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Genetics, Vol. 173, 87-98, May 2006, Copyright © 2006
doi:10.1534/genetics.105.053199
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* Plant Science Initiative and Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68588 and Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, CEP 14040-903 São Paulo, Brazil
1 Corresponding author: Plant Science Initiative, University of Nebraska, The Beadle Center N234, 19th and Vine Sts., P. O. Box 880665, Lincoln, NE 68588-0660.
E-mail: sharri1{at}unlnotes.unl.edu
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ABSTRACT |
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The canonical PARP enzyme, human PARP1, is a 116-kDa protein that consists of three domains: an N-terminal DNA-binding domain containing two zinc fingers and a nuclear localization sequence, a central auto-modification domain that includes a BRCA1 C-terminus domain (BRCT) motif, and a C-terminal catalytic domain (summarized by AME et al. 2004 and KIM et al. 2005). PARP enzymes have been identified throughout the animal and plant kingdoms, with the catalytic domain exhibiting the greatest degree of sequence similarity. Indeed, members of the PARP superfamily are defined by the presence of this domain. By contrast, the DNA-binding and auto-modification domains are not very well conserved and are usually replaced with alternative domains that reflect the function of a specific PARP isoform (AME et al. 2004). The functional characterization of PARP isoforms has typically relied upon the construction of knockout mutants or the alteration of PARP expression (KIM et al. 2005). However, this type of approach has been limited by functional redundancy among PARP isoforms. For example, the functions of PARP1 and PARP2 may overlap during the DNA damage response, but the embryonic lethality of parp1–/– parp2–/– double mutants has precluded systematic attempts to determine the consequences of complete PARP inactivation (MENISSIER DE MURCIA et al. 2003). The existence of several small molecule inhibitors that target the PARP catalytic domain has also permitted the use of chemical genetics, but these studies are potentially compromised by the likelihood that PARP has additional "scaffolding" functions that are not affected by the inhibitors (JAGTAP and SZABO 2005). Although these problems could conceivably be circumvented by the use of microbial genetics, PARP function has not yet been characterized in any microbial system. Notably, no recognizable homolog of PARP exists in the proteome of either Saccharomyces cerevisiae or Schizosaccharomyces pombe.
The filamentous fungus Aspergillus nidulans possesses a sophisticated DNA damage response that ensures the maintenance of genome integrity (reviewed by GOLDMAN et al. 2002). Characterization of this response has revealed that key regulatory proteins such as UvsBATR, AtmAATM, components of the Mre11 complex (ScaANBS1, MreAMRE11, and SldIRAD50), the cdc2-related kinase NpkA, the signalosome, and SepBCTF4 promote DNA repair and cell cycle arrest (HOFMANN and HARRIS 2000; BRUSCHI et al. 2001; SEMIGHINI et al. 2003; FAGUNDES et al. 2004, 2005; LIMA et al. 2005; GYGAX et al. 2005; MALAVAZI et al. 2005, 2006). However, it remains unclear how hyphal cells initially sense the presence of DNA damage and trigger the appropriate repair and checkpoint pathways. Moreover, because A. nidulans hyphae are multicellular (HARRIS 1997), their response to DNA damage is likely to be more complex than the well-characterized response of yeast cells. To determine if A. nidulans possesses DNA damage response functions that are uniquely conserved with multicellular eukaryotes, the recently completed and annotated A. nidulans genome sequence was screened for homologs of repair and DNA damage signaling proteins that are not conserved in S. cerevisiae or S. pombe. This screen uncovered a single PARP homolog that is conserved in all filamentous fungi and is closely related to animal and plant PARP1. THRANE et al. (2004) also noted the existence of this homolog and provided evidence that it is subjected to caspase-mediated cleavage. In this study, we show that the putative A. nidulans PARP homolog, PrpA, is an essential protein that functions early in the DNA damage response. We also demonstrate that PrpA is required for programmed cell death and for asexual development. Our results suggest that PARP-mediated ADP ribosylation may be a broadly important feature of fungal physiology.
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MATERIALS AND METHODS |
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Cloning the prpA gene:
Standard methods were used to prepare genomic DNA and total RNA from lyophilized mycelia obtained from the strain A28. Both genomic and cDNA fragments were fully sequenced using gene-specific oligonucleotide primers (sequence available from authors upon request). The resulting prpA sequences were deposited at GenBank (accession nos. AY349148 and AY347573, respectively). A Northern blot was performed to verify the expression of prpA. Total RNA was extracted from strain A28 and 20 µg was fractionated in a 1.2% agarose formaldehyde gel. After transfer to Hybond N+ membrane (Amersham, Buckinghamshire, UK), hybridization was performed using [
-32P]dCTP primer-labeled cDNA probes spanning the conserved prpA catalytic and regulatory domains at its C terminus.
