Transcription of Cell Wall Mannoproteins-1 gene in Saccharomyces cerevisiae Mutant

Protein phosphatase (PPases) are enzymes to catalyze the phosphate groups removal from amino acid residues of proteins by protein kinases. The PPG1, one of PPases in Saccharomyces cerevisiae has less information in function/role. In this research, the disruption of PPG1::CgHIS3 in FY833 genetic background was successfully constructed by PCR-mediated disruption strategies using pCgHIS3 (EcoRI-HindIII) (=pYMS314) (pUC19 base) and primer pair of PPG1, forward (41 to 100) and reverse (1048 to 1101). A BamHI BamHI fragment 3,28 kb PPG1::CgHIS3 consisting of 1 kb upstream PPG1+ 1.78 kb CgHIS3 + 0.5 down stream of PPG1) was confirmed using PCR and detected using electrophoresis. Phenotypic assay of PPG1::CgHIS3 in FY833 and did not show 200g/ml Calco fluor sensitivity, while another mutant PPG1::CgHIS3 in W303-IA show 100g/ml congo red sensitivity. Furthermore, to confirm whether PPG1 could increase a CWP1 transcriptional level was performed Real Time (RT) PCR analysis using Primer pair Kf (AATTCGGCCTGGTGAGTATCC) and Kr (GTTTCAAAGTGCCGTTATCACT GT). RT-PCR’s data showed that transcriptional level of CWP1 in PPG1::CgHIS3 changed less than two-folds comparing with in wild type strain. This result indicated that disruption of PPG1 in S.cerevisiae did not change CWP1 transcriptional level significantly.


INTRODUCTION
Protein kinase phosphorylates other proteins, while protein phosphatase dephosphorylates other proteins, and this phosphorylation is used to activate or inactivate enzyme activity in cells.The reversible phosphorylation of protein is an essential regulatory mechanism that occurs in eukaryotic cells.Defect either protein kinase or protein phosphatase resulting in abnormal phosphorylation of protein cause many disease (Zolnierowicz and Bollen, 2000).Some advantages reasons why Saccharomyces cerevisiae used in elucidation of protein phosphatase function as follows: 1) S.cerevisiae is the pre-eminent eukaryote for genetic studies; 2) it intensely genetically has been studied; 3) evolutionarily conserved between yeast and human; 4) Study in S.cerevisiae could reveal important new insights about cellular defects associated with human disease (Hermansyah, 2010).
The PPG1, one of protein phosphatases which have been found, has a few an information from previous studies in its function as well as its role.Therefore, study of some functions of PPG1 referenced from old studies.That protein was not essential for cell growth, but it is described that the PPG1 is required for glycogen accumulation through the control of the amount of glycogen synthase (Posas et al., 1993), and required for proper meiosis (Marston, 2004).The ΔPPG1 disruptant showed opposing phenotype with Δrlm1 disruptant where deletion of PPG1 and RLM1 were sensitive and resistant to congored, respectively (Hirasaki et al., 2010).Rlm1, a phosphorylated transcriptional activator, is activated by Slt2, a protein kinase that involved in the PKC1-MAPK-signaling pathway regulating cell wall synthesis and the cell cycle (Gustin et al., 1998).
Calco fluor and congo red can inhibit cell wall construction in fungi (Serrano et al., 2006).Calco fluor induces abnormal septa which apparently fail to develop abscission zones between mother and daughter cells (Roncero and Duran, 1985), a similar effect was produced in S.cerevisiae by congo red (Vannini et al., 1983).The phenotype of inhibited growth by Calco fluor and Congo red has been utilized to screen and isolate cell wall mutants (Ram and Klis, 2006).
CWP1 together with CWP2 are two main genes encoding the cell wall mannoproteins (Van der Vaart et al., 1995).
Deletion of both CWP1 and CWP2 genes encoding cell wall mannoproteins markedly increased cell wall permeability, the effects are apparently synergistic, and inactivation of both CWP genes enhances cell staining by Calco fluor white or Congo red (Zhang et al., 2008).
Based on these previous references, PPG1 protein phosphatase may have relationship with CWP1 transcription in S.cerevisiae.Oligonucletides used as to construct as follows : PPG1::CgHIS3 disruptant in pUC19.YPAD media was prepared from YPAD broth (Sigma-Aldrich Co.) with 0.4 mg/mL adenine.

Yeast Transformation
Yeast transformation was conducted according to previous study (Ausubel et al., 2003).

