Purification and molecular cloning of the "A" chain of a rat heteromeric CCAAT-binding protein. Sequence identity with the yeast HAP3 transcription factor.
CCAAT-binding factor (CBF) is a heteromeric mammalian transcription factor which binds to sequences containing a CCAAT motif in a number of promoters such as those for type I collagen, albumin, MHC Class II, beta-actin, and others. It consists of two different components that are both needed for DNA binding. We have purified the "A" chain of CBF to apparent homogeneity by sequence-specific DNA affinity chromatography followed by Mono S and Mono Q ion-exchange chromatography and obtained the amino acid sequences of tryptic peptides of this polypeptide. Amino acid sequences of two of these tryptic peptides were used to synthesize oligonucleotide primers. The primers served to obtain a small cDNA by the polymerase chain reaction method, which was then further used to obtain larger cDNA clones. DNA sequence analysis of a representative cDNA clone revealed the presence of an open reading frame of 207 amino acids coding for a putative polypeptide of 25 kDa. Transcription of these cDNAs in vitro followed by translation in a reticulocyte lysate produced a polypeptide that migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with the same mobility as the native A chain. The deduced amino acid sequence of the A chain showed a remarkable identity over a length of 90-amino acid residues with a sequence of the Hap3 polypeptide, a component of a heteromeric multisubunit yeast transcription factor.
Vuorio
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Department of Molecular Genetics, University of Texas, M. D. Anderson Cancer Center, Houston 77030.
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de Crombrugghe
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B
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D002467:Cell Nucleus / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000302:isolation & purification; D051381:Rats; D011919:Rats, Inbred Strains; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics*
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D014157:Transcription Factors