한국산 노랑초파리 집단내에 존재하는 transposable P element의 구조와 기능에 관한 연구

황혜진 梨花女子大學校 大學院 1994 국내석사

Identification and Characterization of Transposable Elements of Pleurotus eryngii

레귀방 경상대학교 대학원 2009 국내박사

Main part of this thesis presents a research that was carried out in order to identify and characterize transposable elements (TEs) of edible mushroom Pleurotus eryngii from TE-enriched libraries. The libraries were constructed from PCR products of three degenerated primer sets, which target conserved domains of reverse transcriptase in TEs. A rate of 31% of sequenced clones was found to be TE-related through comparison with their DNA sequences with those in repeat element database, Repbase. After removing redundant sequences, 24 TE-related fragments with significant similarity to TE from other species were identified. Most of them were retrotransposon of gypsy superfamily. One full-length non-autonomous copy of retrotranposon class, LTR order, gypsy superfamily, marY1-like TE from P. eryngii KNR2312, RLG-marY1-PE was determined and characterized with assistance of DNA walking technique. In further study about RLG-marY1-PE and 24 the putative TE-related fragment, RT-PCR and northern blot with total mRNA and fosmid genomic library were carried out. Twenty one primer sets were derived and used for RT-PCR. Sixteen putative TE-related fragments were identified to be expressed in total mRNA by RT-PCR with primer sets target specifically to each fragment. The fosmid genomic library was screened in order to quickly identify full-length sequences of the putative TE-related fragments and other possible copies of RLG-marY1-PE. This research is one of few aimed on the P. eryngii regarding identification and characterization of transposable elements. The achieved results may contribute significant information for future study of the subject and their applications 본 논문은 새송이 버섯의 유전체내에 존재하는 전이인자 (transposable element)들을 발굴하고 동정하여, 이들이 새송이버섯균의 유전체안정성과 균주퇴화에 어떤 영향을 미치는지를 밝히는 것을 목적으로 한다. 이를 위하여 알려진 곰팡이 전이인자들의 염기서열 중 역전사 효소의 보존된 영역을 목표로 하여 프라이머세트를 제작하였고 이들을 이용하여 새송이 유전체내의 전이인자들을 PCR 로 증폭하여 라이브러리를 제작하였다. 제작된 라이브러리의 염기서열을 무작위로 결정해본 결과, 서열 확인된 클론의 31%가 Repbase에서 서열 비교를 통해 전이 인자와 연관되어 있다는 것을 발견하였다. 반복적으로 나타나는 서열을 제거한 후, 총 24개의 독립적인 전이인자와 연관된 조각들을 확인하였으며, 대부분이 Gypsy 계열의 역전이효소 유전자였다. 이들 중 gypsy type 의 유전자 배열을 가진 LTR (long terminal repeat) 레트로트랜스포존 1종의 전체 염기서열을 밝혔으며 이를 명명법에 따라 RLG-marY1-PE명명하였다. RLG-marY1-PE와 24개의 전이인자의 새송이버섯 KNR2312 유전체내의 분포와 발현 및 염기서열을 결정하기 위하여Nothern blot 분석과 RT-PCR, Fosmid genomic 라이브러리 탐색을 수행하였다. RT-PCR 을 통해서는 24개중 총 21개의 프라이머세트를 이용하여 분석결과 16개 전이인자가 mRNA 로 발현됨을 확인하였고 이는 최소 16개의 다른 전이인자들이 새송이 유전체에서 활성을 가진 전이인자임을 나타내는 것이다. 추가적인 Northern blot 분석과 real time PCR 이 수행중이다. 16개의 전이인자의 유전자염기서열을 확인하기 위하여 Fosmid 라이브러리의 스크리닝을 수행하였으며 잠정적으로 6개의 신규 전이인자를 확인하였고 이들의 염기서열도 확인 중에 있다. 본 연구는 새송이버섯 유전체내의 전이 인자들의 동정 및 특성을 확인함으로서, 새송이버섯균의 퇴화현상을 설명하는 중요한 단서를 제공하여 새송이 유전체의 변화연구에 공헌할 것이다.

