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Establishment of the loss-of-function experimental system using the maxizyme in the ascidian embryo.

Research Project

Project/Area Number 11680728
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Developmental biology
Research InstitutionKonan University

Principal Investigator

NISHIKATA Takahito  Konan Univ., Dept. of Biology, Associate Professor, 理学部, 助教授 (80212116)

Project Period (FY) 1999 – 2000
Project Status Completed (Fiscal Year 2000)
Budget Amount *help
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2000: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
KeywordsDNAzyme / ascidian / RT-PCR / 2'-O-methyl modification / maternal mRNA / antisense technology / loss-of-function / マキシザイム / polIIIプロモーター
Research Abstract

It has been very difficult to prove the gene function in ascidians, because the experimental system, which can disturb a certain gene function, has not been established. In this study, two types of the loss-of-function experimental system using ribozymes in the ascidian embryo were intended to develop. One is aimed to abolish zygotically expressed mRNA and to prove the zygotic gene function. The other targets the maternal mRNAs, which are stored in the egg.
1 : The plasmid, which carries the human pol III promoter-driven ribozyme sequence, was micro injected into the ascidian fertilized eggs. The eggs were reared until the tailbud stage, and examined for the expression of maxizyme sequence by RT-PCR.Although enough amount to plasmid was retained in the embryo, the maxizyme RNA was not detected. The same plasmid was also introduced into the fertilized egg by electroporation. In this case, only a trace amount of its expression was observed.
2 : Active form (DZ) and inactive form (IDZ) of deoxyribozyme were designed, and microinjected into unfertilized eggs. The injected eggs were kept for several hours. Although the DZ efficiently decrease the amount of the target RNA, IDZ had similar and more effect on the target RNA.This showed the antisense effect of ribozymes in the ascidian egg. Moreover, when the DZ- or IDZ-injected eggs was fertilized, the embryos were developed abnormally, even in the condition, at which the target mRNA was not depleted. This suggest the high toxicity of the deoxyribozyme.
According to these results, the imperative factors for designing the ribozymes are piled up, and the suitable conditions for introducing the ribozymes was found. Moreover, sensitive RT-PCR method offers a reliable evaluation system, which is indispensable for the establishment of the loss-of-function experiment on the ascidian development.

Report

(3 results)
  • 2000 Annual Research Report   Final Research Report Summary
  • 1999 Annual Research Report
  • Research Products

    (54 results)

All Other

All Publications (54 results)

