Screening of Cell Cycle-Related Genes of Pleurotus eryngii Using Yeast Mutant Strains.

Temperature-sensitive yeast mutants were used to screen for cell cycle-related genes from Pleurotus eryngii genomic DNA. A mushroom genomic DNA library was established and each gene was screened for the ability to rescue seven Saccharomyces cerevisiae temperature-sensitive strains. Hundreds of yeast transformants were selected at restrictive temperatures over 30℃. Plasmids from the transformants that survived were isolated and transformed back into their host strains. The temperature sensitivity of the resulting transformants was tested from 30℃ to 37℃. Ten DNA fragments from P. eryngii were able to rescue yeast temperature-sensitive strains, and their DNA sequences were determined.

Cell division is a process by which a parent cell divides into two genetically identical daughter cells. The entire division process proceeds as a cycle of an S phase, where chromosome duplication occurs, and an M phase, where the two daughter cells separate. The two phases are separated by the gap phases G1 and G2. Mitosis is the process in which the duplicated chromosomes segregate into two daughter nuclei. It is followed immediately by cytokinesis, which divides the cellular components into roughly equal shares for each daughter cell. Mitosis and cytokinesis together define the mitotic (M) phase of the cell cycle [1].

Mitotic phase progression in Saccharomyces cerevisiae is controlled by several proteins, including the ser/thr protein kinases Cdc5 and Cdc15, the protein phosphatase Cdc14, and the GTPase Tem1 [2, 3]. The polo-like kinase Cdc5 plays multiple roles throughout the M phase. It promotes G2/M transition to allow initiation of the mitotic phase [4]. Cdc5 also initiates the separation of chromosomes in the middle of M phase [5], and it plays a role in cytokinesis as part of the mitotic exit network (MEN) [2]. Cdc5 promotes mitotic exit by phosphorylating and inhibiting the Bfa/Bub2 complex, which maintains Tem1 in an inactive state [6]. Inhibition of the Bfa1/Bub2 complex thus allows Tem1 activation by the guanine exchange factor Lte1, which converts inactivate Tem1-GDP to the active Tem1-GTP form. Tem1-GTP then binds to and activates Cdc15 and the MEN [7]. Only upon activation of the MEN can cytokinesis take place and cell division be completed. Therefore, Cdc5, Cdc15, and Tem1 are key modulators of the yeast M phase.

Pleurotus eryngii is one of the most cultivated edible mushrooms worldwide. This mushroom has been studied for years, but research has mostly focused on cultivation and bioremediation [8-10]. Basic molecular biology, such as cellular trafficking of molecules, the mechanism of cell division, and even detailed subcellular structures, etc. remain largely unexplored. In this respect, we have been interested in the mechanism of cell division in P. eryngii. In an effort to investigate genes involved in regulating the P. eryngii cell cycle, we developed a method to screen P. eryngii genes in temperature-sensitive mutants of S. cerevisiae for rescue of cell cycle defects at restrictive temperatures. The yeast cell division cycle is well-studied, and temperature-sensitive S. cerevisiae strains containing mutations of the key cell cycle modulators are readily available. As a result, this system is ideal for identifying P. eryngii orthologs of the yeast cell cycle regulators.

To generate the P. eryngii genomic DNA library, genomic DNA was extracted from a powder of the fruiting bodies of P. eryngii KNR2312 with QIAGEN i-genomic DNA extraction mini kits (INtRON Co., Seoul, Korea). The extracted genomic DNA was cut with the restriction enzyme Sau3A1, which generates a GATC overhang sequence on both ends of the DNA fragment. The restricted DNA fragments, ranging from 2~5 kb in length, were extracted from a 1% agarose gel and purified with a gel extraction kit (LaboPass Gel; Cosmo Gentech Co., Seoul, Korea). The extracted DNA fragments were ligated to the BamHI site of a yeast centromeric vector, pRS315, which contains the LEU2 gene as a selective marker. The optimal ratio between vector and DNA fragments was empirically determined to be 4 : 1. After overnight incubation of the reaction at 16℃, the ligation products were directly transformed into competent Escherichia coli DH5α cells. The number of E. coli clones was 2 × 105, and the average insert size was approximately 1 kb. The size of the P. eryngii genome is estimated to be approximately 40Mb [11], so our library appears to cover the whole genome about 5-fold.

In order to screen the mushroom genomic DNA library in yeast S. cerevisiae, we obtained the temperature-sensitive mutant strains by kind donation from Dr. Kyung Lee at the US National Institutes of Health (NIH). The yeast strains in this study are as follows: KL1546 (wild-type), KL2395 (cdc5-1), KL2158 (cdc15-2), KL2391 (tem1-3), KL2392 (tem1-3 Δbub2), KL2412 (tem1-3 Δbfa1), KL2414 (cdc5-1 Δbfa1) and KL2398 (cdc5-1 Δbub2). All the mutants were derived from the S. cerevisiae W303 strain and show a mitotic arrest phenotype, meaning no growth at restrictive temperatures (above 33℃), because mitotic progress is dependent on the activities of the mutated gene products, which only function normally at permissive temperature (25℃).

