Strains and Culture Conditions
strains used in this study are listed in . The pseudohyphal colony formation and invasive growth of S
were examined as described previously (Liu et al., 1993
; Roberts and Fink, 1994
). Lee's and YPD + 10% bovine serum media were used for hyphal induction (Lane et al., 2001b
). YPS with 1% agar was used for colony morphology assay under embedded conditions (Brown et al., 1999
C. albicans and S. cerevisiae strains used in this study
FLO8 Cloning and Disruption
The clone pCF56 was isolated from a C
genomic library (Liu et al., 1994
) based on its ability to suppress the invasive defect of a S
. cerevisiae flo8
mutant (HLY850) on SC-Ura medium. pCF56 contains a 3.4-kb insert, with a 2454 base pair open reading frame (ORF), corresponding to a protein (Flo8) of 817 amino acids. A 2.4-kb Bgl
I fragment in pCF56 was replaced with a 4-kb Bgl
fragment from pCUB6, generating plasmid pCF56-FLO8NΔ. A 1.5-kb Spe
I fragment from pCF56 was inserted into the Spe
I site of pCF56-FLO8NΔ to generate pCF56-FLO8Δ for disruption of FLO8
. pCF56-FLO8Δ was digested with Xho
I and Sst
I and transformed into CAI4 to produce FLO8/flo8
strains. Spontaneous Ura–
derivatives were selected on 5-fluoro-orotic acid-containing medium. The disruption was confirmed by Southern blotting (Supplemental Figure 1). The plasmids used in this study are listed in . All the clones and plasmids were confirmed by DNA sequencing.
pBA1 was constructed by subcloning an ADH1
promoter fragment with Not
I and Eco
RV at each end into BES116 (Feng et al., 1999
) at the Not
I and Eco
R V site.
pBA1-CaFLO8 for the expression of C. albicans FLO8 in C. albicans was constructed by placing a 2.45-kb PCR fragment containing FLO8 coding sequence into the BglII-ClaI site of plasmid pBA1. Primer 1 and primer 2 were used for PCR amplification.
pBA1-CaFLO8ΔN was constructed by placing a 2.1-kb PCR fragment into the BglII-ClaI site of plasmid pBA1. Primer 5′CTGGGATCCGTATGCTTCCTCTTATACAGCAG and primer 2 in were used for PCR amplification.
pBES116-CaFLO8, a 3-kb Hind
V fragment from pCF56 was inserted into BES116 (Feng et al., 1999
). Then, a 2.4-kb Kpn
III PCR fragment containing C
. albicans FLO8
promoter was amplified (primer 3 and 4) and inserted before the FLO8
For pVTU-CaFLO8, a 2.45-kb FLO8 containing PCR product (primers 7 and 8) was subcloned into the SstI-XhoI site of pVT102U to express the C. albicans FLO8 under the control of ADH1p in S. cerevisiae. The full-length coding sequences of FLO8 and EFG1 were PCR amplified (primers 9–14) and inserted into pEG202 and pJG4–5, generating pEG202-CaFLO8, pEG202-EFG1, and pJGCaFLO8 for the two-hybrid assay. SC5314 genomic DNA was used as template for PCR amplification. All constructs were verified by DNA sequencing. The plasmids used in this study are listed in . The primers used for PCR amplification are listed in .
For CaFLO8-MYC13, a 580-base pair fragment containing 13xMYC was PCR amplified from the S
vector pFA6a-13MYC-HIS3MX6 (Longtine et al., 1998
) with oligonucleotides 5 and 6 (), and cloned into the BamH
I and Sph
I sites of the C
. albicans ACT1p
vector (Umeyama et al., 2002
) to create pPR671. A Not
1 and an Mlu
1 site were introduced between the BamH
I and myc13 sequence in pPR671. The FLO8
gene was amplified from C
genomic DNA (SC5314) using oligonucleotides 15 and 16 (). The 2.5-kb PCR product was digested with BamH
I and Mlu
I and then inserted into the BamH
I sites of pPR671, to produce pCaFLO8–13MYC-FLAG-HIS1
(pPR672). pPR672 was digested with Stu
I to target the integration of the plasmid into the genomic RP10
locus under HIS1
selection. Expression of Flo8p under the ACT1
promoter in YPD at 30°C was verified by Western analysis using an anti c-myc-conjugated peroxidase antibody (Roche Diagnostics, Indianapolis, IN) enabling detection of Flo8p at ~150 kDa.
pMSCTAP was constructed by cloning a codon-optimized TAP-tag that contains two copies of protein A sequence followed by a single copy of the calmodulin binding peptide (Rigaut et al., 1999
) into a BlueScript vector that carries C
. albicans URA3
(Shrivastava and Liu, unpublished data). The TAP-CaURA3 can be used as a cassette for PCR amplification and insertion of the TAP in frame to the C terminus of a gene of interest.