Transcriptional analysis of CWP1
CWP1 transcriptional level was analyzed using real time PCR as described in previous study (Hermansyah et al., 2009).
Both wild type and The PPG1 disruptant plasmid (Kitada, Yamaguchi and Arisawa, 1995).The PPG1 disruptants were selected in YPAD media that has no histidine contain (His + transformant).By disrupt the whole sequence of PPG1 caused no activity of PPG1 protein phosphatase.
Selected transformants were confirmed by PCR method using primer pair that contains   S.cerevisiae, and more specifically, it may directly dephosphorylate Rlm1 (Hirasaki et al., 2010).
Sensitivity to either calco fluor or congo red has been found as a pleiotropic phenotype associated with certain yeast cell wall mutants since both these drugs have affinity for chitin as minor component of yeast cell wall (Imai et al., 2005).(Dielbandhoesing et al., 1998).Yeast cell wall consist of glucans, which constitute the inner layer of cell wall, mannoproteins, which form an external cell wall layer, and chitin (Klis, Boorsma and De Groot, 2006).Another mechanism, a cross talk between the CWI pathway and the signaling networks controlling the aging process might provide more explanation of the complex mechanism of budding (Molon, Woznicka and Zebrowsk, 2018).
by additional BamHI restriction site on 5` both of them.The nucleotide fragment length was detected using 1% agarose gel electrophoresis.
Cells inoculum grown in YPAD by incubation for 3-4 hours at 30 o C with shaking 150 rpm to reach an OD 660 = 1.0.The cells were harvested at 4 o C by centrifugation, and, after being resuspended into 1 ml 0.1 M Pb acetate.Pellet cells was then added 0.24 ml PEG 4.000 50% (w/v), 0.036 mL Li Acetate 1.0 M, 0.005 ml single strand DNA carrier (10 mg/mL), and 0.070 mL, DNA product (0.1-10g) and sterile water, respectively.Heat shock the mixtures for 25 minutes at 42 o C, and dissolved it in 100 L.Spread onto selective media, and incubate the cells at 30 o C for 2-3 days until some transformed cells grow as colonies.Calco fluor and Congo red phenotypic assay S.cerevisiae PPG1 disruptant cells were streaked either on 200g/ml Calco fluor containing YPAD agar or 100g/ml Congo red containing YPAD agar media.Then, incubated at temperature 30 °C for 2 days.Sensitive phenotype indicated that PPG1 gen involved in cell wall construction, while resistant phenotype indicated that PPG1 gen did not involve in cell wall construction in S.cerevisiae.
were cultured in YPAD media to reach exponential phase (OD660 = 1.0), and cells were harvested to obtain the cells.cDNA template for quantitative RT PCR was prepared as following steps: RNA of S.cerevisiae strains was isolated using the hot phenol method, and first -strand cDNA was prepared using a high capacity cDNA archive kit In this study, we used some wild type strains FY833 AND W303-1A since they were common yeast strains widely used in yeast genetics.The disruption of PPG1::CgHIS3 was constructed by PCR-mediated gene disruption as described in Figure1.PPG1 gene disrupted by integration gene replacement cassete harbouring the CgHIS3 gene(Sakumoto et al., 2002).DNA fragment produced from PCR method were utilized to transform both yeast strains FY833 and W3030-1A.Each primer (reverse or forward primer) used in this study consist of 60 nucleotides of PPG1 and 20 nucleotides of CgHIS3 from plasmid derivated from pUC19 downstream and upstream sequence of PPG1 gene open reading frame.PPG1 gene located on chromosome XV consist of 1107 nucleotides (Jones et al., 1997).Confirm insert genes were carried out by PCR amplification using primers forward (-1000 to -979) and (2587 to 2607) by additional BamHI restriction site on 5` both of them.A BamHI -BamHI of PPG1::CgHIS3 is of 3.3 kpb consist of 1 kb upstream PPG1+ 1.8 kb CgHIS3 + 0.5 down stream of PPG1 as shown in Figure 2.

Figure 1 .
Figure 1.Strategy construction of PPG1 gene disruption by PCR mediated disruption.PPG1 gene disrupted by integration gene replacement cassette harbouring the CgHIS3 gene.

Figure 2 .Figure 3 .
Figure 2. Confirm insert genes were carried out by PCR amplification.PPG gene has been disrupted which confirmed by the presence of DNA fragment 3,3 kb.This indicated that PPG1 gene replaced by CgHIS3 gene.

Figure 4 .Fold
Figure 4. RT-PCR data analyzed transcriptional level of CWP1 in ppg1::CgHIS3 / W303-IA with two different concentrations, 10 g/ml DNA sample and 25 g/ml DNA sample