Worm Breeder's Gazette 12(2): 65a (January 1, 1992)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
The elt-1 gene encodes the C. elegans homolog of an erythrocyte-specific transcription factor, GATA-1, which recognizes the sequence (A/T)GATA(A/G). In order to define the DNA binding specificity of the elt-1 protein, we have expressed it in yeast under GAL1 control and asked whether it can activate transcription of a lacZ gene fused to the proposed recognition site. There are 8 (A/T)GATA(A/G) sequences in elt-1 'spromoter region (Spieth et. al., 1991, Mol. Cell. Biol. 11:46514659), suggesting it might be autogenously regulated. Two of these, located between -247 and -230, were used as a test elt-1 binding site. An oligonucleotide containing this sequence was inserted, alone and multimerized up to 5 times, about 200 bp upstream of a lacZ reporter gene under control of a CYC1 promoter lacking its UAS. Evidently yeast doesn't have a protein capable of activating at this sequence (at least under the growth conditions we used), since no ß-galactosidase was produced in the absence of expression of the C. elegans elt-1 protein. We expressed the elt-1 protein under the control of the GAL1 promoter by inserting the entire elt-1 cDNA into a yeast expression vector (Bonner,1991, Gene 104: 113-118). Transformants carrying both the elt-1 -expressingand lacZ plasmids were selected and ß-galactosidase activity was measured.
ß-galactosidase was expressed in abundance when the following conditions were met: 1. the elt-1 cDNA was in the correct orientation in the expression plasmid; 2. elt-1 was induced by growth on galactose; and 3. GATA sequences were present upstream of lacZ. Increasing the number of copies of the GATA-containing oligonucleotide in the reporter plasmid resulted in dramatically increased ß-gal activity. These findings demonstrate that elt-1 is a transcriptional activator that recognizes the (A/T)GATA(A/G) sequence. Furthermore, elt-1 is capable of activation without other cis-elements from the elt-1 promoter and without other C. elegans transcriptional machinery. Since the double GATA sequence that we tested normally resides in the elt-1 promoter, it appears likely that elt-1 is autogenously regulated.