INTERNATIONAL
JOURNAL
OF SYSTEMATIC
BACTERIOLOGY,
Jan. 1981, p. 97-103
0020-7713/81/010097-07$02.00/0
Vol. 31, No. 1
Genetic Relatedness of Filo basidiella neoformans
(Cryptococcus neoformans) and Filo basidiella bacillispora
(Cryptococcus bacillisporus) as Determined by
Deoxyribonucleic Acid Base Composition and Sequence
Homology Studies
HARI S. AULAKH, STEPHEN E. STRAUS,* AND K. J. KWON-CHUNG
Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, National
Institutes of Health, Bethesda, Maryland 20205
Deoxyribonucleic acid (DNA) was extracted and purified from three isolates of
Filobasidiella neoformans,representing serotypes A and D, and from two isolates
of Filobasidiella bacillispora, representing serotypes B and C. Portions of each
DNA pool were labeled in vitro by nick translation. Thermal elution profiles were
determined and were used to calculate the thermal elution midpoint temperature
and moles percent guanine-plus-cytosine content for each DNA. The thermal
elution midpoint temperatures of the five DNAs ranged from 91.3 to 92.9"C, and
the corresponding estimated contents ranged from 53.4 to 57.2 mol%. Hybridizations were performed with a.ll possible pairs of homologous and heterologous
DNAs. The DNAs of serotypes A and D of F. neoformans demonstrated relatedness values of 87.7 to 93.5%. DNAs of serotypes B and C of F. bacillispora
showed 88.5% relatedness. Hybridizations of DNAs of F. neoformans with those
of F. bacillispora, however, yielded relatedness values of only 55.2 to 63%,
indicating that these DNAs are significantly different. Moreover, thermal elution
studies revealed substantial base mismatching in heteroduplexes formed between
DNAs of F. neoformans and F. bacillispora. These data support previous
conclusions suggesting that F. neoformans and F. bacillispora are closely related
but different species.
Recently, Cryptococcus neoformans was
found to contain two perfect states, Filobasidiella neoformans and Filobasidiella bacillispora (15, 16). The types of perfect state were
shown to be closely associated with their serotypes: namely, isolates of serotypes A and D
produced F. neoformans (15), whereas those of
serotypes B and C produced F. bacillispora (16).
The haploid states of the two species, C. neoformans (serotypes A and D) and Cryptococcus
bacillisporus (serotypes B and C), have been
carefully compared for their physiological, biochemical, ecological, epidemiological, and genetic differences. The isolates of C. bacillisporus
were found to utilize I-malic acid, unlike those
of C. neoformans ( 4 ) .The pathway of creatinine
metabolism was the same in the two species, but
the enzyme creatinine deiminase, responsible for
the decomposition of creatinine to ammonia and
methylhydantoin, was under ammonia repression in C. neoformans but not in C.bacillisporus
(30). This suggested that only C. neoformans
has evolved a specific regulatory mechanism of
creatinine metabolism to suit the milieu of pigeon droppings, which are the best known nat-
ural sources for the species (30). The natural
reservoir of C. bacillisporus is not yet known.
Epidemiological studies (3,33) indicated that C.
bacillisporus causes about 50% of the cases of
cryptococcosis in Southern California but rarely
does so in other parts of the United States,
whereas C. neoformans-induced infections are
nationwide in distribution. Another indication of
the differences between the two species was
obtained in genetic studies. An intercross produced mostly sterile basidiospores, and in one
instance, 30%of the spores germinated but signs
of meiosis were not clearly demonstrated (17).
To characterize further the relationship between the two species, the deoxyribonucleic
acids (DNAs) of representative isolates of all
four serotypes have been purified and examined.
We report here the results of base composition
determinations and sequence homology studies,
which reflect upon the genetic relatedness of
these two species.
MATERIALS A M ) METHODS
Yeast strains. Three isolates of F. neoformans and
two isolates of F. bacillispora were used in this study.
97
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INT.J. SYST.BACTERIOL.
AULAKH, STRAUS, AND KWON-CHUNG
Their origins, serotypes, mating types, asexual states,
and some important physiological characteristics are
listed in Table 1. The isolates were grown in yeast
nitrogen base broth (Difco Laboratories, Detroit,
Mich.) (9) with 2% glucose for 48 h on a rotary shaker
at 25OC. Cells were harvested by centrifugation and
washed three times with saline. About 15 g (wet pack)
of each isolate was prepared for DNA extraction.
