1990 Fiscal Year Final Research Report Summary
Roles of the Nuclear Scaffold and Higher Order DNA Structure in Transcription
| Project/Area Number |
01570134
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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 |
General medical chemistry
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| Research Institution | Okayama University |
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Principal Investigator |
TSUTSUI Ken Okayama University, Medical School, Associate Professor, 医学部, 助教授 (70108158)
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| Co-Investigator(Kenkyū-buntansha) |
SEKI Shuji Okayama University, Medical School, Professor, 医学部, 教授 (50032884)
YASUDA Tatsuji Okayama University, Medical School, Professor, 医学部, 教授 (30092357)
WATARAI Shinobu Okayama University, Medical School, Research Associate, 医学部, 助手 (50175139)
TSUTSUI Kimiko Okayama University, Medical School, Research Associate, 医学部, 助手 (70144748)
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| Project Period (FY) |
1989 – 1990
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| Keywords | Nuclear scaffold / Super coiled DNA / Transcription / DNA-binding protein |
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| Research Abstract |
Three nuclear scaffold proteins that appear to be involved in regulation of transcription through maintenance or adjustment of the DNA superstructure have been characterized.
1) Superhelicity of the nuclear DNA loop is sustained by its attachment to the nuclear scaffold, however the mechanism of attachment is still unclear. We identified a 120 kDa scaffold protein that specifically binds to the Scaffold Attachment Region DNA (SAR), thus implying its involvement in the loop anchorage. To further analyze the interaction between this protein and SAR, cDNA cloning was attempted. N-terminal amino acid sequences were determined on CNBr-fragments of the 120K protein purified from rat brain. Degenerate oligonucleotide primers based on the peptide sequences were synthesized. Some of the primer combinations yielded double-stranded cDNA segments by the RT-PCR procedure using total RNA as a template. The product DNA was cloned in pUC18 and cDNA clones with ca. 1 kb inserts were obtained.
2) We also
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identified a 75 kDa scaffold protein that shows specific binding to supercoiled DNA and is likely to be a major component of the supercoiled DNA-binding site localized on the nuclear scaffold. The 75K protein was purified from nuclei to test its effects on transcription. Although the protein is always found associated with the isolated scaffold, it was selectively released by directly treating nuclei with high concentration of ethidium bromide under low-ionic-strength conditions. By monitoring the binding activity with biotin-labeled supercoiled DNA, the solubilized 75K protein was purified to near homogeneity after chromatographic separations on ion exchange (MonoQ) and gel filtration (G3000 SW _<XL>) columns. The molecular weight of the protein in gel filtration was estimated to be 380,000, suggesting that the protein is an oligomer native state.
3) Two isoforms (alpha and beta) of DNA topoisomerase II (topo II) encoded by separate genes have been demonstrated in human cells. To assess the possible involvement of topo II beta in RNA synthesis, we first attempted to clone the cDNA for topo II m-RNA expressed in rat brain. Degenerate oligonucleotides corresponding to conserved regions of topo II were designed by comparing the published amino acid sequences of the human and Drosophila enzymes. By using these as primers in the TR-PCR, topo II cDNA fragments were successfully amplified. After cloning the product, the cDNA inserts were classified into two groups (A and B) with different restriction patterns. Determination of the nucleotide sequences revealed striking sequence similarity to the human counterparts. The deduced amino acid identity between the rat and the human sequences, A vs. alpha and B vs. beta, were 95% and 98%, respectively. Less
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