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5-fluoro-orotic acid induces chromosome alterations in genetically manipulated strains of Candida albicans

     Department of Pediatrics, University of Rochester Medical Center, Rochester, New York 14642

M. Anaul
Kabir Elena Rustchenko ¹

     Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York 14642

	ABSTRACT

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We previously reported the occurrence of chromosome alterations in a Candida albicans prototrophic strain 3153A treated with 5-fluoro-orotic acid (5-FOA). In this study we investigated the mutagenic properties of 5-FOA with two derivatives of C. albicans strain CAF4-2 (ura3/ura3), each containing an ectopic copy of URA3 gene (ura3/ ura3 URA3) on a different chromosome. As expected, after the ura3/ura3 URA3 constructs were applied to 5-FOA containing solid medium, the "pop-outs" that lost URA3 appeared. However most of the "pop-outs" acquired various chromosome alterations. Thus constructs exposed to 5-FOA should be examined for chromosome alterations or the use of 5-FOA should be avoided.

Key words: chromosome instability, 5-FOA resistance, Ura^–mutants

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
We previously reported that treatment with 5-fluoro-orotic acid (5-FOA) of a Candida albicans prototrophic laboratory strain 3153A, which has not been manipulated genetically, produced 5-FOA resistant (Foa^R) mutants of two major types due to two specific chromosome alterations and not due to auxotrophy to uridine (Ura^–). Resistance of these prototrophic mutants depended on either 250 kbp inner enlargement of chromosome 5 or trisomy of chromosome 4. All mutants also acquired high instability of chromosome R and some mutants acquired trisomy of other chromosomes, alterations that are considered to be accompanying the specific alterations (Wellington and Rustchenko 2005). In addition a relatively short 24 h exposure to 5-FOA induced nonspecific changes in lengths of various chromosomes, although the cells remained sensitive to 5-FOA (Foa^S). An obvious and important question for laboratory studies is whether the use of 5-FOA for recycling gene deletion cassettes in C. albicans also induces chromosome alterations in the genetically engineered constructs. Such alterations obviously could inflict multiple phenotypic changes.

We obtained Ura^– Foa^R mutants on 5-FOA plates from two Ura⁺ Foa^S constructs, in which both copies of URA3 gene have been deleted and subsequently one copy re-integrated (ura3/ura3 URA3) on either chromosome 7 or 1. As expected all five randomly chosen mutants lost URA3. However when subclones of the five mutants were analyzed, approximately half of them unexpectedly acquired different alterations of various chromosomes, including those that were indicative of chromosome instability.

	MATERIALS AND METHODS

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Strains.— – Foa^S C. albicans prototrophic strain 3153A, as well as its clonal derivatives 3153A-A and 3153A-B were described by Wellington and Rustchenko (2005). C. albicans Ura^– Foa^R strain CAF4-2 (ura3/ura3) (Fonzi and Irwin 1993) was used previously to derive Ura⁺ Foa^S constructs CA61 and CA88 (ura3/ura3 URA3) (Wang et al 2004). In strain CA61 one copy of the URA3 gene was inserted at the LEU2 locus on chromosome 7 via the 7.6 kbp integrative plasmid pRC3915 (Cannon et al 1992). In strain CA88 one copy of the URA3 replaced one copy of the BMH1 on chromosome 1 via the 4.3 kbp URA3 blaster cassette. Constructs CA61 and CA88 gave rise to five Ura^– Foa^R "pop-outs", CA61F4, CA61F5, CA88F1, CA88F4 and CA88F5, from which two subclones each were prepared. Saccharomyces cerevisiae strain 867 chromosomes were used as size markers for C. albicans chromosomes.

