JOURNAL OF BACTERIOLOGY, Oct. 1991, p. 6586-6596 Vol. 173, No. 200021-9193/91/206586-11$02.00/0Copyright © 1991, American Society for Microbiology

Variations of Candida albicans Electrophoretic KaryotypesELENA P. RUSTCHENKO-BULGAC

Department of Biochemistry, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

Received 11 December 1990/Accepted 13 August 1991

We previously described 14 rare spontaneous morphological mutants of Candida albicans that wereassociated with chromosomal aberrations (E. P. Rustchenko-Bulgac, F. Sherman, and J. B. Hicks, J.Bacteriol. 172:1276-1283, 1990). Improved conditions for separation of chromosonmes, as well as hybridizationprobes, were used to investigate the variation of karyotypes of clinical isolates and additional morphologicalmutants. All 23 newly analyzed morphological mutants, representing frequently occurring and highly unstablecolonial forms, had a variety of altered karyotypes. All chromosomal changes were sinilar to those previouslyobserved in mutants ml to m14. In this study, I particularly noted that the most frequent changes involved thelong chromosome VIII, which carries ribosomal DNA cistrons. Two rates of instability were uncovered byanalyzing the progenies from two highly unstable mutants. An unstable mutant proved to be able tocontinuously produce a large number of altered karyotypes that could result in a wide variety of differentphenotypes. Furthermore, all four independent clinical isolates, FC18, C9, 3153A, and WO-1, commonlaboratory strains, revealed different electrophoretic karyotypes and distinct colonial morphologies on asynthetic medium, similar to spontaneous mutants. The differences of electrophoretic karyotypes observedamong clinical isolates resembled the changes found among different kinds of spontaneous morphologicalmutants. These findings contribute to the understanding of natural karyotypic variability and are in agreementwith the hypothesis that chromosomal alterations observed spontaneously under laboratory conditions providethis amictic species with genetic variability in nature.

We previously demonstrated that standard laboratorystrains of Candida albicans, initially clinical isolates, spon-taneously gave rise to a large spectrum of different single andmultiple chromosomal rearrangements. These altered karyo-types were associated with differently altered colonial mor-phologies, i.e., morphological mutants, which appeared at ahigh frequency of approximately 1.4%. The rearrangementsconstituted changes of chromosome lengths and ploidy and,more rarely, possible translocations. Furthermore, many ofthe morphological mutants were unstable, producing mix-tures of different colonial forms at very high rates indicatingthe continuous production of chromosomal aberrations (27).Because independent clinical isolates were previously

reported to have different electrophoretic karyotypes (elec-tro-karyotypes) (11, 14, 17, 18, 22) and because C. albicansdoes not have a sexual cycle, we suggested that the geneticinstability observed under laboratory condition was thesource of genetic variations of this species. To support thishypothesis and to determine whether laboratory mutants canserve as a meaningful model of natural variability, I havefurther investigated electro-karyotypes of spontaneous mor-phological mutants and compared these mutant karyotypeswith those of some clinical isolates that have been studied inseveral laboratories. Because our initial study involvedprimarily the investigation of rare and unusual morphologi-cal mutants, and because I wished to compare clinicalisolates with frequently arising spontaneous morphologicalmutants, I have extended the study to two other classes ofmutants, those representing the most frequently occurringcolonial types and those representing highly unstable mor-phological mutants.

This study revealed that 23 newly analyzed spontaneousmutants all had differently altered karyotypes. The changesin the patterns of chromosomes were the same as thosepreviously described for some rare forms of morphologicalmutants ml to m14 (27). All altered chromosomal patternsobserved so far also resemble the differences in electro-

karyotypes of clinical isolates which represented naturalvariations. Specifically, the most frequently altered chromo-some was one of the largest, chromosome VIII, whichcontained ribosomal DNA (rDNA) cistrons. Furthermore, itwas found that highly unstable mutants could continuouslyproduce a wide variety of altered karyotypes, which conse-quently could probably result in a wide variety of differentphenotypes. These results explain the way in which naturalvariability of C. albicans isolates could occur and are inagreement with the hypothesis that chromosomal alterationsobserved spontaneously under laboratory conditions pro-vide this sexual species with genetic variability in nature.

MATERIALS AND METHODSStrains. The following C. albicans clinical isolates, which

are frequently used for laboratory studies, were employed inthis study: 3153A (7), recently confirmed to be virulent (5);WO-1, isolated from the blood of a patient with a systemiccandidiasis (30); C9, isolated from feces (13); and FC18,isolated from the cervix of a human patient (33). The mutantswere spontaneously derived from 3153A, of which ml tom14 were previously described (27).Our standard laboratory strain Saccharomyces cerevisiae

867 was used to provide size markers for Candida chromo-somes (27). Its banding pattern was similar to that of strainAB972 of Carle and Olson (4). These authors provided thesizes of the six shortest bands; sizes of the remainder wereprovided by Mortimer and Schild (23). The sizes of the top S.cerevisiae bands, shown in Fig. 3, served as markers for thethree arbitrary groups of C. albicans 3153A chromosomes.

