JOURNAL OF BACTERIOLOGY, Aug. 1990, p. 423842460021-9193/90/084238-09$02.00/0Copyright © 1990, American Society for Microbiology

Vol. 172, No. 8

Rapid Genetic Identification and Mapping of EnzymaticallyAmplified Ribosomal DNA from Several Cryptococcus Species

RYTAS VILGALYS* AND MARK HESTER

Department ofBotany, Duke University, Durham, North Carolina 27706

Received 21 December 1989/Accepted 22 May 1990

Detailed restriction analyses of many samples often require substantial amounts of time and effort for DNAextraction, restriction digests, Southern blotting, and hybridization. We describe a novel ap h t usesthe polymerase chain reaction (PCR) for rapid restriction typing and of DNA from maydifferent isolates. DNA fragments up to 2 kfiobase pairs in length were efficiently I from crude DNAsamples of several pathogenic Cryptococcus species, including C. neoformans, C. alTds, C. lawwnti, and C.unigutudatus. Digestion and electrophoresis of the PCR products by using frequent-cuting enzymes

produced complex restriction phenotypes (fingerprints) that were often unique for each stri or species. We

used the PCR to amplify and analyze restriction pattern variation witn three mnor portions of the ribosomlDNA (rDNA) repeats from these fungi. Detaied mapping of many restriction sites within the rDNA locus wasdetermined by fingerprint analysis of progressively larger PCR fragments sharing a common primer site at oneend. As judged by PCR fingerprints, the rDNA of 19 C. neofornans isdates showed no variation for fourrestriction enzymes that we surveyed. Other Cryptococcus spp. showed varying levels of ic pattern

variation within their rDNAs and were shown to be genetically distinct from C. neoformans. The PCR primersused in this study have also been successfuly applied for amplition of rDNAs from other patenic and

nonpathogenic fungi, including Candida spp., and ought to have wide applicability for cll detection andother studies.

Clinical identification of pathogenic fungi is often ham-pered by a number of significant problems. Morphologicaland cultural characters of many fungi are often simple andmay show considerable phenotypic plasticity. Most zoo-pathogenic fungi also do not produce a sexual stage (teleo-morph) and instead are identified only from their asexualreproductive stage (anamorph). Classification of fungi basedsolely on anamorphic features is often inherently artificial,however, and may not therefore reflect true phylogeneticrelationships. For these reasons, molecular approachesshould be especially useful for genetic identification of fungiand for establishing their taxonomic relationships.The genus Cryptococcus is a heterogeneous group of

nonfermentative, encapsulated yeast species, which in-cludes several important human pathogens. Among these,Cryptococcus neoformans has been increasingly reportedfrom immunocompromised patients, particularly those in-fected with human immunodeficiency virus. Although theperfect state (teleomorph) of C. neoformans has been iden-tified in the basidiomycete genus Filobasidiella, most iso-lates of C. neoformans and other Cryptococcus spp. rarely,if ever, produce sexual stages under laboratory conditions(3, 8, 17).One recent approach for establishing genetic relationships

in medically important fungi is by analysis of restrictionfragment length polymorphisms (RFLPs) in their DNAs. Forexample, different serotypes of Histoplasma capsulatummay be distinguished by their RFLPs in both mitochondrialDNA and ribosomal DNA (rDNA) (21). Magee et al. (13)also reported that restriction site and length variation withinthe rDNA locus from several Candida spp. correspondedwell with previous recognized taxonomic groupings and thatintraspecific variation for restriction phenotypes might evenbe useful for strain identification.

* Corresponding author.

Several limiting features of conventional restriction anal-yses using Southern blot hybridization for routine identifi-cation and systematics are a requirement for relatively cleanDNA samples as well as substantial time and resourcesnecessary for preparing DNA hybridization membranes andprobes. Sensitivity of Southern blots is also sometimesaffected by poor signal/noise ratios caused by poor hybrid-ization of heterologous probes or high background signals onblotting membranes. To circumvent some of these potentialproblems, we have used an approach that allows directanalysis of a desired target sequence without the necessity ofblotting or hybridization. Our approach utilizes the polymer-ase chain reaction (PCR) to enzymatically amplify targetsequences in vitro by a factor of over 108, starting fromminute amounts of genomic DNA template (19, 21). Thelevel of sensitivity, specificity, and adaptability of the PCRhas resulted in many applications, including efficient cloning,rapid direct sequencing, and highly sensitive detection (2,12, 19, 22). The basic PCR protocol involves the use of twooligonucleotide primers (which flank a target DNA se-quence) to initiate DNA synthesis on opposing strands of atarget sequence, using a heat-stable DNA polymerase. Re-peated cycles (20 to 30) of denaturation, primer annealing,and DNA synthesis result in a theoretical doubling of targetsequence during each cycle. Automation of the PCR byusing a thermal cycler device allows simultaneous amplifi-cation ofDNA from many samples, which can then be useddirectly for analysis.

