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Eukaryotic Cell, June 2007, p. 1053-1062, Vol. 6, No. 6
1535-9778/07/$08.00+0     doi:10.1128/EC.00053-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Transcriptome Analysis in Response to Heat Shock and Cadmium in the Aquatic Fungus Blastocladiella emersonii{triangledown} ,{dagger}

Raphaela C. Georg and Suely L. Gomes*

Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, 05508-000, São Paulo, Brazil

Received 23 February 2007/ Accepted 6 April 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The global transcriptional response of the chytridiomycete Blastocladiella emersonii to environmental stress conditions was explored by sequencing a large number of expressed sequence tags (ESTs) from three distinct cDNA libraries, constructed with mRNA extracted from cells exposed to heat shock and different concentrations of cadmium chloride. A total of 6,350 high-quality EST sequences were obtained and assembled into 2,326 putative unigenes, 51% of them not previously described in B. emersonii. To approximately 59% of the unigenes it was possible to assign an orthologue in another organism, whereas 41% of them remained without a putative identification, with transcripts related to protein folding and antioxidant activity being highly enriched in the stress libraries. A microarray chip was constructed encompassing 3,773 distinct ESTs from the B. emersonii transcriptome presently available, which correspond to a wide range of biological processes. Global gene expression analysis of B. emersonii cells exposed to stress conditions revealed a large number of differentially expressed genes: 122 up- and 60 downregulated genes during heat shock and 189 up- and 110 downregulated genes during exposure to cadmium. The main functional categories represented among the upregulated genes were protein folding and proteolysis, proteins with antioxidant properties, and cellular transport. Interestingly, in response to cadmium stress, B. emersonii cells induced genes encoding six different glutathione S-transferases and six distinct metacaspases, as well as genes coding for several proteins of sulfur amino acid metabolism, indicating that cadmium causes oxidative stress and apoptosis in this fungus.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Living organisms possess notable properties of adaptation to physiological changes or to adverse conditions. Unicellular organisms, in particular, must contend with fluctuations in nutrients, pH, temperature, and external osmolarity, as well as exposure to a range of potentially toxic environmental compounds. The cellular response to these environmental challenges involves drastic changes in gene expression. This reprogramming of gene transcription can be unveiled using high-throughput technologies such as expressed sequence tag (EST) sequencing and DNA microarrays, which provide valuable information about the expression patterns of the cells under determined conditions. Characterization of environmentally triggered gene expression changes provides insights into when and how each gene is expressed and offers a glimpse at the physiological response of the cells to variations in their surroundings.

ESTs have historically provided data for gene discovery (18, 51), tissue- or stage-specific gene expression (3, 7, 14), and alternative splicing (27, 63). In fact, EST sequencing is likely to make its greatest impact on understudied genomes where little prior sequence data exist and where full genome sequencing projects may not be undertaken in the near future (36). DNA microarray technology created in the 1990s, using either whole-genome information or selected ESTs, allowed the simultaneous analysis of thousands of genes, as well as the identification of gene expression patterns related to cellular physiology, revolutionizing gene expression analysis (50).

The aquatic fungus Blastocladiella emersonii belongs to the Chytridiomycete class, which is at the base of the fungal phylogenetic tree (28, 60). Its particular taxonomic position makes this fungus an interesting system for consideration when studying the biology of lower fungi. Throughout its life cycle this fungus suffers dramatic biochemical and morphological changes, especially during two distinct stages of cell differentiation: germination and sporulation (39). Both stages can be induced with a high degree of synchrony, and drastic changes in the patterns of RNA and protein syntheses are observed throughout. During the very early stages of B. emersonii zoospore germination, mostly posttranslational events take place, and as germination progresses protein synthesis begins, being directed mainly by mRNAs transcribed near the end of sporulation and stored in the zoospores. Later in germination the majority of the changes in the pattern of protein synthesis are attributed to newly synthesized mRNAs (39, 55). A recent large-scale EST sequencing program revealed a greater diversity of transcripts in sporulation cells, in agreement with the higher mRNA turnover previously observed during this stage (31, 49, 54). All these characteristics make this fungus a suitable model for gene expression studies. In addition, information generated during analyses of B. emersonii ESTs has revealed sequences previously considered specific to animals or plants, and sequences highly divergent relative to other fungi, which makes the study of this fungus even more interesting (48, 49).

The heat shock response has been previously analyzed in B. emersonii and shown to be developmentally regulated, with different subsets of heat shock proteins (Hsps) being synthesized in response to heat stress during sporulation, germination, and vegetative growth (8). In addition, differences have been observed in the degree of induction of B. emersonii heat shock genes, depending on the stage of the fungus life cycle (24, 47, 57). On the other hand, the response to cadmium stress is poorly characterized in this fungus, with preliminary experiments showing increased synthesis of a set of proteins distinct from those characterized during heat shock (R. M. P. Stefani and S. L. Gomes, unpublished data).

For a more comprehensive evaluation of these stress responses in B. emersonii, we carried out a large-scale gene expression study using EST sequencing and DNA microarray hybridization analysis. A total of 6,350 high-quality sequence ESTs were obtained from stress cDNA libraries, representing 2,326 putative unigenes, among which 51% had not been sequenced before. As the complete genome of this fungus is not available, we constructed a 9,216-element DNA microarray containing 3,773 distinct EST sequences obtained from this work and from a previous study (49), and microarray experiments were carried out to investigate the expression pattern of B. emersonii genes in response to heat shock and cadmium stress. A large number of B. emersonii genes were identified as upregulated at least twofold in response to these stresses (122 genes during heat shock and 189 in the presence of cadmium), some being induced during both stresses and some being upregulated only during germination or sporulation stages, suggesting a developmental control superimposed to the stress responses. The upregulated genes were classified into several biological processes of the Gene Ontology Consortium (GO), with protein folding and proteolysis, antioxidant cellular processes, and cellular transport being highly represented. In addition, many genes encoding proteins with no putative function assigned were identified as differentially expressed during exposure to heat shock and cadmium, some of them being upregulated during both stresses, constituting an initial functional characterization of these genes.

