The clp1 Gene of the Mushroom Coprinus cinereus Is Essential for A-Regulated Sexual Development

Corresponding author: Takashi Kamada, Department of Biology, Faculty of Science, Okayama University, Okayama 700-8530, Japan., kamada{at}cc.okayama-u.ac.jp (E-mail)

Communicating editor: R. H. DAVIS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Sexual development in the mushroom Coprinus cinereus is under the control of the A and B mating-type loci, both of which must be different for a compatible, dikaryotic mycelium to form between two parents. The A genes, encoding proteins with homeodomain motifs, regulate conjugate division of the two nuclei from each mating partner and promote the formation of clamp connections. The latter are hyphal configurations required for the maintenance of the nuclear status in the dikaryotic phase of basidiomycetes. The B genes encode pheromones and pheromone receptors. They regulate the cellular fusions that complete clamp connections during growth, as well as the nuclear migration required for dikaryosis. The AmutBmut strain (326) of C. cinereus, in which both A- and B-regulated pathways are constitutively activated by mutations, produces, without mating, dikaryon-like, fertile hyphae with clamp connections. In this study we isolated and characterized clampless1-1 (clp1-1), a mutation that blocks clamp formation, an essential step in A-regulated sexual development, in the AmutBmut background. A genomic DNA fragment that rescues the clp1-1 mutation was identified by transformations. Sequencing of the genomic DNA, together with RACE experiments, identified an ORF interrupted by one intron, encoding a novel protein of 365 amino acids. The clp1-1 mutant allele carries a deletion of four nucleotides, which is predicted to cause elimination of codon 128 and frameshifts thereafter. The clp1 transcript was normally detected only in the presence of the A protein heterodimer formed when homokaryons with compatible A genes were mated. Forced expression of clp1 by promoter replacements induced clamp development without the need for a compatible A gene combination. These results indicate that expression of clp1 is necessary and sufficient for induction of the A-regulated pathway that leads to clamp development.

SEXUAL development in the mushroom Coprinus cinereus is under the control of the A and B mating-type genes (KIMURA 1952 ). Basidiospores (sexual spores), which are produced on the mushroom, germinate to give haploid homokaryons. Homokaryons are normally sterile. When two homokaryons with compatible alleles at both A and B genes are mated, both A- and B-regulated pathways operate to give the fertile dikaryon with the characteristic clamp structure (see RAPER 1966 ; CASSELTON and OLESNICKY 1998 ). Compatible B genes promote exchange and reciprocal migration of the nuclei between the two mating partners for dikaryosis. The dikaryon is a prolonged mycelial stage in which the two nuclei from each mating partner remain paired without fusing in each cell. Compatible A genes promote conjugate mitotic division of the two nuclei with an associated clamp connection. One nucleus divides in the clamp and the other in the hypha just beneath the clamp, which results in the formation of a binucleate tip cell and uninucleate clamp and subterminal cells. B gene compatibility is again required for fusion of the clamp with the subterminal cell to complete the clamp connection, allowing the clamp cell nucleus to enter the subterminal cell. Thus the dikaryotic mycelium carrying the two different nuclei in each hyphal compartment is established. However, the AmutBmut homokaryotic strain of C. cinereus, in which both A- and B-regulated pathways are constitutively activated by mutations, produces, without mating, dikaryon-like fertile hyphae with clamp connections (SWAMY et al. 1984 ). In this strain, two genetically identical nuclei pair in each cell (SWAMY et al. 1984 ).

Molecular analysis in C. cinereus revealed that the A genes encode proteins with homeodomain motifs while the B genes encode pheromones and pheromone receptors (see CASSELTON and OLESNICKY 1998 ). The A mating-type proteins fall into two classes, designated HD1 and HD2, on the basis of conserved but distinct homeodomain motifs (KUES et al. 1992 , KUES et al. 1994 ). A compatible mating is recognized by the ability of allelic forms of HD1 and HD2 proteins to heterodimerize. The heterodimerization brings together potential DNA-binding and activation domains of a putative dikaryon-specific transcription factor (see CASSELTON and OLESNICKY 1998 ). Similar homeodomain proteins are encoded by the A mating-type genes of another homobasidiomycete, Schizophyllum commune (STANKIS et al. 1992 ; SPECHT et al. 1992 ), and the b mating-type genes of the heterobasidiomycetes Ustilago maydis (GILLISSEN et al. 1992 ) and U. hordei (BAKKEREN and KRONSTAD 1993 ). The HD1 and HD2 proteins resemble the a1 and 2 homeodomain mating-type proteins of Saccharomyces cerevisiae (see CASSELTON and OLESNICKY 1998 ). Recently, we identified a C. cinereus gene, pcc1, in which mutation leads to clamp development in the absence of a compatible A gene complement and thus the heterodimeric A protein transcription factor (MURATA et al. 1998 ). The pcc1 gene encodes a high mobility group domain transcription factor (MURATA et al. 1998 ) that is suggested to act as a suppressor in a pathway leading to clamp development without mating (Y. MURATA and T. KAMADA, unpublished results). How the A protein heterodimer activates the pathway leading to clamp development still remains to be elucidated, however.

