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Genetics, Vol. 172, 1557-1566, March 2006, Copyright © 2006
doi:10.1534/genetics.105.052563
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* Department of Molecular Genetics, The Ohio State University, Columbus, Ohio 43210 and Department of Genetics, The University of Melbourne, Parkville, Victoria 3010, Australia
1 Corresponding author: Department of Molecular Genetics, The Ohio State University, 484 W. 12th Ave., Columbus, OH 43210.
E-mail: oakley.2{at}osu.edu
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. We have also developed heterologous markers that are selectable in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuA and the heterologous selectable markers make up a very efficient gene-targeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genomewide gene-targeting projects feasible.
The filamentous fungus Aspergillus nidulans is an important experimental organism. Significant findings in a number of areas have resulted from work with A. nidulans, and it serves as an important model organism for the genus Aspergillus, which includes commercially important fermentation organisms as well as serious pathogens. Gene targeting is possible in A. nidulans, but homologous recombination during transformation is not particularly efficient. Substantial stretches of homologous DNA are required to obtain useful frequencies of homologous integration and, even then, the majority of transforming sequences integrate heterologously. For example, in the study of YU et al. (2004), gene targeting with 29 different fusion PCR products with flanking sequences ranging from 480 bp to 4.3 kb resulted in correct gene-targeting frequencies ranging from 0 to 40% (20% or less in 24/29 cases). In addition, integration into multiple sites often occurs and transforming linear DNA fragments may circularize before integration. As a result, in most cases many transformants must be analyzed to identify one carrying a correct, single homologous targeting event. The A. nidulans genome has now been sequenced (GALAGAN et al. 2005) and the development of a more efficient gene-targeting system not only would facilitate current research with A. nidulans, but also would make possible genomewide gene-targeting projects (e.g., genomewide gene tagging or deletion).
We now report the development of a very efficient gene-targeting system for A. nidulans. Our approach is based on the results of NINOMIYA et al. (2004) who found that the deletion of genes required for nonhomologous end joining DNA repair (homologs of the human KU70 and KU80 genes) increases the frequency of gene replacement in Neurospora crassa. We have identified and deleted the A. nidulans KU70 homolog. This deletion has little or no effect on growth or sensitivity to mutagens, but it dramatically improves gene targeting. We have developed heterologous selectable markers that can be used for gene targeting in A. nidulans, and we have used our system for gene tagging; gene replacement, including replacement of essential genes (gene replacement/heterokaryon rescue); promoter replacement; and targeted integration of circular molecules. In all cases, the great majority of transformants carried a single correct integration. The system is efficient enough to allow genomewide gene-targeting projects. Our data in combination with the data of NINOMIYA et al. (2004) suggest that deletion of Ku homologs may be a generally useful strategy for improving gene targeting.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Methyl methanesulfonate (MMS) sensitivity tests were carried out on solid minimal medium with 10 mg/ml D-glucose as a carbon source and appropriate nutritional supplements [1.0 mg/ml uracil, 2.442 mg/ml (10 mM) uridine, 2.5 µg/ml riboflavin, 1 µg/ml para-aminobenzoic acid, 0.5 µg/ml pyridoxine, 0.5 mg/ml L-arginine]. MMS was obtained from Sigma-Aldrich.
The bar gene and selection:
The source of the bar cassette was Mogens Trier Hansen (Novozymes A/S, Bagsvaerd, Denmark). The bar gene encoding glufosinate resistance was taken from the plasmid pBP1T (STRAUBINGER et al. 1992). The bar gene was then placed between the amdS promoter [containing the I9 and the I66 mutations that give increased expression (see HYNES and DAVIS 2004)] and the Aspergillus niger glucoamylase terminator.
Glufosinate was prepared by chloroform extraction of the commercial herbicide Basta (Hoechst Schering AgrEvo GmbH), which contains 200 g/liter glufosinate–ammonium. The yellowish aqueous phase separated from the chloroform layer containing the blue dye added to this preparation by the manufacturers was taken for use. The aqueous phase was stored in the cold until used and was added at 25 µl/ml of 1% glucose minimal medium with 10 mM ammonium tartrate as sole nitrogen source.