Pulse field gel electrophoresis (PFGE).— – Both orthogonal field alternating gel electrophoresis (OFAGE) and contour-clamped homogenous electrophoretic field (CHEF) versions of PFGE were used. The chromosomes of each strain were separated under at least three different conditions that were optimal for the precise separation of short chromosomes, 5–7; middle-size chromosomes, 3 and 4; and long chromosomes, R, 1 and 2. Rustchenko-Bulgac and Howard (1993), Janbon et al (1998) and Perepnikhatka et al (1999) described the optimal PFGE conditions for different size ranges. Gels were stained with 0.5–1x GelStar (BioWhittaker Molecular Applications, Rockland, Maine) 1 h, distained 1 h to overnight with 0.5x Tris/borate-EDTA electrophoresis buffer and photographed with Polapan 55 PN film (Polaroid Corp., Cambridge, Massachusetts) supplied with negatives.

Various procedures and media.— – Yeast-peptone-dextrose (YPD), synthetic dextrose (SD) and 5-FOA media have been described (Sherman 2002, Rustchenko et al 1994, Wellington and Rustchenko 2005). Cells growth, preservation and maintenance, which were designed by us to prevent induction of chromosome instability, have been described (Perepnikhatka et al 1999, see Rust-chenko and Sherman 2002 for details). Wellington and Rustchenko (2005) have described preparations of cell mass and spot dilution assays that were used to determine phenotype of strains on various solid media. Densitometry was used to estimate the comparative amount of DNA in the bands on PFGE gel as described by Wellington and Rustchenko (2005). Polymerase chain reactions (PCR) were carried out as described by Wang et al (2004). Primers KR70 (CAG TTG AAG AAA GAA ATA GAA) and KR88 (TAT TTA TTC TAC ATA TAT ACA) were used to PCR amplify the coding region of the URA3 gene.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Isolation of Ura^– Foa^R "pop-outs" from ura3/ura3 URA3 C. albicans strains CA61 and CA88.— – The Ura⁺ Foa^S constructs CA61 and CA88 (ura3/ura3 URA3) having an ectopic URA3 on chromosomes 7 and 1, respectively (see Materials and Methods), were spread on 5-FOA plates at a high cell density. After appearance of the presumably Ura^– colonies (see below), five random "pop-outs", CA61F4 and CA61F5 from construct CA61, as well as CA88F1, CA88F4, and CA88F5 from construct CA88, were removed from the plates and streaked for independent colonies on fresh 5-FOA medium for purification. In anticipation of the heterogeneity that we found in the Ura⁺ Foa^R mutant populations derived from the prototrophic strain 3153A (Wellington and Rustchenko 2005), as well as for better comparison with Ura⁺ Foa^R mutants, we also prepared and analyzed two random subclones from each original mutant. The subclones that were denoted CA61F4-1, CA61F4-2, CA61F5-1, CA61F5-2, CA88F1-1, CA88F1-2, CA88F4-1, CA88F4-2, CA88F5-1, CA88F5-2, will be referred to further as "subclones", "mutants" or "pop-outs". The relationship among all strains is illustrated (FIG. 1).

View larger version (10K): [in this window] [in a new window]   FIG. 1. Origins and interrelationships of the sequentially derived strains of C. albicans. Shown are the original Ura– FoaR strain CAF4-2 (Fonzi and Irwine 1993); the Ura+ FoaS constructs CA61 and CA88 (Wang et al 2004); and their Ura– FoaR "pop-outs", which were derived in this study.

Frequencies of original Ura^– Foa^R "pop-outs".— – The frequencies of Ura^– Foa^R "pop-outs" were determined and compared with frequencies of Ura⁺ Foa^R mutants from prototrophic strain 3153A, whose Foa^R phenotype resulted from alteration of either chromosome 4 or 5 (Introduction). Two independent clones were prepared from strains 3153A, CA61, and CA88 and deposited as stocks at –70 C. Cells of each stock culture were prepared as a cell mass and plated on 5-FOA medium at approximately 5 x 10⁶ cfu per plate, either as a single plate or in duplicate. An aliquot of the same suspension was diluted further and plated for independent colonies on YPD plates in duplicate to verify the concentration of the suspension. Plates were incubated 20 d. The majority of the Ura^– Foa^R "pop-outs" appeared in the first 2 d. The approximate average mutant frequencies in CA61 and CA88 were 2 x 10^–5 or 5 x 10^–5, respectively, whereas in 3153A the average frequency was approximately 1 x 10^–6.

