Current Genetics
Current Genetics (1983)7:167-173
© Springer-Verlag 1983
UV-Induced Mitotic Co-Segregation of Genetic Markers in Candida albicans: Evidence for Linkage Marjorie Crandall 1 School of Biological Sciences, University of Kentucky, Lexington, KY 40506, USA
Summary. Parasexual genetic studies of the medically important yeast Candida albicans were performed using the method of UV-induced mitotic segregation. UV-irradiation of the Hoffmann-La Roche type culture of C. albicans yielded a limited spectrum of mutants at a relatively high frequency. This observation suggested natural heterozygosity. Canavanine-sensitive (CanS) segregants were induced at a frequency of 7.6 x 10 -3. Double mutants that were both CanS and methionine (Met-) auxotrophs were induced at a frequency of 7.4 x 10 - 3 . The single Met- segregant class was missing indicating linkage. UV-induced CanS or Met-CanS segregants occurred occasionally in twin-sectored colonies. Analyses of the sectors as well as the observed and missing classes of segregants indicated that genes m e t and can are linked in the cis configuration. The proposed gene order is: centromere - m e t - can. Thus, it is concluded that the Hoffmann-La Roche strain of C. albicans is naturally heterozygous at two linked loci. These findings are consistent with diploidy. Key words: UV - C. albicans - Linkage
Introduction Genetic studies of C. albicans have been impeded because a sexugl cycle has not been observed. Consequently, it has been necessary to use parasexual genetic approaches
Present address: Division of Infectious Diseases, Department
of Medicine E-5, Harbor-UCLA Medical Center, Torrance, CA 90509, USA
to the determination of the ploidy and inheritance of this yeast. In 1969, van der Walt and Pitout reported differences in cell volume and ploidy in derivative cell lines of C. albicans strain CBS 5736. Olaiya and Sogin (1979) using the technique of flow microfluorometry reported that C. albicans strain 3153A is diploid based on a comparison of its DNA content per cell with that of a series of strains of S a c c h a r o m y c e s cerevisiae of known ploidy. Germ tube negative variant clones 06 and 09 derived from 3153A were reported to have half the amount of DNA (Olaiya et al. 1980). However, Buckley et al. (1982) reporting on these same parental and germ tube negative strains found no difference in DNA contents. Clinical isolates of C. albicans studied by fluorescent microscope photometry were found to vary in ploidy (n, 2n, 4n) with diploid strains being observed most frequently and haploid strainsbeinggerm tube negative (Suzuki et al. 1982). Further evidence that most isolates of C. albicans are diploid comes from genetic approaches. Whelan et al. (1980) reported that strain FC18 of C. albicans is heterozygous at a single locus required for the synthesis of both methionine and cysteine based on results of UV-induced mitotic recombination. Subsequent reports by Whelan and Magee (1981) and Whelan et al. (1981) indicated that clinical isolates are frequently heterozygous for auxotrophic and/or antibiotic resistance markers. Riggsby et al. (1982) also reached the conclusion that the most common strains of C. albieans are diploid based on the analysis of the kinetic complexity of single-copy DNA. In contrast to these above-mentioned studies of natural isolates, two reports of parasexual genetic studies using induced mutants conclude that C. albieans is haploid (Sarachek et al. 1981; Poulter et al. 1981). This discrepancy could be explained if their mutagenesis procedures induced nondisjunction resulting in monosomic (2n - 1) strains which would behave like haploids in crosses performed by protoplast fusion.
168 The results presented here confirm the observations o f Whelan and co-workers who demonstrated that clinical isolates o f C albicans are naturally heterozygous for a limited number o f genetic markers. In this paper, studies o f UV-irradiation o f the t y p e culture o f C. albieans from Hoffmann-La Roche (strain 6631) have shown that this strain is naturally heterozygous for two genes linked in the eis configuration. UV-irradiation yielded two phenotypic classes: M e t - C a n S and CanS. Absence o f the single M e t - phenotypic class allowed the ordering o f the genes: centromere - m e t - can. This is the first report of linked genetic markers being present naturally in the heterozygous condition in an isolate of C albicans (M. Crandall, Abs. Ann. Mtg. Amer. Soc. Microbiol. D36, 1981) and represents additional documentation o f diploidy in C albicans.
Materials and Methods Strains. C albicans strain 6631 is the type culture from Hoff-
mann-La Roche and was obtained from Dr. Norman Goodman. A single cell was isolated from this culture by micromanipulation using methods published in Crandall and Caulton (1979). This clonal culture was labeled PY17-1AS (smooth colony type) and was used for all further studies. The species was confirmed by germ tube and chlamydospore formation and standard assimilation tests (Silva-Hutner and Cooper 1980). Strain FC18 of C. albicans was obtained from Dr. William L. Whelan and was used as a control for UV analysis of natural heterozygosity. UV Irradiation. Cultures were grown as a lawn on YPD agar (1% yeast extract, Difco + 2% peptone, Difco + 2% dextrose + 2% Bacto-agar, Difeo). Plates were inoculated with 0.5 ml of a YPD broth preculture and incubated overnight at 37 °C. Cells were resuspended from the plate in 5 ml of sterile deionized water (SDW), washed, counted in a hemocytometer and then diluted in SDW to yield 200 colonies/0.1 ml which was spread on each YPD plate. This population consisted of 66% single cells and the rest were doublets. Saline was not used for dilutions because it promotes self-flocculation with this strain (M. CrandaU, manuscript in preparation). The seeded plates were irradiated for various times with a UV lamp (model R-52 Mineralight, UltraViolet Products, Inc., San Gabriel, CA). The dose rate at the agar surface was 50 ergs mm - 2 s-1 (measurement courtesy of Dr. John Calkins). In a pilot experiment measuring UV survival (data not shown), it was demonstrated that it was not necessary to protect the UVqrradiated plates from visible light confirming a report by Busbee and Saraehek (1969) that C. albicans does not photoreactivate. The irradiated and control plates were incubated at 37 °C for 2 days and then replica plated to the following media: minimal (MIN) agar (0.67% yeast nitrogen base without amino acids, Difco + 2% dextrose + 2% agar), complete minus adenine (C-ADE), complete minus methionine (C-MET), complete (COM), COM minus arginine plus 100 ~g/ml canavaninesulfate, Sigma (CAN), and YPD plus 100 ~g/ml cycloheximide, Sigma (CYH), in that order. COM medium contains 20 amino acids and nucleotide bases each at a final concentration of 20 #g/ml added to MIN. All colonies or sectors identified as mutants on the replica plates were picked from the YPD master plate for streak purification on YPD agar.
