Current Genetics
Curr Genet (1986) 10:685-693
© Springer-Verlag 1986
Segregant-defective heterokaryons of Candida albicans Alvin Sarachek and David A. Weber Department of BiologicalSciences,WichitaState University,Wichita, KS 67208, USA
Summary. Heterokaryons (hets) of the asexual, pathogenic yeast Candida albicans obtained by fusing protoplasts of complementing auxotrophic strains generate large numbers of parental-type auxotrophic monokaryons by random assortment of single nuclei into blastospores, and smaller numbers of monokaryons bearing hybrid nuclei formed through either karyogamy or the transfer of genetic material from one bet nucleus to another. Het populations grown at 30 °C or 37 °C contain high frequencies (approx. 5%-10%) of two kinds of stable variants peculiar specifically for segregation of parental-type monokaryons: NS variants produce inviable auxotrophic monokaryons of one or both parental classes while AT variants yield parental-type monokaryons which grow very slowly. Variant frequencies are not affected by the wild-type strain background of hets, or the auxotrophies used to force heterokaryosis. However, both kinds of variants are induced by growth at 25 °C or by treatments with certain chemical or physical metabolic inhibitors. Evidence is presented that variant nuclei of independent origins carry different nutritionally irreparable recessive lethal (NS) or debilitating (AT) defects acquired in the course of actual or potential internuclear transfers of genetic material within het cells. The high incidence of variants, therefore, indicates considerable intrinsic genetic instability among het nuclei. Significances of these observations for parasexual genetic analyses of C albicans and other yeasts through protoplast fusions are considered. Key words: Candida albicans - Heterokaryons - Nuclear instability - Protoplast fusion
Offprint requests to. A. Sarachek
Introduction
As the most commonly encountered cause of fungal disease in humans, the asexual yeast Candida albicans has been subject to intensive investigation historically (Odds 1979; Shepherd et al. 1985). Yet the genetic bases of its morphological, biochemical and clinical properties have remained obscure since conventional analytical procedures requiring genetic recombinations between strains are not applicable to this naturally anamorphic species. In recent years, however, protoplast fusions have been exploited to develop an artificial parasexual system which can potentially provide for comprehensive genetic analysis of the yeast (Sarachek et al. 1981 ; Poulter et al. 1981). Nutritionally balanced heterokaryons (hets) are first produced by fusing protoplasts of complementing auxotrophic strains. On minimal medium, the hets form minute heterogeneous colonies consisting of a small number of slowly replicating het cells and a preponderance of nongrowing auxotrophic monokaryons. The auxotrophs arise by random assortment of single parental nuclei into blastospores containing biparental cytoplasm and thus may be useful for evaluating transmission of cytoplasmic genetic determinants or the effects of variant cytoplasms on nuclear gene activities. Karyogamy or internuclear transfers of genetic materials within het cells also generate euploid or, more frequently, aneuploid hybrid nuclei (Sarachek and Weber 1984). Monokaryons bearing such nuclei typically appear as rapidly growing sectors or papillae on older het colonies, and recombinations for chromosomal markers in hybrid monokaryons can occur through either spontaneous or induced mitotic crossovers o r chance chromosome losses. In view of the central role of hets in the parasexual system, we have been particularly interested in identifying cultural factors important to their growth and
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A. Sarachek and D. A. Weber: Segregant-defective heterokaryon of C albicans
development. Investigations to data have established that complementation of auxotrophies is essential to force but not to maintain heterokaryosis since hets can be propagated indefinitely on enriched media as well as minimal medium if care is taken to prevent their overgrowth b y monokary0tic segregants (Sarachek et al. 1981). They also demonstrated that complex nutritional enrichments which are stimulatory for monokaryons do not affect growth or stability of bets (Sarachek and Weber 1985), and that the viability of hets, their internuclear genetic transfers, plating efficiencies and tendencies to dissociate into monokaryons each exhibit unique growth temperature dependencies (Sarachek and Rhoads 1982, 1983; Sarachek and Weber 1984). In the course of these studies, we reported on several occasions that het cell populations routinely contain appreciable numbers of stable, nonsegregating (NS) variants which produce clones lacking one or the other, or both, expected classes of parental-type auxotrophic monokaryons. Feulgen stained preparations of clones lacking both parentaltype monokaryons revealed that the behavior of variant hets reflects their production of inviable uninculeate cells, not their mechanical failure to assort single nuclei into blastospores: even complex nutritional enrichments cannot instigate growth of the monokaryons in such populations (Sarachek and Weber 1985). We have also noticed that a small percentage of hets segregating viable monokaryons of b o t h parental types are atypical in that one or both classes of segregants grow very slowly on minimally supplemented or enriched media. These previously undescribed odd hets (AT variants) persistently segregate both normal and abnormal monokaryons in roughtly equal numbers upon repeated subculture. The characteristics of NS and AT variants suggest that NS strains carry nutritionally irreparable recessive lethal nuclear defects and that AT strains bear analogous defects which are merely debilitating when expressed in monokaryotic segregants. We now report observations on the genetic properties of variant hets and the cultural conditions affecting their incidences which corroborate those interpretations and indicate that the defects of variant nuclei are genetic deficiencies arising in association with the temperature dependent process b y which internuclear genetic transfers occur within het cells. Important implications of these findings for genetic analyses of C albicans based on protoplast fusions are discussed.