Preparation of DNA. Freshly prepared pellets of
yeast were suspended in a lysing solution containing
8 M urea, 1%sodium dodecyl sulfate, 1 M sodium
perchlorate (NaClOd),0.24 M sodium phosphate buffer
(PB), pH 7.0 (an equimolar solution of mono- and
dibasic sodium phosphate, NaH2P04/Na2HP04), and
1 mM disodium ethylenediaminetetraacetate, pH 7.5.
The cells were thoroughly homogenized in a Sorvall
Omnimixer with five 1-min pulses alternating with
periods of cooling. The homogenate was shaken for 15
min after addition of an equal volume of chloroformoctonol (24:1, vol/vol). This was then centrifuged at
10,OOO x g for 20 min. The top phase containing DNA
was collected and added to hydroxyapatite (HAP; 2
g/g of initial wet weight of cells) (5,8). The HAP was
centrifuged at 1,OOO rpm for 5 min, and the pellet was
washed five times with 8 M urea in 0.24 M PB to
remove proteins and ribonucleic acid. The HAP pellet
was washed further with 0.03 M PB, pH 7.0, to remove
urea. Finally, the DNA was eluted from the HAP with
0.48 M PB, pH 7.0, and centrifuged at 100,OOO x g for
20 h. The resulting DNA pellet was dissolved in 0.1 M
tris(hydroxymethy1)ainomethane hydrochloride,pH
7.5.
DNA isolated by the method described above is
relatively free of proteins and ribonucleic acid (8). As
a further precaution, however, it was incubated with
100 pg of proteinase K per ml at 65°C for 1 h. The
reaction was stopped by the addition of 1%sodium
dodecyl sulfate and 0.4 M NaC1. A portion of this
"native" DNA was reserved for in vitro labeling by
nick translation. Labeled or unlabeled DNA was
sheared with a French press (American Instrument
Co., Silver Spring, Md.), applying a pressure of 50,OOO
lb/in2. The sheared DNA was cleaned of contaminating press oil by repeated extractions with chloroform.
Finally, the DNA solution was dialyzed against 50 mM
NaC1-1 mM ethylenediaminetetraacetate, pH 7.5, and
concentrated by freeze-drying.
The optical density (OD) of each DNA preparation
was determined, before shearing, at 230, 260, and 280
nm. The DNA concentration was calculated by using
the formula 1.0 OD2W = 50 pg/ml. An absorbance ratio
(OD~W/OD~W)
of 2.0 f 0.1 was considered to indicate
that the DNA preparation was relatively free of ribonucleic acid and protein (6-8). Contaminating carbohydrates are effectively removed from DNA adsorbed
to HAP during high-salt washes.
In vitro labeling of DNA probes. Purified native
DNA was labeled in vitro with all four 3H-labeled
nucleoside triphosphates by nick translation techniques described by Maniatis et al. (21) and Kelly et
al. (13). Reaction mixtures (200 pl) contained 2 pg of
native DNA, 0.027 mM each 3H-labeled nucleoside
triphosphate, 50 mM tris(hydroxymethy1)aminomethane hydrochloride (pH 7.8), 5 mM MgCL, 10 mM
P-mercaptoethanol, 50 pg of bovine serum albumin per
ml, 6 U of Escherichia coli DNA polymerase I (Boehringer Mannheim, GmbH, Mannheim, West Germany), and 2 pl of a 10-7-g/ml deoxyribonuclease I
solution (Millipore Corp., Freehold, N.J.). Incorporation was monitored by determining trichloroacetic
acid-precipitable counts in samples taken from the
reaction mixtures every 10 min. When maximum incorporation was achieved, the reaction was terminated
by the addition of 0.4 M NaCl and 0.4% sodium dodecyl
sulfate. The 3H-labeled DNA was extracted with
phenol-cresol solution and purified by two cycles of
HAP chromatography. By these methods, the DNA
preparations were labeled to specific activities of approximately 2.0 x lo7cpm/pg.
Thermal elution profiles. For determination of
DNA was adsorbed
thermal elution midpoints [Tmce,],
to columns of HAP and washed (five times) with 0.12
M PB and 0.2% sodium dodecyl sulfate. The temperature was raised by sequential 4OC increments from
60 to 10OoC, eluting the column each time with the
same buffer. The radioactivity eluted at each temper
ature was measured in a scintillation counter (Beckman LS 250). The T,(,,was calculated as the temperature at which 50% of total counts were eluted in
single-stranded DNA molecules (24).