Media. YPD medium contained 1% yeast Bacto extract,2% Bacto Peptone, and 2% glucose, with 2% Bacto Agarwhen required for solid medium (29). LBC synthetic medium(15), which was initially developed to monitor yeast-hyphatransition, consisted of 5 g of (NH4)2SO4, 0.2 g ofMgSO4- 7H20, 2.5 g of K2HPO4, 5 g of NaCl, 12.5 g ofglucose, 0.5 g of alanine, 1.3 g of leucine, 1 g of lysine, 0.1 g

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VARIATIONS OF C. ALBICANS ELECTROPHORETIC KARYOTYPES 6587

TABLE 1. Hybridization probes

Chromosome Name or gene Reference

I pBL1-6, undefined 14II BEN4 (Ben') 16III CAGi 21

L YS2 16IV IH399, undefined 24

pBL4-1, undefined 14V ADE2 12

URA3 9SOR2 16

VI HIS3 25VII TRPI 25

GAL] 25TUB2 25

VIII Ca5 (rDNA) or WOL-25 (rDNA) 21SOR9 16

of methionine, 0.0714 g of ornithine, 0.5 g of phenylalanine,0.5 g of proline, 0.5 g of threonine, 1 mg of biotin (SigmaChemical Co.), and 1 liter of distilled water, with 20 g ofBacto Agar when required for solid medium. The biotin wasfilter sterilized and added after the other ingredients wereautoclaved at 120°C for 15 to 20 min.

Maintenance of strains. All strains were streaked on solidYPD medium, grown for 2 days at 30°C, suspended in 15%glycerol, and stored at -70°C.Growth of strains. Strains were grown by transferring a

heavy inoculum from the preserved frozen stock to solid orliquid YPD medium. Because of instabilities, attempts weremade to use a population of cells to represent a particularstrain, and the general practice of subcloning was avoided.Standard conditions for examining colonial morphologies.

Approximately 20 cells were spread on each LBC plate, andapproximately 100 colonies of each strain were examinedafter 4 weeks of incubation at room temperature.DNA sample preparation. Chromosome-size DNA was

prepared from cultures grown to 2 x 107 to 4 x 107 cells perml as described by Carle and Olson (4).

Electrophoretic protocols. Orthogonal-field-alternation gelelectrophoresis (OFAGE) (3) was used to resolve C. albi-cans chromosomes. Three different running conditions ofOFAGE were applied to improve the separation. The firstcondition consisted of a constant 7.5 V/cm, approximately90 mA at the start of the run, a 160-s pulse time, 0.5xTris-borate-EDTA cooling buffer at 10°C (19), 1% agarose,and a 24-h running time. The second condition was the sameexcept for a 250-s pulse time and approximately 40-h runningtime. The third condition, which was applied to separategels, included a constant 3 V/cm, approximately 30 mA atthe start of the run, a 30-min pulse time, 1% agarose, and a

3- to 5-day running time.Hybridization probes and protocols. The probes used for

DNA-DNA hybridization to assign C. albicans chromo-somes are presented in Table 1. Labeling with [a-32P]ATP bythe random priming method, Southern blot hybridizations,and preparation of autoradiograms were carried out bystandard methods (8, 19).

RESULTS

Change of strain 3153A. We previously reported the phe-notype and electro-karyotype of strain 3153A, which was

obtained from J. Hicks and used by us for over 4 years (27).Its current colonial morphology differs from the previously

recorded form (compare Fig. 1 with Fig. Sa and b), probablybecause an altered subclone was chosen during subsequentinvestigations. I attribute this change to at least one visiblekaryotypic change; in contrast to an earlier finding thatchromosome VIIIb and chromosome VII comigrated, chro-mosome VIllb now migrates below chromosome VII (Fig.2a and b; Fig. 3).

Analysis of four clinical isolates. All of the four commonlaboratory strains, 3153A, WO-1, C9, and FC18, originallyisolated from patients, revealed individual colony morphol-ogies under standard conditions (Fig. 1). Strain C9 also hadaltered cell shape and grew slowly, possibly because of thepoor ability of cells to separate. In addition, all strainsdiffered in their electro-karyotypes, which were prepared ontwo gels for better resolution (Fig. 2a and g). The three verylarge chromosomes (top group) of each strain were assignedwith the following probes: CaS or WOL-25 (rDNA), SOR9,TRPI, GAL], TUB2, and HIS3 (Table 1 lists the DNAprobes used). The signals for rDNA and TRPI, whichdistinguished between the two largest chromosomes, VIIIand VII, are shown in Fig. 2b and c, respectively. One cansee that two homologous chromosomes VIII, containingrDNA genes, comigrated only in C9 (Fig. 2a and b). Incontrast, the rDNA-containing homologs deviated in theremaining strains; in these cases, the longer homologs con-tained more rDNA copies, as indicated by the strongersignals. As expected, however, probes other than rDNAproduced equally bright signals in deviated homologs (Fig.2c and the remaining probes in Fig. 2), suggesting thepresence of a single copy in each of the homologous chro-mosomes. Figures 2e, f, and i show signals of three otherprobes which facilitated the distinction of deviated ho-mologs. Dramatic differences between them in WO-1 can beseen in Fig. 2e and i. Slight differences between homologscan be seen in the other strains in Fig. 2e and i. Finalchromosomal assignments, schematically summarized inFig. 3, were based on the 16 hybridization probes presentedin Table 1 and the relative amount of DNA in each band,which was assessed from the intensities of ethidium bromidestaining. S. cerevisiae 867 was used to provide markers forthree conditional groups, bottom, middle, and top, of C.albicans chromosomes (27) (see Materials and Methods fordetails).

