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Journal of Bacteriology, December 1998, p. 6697-6703, Vol. 180, No. 24
Department of Biological Sciences, Center for
Molecular & Cellular Biosciences, The University of Southern
Mississippi, Hattiesburg, Mississippi 39406
Received 12 June 1998/Accepted 16 October 1998
The genome size, complexity, and ploidy of the dimorphic pathogenic
fungus Histoplasma capsulatum was determined by using DNA
renaturation kinetics, genomic reconstruction, and flow cytometry. Nuclear DNA was isolated from two strains, G186AS and Downs, and analyzed by renaturation kinetics and genomic reconstruction with three
putative single-copy genes (calmodulin, The dimorphic fungus
Histoplasma capsulatum is the etiologic agent of
histoplasmosis, a respiratory disease affecting an estimated 500,000 people per year in the United States (10). In the
environment or in vitro at 25°C the organism grows in a multicellular
mold form. In the infected host or in the laboratory at 37°C the
fungus grows as a unicellular budding yeast. Because this mold-yeast conversion is reversible and easily accomplished in the laboratory by
temperature shifts, H. capsulatum serves as an interesting model for cell differentiation in a lower eucaryote. More importantly, the mold-to-yeast conversion is required for pathogenesis
(20).
Very little information regarding the genome of H. capsulatum is available. The nuclear guanine plus cytosine
(G+C) content has been reported to be 45.4 to 49.8%, with an
observed mean of 47.3% (2). Originally, isolates of
H. capsulatum were separated into three classes based on
restriction fragment length polymorphism (RFLP) analysis of the
mitochondrial DNA (27). Subsequent studies with ribosomal
DNA polymorphisms (24), yps-3 RFLP analysis
(15), and arbitrary primer PCR (16)
demonstrated that strains of H. capsulatum are actually
quite diverse. Analysis of chromosome number by field-inversion
gel electrophoresis and contour-clamped homogeneous electric field
electrophoresis also showed major differences between strains. Steele
et al. (25) showed that the Downs strain has at least seven
chromosomes, G186B has at least four, and G217B has at least three.
While these studies have yielded important information regarding the
H. capsulatum genome, the fundamental features in terms of
genome size, complexity, and ploidy have not been reported.
Strains.
The H. capsulatum strain G186AS (a
low-virulence derivative from G186B) was a kind gift from William
Goldman (Washington University Medical School, St. Louis, Mo.). The
H. capsulatum strains Downs (ATCC 38904) and G186B (ATCC
26030) and Escherichia coli K-12 (ATCC 23588) were purchased
from the American Type Culture Collection (Manassas, Va.). Haploid and
diploid strains of Saccharomyces cerevisiae were a kind gift
from Joanne Tornow and George Santangelo (University of Southern
Mississippi). Tetraploid strains of S. cerevisiae were
obtained from the Yeast Genetic Stock Center (Berkeley, Calif.).
Media and growth conditions.
H. capsulatum yeast cells
were grown to mid-log phase in GYE (2% glucose, 1% yeast extract;
Difco Laboratories, Detroit, Mich.) at 37°C. S. cerevisiae
cells were grown to mid-log phase in GYE broth at 30°C. E. coli was grown to mid-log phase in nutrient broth (Difco) at
37°C.
Single-copy genes.
Three genes previously reported to be
present as single copy in H. capsulatum, calmodulin
(6), DNA extraction.
E. coli DNA was extracted and purified
by standard molecular techniques (1). DNA was
extracted from H. capsulatum G186AS by a modification of the
technique of Worsham and Goldman (29). Briefly, yeast cells
in mid-log phase were washed in phosphate-buffered saline (PBS; 4.3 mM Na2HPO4, 1.4 mM
KH2PO4, 2.7 mM KCl, 137 mM NaCl [pH 7.4]) and
resuspended in spheroplasting buffer (1 M sorbitol, 100 mM sodium
citrate, 60 mM disodium EDTA [pH 5.9]), and 120,000 U of
DNA radiolabeling.
DNA was radiolabeled with
[ Reassociation kinetics.
Purified DNA from E. coli
and H. capsulatum was diluted to 0.1 mg per ml in sterile TE
(10 mM Tris [pH 7.4], 1 mM EDTA) and sonicated to yield fragments
between 200 and 2,000 bp in length. The DNA was ethanol precipitated
and redissolved in 2× SSC-1 mM EDTA (pH 7.0) to a concentration of
0.25 µg/µl. The DNA was aliquoted in 10-µl amounts (2.5 µg),
overlayed with sterile mineral oil, denatured by boiling, and placed at
a reassociation temperature of 72°C. After reassociation to the
desired EC0t (equivalent
C0t corrected for nonstandard salt
concentration [4]), the aliquots were quick frozen in
a dry ice-ethanol bath. After thawing of the aliquots, single-stranded
DNA (ssDNA) was digested with S1 nuclease (2 U per 100 ng of DNA) at
37°C for 45 min. The reaction was stopped with 20 µl of termination
buffer (1 M Tris [pH 9.0]-0.1 M EDTA). The renatured DNA was
quantitated by diluting a 50-µl aliquot of each reaction mixture to
1-ml volume with detection buffer (10 mM Tris [pH 7.4], 1 mM EDTA,
0.1 M NaCl, 0.1 µg [per ml] bisbenzimide) followed by fluorescence
measurement (28) in a Hitachi F-2000 fluorescence
spectrophotometer (with excitation at 356 nm and detection at 456 nm).
