INTRODUCTION

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Cryptococcus neoformans is the etiologic agent of cryptococcosis, which is one of the most serious fungal diseases encountered worldwide. Although C. neoformans primarily affects patients with impaired immune systems, people with no known underlying immunodeficiencies are also affected (9).

C. neoformans is a bipolar heterothallic fungus in which completion of the meiotic cycle is dependent upon interaction between cells of MAT and MATa types. Although segregation patterns of the meiotic products yield MAT and MATa progeny in the expected 1:1 ratio for a bipolar heterothallic fungus (12), MAT strains are found far more frequently than MATa strains in clinical as well as environmental isolates among serotype D strains (10). Among serotype A strains, MAT has thus far been the only mating type to be found regardless of isolate source (13). To investigate the relationship between mating type and virulence, Kwon-Chung et al. (11) constructed a pair of congenic MAT (B-4500) and MATa (B-4476) strains of serotype D for genetic analysis. In a mouse systemic infection model, both the parental strain and F₁ progeny of MAT type were found to be significantly more virulent than the MATa parental strain and its F₁ progeny (11). Therefore, molecular and pathobiological studies of C. neoformans serotype D isolates have since been carried out mostly with MAT strains (3, 20).

In 1993, Moore and Edman (16) identified the MAT locus by employing a difference cloning method using the congenic strains B-4500 (JEC21, MAT) and B-4476 (JEC20, MATa). The MAT locus was marked by the presence of the MF gene, which encodes a pheromone precursor. Analysis of MAT-specific phage and cosmid clones led them to conclude that the MAT locus was 35 to 45 kb in size. Subsequently, Wickes et al. discovered haploid fruiting to be a MAT-specific phenomenon in C. neoformans (26). Molecular analysis of haploid fruiting in MAT strains resulted in the cloning of the STE12 gene, a homolog of the Saccharomyces cerevisiae transcriptional activator STE12, and the STE11 gene (26), a homolog of the S. cerevisiae STE11 (MEKK) gene (19, 22). Recently, Wang and Heitman isolated STE20 (accession no. AF162330), a homolog of S. cerevisiae STE20, from H99, a serotype A MAT strain of C. neoformans. Although these genes were reported to be specific for MAT strains, their genomic locations have not been clearly determined. Additionally, STE12 was not found in the MAT-specific region (26) previously reported by Moore and Edman (16), nor was a MAT-specific receptor of the MATa pheromone found, a gene which should be mating type specific.

The STE12 gene recently has been deleted from both serotype D and serotype A strains, and the gene was found to be essential for haploid fruiting but not for mating (4, 28). Furthermore, STE12 was reported to regulate several virulence associated genes in serotype D C. neoformans (4). Mating type-specific mitogen-activated protein (MAP) kinase genes of the signal transduction cascade have not been reported in any other fungi. Since the importance of these genes in the pathobiology of C. neoformans is becoming increasingly evident (4; D. L. Clarke, U. Edman, G. L. Woodlee, C. M. McClelland, T. S. Seymour, J. C. Edman, and B. L. Wickes, unpublished data), we have attempted to determine their genomic locations.

In this paper, we present a physical map of the B-4500 MAT locus which was obtained by analysis of overlapping subclones isolated from a cosmid library of B-4500. The MAT locus, which spans 50 kb, contained several mating type -specific homologs of the pheromone response MAP kinase signal transduction cascade genes, multiple copies of MF, and one copy of the pheromone receptor gene, CPR. Unexpectedly, a myosin gene and a homolog of S. cerevisiae translation initiation factor, PRT1, specific to the MAT strain, were also discovered within the locus.

	MATERIALS AND METHODS

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C. neoformans strains, Escherichia coli plasmids and cosmids, and growth conditions. The C. neoformans strains B-4500 (JEC21, MAT) and B-4476 (JEC20, MATa) are isogenic strains (11). The strains were maintained on YEPD agar medium (1% yeast extract, 2% peptone, 2% dextrose, 1.5% agar) and grown on MIN medium (0.67% yeast nitrogen base without amino acids, 2% glucose) before DNA was extracted. E. coli plasmids and cosmids used in this study are listed in Table 1.

