USF in the Lytechinus sea urchin embryo may act as a transcriptional repressor in non-aboral ectoderm cells for the cell lineage-specific expression of the LpS1 genes

Christopher A. Seid, Jenny M. George, Amy K. Sater, Mark T. Kozlowski, Haemin Lee, Venkatesh Govindarajan, Ravi K. Ramachandran, Craig R. Tomlinson

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

Expression of the aboral ectoderm-specific LpS1 gene in Lytechinus was used to study lineage-specific transcriptional regulation during sea urchin development. Band shift assays using anti-USF antibody showed that a USF-like protein bound the USF core sequence 5'-CACGTG-3' in the promoter of the LpS1 gene. DNA constructs consisting of a wild-type LpS1 promoter and the same LpS1 promoter with a mutated USF binding site fused to the bacterial chloramphenicol acetyltransferase reporter gene were tested. The mutation in the USF binding site caused an increase in chloramphenicol acetyltransferse activity. We selected a clone that encodes USF, LvUSF, from a gastrula-stage cDNA library representing Lytechinus variegatus. Transactivation experiments, in which LvUSF RNA or a DNA construct consisting of the LvUSF cDNA clone fused to the Lytechinus pictus metallothionein promoter coinjected with the wild-type or mutated LpS1 promoter-chloramphenicol acetyltransferase gene construct, showed that chloramphenicol acetyltransferase activity from the wild-type construct was repressed, while the construct mutated at the USF binding site was active. The same wild-type and mutated LpS1 promoter DNA fragments ligated to the green fluorescent protein reporter gene were used to examine spatial expression. The reporter gene constructs containing the mutated USF binding site were expressed inappropriately in all cell types including the gut and oral ectoderm in gastrula and larva stage embryos, while the wild-type constructs were expressed primarily in the aboral ectoderm. USF was expressed in all cells of the early embryo and in all tissues except the aboral ectoderm in later embryos. The data are consistent with a model depicting Lytechinus USF, as a temporal and spatial regulator by repressing LpS1 gene transcription in non-aboral ectoderm cells.

Original languageEnglish
Pages (from-to)7-19
Number of pages13
JournalJournal of Molecular Biology
Volume264
Issue number1
DOIs
StatePublished - Nov 22 1996
Externally publishedYes

Fingerprint

Lytechinus
Ectoderm
Sea Urchins
Cell Lineage
Embryonic Structures
Chloramphenicol O-Acetyltransferase
Gene Expression
Binding Sites
Reporter Genes
Gastrula
Genes
DNA
Clone Cells
Metallothionein
Chloramphenicol
Green Fluorescent Proteins
Gene Library
Transcriptional Activation
Larva
Anti-Idiotypic Antibodies

All Science Journal Classification (ASJC) codes

  • Virology

Cite this

USF in the Lytechinus sea urchin embryo may act as a transcriptional repressor in non-aboral ectoderm cells for the cell lineage-specific expression of the LpS1 genes. / Seid, Christopher A.; George, Jenny M.; Sater, Amy K.; Kozlowski, Mark T.; Lee, Haemin; Govindarajan, Venkatesh; Ramachandran, Ravi K.; Tomlinson, Craig R.

In: Journal of Molecular Biology, Vol. 264, No. 1, 22.11.1996, p. 7-19.

Research output: Contribution to journalArticle

Seid, Christopher A. ; George, Jenny M. ; Sater, Amy K. ; Kozlowski, Mark T. ; Lee, Haemin ; Govindarajan, Venkatesh ; Ramachandran, Ravi K. ; Tomlinson, Craig R. / USF in the Lytechinus sea urchin embryo may act as a transcriptional repressor in non-aboral ectoderm cells for the cell lineage-specific expression of the LpS1 genes. In: Journal of Molecular Biology. 1996 ; Vol. 264, No. 1. pp. 7-19.
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abstract = "Expression of the aboral ectoderm-specific LpS1 gene in Lytechinus was used to study lineage-specific transcriptional regulation during sea urchin development. Band shift assays using anti-USF antibody showed that a USF-like protein bound the USF core sequence 5'-CACGTG-3' in the promoter of the LpS1 gene. DNA constructs consisting of a wild-type LpS1 promoter and the same LpS1 promoter with a mutated USF binding site fused to the bacterial chloramphenicol acetyltransferase reporter gene were tested. The mutation in the USF binding site caused an increase in chloramphenicol acetyltransferse activity. We selected a clone that encodes USF, LvUSF, from a gastrula-stage cDNA library representing Lytechinus variegatus. Transactivation experiments, in which LvUSF RNA or a DNA construct consisting of the LvUSF cDNA clone fused to the Lytechinus pictus metallothionein promoter coinjected with the wild-type or mutated LpS1 promoter-chloramphenicol acetyltransferase gene construct, showed that chloramphenicol acetyltransferase activity from the wild-type construct was repressed, while the construct mutated at the USF binding site was active. The same wild-type and mutated LpS1 promoter DNA fragments ligated to the green fluorescent protein reporter gene were used to examine spatial expression. The reporter gene constructs containing the mutated USF binding site were expressed inappropriately in all cell types including the gut and oral ectoderm in gastrula and larva stage embryos, while the wild-type constructs were expressed primarily in the aboral ectoderm. USF was expressed in all cells of the early embryo and in all tissues except the aboral ectoderm in later embryos. The data are consistent with a model depicting Lytechinus USF, as a temporal and spatial regulator by repressing LpS1 gene transcription in non-aboral ectoderm cells.",
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AU - George, Jenny M.

AU - Sater, Amy K.

AU - Kozlowski, Mark T.

AU - Lee, Haemin

AU - Govindarajan, Venkatesh

AU - Ramachandran, Ravi K.

AU - Tomlinson, Craig R.

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