M. Crandall: Genetic Linkage in C albicans
Results U V - I n d u e e d Segregants
In a pilot experiment using subclone PY17-1AS, a segregation frequency o f about 1% was observed after UV irradiation for 50 s. Of 7 segregants purified, all had lost the natural resistance o f the parent to canavanine becoming canavanine-sensitive (CanS); one was also lysine-negatire (Cans L y s - ) and 5 also required methionine ( M e t CanS) (M. Crandall, Abs. Ann. Mtg. Amer. Soc. Microbiol. D36, 1981). Some o f these mutants also showed an increased self-flocculation reaction and an unusual adenine phenotype (see below). Co-segregation o f genetic markers was suggestive of genetic linkage; therefore, a large scale isolation o f segregants was undertaken. The second UV irradiation experiment yielded 27 segregant colonies (12 CanS + 14 M e t - C a n S + 1 CanS segregant also requiring isoleucine, valine, and leucine) out o f 1998 survivors. The parental strain is naturally resistant to both cycloheximide and canavanine. However, no cycloheximide-sensitive segregants were ever observed indicating that this strain is not heterozygous for cycloheximide sensitivity. The third UV experiment yielded 46 segregants (24 CanS + 22 M e t - C a n S ) out of 2,735 survivors. Since the pattern o f segregation was similar in the second and third experiments, the data were pooled in order to obtain more accurate estimates o f the segregational frequences (Fig. 1). Upon streak-purification and retesting, some o f these segregants (18%) exhibited the wild t y p e phenotype perhaps because of cross-contamination due to spreading of colonies during replica plating. Others died (3%) either on the YPD streak plate or later in the YPD broth subculture. Many mutants (40%) were slow growers. On 14% o f the streak plates o f putative mutants, there was evidence for additional segregational events occurring (sectoring, papillae, large and small colonies). These observations may indicate that UV irradiation caused recessive lethals or other recessive genes to co-segregate in some of the mutants. No spontaneous segregants could be detected ( < 3 x 10 - 4 ) in the unirradiated culture of PY17-1AS (plotted as zero-time in Fig. 1). Hence, it is concluded that UV is inducing the segregation o f CanS and M e t - CanS mutants. At the lowest dosage tested (20 s = 1,000 ergs r a m - 2 ) , the survival was about 30%. No increase in the frequency o f segregants occurred at higher dosages (lower survival), i.e., the same proportions of the two segregant classes were obtained at all three dosages (Fig. 1). Hence, the data for all dosages were pooled in order to calculate the frequencies for b o t h segregants: CanS = 7.6 x 10 - 3 and M e t - C a n S = 7.4 x 10 - 3 .
M. Crandall: Genetic Linkage in C albicans
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complete growth inhibition ( - ) of 7B20 was only obtained with a mixture of all four amino acids (cys + phe + trp + val). Segregant 1720-1U (also Met-CanS) grew slowly (+) in the presence of these four amino acids but was negative on C-ADE indicating that additional amino acids or nucleotide bases in COM are inhibitory. The phenotype of these Met- CanS segregants is leaky on C-ADE and, for this reason, growth is scored in less than 24 h after replica plating. One Met-CanS segregant isolated out of 4733 survivors, 1720-10N, grows on C-ADE but not on C-MET or CAN. However, when additional cys is added to C-ADE plates (final concentration = 40 #g/ ml), even this Ade + segregant and an Ade + Met-CanS revertant, 7B20-R 14, are caused to require adenine. Thus, it seems likely that this unusual Ade- phenotype is related to the Met- phenotype rather than being a separate mutation. Thus, the met locus appears to be a pleiotropic gene perhaps affecting S-adenosylmethionine metabolism, considering the involvement of cysteine, adenine and methionine in this auxotrophic phenotype.
0.1
UV DOSE (Seconds) Fig. 1. UV survival and mitotic segregation of PY17-1AS. For each time period, 15 YPD plates (spread with about 200 cells) were counted to determine survival percentages. Then plates from each time period were replica plated to MIN, C-ADE, CMET, CAN and CYH in that order. After incubation overnight at 37 °C, replica plates were scored for sectored and nonsectored segregant colonies. No segregants were observed at zero time (arrow indicates < 3 x 10-4). Data from two experiments were pooled to calculate the frequency of segregants at 20, 40 and 60 s of irradiation. Symbols: e, % survival; x, CanS; A, Met-CanS
Description of the Methionine-Negative Phenotype These methionine auxotrophs grow on MIN + methionine but not on MIN + cysteine. All of the Met- segregants are also CanS. When these Met - CanS segregants are tested on drop-out media, they do not grow on C-MET (as expected) but they also do not grow on C-ADE (unexpected since the Met-CanS segregants grow on minimal medium supplemented only with methionine). The adenine requirement is due to inhibition by components of COM medium. Individual amino acids and nucleotide bases from complete stock were tested at high concentration added to MIN + MET with Met-CanS segregant 7B20. Cysteine (cys), phenylalanine (phe), tryptophan (trp) and valine (val) each caused some growth inhibition separately but when tested singly at the concentration in COM (20 /ag/ml), none caused complete growth inhibition. However, cys alone slowed growth the most (from 2+ to 1+). Combinations of three (cys + phe + val or cys + trp + val) inhibited growth of 7B20 from 2+ to +-. But
Description of the Canavanine-Sensitive Phenotype The Hoffmann-La Roche strain of C. aIbicans is canavanine-resistant (CanR). Following UV-irradiation, all segregants isolated are sensitive to canavanine. These CanS segregants are scored on CAN agar (= COM - ARG + CAN; see Materials and Methods). They are not arginine auxotrophs because they grow on C-ARG. Likewise, the double segregant Met-CanS grows on MIN + MET also indicating that these segregants are neither arginine nor adenine auxotrophs. The CanS segregants have a leaky phenotype on CAN; no growth ( - ) after one day at 37 °C but slight growth (+) after 2 days compared to fast growth (2+) of the wild type after only one day. Double segregants, Met-CanS, remain negative ( - ) on CAN plates after 2 days. For simplicity, the can gene is assumed to be recessive in C. albicans even though canavanine-sensitivity is dominant in Saccharornyces cerevisiae (Whelan et al. 1979).
Nature of the Isoleucine- Valine-Leucine Requffement Only one segregant out of 4,733 survivors had an IleV a l - L e u - phenotype. This segregant will not grow on C-ILE or C-VAL or MIN + ILE + VAL but will grow on MIN + ILE + VAL + LEU or C-LEU (contains ile + val). Apparently, a scavenger pathway from isoleucine and valine is being used to synthesize leucine when grown on C-LEU. Since no single ( I l e - ; V a l - ; L e u - ) or double (Ile- Val- ; Ile- L e u - ; Val- L e u - ) mutants were isolated, it seems likely that this triple auxotrophic requirement is caused by a mutation at a single locus. Kakar and Wag-
170
M. CrandaU: Genetic Linkage in C albicans
Table 1. Single reversion and co-reversion of markers # of Revertants a
Selected on
Frequency
3 9 3 2
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Segregant 7B20: 1.4 x 10 - 8 2.7 x 10 - 4 5.0 x 10 - 9 8.5 x 10 - 7
2 3 3 3
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Segregant 1720-1U: 1.3 x 10 - 8 2.6 x 10 - 3 6.7 x 10 - 9 3.0 x 10 - 6
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CeUs of two Met-CanS segregants (7B20 and 1720-1U) were precultured on YPD agar, resuspended in SDW at about 109 cells/ml, washed twice in SDW and spread on MIN, C-ADE, C-MET and CAN agar at various dilutions. Spontaneous revertant colonies were streak purified on the same selective medium, clones were inoculated into YPD broth stock culture and then retested by replica plating
ner (1964) described mutations that led to the double requirement I l e - V a l - named ilv mutations (Hawthorne and Mortimer 1976). However, this is the first description o f a putative single mutation leading to a triple requirement for isoleucine + valine + leucine. Since these three amino acids, when present in a mixture, cause multivalent repression of the 5 enzymes involved in the synthesis o f isoleucine and valine (Bussey and Umbarger 1969; Magee and Hereford 1969), it is conceivable that this I l e - V a l - L e u - segregant is an interesting regulatory mutation not previously described.
indicating significantly more lethality introduced b y this second round o f UV irradiation. Of the 19 potential segregants, 6 were CanS u p o n retesting and none was M e t - . Based on a total frequency o f 15 x 103 CanS plus M e t CanS segregants from the initial UV irradiation (Fig. 1), 11 CanS would have been expected. Thus, it is concluded that these sectors are still heterozygous for can and probably for met but the numbers were too small to detect M e t - segregants expected at a lower frequency (7.4 x 10-3).
