Departments of Molecular Genetics and Microbiology, Pharmacology and Cancer Biology, and Medicine, and the Howard Hughes Medical Institute, Duke University Medical Center, Research Drive, Durham, NC 27710, USA1
Author for correspondence: Joseph Heitman. Tel: +1 919 684 2824. Fax: +1 919 684 5458. e-mail: heitm001{at}duke.edu
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ABSTRACT |
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Keywords: gene disruption, PCR overlap, Cryptococcus neoformans
Abbreviations: MAP, mitogen-acitvated protein
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INTRODUCTION |
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In addition to being an important cause of disease in immunocompromised patients, C. neoformans is an excellent model of fungal pathogenesis. Several well-defined virulence factors include the polysaccharide capsule (which promotes intracellular survival in macrophages), the pigment melanin (which prevents oxidation by macrophages), the production of the enzymes urease and phospholipase B, and the ability to grow at 37 °C (Alspaugh et al., 2000a ; Casadevall & Perfect, 1998
; Chen et al., 2000
; Cox et al., 2000
, 2001
; Cruz et al., 2000
; Fox et al., 2001
; Kwon-Chung & Bennett, 1992
; Odom et al., 1997
). Several animal models have been developed, including a mouse tail-vein injection model, rat and mouse inhalation models, and a rabbit meningitis model (Goldman et al., 1994
; Kwon-Chung et al., 1982
; Perfect et al., 1980
). Importantly, C. neoformans is genetically tractable, with a well-characterized sexual cycle involving mating between two haploid mating types, MATa and MAT
(Alspaugh et al., 2000b
; Kwon-Chung, 1975
, 1976
). Efficient transformation and gene-disruption protocols have been developed along with auxotrophic and dominant selectable markers and overexpression plasmids (Davidson et al., 2000
; Edman & Kwon-Chung, 1990
; Hua et al., 2000
; McDade & Cox, 2001
; Sudarshan et al., 1999
; Toffaletti et al., 1993
). Furthermore, genetic analysis has been facilitated by the recent characterization of a stable diploid state, which allows identification and analysis of essential genes (Sia et al., 2000
). Finally, the entire genome sequence is nearing completion for the serotype D strain JEC21, and a sequencing project has been initiated for the clinical serotype A isolate H99 (Stanford, TIGR, UBC, Duke, Whitehead; reviewed by Heitman et al., 1999
). These advances will allow extensive comparative genomics experimentation between C. neoformans strains and other fungal genomes.
The efficient use of the complete genome sequence will require more rapid gene-function testing, a process that is limited by tedious construction of targeting alleles by cloning. PCR-generated targeting alleles have been employed for Saccharomyces cerevisiae and for the human pathogen Candida albicans, which has overcome the necessity for cloning and has allowed more high-throughput genetic analysis (Baudin et al., 1993 ; Eberhardt & Hohmann, 1995
; Lorenz et al., 1995
; Wach, 1996
; Wach et al., 1994
; Wilson et al., 1999
). However, efficient homologous recombination in C. neoformans requires larger regions of homology, which has prevented the application of similar PCR-based strategies.
Here we present a modified application of a technique called PCR overlap to generate targeting alleles with larger regions of homology (Ho et al., 1989 ; Horton et al., 1989
). This technique can be used in any system where homologous recombination requires longer regions than those that can be incorporated into synthetic oligonucleotides, and will effectively eliminate the time-consuming process of searching for convenient restriction sites and cloning targeting alleles.