Data were analyzed with a reassociation kinetics least-squares computer
program kindly provided by William Pearson (21).
Minicot analysis.
Minicot curves are reassociation kinetic
curves for an isolated fraction of DNA. A minicot uses the isolated
fraction which has been radiolabeled as a tracer in a
C0t curve with total unlabeled DNA.
This proves especially useful in estimating the curve for a very small
amount of repetitive DNA (4, 26). H. capsulatum DNA enriched for repetitive sequences was prepared by a modification of
the method of Timberlake (26). A 5-µg aliquot of sonicated H. capsulatum DNA (0.25 µg/µl) in 1× SSC and 1 mM EDTA
was overlayed with sterile mineral oil and denatured by boiling. The
DNA was placed at a reassociation temperature of 52°C until reaching
an EC0t of 1, after which the sample
was quick frozen in a dry ice-ethanol bath. The ssDNA was digested with
S1 nuclease as described above. After ethanol precipitation the
renatured DNA was quantitated by bisbenzimide fluorescence as described
above. This rapidly reassociating fraction was labeled with
Genomic reconstruction.
Genomic reconstruction experiments
were done by a modification of the method of Francis et al.
(8). DNA was quantitated by fluorescence spectrophotometry
as described above. Serial twofold dilutions of H. capsulatum nuclear DNA were prepared to yield 400 to 12.5 ng in
200 µl of TE buffer. Herring sperm DNA was added as a carrier in
increasing amounts so that the amount of total DNA (H. capsulatum DNA plus herring sperm DNA) was constant at 800 ng per
200 µl of aliquot. In a similar manner, DNA from single-copy H. capsulatum genes was prepared to yield 15 to 0.47 pg in 200 µl
of TE buffer. Herring sperm DNA was added to yield 800 ng of total DNA
per 200-µl aliquot. The final samples (200 µl) were mixed with 44 µl of a mixture of 2 M NaOH and 50 mM EDTA, boiled for 10 min,
neutralized with an equal volume of 0.5 M ammonium acetate (pH 7.0),
and vacuum filtered on a nitrocellulose membrane with a slot blot
apparatus. The nitrocellulose filters were baked at 80°C for 2 h
and prehybridized in a mixture of 0.5 M sodium phosphate (pH 7.0) and
5% (wt/vol) SDS for 1 h at 65°C. Approximately 20 ng of each
single-copy gene was radiolabeled with 32P and used to
probe the appropriate filter for 18 h at 65°C. Filters were
washed two times for 15 min each time in 0.1× SSC-0.1% SDS at 65°C
and exposed to X-ray film. Exposures were adjusted to be within the
linear range of the film. Autoradiographic images were digitized with a
Kohu high-resolution monochrome camera attached to a Macintosh Quadra
computer, and hybridization intensities were quantitated by image
analysis with the public domain NIH Image program (developed at the
National Institutes of Health and available from the Internet by
anonymous FTP from zippy.nimh.nih.gov or on floppy disk from the
National Technical Information Service, Springfield, Va. [part no.
PB95-500195GEI]).
Flow cytometry.
H. capsulatum yeast cells were
pelleted by centrifugation at 800 × g for 10 min at
8°C, washed with sterile PBS, and fixed with 70% ethanol for 1 to
2 h at 25°C. The cells were sonicated gently to reduce clumping,
filtered through a 20-µm-pore-size nylon mesh, washed with PBS, and
resuspended in PBS to a concentration of 2 × 106 to
3 × 106 cells per ml. Thirty microliters of the cell
suspension was treated with 150 µl of 1-mg/ml RNase A (Sigma Chemical
Co.) at 37°C for 1 h. The cells were pelleted at 1,100 × g for 3 min and resuspended in 400 µl of 5-mg/ml pepsin
(Sigma Chemical Co.). After 15 min at 25°C, the cells were pelleted
(1,100 × g, 3 min) and resuspended in 1 ml of a
modified Krishan hypotonic sodium citrate staining buffer containing
5-µg/ml propidium iodide, 4 mM sodium citrate, and 0.1% (vol/vol)
Triton X-100 (5, 19). After a 1-h incubation at 25°C in
the dark, the cells were pelleted and resuspended in 1 ml of PBS and
analyzed in a Coulter EPICS-Profile II flow cytometer (Hialeah, Fla.)
according to the manufacturer's recommendations.
Southern blots.
