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Original Article

DNA Methylation of Gene Expression in Acanthamoeba castellanii Encystation

Eun-Kyung Moon1, Yeonchul Hong2, Hae-Ahm Lee3, Fu-Shi Quan1, Hyun-Hee Kong4,*
The Korean Journal of Parasitology 2017;55(2):115-120.
Published online: April 30, 2017

1Department of Medical Zoology, Kyung Hee University School of Medicine, Seoul 02447, Korea

2Department of Parasitology and Tropical Medicine, Kyungpook National University School of Medicine, Daegu 41944, Korea

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*Corresponding author (hhkong@dau.ac.kr)
• Received: February 21, 2017   • Revised: March 13, 2017   • Accepted: April 3, 2017

Copyright © 2017 by The Korean Society for Parasitology and Tropical Medicine

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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DNA Methylation of Gene Expression in Acanthamoeba castellanii Encystation
Korean J Parasitol. 2017;55(2):115-120.   Published online April 30, 2017
DOI: https://doi.org/10.3347/kjp.2017.55.2.115
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DNA Methylation of Gene Expression in Acanthamoeba castellanii Encystation
Korean J Parasitol. 2017;55(2):115-120.   Published online April 30, 2017
DOI: https://doi.org/10.3347/kjp.2017.55.2.115
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DNA Methylation of Gene Expression in Acanthamoeba castellanii Encystation
Image Image Image Image
Fig. 1 Effect of 5-azacytidine on proliferation and encystation. Acanthamoeba trophozoites were cultured in PYG media without 5-azycytidine (A) or with 5-azacytidine (B) for 3 days. Trophozoites were transferred to encystment media and encystation was induced for 3 days without 5-azycytidine (C) or with 5-azacytidine (D). The 10 μM 5-azacytidine has limited effect on the growth of Acanthamoeba (E). However, encystation ratio was reduced in 5-azacytidine treated cells (52.2%) compared to that in control cells (80.5%) (F). Arrows indicate remained trophozoites after the induction of encystation (D). Values indicate means (±SD) from 3 experiments.
Fig. 2 Promoter region of CSCP. Nucleotide sequence of CSCP promoter region was amplified by PCR using GenomeWalker Kit. This DNA is 2,878 bp in length at the 5′-flaking region of CSCP for bisulfite sequencing PCR. CG dinucleotides in promoter region were underlined.
Fig. 3 Bioinformatic analysis of CSCP promoter region. Three CpG islands (island 1, 1,633 bp; island 2, 625 bp; island 3, 367 bp) of CSCP promoter region were detected by bioinformatics analysis (A). The promoter region was divided into 7 fragments (B). Primers for bisulfite sequencing PCR were designed based on Methprimer.
Fig. 4 Methylation status of CpG island 1 of CSCP promoter. Methylation of promoter CpG island 1 of CSCP in trophozoite was compared to that in cyst by bisulfite sequencing PCR. In trophozoite CpG island 1, 8.2% methylation was observed while 7.3% methylation was observed in cyst. CpG island 1 of trophozoite showed higher methylation than that of cyst. T, trophozoite; C, cyst; ●, methylated CpG site; and ○, unmethylated CpG site.

Fig. 1

Fig. 2

Fig. 3

Fig. 4

DNA Methylation of Gene Expression in Acanthamoeba castellanii Encystation

Primers used in this study

Primer name Sequence (5′ → 3′) Product size (bp)
CP-GSP 1 GAAGAAGAAGACGATGTTGATCCTCAT 2,904
CP-GSP 2 CGTGTTGTTCTTTTTGTAGTTTGTGAGT 2,877
Promoter-F1 F: TTGGTATTATAATATATAATATTATTA
R: AAATTACTCACTACACACTAACAACAA		 322
Promoter-F2 F: TTGTTGTTAGTGTGTAGTGAGTAATTT
R: TTTCTCTCCACTTTTCTTTTTCTTAA		 502
Promoter-F3 F: TTTAAGAAAAAGAAAAGTGGAGAGAA
R: TAAACCTTAATATTATAATAACTAAA		 539
Promoter-F4 F: TTTAGTTATTATAATATTAAGGTTTA
R: TTATAAATATAACTCTTCAACTAACCT		 320
Promoter-F5 F: AGGTTAGTTGAAGAGTTATATTTATAA
R: AACAAAAAAACCCACAAATACCAATAA		 455
Promoter-F6 F: TTATTGGTATTTGTGGGTTTTTTTGTT
R: TAATAATTAACTTAAAACTAAATATAA		 421
Promoter-F7 F: TTATATTTAGTTTTAAGTTAATTATTA
R: TATTATTCTTTTTATAATTTATAAATT		 415
Table 1 Primers used in this study

Table 1

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