Materials and methods Strains. The het obtained by fusing protoplasts of the auxotrophic strains WC-5-4 (HIS-, ARG-) and WCR-1-74 (Ade-, Thr-) was the primary test organism: hets prepared by fusing strains WC-5-4 and WD-18-6 (Lys-, Cys/Met-), or WD-4-4 (Leu-,
Ade-) and WD-18-6 were also examined. Segregant defective variant substrains of the WC-5-4 x WCR-1-74 het used for special determinations are described in the appropriate sections under Results. WC and WD parental auxotrophs are ultraviolet (UV) induced derivatives of the prototrophic clinical isolates 207 (serotype A) and 526 (serotype B), respectively. The WCR-1-74 strain produces a distinctive red pigment during growth on limiting adenine due to accumulation of intermediate behind the block in purine biosynthesis responsible for its adenine requirement. Other details of the origins and properties of the parental auxotrophs as well as procedures for preparing and fusing protoplasts, isolating hets, obtaining hybrid monokaryons from bets, and maintaining stocks of all strains are given in Sarachek et al.
(1981). Media. The minimal and complete media of Haught and Sarachek (1985) were used. To support growth of auxotrophs, minimal medium was supplemented with 25 ~zg/ml of each specifically required nutrient. Where indicated, chloramphenicol was added to preautoclaved media cooled to approx. 60 oC; all other metabolic antagonists used were included in media before autoclaving. Het inocula. Unless otherwise specified, het populations used as experimental inocula were prepared by individually resuspending in 1.5 ml of 0.1 M KC1 five three-day-old het colonies grown on minimal plates at 30 °C. The suspensions were pooled for use after 0.05 ml aliquots of each were plated on minimal medium and on appropriately supplemented media to verify that clones selected were normal segregators of both classes of parental-type monokaryons. The test plates were read after three days incubation at 30 °C, and experiments in progress were discarded on those infrequent occasions when starting inocula were found to include a segregationally aberrant clone. Classification and identification of het variants. Although variant hets which fail to produce viable monokaryons do physically assort individual nuclei to form uninucleate blastospores, we refer to them as nonsegregating (NS) variants to denote their inability to yield expected classes of viable monokaryotic segregants. To detect NS variants in populations of the WC-5-4 x WCR-1-74 bet, individual colonies produced by het cells on minimal plates were picked by means of sterile toothpicks and streaked successively on minimal and complete plates. Colonies grown at 25 °C, 30 ° C or 37 °C were picked when 4, 3, or 2 days old, respectively, so as to be roughly comparable in size yet young enough to avoid the likely inclusion of prototrophic hybrid monokaryons: the ratios of parental-type monokaryons to hets in normal colonies were approx. 150 : 1 at 25 °C, 40 : 1 at 30 °C and 15 : 1 at 37 °C (Sarachek and Rhoads 1983). Nine independent colony transfers were accommodated on each standard (100 mm diameter) diagnostic plate, and plates were read after two days incubation at 30 °C. The transfers to minimal medium typically gave rise only to minute, slightly rugose het colonies. On complete plates, inocula from normal het colonies produced mixtures of rapidly growing smooth white WC-5-4 type and red WCR-1-74 type colonies, inocula from colonies lacking a single class of parentaltype monokaryon formed either red or white colonies exclusively and those from colonies containing neither kind of viable monokaryon produced only distinctive het colonies. Atypical hets (AT variants) producing growth deficient parental-type monokaryons were identified by the small size of monokaryotic colonies on complete test plates. Where indicated, comparable surveys for nonsegregating variants in WC-5-4 x WD-18-6 and WD-4-4 x WD-18-6 bet populations were made. However, since a color marker was not present in these strains to distinguish alternate parental-type segregants visually, their colonies were tested by
A. Sarachek and D. A. Weber: Segregant-defective heterokaryon of C. albicans sequential streakings on minimal medium and on separate minireal plates containing paired supplements supporting growth of one or the other possible segregant. Identification of variants by these procedures could be compromised by the presence of prototrophic hybrids within a het clone since the rapidly growing hybrids might be mistaken for parental-type segregants on supplemented diagnostic plates. However, such occurrences were easily recognized by the appearance of large smooth hybrid colonies on corresponding minimal test plates and in the course of our studies were encountered in less than 0.1% of colonies examined; all hybrid contaminated colonies were excluded from consideration. The reliability of our screening procedure was validated for the WC-5-4 x WCR-1-74 het by re-examining 50 het isolates which were presumptive nonsegregators of (2-5-4 type monokaryons, a like number of WCR-1-74 nonsegregators, 20 double nonsegregators and 20 AT variants abnormal for each of the two types of monokaryotic segregants. Only two NS isolates were found to be misdiagnosed, indicating an error well under 5%. Furthermore, five successive replatings of five arbitrarily chosen isolates of each type confirmed that the variant phenotypes are stable, heritable traits.