Real-time RT–PCR reactions:
Conidia of the indicated strains were inoculated into growth media and treated as described in the legends for Figures 4 and 5. Mycelia were harvested by filtration through a Whatman filter no. 1 (Nalgene) and total RNA was extracted and treated with RNAse-free DNAse (Promega, Madison, WI) as described previously (SEMIGHINI et al. 2002). Real-time RT–PCR reactions were performed using an ABI Prism 7700 sequence detection system (Perkin-Elmer Applied Biosystem) and the Taq-ManR EZ RT–PCR kit (Applied Biosystems, Foster City, CA) as described previously (SEMIGHINI et al. 2002). The measured quantities of each sample were normalized using tubC mRNA amplifications run in the same plate. Sequences of all primers and probes used are available from the authors upon request.
Construction of the 
prpA strain:
A fusion PCR-mediated approach was used to construct the prpA gene replacement strain as previously described (YANG et al. 2004). Three initial amplifications generated 5'- and 3'-flanking regions of the prpA gene (using primers GSP1 and GSP2 and GSP3 and GSP4, respectively) and the pyr4 gene (using primers PYR1 and PYR2), which was employed as a selectable nutritional marker. A final fusion PCR using primers GSP1 and GSP4 amplified a full-length deletion cassette containing the three previous fragments. All PCR amplifications were performed using the Expand Long Template PCR kit (Boehringer Mannheim, Indianapolis) and the amplification conditions specified by YANG et al. (2004). The sequences of oligonucleotide primers were as follows: GSP1, 5'-GGTGCTTATGTTTACAACCGTCGC-3'; GSP2, 5'-TATACAGTAGGTCGCTTCGAATGGTTGCCG-3'; GSP3, 5'-ATCAAATCAACACCCGCCCTCGTATGA-3'; GSP4, 5'-TCACAGCACATCATCACCTTGGCTA-3'; PYR1, 5'-CGGCAACCATTCGAAGCGACCTACTGTATAATCTCCTTACGCATCTGTGCG-3' (underlined bases indicate the region of homology to GSP2); PYR2, 5'-TCATACGAGGGCGGGTGTTGATTTGATATGCATCAGAGCAGATTGTACTG-3' (underlined bases indicate the region of homology to GSP3).
Transformation of the pyrG89 strains GR5 and ACS11 was performed using a previously described protocol (OSMANI et al. 1987) and 5 µg of the deletion cassette. Positive transformants were identified on the basis of their ability to grow on selective media. Southern blot analysis demonstrated that one full-length endogenous wild-type copy of prpA was lacking from the genome of one transformant (ACS14 strain).
Construction of the prpA::GFP strain:
The C-terminal 1175 bp of the prpA coding region minus the stop codon was amplified from genomic DNA and cloned into pCR2.1-TOPO (Invitrogen, San Diego). Following digestion with KpnI and BamHI, the resulting fragment was cloned into KpnI/BamHI-digested pCP19 (PEARSON et al. 2004). This generated a C-terminal prpA::GFP fusion fragment that was transformed into strain GR5. Analysis of Southern blots identified transformants that had undergone homologous integration of the construct at the prpA locus. The desired transformants (i.e., strain ACS6) possess a full-length copy of prpA::GFP expressed under the control of endogenous promoter sequences, plus an N-terminal fragment of prpA that is not fused to GFP and possesses no obvious promoter sequences. Expression of the recombinant PrpA::GFP was confirmed by analysis of Western blots using both anti-GFP (Molecular Probes, Eugene, OR) and a commercial PARP antibody (Cell Signaling). Each antibody was used at a concentration of 1 µg/ml, and peroxidase-conjugated goat anti-rabbit (Sigma, St. Louis) was diluted to 1:10,000 was used as the secondary antibody. Because strain ACS6 is indistinguishable from wild-type controls strains in terms of radial growth rates, levels of conidiation, and resistance to DNA-damaging agents, we conclude that the PrpA::GFP fusion is most likely a functional protein.
