Aneuploid chromosomes are highly unstable during DNA transformation of Candida albicans

Abstract

Candida albicans strains tolerate aneuploidy, historically detected as karyotype alterations by pulsed-field gel electrophoresis and more recently revealed by array comparative genome hybridization, which provides a comprehensive and detailed description of gene copy number. Here, we first retrospectively analyzed 411 expression array experiments to predict the frequency of aneuploidy in different strains. As expected, significant levels of aneuploidy were seen in strains exposed to stress conditions, including UV light and/or sorbose treatment, as well as in strains that are resistant to antifungal drugs. More surprisingly, strains that underwent transformation with DNA displayed the highest frequency of chromosome copy number changes, with strains that were initially aneuploid exhibiting approximately 3-fold more copy number changes than strains that were initially diploid. We then prospectively analyzed the effect of lithium acetate (LiOAc) transformation protocols on the stability of trisomic chromosomes. Consistent with the retrospective analysis, the proportion of karyotype changes was highly elevated in strains carrying aneuploid chromosomes. We then tested the hypothesis that stresses conferred by heat and/or LiOAc exposure promote chromosome number changes during DNA transformation procedures. Indeed, a short pulse of very high temperature caused frequent gains and losses of multiple chromosomes or chromosome segments. Furthermore, milder heat exposure over longer periods caused increased levels of loss of heterozygosity. Nonetheless, aneuploid chromosomes were also unstable when strains were transformed by electroporation, which does not include a heat shock step. Thus, aneuploid strains are particularly prone to undergo changes in chromosome number during the stresses of DNA transformation protocols.

FIG. 1.

RNA expression levels correlate with DNA copy number. (A) RNA expression (log₂ ratios) analysis for Chr2 and -3 of fluconazole-resistant isolates (2-80, 8-46, and 12-99) compared to a drug-sensitive isolate (2-79). Lower relative expression of Chr3 suggests monosomy in all three fluconazole-resistant isolates. (B) aCGH analysis (DNA copy number) for Chr2 and -3 of the isolates used for panel A relative to the known diploid SC5314. Higher levels of Chr3 DNA in the drug-sensitive isolate (2-79) indicate trisomy in this isolate, while the fluconazole-resistant isolates (2-80, 8-46, and 12-99) are disomic for Chr3.

FIG. 2.

Proportion of aneuploid strains based on experiment type. (A) Four hundred eleven published expression experiments were analyzed to predict the frequency of aneuploidy in C. albicans strains. Experiment type included DNA transformation, treatment with fluconazole, exposure to UV and/or sorbose, and time course experiments. Numbers indicate the number of each experiment type included in this analysis. (B) The proportion of aneuploid strains from each experiment type was predicted by plotting expression data as a function of chromosome position. High levels of aneuploidy are predicted for UV and/or sorbose exposure, treatment with fluconazole, and DNA transformation, while very few aneuploids are predicted from time course experiments.

FIG. 3.

Aneuploidy is common in strains treated with UV/sorbose or fluconazole and in strains transformed with DNA. The proportions of strains from each experiment type (UV/sorbose exposure, fluconazole exposure, DNA transformation) that exhibit no aneuploidy, a single aneuploid chromosome, and multiple aneuploid chromosomes (A, C, and E) and of chromosomes that became aneuploid (B, D, and F) are shown. Because CAI-4 was aneuploid prior to transformation, genome instability is indicated as a change in chromosome copy number, and this change frequently involved the aneuploid chromosome Chr2 (G).

FIG. 4.

Chromosome copy number changes are frequent in aneuploid strains. Strains RM1000 2, CAI-4 F2, and CAI-4 F3 were transformed by LiOAc-heat shock transformation (A to C), and CAI-4 F3 was transformed by electroporation transformation (D). Chr1 and -2 copy numbers were predicted by quantitative real-time PCR. Because the different strains have different numbers of Chr1 and Chr2 (e.g., CAI-4 F2 parental chromosome copy numbers are two for Chr1 and three for Chr2), purple bars indicate that the transformants have not changed relative to the parental chromosome copy number; green bars indicate that transformants did have a change in chromosome copy number relative to the parent. Over half of the CAI-4 F2 and CAI-4 F3 transformants analyzed had a chromosome copy number that differed from that of the parent, while for nearly all RM1000 transformants, chromosome copy number resembled that of the parent (rightmost columns). CAI-4 F3 transformed by electroporation protocol also exhibited a large number of isolates that had an altered chromosome copy number relative to the parent.

FIG. 5.

Elevated temperature causes genetic instability. (A) CHEF gel of large and small colonies isolated after SC5314 was subjected to heat shock at 50°C for 60, 90, or 120 s, as indicated. Asterisks are located below (Sm-1 at 90 s) or above the chromosomes with suspected alterations in band intensity. C, control (SC5314 with no heat shock); Lg, large colony isolate; Sm, small colony isolate. (B) aCGH analysis of small colony isolate 2 after heat shock at 50°C for 120 s (from panel A) indicates multiple changes in chromosome copy number relative to SC5314 without heat shock: ChrR and -6 have become monosomic, while Chr3 and -7 have become trisomic. Some of these changes are detected by CHEF gel analysis (A). (C) Strain YJB9834 (heterozygous for URA3 on Chr5) was analyzed by fluctuation analysis at 30°C, 39°C, and 42°C. Bars indicate the LOH rate at the URA3 marker for each temperature tested.