Besides the obvious fact that all four strains had differentchromosomal patterns, certain chromosomes remained thesame size. In Fig. 3, the strains are arranged from the left tothe right to illustrate increasing differences. Strains FC18,C9, and 3153A were more similar, all having the same sizesof chromosomes V, VI, and VII and the same sizes of thelonger homologs of chromosomes III and IV. Commondifferences among the strains were deviations of one of thehomologs and deviations and slight changes in chromosomallength (for example, chromosomes I, II, and VIII). Never-theless, all of the chromosomes remained within same majorgroups of sizes, designated bottom, middle, and top.

In comparison with the other strains, WO-1 appeared tohave major rearrangements. For example, one homolog ofchromosome III, identified with CAG1 () in Fig. 3), was

approximately 820 kb long and represented by far thesmallest chromosome. Its remaining segment, approxi-mately 580 kb, could have been fused to the nonidenticalchromosome II, approximately 1,100 kb, resulting in a new

chromosome having the exact size of the longer homolog ofchromosome IV (approximately 1,600 kb) in the middlegroup, as revealed with both the L YS2 (Q) and BEN4 (<)probes. On the other hand, the CAGI ((J3) and LYS2 (C4)

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FIG. 1. Colonial morphologies of clinical isolates C9, 3153A, WO-1, and FC18. The relative sizes of colonies are maintained in thephotographs. Note that the colonial morphology of 3153A difered from the earlier form shown in Fig. Sa and b and previously reported (27)(see Results).

probes indicated that the other homolog of chromosome IIIalso rearranged, probably fused to a 600-kb segment ofunknown sequence for which no probe exists, and produceda chromosome that is found at the exact size ofchromosomeVI in the top group, where presumably one homolog ofchromosome VII, identified with the TRPI, GAL], andTUB2 )] probes, is found as well (see Fig. 2e and c,respectively, for LYS2 and TRPI hybridization signals).(The increased amount of DNA in this band, which has avery intense and wide appearance with ethidium bromidestaining [Fig. 2a] but has the normal hybridization intensity

[for example, Fig. 2c], is consistent with this view.) Anotherexample of a dramatic difference is the shorter homolog ofchromosome IV, which also gave rise to a signal for thepBL1-6 probe, the marker of chromosome I. If the entireapproximately 1,000-kb chromosome I fused to a 400- to500-kb portion of chromosome IV, which encompasses themarker, then the remaining more than 1,000-kb unmarkedchromosome IV could be identified with the unknown bandon the top of the bottom group. Another major differenceoccurred at the position of chromosome V. Because theethidium bromide staining of the band at the position of

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VARIATIONS OF C. ALBICANS ELECTROPHORETIC KARYOTYPES 6589

....... .........

b *. P. 4.__b_0

e

h

c93 s-

CNC

om 0

0

th

i

* 2 _ , ~* 'IO

-t

J ~ -

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_____~~~~~~~~4

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FIG. 2. OFAGE separations of chromosomes of clinical isolates C9, 3153A, WO-1, and FC18. (a) The three longer chromosomes, top

group (T), were separated under improved conditions (see Materials and Methods). The remaining chromosomes were compressed into twothick bands. (b) Signals of chromosome VIII were revealed after hybridization of the filter shown in panel a with the Ca5 probe (rDNA). (c)Signals of chromosome VII were revealed after hybridization of the filter shown in panel a with the TRPI probe. (d) The entire karyotypeshowing well-resolved shorter chromosomes, bottom group (B), and middle-size chromosomes, middle group (M), was obtained by using twoconditions on the same gel as described in Materials and Methods. Note that the shortest WO-1 chromosome ran off the gel. (e and f) Signalsof chromosome III, with the LYS2 probe, and chromosome IV, with the pBL4-1 probe, were revealed after hybridization of the filter shownin panel d. Panels g and h show two independent preparations of entire karyotypes, similar to those in panel d. (i) Signals of chromosome Iwere revealed after hybridization of the filter shown in panel h with the pBL1-6 probe.

chromosome V is more intense but the ADE2 (®) hybrid-ization signal is normal (results not presented), an additional,unknown chromosome appears to be at this position.Thus, WO-1 appears to contain 17 chromosomes instead

of 16 and appears to have been derived by extensive rear-

rangements, including translocations. Despite all of thesedivergences, WO-1 strain preserved the same three struc-tural groups of sizes as did FC18, C9, and 3153A, as well asthe same lengths of certain chromosomes.

Analysis of spontaneous morphological mutants mlS tom20, representing two frequent colonial morphologies. Ourearlier study (27) was primarily concerned with 14 mutants,ml to m14, which had the most unusual morphologies andwhich all turned out to have abnormal karyotypes. Toextend the characterizations of spontaneous morphologicalmutants from our collection, we chose another six mutants,mlS to m17 and m18 to m20. These mutants represented twoof the three most frequent morphological forms, A and C.

Their origins and relative frequencies are schematicallypresented in Fig. 4, and the morphologies are shown in Fig.Sa.

The strains were preserved on YPD slants at 4°C and inglycerol at -70°C. Strains transferred from 4°C basicallyrevealed the expected morphology, with a few colonialvariants; however, the colonies of strains transferred from-70°C usually had drastic changes in morphologies, gener-

ally being smaller with vigorously growing mycelia. Thisphenomenon was previously found for C. albicans (2, 10,32), and its strong ability to be modified in response to a

variety of factors, such as medium composition, pH, oxygen

tension, and redox potential (20, 26), have been reported.Although I have not distinguished between modificationsand possible mutations when the cultures were transferred tothe standard conditions after storage, the electro-karyotypesrepeatedly prepared from the -70°C stocks were identical.

aTt

d

Mf

g

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J. BACTERIOL.6590 RUSTCHENKO-BULGAC

FC18 C9 3153A WO-1

vm X~@'K@'so 0 a. ......

t0 ....