G+C Content. The guanine-plus-cytosine (G+C)
contents were calculated by using the formula of Marmur and Doty (22) corrected for 0.187 M sodium ion
concentration, namely, G+C = 2.44 ( T m - 81.5 - 16.6
log M), where M is the concentration of the cation
(ZO), or moles percent G+C = 2.44 (T, - 69.4) (18,
TABLE1. Clinical or natural isolates of F. neoformans and F. bacillispora studied
Isolate
Seratype
Mating
type
D
D
a
a
a
C. neoformans
C. neoformans
C. neoformans
a
C. bacillisporus
C. bacillisporus
F. neoformans
NIH 12 (type)"
NIH 430 (type)
NIH 371
F. bacillispora
NIH 191 (type)
NIH 444 (type)
A
B
C
a
Asexual state
LMalate"
-
+
+
CGB
Origin
agarb
-
-
Human bone lesions
Danish pigeon droppings
Thailand cuckoo droppings
+
+
Human spinal fluid
Human sputum
Ability to assimilate 1-malate (4).
Formation of blue color on creatinine-glucose-bromothymolblue (CGB) agar (18).
Type strains of the species.
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VOL. 31,1981
GENETIC RELATEDNESS OF FILOBASIDIZLLA SPECIES
20). The T,(,,’s were used in these calculations as close
estimates of the melting teniperature (T,,J,which is
usually determined spectrophotometricdy.
Nucleic acid hybridization. Sheared “-labeled
probe DNA (10,OOO cpm, approximately 5 x
pg)
was hybridized in the presence of a vast excess of
unlabeled sheared DNA (250 ,ug/ml) in a final volume
of 100 p l containing 0.48 M PB, pH 7.0-0.4% sodium
dodecyl sulfate-1 mM ethylenediaminetetraacetate.
This mixture was placed in a sealed capsule, heated to
105 to llO°C for 5 min, and incubated at 65OC for
reassociation. Portions were taken at various times to
determine the extent of hybridization. In preliminary
experiments, no increase in the percentage of hybridization was observed beyond an ECMof about 200. To
insure maximal hybridization, the final samples were
taken when the calculated E m values were greater
than 300. The E c is
~ the product of time (seconds)
and DNA concentration (molesof nucleotide per liter),
corrected for sodium concentration when greater than
that in 0.12 M PB (6). In practice, this value was
estimated by determining the product of DNA concentration in OD260 units and multiplied by time in hours
divided by two. This value is then multiplied by 5.6495
to correct for the sodium concentration of 0.48 M PB
(6). The porportion of counts in single-stranded or
double-stranded DNA molecules was determined by
HAP chromatography as described previously (2,8).
DNA relatedness. The similarity between the
DNAs of two different isolates was measured by the
relatedness statistic, R, which is a function of the
product of the percent hybridization for the pair of
99
heterologous reactions divided by the corresponding
product for the homologous reactions. Specifically,
R=100x
l/-
where, for example, A*B denotes the percent hybridization of labeled strain A DNA probe in the presence
of excess strain B DNA. Ninety-five percent confidence intervals around the relatedness values were
calculated as antilogs of the corresponding intervals in
the log scale (1).
RESULTS
DNA extraction. The recovery of DNA from
the various isolates was variable. Approximately
2 mg of highly purified DNA could be obtained
from each 20-g wet pellet of yeast, except for
isolate 371, which repeatedly yielded only about
one-fourth that amount. The reasons for this
difference are not known. Only about 40% more
DNA could be obtained if the interphase of the
homogenate of isolate 371 was resuspended in
lysing solution, rehomogenized, and extracted
again.
The quality of the DNA from all isolates
appeared to be excellent. After ultracentrifugation, the DNA pellets were crystal clear and
colorless. The absorbance ratios (OD2m/OD2m)
-
B.
/ /
1
FIG.1. Thermal elutionprofiles for the DNAs of Filobasidiella. (A) Elution data for labeled native DNAs.
(B) Elution data for NIH-12 DNA before (native) and after shearing.
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100
INT. J. SYST. BACTERIOL.