In this article, we describe isolation and molecular characterization of the clampless1-1 (clp1-1) mutation that blocks clamp development in the AmutBmut strain (326) of C. cinereus in which both A- and B-regulated pathways are constitutively activated by mutations in both mating-type genes. The clp1 gene encodes a novel protein of 365 amino acid residues. Normal expression of clp1 is dependent on the presence of the A protein heterodimer. Forced expression of clp1 by promoter replacements induced clamp development in the absence of the A protein heterodimer. clp1 is not transcribed in the pcc1-1 mutant, despite the fact that the mutant exhibits clamps. On the basis of these results, we suggest a possible role for Clp1 in the A-regulated pathway.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains and culture conditions:
Strains of C. cinereus listed in Table 1 were used. Malt extract/yeast extract/glucose (MY) medium (RAO and NIEDERPRUEM 1969 ) solidified with 2% (w/v) agar in 9-cm petri dishes was used for routine mycelial cultures. Slants of MY agar medium in test tubes were used for fruiting, the same medium without agar in 9-cm petri dishes for mycelial cultures for extraction of DNA and RNA. MY medium was supplemented with 100 mg/liter of tryptophan for culture of tryptophan requiring strains. The minimal medium was that of SHAHRIARI and CASSELTON 1974 modified by BINNINGER et al. 1987 . Cultures were maintained at 28° under a 12-hr light/12-hr dark regime unless otherwise stated.

Microscopy:
Presence or absence of clamps in mycelial colonies on agar plates was examined under a Nikon bright-field microscope. For micrographs, hyphae were grown on thin MY agar film on glass slides and observed under a Zeiss microscope equipped with phase-contrast optics.

Measurement of oidia number:
A small piece of agar medium with mycelium (1 x 1 x 1 mm) was inoculated in the center of an MY agar plate and incubated for 10 days under a 12-hr light/12-hr dark regime or continuous darkness. Oidia produced were harvested in water by adding 5 ml of water to the surface of the culture and then gently scratching the culture surface with a glass rod. The number of oidia in the suspension was measured with a hemocytometer.

Mutagenesis:
Strain 326 (AmutBmut pab1-1) was mutagenized by restriction enzyme-mediated integration (REMI) using plasmid pPHT1 carrying the hygromycin B resistance gene as a positive selectable marker in C. cinereus (CUMMINGS et al. 1999 ). The plasmid (4 µg) was digested with 60 units of HindIII in 20–27.2 µl of reaction mixture at 37° overnight. Protoplasts from oidia of strain 326 were transformed with the reaction mixture containing linearized pPHT1 and HindIII according to the procedure described by BINNINGER et al. 1987 with minor modifications. They were then spread on regeneration agar plates (BINNINGER et al. 1987 ) supplemented with 10^-4 M p-aminobenzoic acid (PAB). After incubation for 1 day, the plate was overlaid with 5 ml of regeneration medium supplemented with 10^-4 M PAB and 600 µg/ml hygromycin B and incubated for 2 weeks. Hygromycin B-resistant transformants appeared and were transferred onto minimal medium supplemented with 10^-4 M PAB and 150 µg/ml hygromycin B to purify the transformed mycelium.

Genetic techniques:
Crosses were made by laying two inocula (1 x 1 mm) 1 mm apart on MY agar plates. Basidiospore germlings were isolated at random using a chisel-shaped needle under a dissecting microscope (MILES et al. 1966 ).

DNA manipulations:
Cosmid and plasmid DNA were isolated with the FlexiPrep kit (Pharmacia Biotech, Piscataway, NJ). Genomic DNA from C. cinereus was prepared as described by ZOLAN and PUKKILA 1986 .

