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. 2003 Oct;8(4):317–328. doi: 10.1379/1466-1268(2003)008<0317:mcogec>2.0.co;2

Molecular characterization of genes encoding cytosolic Hsp70s in the zygomycete fungus Rhizopus nigricans

Boštjan Černila 1, Bronislava Črešnar 1,1, Katja Breskvar 1
PMCID: PMC514903  PMID: 15115284

Abstract

Previous studies have shown that some stressors, including steroid hormones 21-OH progesterone and testosterone, stimulate the accumulation of heat shock protein 70 (hsp70) messenger ribonucleic acid (mRNA) population in the zygomycete filamentous fungus Rhizopus nigricans. In this study we report the cloning of 3 R nigricans hsp70 genes (Rnhsp70-1, Rnhsp70-2, and Rnhsp70-3) encoding cytosolic Hsp70s. With a Southern blot experiment under high stringency conditions we did not detect any additional highly homologous copies of the cytosolic hsp70 genes in the R nigricans genome. Sequence analyses showed that all 3 genes contain introns within the open reading frame. The dynamics of the R nigricans molecular response to progesterone, 21-OH progesterone, and testosterone, as well as to heat shock, copper ions, hydrogen peroxide, and ethanol was studied by temporal analysis of Rnhsp70-1 and Rnhsp70-2 mRNA accumulation. Northern blot experiments revealed that the Rnhsp70-2 transcript level is not affected by testosterone, whereas mRNA levels of both genes are rapidly increased with all the other stressors studied. Moreover, the decrease of transcript levels is notably delayed in ethanol stress, and a difference is observed between the profiles of Rnhsp70-1 and Rnhsp70-2 transcripts during heat stress.

INTRODUCTION

In response to adverse environmental conditions, prokaryotes and eukaryotes increase the production of a set of proteins, collectively known as heat shock proteins (Hsps), that contribute to the protection and repair of cells under stress. Several families of Hsps have been identified; among them are the Hsp70s, which have a molecular mass of approximately 70 kDa. Although the Hsp70s were initially characterized as stress-inducible proteins (Craig 1985; Lindquist 1986), certain members of the family are expressed under physiological growth conditions as well (Lindquist and Craig 1988). The Hsp70s constitute 1 of the most evolutionarily conserved multigene families in prokaryotic and eukaryotic organisms. They appear to be absent only in some archaeal species (Gribaldo et al 1999; Macario et al 1999). In eukaryotes, the Hsp70s are found in all major cellular compartments and can be divided into 4 main groups: (1) cytosolic Hsp70s, which are considered as “typical” eukaryotic Hsp70s, (2) Hsp70s from endoplasmic reticulum, also known as Grp78 (glucose-regulated proteins) or BIP (immunoglobulin-binding proteins), (3) Hsp70s from mitochondria, which are related to Hsp70s of purple bacteria, and (4) Hsp70s from plastids, which are related to Hsp70s of cyanobacteria (Boorstein et al 1994). They participate in a variety of processes, including protein folding, assembly-disassembly of oligomeric protein complexes, protein translocation across membranes, and protein degradation (Gething and Sambrook 1992; Rassow et al 1997; Jensen and Johnson 1999). Moreover, microbial Hsp70s are also major immunogens during different types of infections (Allendoerfer et al 1996; Bromuro et al 1998).

In the field of mycology, the majority of studies on Hsp70s were performed on ascomycete fungi such as yeast Saccharomyces cerevisiae, which has proved a valuable model for the investigation of Hsp70 multigene family (Boorstein et al 1994; Lopez-Ribot and Chaffin 1996; Rassow et al 1997). In other fungal phyla, the Hsp70s were investigated only in basidiomycete Cryptococcus curvatus, chytridiomycete Blastocladiella emersonii, and zygomycete Rhizopus nigricans (Figs 3 and 4; Table 1).

Fig 3.

Fig 3.

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Intron characteristics in Rhizopus nigricans and other fungal heat shock protein (hsp70) genes. Coding regions of hsp70 genes (horizontal lines) are aligned according to the conserved Hsp70 amino acid sequence -DLGTTYSCV- in the amino terminal adenosine triphosphate–binding domain (gray boxes). The 5′-UTRs of genes are represented as thin horizontal lines. The introns (vertical lines) are characterized by position (single letter amino acid code/codon number) and phase in which they interrupt the reading frame. Phase 0 introns are positioned between 2 codons, phase 1 introns are positioned after the first base of the codon, and phase 2 introns are positioned after the second base of the codon. Lengths of introns are shown in brackets (bp). Intracellular localizations of Hsp70s are indicated: C, cytosolic Hsp70s; R, Hsp70s associated with ribosomes; ER, Hsp70s from endoplasmic reticulum. GenBank accession numbers of gene sequences are shown in brackets

Fig 4.

