Structural and Functional Characterization and Cloning ofXenopusFOSP‐1 (Frog Oviduct‐Specific Protein‐1) Genes

Abstract

As a model for tissue-specific gene expression, our laboratory has been studying the expression of vitellogenin and FOSP-1 (frog oviduct-specific protein-I) genes in Xenopus laevis which are expressed exclusively in the liver and oviduct, respectively, both strictly regulated by estrogen. Whereas the structure and function of Xenopus vitellogenin mRNAs and the upstream regulatory sequences (URS) of their genes are well established, little or no similar information is available for FOSP-1 genes. In this study, using a combination of 5' rapid amplification of cDNA ends (RACE) and reverse-transcriptase PCR, we have identified two gene copies of FOSP-1, termed FOSP-1A and FOSP-1B. Comparison of the sequences of full-length FOSP-1A and partial FOSP-1B cDNAs revealed a high degree of similarity at the 5' end. We next isolated FOSP-1A and FOSP-1B genomic clones. Dot-plot comparison of their URS showed both similarities and differences. Two estrogen-responsive elements (EREs), termed proximal (pERE) and distal (dERE), were identified at -1070/-1082 and -1167/-1179, respectively, in FOSP-IB, but not FOSP-lA, URS. Quantitative electrophoretic mobility shift assay (EMSA) and DNA footprinting with recombinant Xenopus estrogen receptor (xER) expressed in insect Sf9 cells, showed that xER interacted with a higher affinity with dERE than pERE in a hormone-independent manner, and that the two EREs do not act cooperatively. Functional studies involving transient transfection of human MCF-7 cells with a FOSP-1B URS-&CAT construct confirmed that both EREs act as hormone-inducible cis-acting elements. These studies now pave the way for analysis of tissue specificity of estrogen-inducible gene expression in Xenopus liver and oviduct.