Reversion Analysis Analysis o f Sectored Colonies Out o f a total o f 73 segregant colonies (reported in Fig. 1), only 8 segregants came from 6 sectored colonies; 45 colonies were nonsectored and 20 colonies were unclassifiable because they were in a clump. When b o t h sectors o f the 6 sectored colonies were purified and retested, two colonies were Met+CanR:Met+CanS, two colonies were Met+CanR :Met - CanS and two colonies were Met + C a n S : M e t - C a n S . These results indicate that met and can are cis-linked heterozygous genes in a diploid strain o f the genetic constitution ++/met can. To test this hypothesis, the phenotypically wild t y p e sectors from the two sectored M e t + C a n R : M e t - C a n S were analyzed further. Each o f the two Met+CanR sectors (1760-1SP and t760-20Sp1/2) was irradiated for 20 s under standard conditions (resulting in 65% and 27% survival, respectively). Out o f 737 survivors, 29 colonies were scored initially as possible segregants. However, when streak purified, 10 o f these 29 were nonviable
The pattern o f spontaneous reversion o f the M e t - C a n S segregants is complex. Single, double or triple revertants are obtained depending on the medium used to select the revertants (Table 1). F o r example, revertants o f the unusual adenine phenotype are obtained with a high frequency on C-ADE agar as singles from the two M e t - C a n S co-segregants tested. CanR revertants are obtained with an intermediate frequency on CAN medium as singles from segregant 1720-1U and as double revertants from segregant 7B20. Met + revertants are obtained with the same low frequency on both MIN or C-MET as triple revertants from both segregants. These triple co-revertants are unusually fast growers. Not shown in Table 1 are data for CanS L y s - segregant 7A40 in which b o t h CanS and L y s - co-revert at intermediate frequencies(1.2 x 1 0 - 5 when selected on CAN and 1.6 x 10 - 7 when selected on MIN). Co-reversion m a y be explained b y frame-shift mutations since the genes are linked in the cis configuration,
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Fig. 2. UV survival and mitotic segregation of FC18. For each time period, 15 YPD plates (spread with about 200 cells) were counted to determine survival percentages. Then plates from each time period were replica plated to MIN, CAN and CYH in that order. After incubation overnight at 37 °C, replica plates were scored for sectored and nonsectored segregant colonies. Symbols: o, % survival; o, frequency (34 segregants/10.059 survivors) from Whelan et al. (1980); x, Met-=Cys-
or by various suppressor mutations of different specificities and efficiencies. Single revertant classes may be explained by intragenic events. The fact that the single reversions and the co-reversions occur at different but reproducible frequencies indicates that these events are different from each other.
Comparison o f Mitotic Segregation in Strain PY17-1AS to Strain FC18 Whelan et al. (1980) reported a frequency of one cytosine auxotroph and 34 methionine=cysteine (Met-=Cys - ) auxotrophs among 10,059 colonies grown from strain FC18 cells spread and irradiated for 50 s on YPD agar at a dose rate of 12 ergs mm -2 s -1. The survival of FC18 under their conditions was 73%. Since Whelan et al. (1980) did not present their data for FC18 in figure form, I present my data for FC18 in Fig. 2. For the purpose of comparison, their segregational frequency (3 per 103 survivors) for M e t - = C y s - mutants is plotted in my Fig. 2 as an open circle at 12 s because this is the equivalent dos-
age. Points obtained ha my laboratory for segregation frequencies in FC18 are plotted as X's (35 M e t - = C y s auxotrophs, one Met- and one L y s - among 6,401 survivors). The survival at 12 s in my laboratory was estimated to be 74%. These data from two different laboratories could not agree more closely. Whelan et al. (1980) reported a linear relationship between segregational frequency and UV dosage for strain 214R2 (a derivative of FC18 shown in their Fig. l). Compared to 214R2 at the same UV dosage, FC18 and PY17-1AS both yielded an order of magnitude fewer segregants. An explanation for the dosage-independent response shown in my Figs. 1 and 2 could be that at higher UV dosages, additional segregants were being killed off by multiple genetic events such as extensive UV-induced DNA damage or uncovering pre-existing recessive lethais through UV-induced mitotic crossing-over, gene conversion or nondisjunction. Consistent with this idea, many of the colonies developing after UV-irradiation later died or continued to segregate variant colonies. An important difference between our findings is that I observed a measurable spontaneous segregational frequency in FC18 (0.64 M e t - = C y s - segregants per 103 cells) (Fig. 2) in contrast to their report of finding no auxotrophs among 3,958 coloniesgrown fromunirradiated cells (Whelan et ai. 1980). This discrepancy can be explained by the differences in preculturing methods between our laboratories. Dr. Whelan precultured FC18 on MIN prior to UV-irradiation to select against spontaneous auxotrophic segregants whereas I always grew cultures on YPD medium.
Discussion
Clinical isolates of C. albicans are reported to be diploid and heterozygous for a limited number of genetic markers (references cited in the Introduction). The results presented in this paper are in agreement with these reports. Using the technique of UV-induced mitotic segregation of recessive markers, a subclone of the HoffmannLa Roche type culture of C. albicans was found to be heterozygous for two linked genes for methionine auxotrophy and canavanine-sensitivity. Evidence for heterozygosity is based on obtaining a restricted spectrum of segregant classes at a relatively high frequency ( > 0 . 0 0 l ) following low dose UV-irradiation (criteria for heterozygosity defined by Whelan and Magee 1981). The frequencies of the CanS segregants and the Met-CanS cosegregants following UV irradiation were both about 7 x 10 -3. Mutations segregating at lower frequencies may have been induced by the UV irradiation. Additional criteria for heterozygosity in diploid yeast (proposed by Whelan and Magee 1981) include the ob-
172
M. Crandall: Genetic Linkage in C albicans
servation of sectored colonies following UV irradiation and homozygosity of markers within each sector. Sectoring was observed with the Hoffmann-La Roche strain but the wild type sectors were still heterozygous. Hence, it may be concluded that these sectored colonies did not arise from UV-induced mitotic crossing over. One class of sectored colonies could be explained by UV-induced nondisjunction (Parry et al. 1979) leading to the segregation of ++/met can :met can colonies where the met can sector is monosomic. The results may also be explained by UV-induced mitotic gene conversion (Fogel and Mortimer 1971) which would result in two classes of sectored colonies: ++/met can :+ can~met can: and ++/met can: met can/met can. The third class of sectored colonies observed could have resulted from a double event, i.e., UVinduced mitotic recombination followed by nondisjunction leading to ÷ can/met can :met can. In addition to confirming previous reports that recessive mutations preexist in clinical isolates of C albicans, this paper extends this concept by reporting the first evidence for natural genetic linkage in C albicans. The proposal of linkage is based on mitotic co-segregation of methionine auxotrophy and canavanine-sensitivity. Criteria for Linkage Co-segregation. UV-induced mitotic recombination in heterozygous diploid yeast leads to the segregation of recessive markers. Mitotic recombination can be due to either crossing-over or gene conversion (Fogel and Mortimer 1971; Kunz and Haynes 1981). In either case, adjacent genes on one chromosome arm co-segregate. Observation of two phenotypic classes of segregants, CanS and Met-CanS, following UV-irradiation of the Hoffmann-La Roche strain of C albieans indicated linkage. Missing Classes o f Expected Segregants. Evidence for
linkage based on co-segregation is corroborated by the absence of the single mutant class of Met- segregants. Only CanS and Met-CanS segregants were obtained following UV-irradiation. The frequency of the double mutant class of Met-CanS segregants was 7.4 x 10 -3 and the frequency of the single mutant class of CanS segregants was 7.6 x 10 -3. Hence, the total frequency of CanS segregants is 15.0 x 10 -3. This total is proportional the map distance between the centromere and the can locus whereas the number of recombinational events between the centromere and the met locus is proportional to the frequency of the Met-CanS segregants. On the basis of these findings, the proposed gene order and map distance is: centromere
met
o
I
I
7.4
15.0
can
where the map units are expressed in terms of the frequency of mitotic segregants per 103 survivors. Cis-trans Test for Linkage. a) The linked genes met and can are proposed to be coupled in the cis configuration
because all Met- segregants from PY17-1AS are also CanS. b) To test this hypothesis further, attempts were made to isolate two-step revertants of the Met-CanS segregants. Revertants of the genotype ++/met can in the cis configuration should repeat the segregation behavior of the parent when irradiated with UV. On the other hand, if two-step revertants occur in the trans configuration, then it would be predicted that in some cases can and met would segregate from each other as sectors in the same colony following UV-irradiation. Such a result would be additional evidence for linkage. Single revertants from CanS to CanR were.isolated (Table 1). Unfortunately, it has not been possible to isolate the desired class of trans Met + single revertants because all 49 spontaneous Met + revertants isolated co-revert to CanR. This class of co-revertants may represent a frame-shift mutation in a monosomic segregant (Met-CanS) and, therefore, cannot be used for the cis-trans test. Nevertheless, even without this corroborative test, the evidence is sufficient to indicate linkage in coupling between these two genes for canavanine-sensitivity and methionine auxotrophy.
Parasexual Genetics
Even though there is no system for sexual recombination in C. albicans, it is possible to demonstrate linkage as shown by this paper and the report by Sarachek et al. (1981). New mutations may be isolated in diploid yeast by immediately irradiating mutagenized cells with UV causing the segregation of cells that are homozygous for the newly introduced genetic markers as well as for preexisting recessive markers (Kakar and Magee 1982). A test for aUelism by complementation is available using the technique of protoplast fusion (Sarachek et al. 1981 ; PouRer et al. 1981). Studies involving transformation with recombinant DNA are underway in several laboratories. Thus, it should now be possible to identify recessive markers affecting virulence in C. aIbicans.
Medical Relevance
The spontaneous segregation of recessive mutations from heterozygous cells has significant implications to the mechanisms of pathogenesis by this medically important yeast. Occult markers in the heterozygous condition in the yeast genome segregate spontaneously during growth in the laboratory. Conceivably the same segregation pro-
M. Crandall: Genetic Linkage in C. albicans
cess could occur during growth of diploid pathogens in the host. It these recessive markers increased virulence, a more life-threatening infection might result. Chemotherapeutic agents might induce mutations and/or mitotic recombination or select potentially more dangerous atypical strains derived from the normal flora. Acknowledgements. Appreciation is expressed to Dr. William L. Whelan for sharing his results and strains prior to publication and for helpful criticisms of this work. Dr. Russell Poulter and Dr. Bernard Kunz are thanked for their detailed suggestions for revisions. Technical assistance by Pat Holleman is gratefully acknowledged. Special thanks are extended to Dr. John Edwards and Dr. Lucien Guze for providing conditions that allowed this project to be completed. This work was supported by the Biomedical Sciences Research Support Grant to the University of Kentucky and by Public Health Service Grant GM21889-05 from the National Institute of General Medical Sciences to Dr. Marjorie Crandall.
References Buckley HR, Price MR, Daneo-Moore L (1982) Infect Immun 37:1209-1217 Busbee DL, Sarachek A (1969) Arch Mikrobiol 64:289 314 Bussey H, Umbarger HE (1969) J Bacteriol 9 8 : 6 2 3 - 6 2 8 Crandall M, Caulton JH (1979) Genetics 9 3 : 9 0 3 - 9 1 6 Fogel S, Mortimer RK (1971) Ann Rev Genetics 5 : 2 1 9 - 2 3 6 Hawthorne D, Mortimer RK (1976) In: Handbook of Biochemistry and Molecular Biology. Chemical Rubber Company Press, Inc, Cleveland, pp 7 6 5 - 8 3 2