Southern and Northern analysis:
DNA or RNA was transferred to Hybond-N⁺ (Amersham, Arlington Heights, IL) according to SAMBROOK et al. 1989 . The enhanced chemiluminescence (ECL) direct system (Amersham) and the Gene Images system (Amersham) were used for probe labeling and detection for Southern and Northern analysis, respectively.

CHEF electrophoresis of C. cinereus chromosomal DNA:
Plugs for clamped homogeneous electric fields (CHEF) electrophoresis were prepared as described by MURAGUCHI and KAMADA 1998 . To separate larger chromosomes of C. cinereus, CHEF electrophoresis was run using 120 ml of 0.8% chromosomal grade agarose (Bio-Rad) gels at 1.8 V/cm for 144 hr with a pulse time of 1200 sec in 0.5 x TBE (45 mM Tris base, 45 mM boric acid, 1 mM EDTA) at 18°, exchanging the buffer every day.

Chromosome IV-specific cosmid library screening, clp1 gene cloning, and sequencing:
A cosmid library of chromosome IV from the wild-type strain (5302) was constructed as described by ZOLAN et al. 1992 . The vector (LLC5200) contains the C. cinereus trp1 gene as a selectable marker (PUKKILA and CASSELTON 1991 ). The library was composed of 1248 clones (96 clones x 13 plates). Groups of 12 clones were cultured on a plate of Luria broth/ampicillin solid medium, mixed, and subjected to miniprep with the FlexiPrep kit (Pharmacia Biotech). The pooled DNAs (1.0 µg) were used to transform protoplasts from strain CLP1F₁#44 (AmutBmut pab1-1 trp1-1, 1-6 clp1-1) as described by BINNINGER et al. 1987 . Fifty to sixty trp⁺ transformants were isolated with each pooled DNA. They were cultured on minimum medium supplemented with 10^-4 M PAB for 3 days to purify the transformed mycelium and then transferred to MY plates to examine for the presence or absence of clamps. Five of the 52 trp⁺ transformants from a single pool of DNA, 3A, produced clamps. Subsequent sib selection revealed that a single cosmid clone, 3A3, has the rescuing activity.

Clone 3A3 was digested with ApaI, HindIII, PstI, SacI, or XbaI, and each digest was used to transform strain CLP1F₁#44. None of the restriction enzymes destroyed the rescuing activity. ApaI digestion produced six fragments (25, 6, 4.5, 3, 2.5, and 1.5 kb). The 25-kb fragment, which contains the whole sequence of LLC5200, was subcloned by self-ligation and its clp1 activity was examined by transformation. The other fragments were ligated into the ApaI site of pGEM-7zf + (Promega, Madison, WI) and examined for the clp1 activity by cotransformation with pCc1003 carrying the intact trp1 gene (SKRZYNIA et al. 1989 ). The 4.5-kb ApaI fragment had the rescuing activity. This 4.5-kb fragment was then deleted from each side with the nested deletion kit (Pharmacia Biotech) and sequenced with a model 373S DNA sequencer (Perkin-Elmer, Norwalk, CT) using a PRISM dye primer cycle sequencing kit (Perkin-Elmer). The deletion products were also examined for the clp1 activity. The genomic DNA sequencing of clp1 and the deduced amino acid sequence data reported in this article will appear in the DDBJ/EMBL/GenBank databases under accession no. AB034196.

Determination of the clp1-1 mutation site:
To sequence the clp1-1 mutant allele, we carried out PCR on the genomic DNA of the CLP1 mutant using the following five pairs of sense and antisense primers that cover the entire length of the open reading frame (ORF) of the clp1-1 gene: CLS1, CLA1, CLS2, CLA2, CLS3, CLA3, CLS4, CLA4, CLS5, CLA5 (Table 2). To sequence the PCR products directly, we added a -21M13 primer sequence (18 bp) to the end of each sense primer sequence and a M13 reverse primer sequence (18 bp) to that of each antisense primer sequence.

Northern analysis:
For total RNA, mycelia grown in MY liquid medium in 9-cm petri dishes for 7–8 days were harvested on a nylon filter, washed thoroughly with sterile water, and then frozen in liquid nitrogen. The frozen mycelia were ground into fine powder with a mortar and pestle. RNA was extracted from the powder as described by YEAGER STASSEN et al. 1997 . About 15 µg of total RNA was fractionated by electrophoresis in 1.3% agarose formaldehyde gel. The 0.9-kb EcoRI-SalI fragment from the clp1 gene (see Fig 3) was used as the probe.