Two-microgram aliquots of genomic DNA from
H. capsulatum G186AS and Downs were digested to completion
with several six-base-recognition restriction enzymes. The DNA was
separated on a 0.7% agarose gel and downward blotted with 0.4 M NaOH
onto a Hybond N+ positively charged nylon membrane (Amersham) according
to the manufacturer's directions. The blots were probed as described
for the genomic reconstruction slot blots.
Reassociation kinetics.
Three independent reassociation
experiments, each with 30 EC0t values
spanning the range from 0.01 to 1,000, were performed for G186AS and
Downs DNA. Each experiment was done in tandem with E. coli
DNA as a standard. The combined data from each H. capsulatum strain were analyzed by the nonlinear least-squares computer program of
Pearson et al. (21).
C0t1/2 values for
H. capsulatum and E. coli were determined from
the computer model best-fitted curve. Comparison of these values,
using a genome size of 4.7 × 106 bp for
E. coli (18), was used to estimate the genome
size of H. capsulatum. The best-fitted curve for G186AS
(root mean squares [RMS] of 0.046) was a single-component genome of
2.2 × 107 bp (Fig. 1A,
Table 1), and the best-fitted curve for
Downs (RMS of 0.028) was a two-component genome of 3.5 × 107 bp with 92% single-copy sequence and 8% moderately
repetitive component (Fig. 1B, Table 1). The complexity of the
repetitive component was 1.3 × 105 bp with a
repetition frequency of 22.
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Genome Size, Complexity, and Ploidy of the
Pathogenic Fungus Histoplasma capsulatum
and
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-tubulin, and 
-tubulin). G186AS was found to have a genome of approximately 2.3 × 107 bp with less than 0.5% repetitive sequences. The Downs
strain, however, was found to have a genome approximately 40% larger
with more than 16 times more repetitive DNA. The Downs genome was
determined to be 3.2 × 107 bp with approximately 8%
repetitive DNA. To determine ploidy, the DNA mass per cell measured by
flow cytometry was compared with the 1n genome estimate to
yield a DNA index (DNA per cell/1n genome size). Strain
G186AS was found to have a DNA index of 0.96, and Downs had a DNA index
of 0.94, indicating that both strains are haploid. Genomic
reconstruction and Southern blot data obtained with - and
-tubulin probes indicated that some genetic duplication has occurred
in the Downs strain, which may be aneuploid or partially diploid.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-tubulin, and -tubulin (11), were
selected for genomic reconstruction experiments. The H. capsulatum -tubulin (GenBank M28358) and -tubulin (GenBank
L39132) clones were kindly provided by Grace Spatafora (Middlebury
College, Middlebury, Vt.). The H. capsulatum calmodulin gene
was isolated in our laboratory. Restriction fragments containing most
of the amino acid-encoding sequences used as probes were as follows:
CAM1 (GenBank AF072882), a 1-kb HindIII fragment containing exons 3 to 5; -tub, a 900-bp
BamHI/EcoRI fragment containing exons 5 and 6;
and -tub, a 1.1-kb BamHI fragment containing exons 5 to 7.
-glucuronidase (Sigma Chemical Co., St. Louis, Mo.) per ml was
added. The cells were placed at 37°C with gentle shaking for 4 to
6 h. Cells were pelleted, washed three times with breakage buffer
(0.9 M sorbitol, 10 mM EDTA, 0.1 M Tris-HCl [pH 7.5]), and lysed in
lysis buffer (0.5 M Tris [pH 9.0], 20 mM NaCl, 0.2 M EDTA, 3%
[wt/vol] sodium dodecyl sulfate [SDS]). The lysate was extracted
with an equal volume of phenol saturated with 1 M Tris (pH 8.0) on a
rotary shaker at 225 rpm for 2 h. The supernatant was mixed with
an equal volume of Tris-saturated phenol:chloroform and shaken
overnight at 25°C. The aqueous phase was extracted with chloroform,
and the DNA was ethanol precipitated. The DNA pellet was washed with
70% ethanol and dissolved in 10 mM Tris-HCl (pH 7.5)-20 mM
NaCl-1 mM EDTA. Because the Downs strain was recalcitrant to
the -glucuronidase procedure, cells were broken by physical means.
Yeast cells were grown to mid-log phase, pelleted, washed with PBS, and
resuspended in breakage buffer at 4°C. The cells were mechanically
disrupted in a Bead Beater (Biospec Products, Bartlesville, Okla.) by
shaking with 0.5-mm-diameter acid-washed glass beads for 3 min at
4°C. The homogenate was mixed with 40 ml of lysis buffer and an equal
volume of Tris-saturated phenol and was gently shaken at 25°C for 35 min. The homogenate was centrifuged at 3,000 × g for
20 min at 25°C, and the aqueous-phase supernatant was extracted with
an equal volume of Tris-saturated phenol:chloroform (1:1) followed by a
chloroform extraction. The DNA was ethanol precipitated as described
above. DNA from both H. capsulatum strains was purified by
ultracentrifugation through successive cesium chloride-bisbenzimide
gradients (12). The nuclear DNA band was carefully recovered
from each gradient and repurified until no mitochondrial DNA was
visible by fluorescence. This typically required three to five
sequential CsCl gradients. Bisbenzimide was removed by several
extractions with 20× SSC-saturated isopropanol (1× SSC is 0.15 M NaCl
plus 0.015 M sodium citrate [pH 7.0]). Before analysis by
reassociation kinetics, the DNA was ethanol precipitated twice to
eliminate carryover of cesium chloride. Examination of the DNA by
agarose gel electrophoresis showed high-molecular-weight DNA (greater
than 23 kb) with no detectable rRNA or tRNA contamination, even in
overloaded lanes.