Molecular techniques. Standard methods described by Sambrook et al. (21) were used for transformation of E. coli and DNA analysis. Genomic DNA was extracted from C. neoformans essentially as described by Pitkin et al. (18). All Southern blots (except the myosin blot) were hybridized and washed under stringent conditions at 65°C. The myosin blot was hybridized under less stringent conditions at 45°C. For subcloning of DNA fragments >10 kb from the isolated cosmids (containing an ampicillin resistance cassette), 5 µg of cosmid DNA was digested to completion, ethanol precipitated, washed, and resuspended in 20 µl of H₂O. The DNA fragments were cloned into a linearized and dephosphorylated plasmid (pBC KS) containing a cassette for chloramphenicol resistance (Stratagene, La Jolla, Calif.). This cloning strategy eliminated clones derived from religation of the cosmid backbone, since the cosmid selectable marker is ampicillin and recircularized vectors will not grow on chloramphenicol. Positive clones were identified by using colony hybridization and an appropriate DNA sequence as a probe. For sequencing, 0.5 to 1.0 µg of DNA was used with the DNA Sequencing Kit ABI PRISM and an ABI377 DNA sequencer from Perkin-Elmer (PE Biosystems, Warrington, England).

CHEF analysis. Contour-clamped homogeneous electric field (CHEF) blots were prepared as previously described (27) and analyzed by Southern hybridization using probes generated from various regions of the MAT locus. The probes of MAT and MATa have previously been described (5). The probes FUR and MK were generated by PCR, using primers derived from partial sequence analysis of regions III and VI, respectively (see Fig. 4). The oligonucleotide pairs 5'-AATGGGGGAAAACGCACGAG-3' with 5'-CTTCTAAGGACTTGCGGTTCTCAACTC-3' and 5'-CCTGCACGGAAAATATCCAC-3' with 5'-GCAAGATATATGGACCCCTG-3' were used to isolate FUR and MK, respectively.

	RESULTS

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Creation of a physical map and localization of STE12 within the MAT locus. Previous studies suggested that the location of STE12 and STE11 was closely linked to the MAT locus (26). Subsequently, STE20, an -specific homolog of S. cerevisiae STE20, was cloned by Wang and Heitman (accession no. AF162330) although its location in relation to the MAT locus has not been reported. To establish the physical relationship between these MAP kinase signal transduction cascade genes and the MAT locus in C. neoformans, the sequence downstream of STE12 was first used as a probe (Fig. 1, probe I) to screen a cosmid library of B-4500. Several overlapping cosmids were isolated. A physical map of these cosmids was created by using the restriction enzyme BamHI (Fig. 1). Southern blot analysis using a PCR-derived probe of MF1, the original marker for the MAT locus described by Moore and Edman (16), detected a 13-kb BamHI band in several of our cosmids (data not shown). These data indicated that STE12 is physically linked to the MF1 gene, although the exact position of MF1 could not be determined at this stage. To investigate the neighboring sequence of STE12, we subcloned and sequenced the 5.5-kb BamHI fragment (Fig. 1) which contained the downstream sequence of the STE12 gene. We identified a 3.4-kb sequence about 1 kb downstream of STE12 with high similarity to the gene encoding a mitochondrial RNA polymerase of S. cerevisiae (RPO41, accession no. M17539) (7). To determine whether this sequence was shared in both mating types, BamHI-digested genomic DNAs from B-4500 and B-4476 were each hybridized with a probe from this gene (Fig. 1, probe II). The hybridization pattern revealed a single band with different sizes in each mating type (see Fig. 4J). Another probe derived from a 3-kb BamHI fragment, which was located next to the 5.5-kb fragment outside the conserved RPO41 gene (Fig. 1, probe III), also showed a signal in both mating types (data not shown). When several different regions upstream of STE12 (left side of STE12 in Fig. 1) were used as probes, they all showed an -specific pattern (see below). It was therefore clear that not only was STE12 physically associated with the MF1 gene but it was actually located at one end of the MAT locus, close to the boundary.