From Transposable Elements to DNA Methylation: The Role of Genome Regulation in the Evolution of the Jellyfish Medusa

Zhang, Xinhui ProQuest Dissertations & Theses University of Cali 2022 해외박사(DDOD)

One phenomenon that has perplexed biologists for decades is the lack of correlation between genome size and organismal complexity, aka the ‘C-value paradox’. It is now generally accepted that the paradox is caused by repetitive, non-coding sequences such as transposable elements. These ‘selfish’ or ‘parasitic’ sequences have profound impacts on the host genome. For instance, DNA methylation is thought to have originated as a defense mechanism against these genome parasites. As one of the most conserved epigenetic modifications in eukaryotes, DNA methylation plays important regulatory roles in animals. However, the evolution of transposable elements and DNA methylation have rarely been studied in non-bilaterian animals. This dissertation explores genome regulation in Cnidaria, the sister group of Bilateria, and its potential role in the evolution of complex life histories. Chapter 1 studies the distribution of DNA methylation across Cnidaria using published genomes and transcriptomes of over 70 species. By analyzing a proxy of DNA methylation, I show this epigenetic modification is prevalent in Cnidaria with instances of loss in a group of endoparasitic species. I also show cnidarian DNA methylation shares many similarities with bilaterian invertebrates, supporting the hypothesis that gene body methylation is the ancestral state in Eumetazoa. Additionally, cnidarians with complex life cycles tend to have heavier methylation, and both gene body methylation and repeat methylation increase with genome sizes. Chapter 2 examines DNA methylation patterns across the life history of a scyphozoan jellyfish, Aurelia coerulea, via stage-specific whole-genome bisulfite sequencing. This work characterizes the distribution of DNA methylation on the Aurelia genome, the correlation between methylation and expression levels, and highlights that sparsely-methylated genes tend to be expressed dynamically. Moreover, many genes are differentially methylated across life history stages; however, methylation changes do not predict expression changes. This study indicates that the regulatory role of gene body methylation lies more in stabilizing expression, a notion emerging as the primary function of DNA methylation in animals. Chapter 3 studies transposable elements (TEs) in the Aurelia genus. TEs in 12 species are annotated and quantified using whole genome sequencing. One clade shows signatures of ancient TE expansion events, indicating the TEs have undergone different evolutionary histories. Based on the phylogeny within the genus, these expansion events predate the closure of the isthmus of panama which separated the two clades of Aurelia. Additionally, the genome size difference between Aurelia aurita and Aurelia coerulea is unlikely due to activities of known TEs; however, Aurelia genomes harbor many unique TEs that would be worth further exploring. Overall, this dissertation research shows that Cnidaria is one of the most fascinating systems to study the evolution of genome regulation. As the first animals with real tissue layers, neuro-sensory systems, and a wide variety of different life histories, Cnidaria can shed light on how the genome ‘dark matter’ plays a role in the evolution of animal complexity.

Genetic variability due to P transposable element in natural population of Drosophila melanogaster

Kim, Min-su 中央大學校 1991 국내석사

식물 병해 길항균 Pseudomonas sp.의 Transposable Element를 이용한 돌연변이 유기 및 유전자 탐색

서영우 조선대학교 대학원 1990 국내석사

This study compared the inhibiting effects of different concentrations (75, 150, 300 and 600 ppmF) of SnF_(2), NaF and APF on the growth of a cariogenic, fluoride-sensitive strain of Streptococcus mutans. The results were as follows ; 1. The growth inhibiting effect of SnF_(2) was being greater while the concentration of SnF_(2) was varing from 75 ppmF to 600 ppmF and SnF_(2) marked bactericidal effect at 600 ppmF 2. The effect of NaF on the growth of S. mutans was less than SnF_(2) in general but the growth of S.mutans stopped at 600 ppmF 3. The inhibiting effect of APF was greater than SnF_(2) and NaF at 75, 150 ppmF but was less than SnF_(2) at 300, 600 ppmF. 4. The growth-inhibiting effect of SnF_(2), NaF and APF at the same concentration was as follwing ; at 75 ppmF APF → NaF → SnF_(2) (High) (Low) at 150 ppmF APF → SnF_(2) → NaF (High) (Low) at 300, 600 ppmF SnF_(2) → APF → NaF (High) (Low) 5. When the broth cultures were adjusted to pH levels of either 6.0 or 7.5 at 600 ppmF concentration, a greater inhibiting effect was obtained at lower pH condition and differences in growth of cultures were most noteworthy with SnF_(2).

