• Title/Summary/Keyword: expression vectors

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Gene Therapy Using GM-CSF Gene Transferred by a Defective Infectious Single-cycle Herpes Virus in Micro-residual Organotropic Head and Neck Squamous Cell Cancer Model (향장기성 두경부 편평세포암종의 미세잔존암 모델에서 GM-CSF 유전자를 이입시킨 제한복제성 헤르페스바이러스 벡터를 이용한 종양백신의 유전자 치료)

  • Kim Se-Heon;Choi Eun-Chang;Kim Han-Su;Chang Jung-Hyun;Kim Ji-Hoon;Kim Kwang-Moon
    • Korean Journal of Head & Neck Oncology
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    • v.19 no.1
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    • pp.25-33
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    • 2003
  • Background and Objectives: The Herpes Simplex type 2 Defective Infectious Single Cycle virus (DISC virus) is attenuated virus originally produced as viral vaccines but are also efficient gene transfer vehicle. The main goals of this study were to examine the efficiencies of the gene transfer using DISC vectors for various head and neck squamous cell carcinoma cell lines and to evaluate the efficacy of vaccination with DISC virus carrying a immunomodulatory genes (GM-CSF) as cancer therapy in a organotopic oral cavity squamous cell cancer model. Materials and Methods : We determinated the gene transfer efficiency of DISC virus by x-gal stain method and proved gene and protein expression of DISC-GMCSF transfected SCCVII cells by RT-PCR and ELISA method. Also we evaluated the ex vivo vaccination effects of SCCVII/GMCSF (DISC-GMCSF transfected SCCVII vaccine) vaccine on preventing the recurrence of micro-residual tumor. After the vaccination of SCCVII/GMCSF, specific cytotoxic T-cell responses was evaluated by CTL assay. Results: At an MOI of 10 DISC virus showed 64-88% of transfection rates in various head and neck squamous cancer cell lines. SCCVII cells transduced by DISC virus vector (MOI=10) carrying the GM-CSF gene, produced 4.5 nanogram quantities of GM-CSF per $10^6$ cells. In vivo vaccination using tumor cells transduced ex vivo with DISC-GMCSF resulted in better protection rate against subsequent tumor recurrence in organotopic oral cavity cancer model. Although tumor free survival rate was not statistically significantly increased in vaccination group (p=0.078), tumor specific cytotocic T-cell responses were significantly increased in SCCVII/GMCSF vaccination group. Conclusion: These data demonstrate that; 1) The DISC virus vector is capable of efficient gene transfer to various head and neck squamous cancer cell lines, 2) GM-CSF secreting genetically modified tumor vaccine (SCCVII/GMCSF) efficiently protected against tumor recurrence in organotopic micro-residual oral cavity cancer model and produced tumor specific cytotoxic T-cell response. DISC virus-mediated, cytokine gene transfer may prove to be useful as a clinical therapy for head and neck cancers.

Cotransformation of Saccharomyces cerevisiae with Yip and Yep Vectors (YEp 및 YIp 벡터에 의(依)한 Saccharomyces cerevisiae의 Cotransformation)

  • Lee, Sung Bum;Rhee, In Koo
    • Current Research on Agriculture and Life Sciences
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    • v.4
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    • pp.36-41
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    • 1986
  • The transformation of Saccharomyces cerevisiae with YIp26, YRp7 and YEp13 was investigated. Transformation frequences of YIp5, YIp26, YRp7 and YEp13 in Escherichia coli HB101 was $5.1{\times}10^{-4}$, $1.5 {\times}10^{-3}$, $1.3{\times}10^{-3}$, $3{\times}10^{-3}$, respectively. When plasmids were used in covalently closed circular form, transformation frequency of YEp13 was $1.2{\times}10^{-4}$ in S. cerevisiae DBY747 and $3.3{\times}10^{-4}$ in S. cerevisiae MC16 and that of YRp7, YIp26 was $3{\times}10^{-6}$, below $6{\times}10^{-8}$ respectively in S. cerevisiae DBY747 by the method of Ito. Cotransformation of YIp26 and YEp13 in linear form increased the frequency of transformation with efficiences 270-fold higher than transformation of YIp26 only in S. cerevisiae DBY747. In cotransformation of YIp5+YEp13 and YIp26+YRp7 with S. cerevisiae DBY747 by Beggs' method. Expression frequency of YIp5+YEp13 and YIp26+YRp7 was $4{\times}10^{-6}$, $1.5{\times}10^{-6}$, respectively. The recombinant plasmid of cotransformant was thought that YIp26 and YEp13, YIp5 and YEp13, and YIp26 and YRp 7 were ligated in vivo in S. cerevisiae DBY747.

