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Spheroplast fusion in
Wangiella dermatitidis
C.R. Cooper, Jr. and P.J. Szaniszlo |
Index:
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Materials
Protocol
Results
Tips |
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Principle
and General Applications |
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Many fundamental genetic analyses of fungal
organisms are based upon complementation studies involving the mating of compatible
strains. In such studies, a mutant phenotype resulting from a dysfunctional allele
in one strain can be reversed by the product of the wild-type allele donated by the mating
partner. Typically, the fungi involved in these studies are sexual non-pathogens.
However, like many other fungal pathogens of humans, Wangiella dermatitidis
either lacks or has an undiscovered sexual cycle. Thus, it is not amenable to
classical genetic analysis. This limitation prompted us to develop protocols to
establish an artificial parasexual cycle for genetically analyzing this fungus (Cooper and
Szaniszlo, 1993a). This cycle was established based on spheroplast fusion
methodologies similar to thoseused with Candida albicans (Kakar and Magee, 1982;
Poulter et al., 1981; Sarchek et al., 1981). Although temperature-sensitive and
melanin deficient auxotrophs were used in our studies, different types of mutants can also
be employed. For example, complementation groups among several uracil auxotrophs of W.
dermatitidis were also ascertained using this methodology (Cooper and Szaniszlo,
1993). Conceiveably, our protocol can be exploited to investigate the geneticsof
other types of mutants of asexually-reproducing fungi; it is particularly well suited for
use with melanized species because fusion products can be identified visually by their
dark pigmentation. Segregants can also be identified by loss or pigmentation. |
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Materials |
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Media
CDY+AAMU: This complete medium is prepared by
first adding the following components to 800 ml of distilled water: 30% (w/v) NaNO3,
10 ml; 20% (w/v) K2HPO4, 5 ml; 20% (w/v) MgSO4·7H2O,
5 ml; 10% (w/v) KCl, 5 ml; glucose 30 g; and 10 ml portions from each sterile stock
solution (2.4 mg ml-1) of adenine-HCl, arginine-HCl, methionine, and racil.
The basal medium is then brought to a volume of one liter and adjusted to pH 6.5
prior to the addition of 1.0 Bacto-yeast extract (Difco, Detroit, Michigan) and 1 ml of a
freshly-prepared 1% (w/v) FeSO4·7H2O solution (or 1 ml of 1% [w/v]
FeSO4·7H2O in 10% HCl, then readjusting the pH if needed).
This medium is then autoclaved for 20 min at 121ºC.CDN Agar: This minimal medium is
prepared as CDY+AAMU broth except the yeast extract compoenent and the nutritional
supplments (adenine, arginine, methionine, and uracil) are replaced with 3 mg thiamine and
5.3 g NH4Cl for each liter of medium. If necessary, the final pH is
readjusted to 6.5 and 20 g of Bacto-agar (Difco) is added prior to autoclaving for 20 min
at 121ºC.
Buffers and Other reagents
Buffer A: 0.5 M MgSO4 7H20
in 0.1 M Tris, final pH 7.2
Buffer B: 0.5 M CaCl2 in 0.1 M Tris, final pH 7.2.
Zymolyase-20T (ICN Immunobiologicals, Lisle, IL) solution: 20·ml-1Buffer A
containing 100 mM ß-mercaptoethanol
Overlay agar (OLA): 1% [w/v] molten Bacto-agar (Difco) in Buffer B and held at 45-50ºC
Polyethylene glycol solution (PEG) solution: 25% [w/v]PEG-8000 in Buffer B)
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Protocol |
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In the original experiments using our
protocol, mutant strains of W. dermatitidis (strains Mc2W-1 and Mc3W-15) were employed.
These strains are melanin-deficient (albino), auxotrophs possessing complementary
mutations in melanin biosynthesis, different nutritional requirements (Met- and
Ura-, respectively), and complementary temperature-sensitive lesion (cdc)
leading to altered morphological development at 37ºC. A successful fusion of
spheroplasts from each strain should permit the regeneration of strains that produce a
darkly-pigmented colony at 25ºC and 37ºC on minimal medium.