AULAKH, STRAUS, AND KWON-CHUNG
all fell between 1.9 and 2.0 and averaged about
1.95.
Thermal elution profiles. The thermal elution curves for all of the in vitro labeled native
DNAs are depicted in Fig. 1A. The T,(e)'S (Table
2) for four of the isolates ranged between 92.2
and 92.9OC. The T,(e) observed for strain 430
DNA differed slightly, averaging 91.3"C. The
T,(,)of a known standard DNA purified from
3H-labeled adenovirus type 2 averaged 91.8"C,
which agrees with the published value after correction for salt concentration (27,29). Thus, by
comparison with a standard DNA, the T,(,,'s
calculated for the yeast strains probably represent close estimates of their T,'s.
In an effort to explain the disparity between
our T,(,t data and the T , data previously reported (10,28)for DNA recovered from isolates
of C. neoformans (87.3 to 89.7"C),we performed
an additional series of thermal elution experiments. It was apparent that the method of DNA
recovery utilized in at least one of the previous
studies was conducive to extensive shearing of
TABLE2. T,,,(eiand G+C content of native DNA
prepared from strains of Filobasidiella
F. neoformans
12
430
371
F. bacillispora
191
444
92.2 +, 0.8'
91.3 k 0.6
92.7 k 0.1
55.6 +, 2.1'
53.4 f 0.3
56.7 f 0.2
92.5 f 0.4
92.9 +, 0.1
56.3 k 1.0
57.2 f 0.2
a Tm(,,,
Temperature at which 50% of adsorbed, labeled, native, double-strandedDNA is denatured and
elutes as single strands from HAP.
bContent of G+C in labeled, native, doublestranded DNA calculated by using the corrected formula of Marmur and Doty (20, 22).
' Mean f standard deviation for two to four individual determinations.
the DNA molecules in that breakage of yeast
was performed with a pressure of 20,000 lb/in2
(10). For this reason, portions of each pool of
labeled, native DNA were sheared with a French
press and reanalyzed. The thermal elution profdes shifted markedly leftwards as exemplified
by the curve in Fig. 1B. The T,(,,'s of the five
sheared Filobasidiella DNAs (Table 3 ) ranged
between 86.6 and 88.2"C.
G+C content. The G+C contents were calculated by using the T,ce, values of native (unsheared) DNA samples. The calculated G+C
content of the control DNA of adenovirus type
2 agreed with the published value of 55% (27).
Base compositions calculated for the five native
DNAs from the Filobasidiella isolates are
shown in Table 2. The G + C contents ranged
from 53.4 to 57.2mol%. If the G+C calculations
were performed utilizing the T,(e,'S of sheared
DNA, inappropriately low values of 42.2 to 45.9
mol% would be obtained, which are closer to
those previously reported by some investigators
for the DNA of C. neoformans (10).
DNA-DNA hybridization. Figure 2 depicts
the mean results of two to four individual sets of
hybridization experiments performed with all
possible pairs of homologous and heterologous
DNAs. The rapidly reannealing component of
each reaction was determined by assessing the
number of counts in duplex DNA after 15 min
(about 3 to 4%) and was subtracted from the
final percent hybridization observed in each experiment. In the presence of a vast excess (250
pg/ml) of unrelated (calf thymus) DNA, hybridization of the probe ranged between 1 and 2%.
In the presence of a similar excess of fungal
DNA, hybridization of the labeled probe was
augmented considerably, reflecting the increased concentration of related sequences. In
control experiments self-hybridization of 3H-labeled adenovirus type 2 DNA approached 96%.
The average results of hybridization in homolo-
TABLE3. T m ( e ) ' ~of sheared DNA and DNA hybrids prepared from strains of Filobasidiella
Probe
T m f d ("C) of
homodup1ex
ATm(e)
("C) of heteroduplex"
12b
430
371
191
444
+0.3
+0.3
-1.2
-11.0
-11.2
-10.8
-8.9
-9.8
-8.8
F. neoformans
12
430
371
F. bacillispora
191
444
86.6
87.9
87.0
-1.2
-1.0
-0.2
86.9
88.2
-9.4
-10.9
-9.4
-10.9
-9.6
-10.0
-2.8
-6.1
a Difference i
n Tmce,
of heteroduplex formed by annealing of unlabeled DNA (vertical columns) and labeled
DNA probe (horizontal rows) from that of homoduplex formed by self-annealing of sheared, labeled, probe
DNA. For example, the Tmce,of the 12/191 heteroduplexwas 75.6OC, or l l ° C lower than the Tm(e,of the sheared
probe 12 homoduplex.