-32P]dATP or -35S-dATP by the random
primer method with a Deca Prime labeling kit (Ambion, Austin, Tex.)
according to the manufacturer's directions.
-35S-dATP, dissolved in 1× SSC and 1 mM EDTA, and used
as a tracer at 0.01% of the total DNA weight in a reassociation
reaction of H. capsulatum nuclear DNA as described above.
Denaturation, reassociation, and S1 nuclease treatment were the same as
for the reassociation kinetic experiments described above. After S1
nuclease treatment, 200 µl was removed from under the oil, mixed with
an ice-cold mixture of 0.2 M sodium pyrophosphate and 2 M HCl, and
incubated on ice for 10 min. The precipitate was collected onto a
Whatman GF/C glass microfiber filter. The filter was washed four times with 3 ml of a mixture of 0.1 M sodium pyrophosphate and 1 M HCl, washed one time with 3 ml of 95% ethanol, and dried overnight. The
amount of radiolabeled double-stranded DNA (dsDNA) retained on the
filter was determined by liquid scintillation counting.
RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (35K):
[in a new window]
FIG. 1.
Renaturation kinetics. Data shown represent the computer
model best-fitted composite of three independent experiments for each
curve for (A) G186AS DNA and (B) Downs DNA. Circles, E. coli
DNA; squares, H. capsulatum DNA. ss, ssDNA.
TABLE 1.
Reassociation kinetics for nuclear DNA from H. capsulatum strains G186AS and Downsa
Minicot analysis. To increase the sensitivity of the analysis for repetitive DNA, renaturation experiments were performed in triplicate with the following modifications. The salt concentration was reduced to 1× SSC to slow the reassociation rate. A small amount (approximately 0.01% of the total DNA) of radiolabeled repetitive-enriched DNA was used as a tracer. Reassociation was quantitated by acid precipitation and scintillation counting, and the data were analyzed by computer modeling as described above.
DNA from the Downs strain (Fig. 2) was best modeled as a two-component genome (RMS of 0.041) with the faster-reassociating component, representing 63.7% of the radiolabeled DNA, reassociating at a rate (k) of 0.18 M
1
sec1. The remaining 36.3% reassociated at a rate of
0.007 M1 sec1. When enriching for the
repetitive fraction, the amount of DNA renatured at an
EC0t of 1, determined by S1 nuclease
digestion of the ssDNA followed by fluorescence quantitation, was
1.9%. Therefore, the minicot repetitive fraction represents 1.21% of the total genome (0.019 × 0.637 × 100). However, at an
EC0t of 1, only 15.2% of the
repetitive component was renatured, as calculated (4) by the
rate equation {100 × [1 
1/1 + k
EC0t)]} = {100 × [1 1/(1 + (0.18 × 1))]} = 15.2. Therefore, the actual
representation of the repetitive component in the total genome was
8.0% [(0.0121/0.152) × 100]. This is in excellent agreement with
the original renaturation analysis estimate of 8.3%. The copy number
as determined from the ratio of the rate constants was 26, with a
complexity of approximately 108,000 (3.5 × 107 × 0.08/26), in good agreement with the original determinations (Table 1).
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1 sec1. The remaining 89.3%
reassociated at a rate of 0.022 M1 sec1 and
represented the single-copy component. The amount of renatured DNA at
an EC0t of 1 in the tracer enrichment
was 1.0%. Computer modeling showed that 10.7% of this dsDNA
represented the repetitive component. Therefore, the minicot repetitive
fraction represented 0.107% of the total genome (0.01 × 0.107 × 100 = 0.107%). However, at an
EC0t of 1, 48.2% of the repetitive
component was dsDNA {100 × [1 1/(1 + k
EC0t)]} = {100 × [1 1/(1 + (0.93 × 1))]} = 48.2. Thus, the actual
representation of the repetitive component in the genome was
approximately 0.22% [(0.00107/0.482) × 100]. The copy number
determined from the ratio of the rate constants was 42 with a
complexity of approximately 1,000 (2.2 × 107 × 0.002/42). This small fraction of repetitive DNA is below the resolution limit of the original renaturation kinetic analysis (Table
1). Our confidence in the accuracy of the minicot method is about
±0.2%; therefore, our best estimate is that G186AS has less than
0.5% repetitive sequences.
Genomic reconstruction.
As an independent method of genome
size estimation, genomic reconstruction experiments with three
single-copy genes were performed. Carefully quantitated amounts of
genomic DNA and cloned DNA from H. capsulatum
single-copy genes were applied to a nitrocellulose membrane.
