Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo.

Abstract

Previous gene-transfer experiments have identified a 2500-nucleotide 5' domain of the Cyllla cytoskeletal actin gene, which contains cis-regulatory sequences that are necessary and sufficient for spatial and temporal control of Cyllla gene expression during embryogenesis. This gene is activated in late cleavage, exclusively in aboral ectoderm cell lineages. In this study, we focus on interactions demonstrated in vitro between sequences of the regulatory domain and proteins present in crude extracts derived from sea urchin embryo nuclei and from unfertilized eggs. Quantitative gel-shift measurements are utilized to estimate minimum numbers of factor molecules per embryo at 24 hr postfertilization, when the Cyllla gene is active, at 7 hr, when it is still silent, and in the unfertilized egg. We also estimate the binding affinity preferences (K,) of the various factors for their respective sites, relative to their affinity for synthetic DNA competitors. At least 14 different specific interactions occur within the regulatory regions, some of which produce multiple DNA-protein complexes. Values of K^ range from ~2 x 10* to ~2 x 10^ for these factors under the conditions applied. With one exception, the minimum factor prevalences that we measured in the 400-cell 24-hr embryo nuclear extracts fell within the range of 2 x 10^ to 2 x 10^ molecules per embryo, i.e., a few hundred to a few thousand molecules per nucleus. Three developmental patterns were observed with respect to factor prevalence: Factors reacting at one site were found in unfertilized egg cytoplasm at about the same level per egg or embryo as in 24-hr embryo nuclei; factors reacting with five other regions of the regulatory domain are not detectable in egg cytoplasm but in 7-hr mid-cleavage-stage embryo, nuclei are already at or close to their concentrations in the 24-hr embryo nuclei; and factors reacting with five additional regions are not detectable in egg cytoplasm and are low in 7-hr embryo nuclei, i.e., ^10% per embryo of the level they attain in 24-hr embryo nuclei. The rise in concentration of factors of the latter class could provide the proximal cause for the temporal activation of the Cyllla gene at the early blastula stage.

The C-terminal Subdomain Makes an Important Contribution to the DNA Binding Activity of the Pax-3 Paired Domain

“…3B). For each derivative, we quantitated the amount of protein-bound and free probe at each oligonucleotide concentration, generated Scatchard plots (data not shown), and used the slopes to derive an estimate of the relative affinities of Pax-3 for each of the target DNA sequences (28). The intensity of the shifted complexes in Fig.…”

DNA-protein binding assays from a single sea urchin egg: a high-sensitivity capillary electrophoresis method.

“…Specific SpP3A2-DNA binding was assayed with purified recombinant protein (9) from a 300 nM stock solution (rSpP3A2), with nuclear extract of blastula-stage sea urchin (Strongylocentrotus purpuratus) embryos (3) (4). In addition to the wild-type (wt) probes we also constructed a probe on which the strong site was destroyed by mutating its sequence.…”

“…This procedure can be used for a variety of purposes, quantitative and qualitative, including studies involving the effects of antibodies on DNA-protein complexes. The experiments we present were carried out with a sea urchin embryo transcription factor, SpP3A2, that had been cloned and extensively characterized in earlier studies (3,4,7). SpP3A2 is the initial member of a small family of transcription factors that now includes the Drosophila erect wing gene product and human nuclear respiratory factor 1 (NRF-1) (8).…”

Rare maternal mRNAs code for regulatory proteins that control lineage-specific gene expression in the sea urchin embryo.

“…USA 87 (1990) -of polyadenylylation of specific sea urchin embryo mRNAs often changes during early development (7), and the experiments shown necessarily were carried out with poly(A)+ rather than total RNA. The P3A proteins were not detected (at least by an assay requiring binding to target-site DNA) in unfertilized egg extracts (4). Therefore, the P3A factor(s) required for the specification process is probably synthesized on the mRNA forms revealed in Fig.…”

“…1; C.H., F.J.C., and E.H.D., unpublished data), and P3A2 was cloned by sequencing a protein purified from mesenchyme blastula-stage nuclear extracts by affinity chromatography over Sepharose bearing the same DNA target site (F.J.C., D. Teplow, C.H., and E.H.D., unpublished data). The P3A target site appears in the regulatory domains of three known genes active in the early embryo of this species, and in in vitro reactions with the regulatory DNA of these genes this site is bound tightly and specifically by proteins present in embryo nuclear extracts (2)(3)(4). These genes are the CyIla cytoskeletal actin gene (5) and the Specl Ca2+-binding-protein gene (6), which are expressed coordinately, exclusively in the aboral ectoderm lineages (7), and the SM50 skeletal matrix protein gene, which is expressed in skeletogenic lineages (8,9).…”