Even though a number of studies involving stress responses of ascomycetes and basidiomycetes are found in the literature, early diverging fungi are poorly represented; thus, this work can contribute to increase the knowledge of the physiology of this important class of fungi.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
Culture conditions. Cultures of B. emersonii were maintained in solid medium containing 0.13% peptone, 0.13% yeast extract, 0.3% glucose, and 1% agar. For RNA extraction, zoospores were inoculated (3 x 105 cells per ml) in defined DM3 liquid medium (41) and incubated for 16 h at 18°C with agitation. Vegetative cells were then induced to sporulate by filtering the cells through a Nitex cloth, rinsing, and resuspending in sporulation solution (1 mM Tris-maleate, pH 6.8, containing 1 mM CaCl2) at a density of 5 x 105 cells per ml. After incubation for 3.5 to 4 h at 27°C with agitation, sporulation was complete with the release of the zoospores from the cells. Zoospores were separated from the empty zoosporangia by filtering through Nitex cloth. Zoospore germination was initiated by inoculation of these cells at a density of 106 cells per ml in DM3 medium and incubation with agitation at 27°C. The progress and synchrony of both sporulation and germination were monitored by taking samples at different times and examining cell types under a light microscope (39, 56).

RNA isolation. Total RNA was isolated using TRIzol (Invitrogen), and its integrity was verified through 1% agarose-2.2 M formaldehyde gel electrophoresis, followed by ethidium bromide staining and RNA visualization under UV light. For cDNA library construction, mRNA was purified from total RNA using the Oligotex-dT mRNA mini kit (QIAGEN). Possible DNA contamination of RNA samples used in the quantitative reverse transcription-PCR assays was eliminated by incubation with RNase-free DNase (Promega).

Construction of cDNA libraries and DNA sequencing. To construct the cDNA libraries, RNA samples were isolated from sporulating cells exposed to heat shock at 38°C from 30 to 60 min after starvation (HSR library) or to 50 µM CdCl2 during the same period (CDM library) and from sporulating cells exposed to 100 µM CdCl2 from 60 to 90 min after starvation (CDC library). The duration of the stress was chosen based on previous data showing that the peak of mRNA accumulation was after a 30-min exposure, both for heat shock and cadmium (47). cDNA was synthesized with reverse transcriptase (Invitrogen) using 1 to 5 µg of poly(A)+ RNA and size fractionated in Sephacryl S-500 HR (Invitrogen) columns, with fractions containing fragments larger than 500 bp being pooled. The cDNA libraries were not normalized and were constructed in the vector pSPORT1 using the SuperScript plasmid system (Invitrogen), resulting in directional cloning. Sequencing reactions were performed with 100 to 200 ng of plasmid DNA prepared in 96-well format plates at all stages. Reactions were carried out with the ABI Prism BigDye Terminator sequencing kit (Applied Biosystems) and analyzed on ABI377 or ABI3100 automatic sequencer instruments.

EST processing pipeline and annotation. The EST sequence chromatograms were stored, processed, and trimmed through a web-based service (46; http://bioinfo.iq.usp.br/estweb), including base calling and quality control by PHRED (17, 19) and trimming [which included the removal of poly(A) tails and low-quality sequences as well as vector and adapter regions] by Cross Match (26; http://www.phrap.org/phrap_documentation.html). The accepted sequences were chosen to contain a minimum of 150 bases, with at least 75 bases within a window of 100 bases with a PHRED quality value of ≥15. The sequences were filtered using a local BlastN analysis (2) intended to eliminate sequences that matched ribosomal and mitochondrial sequences or that matched bacterial sequences. Assembly of ESTs into contigs was carried out using the Cap3 program (29; http://genome.cs.mtu.edu/cap/cap3.html) set with default parameters. The redundancy was estimated as the number of putative unigenes (contigs plus singlets) divided by the total number of ESTs and transformed to a percentage. Annotation of putative protein products was based on BlastX best hits against a nonredundant database at the National Center for Biotechnology Information (NCBI; http://www.ncbi.nlm.nih.gov/BLAST), and GO terms were automatically assigned (http://www.geneontology.org) to the resulting contigs and singlets using BlastX against curated databases (Swiss Prot and TrEMBL) available at the Swiss Institute of Bioinformatics (http://www.expasy.org). A bit score cutoff of 55 (which corresponds approximately to an E-value of ≤10–6, considering the nonredundant database size) was used for the NCBI annotation, and an E-value of ≤10–6 was used as the BlastX cutoff for the Swiss Prot and TrEMBL annotation.

Microarray construction and hybridization and data analysis. The B. emersonii cDNA microarray consists of a 9,216-element chip containing 3,773 distinct EST sequences, spotted at least in duplicate, representative of 3,773 putative genes obtained during this work (804 cDNAs) and in a previous study (49). These cDNA inserts were PCR amplified with primers T7 forward (5'-TAATACGACTCACTATAGGG-3') and SP6 reverse (5'-TTTAGGTGACACTATAG-3'), and the amplified products were purified with Millipore multiscreen filtration plates (MAFB NOB). DNA was eluted with Tris-HCl 10 mM, pH 8.0, and an equal volume of dimethyl sulfoxide was added. DNA samples (final concentration of 100 to 200 ng/µl) were then spotted onto Star 7 slides (Amersham Biosciences) using a Generation III microarray spotter (Amersham Biosciences).

For microarray hybridization, cDNA synthesis and labeling were performed using the Cy-Scribe postlabeling kit (Amersham Biosciences). Briefly, we used 10 µg of total RNA, oligo(dT) primers, amino allyl-dUTP, 1x buffer, dithiothreitol, and the reverse transcriptase Cy-Scribe, according to the supplier's recommendations. The reaction mixture was incubated at 42°C for 3 h following RNA degradation by the addition of NaOH. The resulting first-strand cDNA was purified using a Millipore multiscreen filtration plate (MAFB NOB) and dried in a speed vacuum apparatus. For cDNA labeling, the Cy-Dyes were suspended in 100 mM sodium bicarbonate (pH 9.0), and this mixture was added to the dried cDNA. The reaction was maintained in the dark during 1 h at room temperature and interrupted by addition of 4 M hydroxylamine. The labeled cDNA was purified and dried as described above.

Labeled cDNA was suspended in water, 50% formamide, and 1x hybridization buffer (Amersham Biosciences). Arrays were hybridized at 42°C during 16 h and washed at 55°C once in 1x SSC (0.15 M NaCl plus 0.015 M sodium citrate), 0.2% sodium dodecyl sulfate during 10 min, followed by two washes in 0.1x SSC, 0.2% sodium dodecyl sulfate during 10 min and one final wash in 0.1x SSC during 1 min. Slides were then dried with N2 vapor, and image acquisition was carried out using a Generation III scanner (Amersham Biosciences). Images were analyzed through the ArrayVision 6.0 program (Image Research), and mean fluorescence intensity and the surrounding median background from each spot were obtained with ArrayVision 6.0 (Imaging Research). Spots presenting mean intensities below two times the standard deviation of its corresponding background simultaneously in Cy3 and Cy5 were eliminated from subsequent analyses. Saturated signals (intensity greater than >990 fluorescence units) were also discarded. Normalization was carried out by LOWESS fitting on an M-versus-S plot, where M is the fluorescence log ratio of the heat shock/cadmium time point relative to the control condition [M = log2(treatment/control)] and S is the log mean fluorescence intensity {S = log2[(treatment/2) + (control/2)]} (64). Each stress condition was analyzed with three independent biological experiments. Since each slide carried two replicates of the arrayed genes, and a total of six intensity readings were generated for each gene in the microarray. The expression ratios shown in the tables represent the median values determined among the valid replicates.