View larger version (6K): [in this window] [in a new window]   FIG. 1.   Physical map of the overlapping cosmids digested by BamHI. Cosmids were isolated by using probe I to screen a cosmid library of B-4500 (JEC21). STE12 is located on the right side of the restriction map, and MF1 is localized on the 13-kb BamHI fragment at the left side. The exact position of MF1 could not be determined at this stage. Shaded bars are locations of genes. I, II, and III represent the regions used as probes, hybridizing to genomic DNA of both mating types.

MF1

MF1

MF1

View larger version (64K): [in this window] [in a new window]   FIG. 2.   Southern blot analysis of MF genes. Cosmid DNA and genomic DNA of B-4500 and B-4476 digested with HaeII were hybridized with the MF1 probe. Lanes 1 and 2 show three MF bands detected in the DNA of the cosmids C12 and C18. The same banding pattern was observed with genomic DNA of B-4500 (lane 3), whereas no signal was obtained with the genomic DNA of B-4476 (lane 4). A single band of 1 kb was detected with DNA of cosmid C5-3 (lane 5).

View larger version (88K): [in this window] [in a new window]   FIG. 3.   Southern hybridization with various probes to a CHEF panel of strains B-4476 and B-4500. Shown are the karyotype (A), hybridizations with the MAT probe (B) and the MATa probe (C) recently described by Chaturvedi et al. (5), and hybridizations with FUR (D) and MK (E) (see Materials and Methods).

View larger version (51K): [in this window] [in a new window]   FIG. 4.   Overview of the MAT locus as well as identification and localization of the genes within the locus. Overlapping cosmids covering the whole mating type locus were isolated and analyzed. Using Southern blot techniques and several probes, the mating type-specific (between IV and V) and shared (I to III, VI) sequences were identified. The brackets mark the regions of the probes, indicated by roman letters. Specific probes were used to hybridize with BamHI-digested genomic DNA of B-4500 (MAT) and B-4476 (MATa) to confirm their mating type specificity. Probes A (GTP binding protein) and J (RNA polymerase) are located outside the mating type locus which hybridized with the DNA of both mating types. (B) MF2/3 are located on the 17-kb BamHI fragment, followed by a mating type-specific translation initiation factor, a homolog of the S. cerevisiae PRT1 (C). Previously described MF1 (D) is located on the same 13-kb BamHI fragment as STE11 (E). The largest BamHI fragment is ~21 kb in size and contains a myosin gene (F), STE20 (G), the pheromone receptor CPR (H), and STE12 (I).