V -)a)z)Q

v) Q 0 Q) )G

IV

Hi .~~~~~..........

rn

a)

I ..........

Top

Middle

,.).......

FIG. 3. Schematic representation of the elefour strains of C. albicans, FC18, C9, 3153A,portion of the electro-karyotype of one strain ofThe three groups of C. albicans chromosomes (btop) were resolved by using three different (applied to two independent gels as describecMethods. Dotted, thin, and thick lines correspo

one, two, and more than two chromosomes,of the lines related to the number of chromosof chromosomes I to VIII are indicated at thetions of chromosome VIII in the different st

by arrows. Chromosomes were assigned u!probes: pBL1-6 (an undefined fragment); )CAGI;®, LYS2;(), pBL4-l and IH399 (undefiADE2;@, URA3 and SOR2 (both tested onliHIS3;®, TRPI, GALI, and TUB2; (), Ca5and SOR9. S. cerevisiae 867 was used for size

Therefore, the miS to m20 electro-karyoered to represent two different colonial foi

In contrast to the previously describem14, all six mutants miS to m20 grew betteithan did parental strain 3153A.

Electro-karyotypes of frequently occuri

mutants. The chromosomes of mlS to m2on two gels to reveal the entire electrowell-resolved bottom, middle (Fig. 6a), z

groups of chromosomes. All six mutantaltered karyotypes, as schematically sumr

Hybridization of frequently occurring ml

with the rDNA probe. Hybridization of theFig. 6b with a sequence for rDNA (Tableidentify and compare one of the longenamed VIII (Fig. 6c and 7). All but one

altered patterns in regard to the positionstheir brightness.

Analyses of colonial instabilities of the

S. cerevisiae highly unstable morphological mutants m500 and m14. To867 continue the study of C. albicans instability, I examined two

highly unstable mutants. Mutant mSOO was chosen from thecollection of spontaneous morphological mutants (Fig. 4)because at least four different colonial forms arose at high

2_Mb frequencies after repeated subclonings. Two of the forms,2 Mb m5 and m6, were previously characterized for their colonial

and cellular morphology as well as for the electro-karyo-types (27).The unstable morphological mutant m14 was previously

detected among the progenies of a spontaneous unstablemorphological mutant, m502, as outlined in Fig. 4. Thecellular and colonial morphologies and electro-karyotype of

1.6 Mb m14 were previously reported (27). m14 was shown to haveabout twice tt amount of DNA per cell as did 3153A and tohave a small 1iaction (2 to 5%) of dikaryon cells.New colonal forms were found after the first round of

plating of mSOO and m14, as summarized in Fig. 4. Mutantsm500 and m14 dissociated, respectively, into 6 and at least11 easily distinguished forms (Fig. 4; Fig. 5b and c). Twodifferent patterns of instability were uncovered after the

-1.2 Mb second and third rounds of plating of m14 and m500. All butone of the m500 subclones produced stable uniform popula-

n tions, whereas all of the m14 subclones continued to producean overwhelming degree of variants.

Electro-karyotypes of subclones from highly unstable mu-tants m500 and m14. Electro-karyotypes were determined

0_82_Mb for subclones mS00-1 to m500-6, derived from the first round0.82Mb of plating of mSOO (Fig. 4 and Sb). Better separations of

ctro-karyotypes of chromosomes were achieved on two gels (Fig. 8a and c) asand WO-l, and a described for Fig. 2.fS. cerevisiae, 867. ecie o i.2

)ottom, middle, and The mSOO cumulative karyotype, which apparently repre-)FAGE conditions sents a mixed population, contains a diffuse band thatI in Materials and dissociated into two well-separated homologs of chromo-nd, respectively, to some VIII of various lengths in the subclones (Fig. 8a and 7);with the thickness this diffuse band is schematically presented as three bands inomes. Assignments Fig. 7. The mSOO cumulative karyotype does not reveal theleft. Various posi- following additional bands, probably because the corre-rains are indicated sponding mutants are represented at too low a frequency:sing the following the increased level of chromosome IVa in m5OO-1; the) BEN4 (Benr); tincreased level of the chromosome V in m5OO-3; the homologined fragments); j' icesdlvlo h hoooeVi 503 h ooo

y with3153A)fat; Ia in m5OO-S; and the homolog Illa in mSOO-6. All sixor WOL-26 (rDNA) subclones of mSOO had different patterns of chromosomes, asmarkers. schematically shown in Fig. 7.

As was done for the mSOO subclones, I determined theelectro-karyotypes of m14-1 to m14-11, which derived fromthe first round of plating of m14 (Fig. 4 and Sc). The

types are consid- chromosomes were separated on two gels (Fig. 9a, b, d, andrms. e) as described for Fig. 2. The m14 cumulative karyotyped mutants ml to (not shown) differed from the one reported earlier in itsr on LBC medium bottom, middle, and top groups of chromosomes. The pre-

vious and current m14 karyotypes are presented schemati-ring m15 to m20 cally in, respectively, the upper and lower parts of Fig. 7.!0 were separated These differences probably occurred because a subclone was'-karyotypes with used for preparing DNA in the early study, whereas a massand top (Fig. 6b) of cells was used in this study. Two pairs of subclones,ts had differently m14-6-ml4-9 and m14-7-ml4-10, had identical chromosomenarized in Fig. 7. patterns. The remaining subclones differed according to the[5 to m20 mutants schematic presentation of the electro-karyotypes in Fig. 7.filter presented in Hybridizing subclones from m500 and m14 with an rDNA1) permitted us to probe. I compared the signals obtained in response tost chromosomes, hybridization of the filter in Fig. 8a with the rDNA sequence,strain, m1S, had as was done for mutants mlS to m20. Similarly, the signalsof homologs and exhibited different patterns in regard to their positions and

brightness. An additional homolog of chromosome VIII wastwo spontaneous revealed in mSOO-3 at an altered position. To establish its