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Denoising Self-Attention Network for Mixed-type Data Imputation (혼합형 데이터 보간을 위한 디노이징 셀프 어텐션 네트워크)

  • Lee, Do-Hoon;Kim, Han-Joon;Chun, Joonghoon
    • The Journal of the Korea Contents Association
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    • v.21 no.11
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    • pp.135-144
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    • 2021
  • Recently, data-driven decision-making technology has become a key technology leading the data industry, and machine learning technology for this requires high-quality training datasets. However, real-world data contains missing values for various reasons, which degrades the performance of prediction models learned from the poor training data. Therefore, in order to build a high-performance model from real-world datasets, many studies on automatically imputing missing values in initial training data have been actively conducted. Many of conventional machine learning-based imputation techniques for handling missing data involve very time-consuming and cumbersome work because they are applied only to numeric type of columns or create individual predictive models for each columns. Therefore, this paper proposes a new data imputation technique called 'Denoising Self-Attention Network (DSAN)', which can be applied to mixed-type dataset containing both numerical and categorical columns. DSAN can learn robust feature expression vectors by combining self-attention and denoising techniques, and can automatically interpolate multiple missing variables in parallel through multi-task learning. To verify the validity of the proposed technique, data imputation experiments has been performed after arbitrarily generating missing values for several mixed-type training data. Then we show the validity of the proposed technique by comparing the performance of the binary classification models trained on imputed data together with the errors between the original and imputed values.

Expression of tissue-type plasminogen activator and its derivative proteins in transgenic alfalfa plants (조직형 플라스미노겐 액티베이터와 관련 변이 단백질들을 발현하는 알팔파 형질전환체)

  • Sim, Joon-Soo;Rhee, Yong;Ko, Hyo-Rim;Pak, Hyo-Kyung;Kim, Hyeong-Mi;Lim, Kyu-Hee;An, Ki-Seong;Kim, Yong-Hwan;Hahn, Bum-Soo
    • Journal of Plant Biotechnology
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    • v.36 no.1
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    • pp.30-37
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    • 2009
  • Tissue-type plasminogen activator (t-PA) is a thrombolytic agent important in fibirn clot lysis. T-PA causes fibirn-specific plasminogen activation. Six binary vectors harboring t-PA and its derivative genes were cloned and expressed in transgenic alfalfa plants. The insertion of the t-PA and its derivative genes in genomic DNA of alfalfa plants was confirmed by PCR. The presence of the t-PA and its derivative transcripts in total RNAs of the transgenic alfalfa leaves was verified by RT-PCR. ELISA experiments demonstrated that the highest level of recombinant t-PA expression was $75.1{\mu}g$/ total soluble protein (mg) in alfalfa plants. The amount of recombinant t-PA and its derivative proteins in transgenic plants was estimated to range from 9.7 to $39.5{\mu}g$/ total soluble proteins (mg). Western blot analysis of the transformed alfalfa leaves revealed bands of approximately 68-kDa recombinant t-PA and its derivative proteins. The fibrinolysis of recombinant t-PA and its derivative proteins was confirmed by a fibrin plate assay (range from 3.2 to 8.1 cm). The results presented provide information for the development of an additional production of recombinant human proteins having pharmaceutical applications using transgenic plants.

Enhanced production of monacolin-K through supplement of monacolin-K precursors into production medium and cloning of SAM synthetase gene (metK) (Precursor제공 및 생합성 관련 유전자의 cloning을 통한 Monacolin-K 생산성 향상)

  • Lee, Mi-Jin;Jeong, Yong-Seob;Chun, Gie-Taek
    • KSBB Journal
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    • v.23 no.6
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    • pp.519-524
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    • 2008
  • Monacolin-K is a strong anti-hypercholesterolemic agent produced by Monascus sp. via polyketide pathway. High-yielding mutants of monacolin-K were developed through rational screening strategies adopted based on understanding of monacolin-K biosynthetic pathway. Through the experiments for investigating various amino acids as putative precursors for the monacolin-K biosynthesis, it was found that production level of monacolin-K was remarkably increased when optimum amount of cysteine was supplemented into the production medium. We suggested that these phenomena might be related to the special roles of SAM (S-adenosyl methionine), a putative methyl group donor in the biosynthetic pathway of monacolin-K, demonstrating close interrelationship between SAM-synthesizing primary metabolism and monacolin-K synthesizing secondary metabolism. Namely, increase in the intracellular amount of SAM derived from the putative precursor, cysteine which was extracellularly supplemented into the production medium might contribute to the significant enhancement in the monacolin-K biosynthetic capability of the highly mutated producers. On the basis of these assumptions derived from the above fermentation results, we decided to construct efficient expression vectors harboring SAM synthetase gene (metK) cloned from A. nidulans, with the hope that increased intracellular level of SAM could lead to further enhancement in the monacolin-K production through overcoming a rate-limiting step associated with monacolin-K biosynthesis. Hence, in order to overcome the plausible rate-limiting step associated with monacolin-K biosynthesis by increasing intracellular level of SAM, we transformed the producer mutants with an efficient expression vector harboring gpdA promoter of the producer microorganism, and metK gene. Notably, from the resulting various transformants, we were able to screen a very high-yielding transformant which showed approximately 3.3 fold higher monacolin-K productivity than the parallel nontransformed mutants in shake flask cultures performed under the identical fermentation conditions.