Fig 4.

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Neighbor-joining phylogenetic tree of fungal heat shock protein 70 (Hsp70s). Alignment of complete deduced amino acid sequences and construction of phylogenetic tree was done with ClustalX program (Thompson et al 1997). For visualization, TreeView 1.6.0 program was used (Page 1996). Intracellular localization of Hsp70s is indicated with letters: M, mitochondria; R, associated with ribosomes; ER, endoplasmic reticulum; C, cytosol. The tree was rooted with mitochondrial Hsp70s. Numbers represent bootstrap scores (out of 100 trials). GenBank accesion numbers of protein sequences are shown in brackets.

Table 1.

GenBank accession numbers of fungal Hsp70 sequences used in this study and numbers of introns in fungal hsp70 genes

graphic file with name i1466-1268-8-4-317-t01.jpg

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The zygomycete filamentous fungus R nigricans (syn. R stolonifer) is known as a steroid-biotransforming organism, able to hydroxylate progesterone, 21-OH progesterone, and testosterone with differing efficiencies (Zakelj-Mavric et al 1989). The reaction is catalyzed by a steroid-inducible multienzyme system containing cytochrome P450 as a terminal oxidase (Breskvar and Hudnik-Plevnik 1978; Makovec and Breskvar 1998). It was also established that steroids that function as inducers of the hydroxylase inhibit fungal growth rate (Breskvar et al 1995) and trigger accumulation of messenger ribonucleic acid (mRNA) for a stress protein, possibly a sugar epimerase (Cresnar et al 1998). On the basis of these results, it was assumed that the exposure of R nigricans to the toxic steroids triggers a complex molecular response in which increased hydroxylase activity, which transforms absorbed lipophilic substrates into more water-soluble products, is likely to be a part of a protective response targeted toward the maintenance of cell integrity. Recently, we characterized 2 complementary deoxyribonucleic acid (cDNA) clones (RnH20/2 and RnH2/3) encoding different cytosolic Hsp70s from R nigricans (Cernila et al 1999), and the sequence of the third cDNA encoding a cytosolic member of R nigricans Hsp70s (RnH2/5) was deposited in GenBank. Furthermore, it was confirmed that under the conditions of steroid hydroxylase induction, 21-OH progesterone and testosterone provoke an accumulation of the hsp70 mRNA population (Cernila et al 1999).

The aim of our present work was to clone and characterize the genes encoding cytosolic members of the Hsp70 gene family of R nigricans and to analyze their transcript levels under different stress conditions. We report the isolation and characterization of 3 intron-containing genes (Rnhsp70-1, Rnhsp70-2, and Rnhsp70-3) that correspond to previously analyzed hsp70 cDNA clones. In the phylogenetic tree of fungal Hsp70s, all the 3 encoded R nigricans Hsp70s are positioned within the cytosolic group. We also present evidence that in R nigricans the cytosolic subgroup of Hsp70s includes 3 members. The Rnhsp70-1 and Rnhsp70-2 mRNA levels are rapidly increased after exposure of the fungus to studied stressors with the exception of testosterone, which has influence only on the level of Rnhsp70-1 transcripts. In addition, the abundances of specific gene transcripts differ during heat stress.

MATERIALS AND METHODS

Organism and treatment of fungal mycelia

Filamentous fungus R nigricans strain ATCC 6227b was obtained from the Fungal Culture Collection of the National Institute of Chemistry (Ljubljana, Slovenia) and was cultivated as described previously (Breskvar and Hudnik-Plevnik 1978). After 18 hours of growth in 200 mL liquid cultures in a rotary shaker at 28°C, fungal mycelia were exposed to stressors for time periods ranging from 10 to 120 minutes. The stress response was triggered by the addition of steroids (progesterone, 21-OH progesterone, and testosterone) dissolved in dimethylformamide (DMF), CuSO4, H2O2, or ethanol to the fungal growth medium to final concentrations of 300 μM, 200 μM, 3 mM, and 5%, respectively. In heat stress experiments, fungal cultures were transferred to 32°C. Control experiments were conducted by growing untreated fungal mycelia in parallel at 28°C or, in the case of a steroid stress, by the addition of DMF (final concentration of 0.1%) only.

