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    ๊ฐ๋งˆ ๋ฐฉ์‚ฌ์„  ๊ฐ•๋„์— ๋”ฐ๋ฅธ ๋ฏธ์ƒ๋ฌผ๊ตฐ์ง‘ ๋ณ€ํ™”์™€ ๊ธฐ๋Šฅ์œ ์ „์ž ๋ฐ˜์‘

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    ํ•™์œ„๋…ผ๋ฌธ (๋ฐ•์‚ฌ)-- ์„œ์šธ๋Œ€ํ•™๊ต ๋Œ€ํ•™์› : ์ž์—ฐ๊ณผํ•™๋Œ€ํ•™ ์ƒ๋ช…๊ณผํ•™๋ถ€, 2019. 2. Waldman, Bruce.์ด์˜จํ™”๋ฐฉ์‚ฌ์„ ์ด ํ™˜๊ฒฝ์— ์ดˆ๋ž˜ํ•˜๋Š” ์˜ํ–ฅ์— ๋Œ€ํ•ด ์•Œ์•„๋ณด๊ธฐ ์œ„ํ•ด์„œ๋Š” ์ƒํƒœํ•™์  ์ ‘๊ทผ์ด ํ•„์š”ํ•˜๋‹ค. ๊ทธ๋Ÿฌ๋‚˜, ์ด์˜จํ™”๋ฐฉ์‚ฌ์„ ์ด ํ† ์–‘ ์ƒ๋ฌผ๊ตฐ์ง‘์— ์ฃผ๋Š” ์˜ํ–ฅ์— ๋Œ€ํ•ด์„œ ์•Œ๋ ค์ง„ ๋ฐ”๊ฐ€ ๋งŽ์ง€ ์•Š๋‹ค. ๋ณธ ์—ฐ๊ตฌ์—์„œ๋Š” 6์ฃผ๊ฐ„ ์ฃผ๋‹น 24์‹œ๊ฐ„ ๋™์•ˆ 3๊ฐ€์ง€ ๋‹ค๋ฅธ ๋†๋„(0.1 kGy/hr [์ €], 1 kGy/hr [์ค‘] and 3 kGy/hr [๊ณ ])์—์„œ ํ† ์–‘์„ 60Co ๊ฐ๋งˆ์„ ์— ๋…ธ์ถœ์‹œ์ผฐ๋‹ค. ์ฒซ ๋ฒˆ์งธ ์—ฐ๊ตฌ์—์„œ๋Š” ํ† ์–‘ ๋‚ด DNA๋ฅผ ์ถ”์ถœํ•œ ํ›„ ์ƒท๊ฑด ๋ฉ”ํƒ€์ง€๋†ˆ ์‹œํ€€์‹ฑ (shotgun metagenome sequencing)์„ ํ•˜๊ณ  MG-RAST๋ฅผ ์ด์šฉํ•˜์—ฌ ๋ถ„์„ํ–ˆ๋‹ค. ๋ฐฉ์‚ฌ์„  ๋…ธ์ถœ์ด ์ฆ๊ฐ€ํ• ์ˆ˜๋ก ๋ถ„๋ฅ˜ํ•™์  ๋‹ค์–‘์„ฑ๊ณผ ๊ธฐ๋Šฅ์  ๋‹ค์–‘์„ฑ ๋ชจ๋‘ ๊ฐ์†Œํ•  ๊ฒƒ์œผ๋กœ ์˜ˆ์ƒํ–ˆ๋‹ค. ๋ฐ•ํ…Œ๋ฆฌ์•„์˜ ๋ถ„๋ฅ˜ํ•™์  ๋‹ค์–‘์„ฑ์€ ๊ฐ์†Œํ•œ ๋ฐฉ๋ฉด, ์˜ˆ์ƒ์™ธ๋กœ ๊ท  ๋ฐ ์กฐ๋ฅ˜์˜ ๋‹ค์–‘์„ฑ์€ ์ฆ๊ฐ€ํ–ˆ๋Š”๋ฐ, ์•„๋งˆ๋„ ๊ฒฝ์Ÿ์œผ๋กœ๋ถ€ํ„ฐ ๋ฒ—์–ด๋‚˜๊ฒŒ ๋œ ๊ฒƒ์ด ์ด์œ ๋กœ ์ถ”์ •๋œ๋‹ค. ๋ฐ•ํ…Œ๋ฆฌ์•„ ๋ฐ ์ „์ฒด ์ƒ๋ฌผ๊ตฐ์˜ ๋ถ„๋ฅ˜ํ•™์  ๋‹ค์–‘์„ฑ์€ ๊ฐ์†Œํ–ˆ์œผ๋‚˜, ๋ฐ•ํ…Œ๋ฆฌ์•„, ๊ท  ๋ฐ ์ „์ฒด ์ƒ๋ฌผ๊ตฐ์˜ ๊ธฐ๋Šฅ์  ์œ ์ „์ž ๋‹ค์–‘์„ฑ์€ ์ฆ๊ฐ€ํ–ˆ๋‹ค. ์ด๋Š” ์ŠคํŠธ๋ ˆ์Šค ํ˜น์€ ๊ต๋ž€์ด ๋‹ค์–‘์„ฑ์„ ์ฆ๊ฐ€์‹œํ‚ค๋Š” ํšจ๊ณผ๊ฐ€ ์žˆ๋‹ค๋Š” ๊ฒƒ์— ๋Œ€ํ•œ ์ƒˆ๋กœ์šด ์˜ˆ์‹œ๋กœ, ๋ฐฉ์‚ฌ์„  ๋…ธ์ถœ์ด ํ† ์–‘ ์ƒ๋ฌผ๋กœ ํ•˜์—ฌ๊ธˆ ๊ธฐ๋Šฅ์ ์œผ๋กœ ๋” ๊ด‘๋ฒ”์œ„ํ•œ ์ „๋žต์„ ์ทจํ•˜๊ฒŒ ํ•œ๋‹ค๋Š” ๊ฒƒ์„ ์•”์‹œํ•œ๋‹ค. ๋ฐ˜๋ณต์ ์ธ ๋ฐ€๋„ ์˜์กด์  ๊ตฐ์ง‘ ์ƒ์žฅ์˜ ๋ถ•๊ดด ๋ฐ ํ™•์žฅ์ด ๋ณต๊ถŒ๊ณผ ๊ฐ™์€ ํšจ๊ณผ (lottery effect)๋ฅผ ์ฃผ์–ด ๊ณต์กด์„ ์ฆ์ง„์‹œํ‚ค๋Š” ๊ฒƒ์ผ ์ˆ˜ ์žˆ๋‹ค. ๋ฐฉ์‚ฌ์„ ์€ ์ „๋ฐ˜์ ์ธ ๊ตฐ์ง‘ ๊ตฌ์„ฑ์— ํฐ ๋ณ€ํ™”๋ฅผ ์ฃผ์—ˆ๋‹ค. ๋ณธ ์—ฐ๊ตฌ์—์„œ๋Š” ๋ฐฉ์‚ฌ์„ ์— ๋‚ด์„ฑ์ด ์žˆ๋Š” ์ƒˆ๋กœ์šด ๋ฏธ์ƒ๋ฌผ ๊ตฐ์„ ์ œ์‹œํ•œ๋‹ค: ์ „์ฒด ๊ตฐ์ง‘์˜ 20%๋ฅผ ์ฐจ์ง€ํ•˜๋Š” Deinococcus-Thermus๋ฟ ์•„๋‹ˆ๋ผ Chloroflexi (๋ฐ•ํ…Œ๋ฆฌ์•„), Basidiomycota์™€ Chytridiomycota(๊ท ), ๊ทธ๋ฆฌ๊ณ  Nanoarcheota (๊ณ ์„ธ๊ท ) ๋˜ํ•œ ๋ฐฉ์‚ฌ์„ฑ์— ๋‚ด์„ฑ์ด ์žˆ๋Š” ๊ฒƒ์œผ๋กœ ๋ณด์ธ๋‹ค. ๋ฐฉ์‚ฌ์„ฑ ๋…ธ์ถœ๋กœ ์ธํ•ด ๋ฐ”์ด๋Ÿฌ์Šค์™€ ์ „์ด์ธ์ž (transposon)์˜ ์ƒ๋Œ€์  ๋นˆ๋„๋Š” ์ฆ๊ฐ€ํ–ˆ๋Š”๋ฐ, ๋ฐฉ์‚ฌ๋Šฅ ์ŠคํŠธ๋ ˆ์Šค๋ฅผ ๋ฐ›์€ ์„ธํฌ๋“ค์˜ ์ €ํ•ญ์„ฑ์ด ๋–จ์–ด์กŒ๊ธฐ ๋•Œ๋ฌธ์œผ๋กœ ์ถ”์ธก๋œ๋‹ค. ์˜ˆ์ƒ์™ธ๋กœ, ์—ด ์ถฉ๊ฒฉ (heat shock), ํ•ด๋…์ž‘์šฉ, ์‚ฐ(acid) ์ŠคํŠธ๋ ˆ์Šค ๋ฐ ์ €์˜จ ์ŠคํŠธ๋ ˆ์Šค ๋“ฑ์˜ ์ŠคํŠธ๋ ˆ์Šค ๊ด€๋ จ ์œ ์ „์ž์˜ ์ƒ๋Œ€์  ๋นˆ๋„๋Š” ๋ฐฉ์‚ฌ๋Šฅ ๋†๋„๊ฐ€ ๊ฐ€์žฅ ๋†’์„ ๋•Œ ์ค„์–ด๋“ค์—ˆ๋‹ค. ๊ทธ๋Ÿฌ๋‚˜, ํœด๋ฉด ๊ด€๋ จ ์œ ์ „์ž(ํผ์‹œ์Šคํ„ฐ ์„ธํฌ (persister cells), ํฌ์ž์˜ ํƒˆ์ˆ˜, ์ƒ์„ฑ ๋ฐ ์ƒ์žฅ ๋“ฑ)์˜ ๋‹ค์–‘์„ฑ ๋ฐ DNA ์ˆ˜๋ฆฌ ๊ด€๋ จ ์œ ์ „์ž๋Š” ์˜ˆ์ƒ๋Œ€๋กœ ์ฆ๊ฐ€ํ–ˆ๋‹ค. ๋‘ ๋ฒˆ์งธ ์—ฐ๊ตฌ๋Š” Deinococcus (๊ฐ๋งˆ์„ ์— ์ €ํ•ญ์„ฑ์ด ์žˆ๋‹ค๊ณ  ์•Œ๋ ค์ง„ ๋ฐ•ํ…Œ๋ฆฌ์•„์˜ ํ•œ ์†(genus))์˜ ์ƒํƒœ์  ํŠน์„ฑ์— ์ดˆ์ ์„ ๋งž์ถ”์–ด ์ง„ํ–‰๋˜์—ˆ๋‹ค. 6์ฃผ๊ฐ„ ๋ฐฉ์‚ฌ์„ ์— ๋…ธ์ถœ๋œ ํ† ์–‘ ๋‚ด์—์„œ DNA๋ฅผ ์ถ”์ถœํ•˜์—ฌ 16S rRNA ์œ ์ „์ž์˜ ์‹œํ€€์Šค ๋ฐ์ดํ„ฐ๋ฅผ ํ™•๋ณดํ–ˆ๊ณ , ๋ฉ”ํƒ€์ง€๋†ˆ (metagenome) ๋ฐ์ดํ„ฐ ๋ฐ ๊ธฐ์กด์— ๋ฐœํ‘œ๋œ Deinococcus์˜ ์ „์ฒด ์œ ์ „์ž ๋ฐ์ดํ„ฐ๋ฅผ ์ด์šฉํ•˜์—ฌ ๋‹ค์Œ ๋ฌผ์Œ์— ๋Œ€ํ•œ ํ•ด๋‹ต์„ ์–ป๊ณ ์ž ํ–ˆ๋‹ค: 1) ๋ฐฉ์‚ฌ์„  ๋…ธ์ถœ์ด ์ฆ๊ฐ€ํ•จ์— ๋”ฐ๋ผ ๋ฐ•ํ…Œ๋ฆฌ์•„์˜ ๊ตฐ์ง‘ ๊ตฌ์กฐ๋Š” ์–ด๋–ป๊ฒŒ ๋ณ€ํ™”ํ•  ๊ฒƒ์ธ๊ฐ€? ๊ทธ๋ฆฌ๊ณ  Deinococcus์— ์†ํ•˜๋Š” ๋‹ค์–‘ํ•œ ์ข…๋“ค์ด ๋ฐฉ์‚ฌ์„  ๋†๋„์— ๋”ฐ๋ผ ์„œ๋กœ ๋‹ค๋ฅด๊ฒŒ ์šฐ์ ํ•  ๊ฒƒ์ธ๊ฐ€? ์ฆ‰, ๋ฐฉ์‚ฌ์„ ์— ๋”ฐ๋ฅธ ์ƒํƒœ์  ์ง€์œ„ (radiation niches)๊ฐ€ ์กด์žฌํ•  ๊ฒƒ์ธ๊ฐ€? 