Localization of other mating type  strain-specific genes in the MAT locus. After isolation of overlapping cosmids spanning the entire MAT locus, further analysis of the locus was made in relation to other -specific genes. We characterized regions near all the BamHI sites in the map. Southern blottings were used to demonstrate the mating type specificity of each characterized region (Fig. 4). The positions of already known mating type-specific genes inside the locus were determined, and new genes were discovered. Sequence data suggested the existence of a sequence encoding a putative GTP-binding protein (accession no. AC019018) between the 1.3- and 6.5-kb BamHI fragments (Fig. 4A) located in the genomes of B-4500 and B-4476. From the HaeII-digested genomic DNA of B-4500 (Fig. 2), we predicted that there were three copies of the MF gene in the MAT locus. By analyzing our cosmid clones, we located all three copies of the MF genes. The previously reported MF gene (16) was located on the 13-kb BamHI fragment (Fig. 4D) and was renamed MF1 (accession no. S56460). The other two MF genes were adjacent to each other and were located in the 17-kb BamHI fragment near the left boundary of the MAT locus (Fig. 4B). We designated these two genes MF2/3. A putative eukaryotic translation initiation factor (accession no. J02674), a homolog of S. cerevisiae PRT1, was located between the 4.5- and 13-kb fragments (Fig. 4C). The STE11 gene (accession no. AF294841) was found (Fig. 4E) in the 13-kb fragment where MF1 is located (Fig. 4D). The genes on the 21-kb BamHI fragment that have been thus far identified include a MAT-specific myosin (accession no. AF267642) gene (Fig. 4F) and STE20 (accession no. AF162330) (Fig. 4G). In the case of myosin, an additional band was detected in both MAT and MATa strains, under low stringency and washing conditions (Fig. 4F). We also identified the CPR gene (accession no. AF259519), a homolog of the Coprinus cinereus pheromone receptor (Fig. 4H) located about 1 kb upstream of STE12 (accession no. AF012924) (Fig. 4I). The 5.5-kb fragment beyond the right border of the MAT locus contained an RNA polymerase gene (accession no. AF295125) (Fig. 4J). Therefore, the entire MAT locus of C. neoformans var. neoformans appears to span about 50 kb on chromosome 3 (27).

	DISCUSSION

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It is known for S. cerevisiae that STE20, STE11, and STE12, components of the MAP kinase pathway that signal the mating pheromone response, are also involved in filamentous morphogenesis in diploid as well as haploid cells (reviewed in reference 15). These genes are required for mating since mutations in these genes cause sterility. Unlike those in C. neoformans, however, the components of the S. cerevisiae MAP kinase pathway which respond to pheromone have no association with mating type. The link between mating type, virulence, and haploid (monokaryotic) fruiting in C. neoformans is unique to this species. It is even more unusual to find that some genes of the MAP kinase pathway are mating type specific. Recent studies have shown that STE12 (4) and STE11 (D. L. Clarke, U. Edman, G. L. Woodlee, C. M. McClelland, T. S. Seymour, J. C. Edman, and B. L. Wickes, unpublished data) play important roles in virulence in the mouse model. In order to clarify the physical relationship between the -specific genes and the MAT locus, we cloned the entire MAT locus and constructed its physical map.

A previous report by Moore and Edman (16) indicated that the entire MAT locus may span between 35 and 70 kb and that one MF gene (MF1) was located within the locus. The present study revealed that the locus is approximately 50 kb in size. We detected 5 to 7 kb of flanking sequence at both ends of the locus which is shared between strains of MAT and MATa, reflecting the boundary of the MAT locus. The MAT locus harbored two additional MF genes, MF2 and MF3, and a putative -pheromone receptor, CPR. The presence of the pheromone genes and pheromone receptor(s) in the mating type locus has been frequently recognized in several heterothallic fungi, such as C. cinereus, Schizophyllum commune, and Ustilago maydis (2, 17, 25).

The three homologs of the S. cerevisiae pheromone response MAP kinase signal transduction cascade genes, STE11, STE12, and STE20, were found to be within a 24-kb region near MF1. Between STE20 and STE11, a MAT-specific sequence homologous to myosin was identified. The existence of MAP kinase signal transduction cascade genes, a mating type-specific gene of a molecular motor, and a translation initiation factor in a MAT locus have never been reported for any other fungi. These MAP kinase signal transduction cascade genes may play important roles in pathogenicity in addition to the mating of the fungus. In fact, a recent study of the role of STE12 suggested that this gene regulates virulence-associated genes, such as the CAP genes and CNLAC1 gene in serotype D strains of C. neoformans (4). What sets STE12 apart from STE12 of S. cerevisiae is that STE12 is not essential for mating but essential for haploid fruiting (4). Such a phenomenon was not expected since STE12 is part of the MAT locus. While STE12 is not essential for mating, ste12 mutants are reduced in mating efficacy by approximately 100-fold (4).