173 Kakar SN, Magee PT (1982) J Bacterio 1151:1247-1252 Kakar SN, Wagner RP (1964) Genetics 4 9 : 2 1 3 - 2 2 2 Kunz BA, Haynes RH (1981) Ann Rev Genet 1 5 : 5 7 - 8 9 Magee PT, Hereford LM (1969) J Bacterinl 9 8 : 8 5 7 - 8 6 2 Olaiya AF, Sogin SJ (1979) J Bacteriol 140:1043-1049 Olaiya AF, Steed JR, Sogin SJ (1980) J Bacteriol 141:12841290 Parry JM, Sharp D, Tippins RS, Parry EM (1979) Mutat Res 61: 37 - 5 5 PouRer R, Jeffery K, Hubbard MJ, Shepherd MG, Sullivan PA (1981) J Bacteriol 1 4 6 : 8 3 3 - 8 4 0 Riggsby WS, Torres-Bauza LJ, Wills JW, Townes TM (1982) Mol Cell Biol 2 : 8 5 3 - 8 6 2 Sarachek A, Rhoads DD, Sehwarzhoff RH (1981)Arch Microbiol 129:1-8 Silva-Hutner M, Cooper BH (1980) In: Lennette EH, Balows A, Hausler WJ, Truant JP (eds) Manual of Clinical Microbiology. 3rd edn. Am Soc Microbiol, Washington, DC, pp 5 6 2 - 5 7 6 Suzuki T, Nishibayashi S, Kuroiwa T, Kanbe T, Tanaka K (1982) J Bacteriol 1 5 2 : 8 9 3 - 8 9 6 van der Walt JP, Pitout MJ (1969) Antonie van Leeuwenhoek 35:227-231 Whelan WL, Magee PT (1981) J Bacteri61 145 : 896-903 Whelan WL, Gocke E, Manney TR (1979) Genetics 9 1 : 3 5 - 5 1 Whelan WL, Partridge RM, Magee PT (1980) Mol Gen Genet 180: 107-113 Whelan WL, Beneke ES, Rogers AL, Soll DR (1981) Antimicrob Ag Chemother 19:1078-1081
C o m m u n i c a t e d b y R. H a y n e s Received January 7, 1983
Current Genetics (1983)7:167-173
© Springer-Verlag 1983
UV-Induced Mitotic Co-Segregation of Genetic Markers in Candida albicans: Evidence for Linkage Marjorie Crandall 1 School of Biological Sciences, University of Kentucky, Lexington, KY 40506, USA
Summary. Parasexual genetic studies of the medically important yeast Candida albicans were performed using the method of UV-induced mitotic segregation. UV-irradiation of the Hoffmann-La Roche type culture of C. albicans yielded a limited spectrum of mutants at a relatively high frequency. This observation suggested natural heterozygosity. Canavanine-sensitive (CanS) segregants were induced at a frequency of 7.6 x 10 -3. Double mutants that were both CanS and methionine (Met-) auxotrophs were induced at a frequency of 7.4 x 10 - 3 . The single Met- segregant class was missing indicating linkage. UV-induced CanS or Met-CanS segregants occurred occasionally in twin-sectored colonies. Analyses of the sectors as well as the observed and missing classes of segregants indicated that genes m e t and can are linked in the cis configuration. The proposed gene order is: centromere - m e t - can. Thus, it is concluded that the Hoffmann-La Roche strain of C. albicans is naturally heterozygous at two linked loci. These findings are consistent with diploidy. Key words: UV - C. albicans - Linkage
Introduction Genetic studies of C. albicans have been impeded because a sexugl cycle has not been observed. Consequently, it has been necessary to use parasexual genetic approaches
Present address: Division of Infectious Diseases, Department
of Medicine E-5, Harbor-UCLA Medical Center, Torrance, CA 90509, USA
to the determination of the ploidy and inheritance of this yeast. In 1969, van der Walt and Pitout reported differences in cell volume and ploidy in derivative cell lines of C. albicans strain CBS 5736. Olaiya and Sogin (1979) using the technique of flow microfluorometry reported that C. albicans strain 3153A is diploid based on a comparison of its DNA content per cell with that of a series of strains of S a c c h a r o m y c e s cerevisiae of known ploidy. Germ tube negative variant clones 06 and 09 derived from 3153A were reported to have half the amount of DNA (Olaiya et al. 1980). However, Buckley et al. (1982) reporting on these same parental and germ tube negative strains found no difference in DNA contents. Clinical isolates of C. albicans studied by fluorescent microscope photometry were found to vary in ploidy (n, 2n, 4n) with diploid strains being observed most frequently and haploid strainsbeinggerm tube negative (Suzuki et al. 1982). Further evidence that most isolates of C. albicans are diploid comes from genetic approaches. Whelan et al. (1980) reported that strain FC18 of C. albicans is heterozygous at a single locus required for the synthesis of both methionine and cysteine based on results of UV-induced mitotic recombination. Subsequent reports by Whelan and Magee (1981) and Whelan et al. (1981) indicated that clinical isolates are frequently heterozygous for auxotrophic and/or antibiotic resistance markers. Riggsby et al. (1982) also reached the conclusion that the most common strains of C. albieans are diploid based on the analysis of the kinetic complexity of single-copy DNA. In contrast to these above-mentioned studies of natural isolates, two reports of parasexual genetic studies using induced mutants conclude that C. albieans is haploid (Sarachek et al. 1981; Poulter et al. 1981). This discrepancy could be explained if their mutagenesis procedures induced nondisjunction resulting in monosomic (2n - 1) strains which would behave like haploids in crosses performed by protoplast fusion.
168 The results presented here confirm the observations o f Whelan and co-workers who demonstrated that clinical isolates o f C albicans are naturally heterozygous for a limited number o f genetic markers. In this paper, studies o f UV-irradiation o f the t y p e culture o f C. albieans from Hoffmann-La Roche (strain 6631) have shown that this strain is naturally heterozygous for two genes linked in the eis configuration. UV-irradiation yielded two phenotypic classes: M e t - C a n S and CanS. Absence o f the single M e t - phenotypic class allowed the ordering o f the genes: centromere - m e t - can. This is the first report of linked genetic markers being present naturally in the heterozygous condition in an isolate of C albicans (M. Crandall, Abs. Ann. Mtg. Amer. Soc. Microbiol. D36, 1981) and represents additional documentation o f diploidy in C albicans.
Materials and Methods Strains. C albicans strain 6631 is the type culture from Hoff-
mann-La Roche and was obtained from Dr. Norman Goodman. A single cell was isolated from this culture by micromanipulation using methods published in Crandall and Caulton (1979). This clonal culture was labeled PY17-1AS (smooth colony type) and was used for all further studies. The species was confirmed by germ tube and chlamydospore formation and standard assimilation tests (Silva-Hutner and Cooper 1980). Strain FC18 of C. albicans was obtained from Dr. William L. Whelan and was used as a control for UV analysis of natural heterozygosity. UV Irradiation. Cultures were grown as a lawn on YPD agar (1% yeast extract, Difco + 2% peptone, Difco + 2% dextrose + 2% Bacto-agar, Difeo). Plates were inoculated with 0.5 ml of a YPD broth preculture and incubated overnight at 37 °C. Cells were resuspended from the plate in 5 ml of sterile deionized water (SDW), washed, counted in a hemocytometer and then diluted in SDW to yield 200 colonies/0.1 ml which was spread on each YPD plate. This population consisted of 66% single cells and the rest were doublets. Saline was not used for dilutions because it promotes self-flocculation with this strain (M. CrandaU, manuscript in preparation). The seeded plates were irradiated for various times with a UV lamp (model R-52 Mineralight, UltraViolet Products, Inc., San Gabriel, CA). The dose rate at the agar surface was 50 ergs mm - 2 s-1 (measurement courtesy of Dr. John Calkins). In a pilot experiment measuring UV survival (data not shown), it was demonstrated that it was not necessary to protect the UVqrradiated plates from visible light confirming a report by Busbee and Saraehek (1969) that C. albicans does not photoreactivate. The irradiated and control plates were incubated at 37 °C for 2 days and then replica plated to the following media: minimal (MIN) agar (0.67% yeast nitrogen base without amino acids, Difco + 2% dextrose + 2% agar), complete minus adenine (C-ADE), complete minus methionine (C-MET), complete (COM), COM minus arginine plus 100 ~g/ml canavaninesulfate, Sigma (CAN), and YPD plus 100 ~g/ml cycloheximide, Sigma (CYH), in that order. COM medium contains 20 amino acids and nucleotide bases each at a final concentration of 20 #g/ml added to MIN. All colonies or sectors identified as mutants on the replica plates were picked from the YPD master plate for streak purification on YPD agar.