Primers EFG1-F and EFG1-R are used to PCR amplify TAP-CaURA3 from pMSCTAP (EFG-TAP-F 5′-CCTTCACCCCAACAACATCAAGCTAATCAATCAGCTAGCACTGTTGCCAAAGAAGAAAAGAACATGGAAAAGAGAAGATGG
-3′; URA3-EFG-R 5′-CGTTCATGTCAATGGATTTGGGAGAAGATTATGATCTATACTATTTCTTTTTTTATTATTCCGCGGTGGCGGCCGCTCTAG
-3′). The underlined regions are the sequences identical to the beginning of TAP and URA3
, respectively. The 5′ 60 bp are homologous to the C-terminal end of EFG1
. The amplified DNA was transformed directly into C
(Wilson et al., 1999
), and the transformants with the TAP fused to the C terminus of Efg1 by homologous recombination were identified by PCR and verified by Western blot.
C. albicans Microarray Construction
microarray containing 6917 elements was printed with a C
70-mer set (QIAGEN Operon, Alameda, CA) on a code-link activated slide (GE Healthcare, Little Chalfont, Buckinghamshire, UK) by Microarray (Nashville, TN). The 70-mer set includes ~6530 ORFs from the Assembly 6 ORFs released by the C
Genome Sequencing Project at Stanford University (Stanford, CA). Oligonucleotide information is available at www.qiagen.com
. The set includes 192 randomly generated 70-mers as negative controls. The mean intensity of the negative controls is used as the basal hybridization intensity in data evaluation.
Preparing cDNA for Microarray Experiments
Total RNA was extracted using the hot acid phenol method with the addition of phase lock gels (Eppendorf, Westbury, NY) and a LiCl precipitation. The quality of RNA was checked by running the samples on a nanochip using the Agilent Bioanalyzer 2100 (University of California Irvine DNA MicroArray Facility, Irvine, CA). Samples of high-quality RNA, as determined by rRNA profiles, were used for cDNA synthesis. For each experiment, RNA from two samples was pooled (12 μg each) and annealed to 10 μg of Oligo(dT) and Random 9mer primers from the Prime-It II kit (Stratagene, La Jolla, CA). cDNA was synthesized using SuperScript II reverse transcriptase (Invitrogen, Carlsbad, CA) in a mixture containing 0.5 mM deoxynucleoside triphosphates (aminoallyl-dUTP:dT in a 3:2 ratio), 5× first strand buffer (Invitrogen), and 0.1 M dithiothreitol (DTT) overnight at 42°C. The RNA was hydrolyzed with 0.2 M NaOH and 0.1 M EDTA at 65°C for 15 min and subsequently neutralized with 0.33 M Tris, pH 7.4. The cDNA was washed with double distilled H2O several times and concentrated to a small volume with a Microcon-3 filter (Millipore, Billerica, MA) and stored at –20°C.
Coupling and Microarray Hybridization
The cDNA was thawed at 42°C for 5 min, resuspended in 0.05 M sodium bicarbonate buffer, pH 9.0, and incubated with Cy3 or Cy5 dye (GE Healthcare) for 1 h at room temperature in the dark. Alternatively, the cDNA was coupled to Alexa Fluor 555 or 647 (Invitrogen) according to the manufacturer's instructions. The QIAGEN PCR purification kit was used to remove any unincorporated dye and eluted with 30 μl of 10 mM Tris-Cl, 5 mM EDTA, pH 8.0, twice. The whole sample was loaded into an Ultravette disposable cuvette (Brandtech Scientific, Essex, CT) and scanned from optical density (OD)200 to OD800 using a spectrophotometer to quantify the amount of cDNA generated as well as the amount of dye coupled to the cDNA. Then, volumes containing equal amounts of cDNA for the appropriate experiments were mixed and concentrated using a Microcon-30 filter. Either 5 or 9 μl of the probe (depending on the size of the coverslip) was heated to 100°C for 2 min. Meanwhile, the microarray slide was washed in 0.2% SDS for 10 min, washed in filtered H2O, dipped in ethanol, and spun dry. A hybridization chamber and Millipore buffer #3 was prewarmed (50°C) and two-thirds volume Millipore buffer was added to the probe. The mixture was spun down before adding to the microarray slide. The slides were hybridized in the hybridization chambers for 16–20 h at 50°C.
