A cDNA encoding a human CCAAT-binding protein cloned by functional complementation in yeast.
We constructed a comprehensive cDNA library from HeLa cell mRNA in a vector that directs expression of the cDNA in Saccharomyces cerevisiae. We used this library to clone the human counterpart of the Sa. cerevisiae CCAAT-binding transcription factor, Hap2, by functional complementation of a hap2 mutation. The cDNA encoding the human Hap2 homolog encodes a protein of 257 amino acids that has a 62-amino acid carboxyl-terminal region 73% identical to the essential core region of Hap2. The amino terminus of the protein is highly enriched in glutamine residues, reminiscent of transcriptional activation domains of several other mammalian transcription factors. Analysis of human Hap2 expression reveals three major transcripts: a 4.1-kilobase species found in all cell types examined, a 7.0-kilobase species specific to B lymphocytes, and a 1.6-kilobase species that is expressed preferentially in HeLa cells and that likely corresponds to our cDNA clone. Thus, the human Hap2 homolog and related factors may play both a constitutive and cell type-specific role in gene expression. The general approach of cloning by complementation should allow the isolation of many human genes for which corresponding yeast mutations exist.
Becker
D M
DM
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Fikes
J D
JD
Guarente
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000235:genetics*; D015723:Gene Library; D005816:Genetic Complementation Test; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D014157:Transcription Factors