Inductions of variants by metabolic inhibitors. Although het cells suspend well in 0.1 M KC1, induction of het variants by chemical inhibitors could not be measured reliably in broth since her cells clump severely during growth in liquid media. Therefore, variant induction in the WC-5-4 x WCR-1-74 het was determined by streaking her cells on minimal plates containing a concentration of inhibitor sufficient to reduce survival of the inoculum by approx. 50-70% during incubation at 37 °C, and examining the colonies eventually formed by survivors. To test the effect of UV or cold temperature, hets spread on minimal plates were either UV irradiated (48 j/M2), as described by Sarachek et al. (1981), or subjected to the lethal effect of holding at 10 °C for 48 h to reduce survival by approx. 50-70% (Sarachek and Rhoads 1982): survivors were then incubated at 37 °C to form colonies for analysis. Since rates of colony development differed for cells damaged by various treatments, in each case colonies were examined when 0.5 to 0.6 mm in diameter, the approximate sizes of two-day-old 37 °C grown control colonies. In order to distinguish between induction and selective survival as causes of increases in variant frequencies which might occur in treated populations, a control mixed population of the NS variant strains NR-3 and NW-5, described under Results, was tested for survival on the complete panel of inhibitors. No notable differences were found in susceptibilities of variant and standard bets to inactivation by any of the agents.
Results Frequencies o f A T and NS variants among genetically different hets Prior studies have shown that the plating efficiencies of hets and their tendencies to dissociate into parental type m o n o k a r y o n s decrease with increasing growth temperatures ranging from 25 °C to 37 °C (Sarachek and Rhoads 1983). Both responses are functions of heterokaryosis per se and are independent of the genetic makeups o f hets. Since our method o f identifying AT and NS het variants was based on qualitative assessments o f the cellular compositions of the colonies they form, it was o f
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interest to determine whether plating temperatures, growth temperatures or genetic backgrounds o f normal het populations influence recovery o f variants. The effects of plating temperatures and genetic constitutions o f hets were tested b y examining colonies produced at 25 °C, 30 °C, or 37 °C b y 30 °C grown populations o f three hets differing in complementing auxotrophies and wild-type strain backgrounds. The findings summarized in Table 1 show that genetic differences between hets do not significantly affect absolute yields o f variants nor recoveries of variants at different plating temperatures. For individual hets, 1) NS variants were approx. 2 to 5 times more c o m m o n than AT variants, 2) variants were found at like frequencies at all three temperatures and 3) NS or AT variants aberrant for one or the other o f the two classes o f parental-type monokaryons occurred with equal probabilities. Since the het populations plated at efficiencies approx. 35% lower at 37 °C than at 25 °C (Sarachek and Rhoads 1983), the equivalent variant frequencies at those two plating temperatures signified that the stress at 37 °C which blocks colony formation b y many otherwise viable normal het cells is not selective for or against variant her cells. Furthermore, persistent segregations by variant isolates o f the same single class o f abnormal monokaryons indicated that variant phenotypes are determined b y nuclear, not cytoplasmic, factors.
Independent origins o f variant defects in het cell nuclei Only one NS variant and no AT variant defective for b o t h classes of possible monokaryotic segregants were found in the collection o f het populations described in Table 1. Considering the limited sample sizes involved and the frequencies of singly defective variants, this could be reasonably ascribed to the improbability o f encountering het cells with coincident defects in b o t h nuclear components. Nevertheless, to determine directly whether an existing variant defect in a h e t cell influences the chance o f detecting a new defect arising in the norreal nuclear component of the same cell, four NS derivatives o f the WC-5-4 x WCR-1-74 bet, NR-3 and NR-4 which segregate only WCR-1-74 monokaryons and NW-3 and NW-5 which segregate only WC-5-4 monokaryons, were examined for acquisition o f AT or NS lesions b y their normal nuclei. An analogous set o f AT derivatives o f WC-5-4 x WCR-1-74, AR-1 and AR-2 producers o f slow growing WC-5-4 monokaryons and AW-1 and AW.2 producers of slow growing WCR-1-74 monokaryons, were screened for appearances of NS defects in their normal and variant nuclei, as well as for AT defects in their normal nuclei. For each test strain, 300 to 400 colonies produced at 30 °C from 30 °C grown inocula were examined for m o n o k a r y o n contents. No revertant clones
A. Sarachek and D. A. Weber: Segregant-defectiveheterokaryon of C. albicans
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Table 1. Frequencies of AT and NS variants detected in 30 °C grown populations of three different heterokaryons by analysis of bet clones produced by plating at 25 °C, 30 °C, or 37 °C Heterokaryon
Plate growth temp (oC)
Number of clones tested
Number (% of total) of variant clones by class of aberrant parental-type monokaryotic segreganta AT variants
WC-5-4 (His-, Arg-) x WCR-1-74 (Ade-, Thr-)
25 30 37
351 356 355b
WC-5-4 (His-, Arg-) x WC-18-6 (Lys-, Met/Cys-)
25 30 37
308 281 288
WD-4-4 (Leu-, Ade-) x WD-18-6 (Lys-, Met/Cys-)
25 30 37
323 339 308
NS variants
WC-5-4 only