Isolation of genomic DNA

Fungal genomic DNA was isolated by a modified method of Moller (Moller et al 1992). Mycelia were washed with extraction solution (150 mM NaCl, 100 mM ethylenediamine-tetraacetic acid [EDTA], pH 8.2), filtered, and powdered in liquid nitrogen as described previously (Breskvar et al 1991). Ten grams of powdered mycelia was suspended in 20 mL boiled extraction solution, incubated for 30 minutes at 68°C with occasional gentle mixing, chilled on ice, and centrifuged at 13 000 × g for 20 minutes at 4°C. DNA from the supernatant was precipitated with ethanol and dissolved in 2 mL of Tris-EDTA-sodium dodecyl sulfate (TES) buffer (100 mM Tris, pH 8.0, 10 mM EDTA, 2% sodium dodecyl sulfate [SDS]). Subsequently, proteinase K (20 mg/mL) was added to a final concentration of 4 mg/mL. The solution was incubated for 30 minutes at 68°C, transferred on ice, and the salt concentration adjusted to 1 M NaCl. After centrifugation at 13 000 × g for 10 minutes at 4°C, DNA was precipitated with ethanol and dissolved in 1 mL Tris-EDTA (TE) buffer (10 mM Tris, pH 8.0, 20 mM EDTA). DNA was then extracted (twice with phenol, twice with phenol:chloroform 1:1, and once with chloroform) and ethanol precipitated. After dissolving in 1 mL TE buffer, NaCl was added to a concentration of 1 M. After treatment with ribonuclease (4 mg/mL) for 1 hour at 37°C, DNA was ethanol precipitated and dissolved in approximately 1 mL of TE buffer. Concentration, purity, and integrity of DNA were established by spectrophotometry and agarose gel electrophoresis.

Construction and screening of a genomic library

Genomic DNA was partially digested with Sau3A and fractionated by centrifugation through a sucrose density gradient using a conventional method (Maniatis et al 1982). Subsequently, fragments between 3 and 7 kb were used for library construction in λ ZAP expression vector, following the manufacturer's instructions (Stratagene, La Jolla, CA, USA). DNA from plaques was transferred to Nylon Hybond N+ membrane (Amersham), as described (Maniatis et al 1982), and hybridized with 0.4-kb PstI fragment of fungal RnH2/3 cDNA (AF188289) encoding the conserved N-terminal part of Hsp70. The probe was radiolabeled with α-[32P]-deoxycytidine 5′-triphosphate (Amersham, Piscataway, NJ, USA) using a Prime-It II Random Primer Kit (Stratagene). Prehybridization was carried out in 6× standard saline citrate (SSC), 0.5% SDS, 5× Denhardt's, and 15 μg/mL salmon sperm DNA for at least 4 hours at 65°C. Hybridization was performed with a final probe concentration of 0.6 × 106 cpm/mL overnight at the same temperature. A final wash was carried out in 1× SSC containing 0.1% SDS for 15 minutes at 65°C.

Selection and sequencing of genomic clones

DNA from phages that hybridized with the probe was isolated according to the manufacturer's protocol (Stratagene). Selected phage clones were converted to the pBK-CMV phagemid with ExAssist helper phage using the in vivo excision protocol (Stratagene). Restriction mapping of the genomic DNA fragments was performed with enzymes (Roche, Basel, Switzerland) BglII, ClaI, EcoRI, EcoRV, HindIII, SspI, StuI, and some combinations of them. The enzymes were chosen according to the restriction maps of Rnhsp70 cDNA clones. Double-stranded sequences of the genomic fragments were determined with ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Boston, MA, USA) in conjunction with phagemid-specific primers and synthetic primers derived from the available sequence on ABI PRISM 310 genetic analyzer (Perkin-Elmer).