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VARIATIONS OF C. ALBICANS ELECTROPHORETIC KARYOTYPES 6591

IndepntaOones Of 3153A.. . . . .~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

985% 0A9% 0.2% 0.2% 0.1%

mlS m16 m17 ml8 ml9 n2

94% 02% 2% 0.2%

82% 4% 4%

1%

52%48%100% 90

30% 30% 15% 10% I

35% 25% 20% 5% 5% 5%

0.1% 0.05% 0.05% 0.05% 0.01% 0.01% 0.01% 0.2%

m502/ sm501 m503 m504

m7 m8 m13 mll m12 m3 m4 ml m2 m5 m6 mlO m9

1% 0.2% 0.2% 0.2% 0.2% 0.1% 62% 13% 13% 6% 4% 2%

A~~~~~1(i0c(\d(l (0)(I) 10% 80%20% 100% 100% 100% 100% 100% 20% 71

10% 5% 58%22% 13%7%

5% 50% 24% 13% 13%

76% 20% 4%

1stplating

85R8% 2%

Q.

*0NSDQQ

3rd

6 to 20 different forms from each colony 100% 100%100%9100%100% p g

90% 6% 3% 83% 17%

FIG. 4. Origins of spontaneous morphological mutants from strain 3153A and patterns of instabilities of m14 and m500. The circled Nrefers to the normal colonial form of parental strain 3153A as shown in Fig. 5a and b. The circled letters and numbers denote variousmorphological types derived from the parental strain 3153A (A to L) and from the unstable mutants m14 (1 to 11) and m500 (1 to 6). The circledL denotes heterogeneous and ambiguous forms. Independently derived mutants belonging to a specific morphological type are indicatedbelow the semicircles at the top. The approximate relative percentages of the different morphological types are indicated below themorphological types.

localization, the filter shown in Fig. 8c was hybridized withrDNA as well. The greatly shortened homolog comigratedwith chromosome V, explaining the increased ethidiumbromide staining. The signals exhibited by chromosome VIIIin strains m14-1 to m14-11 (Fig. 7; Fig. 9c and f) reflected a

variability similar to that observed with mlS to m20 andm500-1 to m500-6.

DISCUSSION

We previously reported a collection of C. albicans spon-taneous mutants that arose from strain 3153A at a highfrequency and that had different types of altered colonies(27). All 14 of these so-called morphological mutants, ml tom14, that were chosen from the collection because of theirrare and unusual morphologies (Fig. 4), were found to havegross single and multiple chromosomal changes (Fig. 7). The

types of alterations included production of aneuploidy,changes of chromosomal length, and possibly cases of trans-locations. In this study, the two different patterns of insta-bility of mutants m5OO and m14 (Fig. 4; see Results) indi-cated that formation of altered karyotypes is even morecomplex because of the subsequent stabilization of some ofthe subclones and the different patterns of instability. Such aphenomenon is reminiscent of mobile elements (1). Althoughfurther analyses of additional unstable mutants undoubtedlywill reveal a larger range of various patterns, it has alreadybeen observed from the results for m14 that a single spon-taneous mutant can continuously produce an overwhelmingrange of altered karyotypes, resulting in a wide variety ofdifferent phenotypes.Mutants mlS to m17 and m18 to m20, representing the A

and C phenotypes, respectively (Fig. 4 and 5a), arose most

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I

|_.~~ ~ ~~~~ XW

IFIG. 5. Colonial morphologies of parental strain 3153A (+) and spontaneous morphological mutants. (a) Most frequently occurring

mutants; (b) colonial forms arising from the unstable mutant m500 after the first round of plating; (c) colonial forms arising from the unstablemutant m14 after the first round of plating. The relative sizes of the colonies are maintained within panel a and within panels b and c. See Fig.1 and Results for variation of the 3153A morphology; see Fig. 4 for the origins of the spontaneous mutants.

h*_ 0

13'

FIG. 6. OFAGE separations of chromosomes of spontaneous morphological mutants ml5 to m20, which represent frequently occurringcolonial forms. (a) Well-separated short (bottom group [B]) and middle-size (middle group [MI) chromosomes of mlS to m17. The bottom andmiddle groups of m18 to m20 chromosomes were normal and are not shown. (b) Well-separated three largest chromosomes (top group [T]).See Fig. 2a and b, respectively, for the conditions of separation used in panels a and b. (c) Signals of chromosomes VIII, revealed afterhybridization of the filter in panel b with the rDNA probe.

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VARIATIONS OF C. ALBICANS ELECTROPHORETIC KARYOTYPES

mSQ3 mS5

ml m2 m3 m4 mS m6-...-- .....

m7 m8 m9,l ~

........

mSOl

mllmlO ml2 m13

A C

m14 mIS m16 m17 mlg m19 m20................ .....

