| Budget Amount *help |
¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1988: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1987: ¥900,000 (Direct Cost: ¥900,000)
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| Research Abstract |
The promoter of human EGF receptor gene contains six pyrimidine clusters; one overlaps a SP1-binding sequence, two were at adifferent position and each of them is contiguous to a SP1-binding sequence, another is within a stem-loop sequence, and remaining two are situated with no relation to these specific sequences. Using pERCAT consisting of the promoter of human EGF receptor gene and a CAT gene, conformational change of DNA structrue and its relation to biological activity of DNA were analyzed employing monoclonal anti-DNA antibodies recognizing higher-order structures of DNA and a transcriptional factor, SP1, for a probe. The results were as follows: 1. SP1 bound to linear DNA as well as superhelical DNA, but it enhanced transcription of the latter much more than that of the former. 2. The Fab fraction of anti-deoxypyrimidine cluster antibody (5H4) bound to pERCAT whose SP1-binding sequences were covered with SP1 but had no effect on template activity of pERCAT. 3. SP1 did not bind
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to pERCAT in which Fab fractions of 5H4 bound to the pyrimidine clusters, and the addition of SP1 did not enhance transcription in the in vitro system consisting of Fab-bound pERCAT as a template. 4. We established 17 hybridomas producing monoclonal anti-DNA antibodies with the following characteristics: (1) reactive to single-stranded DNA; (2) reactive to double-stranded DNA as well as singlestranded DNA; (3) reactive to polydeoxypyrimidine; (4) reactive to stem-loop DNA; (5) reactive to brominated poly (dGdC) and N-acetoxy-2-acetylaminofruorene-treated poly(dG-dC), but not to poly(dG-dC); and (6) reactive to a bending dna fragment, GGG(A_5N_5)_5A_5CGG. We are now studying whether the bound Fab fraction of 5H4 covers the contiguous SP1-binding locus or causes conformational changes of DNA that prevent SP1 binding, and characterizing the interaction between conformational change and the biological activity of DNA using monoclonal antibodies specific to higher-order structures of DNA as a probe. Less
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