M. Crandall: Genetic Linkage in C albicans
Results U V - I n d u e e d Segregants
In a pilot experiment using subclone PY17-1AS, a segregation frequency o f about 1% was observed after UV irradiation for 50 s. Of 7 segregants purified, all had lost the natural resistance o f the parent to canavanine becoming canavanine-sensitive (CanS); one was also lysine-negatire (Cans L y s - ) and 5 also required methionine ( M e t CanS) (M. Crandall, Abs. Ann. Mtg. Amer. Soc. Microbiol. D36, 1981). Some o f these mutants also showed an increased self-flocculation reaction and an unusual adenine phenotype (see below). Co-segregation o f genetic markers was suggestive of genetic linkage; therefore, a large scale isolation o f segregants was undertaken. The second UV irradiation experiment yielded 27 segregant colonies (12 CanS + 14 M e t - C a n S + 1 CanS segregant also requiring isoleucine, valine, and leucine) out o f 1998 survivors. The parental strain is naturally resistant to both cycloheximide and canavanine. However, no cycloheximide-sensitive segregants were ever observed indicating that this strain is not heterozygous for cycloheximide sensitivity. The third UV experiment yielded 46 segregants (24 CanS + 22 M e t - C a n S ) out of 2,735 survivors. Since the pattern o f segregation was similar in the second and third experiments, the data were pooled in order to obtain more accurate estimates o f the segregational frequences (Fig. 1). Upon streak-purification and retesting, some o f these segregants (18%) exhibited the wild t y p e phenotype perhaps because of cross-contamination due to spreading of colonies during replica plating. Others died (3%) either on the YPD streak plate or later in the YPD broth subculture. Many mutants (40%) were slow growers. On 14% o f the streak plates o f putative mutants, there was evidence for additional segregational events occurring (sectoring, papillae, large and small colonies). These observations may indicate that UV irradiation caused recessive lethals or other recessive genes to co-segregate in some of the mutants. No spontaneous segregants could be detected ( < 3 x 10 - 4 ) in the unirradiated culture of PY17-1AS (plotted as zero-time in Fig. 1). Hence, it is concluded that UV is inducing the segregation o f CanS and M e t - CanS mutants. At the lowest dosage tested (20 s = 1,000 ergs r a m - 2 ) , the survival was about 30%. No increase in the frequency o f segregants occurred at higher dosages (lower survival), i.e., the same proportions of the two segregant classes were obtained at all three dosages (Fig. 1). Hence, the data for all dosages were pooled in order to calculate the frequencies for b o t h segregants: CanS = 7.6 x 10 - 3 and M e t - C a n S = 7.4 x 10 - 3 .
M. Crandall: Genetic Linkage in C albicans
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complete growth inhibition ( - ) of 7B20 was only obtained with a mixture of all four amino acids (cys + phe + trp + val). Segregant 1720-1U (also Met-CanS) grew slowly (+) in the presence of these four amino acids but was negative on C-ADE indicating that additional amino acids or nucleotide bases in COM are inhibitory. The phenotype of these Met- CanS segregants is leaky on C-ADE and, for this reason, growth is scored in less than 24 h after replica plating. One Met-CanS segregant isolated out of 4733 survivors, 1720-10N, grows on C-ADE but not on C-MET or CAN. However, when additional cys is added to C-ADE plates (final concentration = 40 #g/ ml), even this Ade + segregant and an Ade + Met-CanS revertant, 7B20-R 14, are caused to require adenine. Thus, it seems likely that this unusual Ade- phenotype is related to the Met- phenotype rather than being a separate mutation. Thus, the met locus appears to be a pleiotropic gene perhaps affecting S-adenosylmethionine metabolism, considering the involvement of cysteine, adenine and methionine in this auxotrophic phenotype.
0.1
UV DOSE (Seconds) Fig. 1. UV survival and mitotic segregation of PY17-1AS. For each time period, 15 YPD plates (spread with about 200 cells) were counted to determine survival percentages. Then plates from each time period were replica plated to MIN, C-ADE, CMET, CAN and CYH in that order. After incubation overnight at 37 °C, replica plates were scored for sectored and nonsectored segregant colonies. No segregants were observed at zero time (arrow indicates < 3 x 10-4). Data from two experiments were pooled to calculate the frequency of segregants at 20, 40 and 60 s of irradiation. Symbols: e, % survival; x, CanS; A, Met-CanS
Description of the Methionine-Negative Phenotype These methionine auxotrophs grow on MIN + methionine but not on MIN + cysteine. All of the Met- segregants are also CanS. When these Met - CanS segregants are tested on drop-out media, they do not grow on C-MET (as expected) but they also do not grow on C-ADE (unexpected since the Met-CanS segregants grow on minimal medium supplemented only with methionine). The adenine requirement is due to inhibition by components of COM medium. Individual amino acids and nucleotide bases from complete stock were tested at high concentration added to MIN + MET with Met-CanS segregant 7B20. Cysteine (cys), phenylalanine (phe), tryptophan (trp) and valine (val) each caused some growth inhibition separately but when tested singly at the concentration in COM (20 /ag/ml), none caused complete growth inhibition. However, cys alone slowed growth the most (from 2+ to 1+). Combinations of three (cys + phe + val or cys + trp + val) inhibited growth of 7B20 from 2+ to +-. But
Description of the Canavanine-Sensitive Phenotype The Hoffmann-La Roche strain of C. aIbicans is canavanine-resistant (CanR). Following UV-irradiation, all segregants isolated are sensitive to canavanine. These CanS segregants are scored on CAN agar (= COM - ARG + CAN; see Materials and Methods). They are not arginine auxotrophs because they grow on C-ARG. Likewise, the double segregant Met-CanS grows on MIN + MET also indicating that these segregants are neither arginine nor adenine auxotrophs. The CanS segregants have a leaky phenotype on CAN; no growth ( - ) after one day at 37 °C but slight growth (+) after 2 days compared to fast growth (2+) of the wild type after only one day. Double segregants, Met-CanS, remain negative ( - ) on CAN plates after 2 days. For simplicity, the can gene is assumed to be recessive in C. albicans even though canavanine-sensitivity is dominant in Saccharornyces cerevisiae (Whelan et al. 1979).
Nature of the Isoleucine- Valine-Leucine Requffement Only one segregant out of 4,733 survivors had an IleV a l - L e u - phenotype. This segregant will not grow on C-ILE or C-VAL or MIN + ILE + VAL but will grow on MIN + ILE + VAL + LEU or C-LEU (contains ile + val). Apparently, a scavenger pathway from isoleucine and valine is being used to synthesize leucine when grown on C-LEU. Since no single ( I l e - ; V a l - ; L e u - ) or double (Ile- Val- ; Ile- L e u - ; Val- L e u - ) mutants were isolated, it seems likely that this triple auxotrophic requirement is caused by a mutation at a single locus. Kakar and Wag-
170
M. CrandaU: Genetic Linkage in C albicans
Table 1. Single reversion and co-reversion of markers # of Revertants a
Selected on
Frequency
3 9 3 2
MIN C-ADE C-MET CAN
Segregant 7B20: 1.4 x 10 - 8 2.7 x 10 - 4 5.0 x 10 - 9 8.5 x 10 - 7
2 3 3 3
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Segregant 1720-1U: 1.3 x 10 - 8 2.6 x 10 - 3 6.7 x 10 - 9 3.0 x 10 - 6
Phenotype MIN
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CeUs of two Met-CanS segregants (7B20 and 1720-1U) were precultured on YPD agar, resuspended in SDW at about 109 cells/ml, washed twice in SDW and spread on MIN, C-ADE, C-MET and CAN agar at various dilutions. Spontaneous revertant colonies were streak purified on the same selective medium, clones were inoculated into YPD broth stock culture and then retested by replica plating
ner (1964) described mutations that led to the double requirement I l e - V a l - named ilv mutations (Hawthorne and Mortimer 1976). However, this is the first description o f a putative single mutation leading to a triple requirement for isoleucine + valine + leucine. Since these three amino acids, when present in a mixture, cause multivalent repression of the 5 enzymes involved in the synthesis o f isoleucine and valine (Bussey and Umbarger 1969; Magee and Hereford 1969), it is conceivable that this I l e - V a l - L e u - segregant is an interesting regulatory mutation not previously described.