Microarray Data Analysis
After hybridization, slides were washed, dried, and then scanned with a GSI Lumonics ScanArray 4000 slide scanner (GMI, Ramsey, MN) using its Scan Array software. The background-subtracted intensity of Cy3 and Cy5 at each spot was determined using the QuantArray software of the scanner. The background-subtracted intensities of Cy3 and Cy5 were used to plot log2
) ratio for each element on a microarray against log10
) (R-I plot) (data not shown). R-I plots were used to predict the quality of hybridization. For example, curved R-I plots (usually because of photobleaching of one dye) or scattered R-I plots (usually indicates degraded RNA) were not used for subsequent data analysis. We routinely carried out four experimental repeats and array hybridizations for each experiment. Data from two high-quality hybridizations for each experiment, as determined by their R-I plots, were used for subsequent data analyses. Because equal amounts of cDNA for each dye were used in hybridization, we used total intensity normalization to normalize the Cy3 and Cy5 for each hybridization. The normalization involved scaling the Cy5 intensities by multiplying them with a normalization factor, which was determined by dividing the sum of Cy3 intensities by the sum of the Cy5 intensities. The ratio of Cy5i to Cy3i was then calculated using the corrected Cy5 values. The ratios that were above 2 times basal cutoff (Cy5i-Cy3i> avgCy5ibasal+avgCy3ibasal) and threefold cutoff were log transformed and clustered with the average linkage cluster in a hierarchical cluster program (Eisen et al., 1998
). The clustered data were viewed in TreeView. The cluster and TreeView programs are at http://rana.stanford.edu/software/
C. albicans cells were grown to 3–5 × 107 cells/ml in 50 ml of YPD, SSA, or Lee's media in yeast or hyphal growth conditions. The cells were harvested by centrifugation at 4°C, washed, and resuspended in 0.5 ml of lysis buffer (10 mM Tris-HCl, pH 8, 250 mM NaCl, 0.1% NP-40, 0.5 mM DTT, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 0.5 μg/ml leupeptin, 1.4 μg/ml pepstatin, 2.4 μg/ml chymostatin, and 17 μg/ml aprotinin) and equal volume of glass beads (Sigma, St. Louis, MO). Cells were lysed at 4°C using a Fast-Prep system (FP120; Thermo Electron, Waltham, MA). Cell lysates were centrifuged for 10 min at 13,000 rpm in a microcentrifuge at 4°C. Protein extract containing 5 mg of protein was subjected to immunoprecipitation using 60 μl of rabbit IgG agarose bead slurry that was preincubated once with 0.2 mg/ml sheared salmon sperm DNA, 0.5 mg/ml bovine serum albumin in phosphate-buffered saline (PBS), and washed once in the lysis buffer. After incubation for 2 h at 4°C, beads were washed six times with 0.5 ml of lysis buffer and once with 1 ml of TE (10 mM Tris-HCl, pH 8, 1 mM EDTA). Bound proteins were eluted from the beads in 60 μl of elution buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, and 1% SDS) by incubation for 10–15 min at 65°C. Proteins were separated by 8% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Hybond; GE Healthcare). After blocking in 3% skim-milk powder in 0.05% PBS, Tween 20, a peroxidase-conjugated anti-c-myc antibody (Roche Diagnostics) was used to probe for myc-tagged proteins, which were then detected using the ECL system (GE Healthcare).
Yeast Two-Hybrid Analysis
Two-hybrid assays were performed as described previously (Gyuris et al., 1993
). Yeast strain EGY48 containing the LexAop-LacZ
reporter plasmid pSH18-34 was cotransformed with pEG202-based plasmids expressing LexA DNA binding domain fusions and pJG4-5-based plasmids containing transcriptional activation domain fusions (Gyuris et al., 1993
RNA extraction and Northern blotting were performed as described by Lane et al
). A 3.4-kb FLO8
fragment from pCF56 was used as a probe for Northern analysis. PCR products for C
. albicans ECE1
, and HSP31
were used for probing Northern blots.
The virulence of C
strains was tested as described by Chen et al
). ICR male mice (18–21 g) from Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China) were used for the virulence assay.
Nucleotide Sequence Accession Number
The GenBank accession number for the C. albicans FLO8 nucleotide sequence is AF414113 and orf19.1093.