To evaluate the experimental variation inherent in our microarray assays, we used self-self hybridization assays. In this type of hybridization, the same RNA sample is labeled separately with both Cy3 and Cy5 dyes. Two distinct self-self experiments were performed, one with RNA from germinating cells and another with RNA from sporulating cells. The HTself program available on the Web (61; http://blasto.iq.usp.br/~rvencio/HTself) was used to determine the intensity-dependent cutoff lines (that delimits genes which do not show variation in their expression levels). Through these cutoff values we were able to determine which genes were differentially expressed under the stress conditions tested. Those genes that presented at least 80% of replicates with expression ratios above or below cutoff lines determined in self-self hybridization experiments were considered induced or repressed, respectively.

Validation of microarray data by quantitative RT-PCR analysis. To verify the level of reliability of the array-based data, we selected eight genes upregulated by heat shock or cadmium, both during sporulation and germination, and analyzed their expression levels by quantitative RT-PCR. All time points tested revealed a coincidence in the direction of gene expression modulation determined with both methods, indicating an overall corroboration between microarray hybridization and quantitative RT-PCR data (see Table S1A in the supplemental material). qRT-PCR experiments were performed using the GeneAmp 5700 sequence detection system (Applied Biosystems) and the Platinum SYBR Green qPCR SuperMix UDG kit (Invitrogen). The thermocycling conditions comprised an initial step at 50°C for 2 min, followed by 95°C for 10 min and 40 cycles of 95°C for 15 seconds and 60°C for 1 min. For each gene analyzed, two independent RNA samples were used. The gene encoding the mitochondrial RNA helicase-like protein was used as the calibrator gene in all experiments. The determination of the expression ratios was carried out using the method 2–{Delta}{Delta}CT, as described by Livak and Schmittgen (38).

Nucleotide sequence accession numbers. All sequences described in this study have been submitted to the GenBank EST section with the accession numbers EE730389 to EE736848.

Microarray data accession number. The microarray data discussed in this work have been deposited in NCBI's Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession number GSE6231 [NCBI GEO] (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE6231).


   RESULTS AND DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Analysis of cDNA stress libraries. Three distinct cDNA libraries were constructed with mRNA extracted from B. emersonii cells submitted to stress conditions (heat shock or cadmium) during sporulation and a large number of clones were analyzed, generating 6,350 high-quality sequences after removal of low-quality sequences, poly(A) tails, and vector and adapter sequences, as well as contaminant bacterial, ribosomal, and mitochondrial DNA (Table 1). The estimated redundancy of the whole project was 63%, a good value for nonnormalized libraries. The range of insert sizes varied from 200 to 2,300 bp, and the average sequence size (571 nucleotides [nt]) indicated that most of the reads extended into the coding region, since the majority of B. emersonii 5' untranslated regions previously identified vary between 65 and 362 nt (49). Assembly of the ESTs into contigs revealed 2,326 putative unique transcripts, 51% (1,198) of them not previously described in B. emersonii.


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  		TABLE 1. Summary of EST sequencing from B. emersonii stress libraries

 
Table 2 shows the 25 most sequenced ESTs from the cDNA stress libraries. Several genes encoding proteins related to protein folding and proteolysis, such as Hsp17, Hsp90, Hsp10, cyclophilin A, polyubiquitin, and metacaspase, are among those which presented the highest number of sequenced ESTs. ESTs encoding a member of the universal stress protein (USP) family and those encoding proteins with antioxidant properties, such as a thioredoxin peroxidase and an NADH-dependent flavin oxidoreductase, were also highly sequenced.


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  		TABLE 2. Top 25 most-sequenced ESTs

 
It was possible to assign an orthologue in another organism to approximately 59% (1,369) of the unigenes (ESTs with matches in GenBank), whereas 41% (957) remained without a putative identification. Clustering of ESTs obtained from the stress libraries together with ESTs previously characterized (49) resulted in a total of 6,071 putative unique transcripts for B. emersonii, roughly 20% of which originated in the present work.

The putative unigenes obtained here were classified according to GO categories, and among the 2,326 unigenes, a total of 1,282 revealed similarity with sequences deposited in the Swiss Prot database and were classified in at least one of the three ontologies of the GO structure (see Fig. S1 in the supplemental material). This number is very similar to the number of genes with matches in the GenBank database (1,369 unigenes), the difference observed being probably due to hypothetical proteins, which were not considered in GO categories. According to GO categorization, 1,113 genes were classified in the molecular function ontology category, 650 in cellular component, and 999 in biological process. Protein metabolism (34%), nucleic acid metabolism (16%), and cell organization and biogenesis (10%) were the subcategories in the biological process classification showing a higher number of representative genes. It is important to notice that in the protein metabolism subcategory, 19% of the putative unigenes corresponded to protein folding (see Fig. S1 in the supplemental material), constituting a significant enrichment compared to the 9% observed for this category among the unigenes previously sequenced from B. emersonii nonstress libraries (49).

Differentially expressed genes in response to heat shock. To assess B. emersonii gene expression on a large scale, we constructed a microarray chip carrying PCR-amplified cDNA representatives of 3,773 EST clusters obtained from B. emersonii libraries (present work and reference 49). Although the number of genes in the B. emersonii genome is still unknown, considering that already sequenced fungal genomes present between 5,000 and 12,000 genes, we estimate that the gene content in the B. emersonii microarray represents a considerable part of its genome. The cDNA amplicons spotted in the arrays ranged from 0.2 to 2.0 kb in length and were annotated according to Gene Ontology classification (see Fig. S2 in the supplemental material), encompassing a large variety of biological functions, which permits an ample investigation of B. emersonii gene expression.

Microarray hybridizations were carried out with cDNA synthesized from RNA extracted from cells heat shocked (38°C) from 30 to 60 min after induction of germination or sporulation. As a reference, we used RNA obtained from cells at the same developmental stages under normal temperature conditions (27°C). After statistical analysis of normalized hybridization data, we identified a total of 101 genes differentially expressed (at least twofold) during heat shock in sporulating cells (61 up- and 40 downregulated) and 115 genes differentially expressed during heat stress in germinating cells (95 up- and 20 downregulated). Among the upregulated genes, only 34 of them were induced in both stages of development. These results suggest a developmental control of the heat shock response in B. emersonii, in agreement with previous data obtained through two-dimensional gel electrophoresis analysis of 35S-labeled protein extracts that showed that different sets of heat shock proteins were synthesized in cells exposed to high temperature according to the stage of the fungus life cycle (8).