PS-341-Induced Apoptosis is Related to JNK-Dependent Caspase 3 Activation and It is Negatively Regulated by PI3K/Akt-Mediated Inactivation of Glycogen Synthase Kinase-$3{\beta}$ in Lung Cancer Cells (폐암세포주에서 PS-341에 의한 아포프토시스에서 JNK와 GSK-$3{\beta}$의 역할 및 상호관련성)

  • Lee, Kyoung-Hee;Lee, Choon-Taek;Kim, Young Whan;Han, Sung Koo;Shim, Young-Soo;Yoo, Chul-Gyu
    • Tuberculosis and Respiratory Diseases
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    • v.57 no.5
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    • pp.449-460
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    • 2004
  • Background : PS-341 is a novel, highly selective and potent proteasome inhibitor, which showed cytotoxicity against some tumor cells. Its anti-tumor activity has been suggested to be associated with modulation of the expression of apoptosis-associated proteins, such as p53, $p21^{WAF/CIP1}$, $p27^{KIP1}$, NF-${\kappa}B$, Bax and Bcl-2. c-Jun N-terminal kinase (JNK) and glycogen synthase kinase-$3{\beta}$ (GSK-$3{\beta}$) are important modulators of apoptosis. However, their role in PS-341-induced apoptosis is unclear. This study was undertaken to elucidate the role of JNK and GSK-$3{\beta}$ in the PS-341-induced apoptosis in lung cancer cells. Method : NCI-H157 and A549 cells were used in the experiments. The cell viability was assayed using the MTT assay and apoptosis was evaluated by proteolysis of PARP. The JNK activity was measured by an in vitro immuno complex kinase assay and by phosphorylation of endogenous c-Jun. The protein expression was evaluated by Western blot analysis. Dominant negative JNK1 (DN-JNK1) and GSK-$3{\beta}$ were overexpressed using plasmid and adenovirus vectors, respectively. Result : PS-341 reduced the cell viability via apoptosis, activated JNK and increased the c-Jun expression. Blocking of the JNK activation by overexpression of DN-JNK1, or pretreatment with SP600125, suppressed the apoptosis induced by PS-341. The activation of caspase 3 was mediated by JNK activation. Blocking of the caspase 3 activation suppressed PS-341-induced apoptosis. PS-341 activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but its blockade enhanced the PS-341-induced cell death via apoptosis. GSK-$3{\beta}$ was inactivated by PS-341 via the PI3K/Akt pathway. Overexpression of constitutively active GSK-$3{\beta}$ enhanced PS-341-induced apoptosis; in contrast, this was suppressed by dominant negative GSK-$3{\beta}$ (DN-GSK-$3{\beta}$). Inactivation of GSK-$3{\beta}$ by pretreatment with lithium chloride or the overexpression of DN-GSK-$3{\beta}$ suppressed both the JNK activation and c-Jun up-regulation induced by PS-341. Conclusion : The JNK/caspase pathway is involved in PS-341-induced apoptosis, which is negatively regulated by the PI3K/Akt-mediated inactivation of GSK-$3{\beta}$ in lung cancer cells.

