• Title/Summary/Keyword: double sequence

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Application of simple and massive purification system of dsRNA in vivo for acute toxicity to Daphnia magna

  • CHOI, Wonkyun;LIM, Hye Song;KIM, Jin;RYU, Sung-Min;LEE, Jung Ro
    • Entomological Research
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    • v.48 no.6
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    • pp.533-539
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    • 2018
  • The RNA interference (RNAi) has been considered as an important genetic tool and applied to develop a new living modified (LM) crop trait which is an improvement of nutrient quality or pest management. The RNAi of DvSnf7 has been used for resistance to LM maize and the Western Corn Rootworm which is a major agricultural pest for the US Corn Belt. Most of the environmental risk assessments (ERA) of double strand RNA (dsRNA) have been performed using in vitro transcript products, and not in vivo expressed product. A large amount of dsRNA was required for the acute toxicity assay of water fleas. Therefore development of massive dsRNA purification techniques is critical. Daphnia, a freshwater microcrustacean, is a model organism for studying cellular and molecular mechanism involved in life history traits and ecotoxicology. In this study, we established the massive dsRNA purification method using Escherichia coli and implemented acute toxicity assays to Daphnia magna. As a result, the present RNase A and DNase I, dsRNA was efficiently purified without any special techniques or equipment. Even though purified dsRNA existed during the acute toxicity test, lethality or abnormal behavior were not observed in D. magna. These results indicated that GFP and DvSnf7 dsRNA were not significantly affected to D. magna due to their lack of sequence matching in its genome. The purification method of dsRNA and the acute toxicity assay of water fleas using purified dsRNA would be suitable for the toxicological studies of LMOs to aquatic non-target organisms.

Observational Feature of Ejecta-Companion Interaction of A Type Ia SN 2021hpr Via The Very Early Light Curve

  • Lim, Gu;Im, Myungshin;Paek, Gregory S.H;Yoon, Sung-Chul;Choi, Changsu;Kim, Sophia;Seo, Jinguk;Kang, Wonseok;Kim, Taewoo;Sung, Hyun-Il;Kim, Yonggi;Yoon, Joh-Na
    • The Bulletin of The Korean Astronomical Society
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    • v.46 no.2
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    • pp.50.3-51
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    • 2021
  • The progenitor of Type Ia supernovae is largely expected as a close binary system of a carbon/oxygen white dwarf (WD) primary and its secondary non-degenerate (single degenerate; SD) or degenerate companion (double degenerate; DD). Here we present a high-cadence monitoring observation of SN 2021hpr in a spiral galaxy, NGC 3147. SN 2021hpr shows typical characteristics as a normal type Ia supernova from its photometric (Δm15(B)=1.01±0.03, dust free MB,max=-19.45±0.02) and spectroscopic data. To investigate its progenitor system, we fit the early part of BVRI-band light curve simultaneously with a combined version of ejecta-companion and simple power-law model. As a result, we found a significant feature of an early excess possibly from a 7.63±0.52R-sized companion at the optimal viewing angle while the fit is not successful at the common viewing angle. No possible red sources brighter than F555W=-7.01 AB mag is detected at the SN location in Hubble Space Telescope (HST) pre-explosion images, excluding massive stars with initial mass of >16M as companions. We suggest the progenitor system of SN 2021hpr can be a fairly large companion such as a main sequence, a low mass subgiant, and a helium giant star. In addition, a possibility of the ejecta-Disk Originated Matter (DOM) interaction for the DD scenario considering linearly-rising early flux still remains.

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The Study of Trnascriptional Regulated Gene, $hrp^{2+}$, in Yeast

  • Choi, In-Soon
    • Journal of Life Science
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    • v.11 no.2
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    • pp.111-115
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    • 2001
  • This study was designed to clone the SNF2/SW12 helicase-related genes from the fission yeast Schizosaccha-romyces pombe and thereafter to elucidate the common functions of the proteins in this family. The $hrp^{2+}$gene was cloned by polymerase chain reaction amplification using degenerative primers from conserved SNF2 motifs within the ERCC6 gene, which encodes a protein involved in DNA excision repair. Like other SNF2/SW12 family proteins, the deduced amino acid sequence of Hrp2 contains DNA-dependent ATPase/7 helicase domains as well as the chromodomain and the DNA binding domain. This configuration is similar to that of mCHD1 (mouse chromo-ATPase/helicase-DNA-dinding protein 1), suggesting that Hrp2 is a S. pombe homolog of mCHD1, which is thought to function in altering the chromatin structure to control the gene expression. To characterize the function of Hrp2, 4 Uracil-Hrp2 fusion protein, it was purified near homogeneity by affinity chromatography on $Ni^{2+}$-NTA agarose, DEAE-Sepharose ion exchange arid Sephacryl S-200 gel filtration chromatographies. The purified fusion protein exhibited DNA-dependent ATPase activity, which was stimulated by both double-stranded and single-stranded DNA. To determine the steady-state level of $hrp^{2+}$ transcripts during growth, cells were cultured in medium and collected at every 2hr to prepare total RNAs. The northern blot analysis showed that the level of $hrp^{2+}$ transcripts reached its maximum before the cells entered the exponential growth phase and then decreased gradually, This result implies that Hrp2 may be required at early stages of cell growth.h.