초파리의 Copia 및 P transposable element의 염색체 분포와 기능

남영미 中央大學校 1990 국내석사

Predictive markers for cancer therapy derived from targeted DNA sequencing and whole transcriptome sequencing

이보람 성균관대학교 일반대학원 2021 국내박사

Background: Newly developed cancer drugs are usually effective only in a subset of patients. Identification of patients for whom the drug is not effective will result in avoidance of adverse drug reactions and reduce inefficient costs. The differences in drug effectiveness between patients usually stem from genetic differences between cancers. Predictive markers for cancer therapy can be identified by analyzing large-scale data containing comprehensive genetic information from a large number of patients. Methods: In part 1, all targeted panel sequencing data for non-small cell lung cancer patients who received EGFR tyrosine kinase inhibitor (TKI) therapies generated at the Samsung Medical Center between January 2014 and May 2017 were retrospectively analyzed. In part 2, I developed a computational tool named ‘rTea’ to detect transposable element-incorporated transcripts (TEITs) from high-throughput short-read RNA sequencing data. Applying this tool to four large-scale consortium data, the landscape of TEITs was analyzed. Results: Seventy-five patients treated with 1st or 2nd generation EGFR TKI (cohort 1) and 82 patients treated with 3rd generation EGFR TKI (cohort 2) were included in part 1. In cohort 1, alterations in TP53 were independently associated with worse progression-free survival in multivariate analysis. In cohort 2, alterations in TP53, RB1, PTEN, and MDM2 were independently associated with worse progression-free survival. In part 2, the detection performance of rTea was validated by in silico simulation and RT-PCR assays. A total of 52,277 cancer-specific and 28,991 normal TEITs were detected in 10,257 cancer samples and 3,190 normal samples. The expression of TEITs was associated with DNA methylation levels. Some TEITs were associated with patient survival in certain cancer types. There was a correlation between the number of TEITs and the immune score calculated from the RNA expression of marker genes. Some of the new peptides derived from cancer-specific TEITs could bind to MHC class I. Conclusions: Possible predictive markers for oncologic drug treatment could be identified from targeted panel sequencing data and whole transcriptome data. In EGFR-mutated non-small cell lung cancer, co-occurring genomic alterations detected by targeted panel sequencing were associated with the clinical outcomes of EGFR-TKI treatment. With the development of rTea, it became possible to identify TEITs that are associated with cancer treatment. 배경: 새로 개발되는 암 치료제는 대개 일부 환자에게만 효과가 있다. 만약 특정 약이 효과적이지 않은 환자들을 식별할 수 있다면, 부정적인 약물 반응을 피하고 낭비되는 비용도 절약할 수 있다. 환자간 약물 효과의 차이는 대개 암의 유전적 차이에서 기인한다. 많은 환자로부터 얻은 대규모 유전체 데이터를 분석하면 암 치료 예측 마커를 효과적으로 발굴할 수 있다. 방법: 1부에서는 2014년 1월부터 2017년 5월까지 삼성서울병원에서 시행한 암 유전자 패널검사 중 EGFR 티로신키나제 억제제 치료를 받은 비소형 세포폐암 환자의 데이터를 모두 분석했다. 2부에서는, 전장 전사체 데이터에서 트랜스포존이 포함된 전사체(TEIT)를 검출할 수 있는 ‘rTea’라는 컴퓨터 분석 툴을 개발했다. 이를 이용해 네 개의 대규모 컨소시엄 데이터에서 TEIT를 분석했다. 결과: 코호트 1에서 TP53의 변이는 다변량 분석에서 더 나쁜 무진행생존과 독립적으로 연관되어 있었다. 코호트 2에서 TP53, RB1, PTEN 및 MDM2의 변이는 독립적으로 더 나쁜 무진행생존과 관련이 있었다. 파트 2에서, rTea의 성능은 컴퓨터 시뮬레이션과 RT-PCR 검사에 의해 검증되었다. 암 시료 10,257건과 정상 시료 3,190건에서 총 5만2,277개의 암 특이 TEIT와 2만8,991건의 정상 TEIT가 검출되었다. TEIT의 발현은 DNA 메틸화 수준과 연관되어 있었다. 일부 TEIT는 특정 암종의 환자 생존과 연관되어 있었고 시료당 검출된 TEIT 수와 마커 유전자 발현으로 계산한 면역점수 사이에 상관관계가 있었다. 암 특이 TEIT에서 발현된 새로운 펩타이드 중에 주조직적합성복합체 I 과 결합이 가능한 것이 있었다. 결론: 암 유전자 패널 데이터와 전장 전사체 데이터를 분석하여 암 치료의 예측 마커의 후보를 얻을 수 있었다. EGFR 변이가 있는 비소세포폐암에서 암 유전자 패널 데이터로 얻은 유전자 변이가 EGFR-TKI 치료의 임상 결과와 관련이 있었다. 또한 rTea의 개발로 인해 암 치료에 영향을 미칠 수 있는 TEIT를 분석할 수 있게 되었다.