2) ์–ด๋–ค ์œ ์ „์  ํŠน์ง•์ด Deinococcus๊ฐ€ ๋ฐฉ์‚ฌ์„ ์ด ๋†’์€ ํ™˜๊ฒฝ์—์„œ๋„ ์ƒ์กดํ•  ์ˆ˜ ์žˆ๊ฒŒ ํ•˜๋Š”๊ฐ€? 3) ๋ฐฉ์‚ฌ์„ ์— ๋…ธ์ถœ๋œ ํ† ์–‘์—์„œ Deinococcus๋Š” ์–ด๋–ค ์˜์–‘ํ•™์  ํŠน์ง•์„ ๊ฐ€์ง€๋ฉฐ, ์ด๋ฅผ ํ†ตํ•ด ๋ฐฉ์‚ฌ์„ ์— ๋…ธ์ถœ๋œ ํ† ์–‘ ๋‚ด ์ƒํƒœ์  ๊ณผ์ •๋“ค์— ๋Œ€ํ•ด ์•Œ ์ˆ˜ ์žˆ๋Š” ๊ฒƒ์€ ๋ฌด์—‡์ธ๊ฐ€? ๋ณธ ์—ฐ๊ตฌ์˜ ๊ฒฐ๊ณผ๋Š” ๋‹ค์Œ๊ณผ ๊ฐ™๋‹ค: 1) ๋ฐฉ์‚ฌ์„  ๋…ธ์ถœ ๋†๋„๊ฐ€ ๋†’์„์ˆ˜๋ก Deinococcus์˜ ์ƒ๋Œ€์  ๋นˆ๋„๋Š” ์ฆ๊ฐ€ํ–ˆ์œผ๋ฉฐ, ๊ฐ€์žฅ ๋†’์€ ๋†๋„์—์„œ๋Š” ์ƒ๋Œ€์  ๋นˆ๋„๊ฐ€ 80%์— ์œก๋ฐ•ํ–ˆ๋‹ค. ๋ฐฉ์‚ฌ์„  ๋†๋„์— ๋”ฐ๋ผ ์šฐ์ ํ•˜๋Š” Deinococcus ์ข…์ด ์ƒ์ดํ–ˆ๋Š”๋ฐ, ์ด๋Š” ๋ฐฉ์‚ฌ์„ ์— ๋”ฐ๋ฅธ ์ƒํƒœ์  ์ง€์œ„๊ฐ€ ์กด์žฌํ•จ์„ ๋‚˜ํƒ€๋‚ธ๋‹ค. 3 kGy/hr์˜ ๋†๋„์—์„  D. ficus๋กœ ์ถ”์ •๋˜๋Š” ๋‹จ ํ•˜๋‚˜์˜ ์กฐ์ž‘๋ถ„๋ฅ˜๋‹จ์œ„ (OTU: operational taxonomic unit)๊ฐ€ ์šฐ์ ํ–ˆ๋‹ค. 2) ๊ธฐ์กด์— ๋ฐœํ‘œ๋œ ๋ฉ”ํƒ€์ง€๋†ˆ ๋ฐ์ดํ„ฐ์—์„œ๋„ D. ficus๊ฐ€ Deinococcus์— ์†ํ•˜๋Š” ๋‹ค๋ฅธ ์ข…๋“ค๋ณด๋‹ค ๋” ๋ณต์žกํ•œ ์œ ์ „์  ๊ตฌ์กฐ๋ฅผ ๊ฐ€์ง€๋ฉฐ, DNA ๋ฐ ๋‰ดํด๋ ˆ์˜คํ‹ฐ๋“œ(nucleotide) ๋Œ€์‚ฌ, ์„ธํฌ๋ฒฝ, ์„ธํฌ๋ง‰ ์‹ ์ƒ, ์„ธํฌ ๋ถ„์—ด ์กฐ์ ˆ, ์„ธํฌ ๋ถ„์—ด ๋ฐ ์—ผ์ƒ‰์ฒด ๋ถ„ํ•  ๊ด€๋ จ ์œ ์ „์ž๊ฐ€ ์ƒ๋Œ€์ ์œผ๋กœ ๋” ๋งŽ์•˜๋‹ค๊ณ  ๋ณด๊ณ ๋˜์—ˆ๋‹ค. ๋˜ํ•œ GC ๋น„์œจ๋„ Deinococcus์— ์†ํ•˜๋Š” ๋‹ค๋ฅธ ์ข…์— ๋น„ํ•ด ๋†’๋‹ค. ์ด๋Ÿฌํ•œ ํŠน์ง•๋“ค์€ ์œ ์ „์ž์˜ ์•ˆ์ •์„ฑ์— ๊ธฐ์—ฌํ•  ๊ฒƒ์œผ๋กœ ์ƒ๊ฐ๋˜๊ณ  ๋ช…๋ฐฑํžˆ ๊ฒฝ์Ÿ๋„๊ฐ€ ๋†’๋‹ค๊ณ  ํ•  ์ˆ˜ ์žˆ๋Š” ๋ฐฉ์‚ฌ์„  ๋…ธ์ถœ์ด ์‹ฌํ•œ ๊ณณ์—์„œ๋„ ์šฐ์ ํ•  ์ˆ˜ ์žˆ๋Š” ๊ทผ๊ฑฐ๋ฅผ ์ œ๊ณตํ•œ๋‹ค. 3) ๊ธฐ์กด์— ๋ฐœํ‘œ๋œ ์œ ์ „์—ฐ๊ตฌ ๋ฐ์ดํ„ฐ๋ฅผ ๊ธฐ๋ฐ˜์œผ๋กœ ํ•  ๋•Œ, D. ficus ๋“ฑ์„ ํฌํ•จํ•˜๋Š” Deinococcus ์†์ด ๋ฐฉ์‚ฌ์„ ์œผ๋กœ ์ธํ•ด ์ฃฝ์€ ๋ฏธ์ƒ๋ฌผ ์„ธํฌ๋กœ๋ถ€ํ„ฐ ์œ ๋ž˜ํ•œ ํƒ„์†Œ๊ณต๊ธ‰์› (์•„๋ผ๋น„๋…ธ์Šค (arabinose), ์ –๋‹น, ์•„์„ธํ‹ธ ๊ธ€๋ฃจ์ฝ”์‚ฌ๋ฏผ (N-acetyl-D-glucosamine) ๋“ฑ C5-C12๋ฅผ ํฌํ•จํ•˜๋Š” ๋ณตํ•ฉ์ฒด)๊ณผ ์‹๋ฌผ๋กœ๋ถ€ํ„ฐ ์œ ๋ž˜ํ•œ ํ† ์–‘ ์œ ๊ธฐ๋ฌผ์งˆ๋“ค(์…€๋ฃฐ๋กœ์˜ค์Šค (cellulose), ํ—ค๋ฏธ์…€๋ฃฐ๋กœ์˜ค์Šค (hemicelluloses) ๋“ฑ)์„ ์ด์šฉํ•  ์ˆ˜ ์žˆ๋‹ค๋Š” ๊ฒƒ์„ ์•Œ ์ˆ˜ ์žˆ์—ˆ๋‹ค. 4) ๋ฐฉ์‚ฌ์„  ๋…ธ์ถœ์ด ๊ฐ€์žฅ ์‹ฌํ–ˆ๋˜ ํ† ์–‘์—์„œ๋„ ํ† ์–‘์‹œ์Šคํ…œ ๋‚ด์—์„œ์˜ ๊ธฐ๋ณธ์ ์ธ ๊ธฐ๋Šฅ๊ณผ ๊ด€๋ จํ•œ ์œ ์ „์ž๋“ค (๋ฆฌ๊ทธ๋‹Œ (lignin) ๋ถ„ํ•ด, ์ธ (P) ๊ฐ€์šฉํ™”, ์งˆ์†Œ ๊ณ ์ • ๋“ฑ)์ด ๋น„๋ก ๋‚ฎ์€ ๋†๋„์ด์ง€๋งŒ ์—ฌ์ „ํžˆ ์กด์žฌํ•œ๋‹ค๋Š” ๊ฒƒ์„ ๋ฉ”ํƒ€์ง€๋†ˆ์„ ํ†ตํ•ด ์•Œ ์ˆ˜ ์žˆ์—ˆ๋‹ค. ๊ณ ๋†๋„๋กœ ๋ฐฉ์‚ฌ์„ ์ด ๋…ธ์ถœ๋œ ํ† ์–‘์˜ ํšŒ๋ณต๋ ฅ ๋ฐ ์ง€์† ๊ฐ€๋Šฅ์„ฑ์— ๋Œ€ํ•œ ์—ฐ๊ตฌ, ๋‹ค์–‘ํ•œ ํ† ์–‘ ์œ ํ˜•์—์„œ์˜ Deinococcus์˜ ์šฐ์ ๋„ ๋ฐ ํ† ์–‘ ๋‚ด ๊ธฐ๋Šฅ ๋ณ€ํ™”์— ๊ด€ํ•œ ์—ฐ๊ตฌ๋„ ํ›„์†์—ฐ๊ตฌ ์ฃผ์ œ๋กœ ํฅ๋ฏธ๋กœ์šธ ๊ฒƒ์œผ๋กœ ์ƒ๊ฐ๋œ๋‹ค. ๋ณธ ์—ฐ๊ตฌ๋ฅผ ํ†ตํ•ด ๊ฐ๋งˆ์„ ์— ๋…ธ์ถœ๋œ ํ† ์–‘ ์ƒ๋ฌผ๊ตฐ์ง‘์˜ ๊ธฐ๋Šฅ์  ์œ ์ „์ž ๋ฐ ๋ถ„๋ฅ˜ํ•™์  ๊ตฌ์„ฑ์— ๋Œ€ํ•ด ์•Œ์•„๋ณผ ์ˆ˜ ์žˆ์—ˆ๋‹ค. ๋ณธ ์—ฐ๊ตฌ๋Š” ํ† ์–‘ ๋ฏธ์ƒ๋ฌผ ๊ตฐ์ง‘์ด ์ด์˜จํ™”๊ฐ๋งˆ์„  (ionizing gamma irradiation)์˜ ๋‹ค์–‘ํ•œ ๋…ธ์ถœ ๋†๋„์—์„œ ์‚ด์•„๋‚จ์„ ์ˆ˜ ์žˆ๋Š” ๋Šฅ๋ ฅ์ด ์žˆ๋‹ค๋Š” ๊ฒƒ์„ ๋ณด์—ฌ์ฃผ์—ˆ๋‹ค. ์ด๋Š” ๊ฐ๋งˆ์„ ์ด ํ† ์–‘ ๋˜๋Š” ๋‹ค๋ฅธ ์ค‘์š” ๋ฌผ์งˆ๋“ค์˜ ๋ฉธ๊ท ๊ณผ์ •์— ๋„๋ฆฌ ์ด์šฉ๋˜๋Š” ๊ฒƒ์— ๋Œ€ํ•œ ์ค‘์š”ํ•œ ์‹œ๊ฐ์„ ์ œ๊ณตํ•˜๊ณ  ์˜ค์—ผ๋œ ํ™˜๊ฒฝ์— ๋Œ€ํ•œ ์ดํ•ด๋ฅผ ๋„์šฐ๋ฉฐ, ์šฐ์ฃผ์—ฌํ–‰์— ๋Œ€ํ•œ ๊ฐ€๋Šฅ์„ฑ ๋ฐ ๋‹ค๋ฅธ ํ–‰์„ฑ์—์„œ์˜ ์ง€๊ตฌ์ƒ๋ช…์ฒด์˜ ์ƒ์กด ๊ฐ€๋Šฅ์„ฑ์„ ์ œ์‹œํ•œ๋‹ค. ๋ฐ˜๋ณต์ ์ธ ์ฃผ๊ธฐ๋กœ ํ† ์–‘์„ ๊ฐ๋งˆ์„ ์„ ๋…ธ์ถœ์‹œํ‚จ ๋ณธ ์—ฐ๊ตฌ์™€๋Š” ๋‹ฌ๋ฆฌ ์ง€์†์ ์œผ๋กœ ํ† ์–‘์„ ๊ฐ๋งˆ์„ ์— ๋…ธ์ถœ์‹œํ‚จ ์—ฐ๊ตฌ, ์‹ค์ œ ๋ฐฉ์‚ฌ์„ ์œผ๋กœ ์˜ค์—ผ๋œ ํ† ์–‘์ƒ˜ํ”Œ์„ ๋Œ€์ƒ์œผ๋กœ ํ•œ ์—ฐ๊ตฌ, ๊ธฐํƒ€ ๋‹ค๋ฅธ ์ฐจ์„ธ๋Œ€์‹œํ€€์‹ฑ (NGS: next generation sequencing)๊ธฐ๋ฒ•๋“ค์„ ์ด์šฉํ•œ ์—ฐ๊ตฌ ๋“ฑ์˜ ํ›„์†์—ฐ๊ตฌ๋“ค์ด ๋“ฑ์žฅํ•  ๊ฒƒ์œผ๋กœ ๊ธฐ๋Œ€๋œ๋‹ค. ์ด๋Š” ์ƒ๋ฌผ์  ํ™˜๊ฒฝ ์ •ํ™” (bioremediation) ๋ฐ ์ •์ฑ… ํ˜•์„ฑ์— ๊ธฐ์—ฌํ•˜๊ณ  ์œ ์šฉํ•œ ์ƒ๋ฌผ, ์œ ์ „์ž, ๋ฐ ์ƒํ™œ์„ฑ (bioactive) ๋ฌผ์งˆ๋“ค์— ๋Œ€ํ•œ ์—ฐ๊ตฌ ๋ฐ ๊ฐœ๋ฐœ์—๋„ ๋„์›€์„ ์ค„ ๊ฒƒ์ด๋‹ค.Ionizing radiation is a unique pollutant that requires novel ecological approaches and concepts to outline the resulting environmental outcome. However, little is known of the effects of ionizing radiation exposure on soil biota. Here, the soil was exposed to weekly 24-hour bursts of 60Co gamma radiation over a six-week period, at three levels of exposure (0.1 kGy/hr [low], 1 kGy/hr [medium] and 3 kGy/hr [high]). In the first study, soil DNA was extracted and shotgun metagenomes were sequenced and characterised using MG-RAST. It was hypothesized that with increasing radiation exposure there would be a decrease in both taxonomic and functional diversity. While bacterial diversity decreased, the diversity of fungi and algae unexpectedly increased, perhaps because of release from competition. Despite the decrease in diversity of bacteria and of biota overall, functional gene diversity of algae, bacteria, fungi and total biota increased. Thus, cycles of radiation exposure may increase the range of gene functional strategies viable in the soil, which is a novel ecological example of the effects of stressors or disturbance events promoting some aspects of diversity. Repeated density-independent population crashes followed by population expansion may allow lottery effects, promoting coexistence. Radiation exposure produced large overall changes in community composition. The study suggests several novel radiation tolerant groups: in addition to Deinococcus-Thermus, which reached up to 20 % relative abundance, the phyla Chloroflexi (bacteria), Basidiomycota and Chytridiomycota (fungi) and Nanoarcheota (archaea) are suggested to include radiation-tolerant members. In addition, virus and transposon abundance increased, perhaps owing to reduced resistance by radiation-stressed cells. Unexpectedly, the relative abundance of stress-related genes decreased at higher radiation doses such as heat shock, detoxification, acid stress and cold shock related genes, but the diversity of dormancy (like persister cells, spore core dehydration, spore germination and sporulation cluster related genes) and DNA-repairs-related genes increased โ€“ as might be expected for selection for DNA repair mechanisms. In the second study, attention was focussed on the ecology of Deinococcus โ€“ a genus of soil bacteria known for their radiation resistance, in the context of gamma radiation. The soil DNA was extracted following six weekly cycles of irradiated and studied using 16S rRNA amplicon data, annotated metagenome data and published whole genome for Deinococcus, to investigate the following questions: 1) How does the bacterial community structure change with increasing radiation exposure, and do different Deinococcus species dominate at different radiation intensities โ€“ suggesting the existence of radiation niches? 2) What features of the genomes of the Deinococcus species that predominate at higher radiation intensities confer greater success at high radiation exposures? 