Phenotypes and electrophoretic karyotypes of "pop-outs".— – All 10 of the above mentioned mutants, CA61F4-1, CA61F4-2, CA61F5-1, CA61F5-2, CA88F1-1, CA88F1-1, CA88F4-1, CA88F4-2, CA88F5-1 and CA88F5-2, were analyzed for growth on SD and 5-FOA solid medium with a spot phenotype assay. As expected the mutants did not grow on uridine lacking SD medium and did grow well on uridine containing 5-FOA medium, which is toxic for Ura⁺ strains (data not presented). The mutants presumably became Ura^– by "popping-out" URA3 (see above) by homologous recombination between the flanking copies of LEU2 in derivatives of CA61 and hisG in derivatives of CA88 (see Materials and Methods). In addition to the verification of the constructs CA61 and CA88 by Wang et al (2004) the integration of URA3 in CA61 and the subsequent eviction of URA3 in its derivatives also is illustrated by change of size of chromosome 7, which carries LEU2. In Ura⁺ strain CA61, chromosome 7b enlarged on targeted integration of 7.6 kbp long plasmid pRC3915 carrying URA3 into LEU2 locus and migrated on PFGE gel higher than usual, immediately underneath chromosome 7a. Chromosome 7b consistently shortened and went down into the original position in Ura^– derivatives of CA61, which lost the integrated plasmid (as schematically presented in FIG. 2A–C). Photographs of the corresponding PFGE gels are not presented. The similar changes in migration pattern of chromosome 1 in Ura⁺ strain CA88 and its Ura^– derivatives that, respectively, incorporated and lost the URA3 blaster cassette, could not be observed in our separations. Approximately 4 kbp length blaster cassette was too small compared with the size of approximately 3 Mbp of chromosome 1. Although we successfully have separated the longest chromosomes, we have not determined the optimal running condition that would clearly resolve the long chromosomes and concomitantly reveal a small difference between them. The electrokaryotypes (shown schematically in FIG. 2) were reconstructed from precise separation of portions of the chromosome pattern on PFGE gels (Materials and Methods). Examples of precise separation are presented with the "pop-outs" from the construct CA88 (FIG. 3A, the short chromosomes or bottom group, B; FIG. 3B, the middle-size chromosomes or middle group, M; and FIG. 3C, the long chromosomes or top group, T). With this approach we could reliably estimate the positions and amount of DNA in the bands. It has to be noted that in most instances the change of chromosome banding pattern also could be identified within poorly separated areas (e.g. B-group in FIG. 3B or B- and M-groups in FIG. 3C). However the correct assignment of an altered chromosome, as well as the estimate of the chromosome copy number, requires precise separation.

View larger version (15K): [in this window] [in a new window]   FIG. 2. Schematic representation of the complete electrophoretic karyotypes of C. albicans original FoaR strain CAF4-2 and its sequential derivatives: FoaS constructs and FoaR "pop-outs". Dotted, thin and thick lines correspond respectively to one, two or three chromosomes. The chromosome numbering referring to the original strain CAF4-2 is presented on the left. The longer and shorter or same size homologues of the same chromosome are assigned respectively "a" and "b". Chromosome 4c refers to the additional chromosome 4 in subclone CA88F5-1. The assignment of individual bands to chromosomes in strain CAF4-2 have been published ( Janbon et al 1998, Rustchenko and Sherman 2002). Also presented on the left are the B-, M-and T-groups of chromosomes. + refers to the presence of 7.6 kbp plasmid pRC3915 on chromosome 7b and 4.3 kbp URA3 blaster cassette on one homologue of chromosome 1 in constructs CA61 and CA88, respectively (see RESULTS for more explanations of the corresponding band migrations). * refers to a band on the gel having an amount of DNA less than one homologue. ** refers to the array of weakly stained inseparable bands of chromosome R on the gel.