Construction of alcA::GFP::prpA strain:
An 852-bp fragment from the 5'-end of the prpA coding region was amplified from genomic DNA. After digestion with AcsI and PacI, the resulting fragment was cloned into AcsI/PacI-digested pMBC17ap (EFIMOV 2003), thereby producing a fusion of alcA(p)::GFP to the 5' half of prpA. Upon transformation into strain GR5, homologous integration at the prpA locus should generate a full-length alcA(p)::GFP::prpA fusion flanked by a truncated version of prpA regulated by native promoter sequences. Transformants were screened by PCR using a forward primer designed to recognize the alcA promoter sequence and a reverse primer designed to recognize the prpA sequence (starting at 1063 bp). Multiple transformants with the desired integration event were recovered, one of which (i.e., strain ACS9) was sequenced to verify homologous integration of the alcA(p)::GFP::prpA fusion. Expression of the fusion under control of the alcA promoter was confirmed by both real-time RT–PCR and the analysis of Western blots. For the real-time RT–PCR analysis, oligonucleotide primers that are capable of binding full-length prpA were used. For the Western analysis, blots of total protein extracts were probed with a 1:1000 dilution of a rabbit polyclonal anti-PARP antibody (Cell Signaling Technologies, no. 9542), followed by a 1:10,000 dilution of an alkaline phosphatase-conjugated anti-rabbit immunoglobulin G antibody (Sigma A3687). Bands were detected using enhanced chemiluminescence (Roche, Indianapolis).
Microscopy:
After 12 hr of germination on coverslips in YGV medium at 30°, strains were treated with DNA-damaging agents, oxidizing agents, or chemical inhibitors as indicated for each experiment. Coverslips were fixed and stained with Hoechst 33258 (Molecular Probes) as described by HARRIS et al. (1994). Slides were viewed using an Olympus BX51 fluorescent microscope and individual images were captured with a Photometrics CoolSnap HQ CCD camera. Confocal images were obtained with an Olympus FW500/BX61 confocal laser-scanning microscope using the following laser lines: 405 nm for Hoechst 33258 and 488 nm for GFP. Images were captured by direct acquisition with a Z step of 1 µm and were subsequently processed using Adobe PhotoShop 6.0.
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RESULTS |
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2.0-kb transcript. Hereafter, we refer to the predicted AN3129.2 protein as PrpA.
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prpA is an essential gene and displays dose-dependent effects:
To determine the function of PrpA, we constructed a complete gene replacement using the fusion PCR-mediated approach described by YANG et al. (2004). Multiple attempts to transform this construct into the haploid strain GR5 yielded no viable transformants that could be subcultured under selective conditions (Figure 2A). Because this observation suggested that a prpA deletion might be lethal, we repeated the transformation using a diploid recipient strain (ACS11). This led to the recovery of a single diploid transformant (ACS14) in which one copy of prpA had been replaced with prpA::pyrG, whereas the other copy remained intact (Figure 2B). Strain ACS14 was haploidized using standard approaches (HASTIE 1970), but the only viable haploid segregants possessing the gene replacement also contained additional copies of prpA as determined by PCR and the analysis of Southern blots (data not shown). Taken together, these observations strongly suggest that prpA is an essential gene in A. nidulans. Although strain ACS14 is heterozygous for the prpA gene replacement (Figure 2C), it displayed a series of phenotypic defects when compared to a wild-type diploid strain (ACS11). First, prpA/+ colonies are approximately one-half the diameter of wild-type controls (Figure 2B; also see Figure 5A). Second, prpA/+ colonies frequently possess sectors (Figure 2B; 61.5%, n = 300) that presumably reflect genome instability (KAFER and UPSHALL 1973). Analysis of these sectors showed that they consist of diploid clones that had presumably undergone homologous recombination leading to loss of heterozygosity for either the wA or the yA spore color markers. Third, the prpA/+ strain displayed sensitivity to numerous chemical compounds that cause DNA or oxidative damage (see Figure 5A). Notably, growing prpA/+ hyphae were extremely sensitive to NCT, which is a known feedback inhibitor of PARP catalytic activity (HAGEMAN and STIERUM 2001). This result suggests that an 50% reduction of PARP activity in the prpA/+ strain sensitized it to further inhibition of activity caused by NCT. Real-time RT–PCR was used to confirm that prpA transcript levels were reduced by 50% in the prpA/+ strain relative to a wild-type diploid (Figure 2D). Collectively, the phenotypes displayed by strain ACS14 are consistent with the notion that prpA is a haplo-insufficient gene in A. nidulans, whereby a single functional copy of the gene cannot support the growth of a diploid strain or a heterokaryon.