Sequence analysis and phylogenetic tree construction

Sequences of 3 R nigricans genomic fragments were analyzed with software tools available on the World Wide Web. For analyses of intron numbers, lengths, and positions in other fungal hsp70 genes, sequences available in GenBank database (http://www.ncbi.nlm.nih.gov/) were used. Deduced amino acid sequences of 3 R nigricans Hsp70s and 39 other fungal Hsp70 protein sequences obtained from GenBank were used in a phylogenetic tree construction. The 39 sequences were selected on the basis of high similarity score in a BLASTP search of the GenBank fungal protein database by using the S cerevisiae SSA1 protein sequence (CAA31393) as the query sequence. Only full-length protein sequences were selected for the phylogenetic tree construction. Multiple sequence alignment and neighbor-joining tree construction were carried out with ClustalX program (Thompson et al 1997). Confidence values for the groupings in a resulting tree were derived with 100 bootstrap trials. Treeview program (Page 1996) was used for visualizing the phylogenetic tree.

Southern analysis of R nigricans genomic DNA

Ten micrograms of R nigricans genomic DNA was digested overnight with enzymes (Roche) chosen according to the restriction maps of the Rnhsp70 genes. The DNA fragments were electrophoresed on a 0.9% agarose gel. After denaturation and neutralization, DNA was transferred to Nylon Hybond N+ membrane (Amersham). The DNA blots were probed with the same radiolabeled cDNA fragment as was used for the genomic library screening. Prehybridization, hybridization, and final wash were carried out as described above.

Preparation of RNA and Northern hybridization

Total RNA from treated and untreated R nigricans mycelia was isolated as described previously (Breskvar et al 1991). Samples of the total RNA (25 μg/lane) were separated by electrophoresis in a 1.2% agarose-formaldehyde gel and transferred to Nylon Hybond N+ membranes (Amersham). The RNA blots were reversibly stained with methylene blue to check for equal sample loading (Herrin and Schmidt 1988). The 0.5-kb 3′ end PvuII fragment of the RnH20/2 cDNA (AF188288) and the 0.3-kb 3′ end HindIII fragment of the RnH2/3 cDNA (AF188289) were used as gene-specific probes in a temporal analysis of Rnhsp70-1 and Rnhsp70-2 mRNA expression, respectively. Radiolabeling of the probes, prehybridization, hybridization, and final wash were carried out as in Southern hybridization experiments.

RESULTS

Analyses of R nigricans cytosolic hsp70 genes and copy number determination

The R nigricans genomic library was screened with the 0.4-kb PstI fragment of the RnH2/3 cDNA encoding the conserved N-terminal part of RnHsp70-2, and several positive clones were obtained. Restriction analysis and partial sequencing of a few genomic fragments revealed that clones designated G17, 702B, and G16 correspond to the previously characterized cDNAs RnH20/2 encoding RnHsp70-1 (AF188288), RnH2/3 encoding RnHsp70-2 (AF188289), and RnH2/5 encoding RnHsp70-3 (AF412312), respectively. Additional sequence analyses and alignments with the cDNAs demonstrated that, in the genomic fragment of the G17 clone (AY147871), the 3′ end of the Rnhsp70-1 gene is missing, whereas the clones 702B (AY147870) and G16 (AY147869) encode full-length RnHsp70-2 and RnHsp70-3, respectively (Fig 1).

Fig 1.

Fig 1.

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Schematic representation of genomic clones G17, 702B, G16 (upper lines) and complementary deoxyribonucleic acid clones RnH20/2, RnH2/3, RnH2/5 (lower lines) containing Rnhsp70-1, Rnhsp70-2, and Rnhsp70-3 genes. GenBank accession numbers of sequences, coding regions (striped boxes), introns (open boxes), and stop codons (asterisks) are indicated

The coding regions of the Rnhsp70-1 and Rnhsp70-3 genes display very high nucleotide sequence identity (94%), whereas the sequence of the Rnhsp70-2 gene exhibits lower nucleotide identity (80%) with the other 2 genes (data not shown).

Alignments of the genomic sequences with the corresponding cDNAs indicated the presence of short introns (55 to 73 bp) in the coding regions of the Rnhsp70 genes. The number of introns in a particular gene varies, Rnhsp70-1 having at least 2 and Rnhsp70-2 and Rnhsp70-3 1 and 3 introns, respectively. In addition, a comparison of the 3′-UTRs of the Rnhsp70 genes or cDNAs (or both) revealed that there is a difference in the length of the 3′-UTRs between the Rnhsp70-1 and the other 2 genes and that the 3′-UTR of Rnhsp70-2 contains consecutive tetranucleotide repeats (Fig 2).

Fig 2.

Fig 2.