Table 3 shows the genes with matches in the GenBank and/or Swiss Prot databases and classified as upregulated (threefold or more) in response to heat shock during sporulation (S) and germination (G) stages. The complete list of upregulated genes is in Table S1B of the supplemental material. Among the induced genes we observed a high number of genes encoding proteins related to protein folding and proteolysis (23% in S and 20% in G), as well as proteins with antioxidant properties (8% in S and 5% in G) and proteins involved in nucleotide (7% in S and 6% in G) and carbohydrate (5% in S and 4% in G) metabolism. In addition, 25% of the upregulated genes in sporulation cells and 36% in germination cells did not present a putative identification (not shown). As observed in Table S1B of the supplemental material, a total of 34 genes were induced during heat stress both in germination and sporulation cells and 18 genes were specific to heat shock, since they were not observed in the microarray analysis of cadmium-treated cells, as described below.


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  		TABLE 3. Genes induced threefold or more in response to heat shock during sporulation and/or germination

 
Protein folding and proteolysis. Among the genes related to protein folding, we found those encoding heat shock proteins Hsp10, Hsp17, Hsp60, Hsp70-2, Hsp70-8, Hsp90, and Hsp100, a protein from the Hsp40 family (Psi1), and several peptidyl-prolyl cis-trans isomerases. Some genes encoding proteins related to proteolysis were also upregulated during heat stress. One of them encodes the protein Der1, which is specifically required for the degradation process of unfolded proteins in the endoplasmic reticulum lumen (33). Another gene related to proteolysis encodes an arginine-tRNA transferase and was also induced in the presence of cadmium, as shown below. This protein is responsible for the addition of arginine to the N terminus of proteins, targeting them to the ubiquitin-dependent proteolysis cascade (43). Two other genes related to ubiquitin-dependent proteolysis were also induced during heat shock, encoding two putative paralogs of an ubiquitin-activating enzyme (E1), which is responsible for the first step in the protein ubiquitinylation pathway. The ubiquitin-activating enzyme (E1) is a key enzyme of the ubiquitinylation process, which targets proteins to the 26S proteasome degradation pathway. Recent studies showed that the simple increase of the ubiquitin- or proteasome-dependent degradation activity could replace the essential functions performed by heat shock proteins during high-temperature stress. These results indicate that accumulation of unfolded or aggregated proteins is the main reason for viability loss due to heat shock (21).

Proteins with antioxidant properties. A total of five genes encoding proteins with antioxidant properties were upregulated during heat stress both in germination cells and sporulation cells. These predicted proteins were glutathione S-transferases (GST) 1, 2, and 3, thioredoxin peroxidase, and peroxiredoxin. In Schizosaccharomyces pombe all these genes are induced in response to many different stresses and are among the core environmental stress response genes (12); as discussed below, they are also among the genes induced by cadmium in B. emersonii. These results agree with reports showing that heat shock causes oxidative stress (15, 32).

Carbohydrate metabolism and cell wall and membrane biosynthesis. Genes encoding proteins involved in carbohydrate metabolism, such as glyceraldeyde 3-phosphate dehydrogenase, phosphorylase B kinase alpha regulatory chain, and enolase, were induced in response to heat shock in B. emersonii. In addition, the gene encoding the enzyme glutamine:fructose- 6-phosphate amidotransferase, which is involved in chitin synthesis, and several genes encoding proteins related to phospholipid biosynthesis were also upregulated during high-temperature stress. These results corroborate the observation that cell wall formation and remodeling are necessary to prevent cell lysis at elevated temperatures (30).

Differentially expressed genes in response to exposure to cadmium. To evaluate global changes in gene expression of B. emersonii cells in response to cadmium, microarray experiments were also performed. Hybridizations were carried out with cDNA synthesized from mRNA obtained from cells exposed to two different concentrations of cadmium (100 µM and 200 µM CdCl2) from 30 to 60 min after induction of germination or sporulation. RNA obtained from 60-min germination or sporulation cells, and not exposed to cadmium, was used as a reference. Statistical analysis of normalized hybridization data led to the identification of 202 differentially expressed genes (at least twofold) in sporulation cells (117 up- and 85 downregulated) and 151 genes in germination cells (100 up- and 51 downregulated). Among the upregulated genes only 28 genes were classified as induced in both stages of the life cycle, suggesting a developmental regulation also for the cadmium response in B. emersonii.

Table 4 shows genes upregulated (threefold or more) in response to cadmium during sporulation and/or germination with matches in GenBank and/or Swiss Prot databases. The complete list of upregulated genes is reported in Table S1C of the supplemental material. We observed a large number of genes encoding proteins with antioxidant properties (9% in S and 18% in G), proteins involved in amino acid metabolism (11% in S and 5% in G), and proteins related to cellular transport (6% in S and 11% in G), as well as proteins related to protein folding and proteolysis (8% in S and 7% in G). In addition, 36% of the upregulated genes in sporulation cells and 32% in germination cells encode proteins of unknown function (see Table S1C). Furthermore, only 28 genes were induced by cadmium treatment both in germination and sporulation cells (see Table S1C), and a total of 58 genes were specific to cadmium treatment (not observed in heat shock-treated cells). Among the downregulated genes during exposure to cadmium, many encode proteins related to transport and a large number encode proteins of unknown function (not shown).


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  		TABLE 4. Genes induced threefold or more in response to cadmium during sporulation and/or germination

 
Proteins with antioxidant properties. The induction of genes encoding proteins with antioxidant properties is expected in cells exposed to cadmium, since increased production of reactive oxygen species and lipid peroxidation are observed after exposure to this metal (5, 9, 58). In agreement, several genes encoding proteins with antioxidant properties were upregulated by cadmium treatment in B. emersonii, such as oxidoreductases, peroxiredoxin, thioredoxin, thioredoxin peroxidase, glutathione peroxidase, glutathione reductase, NADPH oxidase, quinone oxidoreductase, and NADH-dependent flavin oxidoreductase. A high number of sequenced ESTs encoding this last enzyme were also observed in the cDNA stress libraries. In addition, six different genes encoding glutathione S-transferases were upregulated by cadmium treatment, three of them being also induced during high-temperature exposure. GSTs are detoxification proteins present in all aerobic organisms. In Saccharomyces cerevisiae, the glutathione-cadmium complex seems to be involved in the control of cellular cadmium uptake, and it has been proposed that GSTs are the proteins responsible for the formation of this complex (1, 25). In agreement with this hypothesis, the absence of one of the two GSTs from S. cerevisiae leads to low cellular cadmium-glutathione complex concentrations, resulting in increased metal absorption (1).