WCR-1-74 only
WC-5-4 only
WCR-1-74 only
5 (1.4%) 7 (2.0%) 3 (0.8%)
5 (1.4%) 3 (0.8%) 5 (1.4%)
8 (2.3%) 10 (2.8%) 8 (2.3%)
9 (2.6%) 10 (2.8%) 11 (3.1%)
WC-5-4
WD-18-6
WC-5-4
WD-18-6
1 (0.3%) 2 (0.7%) 1 (0.3%)
2 (0.6%) 2 (0.7%) 3 (1.0%)
10 (3.2%) 10 (3.6%) 11 (3.8%)
WC-4-4
WD-18-6
WD-4-4
0 (0.0%) 3 (0.9%) 2 (0.6%)
3 (0.9%) 1 (0.3%) 3 (1.0%)
12 (3.7%) 12 (3.5%) 11 (3.6%)
9 (2.9%) 6 (2.1%) 8 (2.8%) WD-18-6 8 (2.5%) 8 (2.4%) 7 (2.3%)
a Aberrantmonokaryons: AT variant, reduced growth rate; NS variant, inviable b One clone yielded no monokaryonsat all
containing two classes of normal monokaryons were obained from any of the test strains. In the NS series, 2.1% (NR-3), 3.9% (NR-4), 2.8% (NW-3) and 3.3% (NW-5) of colonies contained no viable monokaryons, and 1.4% (NR-3), 1.0% (NR-4), 1.2% (NW-3) and 0.7% (NW-5) of colonies contained AT monokaryons. These values, commensurate with the incidences of AT or NS defects affecting single classes of monokaryons produced by the standard het (Table 1), confirmed that a presumptive recessive lethal NS lesion in one nuclear component of a het does not significantly bias the likelihood of either an AT lesion or a complementing NS lesion arising in the het's other nuclear component. The absence of bias further suggests that noncomplementing NS defects resuiting in inviable her cells undetectable by our screening procedure occur rarely if at all. Among the four AT test strains, total NS variants were found at frequencies of 4.8% (AR-1), 7.1% (AR-2), 6.7% (AW-1) and 6.2% (AW-2), values consonant with that for the standard het: single nonsegregators of the normal or the abnormal AT monokaryon occurred in roughly equal proportions within each strain. Thus, an AT defect in one bet nucleus does not significantly affect the probability of an NS lesion appearing either in the same nucleus or a compa nion nucleus. A total of 14 (1.1%) colonies distributed among the four AT populations examined contained slow growing monokaryons of both parental types. Their overall frequency corresponded well with that for AT variants of a given nuclear type produced by the
standard het (Table 1) and implies, as in the case for NS lesions, that an AT defect in one het nucleus does not significantly affect the probability of a new AT defect arising in another. Moreoever, the hets from doubly defective AT clones produced colonies of about the same size as the standard het when replated, indicating that, as a rule, AT defects of independent origins, like NS defects, complement in het cells.
Confirmation o f recessive lethals in N S variants Colonies produced by variant or normal hets give rise to rapidly growing outgrowths of hybrid monokaryons at about the same time during extended incubation at a given temperature. The hybrids are of two types, either 1) prototrophs, heterozygous for all parental auxotrophies, whose nuclei arose through karyogamy within het cells, or 2) partial hybrids whose nuclei contain the entire genetic content of one bet nucleus supplemented with some genetic material of a complementing nucleus acquired through internuclear transfer (Sarachek and Weber 1984). Unlike karyogamy, transfer events are growth temperature dependent, occurring frequently at 25 °C but seldom at 37 °C, and transfers which render a recipient nucleus heterozygous for only one of its two auxotrophic markers can be identified by selecting for monoauxotrophic hybrid outgrowths on het colonies grown on minimal medium containing a single
A. Saraehekand D. A. Weber: Segregant-defectiveheterokaryon of C. albicans Table 2. Means and ranges of frequencies of AT or NS variants
within sets of 15 independent elonal populations of the WC-5-4 x WCR-1-74heterokaryon (het) grown at 25 °C, 30 °C, or 37 °Ca Het growth temp (°c)
25 30 37
Frequencies (%) of variants AT variants
NS variants
Mean±S.E. R a n g e
Mean±S.E. Range
11.3±3.5 3.3±0.7 2.9±0.5
30.2±7.7 6.1±0.8 6.2±0.6
0.0-65.1 0.8-15.0 0.7-9.2
1.7-80.6 2.4-11.9 2.0-10.4
a Individual populations were assayed by replating at 30 °C and analyzing 120 to 150 het generated colonies
nutrient requirement of each of the complementing het nuclei. The proposition that recessive lethals account for the behavior of NS hets implies certain restrictions on the abilities of those variants to generate monoauxotrophic hybrids. Since the genetic constitution of a partial hybrid derives primarily from a single diaxotrophic het nucleus, a recessive lethal in that nucleus should prevent emergence of partial hybrids expressing either auxotrophic marker of the nucleus, except in rare instances when incidental genetic transfers either cover or compensate for the lethal. Comparisons of individual hybrid outgrowths on nine-day-old, 25 °C grown colonies of the WC-5-5 (His-, Arg-) x WCR-1-74 (Ade-, T h r - ) het, its single segregating variants NR-3 and NW-3, and a double nonsegregating variant, DNS-Y, conformed fully with these expectations. From 400 to 500 hybrid isolates per strain were examined. In addition to prototrophic hybrids, appreciable frequencies of all four possible monoauxotrophic hybrids (Ade-, 35%; Thr-, 54%; His-, 17%, Arg-, 5%) were obtained from colonies of the standard her grown on appropriately supplemented minimal media. In contrast, NR-3, which does not segregate viable WC-5-4-type monokaryons, yielded many Ade- (32%) and Thr- (20%) hybrids but only one Arg- and no His- hybrids, while NW-3, a nonsegregator of WCR-1-74-type monokaryons, produced many Arg- (5%) and His- (9%) hybrids and few Ade- (1%) or Thr- (2%) hybrids: the DNS-Y variant which segregates neither parental type monokaryons gave few or no monoauxotrophic hybrids of any sort (Ade-, 1%; T h r - , 1%; His-, 1%; Arg-, 0%).