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Comparison of 3′-UTRs of Rnhsp70-1, Rnhsp70-2, and Rnhsp70-3. Tetranucleotide CATT elements are indicated

From Table 1 it can be seen that in fungal hsp70 genes encoding Hsp70s from different cellular compartments different numbers of introns are present, ranging from 1 in B emersonii hsp70 gene (L22497) to up to 8 in Emericella nidulans hsp70 gene (X98931) and that in some species, especially in ascomycete yeasts (eg, S cerevisiae, Pichia angusta, Candida albicans, and Schizosaccharomyces pombe), the coding regions of their hsp70 genes are not interrupted with introns. Moreover, from Figure 3 it is evident that the intron positions are not conserved, with the exception of the introns in hsp70 genes of closely related species (eg, Aspergillus spp.). It should also be noted that in almost all intron-containing fungal hsp70 genes known so far, at least 1 intron is present in the amino terminal region in the vicinity of the Hsp70 conserved -DLGTTYSCV- sequence of the adenosine triphosphate–binding domain.

The complete deduced protein sequences of 3 R nigricans Hsp70s together with 39 other fungal Hsp70 sequences that were obtained from GenBank were used for the phylogenetic tree construction. The resulting tree shows 4 distinct branches (Fig 4). Three branches include the Hsp70 family members of different intracellular compartments (cytosol, endoplasmic reticulum, and mitochondria), and the fourth branch represents the ribosome-associated Hsp70s that appear to be structurally different from the other cytosolic Hsp70s. All 3 R nigricans Hsp70s are clustered with the fungal cytosolic Hsp70s. This result and the identification of degenerate repeats of the tetrapeptide GGMP in the C-terminal regions as well as the presence of the cytosolic Hsp70-specific motif -EEVD- at the C-terminus of encoded proteins strongly supports the cytoplasmic localization of the Rnhsp70 gene products (data not shown).

The copy number of cytosolic hsp70 genes in the R nigricans genome was assessed by Southern hybridization at high stringency (Fig 5). The genomic DNA was digested with selected enzymes and probed with the radiolabeled 0.4-kb fragment of RnH2/3 cDNA encoding the conserved N-terminal part of RnHsp70-2. The sizes of DNA fragments that hybridized correspond to those expected from the restriction maps of isolated R nigricans genomic clones. Although it is very likely that the R nigricans genome also contains more diverged genes encoding Hsp70s from other groups (eg, Hsp70s from endoplasmic reticulum and mitochondrial Hsp70), under the conditions used, we could detect only 3 genes highly homologous to the probe. The result of the Southern hybridization thus suggests that the R nigricans genome contains 3 copies of genes encoding Hsp70s of the cytosolic group.

Fig 5.

Fig 5.

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Southern hybridization of Rhizopus nigricans genomic deoxyribonucleic acid (DNA) and partial restriction maps of genomic clones G17, 702B, and G16. (A) Genomic DNA (10 μg/lane) cut with restriction enzymes HindIII (H), HindIII/PstI (HP), HindIII/EcoRI (HE), HindIII/XbaI (HX), and HindIII/SspI (HS) was hybridized with N-terminal fragment of RnH2/3 complementary DNA (0.4 kb) encoding conserved part of Hsp70s. (B) The striped boxes indicate regions in genomic clones that were homologous to the probe used in Southern hybridization. The restriction fragments of individual genomic clones and corresponding hybridization signals are labeled with numbers

Northern analysis of Rnhsp70-1 and Rnhsp70-2 mRNA levels during steroid-induced and other stresses

The changes in transcript levels of 2 cytosolic Rnhsp70s under different stress conditions were studied by Northern blot analysis. Temporal analyses of Rnhsp70-1 and Rnhsp70-2 mRNA abundance were carried out during treatment of fungal mycelium with progesterone (300 μM), 21-OH progesterone (300 μM), testosterone (300 μM), heat stress (32°C), CuSO4 (200 μM), hydrogen peroxide (3 mM), and ethanol (5%) (Fig 6). The Rnhsp70-1 and Rnhsp70-2 mRNA levels are nearly undetectable under normal growth conditions, as well as when the steroid carrier DMF was added to the growth medium. It is evident that no increase in the transcript level of Rnhsp70-2 was detected in response to testosterone, whereas all the other stressors tested influenced the level of both fungal hsp70 transcripts. Despite the rapid and nearly universal increase in transcript levels of Rnhsp70-1 and Rnhsp70-2 within 10 to 20 minutes, the duration of increased mRNA amount is diverse and depends on the transcript species and on the type of stressor. There is an evident difference in the profiles of Rnhsp70-1 and Rnhsp70-2 mRNA abundance during the response of the fungus to heat stress. The Rnhsp70-1 transcripts rapidly decline to the pre–heat-stress level after 40 minutes, whereas the amount of Rnhsp70-2 mRNA still remains above the control level after 120 minutes. In response to various stressors, 3 modes of decrease in the Rnhsp70 mRNA levels could be discerned: rapid, after 20 minutes; moderate, within 40 to 80 minutes; and slow, more than 120 minutes of exposure of the fungus to stressors.