In this study, we examined DNA sequence variation in therDNA of several Cryptococcus spp. by analysis of enzymat-ically amplified rDNA fragments. The various rRNAs andtheir respective DNA coding regions are known for theirvalue as evolutionary markers, since they contain regions ofboth high and low sequence variability (5). The rDNA repeatof C. neoformans is organized similarly to that of most otherfungi and contains coding regions for 17S, 5.8S, and 25S

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PCR FINGERPRINTING OF rDNA IN CRYPTOCOCCUS SPP.

TABLE 1. Cryptococcus and Candida strains used, along with data on their sources and teleomorphs (if known)Taxon and strain

Taxa associated with a Filobasidiella teleomorphC. neoformans 132C. neoformans 101C. neoformans C3DC. neoformans 3501C. neoformans 3502C. neoformans n32C. neoformans n34C. neoformans n33C. neoformans 602C. neoformans D321C. neoformans nlOC. neoformans nllC. neoformans n12C. neoformans n31C. neoformans n16C. neoformans n18C. neoformans n25C. neoformans n27C. neoformans n35

Taxa associated with a Filobasidium teleomorphC. albidus var. aerius 155C. albidus var. albidus 142C. albidus var. diffluens 160C. albidus var. ovalis K6-7C. albidus D322C. uniguttulatus 103.87C. uniguttulatus 162.86

Taxa not associated with a teleomorph (asexual)C. ater 4685C. Iaurentii 139C. Iaurentii D318C. laurentii 108.87C. laurentii 142.89Candida albicans 666Candida albicans C9a AIDS, Acquired immunodeficiency syndrome.

Source

Type culture, serotype D, =ATCC 32719, IFO 0608, CBS 132J. Perfect, serotype AJ. Perfect, serotype A, clinical isolateJ. Perfect, serotype D, mating type a, =ATCC 34873J. Perfect, serotype D, mating type a, = ATCC 34874J. Perfect, serotype BJ. Perfect, serotype CJ. Perfect, serotype CJ. Perfect, serotype A, nonencapsulated strainW. Schell, clinical isolateJ. Perfect, clinical isolate from AIDS' patientJ. Perfect, clinical isolate from AIDS patientJ. Perfect, clinical isolate from AIDS patientJ. Perfect, clinical isolate from AIDS patientJ. Perfect, Busse-Bueske isolate made in 1893J. Perfect, clinical isolate, serotype A or DJ. Perfect, clinical isolateJ. Perfect, clinical isolate, serotype A or DJ. Perfect, serotype B or C

Type culture, =ATCC 10665, NRRL Y-1399, CBS 155Type culture, =ATCC 10666, CBS 142Type culture, =ATCC 12307, IFO 0612, CBS 160, NRRL Y-15Type culture, =ATCC 22460, S. Goto K6-7W. Schell, clinical isolateW. Schell, clinical isolateW. Schell, clinical isolate

Type culture, =ATCC 14247, CBS 4685, NRRL Y-66Type culture, =ATCC 18803, CBS 139, IFO 0609, 0906W. Schell, clinical isolateW. Schell, clinical isolateW. Schell, clinical isolateJ. PerfectJ. Perfect

RNAs along with their respective spacer segments (18). Inthis study, we demonstrate the utility of restriction analysisusing PCR products for (i) amplification and restrictionmapping of rDNA from Cryptococcus species, (ii) identifi-cation of species- and strain-specific RFLPs, (iii) linking ofanamorphic and teleomorphic life stages, and (iv) phyloge-netic analysis.

MATERIALS AND METHODS

DNA preparation. The fungal isolates used are listed inTable 1. DNA from 14 isolates of C. neoformans andCandida albicans was prepared from protoplasted cells (16)provided by John R. Perfect (Department of Medicine, DukeUniversity). The remaining isolates were grown overnight in25 ml of potato dextrose broth with shaking. Because thePCR does not necessarily require high-quality DNA forsuccessful amplification, we adopted a "minimal prep"procedure for simplified cell lysis and DNA extraction (11).Yeast cells were harvested by centrifugation (5,000 x g, 10min, 5°C), washed once with cold distilled water, recentri-fuged, and suspended in 3 to 5 ml of extraction buffer (0.15M NaCl, 50 mM Tris hydrochloride, 10 mM disodiumEDTA, 2% sodium dodecyl sulfate [pH 8.0]). The suspendedcells were then incubated at 65°C for 1 h, extracted one timewith phenol-chloroform-isoamyl alcohol (25:24:1 by vol-ume), and precipitated with 0.6 volume of isopropyl alcohol.