Transformation of each yeast strain by the mushroom genomic DNA library was carried out as described previously [12]. In brief, the yeast cells were cultivated to OD600 = 0.6~0.8 in 100 mL of yeast extract-peptone-dextrose (YPD) culture media containing 1% yeast extract, 2% peptone, and 2% dextrose at 25℃ with shaking at 100 rpm/min. Cells were harvested and suspended in 100 µL of 0.3M lithium acetate (LiAc) in TE buffer (10mM Tris-HCl and 1 mM EDTA, pH 8.0). To this suspension, 3 µL of salmon sperm DNA and 15 µL of the mushroom genomic DNA library were added, and the mixture was allowed to stand at room temperature for 30 min. Afterwards, 300 µL of polyethylene glycol 4000 solution (40% in LiAc-TE buffer) was added to the mixture and then incubated at 42℃ for 15 min. The suspensions were spun twice to remove all the liquid. Finally, the cells were suspended in a small amount of SOS medium (10 mL of 2M sorbitol, 1.34 mL of 5 × YPD, 0.13 mL of 1M CaCl2, 8.53 mL of water).

Primary selection of transformed colonies was carried out on a leucine dropout minimal medium plate (SD-Leu) containing 6.7 g/L of Yeast Nitrogen Base (BIO101 Co., Vista, CA, USA), 0.72 g/L of CSM-Leu (BIO101 Co.), and 2% glucose. To screen for yeast cells that overcame the temperature-sensitive phenotype through the introduced mushroom gene, colonies on the SD-Leu plate were transferred to four YPD plates by a replica plating method. The YPD plates were then incubated at a permissive temperature (25℃) and several restrictive temperatures (30℃, 33℃, and 37℃). All of the cells grew as normal at 25℃, which is suitable for yeast growth. Growth at 30℃ was not much different from growth at 25℃. The temperature 33℃ seemed to be restrictive to some of the transformants because most of them showed only weak growth at this temperature. Almost all the transformants were unable to grow at 37℃ (Fig. 1). We predicted that the transformants that could survive at higher temperatures were rescued from temperature sensitivity by the induced genes from mushroom genomic DNA. From this initial screening, a total of 129 transformants that formed colonies at 33℃ and 37℃ were selected for further screening.

Fig. 1

Suppression of temperature-sensitive phenotypes of Saccharomyces cerevisiae mutant strains by Pleurotus eryngii (P. eryngii) genomic DNA. Cell cycle-defective yeast host strains (tem1-3Δbub2, cdc5-1, and cdc15-2) were transformed with plasmid clones containing P. eryngii mushroom DNA. The transformants were serially diluted with liquid medium (1 ×, 0.5 ×, 0.1 ×, 0.05 ×) and spotted onto 3 yeast extract-peptone-dextrose plates. The plates were incubated at the permissive (25℃) or restrictive (33℃ and 37℃) temperatures. The clone numbers are listed next to each host strain name.

In order to confirm whether survival at the restrictive temperature was due to P. eryngii DNA, we recovered 25 plasmid DNAs from the yeast transformants that exhibited the strongest growth at the restrictive temperature and determined their DNA sequences. The recovered plasmids were also separately transformed back into their original hosts, and their effects on host growth were examined in serially diluted liquid cultures. Among the 25 plasmids selected for further screening, 10 plasmids showed the best growth at 33℃ or 37℃ (Fig. 1). Analysis of the determined sequence revealed that the P. eryngii sequences contained in the plasmids mostly carried DNA fragments with unknown functions (Table 1). The three transformants of the tem1-3Δbub2 strain that carry the plasmids 2-2-4, 2-2-5 and 2-2-7 had good growth at 37℃, whereas the control host cells could hardly survive (Fig. 1). Interestingly, sequences from clone 2-2-5 showed some homology with a γ-tubulin interacting protein of Chlamydomonas reinhardtii (Fig. 2A). Tubulin disassembly is an important process during mitotic exit [13], suggesting a possible mitotic role of the mushroom DNA contained in clone 2-2-5. Clone 2-2-7 contained a sequence with high homology to the putative urea carboxylase of Aspergillus nidulans, but its role in the cell division is largely unknown (Fig. 2C). We were also able to isolate 3 and 2 more mushroom library plasmids that suppressed cdc5-1 and cdc15-2 temperature-sensitive growth arrest, respectively. BLAST analysis of the genes predicted a few homologous proteins with unknown functions. Only clone 10-1, which suppressed temperature sensitivity of the cdc15-2 strain, appeared to show some homology with the hedgehog/intein hint domain protein of Yersinia intermedia (Fig. 2B). Hedgehog has been reported to upregulate cell cycle proteins [14]. For further information, more rigorous analyses of these identified DNA sequences are required in both P. eryngii and S. cerevisiae.

Table 1

Selected Pleurotus eryngii genomic DNA clones and their homologous proteins

Fig. 2

BLAST analysis of three mushroom DNA sequences that suppressed yeast temperature-sensitive phenotypes. A, Clones 2-2-5 and C, 2-2-7 were selected as suppressors of the yeast tem1-3Δbub2 strain. B, Clone 10-1 was a suppressor of the yeast cdc15-2 strain.