Fig 6.

Fig 6.

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Temporal analysis of Rnhsp70-1 and Rnhsp70-2 mitochondrial ribonucleic acid (mRNA) levels during various stress conditions. Filamentous fungus Rhizopus nigricans was exposed for indicated times to different steroid stressors (P, 300 μM progesterone; DOC, 300 μM 21-OH progesterone; T, 300 μM testosterone), steroid carrier dimethylformamide (DMF) alone (C, 0.1% DMF), or other stressors (HS, heat stress 32°C; Cu, 200 μM CuSO4; HP, 3 mM H2O2; Et, 5% ethanol). Total RNA was isolated, blotted to nylon membranes (25 μg/lane), stained reversible with methylene blue (lower panels), and hybridized with radiolabeled 0.5-kb PvuII fragment or 0.3-kb HindIII fragment of complementary deoxyribonucleic acid encoding RnHsp70-1 or RnHsp70-2, respectively (upper panels)

DISCUSSION

We report the identification and molecular characterization of 3 genes (Rnhsp70-1, Rnhsp70-2, and Rnhsp70-3) encoding Hsp70s from the zygomycete filamentous fungus R nigricans. The encoded protein sequences possess multiple degenerate tetrapeptide GGMP repeats and the EEVD motif at the carboxy terminus (data not shown), which are typical structural elements of eukaryotic cytosolic Hsp70s (Boorstein et al 1994; Freeman et al 1995). Moreover, in the phylogenetic tree of fungal Hsp70s, the R nigricans Hsp70s cluster together with the other known cytosolic Hsp70s (Fig 4). These findings strongly suggest that proteins synthesized from the Rnhsp70 genes belong to the cytosolic subgroup of the Hsp70 family.

The coding regions of the R nigricans hsp70 genes are interrupted with different numbers of short introns (Fig 3). The interesting feature of their positions is that the first and the second intron in Rnhsp70-1 are identical according to the disrupted codon number and phase to the first and the third intron in Rnhsp70-3, respectively. This characteristic together with very high percentage (94%) of nucleotide identity in the coding regions of those 2 genes (data not shown) strongly suggests that Rnhsp70-1 and Rnhsp70-3 are paralogs that arose from a common ancestor by duplication. The presence of an additional intron in Rnhsp70-3 therefore could indicate that either this gene recently gained this intron or, alternatively, an intron was lost from the Rnhsp70-1 gene. The presence of introns in hsp70 genes is not unique to R nigricans because several intron-containing stress-responsive and constitutively expressed hsp70 genes were already described in other fungal species (Table 1). On the other hand, coding regions of several yeast hsp70 genes are not interrupted with introns. The comparison of the intron positions in hsp70 genes of different fungal species also revealed that the intron positions are not conserved except in closely related species (eg, Aspergillus spp.). Studies in Drosophila (Yost and Lindquist 1986), yeast (Yost and Lindquist 1991), and human cells (Bond 1988) showed that severe heat stress inhibits mRNA splicing and that most of the hsp70 genes in these organisms are free of introns. Therefore, it was proposed that in contrast to constitutively expressed intron-containing hsp70 genes, stress-inducible ones should be, in accordance with their function in stressed cells, intronless. However, the presence of introns also was reported in stress-inducible hsp70 genes of different organisms, including nematode Caenorhabditis elegans (Heschl and Baillie 1990), plant Spinacia oleracea (Guy and Li 1998), and several fungi such as B emersonii (Stefani and Gomes 1995), Paracoccidioides brasiliensis (da Silva et al 1999), and Trichophyton rubrum (Rezaie et al 2000). Moreover, it was shown that in the above-mentioned fungi splicing is highly thermoresistant. The identification of introns in R nigricans stress-inducible hsp70 genes contributes additional evidence that the absence of introns in these genes is not a general rule.