Effect of retinoic acid on the bystander effect in gene therapy using the Herpes Simplex Virus thymidine kinase (Herpes Simplex Virus thymidine kinase gene을 이용한 유전자 치료에서 retinoic acid가 bystander effect에 미치는 영향)

  • Park, Jae Yong;Kim, Chang Ho;Jung, Tae Hoon;Albelda, Steven M.
    • Tuberculosis and Respiratory Diseases
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    • v.44 no.1
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    • pp.162-174
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    • 1997
  • Background : Metabolic cooperation via gap junctional intercellular communication (GJIC) is an important mechanism of the bystander effect in gene therapy using the Herpes Simplex Virus thymidine kinase/ganciclovir (HSVtk) "prodrug" system. Since retinoids have been reported to increase GJIC by induction of connexin 43 expression, we hyporthesized that treatment of tumor cells with retinoic acid could augment the bystander effect of the HSVtk/GCV system and result in improved tumor cell killing by enhancing GJIC. Methods : We transferred HSVtk gene to SKHep-J cell line that does not express connexin43, and also transferred the gene to human and murine mesothelioma cell lines that express connexin43. We verified that retinoic acid enhanced GJIC utilizing a functional double-dye transfer study and evaluated the effects of retinoic acid on the growth rate of tumor cells. We then tested the effects of retinoic acid on bystander-mediated cell killing. Results : Addition of all-trans retinoic acid (RA) increased GJIC in cell lines expressing connexin 43 and was asspciated with more efficient in vitro bystander killing in cells transduced with HSVtk via adenoviral and retroviral vectors. In contrast, there was no increase in the efficiency of the bystander effect after exposure to RA in a cell line which had no delectable connexin 43. Conclusion : These results provide evidence that retinoids can augment the efficiency of cell killing with the HSVtk/GCV system by enhancing bystander effect and may thus be a promising new approach to improve responses in gene therapy utilizing the HSVtk system to treat tumors.

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Transformation of Populus nigra × P. maximowiczii Using Agrobacterium tumefaciens vectors (Agrobacterium tumefaciens vector를 이용(利用)한 양황철의 형질전환(形質轉換))

  • Son, Suk Gyu;Hyu, Jung Oh
    • Journal of Korean Society of Forest Science
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    • v.87 no.2
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    • pp.164-172
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    • 1998
  • This study was conducted to find the optimum transformation condition using Agrobacterium harboring promoterless GUS gene. The optimal medium for shoot induction from leaves of Populus nigra${\times}$P. maximowiczii was MS medium supplemented with $0.1mg/{\ell}$ NAA, $0.5mg/{\ell}$ BAP(94% regeneration frequency and 11.5 average number of shoot) According to the test using pBI121, the concentration of antibiotics for selection marker gene was $100mg/{\ell}$ kanamycin or $60mg/{\ell}$ geneticin in the SIM(shoot inducing medium) 3. Two weeks later, callus was induced in the SIM 3 and this callus grew up to 0.5-1cm shoots after 6 weeks in the new SIM 3. And the treatment with methylation inhibitor(5-azacytidine) led to a dramatic increase in foreign gene expression rate from 5.7% to 26.7%. The vector systems showed. different transformation efficiencies based on the fluorometric and histochemical GUS assay. In this study the vector systems used for transformation seemed to affect transformation frequency, in which pEHA101 yielded more transformants(35.9%) than LBA4404/pBI121 did(5.7%). This result indicated that pEHA101 was effective to insert the promoterless foreign gene into a poplar genome.

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Molecular Breeding of Tobacco Plants Resistant to TMV and PVY (분자생물학적 TMV 및 PVY 저항성 연초 육종)

  • E.K. Pank;Kim, Y.H.;Kim, S.S.;Park, S.W.;Lee, C.H.;K.H.Paik
    • Proceedings of the Korean Society of Tobacco Science Conference
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    • 1997.10a
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    • pp.134-152
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    • 1997
  • Plant viruses of tobacco including tobacco mosaic virus (TMV) and potato virus Y (PVY) cause severe economic losses in leaf-tobacco production. Cultural practices do not provide sufficient control against the viruses. Use of valuable resistant cultivars is most recommendable for the control of the viruses. However, conventional breeding programs are not always proper for the development of virus-resistant plants mostly owing to the frequent lack of genetic sources and introduction of their unwanted properties. Therefore, we tried to develop virus-resistant tobacco plants by transforming commercial tobacco cultivars, NC 82 and Burley 21, with coat protein (CP) or replicase (Nlb) genes of TMV and PVY necrosis strain (PVY-VN) with or without untranslated region (UTR) and with or without mutation. Each cDNA was cloned and inserted in plant expression vectors with 1 or 2 CaMV 35S promotors, and introduced into tobacco leaf tissues by Agrobacterium tumefaciens LBA 4404. Plants were regenerated in kanamycin-containing MS media. Regenerated plants were tested for resistance to TMV and PVY In these studies, we could obtain a TMV-resistant transgenic line transformed with TMV CP and 6 genetic lines with PVY-VN cDNAs out of 8 CP and replicase genes. In this presentation, resistance rates, verification of gene introduction in resistant plants, stability of resistance through generations, characteristics of viral multiplication and translocation in resistant plants, and resistance responses relative to inoculum potential and to various PVY strains will be shown. Yield and quality of leaf tobacco of a promising resistant tobacco line will be presented.

