The general goal of this project is to characterize upstream promoter factors that regulate murine alpha-globin gene transcription. In published work, four such factors, termed CP1, CP2, GATA-1, and IRP have been described. Each has been purified to apparent homogeneity by DNA sequence affinity chromatography, and the function of each of factor has been assessed using an in vitro transcription assay. In addition cDNA and genomic clones encoding CP1, CP2 and GATA-1 have been obtained. Each of the cloned factors has been expressed in bacteria and factor specific antisera have been (cP2, GATA-1) or are being (CP1) prepared. Because this laboratory has been most recently involved in the cloning of CP2, and because this factor has been less well studied, the specific aims of this proposal emphasize the further characterization of this factor. Our specific goals for the next three years are to: 1. Determine the DNA binding and transcription activation domains of the cloned transcription factor CP2. 2. Obtain complete murine and human genomic clones for CP2, localize the genes to their appropriate chromosomes, determine their intron/exon structures, identify and characterize their promoters. 3. Examine the tissue distribution and expression of CP2 and obtain CDNA clones that encode antigenically related factor present in Xenopus oocytes and yeast. 4. Use antisera against GATA-1 and CP2 and columns of GATA-1 and CP2 affixed to sepharose beads to determine if these factors interact with additional nuclear proteins. Prepare antisera specific for CP1 and determine if additional subunits of this factor must be cloned.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Hematology Subcommittee 2 (HEM)
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Sloan-Kettering Institute for Cancer Research
New York
United States
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Swendeman, S L; Spielholz, C; Jenkins, N A et al. (1994) Characterization of the genomic structure, chromosomal location, promoter, and development expression of the alpha-globin transcription factor CP2. J Biol Chem 269:11663-71
Zhong, F; Swendeman, S L; Popik, W et al. (1994) Evidence that levels of the dimeric cellular transcription factor CP2 play little role in the activation of the HIV-1 long terminal repeat in vivo or following superinfection with herpes simplex virus type 1. J Biol Chem 269:21269-76
Lim, L C; Fang, L; Swendeman, S L et al. (1993) Characterization of the molecularly cloned murine alpha-globin transcription factor CP2. J Biol Chem 268:18008-17
Lim, L C; Swendeman, S L; Sheffery, M (1992) Molecular cloning of the alpha-globin transcription factor CP2. Mol Cell Biol 12:828-35
Kim, C G; Swendeman, S L; Barnhart, K M et al. (1990) Promoter elements and erythroid cell nuclear factors that regulate alpha-globin gene transcription in vitro. Mol Cell Biol 10:5958-66
Kim, C G; Sheffery, M (1990) Physical characterization of the purified CCAAT transcription factor, alpha-CP1. J Biol Chem 265:13362-9
Barnhart, K M; Kim, C G; Sheffery, M (1989) Purification and characterization of an erythroid cell-specific factor that binds the murine alpha- and beta-globin genes. Mol Cell Biol 9:2606-14
Sheffery, M; Kim, C G; Barnhart, K M (1989) Purification of four erythroid cell proteins that bind the promoters of the murine globin genes. Prog Clin Biol Res 316A:343-57
Barnhart, K M; Kim, C G; Banerji, S S et al. (1988) Identification and characterization of multiple erythroid cell proteins that interact with the promoter of the murine alpha-globin gene. Mol Cell Biol 8:3215-26
Kim, C G; Barnhart, K M; Sheffery, M (1988) Purification of multiple erythroid cell proteins that bind the promoter of the alpha-globin gene. Mol Cell Biol 8:4270-81