Additional isopropanol (up to 1 volume) was added to lysatesthat did not produce a visible precipitate after 5 min at roomtemperature. The DNA precipitates were collected by cen-trifugation (10,000 x g, 15 min, 5°C), washed in ice-cold 80%ethanol for 5 min, and dried under vacuum. The final DNApellet was suspended in 500 ,ul of TE (10 mM Tris hydro-chloride [pH 8.0], 1 mM disodium EDTA) and stored at-20°C until use. Concentrations of DNA samples wereestimated by running small samples on agarose minigelsagainst genomic DNA standards.Handling procedures and precautions for using PCR. Be-

cause of the risk of cross-contamination from handling ofmany identical PCR reactions, it was necessary to followstrict precautions as outlined previously (6). Many of theseprecautions simply require good sterile technique and aware-ness of potential contamination sources. Dedicated pipet-tors, electrophoresis chambers, storage space, and reagentsfor handling PCR products were assigned before the exper-iments were performed. Negative controls (omitting DNAtemplate) were also included in each PCR experiment.Enzymatic amplification of DNA with use of the PCR. DNA

samples were diluted before the PCR was performed. Asample of each miniprep DNA (10 to 15 ,ul) was electro-phoresed into low-melting-point agarose gels (0.6% Sea-Plaque agarose; FMC Bioproducts), using Tris-borate-EDTA buffer as described previously (14). After staining

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4240 VILGALYS AND HESTER

TABLE 2. Oligonucleotide primer sequences usedto amplify fungal rRNA

Nucleotide sequence, Position onName 5'--3' S. cerevisiae rRNA

SR1-R TACCTGGTTGATTCTGC 1-17, 17S RNA5.8S CGCTGCGTTCTTCATCG 51-34, 5.8S RNA5.8S-R TCGATGAAGAACGCAGC 34-51, 5.8S RNALR1 GGTTGGTTTCTTTTCCT 73-56, 25S RNALR3 GGTCCGTGTTTCAAGAC 654-638, 25S RNALR5 ATCCTGAGGGAAACTTC 968-952, 25S RNALR6 CGCCAGTTCTGCTTACC 1141-1125, 25S RNALR7 TACTACCACCAAGATCT 1448-1422, 24S RNALR7-R AGATCTTGGTGGTAGTA 1422-1448, 25S RNALR12 GACTTAGAGGCGTTCAG 3126-3110, 25S RNA

with ethidium bromide, the high-molecular-weight bandscorresponding to genomic DNA (100 to 200 ng of DNA) werecut out from the gels, diluted into 1 ml of sterile distilledwater, and dissolved by heating at 65°C for 10 min beforepreparation of the PCR reactions. Oligonucleotide primerswere prepared by using standard phosphoramidite blockingand deblocking chemistries on an Applied Biosystems 380Bsynthesizer. The oligonucleotide primers used and theirlocations on the rDNA are shown in Table 2. The PCRreactions were set up with Amplitaq DNA polymerase (U.S.Biochemicals) in either 100- or 25-Rl volumes, using bufferconditions recommended by the manufacturer. Thirty PCRcycles were performed on an automated thermocycler de-vice (Perkin-Elmer-Cetus), using the following parameters:94°C denaturation step (1 min), 50°C annealing step (45 s), 50to 72°C ramp (1 min), and primer extension at 72°C (1 min).A final 7-min incubation step at 72°C was added after thefinal cycle to ensure complete polymerization of any remain-ing PCR products. PCR products were checked by running 1to 2 p.l of each reaction mixture on agarose minigels.

Restriction analysis of PCR products. After removal of

SR1-R9

5.8S5.8S-R9

LR7LR7-R9

LR1 29

175 RNA 5.8S US RNA

A B C

2l314151FIG. 1. Generic map of a portion of the rDNA repeat showing

the locations of oligonucleotide primer sites (given in Table 1) usedto amplify rDNAs from Cryptococcus spp. and other fungi. Theexpanded region of fragment B shown at the bottom also indicatesthe locations of additional primer sites used to determine the orderof restriction sites by PCR mapping.

FIG. 2. PCR fingerprint analysis of rDNAs from six strains ofCryptococcus and Candida spp. (A) Oligonucleotide primers LR7-Rand LR12 were used to amplify fragment C from Fig. 1 (correspond-ing to the 3' half of the 25S RNA cistron); 5 p.l of each PCR reactionproduct was electrophoretically separated in 0.8% gels. (B) Enzy-matically amplified C fragments from the six strains were digestedwith HhaI and separated on 3% agarose. Restriction analysis revealsspecies- or strain-specific patterns among the six strains.

some of the mineral oil overlay from the PCR reactions, theamplified products were extracted once with 1 volume ofphenol-chloroform-isoamyl alcohol. The aqueous portionwas removed, and DNA was precipitated by addition of 1/10volume of 3 M sodium acetate (pH 5.0), followed by 3volumes of 95% ethanol. Ample time was given for precipi-tation (>1 h at -20°C). The precipitates were collected bycentrifugation for 15 min in a microfuge, washed in 80%ethanol, dried under vacuum, and resuspended in 50 ,ul ofTE. For restriction analysis, 5 to 10 p.l of each PCR productwas digested in a 20-,ul volume, using TaqI, Avall, Hinfl,HhaI, or MspI under conditions supplied by the manufac-turer (Promega Biotec). The restriction reactions were sep-arated by electrophoresis in 3% agarose gels (2.25% NuSieveagarose, 1.75% Seakem GTG agarose; FMC Bioproducts),using 4X174 HaeIII-digested fragments as molecular weightstandards. After staining, the gels were photographed over aUV transilluminator to record the results.