The membrane was hybridized with the appropriate radiolabeled single-copy gene. Autoradiograms within the linear range of the film were digitized and analyzed by image analysis as shown in Fig.
3. A standard curve was calculated to
correlate band intensity with amount (in picograms) of single-copy gene
(Fig. 3). This standard curve was then used to quantitate the amount of
the gene in several hundred nanograms of genomic DNA. With these data
the amount of genomic DNA required to yield one copy of a single-copy gene (i.e., n or haploid genome size) was calculated. In the
representative example shown in Fig. 3, 100 ng of genomic DNA was found
to contain 4.7 pg of the 1-kb H. capsulatum calmodulin
probe; thus, the calculated haploid genome size was 2.1 × 107 bp (0.0047 ng/100 ng = 1,000 bp/n).
Results for each triplicate experiment are shown in Table 2. The genome
size estimate for G186AS when calmodulin was used as the probe was
2.13 × 107 ± 0.09 × 107 bp, that
when -tubulin was used was 2.73 × 107 ± 0.51 × 107 bp, and that when -tubulin was used was 2.35 × 107 ± 0.62 × 107 bp. Therefore, the
average estimated genome size for G186AS based on nine independent
genomic reconstruction analyses is 2.4 × 107 ± 0.3 × 107 bp.
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-tubulin was used was 1.91 × 107 ± 0.29 × 107 bp, and that when -tubulin was used was
2.04 × 107 ± 0.08 × 107 bp.
Therefore, the overall estimated genome size for Downs, based on
nine independent genomic reconstruction analyses, was 2.3 × 107 ± 0.6 × 107 bp. We were concerned
that both the tubulin probes gave 1n genome estimates
significantly lower than those given by the calmodulin probe,
renaturation kinetics, or flow cytometry (see below). Additional replications with new preparations of DNA confirmed the precision of
these measurements.
Since the published data indicating that - and -tubulin are
single copy in H. capsulatum are based on strain G217B
(11), the most simple explanation for the tubulin genomic
reconstruction data in the Downs strain is that these genes are not
present as single copies in Downs. Comparison of the kinetic haploid
genome estimate with the - and -tubulin genomic reconstruction
data is more consistent with two-copy genes (35 Mb/19 Mb = 1.8 for -tub and 35 Mb/20 Mb = 1.75 for -tub). To test this, we
prepared Southern blots from genomic DNA cut to completion with several six-base-recognition restriction enzymes which either do not cut within
the gene or cut only once within the gene but outside the probe
sequence region. The blots were then probed with the same CAM1,
-tub, and -tub probes. Figure 4
shows side-by-side comparisons of several representative lanes from
G186AS and Downs blots. Complete digestion was confirmed in each blot
by stripping the blot and reprobing with CAM1. Blots from both G186AS
DNA and Downs DNA probed with CAM1 had a single band in each
lane, as expected for a single-copy gene. In contrast,
blots probed with -tub and -tub typically showed single
bands in G186AS DNA and two bands in Downs DNA, consistent with a
single-copy gene in G186AS and a two-copy gene in Downs. Based on
this result we excluded the tubulin genomic reconstruction data from
the calculations below for the Downs strain.
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Flow cytometry. To determine the average mass of DNA per cell, H. capsulatum cells were analyzed by fluorescence flow cytometry. Channel number represents the intensity of fluorescence, which is directly proportional to the amount of DNA contained in each cell. The first peak in each graph represents the cells that are in the G0 and G1 phases of the cell cycle. The second peak, which appears at approximately twice the channel number of the G0-G1 peak, represents the cells that are in the G2 and M phases of the cell cycle. Haploid, diploid, and tetraploid strains of S. cerevisiae were analyzed by flow cytometry and used to construct a standard curve (r2 is >0.99). The theoretical amount of DNA in these S. cerevisiae strains was calculated from the recently completed sequence of the S. cereisiae genome (http://www-genome.stanford.edu). Based on a total sequence of 13,026,500 bp per haploid genome (12,057,500-bp unique sequence plus 969,000-bp repetitive sequence) and calculation of an average of 650 Da per bp of DNA, the haploid mass of DNA is 14.06 fg. The channel numbers of the G0-G1 peaks of 1n, 2n, and 4n strains of S. cerevisiae were plotted against the amount of DNA per cell that each strain contains (Fig. 5A). The channel mean of the G0-G1 peak of each Histoplasma strain (Fig. 5B) was then used to calculate the DNA content per Histoplasma cell. These data were then converted to base pairs using an average value of 650 Da/bp.
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Ploidy. Ploidy can be determined by comparing haploid genome size with DNA content per cell to calculate the DNA index (DNA per cell per haploid genome size). A haploid organism should have a DNA index of approximately 1, a diploid organism should have an index of approximately 2, and so forth. To determine ploidy in H. capsulatum we compared the DNA content (flow cytometry data) with the average 1n genome estimate (reassociation kinetics and genomic reconstruction) to determine the DNA index.