3) What are the overall trophic features of the Deinococcus assemblage in radiation-exposed soils, and what might this indicate about ecosystem processes in the irradiated soil? It was observed that 1) increasing radiation dose produced a major increase in relative abundance of Deinococcus, which reached ~80 % of reads at the highest doses. Differing relative abundances of the various Deinococcus species with exposure levels indicate distinct radiation niches. At 3 kGy/hr, a single OTU identified as D. ficus overwhelmingly dominated the mesocosms. 2) Corresponding published genome data show that the dominant species at 3 kGy/hr, D. ficus, has a larger and more complex genome than other Deinococcus species with a greater proportion of genes related to DNA and nucleotide metabolism, cell wall, membrane and envelope biogenesis as well as more cell cycle control, cell division and chromosome partitioning related genes. It also has a higher guanine-cytosine ratio than most other Deinococcus. These features may be linked to genome stability, and explain its greater abundance in this apparently competitive system, under high radiation exposures. 3) Published genome analysis suggests that Deinococcus, including D. ficus, are capable of utilizing diverse carbon sources derived both from microbial cells killed by the radiation (including C5-C12 containing compounds like arabinose, lactose, N-acetyl-D-glucosamine) and plant-derived organic matter in the soil (e.g. cellulose and hemicellulose). 4) Overall, from its metagenome, even the most highly irradiated (3 kGy/hr) soil possesses a wide range of activities necessary for a functional soil system, such as lignin degradation, P solubilisation and N fixation โ€“ although at low abundances. Future studies may consider the resilience and sustainability of such soils in a high radiation environment. In addition to exploring, the extent to which species of Deinococcus dominate depending upon the soil type and the differences this might make to the soil functions. The experimental frameworks of these studies enabled an assessment of functional gene and taxonomic composition of a gamma-irradiated soil community. These studies demonstrated the capacity of soil microbial community to remain viable under differing doses of ionizing gamma irradiation. This consideration is vital in view of the widespread application of gamma radiation to sterilize soil and other important materials, understanding contaminated environments, potential space travel and the viability of Earths lifeforms on other planets. To elaborate on these findings, future studies are anticipated to adopt continuous irradiation of soil as opposed to the repeated burst adopted for the present studies, include samples from actual irradiation contaminated sites and incorporate other next-generation sequencing techniques. Such studies will have greater implications for bioremediation, policy formulation, space exploration and harvesting of useful organisms, genes and bioactive compounds from irradiated soils.Part 1. Enumerating Soil Microbiota Response to Gamma Irradiation using Advanced Sequencing Techniques: An Introduction. 1 1.1. Soil Contamination by Ionizing Radionuclide: Radioecology . 2 1.1.1. The Evolving Field of Radioecology 2 1.1.2. Soil Contamination by Gamma Irradiation . 4 1.2. Application of DNA Sequencing Techniques in Soil Radioecology Studies . 6 1.2.1. -Omics Approaches and Strategies 7 1.2.2. Soil Microbiome Legacy Effects in a Contaminated Environment . 