC cloning technology to a filamentous fungal system demonstrates the usefulness of this new tool for physical analysis of fungal genomes. When considering a large DNA insert vector for construction of a M. grisea genomic library, we chose to use the BAC system instead of YACs because of the great advantage that BACs provide in terms of ease of isolation and manipulation of clones. BAC DNA can be purified from bacterial cultures by standard alkaline lysis methods while YAC clones

Magnaporthe grisea BAC Library

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FIG. 2. Physical map of a region of chromosome 1 with cosmids versus BACs. A 207-kb contig surrounding, and initiated from, cosmid cos72 is represented by a nine-step cosmid library walk. The same region is spanned by 3 BACs that were identified by probing the BAC library with a probe representing the cosmid contig. The BAC clones identified covered a region greater than 207 kb as judged by the hybridization of some BACs only to cosmids at the end of the contig. Cosmids and BACs are indicated below the bars by numbers that indicate their position in the library (i.e., 25G5 indicates microtiter plate 25, rowG, column 5). The BAC clones 8E5, 10F1, 7G5, and 16C8 represent 4 of the 18 BACs that hybridize to the cosmid contig. The ends of each BAC were not mapped, and so are indicated by dashed lines. The number (in kb) above each cosmid bar indicates the distance that that step progressed from the previous step.

cannot be selectively purified from yeast chromosomes and must be cut from pulsed-field gels after embedding and lysing yeast cells in agarose plugs. BAC libraries have also been reported to lack or have a much lower level of chimeric clones than YAC libraries (Shizuya et al., 1992; Woo et al., 1994), which is critical for chromosome walking. While we have not analyzed our BAC clones in detail for chimerism, comparison of the BACs isolated from the chromosome contig around cos72 (Fig. 2) did not reveal obvious chimeric clones (data not shown), nor did we see evidence for chimeric clones in the BACs isolated that contained the LYS1 gene. For fungal DNA libraries the advantages described above are of much greater importance than maximization of insert size, as is possible with YACs, due to the relatively small size of most fungal genomes. An obvious advantage of BAC vectors over cosmid vectors is the opportunity to clone larger DNA inserts. Another potential advantage is the likelihood of increased stability of inserts in BAC clones compared to cosmids, possibly due to the difference in copy number. BACs, which are single-copy vectors, have been reported to be stable over at least 100 generations of serial growth (Shizuya et al., 1992; Woo et al., 1994), while cosmids

(multiple-copy vectors) grown for fewer generations have shown a significant frequency of rearrangements (Kim et al., 1992). We tested the stability of five random BACs from the M. grisea library, ranging from 58 to 95 kb, over a period of 100 generations. We observed no changes in our BACs as judged by HindIII restriction fragment patterns, suggesting that, as in other libraries, the M. grisea BACs appear to be stable (data not shown). The 4128-clone M. grisea library we constructed has a 99% probability of containing any sequence within the genome and is easily stored in 43 96-well microtiter dishes. Preliminary analyses screening for three single-copy genes (LYS1, ILV1, and TUB2) and a single-copy RFLP marker (12B5) suggest that this library is representative of the M. grisea genome. One of our purposes in constructing this library was to isolate clones spanning the AVR1-MARA gene using linked RFLP markers. We have found that there is at least one gap in our library, as we were unable to identify BACs containing sequences adjacent to the grh25 locus which is linked to AVR1-MARA (Sweigard et al., 1993) (M. A. Mandel and M. J. Orbach, unpublished). Interestingly, these sequences are not present in either cosmid or l libraries that we have constructed (V. W.

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FIG. 3. Complementation tests of BACs homologous to the LYS1 cosmid CP866E1. (A) Analysis of BACs that were identified by hybridization to cosmid CP866E1 for size and ability to restore prototrophic growth to the lysine auxotrophic strain 4389-R-2. (B) (Left) The ethidium-bromide-stained agarose gel of duplicate preparations of the CP866E1 homologous BAC clones. (Right) The autoradiogram of the same gel, probed with CP866E1. The identity of each pair of BAC preps is indicated above the preps for both sides of the panel. The size markers visible on the agarose gel are the 1-kb ladder (Life Technologies) in the outermost lanes and l-HindIII in the adjacent lanes.

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Magnaporthe grisea BAC Library

Crouch, M. A. Mandel, and M. J. Orbach, unpublished), suggesting that they may be lethal to E. coli. The advantage of using a BAC library in comparison to a cosmid library in chromosome walking was clearly demonstrated by our coverage of a 207-kb contig developed through nine steps in a cosmid library with three BAC clones. Although the average BAC insert in our library is 66 kb, for each probe we have used we identified BACs at least 90 kb. This is to be expected since our library was constructed in two parts, with one part that represents 70% of the clones having an average insert size of 78 kb. Thus, by using the largest BACs in each chromosome-walk step, it should be possible to cut the number of steps necessary in a walk two- to three-fold. Construction of BAC libraries can be biased toward smaller inserts due to the higher efficiency of electroporation of small clones versus large ones (Sheng et al., 1995; Wang et al., 1995). This transformation bias can be compounded if the size-fractionated insert DNA is contaminated with smaller fragments that did not resolve properly. We found that these fragments can be efficiently removed from the insert DNA by subjecting the size-fractionated insert DNA plugs to a brief pulsed-field gel regime that allows the small fragments to migrate into the gel, while retaining the desired insert segments in the plug. The utility of BACs for gene isolation in M. grisea was demonstrated by our ability to directly complement the M. grisea lys1-3 mutant with miniprep quality BAC DNA. There was no apparent difference in transformation efficiency between the 82-kb BAC, 10D9, and the 113-kb BAC, 14F6, suggesting that we are not near to the size limit of BAC DNAs that can be introduced into M. grisea (data not shown). Considering that the average size of the BACs was twice that of cosmid CP866E1, the two types of clones appear to tranform equally well on a stoichiometric basis. LYS1 transformants were identified both by direct selection for prototrophic growth and by screening colonies where the LYS1 BACs were cotransformed with a plasmid encoding hygromycin resistance. Thus, when trying to isolate a gene in a chromosome walk, it should be possible to test the BACs obtained in a step for the presence of the gene by cotransformation with a selectable marker followed by screening.

ACKNOWLEDGMENTS We thank H. Shizuya for providing the pBeloBAC11 vector. We are grateful to B. Birren for providing unpublished protocols and advice

about BAC library construction. Thanks to M. A. Mandel for assistance and U. Gunawardena and M. A. Mandel for critical reading of the manuscript. This work was supported by the USDA NRICGP (Grant 92-37303-7794).

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