Induction o f variant bets by growth at 25 ° C
To assess effects of growth temperatures on intraclonal frequencies of variants, sets of 15 whole WC-5-4 x WCR-
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1-74 colonies were picked from 25 °C, 30 °C or 37 °C plates when 4, 3 or 2 days old respectively, and individually replated on minimal medium. The 25 °C grown colonies contained approx. 130 to 180 het cells each and were streaked directly onto plates; 30 °C and 37 °C colonies contained roughly 1,000 to 2,500 hets cells, respectively, and were plated at dilutions which provided approx. 150 hets per plate. Colonies arising after three days incubation at 30 °C were then examined for their monokaryon contents. Table 2 summarizes the ranges and averages (-+ standard errors) of total NS or AT variant frequencies per colony for each of the three sets. The values for 30 °C and 37 °C grown colonies were essentially alike. In contrast, those for the 25 °C set indicated that 25 °C grown colonies accumulated much larger and more variable numbers of variants. The exceptional variant frequencies within 25 °C colonies could be attributed either to induction or to preferential growth of variants at the low temperature. To resolve that issue, quantitative comparisons were made as previously described (Sarachek and Rhoads 1983) of the sizes and cell contents of colonies produced at 25 °C (4 days) and at 37 °C (2 days) by the standard het and its NR-3, NW-3 and DNS-Y derivatives. At a given temperature, all strains produced the same size colonies containing about the same number of her cells. Moreover, the number of viable parental-type monokaryons in colonies of the single segregating variants NR-3 and NW-3 were equivalent to the numbers of corresponding monokaryons in colonies produced by the standard het. The absence of any discernable difference in replication of standard and variant het ceils indicated that the high frequencies of variants in 25 °C grown colonies of the standard her was due to their induction, not selection. In addition, the similarities in sizes and het cell contents of standard and variant het colonies under comparable conditions indicated that all het clones produced the same number of monokaryotic cells of each parental-type, whether viable or not. Evidently, the presence of a recessive lethal in a het nucleus does not affect the probability of that nucleus segregating into a uninucbate blastospore.