11 1.2.3. Research Aims and Objectives 13 Part 2. Changes in Soil Taxonomic and Functional Diversity Resulting from Gamma Irradiation 15 2.1. Introduction . 16 2.2. Materials and Methods 21 2.2.1. Study Sites and Sampling . 21 2.2.2. Soil Incubation and Gamma [60Co] radiation treatment 21 2.2.3. Soil Chemical Analysis 23 2.2.4. Total DNA Extraction and Shotgun Metagenomic Sequencing 23 2.2.5. Data Processing . 24 2.2.6. Quantitative Reverse Transcription Polymerase Chain Reaction . 24 2.2.7. Statistical Analysis 25 2.3. Results 26 2.3.1. Soil Chemical Properties 26 2.3.2. Microbial Taxa Abundance and Community Composition . 28 2.3.3. Functional Gene Abundance and Composition 34 2.3.4. Microbial Community and Functional Diversity. 37 2.4. Discussion 46 Effects of ฮณ-Irradiation on Soil Chemistry 46 Hypothesis 1: Soils exposed to ฮณ-irradiation will have a taxonomically distinct biota with lower diversity compared to untreated control samples . 47 Hypothesis 2: Soils exposed to ฮณ-radiation will have a lower diversity of functional genes 50 Hypothesis 3: Changes in abundance of certain groups of genes associated with radiation exposure . 51 2.5. Concluding Remarks 54 Part 3. Community Ecology of a Gamma-Irradiated Deinococcus dominatedsoil 57 3.1. Introduction. 58 3.2. Materials and Methods. 62 3.2.1. Sampling, Soil Collection and DNA Extraction 62 3.2.2. Bacteria 16S rRNA Pyrosequencing and Sequence Processing 62 3.2.3. Statistical Analysis 63 3.3. Results 65 3.4. Discussion 85 3.5. Concluding Remarks 93 Part 4. Conclusion. 95 4.1. Changes in Functional Genes and Taxonomic Composition due to 60Co Gamma Irradiation 96 4.2. Dominance of Radiation-Resistant Taxa in Soil Bacterial Community after 60Co Gamma Irradiation 97 4.3. Ecological Implications of 60Co Gamma Irradiation on Soil Microbial Assemblage. 98 4.3.1. Deterministic Outcomes in Soil Microbiome after 60Co Gamma Irradiation 99 4.3.2. Stochastic Effects on Soil Microbial Community of 60Co Gamma Irradiation 100 4.4. Future Directions and Policy Implications. 102 References 104 Appendices 142 ๊ตญ๋ฌธ์ดˆ๋ก (Abstract in Korean). 166 Acknowledgement 171Docto

    Genome-Wide Transcriptome and Antioxidant Analyses on Gamma-Irradiated Phases of Deinococcus radiodurans R1

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    Adaptation of D. radiodurans cells to extreme irradiation environments requires dynamic interactions between gene expression and metabolic regulatory networks, but studies typically address only a single layer of regulation during the recovery period after irradiation. Dynamic transcriptome analysis of D. radiodurans cells using strand-specific RNA sequencing (ssRNA-seq), combined with LC-MS based metabolite analysis, allowed an estimate of the immediate expression pattern of genes and antioxidants in response to irradiation. Transcriptome dynamics were examined in cells by ssRNA-seq covering its predicted genes. Of the 144 non-coding RNAs that were annotated, 49 of these were transfer RNAs and 95 were putative novel antisense RNAs. Genes differentially expressed during irradiation and recovery included those involved in DNA repair, degradation of damaged proteins and tricarboxylic acid (TCA) cycle metabolism. The knockout mutant crtB (phytoene synthase gene) was unable to produce carotenoids, and exhibited a decreased survival rate after irradiation, suggesting a role for these pigments in radiation resistance. Network components identified in this study, including repair and metabolic genes and antioxidants, provided new insights into the complex mechanism of radiation resistance in D. radiodurans