indicating significantly more lethality introduced b y this second round o f UV irradiation. Of the 19 potential segregants, 6 were CanS u p o n retesting and none was M e t - . Based on a total frequency o f 15 x 103 CanS plus M e t CanS segregants from the initial UV irradiation (Fig. 1), 11 CanS would have been expected. Thus, it is concluded that these sectors are still heterozygous for can and probably for met but the numbers were too small to detect M e t - segregants expected at a lower frequency (7.4 x 10-3).
Reversion Analysis Analysis o f Sectored Colonies Out o f a total o f 73 segregant colonies (reported in Fig. 1), only 8 segregants came from 6 sectored colonies; 45 colonies were nonsectored and 20 colonies were unclassifiable because they were in a clump. When b o t h sectors o f the 6 sectored colonies were purified and retested, two colonies were Met+CanR:Met+CanS, two colonies were Met+CanR :Met - CanS and two colonies were Met + C a n S : M e t - C a n S . These results indicate that met and can are cis-linked heterozygous genes in a diploid strain o f the genetic constitution ++/met can. To test this hypothesis, the phenotypically wild t y p e sectors from the two sectored M e t + C a n R : M e t - C a n S were analyzed further. Each o f the two Met+CanR sectors (1760-1SP and t760-20Sp1/2) was irradiated for 20 s under standard conditions (resulting in 65% and 27% survival, respectively). Out o f 737 survivors, 29 colonies were scored initially as possible segregants. However, when streak purified, 10 o f these 29 were nonviable
The pattern o f spontaneous reversion o f the M e t - C a n S segregants is complex. Single, double or triple revertants are obtained depending on the medium used to select the revertants (Table 1). F o r example, revertants o f the unusual adenine phenotype are obtained with a high frequency on C-ADE agar as singles from the two M e t - C a n S co-segregants tested. CanR revertants are obtained with an intermediate frequency on CAN medium as singles from segregant 1720-1U and as double revertants from segregant 7B20. Met + revertants are obtained with the same low frequency on both MIN or C-MET as triple revertants from both segregants. These triple co-revertants are unusually fast growers. Not shown in Table 1 are data for CanS L y s - segregant 7A40 in which b o t h CanS and L y s - co-revert at intermediate frequencies(1.2 x 1 0 - 5 when selected on CAN and 1.6 x 10 - 7 when selected on MIN). Co-reversion m a y be explained b y frame-shift mutations since the genes are linked in the cis configuration,
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Fig. 2. UV survival and mitotic segregation of FC18. For each time period, 15 YPD plates (spread with about 200 cells) were counted to determine survival percentages. Then plates from each time period were replica plated to MIN, CAN and CYH in that order. After incubation overnight at 37 °C, replica plates were scored for sectored and nonsectored segregant colonies. Symbols: o, % survival; o, frequency (34 segregants/10.059 survivors) from Whelan et al. (1980); x, Met-=Cys-
or by various suppressor mutations of different specificities and efficiencies. Single revertant classes may be explained by intragenic events. The fact that the single reversions and the co-reversions occur at different but reproducible frequencies indicates that these events are different from each other.
Comparison o f Mitotic Segregation in Strain PY17-1AS to Strain FC18 Whelan et al. (1980) reported a frequency of one cytosine auxotroph and 34 methionine=cysteine (Met-=Cys - ) auxotrophs among 10,059 colonies grown from strain FC18 cells spread and irradiated for 50 s on YPD agar at a dose rate of 12 ergs mm -2 s -1. The survival of FC18 under their conditions was 73%. Since Whelan et al. (1980) did not present their data for FC18 in figure form, I present my data for FC18 in Fig. 2. For the purpose of comparison, their segregational frequency (3 per 103 survivors) for M e t - = C y s - mutants is plotted in my Fig. 2 as an open circle at 12 s because this is the equivalent dos-
age. Points obtained ha my laboratory for segregation frequencies in FC18 are plotted as X's (35 M e t - = C y s auxotrophs, one Met- and one L y s - among 6,401 survivors). The survival at 12 s in my laboratory was estimated to be 74%. These data from two different laboratories could not agree more closely. Whelan et al. (1980) reported a linear relationship between segregational frequency and UV dosage for strain 214R2 (a derivative of FC18 shown in their Fig. l). Compared to 214R2 at the same UV dosage, FC18 and PY17-1AS both yielded an order of magnitude fewer segregants. An explanation for the dosage-independent response shown in my Figs. 1 and 2 could be that at higher UV dosages, additional segregants were being killed off by multiple genetic events such as extensive UV-induced DNA damage or uncovering pre-existing recessive lethais through UV-induced mitotic crossing-over, gene conversion or nondisjunction. Consistent with this idea, many of the colonies developing after UV-irradiation later died or continued to segregate variant colonies. An important difference between our findings is that I observed a measurable spontaneous segregational frequency in FC18 (0.64 M e t - = C y s - segregants per 103 cells) (Fig. 2) in contrast to their report of finding no auxotrophs among 3,958 coloniesgrown fromunirradiated cells (Whelan et ai. 1980). This discrepancy can be explained by the differences in preculturing methods between our laboratories. Dr. Whelan precultured FC18 on MIN prior to UV-irradiation to select against spontaneous auxotrophic segregants whereas I always grew cultures on YPD medium.