View larger version (131K): In this window In a new window Download PPT slide   Figure 1. Phase contrast micrographs showing parts of hyphae of the AmutBmut strain 326 (A) and the clp1-1 mutant (B). The arrow indicates a clamp and the arrowhead a septum without a clamp.

View larger version (16K): In this window In a new window Download PPT slide   Figure 2. RFLP analysis of the progeny from the cross CLP1 (AmutBmut pab1-1 clp1-1) x KF2#1 (A91B92 + +). The progeny were scored for the wild-type forming clamps (+) and the clp1-1 mutant not forming clamps (-) in the Amut background. A selection of eight wild type and eight mutant progeny were then scored for RFLP using enzyme BamHI and the chromosome IV marker, Hiroshima II#11.

View larger version (16K): In this window In a new window Download PPT slide   Figure 3. Deletion products of the 4.5-kb ApaI fragment and the activity that rescues the clp1-1 mutation. The values in parentheses are clamp-forming trp+ transformants/total trp+ transformants examined. The thick line, open box, and solid box indicate the region transcribed, a deduced ORF, and the intron, respectively.

5'-RACE and 3'-RACE experiments:
clp1 cDNA was amplified using a cDNA library synthesized with the Marathon cDNA amplification kit (CLONTECH, Palo Alto, CA) by MURAGUCHI and KAMADA 1998 as a template and a nest of gene-specific primers (GSP1, GSP2, NGSP1, NGSP2) (Table 2). PCR was performed with AmpliTaq Gold DNA polymerase (Perkin-Elmer) and the PCR products were cloned into pGEM-T Easy vector (Promega), according to the manufacturer's instructions, and sequenced as described above.

Replacements of the promoter of clp1 with those of C. cinereus ß1-tubulin and actin genes:
The clp1 ORF fused to the promoter of the ß1-tubulin gene was constructed by sequential PCR amplifications using Pfu DNA polymerase (Stratagene, La Jolla, CA). In the first step, 393 bp upstream of the ß1-tubulin coding sequence with a 3' extension of the 5' terminal 18 bp of the clp1 coding sequence and 839 bp from the clp1 start codon to the unique SalI site in clp1 (see Fig 3) with a 5' extension homologous to 18 bp upstream of the ß1-tubulin coding sequence were amplified: the former was amplified using pBl204KS carrying the ß1-tubulin gene (MATSUO et al. 1999 ) as the template and primers TB-SphI and CL1-TB. TB-SphI (27-mer) is the 5' terminal 17 bp of the ß1-tubulin gene promoter with a 10-bp 5' extension having a SphI site, which is located 377–382 bp upstream of the clp1 start codon. CL1-TB (36-mer) is the antisense sequence of 18 bp upstream of the ß1-tubulin start codon with a 5' extension homologous to the 5' terminal 18 bp of the clp1 coding sequence. The latter was amplified using the 4.5-kb ApaI fragment as the template and primers TB-CL1 and CL1-SalI. TB-CL1 is a 5' extension that is homologous to 18 bp upstream of the ß1-tubulin start codon with a 3' extension of the 5' terminal 18 bp of the clp1 coding sequence, and CL1-SalI is the antisense sequence of a unique SalI site in clp1 and its upstream 15 bp. Because primers CL1-TB and TB-CL1 contain complementary 5' extensions, two PCR products containing a region of overlapping homology are generated. In the second step, PCR was performed on the mixture of the two PCR products with primers TB-SphI and CL1-SalI to produce clp1 fused to the ß1-tubulin promoter. The 1.2-kb region extending from the SphI site to the SalI site in the 3.2-kb fragment in pGEM-7zf+, which is a deletion product from the 4.5-kb ApaI fragment in pGEM-7zf+ (see Fig 3), was replaced with the second PCR product digested by SalI and SphI. Sequencing analysis showed that the construct has the intact clp1 fused to the intact ß1-tubulin promoter. Tryptophan-requiring homokaryotic strain 292 was cotransformed with the construct and pCc1003 carrying the intact trp1 gene. As a control, strain 292 was cotransformed with the intact 3.2-kb fragment in pGM-7zf+ and pCc1003.