Amino acid metabolism. Among the upregulated genes related to amino acid metabolism, we observed several genes encoding amino acid transporters, including a high-affinity methionine permease. In response to this metal, S. pombe and S. cerevisiae cells also induce genes encoding amino acid transporters, but not as many as we observed in this work (12, 20).

Genes encoding enzymes of the sulfur amino acid biosynthetic pathway were also observed to be induced by cadmium, such as N5-methyltetrahydropteroyltriglutamate-homocysteine-S- methyltransferase and S-adenosylmethionine synthetase. These two enzymes are responsible for the conversion of L-homocysteine into L-methionine and of this last compound into S-adenosylmethionine, respectively. The metabolite S-adenosylmethionine has been shown to exert protective effects on different experimental pathological models in which free radicals are involved and could be itself an antioxidant molecule (10). Furthermore, S-adenosylmethionine can be later converted via other enzymatic reactions into cysteine, one of the three amino acids necessary for glutathione ({gamma}-Glu-Cys-Gly; GSH) synthesis. GSH is one of the main molecules responsible for metal sequestration inside the cells, and the increase in its cellular levels is related to the cadmium stress response (35). Coherent with these data, among the genes presenting the highest levels of induction in the presence of cadmium we found a gene encoding a putative cystinosin, a lysosomal transmembrane protein involved in exporting of L-cystine out of the lysosome to the cytoplasm. This amino acid could be directed to glutathione synthesis, which is essential to the cellular detoxification of cadmium (5). In fact, human cells with a mutated cystinosin gene presented a significant decrease in GSH levels and required other pathways for cysteine synthesis, indicating that exportation of lysosomal cystine is a natural source of cysteine (13).

In addition, the pathway of cysteine synthesis could also be induced by this heavy metal to increase the levels of cysteine available to GSH synthesis. To investigate this hypothesis, we performed a search in B. emersonii transcriptome data for unigenes encoding other proteins from sulfur amino acid biosynthesis as well as from the glutathione synthesis pathway. Overall, we observed six genes from these metabolic pathways which encode the proteins homocysteine synthase, cystathionine beta-synthase, cystathionine gamma-lyase, and two cysteine synthases (from the sulfur amino acid synthesis pathway) and glutathione synthetase (from the GSH synthesis pathway). Since these genes were not present in the microarray slide, their expression levels were evaluated through quantitative RT-PCR assays using RNA samples from B. emersonii cells exposed to 100 µM cadmium, both during sporulation and germination. We observed that except for the two genes encoding putative cysteine synthases, all four other genes were induced by cadmium treatment, suggesting that cysteine as well as GSH synthesis is upregulated in response to cadmium stress in B. emersonii (Fig. 1).


Figure 1
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  		FIG. 1. Induction of the sulfur amino acid pathway in response to cadmium. Gene expression was analyzed using quantitative RT-PCR, and the induction ratios observed in both phases (sporulation [S] and germination [G]) are indicated between parentheses under the protein names. The genes encoding the proteins inside the dotted squares were observed as induced through microarray experiments. a, the full protein name is N5-methyltetrahydropteroyltriglutamate-homocysteine-S-methyltransferase.

 
Generally, in fungi there are two pathways for de novo cysteine biosynthesis: a cystathionine pathway and an O-acetylserine (OAS) pathway (Fig. 1) (5, 22). In the cystathionine pathway, cysteine is synthesized from homocysteine via the intermediary formation of cystathionine, and the final reaction is catalyzed by cystathionine gamma-lyase. In the OAS pathway, on the other hand, cysteine is generated from serine via the intermediary formation of OAS, and the final reaction is catalyzed by cysteine synthase (5). It seems that B. emersonii possesses both cysteine biosynthetic pathways, and apparently only the cystathionine pathway presented induction by cadmium.

In S. cerevisiae, only some strains present detectable activities of the enzymes involved in the OAS pathway, cysteine being predominantly synthesized via the cystathionine intermediary (22). On the other hand, S. pombe synthesizes cysteine mainly through the OAS pathway, which makes its response to cadmium different from S. cerevisiae (5). The cystathionine pathway as well as glutathione synthesis are highly induced in yeast in response to cadmium treatment, since compartmentalization of the glutathione-cadmium complex into the vacuole is the major mechanism for cadmium detoxification in this fungus (5, 44, 62). In this way, the upregulation in B. emersonii of the genes encoding enzymes from cysteine biosynthesis and glutathione synthetase in response to cadmium suggests that, as observed in S. cerevisiae, the increase in cysteine levels is necessary to elevate GSH synthesis and consequently the availability of this important antioxidant molecule for cadmium detoxification. Furthermore, the induction of the cystathionine pathway in B. emersonii in response to cadmium indicates that this fungus possesses a cadmium response more similar to that described in S. cerevisiae than in S. pombe.

Cellular transport. Another gene category with several members induced by cadmium is cellular transport. Genes in this category mainly encode putative proton ATPases, a putative zinc transporter, and putative transporter proteins from the ABC family. In S. cerevisiae, cadmium is transported into the cell through a promiscuous zinc transporter (25). Once inside the cell, cadmium complexes with glutathione and is transported into the vacuole through Ycf1, an ABC-type protein. This transport and the consequent compartmentalization of cadmium is the major mechanism that allows resistance to cadmium in S. cerevisiae (37). Furthermore, in other fungi, such as S. pombe and Candida glabrata, as well as in plants, this mechanism is also the most important for cadmium resistance (4, 44). Considering the genes induced by cadmium revealed in the present work, there is a strong indication that the cadmium detoxification model proposed for other fungi is also valid for B. emersonii.

Protein folding and proteolysis. The induction of heat shock genes, as well as the increased synthesis of heat shock proteins in response to cadmium, has been described in many organisms (4, 20, 62). In our analysis we observed the upregulation during exposure to cadmium of several genes encoding heat shock proteins, such as Hsp10, Hsp60, Hsp70-2, Hsp70-3, Hsp90, Hsp100, Psi1, and one of the CCT subunits.

