애멸구에서 나타나는 아세틸콜린에스터레이즈1의 점돌연변이와 카보퓨란 저항성 관련 후보 유전자의 동정

학위논문 (석사)-- 서울대학교 대학원 : 농업생명과학대학 농생명공학부, 2019. 2. 이시혁.초록 애멸구(Laodelphax striatellus)는 벼의 주요 해충으로서 어린 유묘를 흡즙하여 고사시킬 뿐만 아니라 벼에 치명적인 식물 바이러스를 매개하는 것으로 알려져 있다. 다양한 계통의 살충제 중, 침투이행성 카바메이트계 살충제인 카보퓨란은 애멸구를 비롯한 벼과 해충 방제에 광범위하게 사용되어 왔으며, 이에 따라 한국 및 다른 동북 아시아 국가에서 광범위하게 그 저항성이 보고되어 왔다. 그러므로 분자 진단 마커의 개발은 살충제 저항 관리를 위한 고효율 스크리닝 시스템을 확립하기 위하여 분자 진단 마커의 개발이 요구되고 있는 실정이다. 본 논문에서는, 카보퓨란으로 도태시킨 저항성 계통의 애멸구에서 카바메이트 저항성과 관련된 type-1 acetylcholinesterase(Lsace1) 내 점 돌연변이를 발견하였다. 미량 국소처리법을 이용하여 9세대 도태시킨SEL9 저항성 계통과 감수성 계통의 표현형 저항성을 비교한 결과, 저항성 비는 14배, estarase 활성은 1.02배, 중간 저해 농도(I50) 값 기반 acetylcholinesterase 불감응성은 4.3배 더 높음을 확인하였다. 이것은 표적 부위의 불감응성이 저항성 인자로서 발생할 수 있음을 시사한다. 또한 5개의 애멸구 계통(SUS, SEL0, SEL3, SEL6, SEL9)의 Lsace1 유전자 서열을 비교하여 2가지 유형의 아미노산 치환체(F330Y와 F331H)를 발견하였다. 또한 정량적 시퀀싱 방법을 사용하여 도태 저항성 계통에서 해당 두 가지 점 돌연변이의 대립 유전자 빈도를 확인하고, Lsace1과 Lsace2, LsCarE1에 대한 유전자 카피 수 및 발현량을 조사하였다. 그 결과, F331H유전자 변이와 저항성 수준은 I50와 밀접한 관련이 있음을 확인하였다. 따라서, F331H 돌연변이와 발현량의 감소는 카바메이트 저항성 발달에 중요한 요인으로 보인다. 또한 해당 점 돌연변이는 양적 시퀀싱과 같은 분자 진단 방법과 함께 신속한 카보퓨란 저항성의 모니터링에 사용될 수 있을 것이다. 한편, 카보퓨란 저항성과 관련된 분자생물학적 요인을 확인하기 위하여 야외 계통(SEL0)과 연속적인 도태를 통하여 획득한 저항성 계통(SEL9)을 사용하여 전사체 분석을 실시하였다. 총 96,185,150개의 read중 62,860,430개의 read가 mapping되었고, 28,332개의 염기서열을 annotation하였다. 차등발현 된 유전자(DEG)를 통계적 조건(p 1, <-1) 하에 조사한 결과, SEL0에 비해 SEL9에서 발현량이 높은 24종의 유전자와 발현량이 낮은 15종의 유전자를 확인하였다. 또한 GO분석을 수행하여, 차등 발현된 유전자들이 높은 비율로 촉매 활성 혹은 다른 분자들과 결합할 수 있는 능력을 지니고 있음을 밝혔다.Abstract Laodelphax striatellus is an important pest of rice due to not only sucking rice seedlings, but also transmitting serious plant viruses. Among various kinds of insecticide groups (carbamates, organophosphorus, neonicotinoids, etc.), carbofuran, a systemic carbamate insecticide, has been most extensively used to control rice pests including L. striatellus, resulting in widespread carbamate resistance in Korea and other Northeast Asia countries. To establish high-throughput screening systems for insecticide resistance management, molecular diagnostic markers are required. Here, we have used the carbofuran selected strains (SEL0 to SEL9) to find a single amino acid point mutation associated with carbamate resistance in the type-1 acetylcholinesterase (Lsace1). When the phenotype resistance of the SEL9 strain, which was selected by carbofuran for 9 generations, was compared with that of the susceptible strain using topical application method, the resistance was determined to be 14-fold higher. In the biochemical enzyme assay, the esterase activity was not significantly different but the median inhibitory concentration (I50) value against acetylcholinesterase (AChE) was 4.3-fold higher. This suggests that insensitive AChE is likely involved as a resistance factor. Comparison of the type-1 acetylcholinesterase (Lsace1) gene sequences of five strains (SUS, SEL0, SEL3, SEL6, SEL9) revealed two types of amino acid substitutions (F330Y and F331H). Correlation analysis between the genotype and phenotype suggested that resistance allele frequency of F331H was strongly correlated with the I50 value. Interestingly, the F331H mutation was negatively associated