-..... -,, .,. ........ ...... . ,, . .............. _ -

m500

5s5>+ 1 2

Jt lab I......,~~~~~~: ...VI

3 4 5 6 m14..... .... . ...........

m14

6 71 2 3 4 5 9 10 8 11

..... .....- .... ...... .........

.......... ..... ............ 00e .... ........ .........a..

LV-

ix Vb - -*: --::IVb

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..... . . .. . . . .. . . . .. . . . .. .................. _ .. ... ..._ . .. .. . ... ......_ .. ... ..._. . .. . . ... ......__

_.. . .. .. . .. .. . .. .. . .. .. . .. .. . .. .. . .. .............................. _ .. . .. .. . .. .. . .. .. . .. ...............

FIG. 7. Schematic representation of the electro-karyotypes of clinical isolates 3153A, FC18, C9, and WO-1 and of the spontaneousmorphological mutants derived from 3153A. The following clonally related pairs of the most morphologically unusual mutants ml to m14 are

shown below braces: m3 and m4; m5 and m6; and mll and m12. Mutants mlS to m17 and m18 to m20 are representatives of the frequentlyoccurring morphological types A and C, respectively. Derivatives of the unstable mutants m500 and m14 from the first rounds of platings are

presented below the corresponding brace. (See Fig. 4 for origins of the strains and Fig. 5 for their morphologies.) Assignments ofchromosomes I to VIII and the homologous pairs, a and b, of strain 3153A are indicated at the left. See the legend to Fig. 3 for explanationsof the different band thicknesses, the probes used for chromosomal assignments, and chromosomal characterizations of clinical isolates C9,FC18, and WO-1. When the intensity of ethidium bromide staining was intermediate between those of single and double copies, indicatinga mixed population, the bands were presented as double copies. The asterisk denotes the band in which a homolog of chromosome VIIIcomigrated with chromosome V.

frequently from the parental strain 3153A. All of these

mutants showed single or multiple chromosomal alterations(Fig. 6) similar to those observed in mutants ml to m14,representing certain rare morphologies (compare the karyo-types shown schematically in Fig. 7). These results indicatedthat no specific or preferred changes were associated with

the A or C phenotype. The fact that chromosome VIII was

solely altered in different ways in all three C mutants, m18 tom20, is difficult to relate exclusively to the C phenotypebecause chromosome VIII was also exclusively altered inmutants mlO to m13, which had distinct and different rare

phenotypes (27). I explain the biased distribution of pheno-

(VI==

-1l .....

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FIG. 8. OFAGE separations of chromosomes of the unstablemutant m5Q0 and its progenies mS00-1 to m500-6. (a and c) Separa-tions performed as described for Fig. 6b and a, respectively. (b andd) Signals of chromosomes VIII, revealed after hybridization of thefilters in panels a and c with the rDNA probe. The absence of therDNA signal for chromosome Vllla of m500-6 in panel b was due topoor blotting.

types in the collection (Fig. 4) by higher growth of thecorresponding strains (see Materials and Methods) and con-sequently higher representation in the steady-state popula-tion.

Despite the different patterns of m500 and m14 instabili-ties, the patterns of karyotypic alterations of the mSOO and

m14 subclones from the first round of plating were similar toeach other and similar to those of ml to m14 mentionedabove and previously described (27) (compare electro-karyo-type photographs and schematics of the m500-1 to m500-6and m14-1 to m14-11 subclones [Fig. 8 and 9] with those ofml to m14 [Fig. 7]). The alterations included mainly changesof lengths of homologous chromosomes and formation ofaneuploidy (see Results), which constituted the mainchanges in karyotypes of ml to m14 as previously reported.The improved separations of the largest chromosomes, theso-called top group, gave me the opportunity to observe thatchromosome VIII was clearly the chromosome most fre-quently altered. A total of 33 out of 38 mutants, or 87%, hadan altered chromosome VIII, compared, for example, with16 out of 38 mutants, or 42%, having an altered chromosomeIV. Altered chromosome VIII probably produces the largestproportion of altered cells in populations, making it difficultto maintain strains with the same karyotype and probablycausing the change of the 3153A strain having an alteredchromosome VIII and colonial morphology (see Results).(However, I do not exclude the possibility of other simulta-neous genetic changes which can be detected after applica-tion of DNA probes, as demonstrated earlier [27].) Presum-ably, recombinational events between multiple copies ofrDNA cistrons are responsible for this high frequency ofchromosome VIII alterations.

Theoretically, no distinction could be made between an-euploidy and mixed cell types present in equal proportionswhen more than two homologs of chromosome VIII werepresent at equal or nearly equal levels (for example, in m20,m500-4, and m14-11). A mixed population was clearly re-flected, nevertheless, in the cumulative karyotype of m500by the presence of a diffuse band at the chromosome VIIIposition. Mixtures of karyotypes also are evident with anumber of mutants, such as m14-3 and m14-4, which havelevels of ethidium bromide staining of chromosomes IVb and

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FIG. 9. OFAGE separations of chromosomes of m14-1 to mi4-11, spontaneous unstable morphological mutants derived from m14. Seelegend to Fig. 6 for separation conditions and probes.

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VOL. 173, 1991 VARIATIONS OF C. ALBICANS ELECTROPHORETIC KARYOTYPES 6595

IVa, respectively (Fig. 9a), that are intermediate to those ofsingle and double copies (e.g., the doublet in IVb of m14-5).On one hand, the ethidium bromide staining of the sepa-

rated chromosomes and the overwhelming production ofnew colonial forms in ml4 subclones indicated mixed popu-lations. On the other hand, there were authentic cases ofmultiple changes within an individual karyotype. This prom-inent feature was found earlier among mutants ml to m14and corroborated in this study, as one can see by comparing,for example, ml, m2, m7, and m8 with m500-3 and -5 andm14-2, -3, and -5.

