Origins and interrelationships of strains clonally derived from C. albicans SGY-243 and used in this study.

Schematic representation of electrophoretic karyotypes of two laboratory strains, SGY-243 and 3153A, and fluconazole-resistant mutants of SGY-243. Three groups of C. albicans chromosomes, bottom (B), middle (M), and top (T), can be resolved singly or in combinations by three different electrophoresis conditions. The chromosomes’ numbers, 1 to 7 and R, and homologues, a or b, are presented. The assignment of chromosomes to individual bands for the reference electrophoretic karyotype of 3153A and their approximate sizes, as well as the rationale for identifying the eight pairs of chromosomes, have been previously published (31, 34, 35). Some of the markers used in this study for hybridizing to 3153A chromosomes are indicated. Chromosomes were identified by the similarity of positions and intensity of bands to those of the reference karyotype of 3153A and by hybridization with the following chromosome probes (numerals in circles): 1, MDR1 (Ben^r); 2, ERG11; 3, CDR3; 4, NMT1; 5, LYS1; 6, CDR1; and 7, CDR2. Each chromosome copy number, presented in square brackets, was determined by densitometry. Dotted, thin, and thick lines correspond, respectively, to one, two, and three or more chromosomes. The array of thin lines for chromosome R represents a cloud of inseparable weakly stained bands. The changes of chromosome R observed in some mutants obtained in this study are not shown. The chromosome patterns of the resistant mutants that differ from SGY-243 are indicated. The ERG11 gene probe did not hybridize with chromosome 5a in strain 3153A (*), possibly due to a deletion. The LYS1 gene probe also hybridized to a band in the top group for SGY-243 (†). A single mutant, fzE5, retained only one homologue of 4a instead of two (‡).

The electrophoretic karyotype of the representative fluconazole-resistant mutant fzD5, obtained after 35 days of exposure to fluconazole. (A) The electrophoretic karyotype obtained by the OFAGE condition that accentuated the separations of the bottom (B) and middle (M) groups of chromosomes. Arrows indicate two specific chromosomal changes, the loss of two of three copies of chromosome 4a and an increase of one copy of chromosome 3. T, top chromosomes; S.c., S. cerevisiae 867 chromosomal size markers. (B) Autoradiogram of blot hybridized with the CDR1 probe, corroborating an increase in the copy number of chromosome 3.

Growth of resistant mutants after 48 h in media containing different concentrations of fluconazole, compared with the growth of the parental strain SGY-243 (▵) and the reference resistant mutant FR2 (▿). (A) Representative mutants fzE9 (●) and fzE10 (■) obtained after a short 7-day exposure to fluconazole. (B) Representative mutants fzD9 (●) and fzD10 (■) obtained after a long 35-day exposure to fluconazole. Arrows indicate the fluconazole concentration at which cell counts were taken for each strain. Note that each pair of resistant mutants is represented by a single curve because of their similar growth responses.

The levels of CDR1, MDR1, ERG11, CDR3, and NMT1 mRNAs in C. albicans strains relative to the levels of ACT1 mRNA. Dots blots of total RNA were hybridized with each of the five radiolabelled probes. Results are the means ± standard deviations (error bars) from three determinations. No CDR2 mRNA was detected in any strain. Four control strains were analyzed, each gave a similar result, and a representative strain, coC1, is shown. Two potential revertant strains were analyzed, both gave the same result, and one, fzG1, is shown.

Electrophoretic karyotype of the parental strain, C. albicans SGY-243. Chromosomes were separated by orthogonal field alternating gel electrophoresis (OFAGE) under conditions to accentuate the separations of either the bottom (B) and middle (M) groups of chromosomes (A) or the bottom but not the other groups (B). These conditions of separation do not resolve the top group (T) of large chromosomes. C. albicans 3153A was used as a reference electrophoretic karyotype, and S. cerevisiae 867 chromosomes (S.c.) were used as size markers. (C) Autoradiogram of chromosome blot hybridized with the MDR1 probe.

Electrophoretic karyotype of the representative fluconazole-resistant mutant fzE5, obtained after 7 days of exposure to fluconazole. Electrophoretic karyotypes obtained under two different OFAGE conditions, which accentuated the separations of different size chromosomes (Fig. 2) are shown (A and B). Arrows indicate chromosome 4a, in which two of three copies are lost. Note that no other detectable chromosomal changes are seen in these separations. Top (T), Middle (M), and bottom (B) chromosomes are indicated. S.c., S. cerevisiae 867 chromosomal size markers. (C) Autoradiogram of blot hybridized with the CDR3 probe, corroborating the loss of two copies of chromosome 4a.

Electrophoretic karyotype of the highly fluconazole-resistant mutant FR2. OFAGE separations of top (T), middle (M), and bottom (B) chromosomes (as described in the legend to Fig. 2). S.c., S. cerevisiae 867. The arrows indicate some visible changes in chromosomal patterns of FR2. (C) Autoradiogram of blot hybridized with the MDR1 probe, indicating a change in the position of chromosome 6b, as well as a reduction in copy number.

Sensitivities of strains to fluconazole. Cell densities are presented for strain SGY-243, the control strains, and the resistant mutant after growth in the presence of 128 μg/ml of fluconazole. Cell densities were determined with a cell-counting chamber. Each value is a mean of two to four determinations for mutants and 17 determinations for SGY-243.