| Project/Area Number |
60570293
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
内科学一般
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| Research Institution | Ehime University |
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Principal Investigator |
SHIOSAKA Takahiko Ehime University, Instructor, 医学部, 講師 (90035486)
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| Project Period (FY) |
1985 – 1986
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| Project Status |
Completed (Fiscal Year 1986)
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| Budget Amount *help |
¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1986: ¥300,000 (Direct Cost: ¥300,000)
Fiscal Year 1985: ¥1,100,000 (Direct Cost: ¥1,100,000)
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| Keywords | Leukemia / Chronic lymphocytic leukemia / gene expression / Cll cDNA library / Colony hybridization / Dot blot hybridization / ノーザン・ブロッティング |
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| Research Abstract |
Human leukemias are considered monoclonal disease viewed as impairing the hematopoietic cell differentiation process. It is reasonable to except that such impairment would be reflect in quantiative changes in mRNA. By using recombinant DNA technology to prepare a cloned library of expressed gene sequences discrete RNA species present in differential amounts in normal and malignant cells can now be examined. The relative abundance of a clonal sequence of cellular RNA can be estimated by nucleic acid hybridization. Selected and analysis of clones containing copies of cDNA transcripts of mRNA was accomplised as shown below.
1.Preparation of cDNA library from total mRNA of CLL cells. And screening of library with [ <^(32)P> ]-cDNA from CLL and placental cells. Selection of 21 clones that react positively with [ <^(32)P> ]-cDNA from CLL but do not react with [ <^(32)P> ]-cDNA from placental cells.
2.The use of these DNA segments to measure the population of mRNA for neoplastic cell specific expressed genes in various leukemic cells by Dot blot hybridization method.
3.Determination of DNA sequence of cDNA insert in each clone.
Total cellular RNAs from a variety of hematopoietic neoplastic cells and normal lymphocytes were analyzed for homology with 21 recombinant DNA segments complementary to the specific mRNA of cells from a patiant with CLL.
Twenty of the clones (except for clone 8-6G) were not significantly represented in the mRNA from normal lymphocytes. The relative concentrations of three clones (6-1E,7-3G and 9-5C) in the RNAs from a variety of normal leukemic leukocytes and human hematopoietic cell lines were measured by Northern blot hybridization. This showed that 7-3G,6-1G and 9-5C RNA were highly abundant in RNA from hematopoietic neoplastic leukocytes. Also, clone 7-2D was highly represented in B cell type hematopoietic neoplastic leukocytes,6-1G was highly represented in Raji cell while clone 6-1E was highly represented in Molt 3 cell.
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