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Evaluation between 3.0 T vs 1.5 T MRI in Detection of Brain Metastasis using Double Dose Gd-DTPA (뇌전이 종양의 발견에 있어서 Doble dose Gd-DTPA를 이용한 3 T MRI와 1.5 T MRI간의 비교연구)

  • Chung, Woo-Suk;Kim, Hyung-Jung;Ahn, Chul-Min;Lee, Jae-Hoon;Hur, Jin;Cho, Eung-Hyuck;Chung, Tae-Sub
    • Investigative Magnetic Resonance Imaging
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    • v.9 no.2
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    • pp.101-108
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    • 2005
  • Purpose : Early detection of small brain metastases is important. The purpose of this study was to compare the detectability of brain metastases according to the size between 1.5 T and 3.0 T MRI. Materials and Methods : We reviewed 162 patients with primary lung cancer who were examined for TNM staging. After administration of double dose of Gd-DTPA, MR imaging was performed with SPGR by 3.0 T MRI and then with T1 SE sequence by 1.5 T MRI. In each patient, three readers performed qualitative assessment. Sensitivity, positive predictive value, and diagnostic accuracy were calculated in 3.0 T and 1.5 T MRI according to size. Using the signal intensity (SI) measurements between the metastatic nodules and adjacent tissue, nodule-to-adjacent tissue SI ratio was calculated. Results : Thirty-one of 162 patients had apparent metastatic nodules in the brain at either 1.5 T or 3.0 T MR imaging. 143 nodules were detected in 3.0 T MRI, whereas 137 nodules were detected at 1.5 T MRI. Six nodules, only detected in 3.0 T MRI, were smaller than 3.0 mm in dimension. Sensitivity, positive predictive value, and diagnostic accuracy in 3.0 T MRI were 100 %, 100 %, and 100 % respectively, and in 1.5 T MRI were 95.8%, 88.3%, and 85.1% respectively. SI ratio was significantly higher in the 3.0 T MRI than 1.5 T MRI (p=0.025). Conclusion : True positive rate of 3.0 T MRI with Gd-DTPA was superior to 1.5 T MRI with Gd-DTPA in detection of metastatic nodules smaller than 3.0 mm.

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Cold-Sensitive Growth of Bacillus subtilis Mutants Deleted for Putative DEAD-Box RNA Helicase Genes (Bacillus subtilis DEAD-Box RNA Helicase 유전자 결손 균주들의 저온 민감성 생장)

  • Oh, Eun-Ha;Lee, Sang-Soo
    • Korean Journal of Microbiology
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    • v.46 no.3
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    • pp.233-239
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    • 2010
  • Four genes (yqfR, yfmL, ydbR, deaD) were identified as putative DEAD-box RNA helicase genes in the genomic sequence of Bacillus subtilis by homology search. To understand the function of these genes, each of the genes was deleted and the constructed strains were tested for their growth charateristics at different temperatures. The growth rate of ydbR deletion mutant ($T_d$=53 min) was a little bit reduced at $37^{\circ}C$ as compared to that of wild type strain (CU1065). But the growth rate of other three (yqfR, yfmL, deaD) deletion mutants ($T_d$=30-40 min) is nearly equal to the growth rate of wild type ($T_d$=32 min). On the other hands, the growth rate of deletion mutants were reduced at $22^{\circ}C$ in order of yqfR ($T_d$=151 min), yfmL ($T_d$=214 min), ydbR ($T_d$=343 min), which showed cold-sensitive phenotype. The deletion mutant of deaD ($T_d$=109 min) grew equally as compared to the growth rate ($T_d$=102 min) of the wild type at $22^{\circ}C$ and did not show cold-sensitive growth. Double, triple and quadruple deletion mutants of these genes were constructed, and growth rate of these mutants were measured at various temperature conditions ($22^{\circ}C$, $37^{\circ}C$, $42^{\circ}C$) using LB broth. Multiple deletion mutations showed more severe cold-sensitive growth than single deletion mutations, and double deletion of ydbR and yfmL ($T_d$=984 min) showed most cold-sensitive growth than any other double mutants. Such a cold-sensitive growth of these mutations is quite similar to the result of csdA or srmB deletion in E. coli and suggested that physiological role of ydbR and yfmL is related with ribosome assembly.