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METHODS |
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The mpk1::URA5 mutant strain was generated by PCR overlap using six primers as outlined (Fig. 1
). The 5' end of the MPK1 gene was amplified with primers JOHE7288 (no. 1) (ACTAGGCGTGCCATTGTTTAC) and JOHE7418 (no. 3) (GGTCGAGCAACTTCGCTCAGGATTGCGTGCCGGACAGTG), the 3' of MPK1 with primers JOHE7419 (no. 4) (CCACCTCCTGGAGGCAAGCACGATGATTATTAGTCTTGC) and JOHE7289 (no. 6) (GCGGACTGGGCAGGAGAAGC), and the URA5 selectable marker with primers JOHE7417 (no. 2) (CACTGTCCGGCACGCAATCCTGAGCGAAGTTGCTCGACC) and JOHE7420 (no. 5) (GCAAGACTAATAATCATCGTGCTTGCCTCCAGGAGGTGG). The three amplified products were run on a 0·6% agarose gel and extracted together using the Qiaquik column method (Qiagen) and were used as templates for the overlap reaction. Primers JOHE7288 (no. 1) and JOHE7289 (no. 6) were used to overlap the three first-round products to yield the mpk1
::URA5 disruption allele. This amplified product was gel-purified, extracted and directly transformed into the serotype D ura5 strain JEC43 by biolistic transformation.
Overlap primer design.
Overlap oligos were approximately 3540 bp in length. Primers were obtained from Integrated DNA Technology and were not PAGE-purified. In the STE11 and MPK1 deletions, the portion of the oligo corresponding to the selectable marker flanked the C. neoformans URA5 gene. In the TOR1 disruption, the portion of the oligo corresponding to the selectable marker was designed to amplify from a plasmid containing URA5. This was done so that any selectable marker cloned into the vector could be easily inserted into the disruption cassette.
The sequence of primer 3 was designed to be completely complementary to the sequence of primer 2, and the sequence of primer 4 was complementary to the sequence of primer 5 as outlined in Fig. 1. This strategy generates fragments with approximately 40 bp of overlap for the final PCR reaction. The 3' ends of primer 3 and primer 4 were designed so that the melting temperature of the oligo fragment corresponding to the targeted gene was similar to the melting temperatures of primers 1 and 6, respectively.
Transformations.
Biolistic transformations were performed by the method previously described by Toffaletti & Perfect (1994) using a Bio-Rad model PDS-1000/He biolistic particle delivery system. Transformants were selected on defined medium (Sherman, 1991
) lacking uracil and containing 1 M sorbitol as an osmotic stabilizer.
PCR amplification.
All PCR amplifications were performed using a Perkin-Elmer Applied Biosystems 9600 thermocycler with Extaq polymerase (Takara). The initial amplification reactions consisted of 35 cycles of 1030 s at 95 °C, 1030 s at 55 °C and 1 min kb1 at 72 °C with an initial denaturation of 2 min at 95 °C and a final extension of 5 min at 72 °C. Importantly, denaturation and annealing times were limited to 1015 s during the final overlap amplification, which reproducibly increases the yield of specific overlap products.
The first-round PCR of the STE11 disruption consisted of an initial denaturation of 2 min at 95 °C, followed by 35 cycles of 15 s at 95 °C, 15 s at 53 °C and 1·5 min at 72 °C, and was completed with a final extension of 5 min at 72 °C. The final round of PCR for the STE11
gene disruption consisted of an initial denaturation of 2 min at 95 °C, followed by 35 cycles of 15 s at 95 °C, 15 s at 53 °C and 4·5 min at 72 °C, and concluded with a final extension of 5 min at 72 °C.
In the final PCR, the three PCR products generated in the first PCR were added in roughly equimolar amounts. The quantity of these products yielding the most efficient overlap in the final PCR varied between constructs, so a gradient of first-round products was added to the reaction (generally 1 ng, 10 ng and 50 ng total template) to obtain the most efficient overlap PCR. The PCR samples were visualized using standard DNA electrophoretic techniques (Sambrook et al., 1989 ).
Southern blot analysis.
Genomic DNA was isolated from JEC21 and JEC20 wild-type, ste11 and mpk1 mutant strains by the method of Pitkin et al. (1996)
. Twenty micrograms genomic DNA was digested with the appropriate enzymes and electrophoresed on a 0·8% TBE agarose gel. Transfer, hybridization and autoradiography were performed as described by Sambrook et al. (1989)
. Fragments of the STE11
and MPK1 genomic ORFs were used as probes for Southern blot hybridization, using [
-32P]dCTP (Amersham) and the Prime-It II random primed labelling kit (Stratagene).
