Protein factors and their gene cloning necessary for the accurate transcription initiation of human beta globin gene

Research Project

Project/Area Number	61480492
Research Category	Grant-in-Aid for General Scientific Research (B)
Allocation Type	Single-year Grants
Research Field	Human genetics
Research Institution	Tokyo Medical and Dental University
Principal Investigator	YASUKOCHI Yukio Professor, Med. Res. Inst., Tokyo Med. & Dent. Univ., 難治疾患研究所, 教授 (60037398)
Project Period (FY)	1986 – 1988
Project Status	Completed (Fiscal Year 1988)
Budget Amount *help	¥4,500,000 (Direct Cost: ¥4,500,000) Fiscal Year 1988: ¥500,000 (Direct Cost: ¥500,000) Fiscal Year 1987: ¥1,500,000 (Direct Cost: ¥1,500,000) Fiscal Year 1986: ¥2,500,000 (Direct Cost: ¥2,500,000)
Keywords	beta globin / In vitro transcription / HeLa細胞 / ヒトβーグロビン / 転写 / ヒトβ-グロビン
Research Abstract	In addition to RNA polymerase II, multiple protein factors from HeLa cells are necessary for the accurate initiation of transcription on minimal promoters in vitro. We have partially purified these factors by chromatographic methods. In addition to RNA polymerase II, six factors (FA, FB, FC, FD, FE snd FF) necessary for initiation at the beta globin promoter start site in vitro have been identified. The Sarkosyl block assays showed that six factors must be incubated with the DNA template and other factors before addition of 0.2% Sarkosyl, indicating they are all initiation factors. Certain of these (FA, FC and FE) have been purified to near homogeneity. The apparent molecular weight of FA was estimated to be 27 KDa on SDS-PAGE. The DNA sequence of cDNA for FA showed a highly basic polypeptide with high content of tyrosine and proline. Comparison of the FC activity with the positions of bands on SDS-PARE suggested that an 80 KDa band might be responsible for the FC activity. The apparent molecular weight of FE was estimated to be 33 KDa on SDS-PAGE. FE did not give any DNA binding activity by gel mobility shift assay and DNase I footprinting assay.