GATA-binding proteins constitute one of transcription factor families, which bind to the consensus target site "WGATAR" (W=A or T and R=A or G). GATA factors, whose distinct feature is the zinc finger motif $Cys-X_2-Cys-X_{17}-Cys-X_2-Cys$, are present in many eukaryotes from yeast to human. GATA factors of vertebrates are shown to be involved in the regulation of globin gene expression or other developmentally related genes. On the other hand in fungi, GATA factors regulate expression of genes involved in the regulation of nitrogen or iron metabolism.
As an attempt toward elucidation for the functions of GATA factors in Shizosaccharomyces pombe, we have isolated and characterized the two GATA factor homologous genes, gaf1 and gaf2. Genomic Southern analysis, with the degenerate oligo probe encoding the well-conserved zinc finger core region, showed the possibility of the presence of at least 2 GATA factor homologs in S. pombe. The gaf1 gene was first cloned by the method of PCR with degenerate primers corresponding to the conserved sites in the zinc finger region. The second GATA factor gene, gaf2, was cloned by the low stringent screening of a λ-S. pombe genomic library using the partial gaf1 gene as a probe. Analysis of the DNA sequences showed that Gaf1 and Gaf2 have the 290- and 564-amino acid ORF, respectively. Northern analysis showed that the sizes of gaf1, gaf2 transcripts are 4.2 kb with 2 minor bands (1.6 and 2.1 kb) and 3.1 kb with a minor band at 1.6 kb, respectively. The transcription levels of gaf1 and gaf2 were not affected by a nitrogen source in media.
Disruption of gaf1 was not lethal and showed no detectable change in the growth rate. The gel mobility shift assay, using the GST-Gaf1 fusion protein and the canonical GATA motif (the DAL7 UAS of S. cerevisiae), showed that Gaf1 binds to GATA motifs with specificity. In addition, the transactivation assay, where the lacZ expression was examined under the GAL4-CYC1 hybrid promoter, showed that Gaf1 functions as a transcription activator. As a consequence, It is certain that Gaf1 is a new member of the GATA family which can function as a transcriptional activator of its presumptive target genes. The function of gaf2 is not known yet, but there is an indication that it might be involved in meiosis pathway of S. pombe.