Interestingly, among the genes that belong to the GO category of protein folding and proteolysis, we observed four genes encoding putative metacaspases and two genes encoding proteins with caspase domains. Metacaspases are proteases of the caspase family found in plants, fungi, and protozoa that are related to apoptosis. The gene encoding one of these metacaspases is also among those with the highest number of ESTs sequenced from B. emersonii stress libraries (Table 2). In human cells cadmium is able to induce apoptosis through the activation of caspases by increasing the production of reactive oxygen species and the modulation of protein kinase and phosphatase activities (6, 42, 53). Caspases are cysteine-proteases essential for the process of apoptosis and are divided into two families: metacaspases, which are found in plants, fungi, and protozoa, and paracaspases, which are observed in metazoa and Dictyostelium discoideum (59). Although cadmium is not a transition metal and therefore is not assumed to directly interfere with cellular oxygen metabolism, it may indirectly contribute to oxidative stress due to an initial depletion of GSH and protective enzymes (52). In yeast, apoptosis can be induced by GSH depletion or by low H2O2 concentrations, indicating that as observed in metazoa, reactive oxygen species are the apoptosis regulators in this organism (40).

Curiously, genes encoding metacaspases were not identified among the upregulated genes in B. emersonii germinating cells exposed to cadmium. The fact that these genes were only induced during sporulation could indicate that cells at this stage are more prone to enter the pathway to apoptosis as they are already facing the stress of nutrient starvation, whereas germinating cells are in a good nutrient environment. In fact, it has been suggested that sporulation conditions can initiate programmed cell death in S. cerevisiae (34). In addition, the induction of metacaspase genes in response to cadmium in B. emersonii is an interesting observation, since there is no description in the literature of other fungi presenting this type of response.

Other genes. The gene encoding a putative pirin was also highly expressed in response to cadmium, both in germination and sporulation cells. In humans, pirin is described as a transcription cofactor. In tomato, pirin mRNA levels increase significantly in cells directed to the programmed cell death pathway (16, 45). Another gene with high levels of induction was the one encoding a super-cysteine-rich protein. This kind of protein performs an important role as a cellular antioxidant (44). We also identified the gene encoding the USP as upregulated in response to cadmium. USP genes are induced in response to many types of stress, but no putative function has been assigned to them yet.

Genes common to both stresses. A total of 28 genes encoding mainly heat shock proteins and proteins related to cellular antioxidant metabolism were induced by both cadmium treatment and heat shock in B. emersonii, and they are possibly part of the core environmental stress response of this fungus (Fig. 2). In fact, genes encoding heat shock proteins as well as antioxidant proteins are induced in response to different stresses and are among the genes of the core environmental stress response in S. pombe and S. cerevisiae (11, 12, 23). In addition, roughly 30% of the genes induced during both stresses in B. emersonii encode proteins with no putative function assigned. These results provide an initial functional characterization of these genes and suggest their importance in environmental stress responses in B. emersonii.


Figure 2
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  		FIG. 2. Genes induced in response to both heat shock and cadmium. The clone identification number of the genes shown can be found in Table 3.

 


   ACKNOWLEDGMENTS
 
This investigation was supported by grants to S.L.G. from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP). R.C.G. was a FAPESP predoctoral fellow. S.L.G. is partially supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico.

We thank Tie Koide and Ricardo Z. Vêncio for their help in microarray analysis and Marilis V. Marques for critical reading of the manuscript.


   FOOTNOTES
 
* Corresponding author. Mailing address: Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes 748, 05508-000, São Paulo, Brazil. Phone: 55 11 3091 3826. Fax: 55 11 3091 2186. E-mail: sulgomes{at}iq.usp.br Back

{triangledown} Published ahead of print on 20 April 2007. Back

{dagger} Supplemental material for this article may be found at http://ec.asm.org/. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 