Polymerase chain reaction:
Several polymerase chain reaction (PCR) polymerases and PCR procedures were used in the participating laboratories. All polymerase chain reactions were performed according to manufacturer's instructions. In some cases PCR was carried out as described by YANG et al (2004). In other cases AccuPrime Pfx DNA Polymerase (Invitrogen, Carlsbad, CA) was used to amplify shorter DNA fragments. AccuPrime Taq DNA Polymerase High Fidelity (Invitrogen, San Diego) was then used in the fusion PCR to obtain the final products. In other cases, Pfu DNA polymerase (Promega, Madison, WI) was used for amplification of short fragments and Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA) was used for long fragments.
Transformation of A. nidulans:
Transformation was carried out as described previously (ANDRIANOPOULOS and HYNES 1998; JUNG et al. 2000; YANG et al. 2004).
Southern hybridizations:
A. nidulans genomic DNA was isolated as described by OAKLEY et al. (1987) or LEE and TAYLOR (1990). Southern hybridization was performed in dried agarose gels (OAKLEY et al. 1987) or as described by YANG et al. (2004) or on Hybond N+ membranes (Amersham, Buckinghamshire, UK) after alkaline capillary transfer with 0.4 M NaOH.
Microscopy:
Images were taken using a 1.3 numerical aperture planfluor objective on an Olympus IX71 inverted microscope equipped with a mercury light source as well as a Uniblitz electronic shutter, a Prior z-axis drive, and a Hamamatsu Orca ER cooled CCD camera controlled by Slidebook software (Intelligent Imaging Innovations, Denver) on an Apple PowerMac G4 computer. Cells were grown and observed using four-chamber Lab-Tek chambered coverglasses (Nalge Nunc International, Naperville, IL). Imaging was through the coverslips at the bottom of the chambers as described previously (HORIO and OAKLEY 2005).
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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We deleted nkuA by replacing it with the A. nidulans argB gene (UPSHALL et al. 1986). We created, by fusion PCR, a fragment in which argB was flanked on each side by 2000 bp of the sequence that flanks nkuA in the genome (Figure 1). This fragment was transformed into strain KJ12. Ten argB+ transformants were screened by Southern hybridizations and two showed a pattern diagnostic for the replacement of nkuA by argB (Figure 1). We will use the abbreviation nkuA for this replacement, which is more completely designated nkuA::argB.
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strains for growth defects and increased sensitivity to mutagens. Growth and conidiation appeared normal in the initial nkuA transformants and in segregants of crosses that carried nkuA (Figure 1). NkuA strains crossed readily to other strains and, thus, apparently are not defective in meiosis.
In N. crassa, deletion of the KU70 homolog mus-51 results in increased sensitivity to the mutagens MMS, ethyl methanesulfonate (EMS), and bleomycin (NINOMIYA et al. 2004). The increased sensitivity to MMS and EMS was somewhat surprising in that these are point mutagens that do not cause double-strand breaks. We tested an nkuA strain for growth sensitivity to MMS over a range from 0.01 to 0.16% and found no difference in growth of the nkuA strain relative to the nkuA+ control (Figure 1). Similarly, we found no inhibition of an nkuA strain relative to an nkuA+ control on 0.5, 1.0, and 2.0 µg/ml bleomycin. This was somewhat surprising because although bleomycin has a complex mechanism of action (HECHT 2000), it does cause double-strand breaks (POVIRK et al. 1977). We also tested growth of an nkuA strain on 15 mM hydroxyurea, a DNA replication inhibitor, and found no inhibition. Finally, we tested the growth of an nkuA strain on 25 µg/ml camptothecin, a topoisomerase I inhibitor that can cause DNA breakage during replication (HSIANG et al. 1985). The nkuA strain showed no inhibition of growth relative to a wild-type control, but a positive control strain carrying a deletion of uvsB [a kinase with a central role in DNA repair (DE SOUZA et al. 1999; HOFMANN and HARRIS 2000)] showed, as expected, a significant reduction of growth relative to the nkuA and wild-type control strains. The fact that nkuA does not enhance the sensitivity to compounds that cause double-strand breaks indicates that A. nidulans has an efficient break repair system independent of nkuA.
Cloning of selectable markers from Aspergillus fumigatus:
Selectable markers from A. nidulans have a disadvantage in gene-targeting experiments in that homologous recombination (or gene conversion in the case of point mutations in selectable marker genes) can occur at the chromosomal copy of the selectable marker as well as at the targeted locus. Initial experiments indicated that this was a concern when targeting genes in nkuA strains (A. OSMANI and S. A. OSMANI, unpublished data). To eliminate this concern, we have cloned the A. fumigatus homologs of the A. nidulans riboB and pyroA genes to use as selectable markers. We have also used the previously cloned A. fumigatus pyrG gene (WEIDNER et al. 1998) and we have constructed a vector that confers resistance to glufosinate (phosphinothricin).