Discussion
Clinical isolates of C. albicans are reported to be diploid and heterozygous for a limited number of genetic markers (references cited in the Introduction). The results presented in this paper are in agreement with these reports. Using the technique of UV-induced mitotic segregation of recessive markers, a subclone of the HoffmannLa Roche type culture of C. albicans was found to be heterozygous for two linked genes for methionine auxotrophy and canavanine-sensitivity. Evidence for heterozygosity is based on obtaining a restricted spectrum of segregant classes at a relatively high frequency ( > 0 . 0 0 l ) following low dose UV-irradiation (criteria for heterozygosity defined by Whelan and Magee 1981). The frequencies of the CanS segregants and the Met-CanS cosegregants following UV irradiation were both about 7 x 10 -3. Mutations segregating at lower frequencies may have been induced by the UV irradiation. Additional criteria for heterozygosity in diploid yeast (proposed by Whelan and Magee 1981) include the ob-
172
M. Crandall: Genetic Linkage in C albicans
servation of sectored colonies following UV irradiation and homozygosity of markers within each sector. Sectoring was observed with the Hoffmann-La Roche strain but the wild type sectors were still heterozygous. Hence, it may be concluded that these sectored colonies did not arise from UV-induced mitotic crossing over. One class of sectored colonies could be explained by UV-induced nondisjunction (Parry et al. 1979) leading to the segregation of ++/met can :met can colonies where the met can sector is monosomic. The results may also be explained by UV-induced mitotic gene conversion (Fogel and Mortimer 1971) which would result in two classes of sectored colonies: ++/met can :+ can~met can: and ++/met can: met can/met can. The third class of sectored colonies observed could have resulted from a double event, i.e., UVinduced mitotic recombination followed by nondisjunction leading to ÷ can/met can :met can. In addition to confirming previous reports that recessive mutations preexist in clinical isolates of C albicans, this paper extends this concept by reporting the first evidence for natural genetic linkage in C albicans. The proposal of linkage is based on mitotic co-segregation of methionine auxotrophy and canavanine-sensitivity. Criteria for Linkage Co-segregation. UV-induced mitotic recombination in heterozygous diploid yeast leads to the segregation of recessive markers. Mitotic recombination can be due to either crossing-over or gene conversion (Fogel and Mortimer 1971; Kunz and Haynes 1981). In either case, adjacent genes on one chromosome arm co-segregate. Observation of two phenotypic classes of segregants, CanS and Met-CanS, following UV-irradiation of the Hoffmann-La Roche strain of C albieans indicated linkage. Missing Classes o f Expected Segregants. Evidence for
linkage based on co-segregation is corroborated by the absence of the single mutant class of Met- segregants. Only CanS and Met-CanS segregants were obtained following UV-irradiation. The frequency of the double mutant class of Met-CanS segregants was 7.4 x 10 -3 and the frequency of the single mutant class of CanS segregants was 7.6 x 10 -3. Hence, the total frequency of CanS segregants is 15.0 x 10 -3. This total is proportional the map distance between the centromere and the can locus whereas the number of recombinational events between the centromere and the met locus is proportional to the frequency of the Met-CanS segregants. On the basis of these findings, the proposed gene order and map distance is: centromere
met
o
I
I
7.4
15.0
can
where the map units are expressed in terms of the frequency of mitotic segregants per 103 survivors. Cis-trans Test for Linkage. a) The linked genes met and can are proposed to be coupled in the cis configuration
because all Met- segregants from PY17-1AS are also CanS. b) To test this hypothesis further, attempts were made to isolate two-step revertants of the Met-CanS segregants. Revertants of the genotype ++/met can in the cis configuration should repeat the segregation behavior of the parent when irradiated with UV. On the other hand, if two-step revertants occur in the trans configuration, then it would be predicted that in some cases can and met would segregate from each other as sectors in the same colony following UV-irradiation. Such a result would be additional evidence for linkage. Single revertants from CanS to CanR were.isolated (Table 1). Unfortunately, it has not been possible to isolate the desired class of trans Met + single revertants because all 49 spontaneous Met + revertants isolated co-revert to CanR. This class of co-revertants may represent a frame-shift mutation in a monosomic segregant (Met-CanS) and, therefore, cannot be used for the cis-trans test. Nevertheless, even without this corroborative test, the evidence is sufficient to indicate linkage in coupling between these two genes for canavanine-sensitivity and methionine auxotrophy.
Parasexual Genetics
Even though there is no system for sexual recombination in C. albicans, it is possible to demonstrate linkage as shown by this paper and the report by Sarachek et al. (1981). New mutations may be isolated in diploid yeast by immediately irradiating mutagenized cells with UV causing the segregation of cells that are homozygous for the newly introduced genetic markers as well as for preexisting recessive markers (Kakar and Magee 1982). A test for aUelism by complementation is available using the technique of protoplast fusion (Sarachek et al. 1981 ; PouRer et al. 1981). Studies involving transformation with recombinant DNA are underway in several laboratories. Thus, it should now be possible to identify recessive markers affecting virulence in C. aIbicans.
Medical Relevance
The spontaneous segregation of recessive mutations from heterozygous cells has significant implications to the mechanisms of pathogenesis by this medically important yeast. Occult markers in the heterozygous condition in the yeast genome segregate spontaneously during growth in the laboratory. Conceivably the same segregation pro-
M. Crandall: Genetic Linkage in C. albicans
cess could occur during growth of diploid pathogens in the host. It these recessive markers increased virulence, a more life-threatening infection might result. Chemotherapeutic agents might induce mutations and/or mitotic recombination or select potentially more dangerous atypical strains derived from the normal flora. Acknowledgements. Appreciation is expressed to Dr. William L. Whelan for sharing his results and strains prior to publication and for helpful criticisms of this work. Dr. Russell Poulter and Dr. Bernard Kunz are thanked for their detailed suggestions for revisions. Technical assistance by Pat Holleman is gratefully acknowledged. Special thanks are extended to Dr. John Edwards and Dr. Lucien Guze for providing conditions that allowed this project to be completed. This work was supported by the Biomedical Sciences Research Support Grant to the University of Kentucky and by Public Health Service Grant GM21889-05 from the National Institute of General Medical Sciences to Dr. Marjorie Crandall.
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173 Kakar SN, Magee PT (1982) J Bacterio 1151:1247-1252 Kakar SN, Wagner RP (1964) Genetics 4 9 : 2 1 3 - 2 2 2 Kunz BA, Haynes RH (1981) Ann Rev Genet 1 5 : 5 7 - 8 9 Magee PT, Hereford LM (1969) J Bacterinl 9 8 : 8 5 7 - 8 6 2 Olaiya AF, Sogin SJ (1979) J Bacteriol 140:1043-1049 Olaiya AF, Steed JR, Sogin SJ (1980) J Bacteriol 141:12841290 Parry JM, Sharp D, Tippins RS, Parry EM (1979) Mutat Res 61: 37 - 5 5 PouRer R, Jeffery K, Hubbard MJ, Shepherd MG, Sullivan PA (1981) J Bacteriol 1 4 6 : 8 3 3 - 8 4 0 Riggsby WS, Torres-Bauza LJ, Wills JW, Townes TM (1982) Mol Cell Biol 2 : 8 5 3 - 8 6 2 Sarachek A, Rhoads DD, Sehwarzhoff RH (1981)Arch Microbiol 129:1-8 Silva-Hutner M, Cooper BH (1980) In: Lennette EH, Balows A, Hausler WJ, Truant JP (eds) Manual of Clinical Microbiology. 3rd edn. Am Soc Microbiol, Washington, DC, pp 5 6 2 - 5 7 6 Suzuki T, Nishibayashi S, Kuroiwa T, Kanbe T, Tanaka K (1982) J Bacteriol 1 5 2 : 8 9 3 - 8 9 6 van der Walt JP, Pitout MJ (1969) Antonie van Leeuwenhoek 35:227-231 Whelan WL, Magee PT (1981) J Bacteri61 145 : 896-903 Whelan WL, Gocke E, Manney TR (1979) Genetics 9 1 : 3 5 - 5 1 Whelan WL, Partridge RM, Magee PT (1980) Mol Gen Genet 180: 107-113 Whelan WL, Beneke ES, Rogers AL, Soll DR (1981) Antimicrob Ag Chemother 19:1078-1081
C o m m u n i c a t e d b y R. H a y n e s Received January 7, 1983
