Two 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 stimulatory factor (USF) in the sea urchin embryo of Lytechinus acts as a transcriptional repressor for the aboral ectoderm-specific expression of the LpS1 genes. Disruption of the extracellular matrix (ECM) arrests development prior to gastrulation and inactivates the LpS1 genes. We wanted to determine whether the inactivation of the LpS1 genes by ECM disruption may be due to an increase in USF expression. In the course of the investigation, a second L, variegatus USF cDNA clone (LvUSF2) was isolated and sequenced. The deduced amino acid sequence of LvUSF2 is nearly identical to LvUSF1 except at the amino end, where they are sharply divergent. Like LvUSF1, LvUSF2 has a USF-specific, a basic/hefixloop-helix, and a leucine zipper domain. Genomic DNA blots indicated that 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, respectively, are the result of differential RNA splicing. ECM disruption in Lytechinus embryos caused a relative drop in USF RNA accumulation levels to approximately 60% of control embryos, while LpS1 RNA accumulation levels dropped to less than 5%. USF protein levels and DNA binding 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 disrupted embryos. These results suggest that the induced drop in LpS1 gene expression by ECM disruption is not due to an increase in the repressive activity of USF.
George
J M
JM
Department of Biology, University of Houston, Texas 77204-5513, USA.
Seid
C A
CA
Lee
H
H
Tomlinson
C R
CR
D000595:Amino Acid Sequence; D000629:Aminopropionitrile / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000235:genetics*; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics; D008969:Molecular Sequence Data; D012313:RNA / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics; D012617:Sea Urchins / Q000737:chemistry / Q000196:embryology*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D051720:Upstream Stimulatory Factors
D002135:Calcium-Binding Proteins; D004268:DNA-Binding Proteins; C099544:LpS1 protein, Lytechinus pictus; D012097:Repressor Proteins; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; D000629:Aminopropionitrile; D012313:RNA; D015500:Chloramphenicol O-Acetyltransferase