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104-fold reduction in spore production (A28, 3.9 x 107 spores/ml; ACS9, <1 x 103 spores/ml; average of two independent experiments). Microscopic examination of the fluffy colonies showed that they largely consist of highly vacuolated aerial hyphae (Figure 4A).
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A core transcriptional regulatory pathway controls developmental gene expression during conidiation in A. nidulans (reviewed by ADAMS et al. 1998). The two key transcriptional factors in this pathway are BrlA and AbaA, which bind to specific sequences in the promoters of developmentally regulated genes (CHANG and TIMBERLAKE 1993; ANDRIANOPOULOS and TIMBERLAKE 1994). By scanning the 2000 bp located immediately upstream of the predicted prpA translational start site, we identified a single consensus BrlA-response element (–832CAAGGGG–826) and two consensus AbaA-binding sites (–1825CATTCT–1820; –1657CATTCC–1652). To determine if AbaA regulates the developmental induction of prpA, we employed a strain in which abaA expression is placed under control of the heterologous alcA promoter. When induced in submerged hyphal cultures that are normally not permissive for development, alcA::abaA causes morphological changes that mimic conidiation (MIRABITO et al. 1989). Using real-time RT–PCR, we found that induction of alcA::abaA under these conditions (i.e., in MN–ethanol) triggered an approximately sixfold increase in prpA transcript levels (Figure 4C). These observations strongly suggest that prpA is a target of the core transcriptional pathway that regulates gene expression during conidiation.
Function of PrpA in the DNA damage response:
In animal cells, PARP is involved in signaling the presence of DNA damage and also regulates the recruitment of DNA repair enzymes to sites of damage (AME et al. 2004). The presence of a single PARP homolog in A. nidulans provides a unique opportunity to determine the specific role(s) of PARP in the DNA damage response of a microbial system. We first confirmed that PrpA is involved in the A. nidulans DNA damage response by testing heterozygous prpA/+ hyphae for sensitivity to a number of DNA-damaging agents. As shown in Figure 5A, the haplo-insufficient prpA/+ mutant is particularly sensitive to PLM, which primarily causes double-strand breaks, and the UV mimetic agent 4-NQO. Although the mutant does not appear to be sensitive to other agents, such as CPT, HU, and MMS (Figure 5A), this may be because a single copy of prpA is sufficient for a normal response to these agents. At the very least, these observations implicate PrpA in the fungal DNA damage response.
To further assess the role of PrpA in the DNA damage response, we used real-time RT–PCR to examine transcript levels in hyphae exposed to DNA-damaging agents. Notably, expression of prpA was dramatically induced (i.e., 10- to 120-fold) in response to MMS, BLEO, or 4-NQO (Figure 5B). Moreover, this response was rapid, in that induction was evident within 10 min of treating hyphae with the DNA-damaging agent (Figure 5B). By contrast, other A. nidulans genes encoding proteins involved in DNA damage signaling and repair (i.e., scaANBS1, mreAMRE11, sldIRAD50) generally display peak transcript levels 1–2 hr after exposure (SEMIGHINI et al. 2003). This observation suggests that PrpA may function early in the fungal DNA damage response.
We have recently demonstrated that A. nidulans proteins involved in signaling the presence of DNA damage and in DNA repair (i.e., UvsCRAD51 and ScaANBS1) are recruited to nuclei following exposure to DNA-damaging agents (FAGUNDES et al. 2005; GYGAX et al. 2005). To determine if PrpA displays a similar localization pattern, and to examine its kinetics, we constructed a functional PrpA::GFP fusion that is expressed under control of native promoter sequences (see MATERIALS AND METHODS). In the absence of DNA damage, PrpA::GFP did not localize to any discrete structure in growing hyphae (Figure 5, C and D). However, upon exposure to a DNA-damaging agent (i.e., 0.025% MMS), PrpA::GFP localized to nuclei (Figure 5, C and D). Most importantly, nuclear localization peaked at 10 min following exposure to DNA damage and had largely disappeared by 30 min (Figure 5D). By comparison, UvsCRAD51 typically cannot be detected in nuclei until at least 60 min after exposure (GYGAX et al. 2005). These results provide additional support for the notion that PrpA acts early in the fungal DNA damage response. Moreover, they raise the possibility that PrpA may be recruited to sites of DNA damage.
Although the analysis of the prpA/+ mutant did not reveal any sensitivity to oxidizing agents (i.e., H2O2, MENA, T-BUT; Figure 5A), we found that PrpA also localized to nuclei within 10 min of exposure to H2O2 (Figure 5, C and D). Furthermore, real-time RT–PCR analysis also revealed that prpA transcript levels increase 10-fold upon exposure to H2O2 (Figure 5B). These results may indicate that prpA is involved in the response to oxidative DNA damage.