  1. Adamis, P. D., D. S. Gomes, M. L. Pinto, A. D. Panek, and E. C. Eleutherio. 2004. The role of glutathione transferases in cadmium stress. Toxicol. Lett. 154:81-88.[CrossRef][Medline]
  2. Altschul, S. F., T. L. Madden, A. A. Schaffer, J. Zhang, Z. Zhang, W. Miller, and D. J. Lipman. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25:3389-3402.[Abstract/Free Full Text]
  3. Audic, S., and J. M. Claverie. 1997. The significance of digital gene expression profiles. Genome Res. 7:986-995.[Abstract/Free Full Text]
  4. Bae, W., and X. Chen. 2004. Proteomic study for the cellular responses to Cd2+ in Schizosaccharomyces pombe through amino acid-coded mass tagging and liquid chromatography tandem mass spectrometry. Mol. Cell Proteomics 3:596-607.[Abstract/Free Full Text]
  5. Baudouin-Cornu, P., and J. Labarre. 2006. Regulation of the cadmium stress response through SCF-like ubiquitin ligases: comparison between Saccharomyces cerevisiae, Schizosaccharomyces pombe and mammalian cells. Biochimie 88:1673-1685.[Medline]
  6. Beyersmann, D., and S. Hechtenberg. 1997. Cadmium, gene regulation, and cellular signalling in mammalian cells. Toxicol. Appl. Pharmacol. 144:247-261.[CrossRef][Medline]
  7. Bonaldo, M. F., G. Lennon, and M. B. Soares. 1996. Normalization and subtraction: two approaches to facilitate gene discovery. Genome Res. 6:791-806.[Abstract/Free Full Text]
  8. Bonato, M. C. M., A. M. Silva, S. L. Gomes, J. C. C. Maia, and M. H. Juliani. 1987. Differential expression of the heat-shock proteins and spontaneous synthesis of Hsp70 during life cicle of Blastocladiella emersonii. Eur. J. Biochem. 163:211-220.[Medline]
  9. Brennan, R. J., and R. H. Schiestl. 1996. Cadmium is an inducer of oxidative stress in yeast. Mutat. Res. 356:171-178.[CrossRef][Medline]
  10. Castillo, C., V. Salazar, C. Ariznavarreta, M. Fossati, J. A. Tresguerres, and E. Vara. 2005. Effect of S-adenosylmethionine on age-induced hepatocyte damage in old Wistar rats. Biogerontology 6:313-323.[CrossRef][Medline]
  11. Causton, H. C., B. Ren, S. S. Koh, C. T. Harbison, E. Kanin, E. G. Jennings, T. I. Lee, H. L. True, E. S. Lander, and R. A. Young. 2001. Remodeling of yeast genome expression in response to environmental changes. Mol. Biol. Cell 12:323-337.[Abstract/Free Full Text]
  12. Chen, D., W. M. Toone, J. Mata, R. Lyne, G. Burns, K. Kivinen, A. Brazma, N. Jones, and J. Bahler. 2003. Global transcriptional responses of fission yeast to environmental stress. Mol. Biol. Cell 14:214-229.[Abstract/Free Full Text]
  13. Chol, M., N. Nevo, S. Cherqui, C. Antignac, and P. Rustin. 2004. Glutathione precursors replenish decreased glutathione pool in cystinotic cell lines. Biochem. Biophys. Res. Commun. 5:231-235.
  14. Claverie, J. M. 1999. Computational methods for the identification of differential and coordinated gene expression. Hum. Mol. Genet. 8:1821-1832.[Abstract/Free Full Text]
  15. Davidson, J. F., and R. H. Schiestl. 2001. Mitochondrial respiratory electron carriers are involved in oxidative stress during heat stress in Saccharomyces cerevisiae. Mol. Cell. Biol. 21:8483-8489.[Abstract/Free Full Text]
  16. Dechend, R., F. Hirano, K. Lehmann, V. Heissmeyer, S. Ansieau, F. G. Wulczyn, C. Scheidereit, and A. Leutz. 1999. The Bcl-3 oncoprotein acts as a bridging factor between NF-{kappa}B/Rel and nuclear co-regulators. Oncogene 18:3316-3323.[CrossRef][Medline]
  17. Ewing, B., and P. Green. 1998. Base-calling of automatic sequencer traces using phred. II. Error probabilities. Genome Res. 8:186-194.[Abstract/Free Full Text]
  18. Ewing, B., and P. Green. 2000. Analysis of expressed sequence tags indicates 35,000 human genes. Nat. Genet. 25:232-244.[CrossRef][Medline]
  19. Ewing, B., L. Hillier, M. C. Wendl, and P. Green. 1998. Base-calling of automatic sequencer traces using phred. I. Accuracy assessment. Genome Res. 8:175-185.[Abstract/Free Full Text]
  20. Fauchon, M., G. Lagniel, J. C. Aude, L. Lombardia, P. Soularue, C. Petat, G. Marguerie, A. Sentenac, M. Werner, and J. Labarre. 2002. Sulfur sparing in the yeast proteome in response to sulfur demand. Mol. Cell 9:713-723.[CrossRef][Medline]
  21. Friant, S., K. D. Meier, and H. Riezman. 2003. Increased ubiquitin-dependent degradation can replace the essential requirement for heat shock protein induction. EMBO J. 22:3783-3791.[CrossRef][Medline]
  22. Fujita, Y., and K. Takegawa. 2004. Characterization of two genes encoding putative cysteine synthase required for cysteine biosynthesis in Schizosaccharmomyes pombe. Biosci. Biotechnol. Biochem. 68:306-311.[CrossRef][Medline]
  23. Gasch, A. P., P. T. Spellman, C. M. Kao, O. Carmel-Harel, M. B. Eisen, G. Storz, D. Botstein, and P. O. Brown. 2000. Genomic expression programs in the response of yeast cells to environmental changes. Mol. Biol. Cell 11:4241-4257.[Abstract/Free Full Text]
  24. Georg, R. C., and S. L. Gomes. 2007. Comparative expression analysis of members of the Hsp70 family in the Chytridiomycete Blastocladiella emersonii. Gene 386:24-34.[CrossRef][Medline]
  25. Gomes, D. S., L. C. Fragoso, C. J. Riger, A. D. Panek, and E. C. Eleutherio. 2002. Regulation of cadmium uptake by Saccharomyces cerevisiae. Biochim. Biophys. Acta 1573:21-25.[Medline]
  26. Green, P. 1996. PHRAP documentation. University of Washington, Seattle. http://bozeman.mbt.washington.edu/phrap.docs/phrap.html.
  27. Gupta, S., D. Zink, B. Korn, M. Vingron, and S. A. Haas. 2004. Genome wide identification and classification of alternative splicing based on EST data. Bioinformatics 20:2579-2585.[Abstract/Free Full Text]
  28. Heckman, D. S., D. M. Geiser, B. R. Eidell, R. L. Stauffer, N. L. Kardos, and S. B. Hedges. 2001. Molecular evidence for the early colonization of land by fungi and plants. Science 293:1129-1133.[CrossRef][Medline]
  29. Huang, X., and A. Madan. 1999. CAP3: a DNA sequence assembly program. Genome Res. 9:868-877.[Abstract/Free Full Text]
  30. Imazu, H., and H. Sakurai. 2005. Saccharomyces cerevisiae heat shock transcription factor regulates cell wall remodeling in response to heat shock. Eukaryot. Cell 4:1050-1056.[Abstract/Free Full Text]
  31. Johnson, S. A., and J. S. Lovett. 1984. Gene expression during development of Blastocladiella emersonii. Exp. Mycol. 8:132-145.[CrossRef]
  32. Kim, I. S., H. Y. Moon, H. S. Yun, and L. Jin. 2006. Heat shock causes oxidative stress and induces a variety of cell rescue proteins in Saccharomyces cerevisiae KNU5377. J. Microbiol. 44:492-501.[Medline]
  33. Knop, M., A. Finger, T. Braun, K. Hellmuth, and D. H. Wolf. 1996. Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast. EMBO J. 15:753-763.[Medline]
  34. Knorre, D. A., E. A. Smirnova, and F. F. Severin. 2005. Natural conditions inducing programmed cell death in the yeast Saccharomyces cerevisiae. Biochemistry (Moscow) 70:264-266.[CrossRef][Medline]