Induction o f het variants by metabolic inhibitors
Heterokaryosis is an unnatural condition for C albicans and hets have been shown to differ physiologically from monokaryotic cells in a number of respects. If the frequent occurrences in hets of nutritionally irreparable, recessive AT and NS defects are simply expressions of nonspecific genetic instability peculiar to such ceils, het nuclei might also be expected to accumulate subsidiary nutritionally reparable auxotrophic mutations. That possibility was examined by screening 2545 30 °C grown
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Table 3. Inductions of AT and NS variants in populations of the WC-5-4 x WCR-1-74 heterokaryon by metabolic inhibitors Treatment
Number of het clones screened
Frequencies (%) of variant clones AT variants
NS variants
Amino acid analogues None dl aUyl glycine (1 mg/ml) a dl ethionine (40 #g/ml) a Thiazolidine 4-carboxylic acid (1 mg/ml)
348 405 376 350
1.7 1.5 2.2 9.2
5.5 6.2 6.6 28.0
Nucleobase analogues None 6-azauracil (30 ug/ml) Caffeine (300 t~g/ml)a 5-fluorocytosine (0.07 tzg/ml)a
335 315 344 355
1.5 38.0 1.9 1.7
7.8 38.1 9.3 8.7
Mitoehondrial inhibitors None Acriflavine (20 ~g/ml) Ethidium bromide (8 #g/ml) Nalidixic acid (120 ~zg/ml) Chloramphenicol (2 mg/ml)
355 369 357 407 351
2.4 5.4 6.7 13.3 5.6
6.2 18.2 14.0 16.5 14.5
UV irradiation None 48 J/m 2
310 115
1.3 13.0
6.5 27.0
Cold-holding (10 ° C) None 48 h
414 220
2.8 5.5
8.2 27.7
a X2 tests of homogeneity between each treated population and its control indicated that incidences of variants are not affected by these treatments (P > 0.7), but are significantly increased by all others (P ~<0.001)
WC-5-4 x WCR-1-74 colonies for parental-type monokaryons recoverable on complete medium but not on minimal medium supplemented only for the het's parental auxotrophies. Of 163 single nonsegregating and 4 double nonsegregating clones identified on supplemented minimal medium, each showed identical characteristics on complete medium. The apparent absence of any newly arisen auxotrophies indicated that the lethals in variant nuclei constitute a special form of genetic damage instigated by heterokaryosis. To probe for specific metabolic processes which might contribute to that damage, various metabolic inhibitors were tested for ability to induce variants of the standard WC-5-4 x WCR-1-74 het under growth conditions. The agents were normalized for biological effectiveness by use at levels inactivating 50% to 70% of the treated population, and experiments were conducted at 37 °C since both the natural stability of hets and their sensitivities to growth inhibitors are greater at 37 °C than at lower temperatures (Sarachek and Rhoads 1983). The results in Table 3 show that variants are induced by a variety of inhibitors, many of which are not usually con-
sidered genotoxic. Each inducing treatment increases frequencies o f both AT and NS variants. Some, but not all, amino acid or nucelobase analogues tested are inducers: the proline analogue, thiazolidine-4-carboxylic acid and the uracil analogue, 6-azauracil are especially potent in this regard. Among the other effective agents, 1) chloramphenicol specifically inhibits mitochondrial protein synthesis, 2) nalidixic acid preferentially impedes mitochondrial nucleic acid synthesis, but also inhibits a number of cytoplasmic enzyme functions in yeasts (Haught and Sarachek 1985), 3) UV, acrifiavin and ethidium bromide impair nucleic acid functions, generally and 4) the trauma resulting from incubation at 10 °C has been associated with impairment o f proper processing and assembly of het ribosomes (Sarachek and Rhoads 1982). These observations do not point to a specific mechanism by which variant nuclei arise, but they do document that the process is exceptionally sensitive to the metabolic status of the het cell. The concommitent inductions of NS and AT variants by particular metabolic antagonists, as well as by het growth at 25 °C (Table 2), also indicates that both kinds of variants arise
A. Sarachek and D. A. Weber: Segregant-defective heterokaryon of C. albicans
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Table 4. Effects of metabolic inhibitors on frequencies of monoauxotrophic partial hybrids produced by the WC-5-4 (His-, Arg-) x WCR-1-74 (Ade-, Thr-) heterokaryon (her) grown at 37 °C on minimal media supplemented with single requirements of het nuclei Her growth conditions
Number (% of total) of monoauxotrophic hybrids a
Supplement
Number of hybrids tested
ade, his arg, thr
213 202
10 (4.7%)
ade, his arg, thr
203 194
8 (3.9%)
ade, his arg, thr
197 216
34 (17.3%)
ade, his arg, thr
0e 206
ade, his arg, thr
204 224
12 (5.9%)
None
ade, his arg, thr
198 210
6 (3.0%)
4 (2.0%)
UV-irradiation (48 J/m 2)
ade, his arg, thr
209 200
31 (i4.8%)
9 (4.3%)
ade, his arg, thr
196 217
36 (18.4%)
Treatment b
Ade-
His-
Arg-
Thr-
0 (0.0%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
1 (0.5%)
3 (1.4%)
3 (1.5%)
7 (3.5%)
1 (0.4%)
0 (0.0%)
0 (0.0%)
0 (0.0%)
2 (1.0%)
4 (2.0%)
0 (0.0%)
5 (2.3%)
Amino acid or pyrimidine analogues None dl ally1 glycine (1 rag/m1) Thiazolidine (1 mg/ml) 6-azauracil (30 #g/ml) 5-fluorocytosine (0.07/zg/ml)
5 (2.3%) 6 (3.0%) 12 (6.1%)
6 (2.9%)
Physical agents
cold-holding (10 °C, 48 h)
16 (8.2%)
a All other hybrid isolates were prototrophic b All plates containing metabolic analogues were inoculated from the same het cell suspension: a different suspension was used for both UV and cold-holding treatments c Hets failed to grow on adenine- and histidine-containing medium in the presence of 6-azauracil
by the same mechanism. A reasonable supposition would be that the moderate, growth inhibiting AT defects represent intermediate steps in formation of lethal NS lesions. However, our prior observation that AT variants are not predisposed to spontaneous conversion into NS variants indicates that AT damage is not a precursor of NS damage.
6-azauracil, thiazolidine-4-carboxylic acid, cold holding and UV, caused distinct increases in yields of partial hybrids (Table 4). This evidence for common events participating in inductions of het variants and partial hybrids suggests that the defective nuclei of NS and AT variants may arise as products of actual or abortive internuclear genetic transfers.