Strain number.
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VOL. 31,1981
GENETIC RELATEDNESS OF FILOBASIDIELLA SPECIES
DNA HYBRIDIZED
PROBE
12
430
371
191
444
101
marked homology. Similarly, DNAs from strains
191 and 444 of F. bacillispora were very homologous. Only about 50 to 60% annealing was observed, however, in hybridizations of DNAs of
F. neoformans isolates with those of F. bacillispora.
Statistical analysis (1)of these hybridization
results was performed to define more accurately
the DNA relatedness among the five isolates
(Table 4). The DNAs of F. neoformans strains
12, 430, and 371 proved to be closely related to
each other (87.8 to 93.5%), as did those of F.
bacillispora strains 191 and 444 (88.5%). The
DNAs of F. neoformans, however, were sign&
cantly different from the DNAs of F. bacillispora in that only 55.2 to 63.0%relatedness was
observed.
DNA relatedness is also reflected in the thermal elution profiles of DNA heteroduplexes. Sequences that are sufficiently similar to anneal
may still contain regions of base mismatches.
This would be manifested as denaturation and
elution from HAP at lower temperatures. Table
3 displays the Tm(e)’sfor homoduplexes of the
five sheared DNA isolates. Also shown are the
differencesin Tm(e)’s[ATmce)]
for all combinations
of heteroduplexes. It is readily appreciated from
the data that the absolute magnitudes of the
ATm(e)’s
can be distributed into the same two
clusters that were defined by the hybridization
analysis. Heteroduplexes formed by reannealing
of DNAs from isolates within the same species
demonstrated small differences in Tmce) from
that of the respective homoduplexes. Heteroduplexes formed by reannealing of DNAs representing different species had Tm(e)’S 10 to 11°C
below that of their respective homoduplexes,
indicating about a 15% base mismatch within
hybridizing fragments (7).
DISCUSSION
FIG. 2. DNA reassociation results
of all possible
homologous or heterologous reactions. The data are
displayed as mean percentages of hybridization (&
standard deviation) relative to those of the homologous reactions (normalized to 1m)
The
. actual percentages of hybridization in the homologous reactions are shown above their respective bars.
gous reactions with the Filobasidiella DNAs
ranged between 78.6 and 85.5%.The results for
each of the sets of heterologous reactions were
compared with those of the individual homologous hybridizations after normalization to 100%
(Fig. 2). The relative percentages of hybridization ranged between 46 and 98.5%.Two distinct
clusters of results are noted. DNA from strains
of F. neoformans,viz., 12,430, and 371, exhibited
These nucleic acid composition and homology
studies were undertaken in an effort to augment
the morphological, genetic, biochemical, and serological data upon which the classification of
two FiZobasidiella species is currently founded.
The previous studies (3, 4, 15-18) summarized
in Table 1 indicated that the genus Filobasidiella includes two closely related but different
species: F. bacillispora and F. neoformans.
The present experimental results are in general agreement with the previously published
data. By base composition analysis, representatives of all four serotypes appear to be related.
The calculated G+C contents range from 53.4 to
57.2 mol%.These values are substantially higher
than those previously reported for C. neoformans (43.0 to 51.5 mol%) (10, 28, 32). Three
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INT. J. SYST.BACTERIOL.
AULAKH, STRAUS, AND KWON-CHUNG
TABLE4. DNA relatedness (R) and 95% confidenceintervals for strains of Filobasidiella
R (95% confidence interval)"
~~
F, neoformans
Strain no.
12
430
371
191
~
F. bacillispora
12
430
371
191
444
100b
93.0
(89.6-96.5)
100.0
93.5
(90.1-97.1)
87.7
(84.5-91.0)
100.0
56.7
(54.6-58.9)
55.2
(53.1-57.3)
59.3
(57.2-61.6)
100.0
63.0
(60.7-65.4)
59.4
(57.2-61.7)
58.4
(56.2-60.6)
88.5
(85.3-91.9)
100.0
444
a R was calculated by the method of Alling (1) from hybridization data obtained in reactions with heterologous
DNAs. In this example, R has been calculated by using the results of experiments in which labeled probe 12 was
annealed in the presence of excess unlabeled strain 430 DNA and labeled probe 430 was annealed in the
presence of excess, cold isolate type 12 DNA.