Identification and characteristics of DDX3 gene in the earthworm, Perionyx excavatus (팔딱이 지렁이(Perionyx excavatus) DDX3 유전자의 동정 및 특성)

  • Park, Sang Gil;Bae, Yoon-Hwan;Park, Soon Cheol
    • Journal of the Korea Organic Resources Recycling Association
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    • v.23 no.1
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    • pp.70-81
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    • 2015
  • Helicases are known to be a proteins that use the chemical energy of NTP binding and hydrolyze to separate the complementary strands of double-stranded nucleic acids to single-stranded nucleic acids. They participate in various cellular metabolism in many organisms. DEAD-box proteins are ATP-dependent RNA helicase that participate in all biochemical steps involving RNA. DEAD-box3 (DDX3) gene is belonging to the DEAD-box family and plays an important role in germ cell development in many organisms including not only vertebrate, but also invertebrate during asexual and sexual reproduction and participates in stem cell differentiation during regeneration. In this study, in order to identify and characterize DDX3 gene in the earthworm, Perionyx excavatus having a powerful regeneration capacity, total RNA was isolated from adult head containing clitellum. Full length of DDX3 gene from P. excavatus, Pe-DDX3, was identified by RT-PCR using the total RNA from head as a template. Pe-DDX3 encoded a putative protein of 607 amino acids and it also has the nine conserved motifs of DEAD-box family, which is characteristic of DEAD-box protein family. It was confirmed that Pe-DDX3 has the nine conserved motifs by the comparison of entire amino acids sequence of Pe-DDX3 with other species of different taxa. Phylogenetic analysis revealed that Pe-DDX3 belongs to a DDX3 (PL10) subgroup of DEAD-box protein family. And it displayed a high homology with PL10a, b from P. dumerilii.

Numerical Design of Double Quantum Coherence Filter for the Detection of Myo-Inositol In vivo (인체 내 myo-Inositol 검출을 위한 수치해석적 이중양자 필터 디자인)

  • Lee, Yun-Jung;Jung, Jin-Young;Noh, Hyung-Joon;Yu, Ung-Sik;Kim, Hyeon-Jin
    • Investigative Magnetic Resonance Imaging
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    • v.13 no.2
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    • pp.117-126
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    • 2009
  • Purpose : A numerical method of designing a multiple quantum filter (MQF) is presented for the optimum detection of myo-inositol (mI), an important brain metabolite, by using in vivo proton nuclear magnetic resonance spectroscopy ($^1$-HMRS). Materials and Methods : Starting from the characterization of the metabolite, the filter design includes the optimization of the sequence parameters such as the two echo times (TEs), the mixing time (TM), and the flip angle and offset frequency of the 3rd $90^{\circ}$ pulse which converts multiple quantum coherences (MQCs) back into single quantum coherences (SQCs). The optimized filter was then tested both in phantom and in human brains. Results : The results demonstrate that the proposed MQF can improve the signal-to-background ratio of the target metabolite by a factor of more than three by effectively suppressing the signal from the background metabolites. Conclusion : By incorporating a numerical method into the design of MQFs in $^1$-HMRS the spectral integrity of a target metabolite, in particular, with a complicated spin system can be substantially enhanced.

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PKA-Mediated Regulation of B/K Gene Transcription in PC12 Cells

