| Project/Area Number |
01580190
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
代謝生物化学
|
|---|
| Research Institution | Osaka University |
|---|
Principal Investigator |
YAMADA Michiyuki Osaka University, Institute for Protein Research, Associate Professor, たんぱく質研究所, 助教授 (10076995)
|
|---|
| Project Period (FY) |
1989 – 1990
|
| Project Status |
Completed (Fiscal Year 1990)
|
| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1990: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1989: ¥1,300,000 (Direct Cost: ¥1,300,000)
|
|---|
| Keywords | Myeloperoxidase / Regulation of Gene Expression / Transfection of Recombinant Plasmid / Cis-element of gene / ミエロペルオキシダ-ゼ |
|---|
| Research Abstract |
Myeloperoxidase (MPO) is a heme containing glycoprotein in polymorphonuclear leukocytes and is involved in the Cl^- -mediated bactericidal function of the cells. The enzyme is mostly synthesized at an early stage of promyelocyte during granulocyte development and then its synthesis was stopped during further development. The enzyme is also synthesized in human promyelocytic leukemia cell HL-60, but not in the cells induced to differentiate into granulocytes by retinoic acid or macrophages by a phorbol ester, TPA. Previously we reported that MPO synthesis in HL-60 cells is regulated at a transcriptional level. In this work I initiated to clarify a regulatory element of MPO gene.
Human MPO genomic clones lombda MPO18 and lambda MPO14 were prepared as described Previously. The two clones taken together encompass MPO gene of approximately 17 kbp including from an upstream of the gene to a downstream. For determination of a regulatory element of MPO gene, the transient expresst expresstion o
… More
f SV40 early promoter dependent bacterial Chloramphenicol Acetyl-transferase (CAT) gene in HL-60 cells was used.
First, the methods of gens transfection were tested for HL-60 cells using pSV2CAT as a standard. The DEAE-dextran method used for various kinds of floating cells and adherent cells was not useful at all for HL-60 cells. The electroporation method was shown to be effective for HL-60 cells. High amount of CsCl density gradient-purified plasmid was needed by this method.
Next, the inserts of the genomic clones were digested with restriction enzymes and then the restriction fragments were inserted to pSVPCAT which was prepared from pSV2CAT by deleting a portion of the enhancer of SV40 early promoter. The recombinant plasmids obtained were transfected to HL-60 cells by the electroporation method and afrer 2-3 days, CAT activity was determined. The results of the CAT assay indicated that two fragments out of the various restriction fragments tested had a weak enhancer activity. Therefore, the nucleotide sequence of the two fragments were determined by the dideoxy chain termination method. Interaction between the fragments and protein was studied using the gel mobility shift assay. ^<32>P-labeled fragment was incubated with nuclear extracts of HL-60 cells and electrophoresed on a native polyacrylamide gel. After electrophoresis the gel was exposed to a X-ray film with intensifying screens at -70^ﾟC. The autoradiogram revealed that the probe-protein complex was formed. However, competition experiments with unlabeled cognate DNA and unrelated DNA could not show any specificity of DNA sequence. Furthermore, DNase I-foot printing experiments and methylation interference experiments failed to prove that specific sequences of these fragments were involved in these DNA-protein interactions.
Purifications of transcription factors from nuclear extracts of HL-60 cells and further fragmentation of these fragments will be required to clarify the sequencespecific DNA protein interaction. Less
|