  • [Publications] Nishikata,T.: "RNA-binding proteins in ascidian development.""Biology of Ascidian" Sawada et al., Eds., Springer-Verlag. 178-185 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Tanaka,K.J.: "Functional analysis of Ciona intestinalis Y-box protein.""Biology of Ascidian" Sawada et al., Eds., Springer-Verlag. 186-192 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Makabe,K.W.: "Maternal genetic information stored in fertilized eggs of the ascidian, Halocynthia roretzi.""Biology of Ascidian" Sawada et al., Eds., Springer-Verlag. 166-177 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hatano,K.: "Zoospore-specific antigen as a useful marker for molecular analysis of net formation in Hydrodictyon reticulatum (Chlorococcales, Chlorophyceae)."Phycological Resesch. 48. 143-148 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Matsugu,H.: "Improved antigen detection on Western-blots."Technical Tips OnLine. T02043 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Tanaka,K.J.: "The transcript coding for an RNA-binding protein is localized in the anterior side of the ascidian 2-cell embryo."Development, Genes and Evolution. 210. 464-466 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Tanaka,K.J.: "An ascidian glycine-rich RNA-binding protein is not induced by temperature stress but is expressed under a genetic program during embryogenesis."Gene. 243. 207-214 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kawashima,T.: "Expression patterns of musashi homologus of the ascidians, Halocynthia roretzi and Ciona intestinalis."Development, Genes and Evolution. 210. 162-165 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Tanaka,K.J.: "A non-radioactive get shift protocol enables recovery of RNA-binding proteins."Technical Tips OnLine. T01794 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakamori,R.: "Characterization of MTR1 : A novel protein correlated with the myoplasmic movement during ascidian ooplasmic segregation."Memoirs of Konan University, Science Series. 46. 25-33 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Okubo,M.-A.: "Generation and characterization of a monoclonal antibody, mH1, raised against mitotic HeLa cells."Development, Growth & Differentiation. 41. 381-389 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nishikata,T.: "The CAB (Centrosome-Attracting Body), microtubule system, and posterior egg cytoplasm are involved in positioning of cleavage planes in the ascidian embryo."Developmental Biology. 209. 72-85 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Chiba,S.: "Interactions between cytoskeletal components during ooplasmic segregation in the myoplasm of ascidian eggs."Development, Growth & Differentiation. 41. 265-272 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Wada,M.R.: "The expression of the protochordate homologue of the proteasome regulatory subunit Rpn12 is transcriptionally and post-translationally regulated during cleavage stage."Zoological Science. 16. 125-129 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nishida,H.: "Maternal cytoplasmic factors for generation of unique cleavage patterns in animal embryos."Current Tbpics in Developmental Biology. 34. 1-37 (1999)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nishikata, T., Wada, M.R.and Tanaka, K.J.: "RNA-binding proteins in ascidian development.""The Biology of Ascidians" Sawada, H.et al. eds, Springer-Verlag, New York. 178-185 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Tanaka, K.J.and Nishikata, T.: "Functional analysis of Ciona intestinalis Y-box protein.""The Biology of Ascidians" Sawada, H.et al. eds, Springer-Verlag, New York. 186-192 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Makabe, K.W., Kawashima, T., Kawashima, S., Sasakura, Y., Ishikawa, H., Kawamura, H., Kanehisa, M., Nishikata, T.and Nishida, H.: "Maternal genetic information stored in fertilized eggs of the ascidian, Halocynthia roretzi.""The Biology of Ascidians" Sawada, H.et al. eds, Springer-Verlag, New York. 165-177 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Hatano, K., Yamamoto, M., Yamada, Y.and Nishikata, T.: "Zoospore-specific antigen as a useful marker for molecular analysis of net formation in Hydrodictyon reticulatum (Chlorococcales, Chlorophyceae)."Phycological Resarch. 48. 143-148 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Matsugu, H.and Nishikata, T.: "Improved antigen detection on Western-blots."Technical Tips Online T02043. (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Tanaka, K.J., Kawamura, H.and Nishikata, T.: "The transcript coding for an RNA-binding protein is localized in the anterior side of the ascidian 2-cell stage embryo."Development Genes and Evolution. 210. 464-466 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Tanaka, K.J., Kawamura, H., Matsugu, H.and Nishikata, T.: "An ascidian glycine-rich RNA -binding protein is not induced by temperature-stress but is expressed under a genetic program during embryogenesis."Gene. 243. 207-214 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Kawashima, T., Murakami, A., Ogasawara, M., Tanaka, K.J., Isoda, R., Sasakura, Y., Nishikata, T., Okano, H.and Makabe, K.W.: "Expression patterns of musashi homologus of the ascidians, Halocynthia roretzi and Ciona intestinalis."Development Genes and Evolution. 210. 162-165 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Tanaka, K.J.and Nishikata, T.: "A non-radioactive gel shift protocol enables recovery of RNA-binding proteins."Technical Tips Online T01794. (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nakamori, R., Murakami, A.and Nishikata, T.: "Characterization of MTR1 : A novel protein correlated with the myoplasmic movement during ascidian ooplasmic segregation."Mem.Konan Univ., Sci.Ser.. 46. 25-33 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Okubo, M.-A., Chiba, S., Nishikata, T., Matsuno A.and Hosoya H.: "Generation and characterization of a monoclonal antibody, mH1, raised against mitotic HeLa cells."Devl.Growth Differ.. 41. 381-389 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nishikata, T., Iseto, T., Hibino, T.and Nishida, H.: "The CAB (Centrosome-Attracting Body), microtubule system, and posterior egg cytoplasm are involved in positioning of cleavage planes in the ascidian embryo."Devl.Biol.. 209. 72-85 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Chiba, S., Miki, Y., Ashida, K., Wada, M.R., Tanaka, K.J., Shibata, Y., Nakamori, R.and Nishikata, T.: "Interactions between cytoskeletal components during ooplasmic segregation in the myoplasm of ascidian eggs."Devl.Growth Differ.. 41. 265-272 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Wada, M.R., Murakami, A.R., Kawamura, H., Nakamori, R., Tanaka, K.J., Nakayama, H.and Nishikata, T.: "The expression of the protochordate homologue of the proteasome regulatory subunit Rpn12 is transcriptionally and post-translationally regulated during cleavage stage."Zool.Sci.. 16. 125-129 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nishida, H., Morokuma, J.and Nishikata, T.: "Maternal cytoplasmic factors for generation of unique cleavage patterns in animal embryos."Curr.Top.Dev.Biol.. 34. 1-37 (1999)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2000 Final Research Report Summary
  • [Publications] Nishikata,T.: "RNA-binding proteins in ascidian development.""Biology of Ascidian" Sawada et al.,Eds.,Springer-Verlag. (In press). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Tanaka,K.J.: "Functional analysis of Ciona intestinalis Y-box protein.""Biology of Ascidian" Sawada et al.,Eds.,Springer-Verlag. (In press). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Makabe,K.W.: "Maternal genetic information stored in fertilized eggs of the ascidian, Halocynthia roretzi.""Biology of Ascidian"Sawada et al.,Eds.,Springer-Verlag. (In press). (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] Hatano,K.: "Zoospore-specific antigen as a useful marker for molecular analysis of net formation in Hydrodictyon reticulatum (Chlorococcales, Chlorophyceae)."Phycological Research. 48. 143-148 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Matsugu,H.: "Improved antigen detection on Western-blots."Technical Tips OnLine. T02043 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Tanaka,K.J.: "The transcript coding for an RNA-binding protein is localized in the anterior side of the ascidian 2-cell stage embryo."Development, Genes and Evolution. 210. 464-466 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Tanaka,K.J.: "An ascidian glycine-rich RNA-binding protein is not induced by temperature stress but is expressed under a genetic program during embryogenesis."Gene. 243. 207-214 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Kawashima,T.: "Expression patterns of musashi homologus of the ascidians, Halocynthia roretzi and Ciona intestinalis."Development, Genes and Evolution. 210. 162-165 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] Tanaka,K.J.: "A non-radioactive gel shift protocol enables recovery of RNA-binding proteins."Technical Tips OnLine. T01794 (1999)