Previously, we usually observed an aneuploidy of the 2n +x type. Here I also observed two instances of loss (2n - x).Chromosomes VIII of ml and Ia (or lIb) of m14-8 wereabsent (compare banding patterns in Fig. 7), implying rarecases of monosomy. In addition, trisomy of chromosome IIIin m500-6, the sole change in this, the only unstable subcloneof m500, may be a source of instability other than chromo-some VIII.Because we previously suggested that the high rate of

chromosomal rearrangements provides amictic C. albicanswith genetic variability (27), I compared clinical isolatesderived from patients with mutants derived under laboratoryconditions. Four clinical isolates, 3153A, FC18, C9, andWO-1, all commonly used laboratory strains, had individualcolonial morphologies under standard growth conditions(Fig. 1) (see Materials and Methods). In addition, C9 culturegrown in liquid YPD medium at 30°C had unusual cell shapesand slow growth, and the cells had a poor ability to separate,similar to morphological mutants arising spontaneously athigh frequency (27). Previously, others (6, 31, 32) reportedthat many freshly isolated strains from patients had distinctand characteristic colonial forms, also similar to spontane-ous morphological mutants (see reference 27 and referencestherein).The electro-karyotypes of all four strains also differed

from each other (Fig. 2a and g; Fig. 3), similar to earlierreports on many clinical isolates, including some of thosepresented herein (14, 17, 22). Furthermore, karyotypic vari-ation observed among clinical isolates resembled thechanges in spontaneous morphological mutants. Specifi-cally, all four clinical isolates differed in the homologs ofchromosome VIII, as did the majority of spontaneous mu-tants (Fig. 2a and b, 3, and 7). Also, strains FC18, C9, and3153A differed among themselves in a pattern similar to thatof the main types of alterations seen in the spontaneousmutants; i.e., either two identical homologs deviated, ordiverged homologs became identical in length (Fig. 3 and 7).In contrast, strain WO-1 varied dramatically but still resem-bled certain rare spontaneous mutants (21, 26). BecauseWO-1 was previously tested with Ca3 and Ca7 DNA hybrid-ization probes, we are confident that it is truly C. albicans(28). The variation between WO-1 and the other clinicalisolates probably involved translocations, since the hybrid-ization probes indicated different arrangements of genes(Fig. 2f, h, and i; Fig. 3). Whereas strains 3153A, FC18, andC9 appeared to be diploid, with 8 pairs of chromosomes, aswas previously reported for strain 3153A (27), strain WO-1appeared to have a total of 17 or more chromosomes, asrevealed by ethidium bromide staining of electrophoreticseparations and hybridization signals (see Results). Thecomplete correspondence between the chromosomes ofWO-1 and those of the other clinical isolates remains to bedetermined. Surprisingly, WO-1 contained a number ofnonhomologous, comigrating chromosomes, three of themfound in the position of chromosome VI and two found in the

position of chromosome IV, a finding difficult to explain.Interestingly and in contrast to findings for the other mu-tants, chromosome V and a shortened homolog VIII werethe same size in the spontaneous mutant m500-3 (Fig. 7 and8). Despite high frequencies of chromosomal rearrangementsand the lack of meiosis, both clinical isolates and spontane-ous mutants retained the general structure of their electro-karyotypes and lengths of chromosomes. However, becauseclinical isolates differed slightly in their patterns of restric-tion fragments when probed with Ca3 whereas spontaneousmutants ml to m14 did not (27, 28), clinical isolates haveundergone additional evolutionary divergence.

In conclusion, I have corroborated our previous findingthat naturally occurring strains of C. albicans are commonlydiploid, with eight pairs of chromosomes; however, anapparent trisomic aneuploid strain with possible transloca-tions was found. Genetic instability of spontaneous mutantsis a poorly understood, complex phenomenon. Although itinvolves rearrangements of all chromosomes, the most fre-quent alteration is due to changes of the length of chromo-some VIII, which encompasses the rDNA cistrons. Compar-ative analysis of variability of the electro-karyotypes of fournatural isolates and of 39 altered spontaneous mutantsrevealed a similarity in patterns, thus indicating that muta-genesis under laboratory conditions can be a valuable exper-imental model, and provides further evidence that chromo-somal aberrations are a natural means for genetic variability.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant R01AI29433 to F. Sherman from the National Institutes of Health.

I thank P. Magee and B. Magee (University of Minnesota, St.Paul), B. Lasker (Centers for Disease Control, Atlanta, Ga.), and M.McEachern (Scripps Clinic and Research Foundation, La Jolla,Calif.) for providing strains C9 and FC18 and hybridization probes,respectively. I warmly thank F. Sherman (University of Rochester,Rochester, N.Y.) for valuable discussions and assistance in prepa-ration of the manuscript.

REFERENCES1. Berg, D. E., and M. M. Howe (ed.). 1989. Mobile DNA.

American Society for Microbiology, Washington, D.C.2. Brown-Thomsen, J. 1968. Variability in Candida albicans. J.

Hered. 60:355-398.3. Carle, G. F., and M. V. Olson. 1984. Separation of chromosomalDNA molecules from yeast by orthogonal-field-alternation gelelectrophoresis. Nucleic Acids Res. 12:5647-5664.