with the transcript level of Lsace1. This suggests that the selection pressure might result in a reduction of the target gene. We also conducted transcriptome analysis and compared the results from the resistant SEL9 and SEL0 (susceptible control) strains to reveal molecular biological factors involved in carbofuran resistance. A total of 96,185,150 reads were analyzed, of which 62,860,430 reads were mapped. From these reads, 28,332 transcripts were annotated. A total of 24 up-regulated and 15 down-regulated genes were identified in the resistant SEL9 strain compared to SEL0 strain by DEG analysis in statistical condition (p 1, < -1). As a result of gene ontology (GO) analysis, we determined the composition of GO terms in biological process group, cellular component group and molecular function group. Overall, there was no significant difference between up-regulated and down-regulated genes in the composition of GO terms. But it was found that GO terms in catalytic activity and binding group occupied a high portion were, suggesting that many genes belonging to these groups can be important factors associated with carbofuran resistance.CONTENTS ABSTRACT ⅰ LIST OF TABLES ⅶ LIST OF FIGURES ⅷ LITERATURE REVIEW 1 1. What is Laodelphax striatellus 1 2. Insecticide resistance and its problem in Laodelphax striatellus 1 3. Resistance mechanism study in Laodelphax striatellus 2 CHAPTER 1. Carbofuran resistance mechanism mediated by target site insensitivity in L. striatellus 5 Abstract 6 1. Introduction 8 2. Materials and methods 10 2.1. Strains and Insects raering 10 2.2. Bioassay 11 2.3. Carbofuran selection 11 2.4. Protein extraction 13 2.5. Biochemical analysis and enzyme activity 13 2.6. Total RNA and genomic DNA extraction 14 2.7. Mutation screening of cloned Lsace1 from L. striatellus and haplotypes 15 2.8. Establishment of quantitative sequencing 18 2.9. Determination of transcription level, gene copy number, and mutation allele frequency 18 2.10. Statistical analysis 19 3. Results 19 3.1. Carbofuran resistance development 19 3.2. Comparison of enzymatic activity 21 3.3. Carbofuran inhibition against AChE 21 3.4. Point mutation screening and haplotype analysis of Lsace1 22 3.5. QS regression and allele frequency dynamics of the two mutations in the selection strains 25 3.6. Dynamics of Lsace1, Lsace2, and LsCarE1 based on mRNA levels and gene copy number 27 3.7. Correlation analysis of phenotypic resistance and the geneotypic strains 29 4. Discussion 29 CHAPTER 2. Identification of putative genes associated with carbofuran resistance in L. striatellus 34 Abstract 35 1. Introduction 36 2. Materials and methods 37 2.1. Strains and insects rearing 37 2.2. Transcriptome samples preparation 38 2.3. Total RNA quality check, library construction, and sequencing 38 2.4. Transcriptome data filtering and sequence alignment 39 2.5. Gene expression estimation and differentially expressed gene (DEG) analysis 39 3. Results 40 3.1. Reference-based mapping assembly of transcriptome data 40 3.2. GO profiles of DEGs 41 3.3. DEGs in carbofuran resistance strain 43 4. Discussion 45 LITERATURE CITED 49 KOREAN ABSTRACT 55Maste