100%related by definition.
factors may explain these discrepancies. First, edness). Similarly, isolates of F. bacillispora are
some investigators may have utilized techniques closely related to each other (88.5%relatedness)
which resulted in preparations of DNA which (Fig. 2, Tables 3 and 4). About 60% of the sewere grossly contaminated with ribonucleic acid, quences of DNAs from members of one group of
protein, and especially cell wall polysaccharides. isolates can stably anneal with DNAs of the
Erke and Schneidau (10) acknowledged that other group. However, within these annealed
contaminating substances which absorb at 260 heterologous sequences there are regions of
nm were probably responsible for the aberrant about 15% base mismatch, as indicated by
melting profile data upon which estimates of ATmc,)'sof 10 to 11°C (Table 3).
their G+C contents were based. The method of
DNA homology studies have been utilized
DNA extraction and purification used in this increasingly to clarify the taxonomy of yeasts as
study yielded DNA with optimal O D ~ W / O Dwell
~ ~ as that of other organisms (2,lO-12,22,25ratios and sharp, reproducible thermal elution 29,31,32). Although it is difficult to select DNA
profiles (Fig. 1A). A second cause for the lower relatedness values which can be used for delinestimates of G+C content in previous studies eation of species, it has been generally accepted
may have been the use of sheared DNA. Erke that values of 25% or less characterize different
and Schneidau (10) utilized pressures of 20,000 species, whereas values of 80 to 100%indicate
lb/in2 to disrupt the cells. Figure 1B and Table that the strains belong to the same species (31).
3 amply demonstrate that the T m ( e ) of sheared
The relatedness values for heterologous reacDNA is much lower than that of native mole- tions between members of the two different specules. The formula of Marmur and Doty (22) cies studied here are all about 60% (Table 4).
results in significant underestimates of the G+C Intermediate levels of relatedness such as these
content if melting studies are performed with have been reported infrequently. Similar data,
extensively sheared DNA (19). Finally, it must however, have been presented by Meyer et al.
be pointed out that the present estimates of (26) from studies of Hanseniaspora and KloeckG+C content are based upon T m ( e , determina- era species. DNA reassociations amang strains
tions, which may differ slightly from T, values of H. osmophilia and K. cortis with H. vineae
derived from spectrophotometric studies (23). and K . africana were found to range between 38
Lower estimates of G+C content might have and 6076, indicating that these yeasts are more
been obtained if native DNA of equivalent pu- closely related to one another than some other
rity was analyzed by the more traditional buoy- species of Hanseniaspora or KZoeckera are to
ant density or spectrophotometric methods. The each other.
control experiments with viral DNA suggest,
The interpretation of intermediate levels of
however, that the present methodology for T m ( e , DNA relatedness has been discussed recently by
determinations can yield reasonable estimates of Kurtzman et al. (14). They argue cogently that
DNA relatedness is an important determinant
G+C content.
Hybridization analysis revealed two groups of of species boundaries but that it must be viewed
DNAs. The tested isolates of F. neoformans in the context of the entire set of studies on the
possess extensive homology (87.7 to 93.5%relat- organisms in question, particularly those perDownloaded from www.microbiologyresearch.org by
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VOL. 31,1981
GENETIC RELATEDNESS OF FILOBASIDIELLA SPECIES
taining to mating. Based upon the levels of relatedness observed here, one may propose that
F. neoformans and F. bacillispora are two varieties of the same species. However, ecological,
epidemiological,genetic, and biochemical differences argue that classification within the same
species is inappropriate. It appears that a varietal status can be mandated only if future studies
detect evidence of meiosis in interspecific
crosses. For the present, it must be concluded
that F. neoformans and F. bacillispora are
closely related but distinct species.
ACKNOWLEDGMENTS
We are indebted to David Alling for performing the statistical analyses utilized in this study and to John E. Bennett for
reviewing this manuscript.
REPRINT REQUESTS
Address reprint requests to: Dr. Stephen E. Straus, Medical
Virology Section, Laboratory of Clinical Investigation, NIAID,
National Institutes of Health, Building 10, Room 11N-113,
Bethesda, MD 20205.
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