Restriction analysis of rDNA by PCR mapping. To maprestriction sites within the rDNA fragments, we used a novelapplication of the PCR which greatly facilitates restrictionmapping by obviating the need for Southern blotting andprobe hybridization. This method, which we call PCR map-ping, is based on restriction analysis of incrementally largerPCR fragments sharing a common endpoint (Fig. 1). Thepositions of different restriction fragments (and their termi-nal restriction sites) from the largest PCR product aredetermined from their serial appearance in digests of succes-sively larger fragments. The B fragments from several Cryp-

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PCR FINGERPRINTING OF rDNA IN CRYPTOCOCCUS SPP.

_c'JC

C') C'c) LO LO C_ D

00

0A 0

0o 0o 0o 0o 0o _ _ 0_

E E E E E E Eo cE CX0 0 0 0 0: 0. O. - 0-

0 0 0 0 0 0m m 0 co co

C Ca co Ca c= Cc X >

U) c CD CO) U) CD cn U) ) C C D Occccca c L.: .: L: L. *.- O. 0,.

mmco c co000Om

FIG. 3. PCR fingerprint analysis of rDNA (fragment B) from 18 reference strains of Cryptococcus and Candida spp. (A) PCR fingerprintsproduced after digestion with Avall; (B) PCR fingerprints produced after digestion of the same 18 fragments with HhaI.

tococcus strains were mapped in this manner, using theprimer pairs 5.8S-R/LR-1, 5.8S-R/LR-3, 5.8S-R/LR-5, 5.8S-R/LR-6, and 5.8S-R/LR7. The positions of restriction sites inseveral strains could also be inferred from the additivity ofpairs of smaller restriction fragments relative to the largerrestriction fragments present in another strain.

Estimation of genetic relationships between taxa andstrains. For estimating genetic relationships between dif-ferent rDNA sequences, a distance metric (D) was calcu-lated by using the equation suggested by Nei and Li (15), D= 1 - 2(ne,)I(nx + ny), in which n, is the number ofrestriction fragments shared in common between two strainsand nx and ny represent the total number of restrictionfragments in each strain. Two important assumptions regard-ing D as an estimate of genetic similarity are (i) identity ofcomigrating restriction fragments from different strains and(ii) the absence of significant length variation within therDNA region being analyzed (which would result in violationof assumption i).

Estimates of overall restriction site similarity were used toconstruct an unrooted tree (network) by the FITCH optionof the computer package PHYLIP (4).

RESULTS

Amplification of rDNA from Cryptococcus spp. The loca-tions of primer sites and strategies for PCR amplification ofrDNA from fungi are shown in Fig. 1. These oligonucleotideprimers themselves represent highly conserved consensusregions between published rRNA sequences availablethrough GenBank for several fungi and eucaryotes, includingSaccharomyces cerevisiae (yeast), Neurospora crassa,Oryza sativa (rice), Mus domesticus (mouse), and humans(Table 1). When small differences in the primer sites werefound (mostly base substitutions), the yeast sequence wasused for primer synthesis. These primers have been success-fully used in our laboratory to amplify rDNAs from a varietyof fungi, plants, and animals (unpublished data).

Six strains of Cryptococcus and Candida spp. were cho-sen initially to amplify three portions of the rDNA repeat (A,B, and C in Fig. 1) spanning almost the entire rDNA codingregion. Figure 2A shows samples of six C fragments ob-tained after PCR amplification from all six strains. All threefragments (A, B, and C) were successfully amplified from allstrains tested, with a typical yield of 1 to 2 ,ug per 100-,ul

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4242 VILGALYS AND HESTER

reaction. The lengths of the A and C fragments were 2.0 and1.6 kilobase pairs (kb), respectively, in all six strains. Minorlength heterogeneity was observed for the B fragments fromC. neoformans 132 and n32 (1.85 kb), Cryptococcus albidus142 (1.85 kb), Cryptococcus uniguttulatus 103.87 (1.80 kb),Cryptococcus laurentii 139 (1.80 kb), and Candida albicans666 (1.75 kb).RFLPs between species. Samples of the A, B, and C

