Mapping of the Cryptococcus neoformans MATalpha locus: presence of mating type-specific mitogen-activated protein kinase cascade homologs

Abstract

In this study we investigated the relationship between the MATalpha locus of Cryptococcus neoformans and several MATalpha-specific mitogen-activated protein (MAP) kinase signal transduction cascade genes, including STE12alpha, STE11alpha, and STE20alpha. To resolve the location of the genes, we screened a cosmid library of the MATalpha strain B-4500 (JEC21), which was chosen for the C. neoformans genome project. We isolated several overlapping cosmids spanning a region of about 71 kb covering the entire MATalpha locus. It was found that STE12alpha, STE11alpha, and STE20alpha are imbedded within the locus rather than closely linked to the locus. Furthermore, three copies of MFalpha, the mating type alpha-pheromone gene, a MATalpha-specific myosin gene, and a pheromone receptor (CPRalpha) were identified within the locus. We created a physical map, based on the restriction enzyme BamHI, and identified both borders of the MATalpha locus. The MATalpha locus of C. neoformans is approximately 50 kb in size and is one of the largest mating type loci reported among fungi with a one-locus, two-allele mating system.

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). STE12α is located on the right side of the restriction map, and MFα1 is localized on the 13-kb BamHI fragment at the left side. The exact position of MFα1 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.

FIG. 2

Southern blot analysis of MFα genes. Cosmid DNA and genomic DNA of B-4500 and B-4476 digested with HaeII were hybridized with the MFα1 probe. Lanes 1 and 2 show three MFα 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).

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 MATα 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).

FIG. 4

Overview of the MATα 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 (MATα) 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) MFα2/3 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 MFα1 (D) is located on the same 13-kb BamHI fragment as STE11α (E). The largest BamHI fragment is ∼21 kb in size and contains a myosin gene (F), STE20α (G), the pheromone receptor CPRα (H), and STE12α (I).