Characterization of the remaining MAP kinase signal transduction genes within the MAT locus should clarify whether STE12 functions in the same pathway as the other two kinases. Sequence analysis of the pheromone receptor which was found adjacent to STE12 showed a high degree of homology to seven previously characterized transmembrane pheromone receptors from other fungi (e.g., C. cinereus, U. maydis, S. commune) (2, 17, 25). The role of CPR in mating as well as in cryptococcal pathogenicity is presently being characterized in our laboratory.

Discovery of MAT-specific myosin in the MAT locus is unprecedented and unexpected. Since myosin is an important protein for maintenance of morphological structure, this gene may be responsible for the ability to form hyphae during mating or during haploid fruiting, which is specific to mating type  strains. An additional smaller band was detected in both mating types with the myosin probe, perhaps due to cross-hybridization, since low-stringency conditions were used. These bands may suggest the presence of an additional non-mating type-specific myosin gene. Functional analysis of the mating type-specific myosin gene should reveal its role in morphogenesis.

The cloning of the complete MAT locus not only will allow us to analyze the function of remaining genes located within the locus, such as the translation initiation factor, but also will allow us to identify their counterparts in the MATa locus. A comparison of the genomic arrangement between the two loci may lead to a further understanding of the mating system and its role in pathobiology of C. neoformans. Our recent characterization of the a mating type-specific pheromone receptor gene, CPRa (M. Karos, Y. C. Chang, and K. J. Kwon-Chung, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. F76, 2000), and STE12a (Y. C. Chang and K. J. Kwon-Chung, Abstr. 99th Gen. Meet. Am. Soc. Microbiol., abstr. F63, 1999) revealed that the relative arrangement of these two genes in the MATa locus is different from that of the MAT homologs (data not shown). It is known from the genomic arrangement of mating loci in other organisms that rearrangement of homologous genes in opposite mating types serves as a preventive measure for recombination or gene conversion, thus protecting the genetic organization of the locus (6). Another important feature of mating type loci is to make sure that the locus is inherited as a single intact unit in order to maintain heterothallism. In fact, conservation of mating type genes within and between species has been well documented for other heterothallic fungi, such as Pyrenopeziza brassicae and Tapesia yallundae, which are plant pathogenic discomycetes (23).

It is possible that an essential gene within the locus may ensure the stable inheritance of the locus. The PRT1 homolog, present in the C. neoformans MAT locus, may serve this function since this gene is essential in S. cerevisiae (8). If this assumption is correct, there must be a gene in the MATa locus which has the same function as the PRT1 gene. Analysis of the MATa locus will clarify this question.

The MAT locus in C. neoformans is one of the largest MAT loci among fungi with a one-locus, two-allele heterothallism. It is known that the MAT locus of Ustilago hordei, a bipolar fungus, is even larger, spanning a region of about 500 kb (14). Though bipolar in phenotype, the MAT locus of U. hordei, however, is composed of two distinct but closely linked loci, a and b. The a locus encodes mating-type specific pheromones (Uhmfa) as well as the pheromone receptors (Uhpra). The b locus is multiallelic and contains two divergently transcribed genes, bE (bEast) and bW (bWest). Furthermore, the b locus governs pathogenicity and completion of the life cycle. The a and b loci are separated by a spacer region in which recombination is probably suppressed, and thus, the bipolarity of the species is maintained (14). The MAT loci in U. maydis or S. commune are much more complex. U. maydis has, in contrast, a tetrapolar mating system because the a and b loci are on separate chromosomes and therefore segregate independently during meiosis (1), whereas S. commune has, in addition to its tetrapolar mating system, a multiallelic B locus (24). Therefore, the MAT locus in C. neoformans is not only different from MAT loci in these fungi in its organization but also is one of the largest known mating type loci. The presence of numerous mating type-specific genes in the MAT locus distinguishes C. neoformans from all other heterothallic fungi thus far investigated.