4. Carle, G. F., and M. V. Olson. 1985. An electrophoretickaryotype of yeast. Proc. Natl. Acad. Sci. USA 82:3756-3759.

5. Di Domenico, E. Unpublished data.6. Di Menna, M. E. 1952. Natural occurrence of rough variant of a

yeast, Candida albicans. Nature (London) 169:550-551.7. Evans, E. G. V., F. C. Odds, M. D. Richardson, and K. T.

Holland. 1974. The effect of growth medium on filament produc-tion in Candida albicans. Sabouraudia 12:112-119.

8. Feinberg, A. P., and B. Vogelstein. 1983. A technique forradiolabeling DNA restriction endonuclease fragments to highspecific activity. Anal. Biochem. 132:6-13.

9. Gilium, A. M., E. Y. M. Tsay, and D. R. Kirsch. 1984. Isolationof the Candida albicans gene for orotidine 5' phosphate decar-boxylase by complementation of S. cerevisiae ura3 and Esche-richia coli pyrF mutations. Mol. Gen. Genet. 198:179-182.

10. Howard, D. Unpublished data.11. Iwaguchi, S.-I., M. Homma, and K. Tanaka. 1990. Variation in

the electrophoretic karyotype analysed by the assignment ofDNA probes in Candida albicans. J. Gen. Microbiol. 136:2433-2442.

12. Kurtz, M. B., M. W. Cortelyou, and D. R. Kirsch. 1986.Integrative transformation of Candida albicans, using a cloned

on February 10, 2021 by guest

http://jb.asm.org/

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nloaded from

6596 RUSTCHENKO-BULGAC J. BACTERIOL.

Candida ADE2 gene. Mol. Cell. Biol. 6:142-149.13. Kwon-Chung, K. J., D. Lehman, C. Good, and P. T. Magee.

1985. Genetic evidence for role of extracellular proteinase invirulence of Candida albicans. Infect. Immun. 49:571-575.

14. Lasker, B. A., G. F. Carle, G. S. Kobayashi, and G. Medoff.1989. Comparison of the separation of Candida albicans chro-mosome-sized DNA by pulsed-field gel electrophoresis tech-niques. Nucleic Acids Res. 17:3783-3793.

15. Lee, K. L., H. R. Buckley, and C. C. Campbell. 1975. An aminoacid liquid synthetic medium for the development of mycelialand yeast forms of Candida albicans. Sabouraudia 13:148-153.

16. Magee, B. B., Y. Koltin, J. A. Gorman, and P. T. Magee. 1988.Assignment of cloned genes to the seven electrophoreticallyseparated Candida albicans chromosomes. Mol. Cell. Biol.8:4721-4726.

17. Magee, B. B., and P. T. Magee. 1987. Electrophoretic karyo-types and chromosome numbers in Candida species. J. Gen.Microbiol. 133:425-430.

18. Mahrous, M., T. J. Lott, S. A. Meyer, A. D. Sawant, and D. G.Ahearn. 1990. Electrophoretic karyotyping of typical and atyp-ical Candida albicans. J. Clin. Microbiol. 28:876-881.

19. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual. Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y.

20. McClary, D. 0. 1952. Factors affecting the morphology ofCandida albicans. Ann. Mo. Bot. Gard. 39:137-164.

21. McEachern, M. J., et al. Unpublished data.22. Merz, W. G., C. Conneily, and P. Hieter. 1988. Variation of

electrophoretic karyotypes among clinical isolates of Candidaalbicans. J. Clin. Microbiol. 26:842-845.

23. Mortimer, R. K., and D. Schild. 1985. Genetic map of Saccha-romyces cerevisiae, edition 9. Microbiol. Rev. 49:181-213.

24. Poulter, R. T. M. Unpublished data.25. Rosenbluh, A., M. Mevarech, Y. Koltin, and J. A. Gorman.

1985. Isolation of the genes from Candida albicans by comple-mentation in Saccharomyces cerevisiae. Mol. Gen. Genet.200:500-502.

26. Rustchenko-Bulgac, E. Unpublished data.27. Rustchenko-Bulgac, E. P., F. Sherman, and J. B. Hicks. 1990.

Chromosomal rearrangements associated with morphologicalmutants provide a means for genetic variation of Candidaalbicans. J. Bacteriol. 172:1276-1283.

28. Sadhu, C., M. J. McEachern, E. P. Rustchenko-Bulgac, J.Schmid, D. R. Soil, and J. B. Hicks. 1991. Telomeric anddispersed repeat sequences in Candida yeasts and their use instrain identification. J. Bacteriol. 173:842-850.

29. Sherman, F., G. R. Fink, and J. B. Hicks. 1986. Laboratorycourse manual for yeast genetics and molecular biology. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

30. Slutsky, B., M. Staebeli, J. Anderson, L. Risen, M. Phaller, andD. R. Soil. 1987. "White opaque transition:" a second high-frequency switching system in Candida albicans. J. Bacteriol.169:189-197.

31. Soll, D. R., C. L. Langtimm, J. McDowell, J. Hicks, andR. Galask. 1987. High frequency switching in Candida strainsisolated from vaginitis patients. J. Clin. Microbiol. 25:1611-1622.

32. Vogel, R. A., and R. S. Sponcler. 1970. The study and signifi-cance of colony dissociation in Candida albicans. Sabouraudia7:273-278.

33. Whelan, W. L., R. M. Partrige, and P. T. Magee. 1980.Heterozygosity and segregation in Candida albicans. Mol. Gen.Genet. 180:107-113.

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