To replace the promoter of clp1 with that of a C. cinereus actin gene, the sequence from the clp1 start codon to the end of the clp1 terminator with the modification of the sequence around the start codon from CAATGC to the NcoI site, CCATGG, and with a 3' extension with an NcoI site was amplified using the 4.5-kb ApaI fragment as the template and primers act1 and act2 (Table 2). The amplified product was digested with NcoI and ligated to the NcoI site of plasmid pLJ2, which carries the promoter of the actin gene and the intact trp1 gene, so that the 5' terminal of the clp1 coding sequence is connected to the actin gene promoter (L. A. CASSELTON and W. J. CUMMINGS, unpublished data). Strain 292 was transformed with the construction.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation and genetic analysis of the clp1-1 mutation:
The clp1-1 mutant that fails to form clamps was found among 3225 hygromycin-resistant transformants of strain 326 (AmutBmut pab1-1) after REMI mutagenesis (Fig 1). The mutant did not exhibit any visible sign of fruiting although the parental strain formed fertile fruit bodies constitutively. Activation of the A-regulated pathway in sexual development has been correlated with repression of asexual sporulation (TYMON et al. 1992 ) and the AmutBmut strain from which the clp1 mutant was derived produces few asexual oidia. The clp1 mutation led to a marked increase in the numbers of oidia produced (Table 3).

When CLP1 (AmutBmut pab1-1 clp1-1) was mated to the wild-type homokaryon, KF₂#1, dikaryotic hyphae with clamp connections emerged from the margin of the colony of KF₂#1, indicating that the mutation is recessive. We isolated 111 progeny from the dikaryon. Of the 111 progeny, 62 were PAB requiring and 49 were prototrophic. Most of the PAB-requiring progeny are considered to carry Amut because the A mating-type locus is closely linked to pab1 at 0.5 map units (TAKEMARU 1982 ). We examined the PAB-requiring progeny for the presence or absence of clamps. Of 62, 29 exhibited clamps in their hyphae whereas 33 did not. This result shows that the clampless phenotype in the Amut background is due to a mutation in a single chromosomal gene, which we designated clp1. We then examined the 62 progeny for hygromycin resistance and found that hygromycin resistance and the clampless phenotype segregated independently in the progeny (date not shown). The clp1-1 mutation is not, therefore, a direct result of plasmid insertion. We carried out genetic linkage analyses using chromosome markers and found that clp1 was linked to a restriction fragment length polymorphism (RFLP) marker, Hiroshima II #11 (Fig 2), which is located on chromosome IV, as separated by a CHEF gel (PUKKILA 1993 ).

Cloning of clp1:
To clone the clp1 gene, we constructed a chromosome IV-specific library from a wild-type strain, 5302, and screened it for a DNA fragment that complements the clp1-1 mutation. We identified a single cosmid clone, 3A3, that rescues the clp1-1 mutation. Sixty-three percent (12/19) of trp⁺ transformants with clone 3A3 exhibited clamps. We scored the 16 progeny used for the experiment shown in Fig 2 for RFLP using the the clone 3A3 as a probe, which showed that the 3A3 region is closely linked to the clp1 locus (recombinants: 0/16). This strongly suggests that the fragment does not contain an extragenic suppressor of the clp1-1 mutation, but clp1 itself. Digestion of clone 3A3 with ApaI followed by tests of the clp1 rescuing activity revealed that a 4.5-kb ApaI fragment from 3A3 contained clp1. We further localized clp1 to 1479 bp in the 4.5-kb fragment by deletions (Fig 3 and Fig 4). The 1479 bp contain 404 bp upstream of the start codon and 49 bp downstream of the stop codon (see below).

View larger version (62K): In this window In a new window Download PPT slide   Figure 4. Nucleotide and predicted amino acid sequences of the clp1 gene of C. cinereus. The number of the first nucleotide of each line is indicated on the left margin, and the last amino acid of each line is indicated on the right margin of the figure. The intron sequence is shown in lowercase letters. The four nucleotides deleted in the clp1-1 mutant allele are double underlined. The 18 nucleotides that show similarity to the conserved hsg in S. cerevisiae (GOUTTE and JOHNSON 1988 ) and a TATA box in the promoter region are underlined.