The estimated genome size of G186AS as calculated by reassociation kinetic analysis was 22 Mb (Table 1), and that calculated by genomic reconstruction was 24 Mb (Table 2). The average size obtained by these two methods was 23 Mb. Analysis by flow cytometry estimated the amount of DNA per cell to be 22 Mb. The DNA index is 0.96 (22 Mb/23 Mb), indicating that H. capsulatum G186AS is haploid.
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Genome size. Since both strains of H. capsulatum appear to be haploid we can directly include the flow cytometry 1n value in the overall genome size estimate. Our best estimate for the genome size derived from kinetic analysis (22 Mb), genomic reconstruction (24 Mb), and flow cytometry (22 Mb) in G186AS is 23 ± 1 Mb. We estimate the Downs genome size, based on the average by kinetic analysis (35 Mb), calmodulin genomic reconstruction (30 Mb), and flow cytometry (30 Mb), to be 32 ± 3 Mb.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Our goal in this work was to determine the basic features of the H. capsulatum genome regarding genome size, complexity, and ploidy. Because of the laborious nature of these experiments, most studies of this type have used only a single strain of the test organism. Considering the apparent diversity of H. capsulatum, we selected two strains for this analysis: the G186AS strain, because it is currently the most amenable to molecular manipulation, and the Downs strain, because a large body of dimorphism research has used this strain. These strains have no close association to each other aside from being clinical isolates. Downs was isolated in 1969 in Illinois (9), and the parent strain of G186AS (G186B) was isolated in Panama in 1967 (3).
Data from reassociation kinetic experiments with H. capsulatum G186AS DNA indicated a haploid genome size of 22 Mb (Fig. 1, Table 1) with less than 0.5% repetitive DNA (Fig. 2). This result is similar to that seen with Aspergillus nidulans DNA, which has a haploid genome size (based on kinetic analysis) of 2.6 × 107 bp and only slightly over 2% repetitive sequences (26). This similarity is not surprising since molecular phylogenetic analysis of several H. capsulatum genes isolated in our lab (6, 7, 23) indicate that the gene structure of H. capsulatum is remarkably similar to that of A. nidulans. The repetitive fraction in H. capsulatum most likely represents ribosomal DNA, as is the case for A. nidulans (26). Genomic reconstruction and flow cytometry gave average estimates of 24 Mb and 22 Mb, respectively, both of which closely accord with the renaturation kinetic estimates. The average estimate of haploid genome size by all three methods is 23 Mb. Data comparing haploid genome size and DNA content per cell clearly indicate that the organism is haploid (DNA index of 0.96).
The data from Downs DNA indicated that this strain has the larger genome and more repetitive DNA. Reassociation kinetic experiments indicated a haploid genome size of 35 Mb with 8.3% repetitive DNA (Fig. 1, Table 1). Minicot analysis (Fig. 2) indicated approximately 8% repetitive sequence, in close agreement with the primary reassociation data. The size derived from the calmodulin probe genomic reconstruction 1n estimate was 30 Mb. Flow cytometry gave an estimate of 30 Mb. The average estimate of haploid genome size by all three methods was 32 Mb. Data comparing haploid genome size and DNA content per cell indicate that Downs is also haploid (DNA index of 0.94).
The overall genome size estimates of G186AS (23 Mb) and Downs (32 Mb) were significantly larger than that of the ascomycetous yeast S. cerevisiae (13 Mb; see Materials and Methods) and similar to those of the filamentous ascomycetes such as A. nidulans (26 Mb [26]), Penicillium paxilli (23 Mb [13]), Podospora anserina (34 Mb [14]), and Neurospora crassa (43 to 45 Mb [22]). This result is consistent with our data indicating that H. capsulatum is more closely related to filamentous ascomycetes than to ascomycetous yeasts, as discussed above.
The difference in genome size in G186AS and Downs is certainly intriguing. Apparently Downs has a genome approximately 40% larger and with over 16 times more repetitive DNA than G186AS. What is this "extra" 9-Mb amount of genomic DNA in Downs? The repetitive DNA (approximately 2 to 3 Mb) is sufficient to account for only about one third of this amount. Steele et al. (25) showed that Downs apparently has more chromosomes (at least seven) than does G186B (at least four). A possible explanation is that chromosomal duplication has occurred in Downs. A single repeat of a few large regions of the chromosome(s) would be detected by neither renaturation kinetics nor genomic reconstruction unless a DNA probe within the duplicated region was fortuitously selected. Alternatively, it is possible that G186AS has lost 6 to 9 Mb of DNA and that the Downs genome more closely represents the "normal" H. capsulatum genome. It would be unlikely, however, that a haploid organism could lose such a large fraction of its genome with no apparent phenotypic effect. We have noted no major differences between the two strains in terms of their growth rates, nutritional requirements, or dimorphic transition during several years of work with these organisms. In addition, other empirical data indicate that Downs is a somewhat unusual strain, as discussed below.