PrpA is required for farnesol-induced cell death:
We have recently demonstrated that the small-molecule lipid farnesol causes apoptosis in A. nidulans in a manner that depends on functional mitochondria and the production of reactive oxygen species (SEMIGHINI et al. 2006). Because PARP activity has been implicated in an apoptosis pathway that requires mitochondria (YU et al. 2002), we tested the role of PARP in farnesol-induced apoptosis. First, using nuclear condensation as a marker for cell death, we found that the PARP feedback inhibitor NCT suppressed the effects of farnesol (Figure 6A). Second, in a more specific test, we found that the ability of farnesol to trigger apoptosis was severely compromised in the prpA/+ mutant (Figure 6B). In particular, a farnesol concentration (100 µM) that typically causes nuclear condensation in 90% of diploid wild-type hyphae caused a similar effect in <20% of prpA/+ hyphae. This observation implicates PrpA in at least one fungal cell death pathway.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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Genetics of PrpA:
A defining feature of the PARP superfamily of proteins is the presence of a highly conserved catalytic domain usually located at the C terminus (reviewed by AME et al. 2004). Because the C-terminal half of PrpA possesses adjacent PARP catalytic (pfam00644) and PARP regulatory (pfam02877) domains, and because this is the region with the highest homology to other PARP homologs, we are extremely confident that PrpA is a bona fide PARP homolog. Furthermore, the presence of the BRCT motif and the WGR domain suggests that PrpA is more closely related to PARP1 and PARP2 than it is to other members of the PARP superfamily (AME et al. 2004). However, several important questions remain unanswered. First, an N-terminal zinc-finger DNA-binding domain is conspicuously absent from PrpA. Accordingly, it is not clear how PrpA binds DNA. This function could be mediated by the WGR domain, which has been implicated in the binding of nucleic acids (i.e., see http://www.sanger.ac.uk/cgi-bin/Pfam/getacc?PF05406). Alternatively, PrpA may not bind DNA directly and may instead interact with a distinct zinc-finger DNA-binding protein. Second, PARP1 possesses a caspase cleavage site located in the DNA-binding domain (KAUFMANN et al. 1993), and cleavage of PARP is recognized as an early marker for caspase-mediated apoptosis. However, no obvious caspase cleavage site is present in PrpA. Although it was previously reported that A. nidulans PARP could be cleaved by caspases in vitro (THRANE et al. 2004), we have not been able to confirm this observation using the same commercial antibody on in vivo extracts (C. SEMIGHINI and S. HARRIS, unpublished observations). Thus, if PrpA is cleaved at all, the relevant protease presumably acts at a novel site. Third, in animals and plants, PARP1 activity is countered by PARG (D'AMOURS et al. 1999; DAVIDOVIC et al. 2001), which cleaves ADP-ribose moieties from PARP substrates. Surprisingly, no obvious PARG homolog could be detected in the genome of A. nidulans or in any other sequenced filamentous fungal genome, despite exhaustive searches. Therefore, an alternative glucohydrolyase may be responsible for this function in filamentous fungi. Finally, it remains to be shown that PrpA does indeed possess PARP catalytic activity. If so, A. nidulans will provide a powerful system for the identification of relevant PARP substrates.
The functional characterization of PrpA is complicated by the observation that it appears to be an essential protein. Furthermore, attempts to generate a conditional allele were foiled by the failure of glucose to repress the expression of alcA(p)-regulated prpA. At this time, we have no explanation for the failed repression, but it has been observed in multiple transformants in different strain backgrounds (C. SEMIGHINI and S. HARRIS, unpublished observations). Therefore, the future functional characterization of PrpA will likely depend on the construction of specific mutations that affect its catalytic activity or possible scaffolding functions.