    • Related Report
      2000 Annual Research Report
  • [Publications] Nakamori,R.: "Characterization of MTR1 : A novel protein correlated with the myoplasmic movement during ascidian ooplasmic segregation."Memoirs of Konan University, Science Series. 46. 25-33 (1999)

    • Related Report
      2000 Annual Research Report
  • [Publications] Okubo,M.-A.: "Generation and characterization of a monoclonal antibody, mH1, raised against mitotic HeLa cells."Development, Growth & Differentiation. 41. 381-389 (1999)

    • Related Report
      2000 Annual Research Report
  • [Publications] Nishikata,T.: "The CAB (Centrosome-Attracting Body), microtubule system, and posterior egg cytoplasm are involved in positioning of cleavage planes in the ascidian embryo."Developmental Biology. 209. 72-85 (1999)

    • Related Report
      2000 Annual Research Report
  • [Publications] Chiba,S.: "Interactions between cytoskeletal components during ooplasmic segregation in the myoplasm of ascidian eggs."Development, Growth & Differentiation. 41. 265-272 (1999)

    • Related Report
      2000 Annual Research Report
  • [Publications] Wada,M.R.: "The expression of the protochordate homologue of the proteasome regulatory subunit Rpn 12 is transcriptionally and post-translationally regulated during cleavage stage."Zoological Science. 16. 125-129 (1999)

    • Related Report
      2000 Annual Research Report
  • [Publications] Nishida,H.: "Maternal cytoplasmic factors for generation of unique cleavage patterns in animal embryos."Current Topics in Developmental Biology. 34. 1-37 (1999)

    • Related Report
      2000 Annual Research Report
  • [Publications] Tanaka,K.J.: "The transcript coding for an RNA-binding protein is localized in the anterior side of the ascidian 2-cell stage embryo"Development,Genes and Evolution. 210(in press). (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Tanaka.K.J.: "An ascidian glycine-rich RNA-binding protein is not induced by temperature-stress but is expressed under a genetic program during embryogenesis"Gene. 243. 207-214 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Kawashima,T.: "Expression patterns of musashi homologus of the ascidians,Halocynthiaroretzi and Ciona intestinalis"Development,Genes and Evolution. 210. 162-165 (2000)

    • Related Report
      1999 Annual Research Report
  • [Publications] Tanaka,K.J.: "A non-radioactive gel shift protocol enables recovery of RNA-binding proteins"Technical Tips Online. T01794 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Nakamori,R.: "Characterization of MTR1 : A novel protein correlated with the myoplasmic movement during ascidian ooplasmic segregation"Memoirs of Konan University,Science Series. 46. 25-33 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Nishikata,T.: "The CAB(Centrosome-Attracting Body),microtubule system,and posterior egg cytoplasm are nvolved in positioning of cleavage planes in the ascidian embrvo"Developmental Biology. 205. 72-85 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Okubo,M.A.: "Generation and characterization on of a monoclonal antibody,mH1,raised against mitotic HeLa cells"Development,Growth&Differentiation. 41. 381-389 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Chiba,S.: "Interactions between cytoskeletal components during ooplasmic segregation in the myoplasm of ascidian eggs"Development,Growth&Differentiation. 41. 265-272 (1999)

    • Related Report
      1999 Annual Research Report
  • [Publications] Wada,M.R.: "The expression of the protochordate homologue of the proteasome regulatory subunit Rpn12is transcriptionally and post-translationally regulated during cleavage stage"Zoological Science. 16. 125-129 (1999)

    • Related Report
      1999 Annual Research Report

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Published: 1999-04-01   Modified: 2016-04-21  

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