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PubMed1983

Structural analysis of three subunits of laminin from teratocarcinoma-derived parietal endoderm cells.

The structure of the three polypeptide chains of the laminin subunits and the number of glycosylation sites in each polypeptide chain were determined using peptide mapping by high-performance liquid chromatography. Analysis of the [35S]methionine-labeled underglycosylated laminin isolated from tunicamycin (TM)-treated cells revealed that the three subunits of laminin contain unique polypeptide chains. Analysis of [3H]glucosamine-labeled glycosylated laminin subunits showed that they are sialylated and that each subunit has 11-14 glycosylation sites.

Animals↗
PubMed1994

Complexity and organization of DNA-protein interactions in the 5'-regulatory region of an endoderm-specific marker gene in the sea urchin embryo.

This study concerns the organization of sites of specific DNA/protein interaction within the regulatory domain of the Endo16 gene of Strongylocentrotus purpuratus. Earlier work had displayed a complex pattern of expression of this gene during embryogenesis. Endo16 transcripts are confined to the definitive vegetal plate in blastula stage embryos; at gastrula stage this gene is expressed throughout the archenteron, but later only in the midgut. In this work we exploited the exceptional experimental accessibility of the sea urchin embryo, with respect to both functional assays of gene regulatory systems and to characterization of transcription factors, in order to approach a complete description of potential Endo16 regulatory interactions. Accurate expression of an Endo16 fusion gene was obtained with a 2200-nucleotide (nt) upstream fragment of the gene. We present a map locating high specificity target sites for DNA-binding proteins within the 2200-nt Endo16 regulatory domain, and an assessment of the complexity of the set of putative Endo16 transcription factors that we have been able to recover from 24-h (blastula stage) nuclear extract. Protein binding sites were initially mapped by gel shift reactions carried out on nested sets of end-labeled restriction fragments, and then to finer resolution by oligonucleotide gel shift competitions. Thirty-eight sites of high specificity DNA-protein interaction were thus identified. Appropriate oligonucleotides were then used for partial purification of the DNA-binding proteins by affinity chromatography. DNA-binding proteins specific for each target site were identified by molecular weight, using southwestern blotting procedures and two-dimensional gel shift separations, and by directly renaturing and reacting with oligonucleotide probes specific proteins that had been resolved by SDS-PAGE from selected affinity column fractions. A complete series of gel shift cross-competitions amongst the target sites was carried out. We conclude that nine different protein factors are bound at unique sites within the Endo16 regulatory domain. Multiple target sites for five other proteins account for the remaining binding site locations. The target sites appear to be organized in a sequence of clusters, focused on the unique factors. The high complexity of the Endo16 gene regulatory system may be characteristic for genes that are spatially regulated in early embryonic development.

Animals↗
PubMed2003

Differential requirement of EGFR signaling for the expression of defective proventriculus gene in the Drosophila endoderm and ectoderm.

A homeobox gene, defective proventriculus (dve), is expressed in various tissues including the ventral ectoderm and midgut. Here, we show the expression pattern of dve in the ventral ectoderm, in which dve expression is induced by Spitz, a ligand for Drosophila epidermal growth factor receptor (EGFR). In spitz mutants, dve expression is only lost in the ventral ectoderm and overexpression of Spitz induces ectopic dve activation in the ventral ectoderm. Dve expression in the middle midgut depends on Decapentaplegic (Dpp) signaling, while expression of a dominant-negative form of Drosophila EGFR (DER(DN)) also causes a marked decrease in dve expression in the middle midgut. Furthermore, heterozygous mutation of thick veins (tkv), a Dpp receptor, strongly enhances the effect of DER(DN). These results indicate that EGFR signaling is crucial for dve expression in the ventral ectoderm and is required in the middle midgut where it cooperates with Dpp signaling.

Animals↗
PubMed2002

Functional characterization of Ets-binding sites in the sea urchin embryo: three base pair conversions redirect expression from mesoderm to ectoderm and endoderm.

Because of the limited knowledge of target genes for the ets family of transcription factors, it is yet unclear how specificity of biological function among different members is achieved in this class of proteins. In the present study, we compared two Ets-binding sites in two differentially expressed genes of the sea urchin embryo. The first gene examined is the cytoskeletal actin CyIIa, which is transiently expressed in skeletogenic and secondary mesenchyme and in its terminal and permanent phase in the gut. The second one encodes the hatching enzyme gene of Strongylocentrotus purpuratus, and is regulated cell-autonomously and asymmetrically along the maternally determined animal-vegetal axis. The Ets sites within the regulatory regions of these two genes interact and form different binding complexes with proteins present in the nuclei of mesenchyme blastula embryos. We also demonstrated that the DNA binding specificity of the CyIIa Ets-binding site can be converted to the other type of Ets site, as in the hatching enzyme promoter, by changing only three base pairs near the Ets core sequence. Switching of these three base pairs near the central GGA trinucleotide motif characteristic of all Ets-binding targets was also sufficient to redirect expression of a reporter gene construct containing a heterologous basal promoter from mesenchyme to non-mesenchyme cell type in transgenic sea urchin embryos. These observations suggest that binding affinity of ets transcription factors plays an important role in determining cell type-specific gene expression.

Actins↗
PubMed2003

Plant cell cycle: cellularisation of the endoderm needs spätzle.

Early development of the endosperm of flowering plants involves the formation of a syncytium through successive rounds of nuclear division without cell wall synthesis. New data reveal that cellularisation of this syncytium requires the SPATZLE protein and involves the formation of cell walls similar to those of somatic tissues.

Cell Cycle↗