We partially sequenced the A. nidulans riboB gene that has previously been cloned (OAKLEY et al. 1987), searched the A. nidulans database, and found that the riboB gene corresponds to AN0670.2. We then carried out a blast search of the A. fumigatus genome (http://tigrblast.tigr.org/er-blast/index.cgi?project=afu1) and identified the riboB homolog (Afu1g13300, blast value 5.1 e-106). We used the sequence information to design PCR primers to amplify the gene from A. fumigatus genomic DNA and cloned the fragment into pBlueScript SK+, creating plasmid pTN2. Similarly, we identified Afu5g08090 as the A. fumigatus pyroA homolog through a blast search with the published pyroA sequence (OSMANI et al. 1999) (blast value 1.2 e-87), amplified the gene by PCR, and cloned the amplified fragment into pBlueScript SK+, creating plasmid pTN1. The A. fumigatus genes complement A. nidulans riboB2 and pyroA4 mutations but in numerous experiments (discussed subsequently and in our unpublished data) have not directed integration at these loci.
When plasmids pTN1 and pTN2 carrying the A. fumigatus pyroA and riboB genes (and no A. nidulans sequences) were used to transform nkuA strains, only small, abortive colonies were obtained. These did not continue to grow when subcultured onto selective medium. When an nkuA strain was transformed with a plasmid carrying A. fumigatus pyrG, larger, nearly normal colonies were obtained. Conidia from these colonies did not grow, however, to form colonies on selective medium. Stable transformants thus were not obtained with any of the three plasmids. Our interpretation is that nkuA prevents heterologous integration into the genome such that the transforming plasmids are eventually lost. The fact that the colonies obtained with the plasmid carrying A. fumigatus pyrG are much larger than the colonies obtained with the other plasmids is interesting and merits further study.
We have also used a vector (pMT1612) conferring glufosinate resistance, in which the bar (glufosinate resistance) gene of Streptomyces hygroscopicus from plasmid pBP1T (STRAUBINGER et al. 1992) is downstream of the A. nidulans amdS promoter [containing the I9 and I66 mutations that give increased expression (HYNES and DAVIS 2004)] and upstream of the A. niger glucoamylase terminator (M. T. HANSEN, personal communication).
Using this plasmid (or other circular plasmids containing this bar cassette as the selectable marker) results in strong resistant transformants observable on a background of "abortive" weaker resistant transformants that do not form stable resistant colonies when picked to new selective media. Thus transient expression of the bar gene from nonintegrated plasmids would seem to be sufficient to give glufosinate resistance initially. Unstable transformants are not seen with linear fragments containing bar.
NkuA dramatically improves the frequency of correct gene targeting:
We initially tested the effects of nkuA on gene targeting using a linear DNA fragment generated by fusion PCR to create a C-terminal histone H1-monomeric red fluorescent protein (mRFP) (CAMPBELL et al. 2002; TOEWS et al. 2004) fusion. We chose this test system because histone H1-mRFP fusions are easily scored by fluorescence microscopy. The strategy and results are shown in Figure 2. We initially transformed a control nkuA+ strain (LO 1180) and an nkuA strain (TN02A7) with a fusion PCR product consisting of the mRFP and A. fumigatus pyrG flanked on each side by 
2000 bp of DNA from the histone H1 gene and from the histone H1 3' untranslated region. The fusion PCR product was purified from an agarose gel. In the nkuA+ strain, we obtained 15 transformants, 10 of which had red fluorescent nuclei, indicating that they have a correct histone H1 mRFP fusion. This is an unusually high frequency of homologous integration, among the highest frequencies that we have obtained in an nkuA+ strain. One of the transformants grew poorly and was not used in subsequent experiments. We examined the remaining 9 histone H1 mRFP-positive transformants by Southern hybridizations and found that 7 had extra bands of hybridization in addition to the bands expected for a single correct homologous integration. Thus, only 2 of 15 transformants had a correct single integration.
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strain, 54 of 60 transformants tested (90%) were histone H1 mRFP-positive (Figure 2). We carried out Southern hybridizations on 12 positive transformants and each carried a single correct integration (Figure 2). NkuA thus increased the frequency of the desired targeting event from 13 to 90%.