Chromosomal Localization of Ribosomal DNAs, Telomeric Tandem Repeats, and Transposable Elements on Bienertia sinuspersici Akhani using Fluorescence in situ Hybridization

Samantha S. Sevilleno Sahmyook University, Sahmyook University Graduate 2020 국내석사

C4 식물은 C3 식물에 비해 탄소고정 능력이 탁월하고 광호흡 제어능력이 뛰어나다. Bienertia sinuspersici Akhani는 Amaranthaceae에 속하며 크란츠 해부 구조를 지니지 않고도 엽육조직에서 C4 광합성을 하는 몇 안되는 종이다. B. sinuspersici는 염분이 많고 고온 다습한 기후에 잘 적응하는 것으로 알려져 있다. B. sinuspersici에 대한 게놈 염기서열분석이 진행중에 있으나 현재까지 이 종에 대한 세포유전학적 보고는 매우 적다. 큰 사이즈의 게놈을 연구하기 위해 염기서열 분석 방법만을 이용하는 것은 최종 염기서열분석에서 물리적 공백을 야기한다. 본 연구에서는 반복염기서열 5S, 45S rDNA와 Arabidopsis-type telomeric repeats을 이용하여 FISH 핵형분석을 수행하였다. 세포유전학 분석 결과, B. sinuspersici는 2배체(2n=2x=18)로 9쌍의 중부동원체형의 염색체로 구성되었다. 진한 DAPI 밴드의 확인으로 AT-rich sequence들이 동원체 영역에 분포됨이 확인되었다. 1쌍의 45S rDNA가 7번 염색체 단완의 말단에서 관찰되었다. 9개의 5S rDNA가 염색체 1, 3, 4, 6, 8번 동원체 주변에서 관찰되었으며 Arabidopsis-type telomeric repeat과 같은 위치에서 관찰되었다. 이 중 8번 염색체는 쌍을 이루지 않고 상동염색체 하나에서만 5S rDNA 시그널이 관찰되었다. 또한 염색체 5, 8번 단완 interstitial region에서 4개의 5S rDNA가 관찰되어 총 13개(13 loci)의 5S rDNA가 확인되었다. C0t 분석을 통해 게놈 연구에 추가적인 데이터를 제공하고 반복염기서열 분포를 확인하고자 수행하였다. C0t-1과 C0t-100 DNA를 이용한 FISH 분석을 통해 B. sinuspersici 게놈상에 많은 부분이 반복염기서열로 구성되어 있음을 확인하였다. B. sinuspersici 게놈상에 존재하는 다양한 반복염기서열의 분포를 확인하고자 FISH 분석을 수행하였다. FISH signal의 정량화는 반복염기서열의 가시화 이외에 유용한 방법으로 이용될 수 있다. 각 반복염기서열의 정량화는 Image J 소프트웨어를 통해 확인하였다. 그 결과, 815 copy로 이루어진 satellite DNA는 1쌍이 염색체 단완에서 관찰되었으며 RC/Helitron transposons, LTR/Gypsy, LINE retroelements은 염색체에 산재하여 분포하였으나 동원체 주변과 염색체 말단부위에서는 매우 약하게 시그널이 관찰되었다. 데이터베이스상에서 일치되지 않는 unknown repeat family 가운에 rnd-5_family-1128를 제외하고 모두 염색체 전반에 걸쳐서 산재하여 관찰되었다. 본 연구를 통해 수집된 데이터는 B. sinuspersici의 게놈을 이해하는데 도움을 줄 것으로 판단된다. C4 plants are efficient in suppressing photorespiration and enhancing carbon gain as compared to C3 plants. Bienertia sinuspersici Akhani, a member of the Amaranthaceae family, is one of the few species that can perform C4 photosynthesis within individual chlorenchyma cells, without having the conventional Kranz anatomy in its leaf. This plant is known to be well-adapted in hot and humid climate with high salt environment. The sequencing of its genome is currently in progress however, to date, there has been no reported cytogenetic analyses yet on this species. Studying the genome organization of plants having medium to large genomes using sequencing methodologies alone will cause physical gaps in the assembly of its final genome sequence. In this study, fluorescence in situ hybridization (FISH) karyotype