Molecular cloning of the alpha-globin transcription factor CP2.
CP2, a transcription factor that binds the murine alpha-globin promoter, was purified and subjected to amino acid sequence analysis. Oligonucleotide primers derived from the sequence were used to obtain murine and human cDNA clones for the factor. The murine cDNA spans approximately 4 kb and contains two coextensive open reading frames (ORFs) which encode deduced polypeptides of 529 (ORF-1; molecular weight, 59,802) and 502 (ORF-2; molecular weight, 56,957) amino acids, slightly smaller than the purified factor as estimated from its mobility in sodium dodecyl sulfate-polyacrylamide gels (64,000 to 66,000). The human cDNA contains a single ORF of 501 amino acids that is nearly contiguous with murine ORF-2. Indeed, comparison of deduced human and murine amino acid sequences shows that the two polypeptides are 96.4% identical. A strictly conserved region is rich in serine and threonine (17.5%) and in proline (11%) residues (S-T-P domain). This S-T-P domain is immediately amino terminal to a string of 10 glutamines (in the human sequence) or a tract of alternating glutamine and proline residues (in the mouse sequence). Bacterial expression of the full-length (502-amino-acid) murine factor or of a core region comprising amino acids 133 to 395 generated polypeptides with the DNA binding specificity of CP2. These results confirmed the cloning of CP2 and delimited the region sufficient for specific DNA sequence recognition. Antisera produced against the core region recognized polypeptide species with Mrs of 64,000 and 66,000 in immune blots of nuclear extracts prepared from both murine and human cell lines, consistent with the size of the purified factor. Lastly, a data base search revealed that amino acids 63 to 270 of the murine factor are distantly related to a domain in the Drosophila gene regulatory factor Elf-1.
Lim
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DeWitt Wallace Research Laboratory, Memorial Sloan-Kettering Cancer Center, New York, New York 10021.
Swendeman
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Sheffery
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D005914:Globins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D016601:RNA-Binding Proteins; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*
D004268:DNA-Binding Proteins; D016601:RNA-Binding Proteins; C501906:TFCP2 protein, human; C501905:Tfcp2 protein, mouse; D014157:Transcription Factors; D005914:Globins