Identification of the clp1 ORF:
Genomic DNA sequencing, together with 5'-RACE and 3'-RACE experiments, identified an ORF interrupted by one intron of 53 nucleotides (nt), which is predicted to encode a protein of 365 amino acids (Fig 4). The 5' splice site, GTAAGT, agrees with the consensus sequence GTRNGT found for filamentous fungi and the 3' splice site, TAG, with the consensus sequence YAG (GURR et al. 1987 ). The predicted protein contains no obvious structural motifs nor shows extensive similarity to any sequences represented in the database. The clp1 mRNA is predicted to have a 63-nt 5'- and a 315-nt 3'-untranslated region. The promoter region of clp1 contains a TATA box 45–39 bp upstream of the predicted translational start site. A total of 110 bp upstream of the TATA box there was a sequence (GATG, 11-nt spacing, ACA) similar to the conserved hsg motif (GATG, 9-nt spacing, ACA) that exists in the promoter regions of the haploid-specific genes in S. cerevisiae (GOUTTE and JOHNSON 1988 ).

clp1-1 mutant allele:
We performed PCR amplifications on the genomic DNA of the clp1-1 mutant using five pairs of primers that were designed on the basis of the wild-type clp1 sequence and sequenced the PCR products directly. Comparison of the sequence of the clp1-1 mutant allele with that of the wild-type gene revealed that the four bases at positions 661–664 (GTGA) were deleted in the clp1-1 mutant allele, which is predicted to cause elimination of codon 128 and frameshifts thereafter (Fig 4).

Developmental regulation of clp1 transcription:
Using a 1.0-kb EcoRI-SalI fragment from the clp1 gene (see Fig 3) as a probe, we examined total RNAs from various strains (Fig 5). As expected from the clp1 cDNA analysis, we identified a transcript at 1.5 kb, in the parental AmutBmut strain, the clp1-1 mutant, the dikaryon, and an A-on homokaryon. Transcription of clp1 was clearly increased in the clp1-1 mutant as compared with the parental AmutBmut strain. The clp1 transcript was not detected in the wild-type homokaryon or in a B-on homokaryon. We also found that clp1 was not transcribed above the level of detection in the pcc1-1 mutant, which exhibits clamps constitutively (MURATA et al. 1998 ).

View larger version (27K): In this window In a new window Download PPT slide   Figure 5. Expression of the clp1 gene. For each lane, 15 µg of total RNAs from the following were electrophoresed: wild-type homokaryon 5302 (lane 1), wild-type dikaryon 5302 x 5401 (lane 2), the AmutBmut strain 326 (lane 3), the AON1 strain (lane 4), the BON1 strain (lane 5), the CLP1 mutant (lane 6), the pcc1-1 mutant 5337#1 (lane 7), and homokaryon 292, in which clp1 fused to the ß1-tubulin gene promoter was introduced (lane 8). After the ribosomal RNA was visualized under UV, the gel was blotted and hybridized. The probe was the 1.0-kb EcoRI-SalI fragment (see Fig 3). After hybridization, the blot was exposed to X-ray film.

Forced expression of clp1:
The above results suggest a pathway in which the A protein heterodimer promotes the expression of clp1, which in turn induces clamp development. To examine whether the expression of clp1 induces clamp development in the absence of the A protein heterodimer, we produced a construct in which the promoter region of clp1 was replaced with that of the C. cinereus ß1-tubulin gene and introduced it into a tryptophan-requiring homokaryon 292 by cotransformation with pCc1003 carrying the intact trp1. We found that 26% (12/46) of trp⁺ transformants exhibited clamps. The clamps formed, however, were unfused ones because the B-regulated pathway is not activated in the transformants. Northern blot analysis showed that clp1 was transcribed in a transformant exhibiting clamps (Fig 5, lane 8). A similar result was obtained with clp1 connected with the promoter of a C. cinereus actin gene: 35% (24/68) of trp⁺ transformants exhibited clamps. As a control, we introduced the intact clp1 gene into the homokaryon and found that no trp⁺ transformants exhibited clamps (0/31).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