fragments were digested with either HhaI or AvaIl andresolved by electrophoresis in 3% agarose. Restriction di-gests for each set of digested PCR fragments yielded similarpatterns of variation among the six strains tested (Fig. 2B;only the HhaI-digested C fragments are shown). We haveoccasionally observed partial digestion of PCR fragmentswith use of some enzymes, suggested by the presence ofsecondary bands of lighter intensity in the photos. Theprimary restriction patterns are clearly obvious in mostcases, however, and allow unambiguous assignment ofstrains to a particular restriction phenotype class. Both C.neoformans strains (representing the two varieties, neofor-mans and gattii) produce identical restriction patterns witheither enzyme for all three fragments. C. albidus 142 and C.uniguttulatum 103.87 shared identical restriction patterns fortwo of the six enzyme-fragment combinations tested. C.laurentii 139 and Candida albicans 666 each producedunique restriction patterns for each fragment with use ofeither enzyme.The rDNA region corresponding to fragment B (Fig. 1)

was chosen for more detailed and extensive analysis using anexpanded set of isolates. Purified B fragments were amplifiedby PCR from 16 reference strains (including several typestrains) representing Cryptococcus taxa known to be patho-genic. DNAs from two strains of Candida albicans were alsoincluded in the experiment as taxonomically unrelated con-trols. This collection of B fragments was digested with fourseparate enzymes (TaqI, AvaIl, HhaI, and Hinfl). Photo-graphs of the AvaIl and HhaI digests are shown in Fig. 3.The restriction phenotypes identified for the various en-zyme-strain combinations are listed in Table 3. A pattern oftaxon-specific restriction phenotypes (i.e., fingerprints) wasclearly evident for all four enzymes examined. A fifthenzyme, MspI, also showed similar taxon-specific variationbut was not surveyed in all strains (data not shown). All ofthe C. neoformans isolates had identical PCR fingerprints forall four enzymes. Similarly, both strains of C. uniguttulatushad identical restriction phenotypes for every enzyme sur-veyed. Intraspecies polymorphism was evident among sev-eral type strains representing different varieties of C. albidusas well as between the two strains of Candida albicans. Byscoring the 18 strains for their restriction patterns producedwith each enzyme, 10 unique genotypic classes could beidentified (Table 3). All of the C. neoformans strains, includ-ing its two varieties gattii and neoformans (8, 10), hadidentical PCR fingerprints for each enzyme tested (class I inTable 3). Both strains of C. uniguttulatum were also identi-cal for all four enzymes (class VI). The type strains ofCryptococcus ater (class VII) and C. Iaurentii (class VIII)were each characterized by unique fingerprints. Strains fromthe remaining taxa were polymorphic for one or moreenzymes as follows: several varieties of C. albidus com-prised classes II, III, IV, and V, and two strains of Candidaalbicans made up classes IX and X.

Identification of isolates by using PCR fingerprints. Anadditional 15 Cryptococclus strains representing clinical iso-lates were tested to determine whether their PCR finger-prints could be assigned to one of the reference classes

TABLE 3. Genotypic classes and restriction phenotypes ofCryptococcus and Candida spp. revealed by PCR fingerprints

Restriction phenotype ofClass Strain B fragment digested with:

TaqI Hinfl Avall HhaI

Reference strainsI C. neoformans 132' A A A AI C. neoformans 3501 A A A AI C. neoformans 3502 A A A AI C. neoformans 101 A A A AI C. neoformans C3D A A A AI C. neoformans n33 A A A AI C. neoformans n34 A A A AI C. neoformans n32 A A A AII C. albidus var. aerius 155a B B B BIII C. albidus var. albidus 142" C -b BIV C. albidus var. diffluens 160a D C C CV C. albidus var. ovalis K6-7' C D D DVI C. uniguttulatus 103.87 B E E DVI C. uniguttulatus 162.86 B E E DVII C. ater 4685" E F F DVIII C. laurentii 139' F - G EIX Candida albicans 666 G G H FX Candida albicans C9M H I

Clinical and other strainsI C. neoformans niOb A A A AI C. neoformans nllb A A A AI C. neoformans nl2b A A A AI C. neoformans n3lb A A A AI C. neoformans n16 A A A AI C. neoformans n18 A A A AI C. neoformans n25 A A A AI C. neoformans n27 A A A AI C. neoformans 602 A A A AI C. neoformans D321 A A A AI C. neoformans n35 A A A AXI C. albidus D322 B D B DXII C. laurentii D318 F - AXIII C. laurentii 108.87 C I K GXI C. laurentii 142.89 B D B D

aType culture for species or variety.b-, Restriction phenotype not determined.Isolate from patient with acquired immunodeficiency syndrome.