Induction of internuclear genetic transfers by inducers of het variants
Discussion
Segregational variants and monoauxotrophic partial hybrids resulting from transfers of genetic material from one het nucleus to another are each manifestations o f instabilities of het nuclei promoted by growth at 25 °C. The possibility that the two phenomena might be interrelated was examined by comparing metabolic inhibitors which do or do not induce het variants for abilities to elevate the normally low production of partial hybrids b y the WC-54 x WCR-1-74 het at 37 °C. The noninducers used, allyl glycine and 5-fluorocytosine, showed no effect on partial hybridization while all inducers tested,
Nuclei in hets o f C albicans produced through protoplast fusions engage in unidirectional genetic exchanges and also sustain heritable recessive lethal or growth debilitating genetic damages at high frequencies, spontaneously. Both behaviors are strikingly similar to ones described earlier for transient hets of the sexual yeast, Saccharomyces cerevisiae, formed b y conventional cytogamy between strains carrying a kar-1 mutation. Dutcher (1981) reported that S. cerevisiae hets transfer one or few intact chromosomes from one nucleus to another at rates inversely proportional to the relative sizes of chromosomes, but that as much as approx. 12% of all
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A. Sarachekand D. A. Weber: Segregant-defectiveheterokaryon of C. albicans
genetic transfers between nuclei involve only parts of chromosomes. The internuclear transfers occur equally well in either direction, can result in either additions to or replacements of homologues in recipient nuclei, and account for the appearance of extrinsic chromosomal material in approx. 2% of all het nuclei. In addition, she noted that 38% of buds produced by heterokaryotic zygotes constructed with monoploid nuclei are inviable, whereas only 8.9% of buds from similar hets bearing diploid nuclei are inviable. She concluded that bud inviability denotes recessive lethal conditions in het nuclei resulting from chromosome losses: the monoploid system indicated that 38% of nuclei suffered such losses and that the probability of capture of freed chromosomes by potentical recipient nuclei approaches 0.05. Although insufficient linkage information exists to document transfers of whole chromosomes in C. albicans hers, we have previously demonstrated that internuclear transfers of genetic material in this organism can occur in either direction between complementing nuclei and can result in genetic additions to recipient nuclei (Sarachek and Weber 1984). The present study established that, under standard growth conditions approx. 5%-10% of cells in het populations are stable variants abnormal for segregation of monokaryons. The majority, NS variants, have recessive lethal defects in one or the other or both complementing nuclei which prevent their formation of viable monokaryons bearing affected nuclei: the remainder, AT variants, carry analogous recessive growth debilitating defects responsible for their segregation of very slow growing monokaryons. It was shown that AT and NS defects arise independently of each other and that each kind of defect occurs in either of the nuclear components of a het cell with equal probability. Furthermore, evaluations of the effects of growth temperatures and of specific metabolic antagonists on inductions of both nuclear defects and internuclear genetic transfers indicated that, as in S. cerevisiae, the two phenomena are interconnected (Table 3 and 4). A variety of molecular and genetic evidences affirm that natural strains of C albicans are diploid, with nuclear DNA contents comparable to that of diploid S. cerevisiae (Kurtz e t al. 1986; Shepherd et al. 1985). Although the number of chromosomes in C. albicans is unknown, our finding that approx. 6% of cells in C albicans het populations carry recessive lethals in a nuclear component (Table 1) is of the same order as Dutcher's estimated 8.9% of nuclei expressing recessive lethals in diploid hets of S. cerevisiae. These parallels between hets of C albicans and of the genetically better defined S. cerevisiae argue convincingly for the natural occurrence of considerable nuclear heterogeneity among forced hets of both yeasts as a consequence of chromosomal losses and gains. For the diploid C albicans, the recessive lethals of NS variants could reasonalby be ascribed to losses leading to nulliso-
mies or to functionally severe aneuploidies or deletions of parts of chromosomes. The growth limiting defects of AT variants might represent less significant chromosomal deficiencies or, possibly, functional imbalances effected in genetically supplemented recipient nuclei. However, it is most likely that both categories of variants arise through losses which are random in kind and modest in extent since independently derived AT and NS defects present in each of the nuclear components of a het are typically expressed as recessive and complementing traits. If so, even the combined AT and NS variant frequencies would probably underestimate the actual incidences of losses among hets since many limited aneuploidies or chromosomal deletions in diploid nuclei might well be expected to be phenotypically cryptic. Instabilities of bet nuclei have special implications for certain common but perplexing features of protoplast fusion crosses in yeasts. Crosses customarily