2 DISTINCT FORMS OF USF IN THE LYTECHINUS SEA-URCHIN EMBRYO DO NOT PLAY A ROLE IN LPS1 GENE INACTIVATION UPON DISRUPTION OF THE EXTRACELLULAR-MATRIX

Recent studies in our laboratory indicated that the upstream stimulato ry factor (USF) in the sea urchin embryo of Lytechinus acts as a trans criptional repressor for the aboral ectoderm-specific expression of th e LpS1 genes. Disruption of the extracellular matrix (ECM) arrests dev elopment prior to gastrulation and inactivates the LpS1 genes. We want ed to determine whether the inactivation of the LpS1 genes by ECM disr uption may be due to an increase in USF expression. In the course of t he investigation, a second L. variegatus USF cDNA clone (LvUSF2) was i solated and sequenced. The deduced amino acid sequence of LvUSF2 is ne arly identical to LvUSF1 except at the amino end, where they are sharp ly divergent. Like LvUSF1, LvUSF2 has a USF-specific, a basic/helix-lo op-helix, and a leucine zipper domain. Genomic DNA blots indicated tha t the two cDNA clones are derived from one gene, which suggested that the Lytechinus USF1 and USF2 mRNAs, of approximately 6.0 and 4.0 kb, r espectively, are the result of differential RNA splicing. ECM disrupti on in Lytechinus embryos caused a relative drop in USF RNA accumulatio n levels to approximately 60% of control embryos, while LpS1 RNA accum ulation levels dropped to less than 5%. USF protein levels and DNA bin ding activities in ECM-disrupted embryos also dropped to approximately 60% to that of control embryos. A mutation at the USF binding site in an LpS1 promoter-chloramphenicol acetyl transferase (CAT) fusion DNA construct did not cause a relative increase in CAT activity in ECM dis rupted embryos. These results suggest that the induced drop in LpS1 ge ne expression by ECM disruption is not due to an increase in the repre ssive activity of USF. (C) 1996 Wiley-Liss, Inc.