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RESULTS |
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Targeted disruption of the MPK1 gene
The gene encoding the MAP kinase homologue Mpk1 was identified by comparative genomics using the Stanford C. neoformans genome sequence database (P. R. Kraus and others, unpublished). A complete sequence of the putative gene was assembled using the Stanford sequence as well as that from the TIGR C. neoformans sequencing project. To assess the function of this MAP kinase homologue, we generated an allele for gene disruption using PCR overlap. As described above, primers were directed against the 5' and 3' flanking regions of the MPK1 gene and the URA5 selectable marker, and three products were amplified (Fig. 4a, b
, lanes 13). These products were overlapped into a single product with the portions of the MPK1 gene flanking URA5 (Fig. 4b
, lane 4). The mpk1
::URA5 PCR-generated disruption allele was transformed into a serotype D ura5 strain of C. neoformans (JEC43), and Ura+ colonies were selected. Two mpk1
::URA5 mutant strains were identified by PCR out of 20 screened (10%) (Table 1
), which was confirmed by a Southern blot (Fig. 4c
).
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DISCUSSION |
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We describe a modified use of the PCR overlap technique originally described by Ho et al. (1989) and Horton et al. (1989)
to generate disruption constructs without the need for cloning. This method can be used to generate both partial and complete deletions of the targeted genes ORF. Using this method, we have disrupted three genes, STE11
(encoding a MAP kinase kinase kinase homologue), TOR1 (encoding a Tor kinase homologue) and MPK1 (encoding a MAP kinase homologue), at efficiencies consistent with previous studies (Davidson et al., 2000
). We also report the disruption of 11 other genes using the same method and a variety of recipient strains, including the recently described serotype D diploids (Sia et al., 2000
). Moreover, both the auxotrophic URA5 and dominant NAT1 selectable markers were used, which allows generation of double mutant strains or the use of prototrophic strains. In all cases, the efficiency of targeted integration was in the range 210% or higher, consistent with previous studies using biolistic transformation in C. neoformans (Alspaugh et al., 1997
; Davidson et al., 2000
; Toffaletti et al., 1993
; Wang et al., 2000
). These findings indicate that generation of constructs by two rounds of PCR does not seem to inhibit homologous recombination significantly.
Additional screening tests can make the PCR-disruption method even more efficient. Jennifer Lodge and coworkers recently found that a significant proportion of initial transformants are unstable, in accord with earlier results (Edman & Kwon-Chung, 1990 ), and by implementing an initial screening step for stable transformants they found that the efficiency of homologous targeting can be increased (Nelson et al., 2002
). We have confirmed these observations in the background of our PCR-based approach to gene disruption and found that approximately 33% of transformants are stable. In the case of the gno1::NAT1 disruption in strain JEC34, the frequency of homologous integration was increased from 10 to 23%, and, in the case of the gno1::NAT1 disruption in strain H99, from 28 to 77%, by first screening for stable transformants. Thus, implementing this important advance of Lodge and coworkers, together with the PCR overlap approaches described here, should result in even more efficacious gene-disruption frequencies.
Implementation of this PCR-based gene-disruption technique will preclude the generation of mutant strains from being the rate-limiting step in performing genetic analyses. The recently reported RNAi and antisense methods for disrupting gene function and the ability to generate panels of strains containing randomly inserted signature tags should also aid in accelerating the analysis of gene function (Gorlach et al., 2002 ; Liu et al., 2002
; Nelson et al., 2001
). However, the PCR-based method proposed here has the potential to be applied to situations in which the ability to alter targeted genomic sequence is necessary. For instance, larger deletions that span multiple genes can be performed using this method. In addition to disruption mutations, this technique can also be applied to the efficient generation of other targeted insertions, including site-directed mutations and the insertion of regulatable promoters or epitope tags.
In conclusion, we show that this PCR-based gene-disruption approach is generally applicable for different genes using a variety of strains and genetic markers, and this should allow more efficient analysis of gene function in cases where the complete sequence is available.
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ACKNOWLEDGEMENTS |
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Received 27 February 2002;
revised 27 March 2002;
accepted 18 April 2002.