analysis was conducted using the metaphase chromosomes of B. sinuspersici to characterize the chromosomal distribution of the repetitive sequences, 5S and 45S rDNAs and Arabidopsis-type telomeric repeats. Results of the cytogenetic analysis confirmed that B. sinuspersici has 2n=2x=18 consisting of 9 pairs of metacentric chromosomes. DAPI staining showed that AT-rich sequences are distributed in the centromeric regions. Two loci of 45S rDNA are found on the distal regions of the short arm of chromosome 7. Nine loci of 5S rDNA are found in the pericentromeric regions of chromosomes 1, 3, 4, 6, and 8, which also co-localized with Arabidopsis-type telomeric repeats; while four loci in the interstitial regions of chromosome 5 and 8 can be observed. The single locus of 5S rDNA that was found in chromosome 8 appears to be hemizygous. C0t analysis was also performed to provide additional data for its genome and locate its major repetitive sequences. C0t-1 and C0t-100 DNA FISH analyses revealed the distribution of FISH signals throughout the entire chromosomes, which confirmed that the genome of this species contained a large fraction of repetitive sequences. In addition to the localization of the major rDNA and telomeric repeats, the FISH technique was utilized to analyze the chromosomal distribution patterns of the main repetitive and several unknown DNA sequences in B. sinuspersici. Quantification of the FISH signal distribution was also conducted and can be considered as a useful tool in addition to visual analysis for molecular cytogenetic studies. The estimate of the signal distribution of each repetitive DNA sequences in the chromosomes was quantified using the ImageJ software. Results revealed that the satellite DNA with 814 copies have co-localized with the 5S rDNA loci in six pairs of chromosomes. RC/Helitron transposons, LTR/Gypsy and LINE retroelements were shown to have strong and dispersed hybridization signals in the interstitial regions of the chromosomes but were absent both in the centromeric and telomeric regions. The major repeat groups that were not classified in the sequence database were observed to be scattered throughout the chromosomes except for one unknown repeat family (rnd-5_family-1128) that hybridized in the telomeric regions of the chromosomes. The gathered data in this study will serve as a benchmark for further cytogenetic and genomic studies on B. sinuspersici and will be useful in understanding the genetic constitution of this species.