Constriction of the mitochondrial inner compartment is a priming event for mitochondrial division.

Mitochondrial division is critical for the maintenance and regulation of mitochondrial function, quality and distribution. This process is controlled by cytosolic actin-based constriction machinery and dynamin-related protein 1 (Drp1) on mitochondrial outer membrane (OMM). Although mitochondrial physiology, including oxidative phosphorylation, is also important for efficient mitochondrial division, morphological alterations of the mitochondrial inner-membrane (IMM) have not been clearly elucidated. Here we report spontaneous and repetitive constriction of mitochondrial inner compartment (CoMIC) associated with subsequent division in neurons. Although CoMIC is potentiated by inhibition of Drp1 and occurs at the potential division spots contacting the endoplasmic reticulum, it appears on IMM independently of OMM. Intra-mitochondrial influx of Ca2+ induces and potentiates CoMIC, and leads to K+-mediated mitochondrial bulging and depolarization. Synergistically, optic atrophy 1 (Opa1) also regulates CoMIC via controlling Mic60-mediated OMM-IMM tethering. Therefore, we propose that CoMIC is a priming event for efficient mitochondrial divisionope

Hepatitis C virus entry is impaired by claudin-1 downregulation in diacylglycerol acyltransferase-1-deficient cells.

Diacylglycerol acyltransferase-1 (DGAT1) is involved in the assembly of hepatitis C virus (HCV) by facilitating the trafficking of the HCV core protein to the lipid droplet. Here, we abrogated DGAT1 expression in Huh-7.5 cells by using either the transcription activator-like effector nuclease (TALEN) or lentivirus vector short hairpin RNA (shRNA) and achieved complete long-term silencing of DGAT1. HCV entry was severely impaired in DGAT1-silenced Huh-7.5 cell lines, which showed markedly diminished claudin-1 (CLDN1) expression. In DGAT1-silenced cell lines, the forced expression of CLDN1 restored HCV entry, implying that the downregulation of CLDN1 is a critical factor underlying defective HCV entry. The expression of the gene coding for hepatocyte nuclear factor 4α (HNF4α) and other hepatocyte-specific genes was also reduced in DGAT1-silenced cell lines. After DGAT1 gene rescue, CLDN1 expression was preserved, and HCV entry was restored. Strikingly, after DGAT1 silencing, CLDN1 expression and HCV entry were also restored by low-dose palmitic acid treatment, indicating that the downregulation of CLDN1 was associated with altered fatty acid homeostasis in the absence of DGAT1. Our findings provide novel insight into the role of DGAT1 in the life cycle of HCV. IMPORTANCE: In this study, we report the novel effect of complete silencing of DGAT1 on the entry of HCV. DGAT1 was recently reported as a host factor of HCV, involved in the assembly of HCV by facilitating the trafficking of the HCV core protein to lipid droplets. We achieved complete and long-term silencing of DGAT1 by either TALEN or repeated transduction of lentivirus shRNA. We found that HCV entry was severely impaired in DGAT1-silenced cell lines. The impairment of HCV entry was caused by CLDN1 downregulation, and the expression of HNF4α and other hepatocyte-specific genes was also downregulated in DGAT1-silenced cell lines. Our results suggest new roles of DGAT1 in human liver-derived cells: maintaining intracellular lipid homeostasis and affecting HCV entry by modulating CLDN1 expression.ope

Targeted Genome Engineering to Control VEGF Expression in Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells: Potential Implications for the Treatment of Myocardial Infarction

Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) exhibit potency for the regeneration of infarcted hearts. Vascular endothelial growth factor (VEGF) is capable of inducing angiogenesis and can boost stem cell-based therapeutic effects. However, high levels of VEGF can cause abnormal blood vessel growth and hemangiomas. Thus, a controllable system to induce therapeutic levels of VEGF is required for cell therapy. We generated an inducible VEGF-secreting stem cell (VEGF/hUCB-MSC) that controls the expression of VEGF and tested the therapeutic efficacy in rat myocardial infarction (MI) model to apply functional stem cells to MI. To introduce the inducible VEGF gene cassette into a safe harbor site of the hUCB-MSC chromosome, the transcription activator-like effector nucleases system was used. After confirming the integration of the cassette into the locus, VEGF secretion in physiological concentration from VEGF/hUCB-MSCs after doxycycline (Dox) induction was proved in conditioned media. VEGF secretion was detected in mice implanted with VEGF/hUCB-MSCs grown via a cell sheet system. Vessel formation was induced in mice transplanted with Matrigel containing VEGF/hUCB-MSCs treated with Dox. Moreover, seeding of the VEGF/hUCB-MSCs onto the cardiac patch significantly improved the left ventricle ejection fraction and fractional shortening in a rat MI model upon VEGF induction. Induced VEGF/hUCB-MSC patches significantly decreased the MI size and fibrosis and increased muscle thickness, suggesting improved survival of cardiomyocytes and protection from MI damage. These results suggest that our inducible VEGF-secreting stem cell system is an effective therapeutic approach for the treatment of MI.ope

Doxycycline enhances survival and self-renewal of human pluripotent stem cells

We here report that doxycycline, an antibacterial agent, exerts dramatic effects on human embryonic stem and induced pluripotent stem cells (hESC/iPSCs) survival and self-renewal. The survival-promoting effect was also manifest in cultures of neural stem cells (NSCs) derived from hESC/iPSCs. These doxycycline effects are not associated with its antibacterial action, but mediated by direct activation of a PI3K-AKT intracellular signal. These findings indicate doxycycline as a useful supplement for stem cell cultures, facilitating their growth and maintenance.ope