The surprising data obtained with the tubulin probes in the Downs
strain genomic reconstruction experiments also suggest some duplication
of chromosomal DNA. The DNA index calculation indicates that Downs,
like G186AS, is haploid, but the data obtained by genomic
reconstruction and Southern blotting with the - and -tubulin probes indicate that these genes are present in two copies in Downs
while G186AS (Table 2, Fig. 4) and G217B (11) have a single
copy of each tubulin gene.
Since Downs is clearly not diploid it either (i) has extra copies of at
least two chromosomes (since - and -tubulin probes hybridize to
different bands on chromosome gels [25]) and is thus
aneuploid or (ii) has intrachromosomal repeats in at least two
chromosomes and is partially diploid. Since Downs has more chromosomes
(25) and aneuploid strains are found in other filamentous fungi (17, 30), it is certainly tempting to favor the
aneuploidy hypothesis. Either hypothesis is consistent with our data,
however. We are currently conducting studies to test these hypotheses.
Our data support the concept that H. capsulatum strains are quite diverse in their genomic makeup. Studies that have examined more than one strain have shown that a number of fungi have a wide range of chromosome numbers and genome sizes in natural isolates (reviewed in reference 30). Fusarium oxysporum isolates, for example, have a genome size of 41 to 51 Mb with 11 to 14 chromosomes. We do not know if either of the two strains used in this study is more representative of H. capsulatum or if there is a single isolate that is most typical of the species. Some of our preliminary flow cytometry data (not shown) indicate that H. capsulatum G186B and G217B have the same DNA mass per cell as does G186AS. There are some data to indicate that Downs may be a somewhat unusual strain, however. Vincent et al. (27) grouped 23 H. capsulatum isolates into three classes based on mitochondrial RFLP analysis. Class 3 had 6 representatives, class 2 had 16 representatives, and Downs was the only member of class 1. More recently, Keath et al. examined 76 clinical and soil isolates and found most to belong to class 2 (15). Only four isolates, all from AIDS patients, were grouped in class 1 with the Downs strain, which is more heat sensitive and less virulent than most H. capsulatum isolates.
The work presented here provides data from two common laboratory stains of H. capsulatum. It would be interesting to characterize the genomes of several recent clinical and soil isolates to determine whether long-term laboratory culture has altered their chromosomal structure or if H. capsulatum is as variable in nature as lab strains apparently are. Nothing is known about chromosome number or genome size of H. capsulatum in wild isolates, and arguments can be made both for and against the survival value of a highly variable genome. As more strains are characterized, we will be able to determine the true genetic variability of this important human pathogen.
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ACKNOWLEDGMENTS |
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We thank Elizabeth Keath for providing additional Downs strain DNA and George Santangelo for critical reading of the manuscript.
This work was supported in part by grants from the National Institutes of Health (AI31192) and the Mississippi Lung Association (RG-032-L).
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FOOTNOTES |
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* Corresponding author. Mailing address: The University of Southern Mississippi, Department of Biological Sciences, Center for Molecular & Cellular Biosciences, Box 5018, Hattiesburg, MS 39406. Phone: (601) 266-4722. Fax: (601) 266-5797. E-mail: glen.shearer{at}usm.edu.
Present address: William Beaumont Hospital, Dept. of Clinical
Pathology, Royal Oak, MI 48073.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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| 1. | Ausubel, F. M., R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl. 1993. Current protocols in molecular biology. John Wiley & Sons, New York, N.Y. |
| 2. |
Bawdon, R. E.,
R. G. Garrison, and L. R. Fina.
1972.
Deoxyribonucleic acid base composition of the yeastlike and mycelial phases of Histoplasma capsulatum and Blastomyces dermatitidis.
J. Bacteriol.
111:593-596 |
| 3. | Berliner, M. D. 1968. Primary subcultures of Histoplasma capsulatum: I. macro- and micromorphology of the mycelial phase. Sabouraudia 6:111-118[Medline]. |
| 4. | Britten, R. J., D. E. Graham, and B. R. Neufeld. 1974. Analysis of repeating DNA sequences by reassociation. Methods Enzymol. 29:363-418[Medline]. |
| 5. | Dressler, L. G., R. L. C. Seame, M. A. Owens, G. M. Clark, and W. L. McGuire. 1988. DNA flow cytometry and prognostic factors in 1331 frozen breast cancer specimens. Cancer 61:420-427[Medline]. |
| 6. | El-Rady, J., and G. Shearer. 1996. Isolation and characterization of a calmodulin-encoding cDNA from the pathogenic fungus Histoplasma capsulatum. J. Med. Vet. Mycol. 34:163-169[Medline]. |
| 7. | El-Rady, J., and G. Shearer. 1997. Cloning and analysis of an actin-encoding cDNA from the dimorphic pathogenic fungus Histoplasma capsulatum. J. Med. Vet. Mycol. 35:159-166[Medline]. |
| 8. | Francis, D. M., S. H. Hulbert, and R. W. Michelmore. 1990. Genome size and complexity of the obligate fungal pathogen, Bremia lactucae. Exp. Mycol. 14:299-309. |
| 9. |
Gass, M., and G. S. Kobayashi.