Having established that gene targeting with a fragment with 2000 bp of flanking DNA is efficient in an nkuA strain, we wanted to determine if targeting is efficient with smaller flanking sequences. There are two advantages in being able to use small flanking regions. First, the fusion PCR is more efficient because the fragment is shorter. Second, because the flanks are shorter, there is less chance of PCR creating a mutation in the gene to be targeted or a nearby gene that overlaps with the 3' flanking region. We consequently tested the efficiency of gene targeting in an nkuA strain (TN02A7) using fusion PCR products with 1000- and 500-bp flanking regions. These products were not band purified from gels but were simply purified with Amicon YM30 filters to remove primers, nucleotides, etc. In both cases, 90% of the transformants were histone H1 mRFP positive (Figure 2). To determine if the transformants carried a single correct integration, we carried out Southern hybridizations on DNA from 10 transformants obtained with the fusion PCR product with 500-bp flanking regions. All transformants had a single correct homologous integration. In a transformation of an nkuA+ control (LO1180) with the fusion PCR product with 500-bp flanking regions, 19 of 50 transformants were histone H1 mRFP positive. Southern hybridizations were carried out on 12 of the histone H1 mRFP-positive transformants and 6 of them showed a single correct gene replacement. These data indicate that nkuA dramatically decreases the frequency of nonhomologous integrations, resulting in much more efficient gene targeting. They also indicate that 500-bp flanking regions are large enough to allow efficient gene targeting in an nkuA strain. It is worth noting that the histone H1-mRFP, nkuA strains grew robustly, at a rate indistinguishable from controls, and that the histone H1 allowed visualization of chromatin and chromosomes through the cell cycle.
Although gene targeting was efficient with 500-bp flanking regions, fewer transformants were obtained than with 2000- and 1000-bp flanking regions. We also attempted to GFP tag An-nsp1, the A. nidulans homolog of the Saccharomyces cerevisiae NSP1 nucleoporin (DE SOUZA et al. 2004), with a fragment containing 30-bp flanking regions. The advantage of such small flanking regions is that they can be synthesized as part of the PCR primer and this eliminates the need for fusion PCR. Few transformants were obtained and none carried a GFP-tagged An-nsp1. The picture that emerges is that nkuA greatly facilitates gene targeting by decreasing the frequency of nonhomologous integration during transformation. Transformation is less efficient with smaller homologous flanking sequences, but 500 bp of flanking DNA is adequate to give acceptable numbers and frequencies of correct targeting events
An obvious question is whether nkuA is useful for targeting other loci. We have now attempted to GFP tag the C termini of 28 genes of diverse functions in strains carrying nkuA. In 24 cases, tagging was successful and in 4 cases it was unsuccessful. The unsuccessful cases are probably due to lethality of the fusion protein. We have used fusion PCR fragments with 500-bp flanks in 12 of the 28 cases. In 10 cases, the tagging was successful and in the 2 other cases the C-terminal GFP fusion was apparently lethal. Fusion PCR products with 500-bp flanks thus are effective for targeting a variety of genes, although it is often advantageous to use larger flanking sequences because they give higher transformation frequencies. Interestingly, even in instances in which the fusion proteins are lethal, nkuA is advantageous in that many of the transformants are balanced heterokaryons (see below) and this can be a useful indication that the fusion is lethal. While we have not carried out a detailed analysis of each targeted gene, 114 transformants from 20 tagging experiments were examined by diagnostic PCR and 107 carried correct integration events (94%). We further examined 20 An-nsp1-GFP transformants by Southern hybridizations. All transformants examined carried a single correct integration event. (However, one transformant was not selected for further analysis because it grew poorly and thus might have carried an incorrect integration event.) We have also attempted to S tag (reviewed by TERPE 2003) the C termini of 14 proteins and were successful in each case. Forty-one transformants were examined by diagnostic PCR and 38 carried correct integrations (93%). NkuA thus seems to be of great value in tagging a wide variety of genes.
NkuA allows efficient gene targeting with suboptimal fusion PCR products:
Although fusion PCR is a remarkably useful technique, production of the correct, full-length product is often inefficient. In addition to the large, complete fusion PCR product, smaller incomplete products are present. In this case, the correct band must be purified from a gel or time and effort must be spent in optimizing PCR conditions for the particular fragment being amplified. We reasoned that if nonhomologous integration were reduced by nkuA, the smaller fragments might not be a significant problem. They would integrate homologously, simply replacing chromosomal sequences with identical sequences generated by PCR.