Heroes of peer review: Hyongbum (Henry) Kim

Peer reviewers are the unsung heroes of science. We celebrate reviewers through a series of interviews with people who have made particularly strong recent contributions to Genome Biology as reviewers. The first interview is with Hyongbum (Henry) Kim, an Associate Professor at Yonsei University College of Medicine in South Korea.ope

Programmable Nuclease-Based Integration into Novel Extragenic Genomic Safe Harbor Identified from Korean Population-Based CNV Analysis

Here, we found two genomic safe harbor (GSH) candidates from chromosomes 3 and 8, based on large-scale population-based cohort data from 4,694 Koreans by CNV analysis. Furthermore, estimated genotype of these CNVRs was validated by quantitative real-time PCR, and epidemiological data examined no significant genetic association between diseases or traits and two CNVRs. After screening the GSH candidates by in silico approaches, we designed TALEN pairs to integrate EGFP expression cassette into human cell lines in order to confirm the functionality of GSH candidates in an in vitro setting. As a result, transgene insertion into one of the two loci using TALEN showed robust transgene expression comparable to that with an AAVS1 site without significantly perturbing neighboring genes. Changing the promoter or cell type did not noticeably disturb this trend. Thus, we could validate two CNVRs as a site for effective and safe transgene insertion in human cells.ope

Generation of a more efficient prime editor 2 by addition of the Rad51 DNA-binding domain

Although prime editing is a promising genome editing method, the efficiency of prime editor 2 (PE2) is often insufficient. Here we generate a more efficient variant of PE2, named hyPE2, by adding the Rad51 DNA-binding domain. When tested at endogenous sites, hyPE2 shows a median of 1.5- or 1.4- fold (range, 0.99- to 2.6-fold) higher efficiencies than PE2; furthermore, at sites where PE2-induced prime editing is very inefficient (efficiency < 1%), hyPE2 enables prime editing with efficiencies ranging from 1.1% to 2.9% at up to 34% of target sequences, potentially facilitating prime editing applications.ope

Deficiency in DGCR8-dependent canonical microRNAs causes infertility due to multiple abnormalities during uterine development in mice

DGCR8 is an RNA-binding protein that interacts with DROSHA to produce pre-microRNA in the nucleus, while DICER generates not only mature microRNA, but also endogenous small interfering RNAs in the cytoplasm. Here, we produced Dgcr8 conditional knock-out mice using progesterone receptor (PR)-Cre (Dgcr8(d/d)) and demonstrated that canonical microRNAs dependent on the DROSHA-DGCR8 complex are required for uterine development as well as female fertility in mice. Adult Dgcr8(d/d) females neither underwent regular reproductive cycles nor produced pups, whereas administration of exogenous gonadotropins induced normal ovulation in these mice. Interestingly, immune cells associated with acute inflammation aberrantly infiltrated into reproductive organs of pregnant Dgcr8(d/d) mice. Regarding uterine development, multiple uterine abnormalities were noticeable at 4 weeks of age when PR is significantly increased, and the severity of these deformities increased over time. Gland formation and myometrial layers were significantly reduced, and the stromal cell compartment did not expand and became atrophic during uterine development in these mice. These results were consistent with aberrantly reduced stromal cell proliferation and completely failed decidualization. Collectively, we suggest that DGCR8-dependent canonical microRNAs are essential for uterine development and physiological processes such as proper immune modulation, reproductive cycle, and steroid hormone responsiveness in mice.ope

En bloc and segmental deletions of human XIST reveal X chromosome inactivation-involving RNA elements

The XIST RNA is a non-coding RNA that induces X chromosome inactivation (XCI). Unlike the mouse Xist RNA, how the human XIST RNAcontrols XCI in female cells is less well characterized, and its functional motifs remain unclear. To systematically decipher the XCI-involving elements of XIST RNA, 11 smaller XIST segments, including repeats A, D and E; human-specific repeat elements; the promoter; and non-repetitive exons, as well as the entire XIST gene, were homozygously deleted in K562 cells using the Cas9 nuclease and paired guide RNAs at high efficiencies, followed by high-throughput RNA sequencing and RNA fluorescence in situ hybridization experiments. Clones containing en bloc and promoter deletions that consistently displayed no XIST RNAs and a global up-regulation of X-linked genes confirmed that the deletion of XIST reactivates the inactive X chromosome. Systematic analyses of segmental deletions delineated that exon 5 harboring the non-repeat element is important for X-inactivation maintenance, whereas exons 2, 3 and 4 as well as the other repeats in exon 1 are less important, a different situation from that of mouse Xist. This Cas9-assisted dissection of XIST allowed us to understand the unique functional domains within the human XIST RNA.ope