[Revisions of the cDNA and primary protein structure of human transcription factor GCF].
GC factor (GCF) was reported as a transcriptional regulator that binds to specific GC-rich sequences in the epidermal growth factor receptor (EGFR) gene promotor, repressing its transcription (Kageyama R. and Pastan I. Cell, 59: 815-825, 1989). In this paper, the author presents revisions of the cDNA and the amino acid sequences of the GCF. 1) 5' rapid amplification of cDNA end (5'RACE) for analysis of RNA of a cancer cell line, A431, was performed, which revealed that the 5' end of GCF cDNA was fused to a 308 bp fragment of other cDNA; simultaneously, the real 5' end cDNA sequence with 31 bp was identified. RNase protection assay presented a main protected band, which was consistent with the result of the RACE analysis. 2) T at the position 787 of the previously reported GCF cDNA was absent from RT-PCR on A431 total RNA. 3) A new sequence with 114 bp was observed on A431 RNA between the positions 851 and 852 of the already reported cDNA by RT-PCR. These observations were confirmed by RT-PCR analyses of RNAs prepared from several other human cell lines, including a non-transformed one (HFL), and white blood cells derived from a normal person. 4) Sequence of genomic GCF DNA was consistent with the new cDNA sequence but not with the previously reported one. 5) The remaining sequence of GCF cDNA was found to be identical to that of the previously reported GCF, based on the results of RT-PCR analyses of RNA prepared from human white blood cells. 6) By the corrections, the GCF cDNA consisted of 2661 bp nucleotides. This revised GCF cDNA (the wild type) encodes a protein of 781 amino acids, including two new sequence regions of 186 amino acids on the N-terminal side of this protein. The revisions eliminated the highly basic region of the amino-terminus of the previously reported GCF, while the other three fourth amino acid sequences of the GCF protein that contained leucine-zipper-like domain had not changed. The revised GCF had no highly homologous protein in the database except the previously reported GCF. 7) The author has developed a specific antibody to human GCF protein. This antibody specifically recognized a protein with a molecular weight of approximately 100 kDa present in the extracts from human cell lines, as confirmed by immunoprecipitation followed by Western blotting. 8) Indirect immunofluorescence of A431 and HeLa cells using the anti-GCF antibody showed that the GCF protein was localized in the nucleus, suggesting that the revised GCF is a nucleoprotein.
Mao
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Division of Gene Regulation, Cancer Institute, Hokkaido University School of Medicine, Sapporo, Japan.
D000595:Amino Acid Sequence; D018076:DNA, Complementary / Q000737:chemistry*; D006801:Humans; D009698:Nucleoproteins / Q000032:analysis; D016133:Polymerase Chain Reaction; D012334:RNA, Neoplasm / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics*; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; C072278:GCFC2 protein, human; D009698:Nucleoproteins; D012334:RNA, Neoplasm; D012097:Repressor Proteins