The Southern blot analysis of genomic DNA suggested that the genome of the fungus R nigricans contains 3 copies of hsp70 genes encoding cytosolic Hsp70s (Fig 5). In different fungal species the copy numbers of these genes are different. Until now, the copy numbers were determined in B emersonii (Stefani and Gomes 1995) and T rubrum (Rezaie et al 2000), where a single-copy cytosolic hsp70 gene was reported, whereas there are 4 copies of differentially regulated cytosolic hsp70 genes in the genome of S cerevisiae (Boorstein et al 1994). The high degree of sequence conservation of Hsp70s makes them particularly useful in investigating deep phylogenetic relationships (Borchiellini et al 1998). However, when inferring organismal phylogenies from Hsp70s, the existence of several paralogous genes in a species, as was observed in R nigricans, should be considered (Martin and Burg 2002).

Our previous (Cernila et al 1999) and present Northern analyses revealed that under the conditions used, no levels of Rnhsp70-1 and Rnhsp70-2 mRNAs were detected in the fungal mycelia under normal growth conditions. However, the amounts of Rnhsp70-1 and Rnhsp70-2 transcripts are rapidly increased, not only with generally studied stressors, of which ethanol is the most toxic agent, but also with steroids, including progesterone, 21-OH progesterone, and testosterone (Fig 6). The observation that only the Rnhsp70-1 mRNA level is increased on testosterone treatment of the fungus suggests that the mechanism of regulation of Rnhsp70-1 and Rnhsp70-2 by testosterone is different from that of progesterone and 21-OH progesterone. An upregulation of hsp70 mRNA with a steroid hormone was previously observed only in oomycete Achlya ambisexualis (Silver et al 1993; Brunt et al 1998). Because of their filamentous growth, oomycetes were considered as a group of fungi, but according to the phylogenetic analysis of 16S ribosomal RNA, they are evolutionarily more closely related to organisms such as diatoms and brown algae (Sogin and Silberman 1998). In A ambisexualis the steroid hormone antheridiol acts as a pheromone and induces developmental changes that lead to sexual reproduction. On the other hand, in zygomycetes, the pheromone system includes trisporic acid instead of steroids (Czempinski et al 1996). Therefore, in the fungus R nigricans, steroids progesterone, 21-OH progesterone, and testosterone most likely represent xenobiotics, which are, in turn, hydroxylated to less toxic hydrophobic products by inducible multienzyme system (Breskvar et al 1995). Recently, steroid-binding sites with high affinity for progesterone and lower affinity for 21-OH progesterone and testosterone were detected in the enriched R nigricans plasma membrane fractions (Lenasi et al 2002). Moreover, the results of that study suggested that progesterone receptors are coupled to G-proteins, that could be involved in the activation of different intracellular signaling pathways.

There was also an evident difference in the downregulation of Rnhsp70-1 and Rnhsp70-2 mRNA levels in the heat-stressed fungus. In response to the elevated temperature, the level of Rnhsp70-1 transcripts declined more rapidly than the level of Rnhsp70-2 mRNA. Furthermore, the analyses of R nigricans hsp70 cDNAs revealed that the 3′-UTR of Rnhsp70-2 differs from that of Rnhsp70-1 in length and in the presence of consecutive CATT tetranucleotide repeats (Fig 2). These results possibly indicate a difference in the mRNA stability. Although the expression of stress-inducible hsp70 genes is primarily regulated at the transcriptional level (Mason and Lis 1997; Morimoto 1998), regulation at the level of mRNA stability has also been reported. In Drosophila, a rapid degradation of hsp70 mRNA at normal temperatures and greater stability during heat stress was observed (Petersen and Lindquist 1988), and the 3′ end of the hsp70 mRNA was shown to be important in the regulation of its turnover (Simcox et al 1985). The effects of 3′-UTR on hsp70 transcript stability also were described in Trypanosoma brucei (Lee 1998) and pigs (Schwerin et al 2002). However, to our knowledge, the CATT element has not yet been associated with mRNA stability.

Analyses of the promoter regions of Rnhsp70-1, Rnhsp70-2, and Rnhsp70-3 genes and the 3′-UTRs of their transcripts will give further insights into the regulation of expression of steroid-inducible cytosolic hsp70 genes in zygomycete R nigricans.

Acknowledgments

This work was financially supported by the Slovenian Ministry of Education, Science, and Sport. The authors are grateful to Savica Soldat for skillful technical assistance.

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