To mRFP tag the C terminus of 
-tubulin, we generated a fusion PCR product that contained an mRFP/A. fumigatus pyrG cassette flanked by 500-bp sequences from the -tubulin gene and its 3' untranslated region. The PCR fusion product also contained a prominent lower band and several additional bands in addition to the desired band (Figure 3). We transformed with this PCR product and examined transformants by fluorescence microscopy to determine if they carried a -tubulin mRFP fusion (easily scored because -tubulin localizes to the spindle pole body). Of 20 transformants tested, 13 contained a -tubulin mRFP fusion. These were analyzed by Southern hybridizations; 12 of the 13 contained a single correct integration and 1 contained a correct integration plus an additional band of hybridization. All -tubulin mRFP fusion strains grew indistinguishably from the wild type. These data demonstrate that nkuA allows reasonably efficient targeting with suboptimal fusion PCR products. This can be a considerable practical advantage in that it can save the time required to optimize fusion PCR or purify the desired fragment.
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facilitates promoter replacements, gene replacements, and heterokaryon gene replacements: strains for other types of gene targeting. It is often useful to regulate the expression of genes, and the alcA promoter of A. nidulans is highly regulatable, allowing gene expression to be turned down to very low levels or up to very high levels (ADAMS et al. 1988; WARING et al. 1989; KENNEDY and TURNER 1996). We tested the efficacy of fusion PCR and nkuA for replacement of the promoter of the A. nidulans MAD2 homolog, md2A, with the alcA promoter. We used the A. fumigatus riboB gene as a selectable marker and 500-bp flanking sequences. The fusion PCR product was not band purified before transformation. To test transformants for promoter replacements, we used the fact that deletion or underexpression of MAD2 homologs in many organisms causes hypersensitivity to antimicrotubule agents such as benomyl, and this is the case with a deletion of md2A in A. nidulans (PRIGOZHINA et al. 2004). We tested 10 transformants for benomyl sensitivity on YAG medium, which represses the alcA promoter (Figure 4), and on a minimal medium containing threonine as an alcA inducer. In instances in which the md2A promoter was replaced by the alcA promotor, the transformant should be benomyl hypersensitive on repressing medium but not on inducing medium. We found that this was the case with 10 of 11 transformants tested (Figure 4). We carried out Southern blots on the 10 positive transformants and found that all 10 had a single correct replacement of the md2A promoter by the alcA promoter.
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for gene replacements, we targeted two A. nidulans genes, AN5843.2 and AN2667.2. For these experiments, we used the bar (glufosinate resistance) cassette as the selectable marker and transformed strain TN02. The transforming fragments in these cases were constructed by normal recombinant DNA techniques rather than by fusion PCR. For AN5843.2, we used 1967 bp of 5' flanking DNA and 1127 bp of 3' flanking DNA. Five transformants were screened by Southern hybridizations and all carried a correct gene replacement. For AN2667.2, we used 1152 bp of 5' flanking DNA and 1721 bp of 3' flanking DNA. Eight transformants were screened by Southern hybridizations and all carried the correct gene replacement. None of these targeted deletions resulted in an observable phenotype. Targeted deletion therefore is highly efficient in an nkuA strain even when screening for an observable phenotype is not possible. Another extremely useful technique in A. nidulans is gene replacement/heterokaryon rescue (OSMANI et al. 1988; OAKLEY et al. 1990; MARTIN et al. 1997; JUNG et al. 2000). Replacement of essential genes with selectable markers during transformation is normally lethal, but balanced heterokaryons forming during transformation carry nuclei with the gene replacement as well as untransformed nuclei. These heterokaryons grow on the selection medium because the untransformed nuclei carry a functional copy of the targeted gene, while the transformed nuclei carry the selectable marker (replacing the targeted gene). Since conidia (asexual spores) are uninucleate, the conidia produced by a gene replacement heterokaryon will not grow to form colonies on selective medium. The phenotypes of the lethal gene disruptions or replacements can be determined, moreover, in the conidia produced by the heterokaryon. Gene replacement/heterokaryon rescue is not normally an efficient technique because two events must occur together—correct gene replacement and heterokaryon formation. On the other hand, if a transforming fragment integrates heterologously and does not disrupt an essential gene, the transformant can grow without heterokaryon formation. Thus there is a partial selection for heterologous integration.