- Strains are innoculated from fresh stock culture slants into
CDY+AAMU and incubated 3-4 days in a 25ºC rotary water-bath shaker (Model G-76; New
Brunswick Scientific Co., Edison, NJ) operating at 150 rpm. Growth is monitored by
determining the OD600 using a Coleman Junior IIA or a Perkin-Elmer Junior Model
35 spectrophotometer. This culture is used to innoculate 100 ml broth media in a 300
ml Nephelo flask (Bellco Glass, Inc., Vineland, NJ) to an OD600 approximately
equal to 0.02. The latter culture is then incubated overnight (12-18 hr) and used to
innoculate a subculture in the same manner.
- Subcultures of strains to be fused are innculated in
CDY+AAMU, as described above (step 1) and are grown 2-4 generations (approximately 12-18
hr).
- The cells from Step 2 are collected by centrifugation (5 min
at 1,600 x G) and resuspended to an OD600 = 0.40 (approximately 5 x 107
CFU ml-1) in CDY+AAMU.
- Cells from the above suspension are again collected by
centrifugation washed once in Buffer A, then resuspended in an equal volume of the same
buffer.
- From each washed cell suspension, 9.0 ml are placed in
separate flasks and ß-mercaptoethanol is added to a final concentration of 100 mM.
The cells are incubated for 30 min at 37ºC in a rotary water-bath shaker operating at 150
rpm.
- After adding 1.0 ml of freshly-prepared Zymolyase-20T
solution, the cell suspension is incubated for an additional 45 min, but at a slower
shaker speed (60 rpm).
- Each preparation is then gently transferred to a 50-ml
conical centrifuge tube (polypropylene). Spheroplasts are collected by
centrifugation for 3 min at 1,600 x G, washed once in Buffer B, and finally resuspended in
10 ml of the same buffer using an alcohol-sterilized glass rod to gently break the clump
of spheroplasts into smaller pieces.
- One ml of each spheroplast suspension is placed in another
50-ml conical centrifuge tube to which 4.0 ml of the fusion enhancer PEG-800 is added.
The spheroplasts are gently mixed by hand and then allowed to stand at room
temperature for 15 min with intermittant gentle agitation.
- Next, the spheroplast mixture is centrifuged for 3 min at
1600 x G and the resulting pellet gently resuspended in 6.0 ml Buffer B. One ml
aliquots of the latter suspension are mixed with 10 ml of OLA, then layered on CDN agar.
- After the agar solidifies, the plates are incubated at 37ºC
for 1-2 weeks.
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Results |
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Fusion products resulting from the protocol
are identified as darkly-pigmented colonies growing on minimal medium (Fig. 1; Cooper and
Szaniszlo, 1993a). Individual products can be cloned for further study by streaking
cells on CDN agar and subsequently isolating a single black colony. Analysis of such
clones led to the confirmation that at least two different cell -division-cycle (CDC)
genes govern bud-emergence during yeast development in W. dermatitidis.
Interestingly, karyogamy appears to occur soon after plasmogamy with this fungus.
The absence of heterokaryon formation stands in contrast to those results obtained with
appropriately marked strains of C. albicans using similar methodology. |
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Tips |
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Although this protocol provides a powerful tool
for establishing complementation groups, there are several considerations an investigator
should take into account when analyzing the results of such studies (Magee et al.,
1988). First, false positive complementation may occur as a result of suppressors or
gene conversion if the complementing phenotype is part of the selection procedure for
fusion products. Second, false negative complementation can arise by reversion of
the forcing marker or by chromosome transfer in which only the functional forcing marker
is carried into a regnerating fusion product. Hence, it is advisable to base
conclusions from complementation studies on the phenotype of a number of fusion products.
Also, the medium described above was used for historical reasons. Unpublished
observations from our laboratory indicate that other growth media may be substituted for
CDY+AAMU and CDN, e.g., YPD and SD (Sherman et al., 1983). In addition, the
above protocol can be shortened by mixing cells to be fused in a single flask or
centrifuge tube prior to spheroplast formation (see Method B in Cooper and Szaniszlo,
1993). The remainder of the protocol is unchanged. |
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This page updated on:
Monday, March 03, 2003 11:34:43 PM |
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