identified previously. Each of the clinical isolates had beententatively identified by its source to species on the basis ofmorphological or cultural features. The B fragments of eachstrain were amplified and digested with the four enzymesused previously. An example of one of the restriction digests(for Avall) is shown in Fig. 4. Eleven clinical isolatesidentified as C. neoformans (including four strains isolatedfrom patients infected with human immunodeficiency virus)produced PCR fingerprints characteristic of genotypic classI, to which all of the reference strains of C. neoformansbelong. Four isolates identified as either C. albidus (oneisolate) or C. laurentii (three isolates) produced PCR finger-prints that were similar to some but not all of the previousreference strains. Two of these clinical isolates (C. Iaurentii318 and C. Iaurentii 108.87) produced unique restrictionpatterns not encountered in any of the previous referencestrains, whereas the other two isolates possessed a uniquearray of restriction patterns for the four enzymes tested. Asa result, it was necessary to assign these four clinical isolatesto three additional genotypic classes (XI, XII, and XIII).PCR mapping of restriction sites in rDNA. Figure 3B shows

six HhaI restriction fragments produced after digestion ofthe B fragment of C. neoformans 132. Figure 5 shows results

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PCR FINGERPRINTING OF rDNA IN CRYPTOCOCCUS SPP.

a) 1) 0) a)

0 0 0 0Xr co Xn XCYo o co o

ol U\, U,- IC)cL C\ \

C C CCe CC)en en en) CO

o) (A ( A A ( ( o (co (AC\0r-ojC\Jco co coX CoC C C 0CC

E E E E E E E E E E Eo. 0 0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0 0 00) 0 e 0) 0) 0 0) 0)C C Cc C

OC

od i: o O

FIG. 4. PCR fingerprinting used to identify taxonomic identity of 15 Cryptococcus strains. Enzymatically amplified rDNA (fragment B)from each strain was digested with Avall and analyzed in 3% agarose. Eleven clinical isolates of C. neoformans produce identical restrictionphenotypes and belong to a single genotypic class. Four remaining strains have fingerprints that are either unique or characteristic of othergenotypic classes.

of a mapping experiment used to determine the positions ofthe six HhaI fragments from this strain. Five incrementalPCR fragments ranging from 350 to 1,750 base pairs (bp)were amplified (Fig. 5A) and digested simultaneously withHhaI. The resulting restriction fragments were run adja-cently on a 3% agarose gel (Fig. 5B). The smallest restrictionfragment (50 bp, indicated by an arrow) is present in all ofthe incremental PCR products, indicating its location within5' end of the B fragment. The large 550-bp restrictionfragment appears in the next and all subsequent lanes, and soits position must be next after the 50-bp fragment. In asimilar fashion, the positions of the remaining four restric-tion fragments of the B fragment can be determined by theirserial appearance from left to right in the five restrictiondigests. The resulting map positions of the HhaI restrictionsites within the B fragment of C. neoformans 132 are shownin Fig. 6, along with sites for AvaIl, TaqI, and the PCRprimers themselves (LR1 through LR7). Restriction maps ofall of the C. neoformans strains were identical for these threeenzymes. Additional restriction site differences between C.neoformans and the other Cryptococcus strains were deter-mined by PCR mapping or by inferring site changes on thebasis of different restriction patterns, indicated below eachmap in Fig. 6. The order of several smaller restrictionfragments generated by TaqI, as well as the numerous Hinflfragments present in most strains, could not be mappedaccurately because of limited resolution of 3% agarose gelsfor fragments of less than 50 bp. Some strains of C. albidusand C. laurentii also yielded an apparent excess number ofrestriction fragments that could not be mapped unequivo-cally, since their summed sizes exceeded the total length ofthe B fragment. We suspect that the presence of additionalfragments in some strains may be due to minor sequence

_ B

AI

0D C U) n o U) co cNX4 >J >J I >J I

a a: D a: a:C/) C/) (l) C/) (D) D - Ci) Cl) CCl)Co Co Co Co Co E ><~ co co co co co

LO LO UO LO U)UDU)U) U) U) X LO LO LO LO LO -e,

<-- 50bp

FIG. 5. PCR-assisted mapping of restriction sites (PCR mapping)in C. neoformans 132. (A) Fragment ladder produced after PCRamplification of five incrementally larger fragments of rDNA (frag-ment B), starting from a common endpoint; (B) restriction patternsof the same five PCR fragments digested simultaneously with HhaI.The order of restriction fragments in the largest fragment (5) can bedetermined by their serial appearance from left to right in each of thefive lanes. The position of a faint 50-bp fragment present in all lanesis indicated by the arrow.

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Hhal

11 I xiii vil I

Avail

vll 11 IIII XIITaql Iv Vwil

I xi 414 4 4

**lV IV VI VlI

O O O? ? Q35.BS-R LR1 LRS LNS LRO LA?

500 bpFIG. 6. Partial restriction maps for one region of the rDNA

repeat (fragment B) from C. neoformans 132 and other Cryptococ-cus spp. Locations of conserved restriction sites present in allstrains are shown above the lines; variable sites are indicated belowwith the respective genotypic class.

heterogeneity among the numerous rDNA repeats that existas a multigene family.