1969.
Histoplasmosis. An illustrative case with unusual vaginal and joint involvement.
Arch. Dermatol.
100:724-727 |
| 10. | Hammerman, K. J., K. E. Powell, and F. E. Tosh. 1974. The incidence of hospitalized cases of systemic mycotic infections. Sabouraudia 12:33-45[Medline]. |
| 11. | Harris, G. S., E. J. Keath, and J. Medoff. 1989. Characterization of the alpha and beta tubulin genes in the dimorphic fungus Histoplasma capsulatum. J. Gen. Microbiol. 135:1817-1832[Medline]. |
| 12. | Hudspeth, M. E. S., D. S. Shumard, K. M. Tatti, and L. I. Grossman. 1980. Rapid purification of yeast mitochondrial DNA in high yield. Biochim. Biophys. Acta 610:221-228[Medline]. |
| 13. | Itoh, Y., R. Johnson, and B. Scott. 1994. Integrative transformation of the mycotoxin-producing fungus Penicillium paxilli. Curr. Genet. 25:508-513[Medline]. |
| 14. | Javerzat, J. P., C. Jacquier, and C. Barreau. 1993. Assignment of linkage groups to the electrophoretically-separated chromosomes of the fungus Podospora anserina. Curr. Genet. 24:219-222[Medline]. |
| 15. |
Keath, E. J.,
G. S. Kobayashi, and G. Medoff.
1992.
Typing of Histoplasma capsulatum by restriction fragment length polymorphisms in a nuclear gene.
J. Clin. Microbiol.
30:2104-2107 |
| 16. |
Kersulyte, D.,
J. P. Woods,
E. J. Keath,
W. E. Goldman, and D. E. Berg.
1992.
Diversity among clinical isolates of Histoplasma capsulatum detected by polymerase chain reaction with arbitrary primers.
J. Bacteriol.
174:7075-7079 |
| 17. | Kistler, H. C., U. Benny, W. A. Boehm, and T. Katan. 1995. Genetic duplication in Fusarium oxysporum. Curr. Genet. 28:173-176[Medline]. |
| 18. | Kohara, Y., K. Ariyama, and K. Isono. 1987. The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell 50:495-508[Medline]. |
| 19. |
Krishan, A.
1975.
Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining.
J. Cell Biol.
66:188-193 |
| 20. |
Medoff, G.,
M. Sacco,
B. Maresca,
D. Schlessinger,
A. Painter,
G. S. Kobayashi, and L. Carratu.
1986.
Irreversible block of the mycelial to yeast phase transition of Histoplasma capsulatum.
Science
231:476-479 |
| 21. |
Pearson, W. R.,
E. H. Davidson, and R. J. Britten.
1977.
A program for least squares analysis of reassociation and hybridization data.
Nucleic Acids Res.
4:1727-1735 |
| 22. | Radford, A., and J. H. Parish. 1997. The genome and genes of Neurospora crassa. Fungal Genet. Biol. 21:258-266[Medline]. |
| 23. |
Shearer, G.
1995.
Cloning and analysis of cDNA encoding an elongation factor 1 from the dimorphic fungus Histoplasma capsulatum.
Gene
161:119-123[Medline].
|
| 24. |
Spitzer, E. D.,
B. A. Lasker,
S. Travis,
G. S. Kobayashi, and G. Medoff.
1989.
Use of mitochondrial and ribosomal DNA polymorphisms to classify clinical and soil isolates of Histoplasma capsulatum.
Infect. Immun.
57:1409-1412 |
| 25. |
Steele, P. E.,
G. F. Carle,
G. S. Kobayashi, and G. Medoff.
1989.
Electrophoretic analysis of Histoplasma capsulatum chromosomal DNA.
Mol. Cell. Biol.
9:983-987 |
| 26. |
Timberlake, W. E.
1978.
Low repetitive DNA content in Aspergillus nidulans.
Science
202:973-975 |
| 27. |
Vincent, R. D.,
R. Goewert,
W. E. Goldman,
G. S. Kobayashi,
A. M. Lambowitz, and G. Medoff.
1986.
Classification of Histoplasma capsulatum isolates by restriction fragment polymorphisms.
J. Bacteriol.
165:813-818 |
| 28. | Vytasek, R. 1982. A sensitive fluorometric assay for the determination of DNA. Anal. Biochem. 120:243-248[Medline]. |
| 29. | Worsham, P. L., and W. E. Goldman. 1990. Development of a genetic transformation system for Histoplasma capsulatum: complementation of uracil auxotrophy. Mol. Gen. Genet. 221:358-362[Medline]. |
| 30. |
Zolan, M. E.
1995.
Chromosome-length polymorphism in fungi.
Microbiol. Rev.
59:686-698 |
Journal of Bacteriology, December 1998, p. 6697-6703, Vol. 180, No. 24
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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