are made by fusing protoplasts of complementing doubly auxotrophic strains and selecting for prototrophs. In the process, aggregates of protoplasts first fuse into syncytia from which persistent or transient het cells regenerate (Ferenczy 1985). Monokaryons emerging from the system heterozygous for all parental markers and with DNA contents greater than either parent have been considered products of karyogamy between normal parental nuclei. Recently, however, molecular analyses have cast doubt on these assumptions. By means of DNA density and two dimensional electrophoretic protein determinations, Groves and Oliver (1984) have shown that monokaryotic hybrids from fusion crosses between Yarrowia lipolytica and Kluyveromyces lactis may carry all parental markers heterozygously and contain DNA approximating twice the parental levels, yet derive almost all of their DNA from only one of the parents. Similar findings, based on DNA reassociation kinetics, were obtained for hybrids from interspecific crosses between a number of Candida, Hansenula~ Saccharomyces and Torulopsis species (Spencer et al. 1985), and BiJttner et al. (1985), using isoenzyme markers, have shown that individual fusion hybrids from crosses ofP. guilliermondii have different, disproportionate amounts of parental genetic information. While sexually produced yeast hybrids normally result from karyogamybetween the single intact nuclei in a mating pair of monokaryotic cells, hybrids formed through protoplast fusions arise from a pool of nuclei subject to various kinds of genetic interactions within a multikaryotic cell. The immediacy and magnitude of possible interactions is indicated by Dutcher's (1981) estimate of extensive losses and gains of whole or partial chromosomes among nuclei of newly formed S. cerevisiae hets. Since a given nucleus could serve as both donor of some chromosomes and recipient of others, the range of variation among progenitors of hybrid nuclei is broad. Klinner and B6ttchers' (1985) ob-
A. Sarachek and D. A. Weber: Segregant-defectiveheterokaryon of C albicans servation that protoplast fusion causes structural rearrangements of chromosomes in Candida maltosa adds still another source of possible genetic variation to the outcome of fusion crosses for some yeasts at least. These considerations pose obvious caveats for the genetic analysis of C albicans by means of protoplast fusions. Although many hybrid monokaryons heterozygous for parental markers obtained from fusion crosses have approximately twice the parental DNA content, others contain distinctly aneuptoid amounts of DNA (Sarachek et al. 1981). This has been construed to signify that karyo'gamy between parental diploid nuclei yields true tetraploid nuclei which are liable to subsequent loss of chromosomes (Hilton et al. 1985; Whelan et al. 1985). However, for reasons cited above, DNA values and heterozygosities for small numbers of selected markers are not adequate bases for judging the constitutions or origins of hybrid nuclei, and our evidence for considerable genetic variation among het nuclei directly contradicts the presumption that hybrids are uniform, tetraploid products of homogeneous populations of parental nuclei. These complications do not negate the utility of protoplast fusions for genetic studies of thi s very important yeast. Indeed, the procedure has already proved valuable in identifying a few gene !inkages and polysomic conditions in C albicans, (Sarachek et al. 1981; Sarachek and Weber 1984; Hilton et al. 1985), and hets formed by fusions offer unique possibilities for studies of extrachromosomal determinants. But, they do emphasize that unequivocal resolution of the composition of hybrid nuclei emerging from fusion crosses will require much more extensively marked parental strains than have been used heretofore, as well as appreciation of the potential for nuclear instabilities peculiar to the intermediary multinucleate cells which generate those hybrids.
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References
Biittner M, Birnbaum D, Bottcher A (1985) J Basic Microbiol 25:83-94 Dutcher SK (1981) Mol Cell Biol 1:245-253 Ferenczy L (1985) Protoplast fusion in yeasts. In: Perberdy JF, Ferenczy L (eds) Fungal protoplasts. Dekker, New York, pp 279-306 Groves, DP, Oliver SG (1984) Curr Genet 8:49-55 Haught MA, Sarachek A (1985) Mutat Res 152:15-23 Hilton C, Markie D, Corner B, Rikkerlink E, Poulter R (1985) Mol Gen Genet 200:162-168 Klinner U, B6ttcher F (1985) Curt Genet 9:619-621 Kurtz MB, Cortelyou MW, Kitsch DR (1986) Mol Cell Biol 6: 142-149 Odds FC (1979) Candida and Candidosis. University Park Press, Baltimore Poulter R, Jeffrey K, Hubbard MJ, Shepherd MG, Sullivan PA (1981) J Bacteriol 146:833-840 Sarachek A, Rhoads DD (1982) Sabouraudia 20:251-260 Saraehek A, Rhoads DD (1983) Mycopathologia83:87-95 Sarachek A, Weber DA (1984) Curr Genet 8:181-187 Sarachek A, Weber DA (1985) Mycopathologia92:121-123 Sarachek A, Rhoads DD, SchwarzhoffRH (1981) Arch Microbiol 129:1-8 Shepherd MG, PouRer RTM, Sullivan PA (1985) Candida albicans: biology, genetics and pathogenicity. In: Ornston LN, Balows A, Bauman P (eds) Annual review of microbiology, vol 39. Annual Revieus Inc., Palto Alto, CA, pp 579-614 Spencer JFT, Spencer DM, Bizeau C, Martini AV, Martini A (1985) Curr Genet 9:623-625 Whelan WL, Markie DM, Simpkin KG, PouRer RM (1985) J Bacteriol 161:1131-1136
Communicated by U. Leupold Received February 17, 1986