    Structural studies of putative general stress and related proteins from Deinococcus radiodurans

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    This study describes the cloning, expression, purification, biophysical characterisation and crystallisation of DR_1146; a putative general stress protein from the extremophilic bacterium Deinococcus radiodurans (R1). The extraordinary ability of D. radiodurans to resist mutation or apoptosis on exposure to high does of ionising radiation has formed the basis of a structural genomics project underway at the European Synchrotron Radiation Facility (ESRF), Grenoble, France. The work presented in this study forms part of the ESRFโ€™s D. radiodurans initiative, and was funded by the Biotechnology and Biological Sciences Research Council (BBSRC) and the ESRF as an Industrial Cooperative Award in Science and Engineering (CASE) PhD studentship. A period of one-year was spent on secondment at the ESRF, working within the Macromolecular Crystallography Group. Several constructs of the dr_1146 gene have been successfully overexpressed in E. coli cells to give high yields of target protein. Purification by immobilised metal affinity chromatography (IMAC) was facilitated by the incorporation of a 6xHis tag and supplemented by a final gel filtration step. Although high purity levels were achieved, imaging by SDS-PAGE analysis identified that DR_1146 was susceptible to stringent proteolysis. It is thought that initial crystallisation trials were unsuccessful due to inhomogeneity of the sample caused by reported degradation of the target protein. Biophysical characterisation of DR_1146 by isothermal titration calorimetry (ITC) and fluorescence spectroscopy (FS) identified a moderate affinity of 4-11 ฮผM for the flavin molecules, riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Differential scanning calorimetry (DSC) and circular dichroism (CD) experiments demonstrated an increase in chemical and thermal stability of the protein on binding to the flavin molecule, FMN. Analytical ultracentrifugation (AUC) and Nuclear magnetic resonance (NMR) spectroscopy were employed to investigate the solution behaviour of DR_1146 in the presence of FMN. AUC results uncovered a monomer-dimer equilibrium; with DR_1146 self-associating to form a dimer at a concentration of 7.67 ฮผM. NMR spectroscopy depicted that global changes occur within the structure of DR_1146 on binding to FMN. The high quality of spectra obtained showed potential for 3-D structure determination by NMR if ordered crystals could not be obtained for X-ray diffraction. Interestingly, analysis of NMR spectra proved to be integral to identifying a homogenous sample for successful crystallisation of DR_1146. By monitoring chemical shifts it was possible to determine the time needed for degradation of DR_1146 to cease, and the amount of FMN needed to ensure saturation of binding sites. From this particular sample, a stable 28 kDa fragment was isolated by gel filtration. Automated sitting-drop vapour-diffusion experiments resulted in the growth of yellow DR_1146-FMN crystals for which, although poor in quality, X-ray