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Studies on the Occurrence, Host Range, Transmission, and Control of Rice Stripe Disease in Korea (한국에서의 벼 줄무늬잎마름병의 발생, 피해, 기주범위, 전염 및 방제에 관한 연구)

  • Chung Bong Jo
    • Korean journal of applied entomology
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    • v.13 no.4 s.21
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    • pp.181-204
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    • 1974
  • The study has been carried out to investigate the occurrence, damage, host range, transmission and control of rice stripe virus in Korea since 1965. 1 Disease occur「once and damage : The virus infection during the seedling stage ranged from 1.3 to $8\%$. More symptom expression was found in regrowth of clipped rice than infected intact plants, and the greater infection took place in early seasonal culture than in ordinary seasonal culture. A higher incidence of the disease was found on the rows close to the bank, and gradually decreased toward the centre of the rice paddy. Disease occurrence and plant maturity was highly correlated in that the most japonica rice types were diseased when they were inoculated within 3 to 7 leaf stage, and$50\%$, $20\%$ and no diseaseb were found if they were inoculated at 9, 11 and 13 leaf stages, respectively. Symptom expression required 7-15 days when the plants were inoculated during 3-7 leaf stages, while it was 15-30days in the plants inoculated during 9-15 leaf stages. On Tongil variety the per cent disease was relatively higher when the plants were infected within 1.5-5 leaf stages than those at 9 leaf stage, and no disease was found on the plants infected after 15 leaf stage. The disease resulted in lowered growth rates, maturity and sterility of Tongil variety although the variety is known as tolerant to the virus. 2. Host range: Thirty five species of crops, pasture grasses and weeds were tested for their susceptibility to the virus. Twenty one out of 35 species tested were found to be susceptible. and 3 of them, Cyperus amuricus Maximowics var. laxus, Purcereus sanguinolentus Nees and Eriocaulon robustius Makino, were found as new hosts of the virus. 3. Transmission: The vector of the virus, Laodelphax striatellus, produces 5 generations a year. The peak of second generation adults occurred at June 20th and those of third was at about July 30th in Suweon area. In Jinju area the peak of second generation adult proceeded the peak at Suweon by 5-7 days. The peaak of third generation adult was higher than the second at Jinju, but at Suweon the reverse was true. The occurrence of viruliferous Laodelphax striatellus was 10-15, 9, 17, 8 and about $10\%$ from overwintered nymph, 1st generation nymph, 2nd generation adult, End generation nymph and the remaining generations, respectively. More viruliferous L. striatellus were found in the southern area than in the central area of Korea. The occurrence of viruliferous L. striatellus depended on the circumstances of the year. The per cent viruliferous vectors gin 2nd and 3rd generation adult, however, was consistantly higher than that of other generations. Matings of viruliferous L. striatellus resulted in $90\%$ viruliferous progenies, and the 3rd, 4th and 5th instars of the vector had higher infectiviey than the rest of the vector stages. The virus acquisition rate of non-viruliferous L. striatellus was $7-9\%$, These viruliferous L. striatellus, however, could not transmit the virus for more than 3 serial times. The optimum temperature for the transmission of the viru3 was $25-30^{\circ}C$, while rare transmission occurred when the temperature was below $15^{\circ}C$. The per cent of L. striatellus parasitization by Haplogonatopus atratus were $5-48\%$ during the period from June to the end of August, and the maximum parasitization was $32-48\%$ at around July 10. 4. Control: 1) Cultural practices; The deeper the depth of transplanting more the disease occurrence was found. The higher infection rate, $1.5-3.5\%$, was observed during the late stages of seedling beds, and the rate became lower, $1.0-2.0\%$, in the early period of paddy field in southern area. Early transplanting resulted in more infection than early seasonal culture, and the ordinary seasonal culture showed the lowest infection. The disease also was favored by earlier transplanting even under tile ordinary seasonal culture. The higher the nitrogen fertilizer level the more the disease occurrence was found in the paddy field. 2) Resistant varieties; Tongil varieties shelved the resistant reaction to the virus in greenhouse tests. In the tests for resistance on 955 varieties most japonica types shelved susceptible reactions, while the resistant varieties were found mostly from introduced varietal groups. 3) Chemical control; Earlier applications of chemicals, Disyston and Diazinon, showed better results when the test was made 4 days after inoculation in the greenhouse even though none of the insecticides shelved the complete control of the disease. Three serial applications of chemicals on June 14, June 20 and June 28 showed bettor results than one or two applications at any other dates under field conditions.

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