To determine if heterokaryon gene replacement is facilitated by nkuA, we transformed an nkuA strain with a fusion PCR fragment consisting of the A. fumigatus pyrG gene surrounded on each side by 1000 bp of mipA flanking DNA. In two experiments, 54 of 93 transformants tested (58%) were balanced heterokaryons.
The efficiency of gene targeting in nkuA strains allows one to determine easily and rapidly if a gene is essential or not. If it is not essential, very few transformants will be heterokaryons. If it is essential, a substantial fraction of the transformants will be balanced heterokaryons in which the hyphae are able to grow on selective medium, but the conidia produced from the hyphae will not be able to grow on selective medium. We have used this approach in an ongoing project to examine the function of genes involved in nuclear transport. To date we have relatively complete data on 30 genes. In 18 cases, transformation with fusion PCR products designed to delete target genes produced high frequencies of balanced heterokaryons and were determined by diagnostic PCR to have deletions of the desired genes. These 18 target genes thus are essential. In 12 additional cases, heterokaryons were not produced and diagnostic PCR revealed that the target genes had been deleted. These genes thus are not essential. An added benefit of this approach is that the phenotype of lethal genes can be observed by germinating spores from the balanced heterokaryons.
Finally, we have found that cotransformation of two different transforming molecules (plasmid/linear, linear/linear) occurs in nkuA strains. However, nkuA does not improve the frequency of cotransformation (results not shown).
Gene targeting with circular plasmids:
Although fusion PCR is a powerful technique, it is sometimes advantageous to target plasmids for integration at particular sites in the genome. To determine if nkuA facilitates homologous integration of plasmids, we transformed strain TN02A25 with a plasmid carrying the bar gene flanked by flanking sequences for the acuE (malate synthase) gene (AN6653.2) (1313 bp of the 5' flank and 1123 bp of the 3' flank). Five transformants were screened by Southern hybridization. All five showed homologous recombination at the acuE locus. In four cases, the plasmid integrated by homologous recombination in the 5' flanking region, and in one case, crossing over in the 5' and 3' regions resulted in replacement of the acuE gene by bar. NkuA, thus, as expected, facilitates targeting of circular molecules.
We have used this technique to create a strain in which nkuA is replaced by bar. Strain TN02A1, which carries a replacement of nkuA by the A. nidulans argB, was transformed with a plasmid carrying the bar cassette flanked by 2288 bp of nkuA 5' flanking DNA and 2030 bp of nkuA 3' flanking DNA. One transformant carried a replacement of nkuA::argB by nkuA::bar. In addition, transformation of this strain with a linear fragment also generated nkuA::bar replacements and two of these were confirmed by Southern blot analysis (Figure 5). This strain (MH1046) is useful because the nkuA deletion can be followed in crosses by scoring for glufosinate resistance, eliminating the need for having argB2 in the strains to which the nkuA strain is crossed.
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We deleted nkuB by replacing it with the A. fumigatus riboB gene. We created, by fusion PCR, a fragment in which A. fumigatus riboB was flanked on each side by 1000 bp of the sequence that flanks nkuB in the genome. This fragment was transformed into the nkuA strain TN02A7. Transformants in which nkuB was replaced by riboB were identified by Southern hybridizations. For brevity, we will refer to this replacement as nkuB. A more complete, but cumbersome, designation is nkuB::A. fumigatus riboB.
The nkuA, nkuB double mutant grew at the same rate as the nkuA single-mutant strain and the parental strain (Figure 1). As with the nkuA mutant, the nkuA, nkuB mutant did not show enhanced sensitivity to MMS (Figure 1).