diffraction was obtained. Overall this study reflects the importance and advantage of incorporating information gained from biophysical characterisation into the strategies employed for successful protein crystallisation. The characterisation of DR_1146 as a flavoprotein points towards a possible role in electron transfer due to the extensive redox capacity of flavin. This could implicate the protein in the production of damaging reactive oxygen species (ROS) as a result of irradiation, contributing to oxidative stress levels. Alternatively, if DR_1146 is identified as a FMN-binding pyridoxine 5'-phosphate oxidase (PNPOx) enzyme, as sequence homology suggests, it could play a role in detoxification and stress response through production of pyridoxal 5'-phosphate (PLP), a known scavenger of ROS. Only further characterisation and elucidation of a 3-D structure would confirm or dispel these functional hypotheses and ultimately provide a greater understanding of how D. radiodurans is able to deal with such oxidising conditions. Simultaneously, experiments were carried out on other soluble and membrane protein targets from D. radiodurans and their corresponding homologues from Streptococcus pneumoniae (TIGR4). The aim of comparable studies was to identify key structural or functional differences between the two Gram-positive bacterial strains. Identification of features unique to D. radiodurans, but unconserved in S. pneumoniae, could contribute to further understanding of bacterial radioresistance. SP_1651 is a thiol peroxidase which forms part of the Mn-ABC transport system in S. pneumonia. Its homologue from D. radiodurans, DR_2242 is a putative thiol-specific antioxidant protein, the structure of which has been solved by Dr. Dave Hall as part of the ESRFโ€™s structural genomics project (unpublished). The aim of this part of the project was to elucidate the structure of SP_1651 so that a comparison with DR_2242 could be made. The sp_1651 gene (psaD) was successfully expressed and purified to homogeneity by IMAC and gel filtration. After the proteolytic removal of a 6xHis tag, the purified protein was crystallised by sitting-drop vapour-diffusion. Preliminary diffraction with a resolution limit of 3.2 ร… was obtained, however data showed high mosaic spread. Unfortunately, attempts to reproduce initial crystals failed and hence, structural comparisons with DR_2242 could not be made. DR_0463 is a 108 kDa maltooligosyltrehalose synthase (MTSase) which has been shown to catalyse the breakdown of maltooligosaccharide (or starch) into the disaccharide, trehalose. The full length gene was expressed in BL21(DE3)pLysS cells, producing large yields of insoluble target protein. DR_0463 was solubilised with 8 M Urea and then purified by IMAC in the presence of the denaturant. The low affinity of DR_0463 for the Ni2+ matrix of the HisTrap column proved to be problematic when trying to obtain homogeneity. However, by sequentially repeating IMAC purification up to three times with the same protein sample, a large proportion of impurities were removed. SP_1648 (PsaB) is an ATP-binding protein that forms part of the Mn-ATP transport system in S. pneumoniae and its homologue from D. radiodurans, DR_2284 is predicted to share similar function. Purification of soluble SP_1648, expressed in B834(DE3) cells, was complicated by an inability