Genetic relationships between rDNAs from different Cryp-tococcus spp. Differences in the number of common restric-tion fragments among PCR fingerprints (Fig. 3 and 4) suggestthat some strains may be closely related. To estimate thelevel of divergence between PCR-amplified rDNA fragmentsfrorn different strains, a matrix of pairwise genetic distanceswas calculated for the genotypic classes defined in Table 3(restriction patterns of the two Candida albicans strainsshared little or no similarity with each other and with theother strains and were therefore not included in this analy-sis). The distance matrix (Table 4) was used to construct abranching network based on the Fitch-Margoliash algorithm,using the FITCH routine of the PHYLIP computer package(Fig. 7). This unrooted network divides the strains intoroughly three equidistant groups, with all of the C. neofor-mans strains at one end and most of the other taxa placed onnumerous terminal branches at the opposite end. Twostrains of C. laurentii belonging to genotypic classes VIIIand XII were placed roughly equidistant between the twogroups (Fig. 7).

DISCUSSIONAnalysis of rDNA frm fuing by using the PCR.

Successful amplification of rDNA was obtained for every

neoformans

Villlaurentli

0.1

XIII

IlurentliIV

dlffluens

FIG. 7. Genetic relationships among different rDNA genotypicclasses identified by PCR fingerprinting. A distance matrix based onrestriction fragment analyses of rDNA fragment B (Fig. 1) was usedto construct an unrooted network, using the FITCH procedure ofPHYLIP.

strain of Cryptococcus and Candida spp. tested by using thePCR primers listed in Table 2. We have successfully usedthese primers to amplify rDNA in numerous other fungi,including ascomycotina and basidiomycotina (data notshown). By using the same primer sequences or their com-plementary strands, amplification of other rDNA regionsshould also be attainable (e.g., the nontranscribed spacerregion). The ability of the PCR to amplify known variableregions of DNA, as well as more conserved tracts, shouldpermit analysis at almost any organismal level, from individ-ual strains to higher taxa.The method for extraction of genomic DNA from yeast

cells had little apparent effect on the yield and quality ofPCRproducts. Purified template DNA was not found to benecessary for efficient amplification, which permitted the useof simplified extraction protocols. We found it convenient togel purify DNA minipreps before use by running a portion ofeach sample into low-melting-point agarose, followed bydilution of the excised genomic DNA band into 1 ml ofdistilled water. This procedure effectively removed potentialinhibitors in every case. Precise quantitation of DNA inthese dilutions was not required, the primary criterion beingthe presence of a visible high-molecular-weight DNA band inthe gel.PCR fingerprinting for identification of Cryptococcns species

and varieties. Analysis of restriction patterns for three sep-arate regions of the rDNA repeat (A, B, and C in Fig. 1)revealed the presence of considerable sequence variationamong the strains studied and provides a convenient methodfor fingerprinting of species, varieties, and even individualstrains. Using four restriction enzymes with 4-base recogni-tion sequences, we were able to recognize 13 distinct geno-

TABLE 4. Genetic distance matrix among 11 genotypic classes of Cryptococcus spp. based on restrictionpattern analysis of rDNA fragment B

II I III IV V VI VII VIII XI XII

XII 0.532 0.362 0.333 0.370 0.362 0.447 0.469 0.455 0.333 0.400XII 0.294 0.333 0.400 0.300 0.333 0.294 0.333 0.111 0.333XI 0.535 0.256 0.182 0.200 0.070 0.209 0.289 0.400VIII 0.263 0.400 0.455 0.364 0.400 0.368 0.300VII 0.545 0.364 0.455 0.294 0.318 0.136VI 0.571 0.286 0.333 0.265 0.238V 0.571 0.286 0.182 0.224IV 0.592 0.306 0.333III 0.524 0.182II 0.429

VIlnter I I

serlusVI III

uniguttulatus albidus

laurentil v ovals-XI albidus, surentil

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PCR FINGERPRINTING OF rDNA IN CRYPTOCOCCUS SPP.

typic classes among the 33 strains surveyed (Table 3). Byamplifying a relatively large PCR fragment (-1 to 2 kb) fromseveral strains, genetic differences can be easily determinedby digesting PCR products with different frequent-cuttingenzymes. Subsequent blotting and probing with labeledDNA probes is not necessary, and the restriction patternsare directly observable during electrophoresis. Another sig-nificant advantage to using PCR-generated fragments forrestriction analysis is the avoidance of poor digestion ofgenomic DNA by some restriction endonucleases caused byDNA base modification (i.e., methylation).Although we were able to detect genetic variation among

most Cryptococcus species and strains from this study, nodifferences were observed among the 19 strains of C. neo-formans with use of the four restriction enzymes. Geneticuniformity within C. neoformans was unexpected, since ourstrains represent two well-characterized varieties (gattii andneoformans) that differ in a number of biologically significantfeatures, including serotype, colony morphology, nutrition,and DNA-DNA hybridization (1, 3, 9, 10). By surveyingother regions of the genome or using a wider array ofrestriction enzymes, it should be possible to identify diag-nostic PCR fingerprints among the varieties and serotypes ofC. neoformans as well. We have obtained preliminary dataindicating that the two C. neoformans varieties differ by anAluI polymorphism within the B fragment (unpublisheddata).