  • Choi, Mi-Hyun;Kim, Ho-Shik;Choi, Sung-Ho;Kim, Mi-Young;Jang, Yoon-Seong;Jang, Young-Min;Lee, Jeong-Hwa;Jeong, Seong-Whan;Kim, In-Kyung;Kwon, Oh-Joo
    • The Korean Journal of Physiology and Pharmacology
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    • v.9 no.6
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    • pp.333-339
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    • 2005
  • B/K protein is a novel protein containing double C2-like domains. We examined the specific signaling pathway that regulates the transcription of B/K in PC12 cells. When the cells were treated with forskolin ($50{\mu}M$), B/K mRNA and protein levels were time-dependently decreased, reaching the lowest level at 3 or 4 hr, and thereafter returning to the control level. Chemicals such as dibutyryl-cAMP, cellpermeable cyclic AMP (cAMP) analogue and CGS21680, adenosine receptor $A_{2A}$ agonist, also repressed the B/K transcription. However, 1,9-dideoxyforskolin did not show inhibitory effect on B/K transcription, suggesting direct involvement of cAMP in the forskolin-induced inhibition of B/K transcription. Effect of forskolin, dibutyryl cAMP and CGS21680 was significantly reduced in PKA-deficient PC12 cell line (PC12-123.7). One cAMP-response element (CRE)-like sequence (B/K CLS) was found in the promoter region of B/K DNA, and electrophoretic mobility shift assay indicated its binding to CREM and CREB. Forskolin significantly suppressed the promoter activity in CHO-K1 cells transfected with the constructs containing B/K CLS, but not with the construct in which B/K CLS was mutated (AC:TG). Taken together, we suggest that the transcription of B/K gene in PC12 cells may be regulated by PKA-dependent mechanism.

Clinical Observation of Congenital Urinary Tract Anomalies (소아 요로계 기형에 대한 임상적 고찰)

  • Chang Soo-Hee;Kim Sun-Jun;Lee Dae-Yeol
    • Childhood Kidney Diseases
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    • v.1 no.1
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    • pp.67-72
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    • 1997
  • Purpose : Congenital urinary tract anomaly is the most common anomaly in the childhood and progress to chronic renal failure and growth retardation. Therefore, early diagnosis arid treatment of urinary tract anomaly are important. Method : We reviewed medical records of 124 patients who had urinary tract anomalies on radiologic studies from Jan. 1986 to Dec. 1996. We analyzed demography and clinical characteristics of urinary tract anomalies. Results : 1) The age distributions were as follows ; 61 cases of 124 patients (49%) were under 1 year, 11 cases (8.8%) from 1 to 3 years, 20 cases (16%) from 4 to 6 years, 10 cases (8%) from 7 to 9 years, 9 cases (7.2%) from 10 to 12 years, 10 cases (8%) from 13 to 15 years, and 3 cases (2.4%) from 16 to 18 years. 2) Chief complaints in patients with urinary tract anomalies were fever, flank pain, prenatally diagnosed hydronephrosis, abdominal mass, dysuria and hematuria. 3) Of 124 patients, 68 cases(54.8%) were combined with urinary tract infection, and main causative organism was E.coli, and the most frequently associated anomaly was vesicoureteral reflux. 4) Most of the urinary tract anomalies were VUR, UPJ obstruction, congenital hydronephrosis and double ureter in order of sequence. 5) Whereas the frequency of simple urinary tract anomaly was 87.9%, that of complex anomaly was 12%. 6) Operative corrections were needed in 47 cases and 7 cases were progressed to renal insufficiency. Conclusion : We emphasize that early detection of urinary tract anomaly, appropriate treatment and regular follow-up are needed.

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DNA-Independent ATPase Activity of Deinococcus radiodurans RecA Protein Is Activated by High Salt (고농도 염에 의한 Deinococcus radiodurans RecA 단백질의 DNA 비의존성 ATPase 역가의 활성화)

  • Kim, Jong-Il
    • Korean Journal of Microbiology
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    • v.46 no.4
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    • pp.313-318
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    • 2010
  • Deinococcus radiodurans RecA protein, when bound to DNA, exhibits a DNA-dependent ATPase. In the absence of DNA, the rate of RecA protein-promoted ATP hydrolysis drops 1,000-fold under the physiological concentrations of salt. This DNA-independent activity can be stimulated to levels approximating those observed with DNA by adding high concentrations (approximately 1.6 M) of a wide variety of salts. This effect was characterized by varying salt concentration and comparing the effects of different ion types. The higher concentrations of salt stimulated the ATP hydrolysis by RecA protein in the absence of DNA. At 1.6 M chloride, the observed stimulation showed the following cation trend $K^+{\geq}Na^+$ > $NH_4^+$ and the following anion sequence was observed: $glutamate^- \; > \; C1^- \;> \; acetate^-\; > \;PO_4^-$ at 1.6 M $K^+$. The catalytic properties of the salt-stimulated ATP hydrolysis reaction was optimal between pH 7.0 and 8.0, which was similar to the double stran nded DNA-dependent ATPase activities of Deinococcus radiodurans RecA protein. In the absence of DNA the active species for ATP hydrolysis by RecA protein was shown to be an aggregate of three RecA protein molecules.