C. cinereus is a heterothallic species in which mating is under the control of the A and B mating-type loci. The A- and B-regulated pathways are normally activated to produce the fertile dikaryon when two homokaryons with different A and B alleles are mated (see CASSELTON and OLESNICKY 1998 ). In the present study we took advantage of the fact that the AmutBmut homokaryotic strain carrying mutations in both mating-type loci produces dikaryon-like, fertile hyphae with clamp connections without the need for mating. We identified a mutation, clp1-1, that blocks clamp formation, an essential step in A-regulated sexual development. The clp1 gene was cloned as a DNA fragment that rescues the clp1-1 mutant phenotype. An ORF that is predicted to encode a novel protein of 365 amino acid residues was identified within this fragment. clp1 transcripts were detected only in mycelia containing compatible A genes and thus able to generate the A protein heterodimer predicted to activate the A pathway in sexual development. We showed that clp1 transcripts are present in dikaryons, A-on mycelia, and homokaryons carrying the A mutation present in strain 326, which is known to generate a chimeric protein with heterodimer activity (KUES et al. 1994 ), but are absent in the pcc1 mutant that activates the A developmental pathway in the absence of a compatible combination of A genes. Significantly, we found that forced expression of clp1 by promoter replacements bypassed the requirement for compatible A genes. Taken together, these results indicate that clp1 is working downstream of the A genes and that it is a likely target for the A protein heterodimer. clp1 transcription is necessary and sufficient for induction of clamp formation in the A pathway.

The element in the promoter region responsible for activation of the clp1 expression remains to be characterized and its ability to bind the A protein heterodimer still has to be tested. However, it seems significant that an 18-bp sequence (GATG, 11-nt spacing, ACA) was identified 218–201 bp upstream of the clp1 start codon that is similar to the conserved hsg motif (GATG, 9-nt spacing, ACA) in S. cerevisiae (GOUTTE and JOHNSON 1988 ). The hsg motif has been shown to bind the heterodimer of MATa1 and MAT2 encoded by the a and mating-type genes in yeast (GOUTTE and JOHNSON 1988 ), proteins that resemble the proteins encoded by the A mating-type genes in C. cinereus (see CASSELTON and OLESNICKY 1998 ). The MATa1/MAT2 heterodimer is a transcriptional repressor so it is particularly relevant that a target gene for the corresponding bE/bW protein heterodimer of U. maydis has been identified recently. The bE/bW heterodimer binds an hsg-like element (GATG, 9-nt spacing, ACA) in the promoter of this gene and acts as a transcriptional activator (ROMEIS et al. 2000 ). The interaction between two homeodomain proteins is obviously a conserved mechanism in regulating sexual development in S. cerevisiae and basidiomycete fungi, but whether the heterodimer acts as a repressor or as an activator depends on other functional domains within the proteins.

The clp1-1 mutation led to increased levels of clp1 transcription. This suggests a feedback mechanism that regulates the level of clp1 transcription, in which Clp1 and/or some downstream factor(s) may be involved. The presumed regulating factor(s) as well as element(s) in the promoter region responsible for this regulation remain to be identified.

The Clp1 protein does not show extensive similarity to any sequences represented in the database and does not contain obvious structural motifs. At the present time it is not known what function Clp1 plays in the A pathway. We have previously suggested that the clamp pathway is repressed in homokaryons by the HMG-domain protein encoded by pcc1. This was based on the fact that loss-of-function mutations in pcc1 lead to clamp formation without mating (MURATA et al. 1998 ; Y. MURATA and T. KAMADA, unpublished results). The A protein heterodimer may in some way release the A pathway from Pcc1 repression. clp1 is not transcribed in the pcc1-1 mutant, despite the fact that the mutant exhibits clamps, suggesting that loss of Pcc1 function bypasses a normally essential A protein-dependent step involving Clp1. If our interpretation is correct, Clp1 cannot relieve Pcc1 repression at the transcriptional level because pcc1 transcripts are still detected in A-on and dikaryotic mycelia (MURATA et al. 1998 ) but this would not preclude a post-translational mechanism.

	ACKNOWLEDGMENTS

We thank Lorna A. Casselton for critical reading of the manuscript, Tsutomu Morinaga for generous gifts of chromosome-specific clones, Miriam E. Zolan for plasmid pPHT1, and Lorna A. Casselton and W. Jason Cummings for the gift of plasmid pLJ2. This work was supported in part by a Grant-in-Aid for Scientific Research (10640652) from the Ministry of Education, Science, Sports and Culture of Japan.

Manuscript received August 14, 2000; Accepted for publication September 21, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

BAKKEREN, G. and J. W. KRONSTAD, 1993  Conservation of the b mating-type gene complex among bipolar and tetrapolar smut fungi. Plant Cell 5:123-136[Abstract/Free Full Text].

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