  35. Lee, S. U., D. Yoo, J. Son, and K. Cho. 2006. Proteomic evaluation of cadmium toxicity on the midge Chironomus riparius Meigen larvae. Proteomics 6:945-957.[CrossRef][Medline]
  36. Li, L., B. P. Brunk, J. C. Kissinger, D. Pape, K. Tang, R. H. Cole, J. Martin, T. Wylie, M. Dante, S. J. Fogarty, D. K. Howe, P. Liberator, C. Diaz, J. Anderson, M. White, M. E. Jerome, E. A. Johnson, J. A. Radke, C. J. Stoeckert, Jr., R. H. Waterston, S. W. Clifton, D. S. Roos, and L. D. Sibley. 2003. Gene discovery in the apicomplexa as revealed by EST sequencing and assembly of a comparative gene database. Genome Res. 13:443-454.[Abstract/Free Full Text]
  37. Li, Z. S., Y. P. Lu, R. G. Zhen, M. Szczypka, D. J. Thiele, and P. A. Rea. 1997. A new pathway for vacuolar cadmium sequestration in Saccharomyces cerevisiae: YCF1-catalyzed transport of bis(glutathionato) cadmium. Proc. Natl. Acad. Sci. USA 94:42-47.[Abstract/Free Full Text]
  38. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2–{Delta}{Delta}CT method. Methods 25:402-408.[CrossRef][Medline]
  39. Lovett, J. S. 1975. Growth and differentiation of the water mold Blastocladiella emersonii: cytodifferentiation and the role of ribonucleic acid and protein synthesis. Bacteriol. Rev. 39:345-404.[Free Full Text]
  40. Madeo, F., E. Frohlich, M. Ligr, M. Grey, S. J. Sigrist, D. H. Wolf, and K. U. Frohlich. 1999. Oxygen stress: a regulator of apoptosis in yeast. J. Cell Biol. 145:757-767.[Abstract/Free Full Text]
  41. Maia, J. C., and E. P. Camargo. 1974. c-AMP phosphodiesterase activity during growth and differentiation in Blastocladiella emersonii. Cell. Differ. 3:147-155.[CrossRef][Medline]
  42. McAleer, M. F., and R. S. Tuan. 2001. Metallothionein protects against severe oxidative stress-induced apoptosis of human trophoblastic cells. In. Vitro Mol. Toxicol. 14:219-231.[CrossRef][Medline]
  43. Meinnel, T., A. Serero, and C. Giglione. 2006. Impact of the N-terminal amino acid on targeted protein degradation. Biol. Chem. 387:839-851.[CrossRef][Medline]
  44. Mendoza-Cozatl, D., H. Loza-Tavera, A. Hernandez-Navarro, and R. Moreno-Sanchez. 2005. Sulfur assimilation and glutathione metabolism under cadmium stress in yeast, protists and plants. FEMS Microbiol. Rev. 29:653-671.[CrossRef][Medline]
  45. Orzaez, D., A. J. de Jong, and E. J. Woltering. 2001. A tomato homologue of the human protein PIRIN is induced during programmed cell death. Plant Mol. Biol. 46:459-468.[CrossRef][Medline]
  46. Paquola, A. C., M. Y. Nishyiama, Jr., E. M. Reis, A. M. da Silva, and S. Verjovski-Almeida. 2003. ESTWeb: bioinformatics services for EST sequencing projects. Bioinformatics 19:1587-1588.[Abstract/Free Full Text]
  47. Pugliese, L. 2003. Estudo da expressão dos genes de choque térmico hsp90, hsp60 e hsp10 do fungo aquático Blastocladiella emersonii. Ph.D. thesis. Universidade de São Paulo, São Paulo, Brazil.
  48. Ribichich, K. F., R. C. Georg, and S. L. Gomes. 2006. Comparative EST analysis provides insights into the basal aquatic fungus Blastocladiella emersonii. BMC Genomics 7:177.[CrossRef][Medline]
  49. Ribichich, K. F., S. M. Salem-Izacc, R. C. Georg, R. Z. N. Vêncio, L. D. Navarro, and S. L. Gomes. 2005. Gene discovery and expression profile analysis through sequencing of expressed sequence tags from different developmental stages of the chytridiomycete Blastocladiella emersonii. Eukaryot. Cell 4:455-464.[Abstract/Free Full Text]
  50. Schena, M., D. Shalon, R. W. Davis, and P. O. Brown. 1995. Quantitative monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467-470.[Abstract/Free Full Text]
  51. Schmitt, A. O., T. Specht, G. Beckmann, E. Dahl, C. P. Pilarsky, B. Hinzmann, and A. Rosenthal. 1999. Exhaustive mining of EST libraries for genes differentially expressed in normal and tumour tissues. Nucleic Acids Res. 27:4251-4260.[Abstract/Free Full Text]
  52. Schutzendubel, A., and A. Polle. 2002. Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by mycorrhization. J. Exp. Bot. 53:1351-1365.[Abstract/Free Full Text]
  53. Shimoda, R., T. Nagamine, H. Takagi, M. Mori, and M. P. Waalkes. 2001. Induction of apoptosis in cells by cadmium: quantitative negative correlation between basal or induced metallothionein concentration and apoptotic rate. Toxicol. Sci. 64:208-215.[Abstract/Free Full Text]
  54. Silva, A. M., J. C. C. Maia, and M. H. Juliani. 1986. Developmental changes in translatable RNA species and protein synthesis during sporulation in the aquatic fungus Blastocladiella emersonii. Cell Differ. 18:263-274.[CrossRef][Medline]
  55. Silva, A. M., J. C. C. Maia, and M. H. Juliani. 1987. Changes in the pattern of protein synthesis during zoospore germination in Blastocladiella emersonii. J. Bacteriol. 169:2069-2078.[Abstract/Free Full Text]
  56. Soll, D. R., R. Bromberg, and D. R. Sonneborn. 1969. Zoospore germination in the water mold. Blastocladiella emersonii. I. Measurement of germination and sequence of subcellular morphological changes. Dev. Biol. 20:183-217.[CrossRef][Medline]
  57. Stefani, R. M., and S. L. Gomes. 1995. A unique intron containing hsp70 gene induced by heat shock and during sporulation in the aquatic fungus Blastocladiella emersonii. Gene 152:19-26.[CrossRef][Medline]
  58. Stohs, S. J., and D. Bagchi. 1995. Oxidative mechanisms in the toxicity of metal ions. Free Radic. Biol. Med. 18:321-336.[CrossRef][Medline]
  59. Uren, A. G., K. O'Rourke, L. A. Aravind, M. T. Pisabarro, S. Seshagiri, E. V. Koonin, and V. M. Dixit. 2000. Identification of paracaspases and metacaspases: two ancient families of caspase-like proteins, one of which plays a key role in MALT lymphoma. Mol. Cell 6:961-967.[Medline]
  60. Van der Auwera, G., and R. De Wachter. 1996. Large-subunit rRNA sequence of the chytridiomycete Blastocladiella emersonii, and implications for the evolution of zoosporic fungi. J. Mol. Evol. 43:476-483.[Medline]
  61. Vencio, R. Z., and T. Koide. 2005. HTself: self-self based statistical test for low replication microarray studies. DNA Res. 12:211-214.[Medline]
  62. Vido, K., D. Spector, G. Lagniel, S. Lopez, M. B. Toledano, and J. Labarre. 2001. A proteome analysis of the cadmium response in Saccharomyces cerevisiae. J. Biol. Chem. 276:8469-8474.[Abstract/Free Full Text]
  63. Wolfsberg, T. G., and D. Landsman. 1997. A comparison of expressed sequence tags (ESTs) to human genomic sequences. Nucleic Acids Res. 25:1626-1632.[Abstract/Free Full Text]
  64. Yang, Y. H., S. Dudoit, P. Luu, D. M. Lin, V. Peng, J. Ngai, and T. P. Speed. 2002. Normalization for cDNA microarray data: a robust composite method addressing single and multiple slide systematic variation. Nucleic Acids Res. 30:e15.[Abstract/Free Full Text]

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