Considerable variability was observed among PCR finger-prints in the remaining 12 Cryptococcus and two Candidastrains examined; these variations could be grouped into 12genotypic classes (Table 3). Although we did not attempt tocharacterize genetic differences between the two strains ofCandida albicans from this study, inspection of their respec-tive PCR fingerprints suggests that the two strains probablydiffer by length mutations rather than any specific restrictionsite loss or gain. A recent survey of rDNA polymorphism inCandida spp. also demonstrated considerable length heter-ogeneity in rDNA among strains of C. albicans (13). Byusing the PCR approach described here, it ought to bepossible to rapidly identify taxa and strains of Candida spp.as well as of other medically important fungi.Type strains of all four varieties of C. albidus were each

characterized by a unique combination of restriction pat-terns with use of the four enzymes. Each type strain of C.ater and C. laurentii was also assignable to its own uniquegenotypic class. Although additional evidence is requiredbefore any taxonomic conclusions can be reached, it appearsthat C. albidus and its varieties, as well as C. laurentii, aregenetically variable as currently circumscribed.

Linking of sexual and asexual life histories and the geneticrelationships of Cryptococcus spp. One powerful applicationof PCR fingerprints is for establishing biological identitybetween asexual and sexual stages of the fungal life cycle.The complex life cycles of many fungi such as Cryptococcusspp. have resulted in separate teleomorphic (sexual) andanamorphic (asexual) taxonomic systems that are not wellintegrated. Of the 19 C. neoformans strains that we sur-veyed, only several have been induced to produce a Filoba-sidiella teleomorph in culture. The uniformity of genotypicclass I based on PCR fingerprint analysis strongly suggestsan association for all of these strains with the genus Filoba-sidiella. In a similar fashion, a direct relationship betweenmost of the other Cryptococcus strains and the basidio-mycete genus Filobasidium is suggested by their PCR fin-gerprints. A high level of genetic relatedness is indicated inFig. 7 for different strains in the C. albidus group, which

includes strains known to have a Filobasidium teleomorph.Strains associated with this Filobasidium complex includeC. ater and C. uniguttulatus as well as two clinical isolatesidentified as C. laurentii. Of these three taxa, only C.uniguttulatus has previously been shown to have a teleo-morph in the genus Filobasidium (10).The analysis of genetic distances indicated by PCR finger-

printing grouped the 11 genotypic classes into three majorgroups (Fig. 7). The most distant grouping includes all of theC. neoformans strains belonging to genotypic class I. Thisarrangement supports previous conclusions that C. neofor-mans and its Filobasidiella teleomorph are phylogeneticallyunique from other taxa in the family Filobasidiaceae (7, 9,10).Most of the remaining Cryptococcus strains belong to a

genetically variable group characterized by a Filobasidiumteleomorph, which includes the different varieties of C.albidus as well as C. ater and C. uniguttulatus. The geneticrelationship of C. laurentii with the other Cryptococcus spp.is less certain. Two strains identified as C. laurentii (geno-typic classes XI and XIII) clearly belong in the Filobasidiumgroup identified above. Two remaining strains of C. laurentii(classes VIII and XII, including the type strain) occupy anintermediate position in Fig. 7 between the C. neoformansgroup representing the genus Filobasidiella and the Filoba-sidium group, which includes C. albidus. These observationsare intriguing, since C. laurentii has been taxonomicallydistinguished from the C. albidus group by its inability toassimilate nitrate and the fact that it is not associated with aknown teleomorph. Although nitrate assimilation has beenwidely used in yeast systematics, its value as a phylogenet-ically informative character has recently come under ques-tion (5). This apparent genetic relatedness of certain C.laurentii strains with C. albidus warrants further scrutiny ofnitrate assimilation as a taxonomic character in this group.

Further study of sequence variation within the rDNAs ofthese strains should also be useful for understanding theevolutionary history of these fungi. The genetic relationshipsindicated in Fig. 7 therefore also represent a phylogenetichypothesis. Additional study using outgroup taxa as well asa larger set of strains should aid in resolving the proposedphylogeny further. Sequence data obtained by using the PCR(2, 19, 22) ought to be especially valuable in this regard.

ACKNOWLEDGMENTS

We thank John Perfect, Wiley Schell, and S. C. Jong (AmericanType Culture Collection) for providing strains used in this study andalso Steve Rehner, Margaret Carroll, and Thomas Mitchell forhelpful discussions and comments. The assistance of StephenJohnston in synthesizing oligonucleotides is also appreciated.

This work was supported in part by funding from NationalScience Foundation grant BSR-88-06655.

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