View larger version (34K): [in this window] [in a new window]   FIG. 3. PFGE analyses of precisely separated portions of the chromosome pattern of C. albicans FoaS parental construct CA88 and its FoaR "pop-outs". The different running conditions favored the precise separation of: (A) short chromosomes 5–7 comprising the bottom group B, separated with OFAGE; (B) middle-size chromosomes 3 and 4, middle group M, separated with OFAGE; and (C) long chromosomes R, 1 and 2, top group T, separated with CHEF. Note that the conditions for the precise separation of one group of chromosomes did not resolve or poorly resolved the other chromosomes (viz. M- and T-groups are compressed in [A]; T-group is compressed and B-group is poorly separated in [B], although the alteration of chromosomes 5 and 6 still can be seen in B-group; and B- and M-groups are poorly separated in [C]). On the left side of the gels in (A) and (B), some of the marker-chromosomes of S. cerevisiae can be seen. Black arrows refer to altered chromosomes. White arrows point the changes on a gel in a poorly separated B-group. (See FIG. 2 for the reconstruction of full electrokaryotypes as well as chromosome nomenclature).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Genetic manipulations and exposure to toxic compounds of C. albicans can produce undesired genomic changes resulting in misleading phenotypes (Wang et al 2004, Kabir et al 2005). In one instance we have established that confluent growth on sorbose medium of a certain construct was not due to deletion of one copy of BMH1 gene, for example, but due to the 5-FOA exposure that induced monosomy of chromosome 5 leading to sorbose use (Wang et al 2004). As a part of our more general analysis of the mutagenic properties of 5-FOA (Introduction), the preliminary observation was extended to a systematic investigation of the relationship between exposure to 5-FOA and chromosome alterations in constructs having a genetic background of strain CAF4-2. We prepared five post-5-FOA "pop-outs", which were chosen randomly and in which a single ectopic copy of URA3 was lost from either chromosome 1 or 7b. In this respect our "pop-outs" resembled the other "pop-outs" that are prepared routinely in many laboratories. The populations of most of the original Ura^– Foa^R "pop-outs" were unstable, a condition that was reflected in their electrokaryotypes by disproportional staining of some bands (see Rustchenko-Bulgac and Howard 1993, Rustchenko and Sherman 2002, and Rustchenko 2003 for the cumulative karyotypes of unstable populations). A similar problem was encountered with Foa^R derivatives from a prototrophic strain, and this problem was solved by analyzing independent subclones (Wellington and Rustchenko 2005). Two subclones thus were investigated from each of the original "pop-out" that also provided a better comparison with the previously published prototrophic Foa^R mutants.

Subclones of one original "pop-out", CA61F5, showed no changes (i.e. their electrokaryotypes resembled the electrokaryotype of the original strain CAF4-2) (FIG. 2A and C, column 1). The two original "pop-outs", CA61F4 and CA88F1, each were represented by one altered and one normal electrokaryotype (FIG. 2C, columns 1 and 2). Every subclone of the remaining "pop-outs", CA88F4 and CA88F5, showed differently altered electrokaryotype (FIG. 2C, column 3). In summary, of the ten electrokaryotypes studied, six were altered, which is approximately one-half of the "pop-outs" frequencies or approximately 1 x 10^–5, which is an order of magnitude greater than frequency of the specific chromosome alterations in prototrophic Foa^R mutants. However the frequency of 1 x 10^–5 is somewhat misleading because most of subclones from the same mutant were not the same, which is indicative of chromosome instability, as well as because the frequency of alterations can be strain-dependent.