To determine if gene targeting is enhanced in the nkuA, nkuB double mutant relative to the nkuA single mutant, we transformed the double mutant with a fusion PCR product consisting of the mRFP and A. fumigatus pyrG flanked on each side by 2000 bp of DNA from the histone H1 gene and from the histone H1 3' untranslated region (the same linear construct that we used to transform the nkuA single mutant). Of 100 transformants, 87 were positive for histone H1 mRFP. This value is very similar to the value obtained with the nkuA single mutant (Figure 2) and gene targeting thus does not appear to be enhanced in the double mutant relative to the single mutant.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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, a deletion of a gene required for nonhomologous end joining, in combination with heterologous selectable markers. In numerous targeting experiments involving many different genes, 90% of transformants have been correctly targeted. This is true of GFP tagging, mRFP tagging, promoter replacement, and replacement of nonessential genes. It does not seem to be dependent on the transformation procedure since good results were obtained with three somewhat different transformation procedures. Replacement of essential genes rescued by the formation of balanced heterokaryons is somewhat less efficient, but is much more efficient than has been possible previously. Even transformation with crude PCR products containing multiple bands gives a majority of transformants with correct gene-targeting events. As importantly, integration events in additional to the correct targeting event are very rare. We have carried out Southern hybrizations on 83 transformants in nkuA strains to date. Only one carried transforming DNA sequences in addition to the correct targeting event and this transformant was from a transformation with a crude fusion PCR preparation. While there will certainly be variations among organisms, our data, in combination with the data of NINOMIYA et al. (2004), suggest that deletion of KU homologs may be a generally useful strategy for improving gene targeting.
The nkuA strains transform well and grow robustly. In experiments to date, we have seen no evidence for interactions of nkuA with tagged, deleted, or promoter replaced versions of a variety of genes involved in mitosis, nuclear transport, or cell cycle regulation. One would anticipate that nkuA would interact with mutations in genes involved in DNA repair, of course, and if one is concerned that nkuA will affect a process under study, it can be removed by a simple cross.
The gene-targeting system that we have developed is efficient enough that only a few transformants need to be obtained to be certain of having at least one with the correct targeting event. Assuming 90% to be the probability that each transformant carries a single correct targeting event, if one obtains even five transformants, then the probability that at least one will carry the desired targeting event is 0.9999. Since one need obtain only a few transformants to be certain of obtaining one with the correct integration, one can transform with a reduced number of protoplasts and a reduced amount of DNA. More usefully, it is quite practical to target 10 or more genes in a single transformation experiment with a protoplast preparation no larger than one usually uses to target a single gene in an nkuA+ strain. Fusion PCR, moreover, allows 10 or more transforming fragments to be generated quite quickly. The efficiency of gene targeting in the nkuA strains also allows one to determine quickly if a targeting event is lethal. For example, if a particular GFP fusion is lethal, a large fraction of transformants will be balanced heterokaryons (discussed below).
The rapidity of fusion PCR, coupled with the efficiency of gene targeting in nkuA strains, makes genomewide gene-targeting projects quite feasible. Indeed, if one can target 20 genes/day, one could target every gene in the A. nidulans genome in <500 working days. This should make genomewide gene tagging, promoter replacement, and gene knockout experiments feasible.
While the function of many genes in the A. nidulans genome can be guessed from sequence similarities, the functions of the majority of the genes are unknown. The combination of fusion PCR and nkuA provide a powerful system for knocking out these genes. A. nidulans has particular advantages, moreover, with respect to gene knockouts. The presence of a high frequency of balanced heterokaryons (the conidia of which do not grow to form colonies on selective medium) among gene knockout transformants is a strong indication that a gene is essential. In addition, one can usually determine the phenotype of the knockout by observing the germination and growth of spores produced by the heterokaryon. Likewise, the existence of excellent regulatable promoters in A. nidulans (ADAMS et al. 1988; WARING et al. 1989; KENNEDY and TURNER 1996; PACHLINGER et al. 2005) makes it possible, in principle, to repress or overexpress essentially every gene in the genome.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Note added in proof: After the online-ahead-of-print publication of this manuscript, two articles were published that indicate that deletion of KU homologs also improves the frequency of gene targeting in Aspergillus fumigatus (M. E. DA SILVA FERREIRA, M. R. KRESS, M. SAVOLDI, M. H. GOLDMAN, A. HARTL et al., 2006, The akuB(KU80) mutant deficient for nonhomologous end joining is a powerful tool for analyzing pathogenicity in Aspergillus fumigatus. Eukaryot. Cell 5: 207–211; S. KRAPPMANN, C. SASSE and G. H. BRAUS, 2006, Gene targeting in Aspergillus fumigatus by homologous recombination is facilitated in a nonhomologous end-joining-deficient genetic background. Eukaryot. Cell 5: 212–215).
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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used as a molecular weight standard. Lane 2 is the fusion PCR product used for transformation. The 4.0-kb fragment designated with an arrow is the correct fusion PCR product. (C) Fluorescence microscopy of a transformant germling shows the spindle pole body localization (arrows) characteristic of 