Haplo-insufficiency is a common genetic phenomenon that in most cases has been attributed to gene dosage effects (SEIDMAN and SEIDMAN 2002). Indeed, a recent comprehensive study in yeast found that 3% of 5900 tested genes were haplo-insufficient (DEUTSCHBAUER et al. 2005). Notably, most of the haplo-insufficient genes were implicated in basic cellular processes, thereby suggesting that insufficient protein production was the underlying cause. We suspect that this is also the basis for the haplo-insufficient phenotypes displayed by the prpA/+ mutant. The alternative hypothesis that haplo-insufficiency is caused by the altered stoichiometry of components within a multi-protein complex predicts that overexpression and haplo-insufficient phenotypes would be the same (PAPP et al. 2003), which was not observed in our case. Strikingly, PARP1 haplo-insufficient phenotypes have been observed in mammalian cells (HANDE 2004) and other important DNA damage signaling proteins (i.e., ATM and BLM; GOSS et al. 2002; SPRING et al. 2002) also display gene dosage effects. These observations suggest that the levels of key regulatory proteins may be precisely calibrated during the DNA damage response.
Role of PrpA in the responses to DNA damage and cellular stress:
Three observations suggest that PrpA is involved in the fungal DNA damage response. First, the prpA/+ mutant is sensitive to the DNA-damaging agents PLM and 4-NQO. Second, prpA transcript levels accumulate within 10 min of exposure to DNA-damaging agents. Third, PrpA transiently localizes to nuclei within a 10- to 20-min window after exposure to DNA damage. Because the increased expression and nuclear localization of other DNA damage signaling and repair proteins typically occurs within 1–2 hr after DNA damage (SEMIGHINI et al. 2003; FAGUNDES et al. 2005; GYGAX et al. 2005), these observations imply that PrpA performs an early signaling function in the DNA damage response. By analogy to PARP1 (D'AMOURS et al. 1999), PrpA may be rapidly recruited to DNA damage sites, where it initiates signaling events that lead to the subsequent recruitment of DNA damage checkpoint and repair complexes. Given the known roles of PARP1 in the regulation of chromatin structure (TULIN and SPRADLING 2003; KIM et al. 2005), the key early function of PrpA may be chromatin remodeling at DNA damage sites. Although the relationship between PARP1 and other DNA damage signaling proteins appears complex (WATANABE et al. 2004), our preliminary observations show that the A. nidulans ATM homolog is required for PrpA nuclear localization (C. SEMIGHINI and S. HARRIS, unpublished observations). Additional dependency studies and double-mutant analyses will help to clarify the roles of PrpA in DNA damage signaling and repair.
A variety of cellular stresses are capable of causing apoptosis in the filamentous fungi (CHENG et al. 2003; MOUSAVI and ROBSON 2004; CHEN and DICKMAN 2005; LEITER et al. 2005). We have recently shown that farnesol, a small 15-carbon lipid, triggers apoptosis in A. nidulans via a mechanism that depends on the accumulation of reactive oxygen species and requires functional mitochondria (SEMIGHINI et al. 2006). PARP1 mediates a caspase-independent form of cell death by promoting the transfer of AIF from mitochondria to nuclei, where it triggers the hydrolysis of chromatin (YU et al. 2002). Accordingly, the role of PrpA in the farnesol-induced response suggests that a functionally analogous pathway involving an AIF homolog may control cell death in A. nidulans (see CHENG et al. 2003).
Role of PrpA in asexual development:
Our observations show that the core BrlA- and AbaA-dependent transcriptional regulatory circuit directs the induction of prpA expression during asexual development. When this regulatory circuit is broken by the fusion of prpA to the alcA promoter, conidiation does not proceed and instead fluffy aconidial colonies are formed. These results imply that the proper regulation of PrpA function is crucial for subsequent development. This may reflect a role for PARP modification in controlling the expression and/or activity of important development proteins. For example, PrpA-mediated chromatin modification may control the derepression of sporulation-specific gene clusters such as spoC1 (MILLER et al. 1987). Alternatively, PrpA expression may affect the cytoskeleton or other cytoplasmic functions important for cellular morphogenesis during asexual development. Notably, in Drosophila, overexpression of PARP1 disrupts actin organization by suppressing Rho GTPase function (UCHIDA et al. 2002).
Finally, there is a striking correlation between the conservation of PARP and the ability of fungi to form multicellular hyphae and/or developmental structures. PARP homologs are not present in S. cerevisiae and S. pombe and also appear to be missing from the dimorphic basidiomycetes U. maydis and C. neoformans. This observation may be instructive in that it emphasizes the correlation between PARP function and a multicellular lifestyle. Another protein that displays a similar conservation pattern in fungi is NADPH oxidase (LALUCQUE and SILAR 2003), which has been implicated in intercellular signaling during sexual development in A. nidulans (LARA-ORTIZ et al. 2003). Thus, A. nidulans may provide an attractive model system for elucidating the ancestral function of proteins whose importance is linked with multicellularity.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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FOOTNOTES |
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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