One subclone, CA88F5-1, was similar to prototrophic Foa^R mutants because it contained a trisomy of chromosome 4, as well as an accompanying alteration, a high instability of chromosome R (FIG. 2C, column 3) (see Introduction). Such condition of chromosomes 4 and R clearly indicated that two independent mechanisms of resistance, one based on the auxotrophy to uridine and another based on a chromosome 4 trisomy, can operate concomitantly in "pop-outs". It is plausible that the second subclone CA88F5-2 from the same original "pop-out" initially also possessed chromosome 4 trisomy, which was replaced by normal disomy after prolonged growth. Chromosome Ra, however, retained instability as a relic of the initial state (FIG. 2C, column 3). Return to the normal disomy of chromosome 4 or, alternatively, loss of an enlarged chromosome 5 leading to chromosome 5 monosomy, has been shown previously in phenotypic revertants to Foa^S phenotype. In this regard the chromosome 5 monosomy in the clonally related CA88F4-1 and CA88F4-2 could be explained by the loss of an enlarged chromosome 5 (FIG. 2C, column 3). The high instability of chromosome R could have been maintained in only subclone CA88F4-1. The loss of the specifically altered chromosome 4 or 5 could occur, first, after the "pop-outs" were transferred from the original 5-FOA plate to a fresh plate for the mutants’ purification. At this point there was no selective pressure to retain specific alteration that confers Foa^R phenotype, in Ura^– Foa^R "pop-outs". Another opportunity arose during propagation in rich medium as a part of the procedure for preparing native chromosomes.

Of two remaining subclones one contained the shortening of chromosome Ra, which has been attributed to general mutagenic properties of 5-FOA (CA61F4-1 in FIG. 2C, column 2), and another one contained chromosome 7 alteration, which has not been seen before (CA88F1-2 in FIG. 2C, column 2).

We previously have investigated the stability of electrokaryotype during growth in various control rich media for different incubation times. No chromosome alterations were found (Rustchenko-Bulgac et al 1990, Rustchenko et al 1993). The routine strain cultivation in our laboratory usually does not lead to the high frequency alteration of electrokaryotype in subclones or induces high frequency instability. We believe that chromosome alterations that we report here derived by exposure to 5-FOA and not simply due to growth.

Although loss, enlargement and shortening of chromosome that underwent URA3 eviction in S. cerevisiae were reported (Hiraoka et al 2000) we did not observe these changes, except for the anticipated change in size of chromosomes 1 or 7 due to insertion and eviction of an integrative sequence (see Results). Further study might reveal similarities and differences in the action of 5-FOA on these two fungi. Also we did not observe either trisomy or monosomy of chromosome 1 that was deduced by study of Ura⁺ derivatives of strain CAF4-2 exposed to liquid 5-FOA medium by Chen et al (2004). The differences could be due to the experimental approaches or due to the handling the strains in laboratory. In addition future chromosome separations of the derivatives that were reported by Chen et al (2004) might clarify the matter.

The work presented herein, supports our earlier suggestion that strains treated with 5-FOA should be examined for their electrokaryotypes or on the other hand the use of 5-FOA should be avoided. Nevertheless strains with changed ploidy or with altered chromosomes can be "cured" by cultivation in YPD medium, which helps to enrich population with balanced euploids ( Janbon et al 1998, Wang et al 2004). On the other hand two subsequent manipulations can be performed with the URA3-flipper, which can be recycled with serum (Morschhäuser et al 1999), although effect of serum exposure on the electrokaryotypes remains to be determined. Another alternative would be the use of C. albicans strains that have a different marker, as for example GAL1 (Gorman et al 1991, Forche et al 2003). A different approach to two subsequent manipulations can be achieved with two different cassettes (e.g. the URA3 blaster or URA3-flipper and MPA^R-flipper) (Wirsching et al 2000). The use of two cassettes would allow a desired omission of one manipulation, cell exposure to toxic 5-FOA or mycophenolic acid, designated to recycle the cassette. Furthermore recently developed strains that are double auxotrophs for His and Leu or His and Arg, and triple auxotrophs for His, Leu and Arg, as well as the complementing plasmids, also allow the omission of cassette eviction and exposure to toxic substances (Forche et al 2003, Noble and Johnson 2005).

	ACKNOWLEDGMENTS

 
This work was supported by NIH research grant AI29433, the James P. Wilmot Cancer Research Fellowship Program and NIH training grant T32 AI07464.

	FOOTNOTES

 
Accepted for publication March 24, 2006.

¹ Corresponding author. E-mail: elena_bulgac{at}urmc.rochester.edu

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