노랑초파리 수컷 생식줄기세포 stemness 유지에 필요한 rhino 역할의 유전학적 연구

이유정 강원대학교 대학원 2023 국내석사

진화 과정에서, 생식세포는 PIWI-interacting RNAs를 사용하여 mobile elements로부터 genome을 보호해왔다. 유전체 내 특정 heterochromatin 지역에서 heterochromatin protein 1d(HP1d, rhino)에 의해 piRNA precursor 전사가 유도된다고 알려져 있다. Female germline에서 rhino는 piRNA 생산을 통해 transposon을 silencing함으로써 genome 안정성 유지에 기여한다고 알려져 있다. Rhino 기능에 결함이 생길 경우 불임 형질을 보이는데, 아직 수컷 생식선에서의 역할에 대한 체계적 연구가 미흡하다. 본 연구에서는 male germline에서 rhino mutant의 역할을 집중적으로 분석하였는데, 흥미롭게도 germ cell과 germline stem cells(GSCs)의 loss가 나타남을 관찰하였다. 성체로의 우화 직후 생식세포를 거의 찾아볼 수 없는 female과 달리, male rhino mutant는 우화 직후 생식세포 수의 큰 변화가 나타나지는 않으나, 7일 이내 점차 사라지는 형질을 발견하였다. 원인을 알기 위해 male GSCs 수를 관찰하였는데, pupa 시기부터 우화 후 며칠 뒤까지 급감하는 형질을 확인하였다. 따라서, 성체로의 우화 후 관찰되는 생식세포 감소는 GSCs 수의 감소 때문인 것을 알 수 있다. 정상 rhino 유전자를 이용한 rescue construct를 지닌 초파리 line에서는 생식줄기세포 수가 회복되는 것을 보아 GSCs 수의 감소가 rhino 유전자 기능 상실 때문임을 알 수 있다. 수컷 GSCs 수 감소가 세포 사멸 때문인지 알기 위하여 세포 사멸 marker인 TUNEL 실험을 수행한 결과, TUNEL positive GSCs 를 전혀 발견할 수 없었다. 수컷 rhino 돌연변이체의 생식줄기세포는 세포 사멸이 아닌 다른 가능성 때문임을 시사하는데, age-dependent한 GSCs 감소가 GSCs의 self-renewal activity의 결함 때문인지 알기 위해 FRT/FLP를 이용한 mosaic clone 실험을 수행하였다. 흥미롭게도, rhino 돌연변이 clone은 나이가 들어감에 따라 감소하였다. 뿐만 아니라, 생식줄기세포의 유지에 중요한 JAK-STAT pathway의 핵심 요소인 STAT의 양이 rhino mutant에서 극적으로 감소한 것을 확인하였다. 결론적으로, rhino 유전자는 수컷생식줄기세포 유지에 중요한 STAT 단백질의 양을 조절하여 생식세포 항상성에 기여한다. Rhino와 STAT 사이의 분자적인 조절 기작에 대한 추후 연구가 필요하다. During evolution, cells have employed unique molecular strategies such as PIWI-interacting RNAs (piRNAs) to safeguard their genome against mobile elements. In this regard, a heterochromatin protein 1d (rhino) together with Deadlock (Dead) and Cutoff (Cuff) plays an essential role to license heterochromatin-dependent transcription for the piRNA production in female germline. rhino protects genome stability by piRNA mediated-TEs silencing in female germline. If there is mutation in rhino gene, it becomes sterile as soon as eclosion. Interestingly, it is not well understood the role of rhino in male germline. We observed germ cells and germline stem cells (GSCs) loss in male rhino mutant germline. Unlike female mutant that almost loss germ cells after eclosion, male rhino mutant does not show a significant change in the number of germ cells immediately after eclosion, however, all of them disappear within 7 days. This phenotype was resulted from reduction of GSCs. The number of GSCs rapidly decreased since from pupa stage to few days after eclosion. The recovery of these phenotype in rescue construct suggests that the decrease in GSCs is due to rhino defect. Therefore, rhino has a different role in male. Cell death marker TUNEL-positive GSCs were not found in male rhino mutant testis. This suggests that the reduction in the number of GSCs is not caused by cell death. We observed that GSCs with rhino mutation were decreased over time by mosaic clone experiment using FLP/FRT system, suggesting that the age-dependent reduction in GSCs is due to defects in self-renewal activity of GSCs. In addition, it was confirmed that the JAK-STAT pathway involved in GSCs maintenance was dramatically reduced in male rhino mutant germline by antibody staining for STAT. Therefore, the reduction in time-dependent GSCs of male rhino mutant seems to be a phenomenon due to the decrease in STAT expression, and further research is required on the molecular regulation mechanism between Rhino and STAT.

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전소연 단국대학교 일반대학원 2022 국내석사