to bind the protein to the column matrix for IMAC. In the case of DR_2284, expression trials yielded only a minute amount of insoluble protein in BL21-AI competent cells. The bottlenecks in early expression and purification stages provided valuable experience in dealing with problematic proteins. As an introduction to molecular cloning, two genes predicted to encode integral membrane proteins from D. radiodurans, were cloned for preliminary expression trials. This work was carried out at the ESRF and contributed to an extension of the structural genomics project, to incorporate membrane protein targets from D. radiodurans. Full length forms of the genes thought to encode an undecaprenyl diphosphatase (UDP) and a diacylglycerol kinase (DGKA) were successfully cloned in to pET-28b, with incorporation of separate N- and C- terminal 6xHis tags

    Bioremediation of Metals and Radionuclides: What It Is and How It Works (2nd Edition)

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    REPSA Directed Assessment of Native Cleavage Resistance of DNA to Type IIS Restriction Endonucleases and Modification of REPSA for High Temperature Application

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    We have modified the combinatorial selection method Restriction Endonuclease Protection and Selection Assay (REPSA) to work in high temperature conditions for the discovery of new DNA-binding proteins in thermophiles (HT-REPSA). We utilized Thermus thermophilus (HB-8/ATCC 27634/DSM 579) as a test organism due to its amenable nature in a laboratory setting and current status as a model thermophilic organism. We used a TetR Family (TFR) transcription factor SbtR as the model protein for optimization of HT-REPSA protocols, as data had previously been obtained regarding SbtR physical characteristics and DNA-binding properties. REPSA was conducted until a cleavage resistant species arose after 7 rounds. Massively parallel sequencing of the selected DNAs and bioinformatics analysis yielded a consensus binding sequence of 5\u27-GA(t/c)TGACC(c/a)GC(t/g)GGTCA(g/a)TC, a 20base pair palindromic site comparable to that described in the literature. Taken together, our data provide a proof-of-concept that HT-REPSA can be successfully used to identify the preferred DNA-binding sequences of transcription factors from extreme thermophilic organisms

    Evolution of Ionizing Radiation Research

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    The industrial and medical applications of radiation have been augmented and scientific insight into mechanisms for radiation action notably progressed. In addition, the public concern about radiation risk has also grown extensively. Today the importance of risk communication among stakeholders involved in radiation-related issues is emphasized much more than any time in the past. Thus, the circumstances of radiation research have drastically changed, and the demand for a novel approach to radiation-related issues is increasing. It is thought that the publication of the book Evolution of Ionizing Radiation Research at this time would have enormous impacts on the society. The editor believes that technical experts would find a variety of new ideas and hints in this book that would be helpful to them to tackle ionizing radiation
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