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

Calzone, Frank J. and Thézé, Nadine and Thiebaud, Pierre and Hill, Ronald L. and Britten, Roy J. and Davidson, Eric H. (1988) Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo. Genes and Development, 2 (9). pp. 1074-1088. ISSN 0890-9369. doi:10.1101/gad.2.9.1074.

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Previous gene-transfer experiments have identified a 2500-nucleotide 5' domain of the CyIIIa cytoskeletal actin gene, which contains cis-regulatory sequences that are necessary and sufficient for spatial and temporal control of CyIIIa 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 CyIIIa gene is active, at 7 hr, when it is still silent, and in the unfertilized egg. We also estimate the binding affinity preferences (K_r) 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_r range from approximately 2 x 10^4 to approximately 2 x 10^6 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 10^5 to 2 x 10^6 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 CyIIIa gene at the early blastula stage.

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Additional Information:© 1981 Cold Spring Harbor Laboratory Press. The Authors acknowledge that six months after the full-issue publication date, the Article will be distributed under a Creative Commons CC-BY-NC License (Attribution-NonCommercial 4.0 International License, Received April 4, 1988; revised version accepted July 6, 1988. We thank Mr. Patrick Leahy for his expert assistance with egg and embryo preparations, and Drs. Norman Davidson and Ellen Rothenberg for helpful and critical reviews of the manuscript. This research was supported by grants from the Lucille P. Markey Charitable Trust and from the National Institutes of Health (HD-05753).
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Lucille P. Markey Charitable TrustUNSPECIFIED
Subject Keywords:Transcription factors; development; sea urchin
Issue or Number:9
Record Number:CaltechAUTHORS:20160518-080831122
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Official Citation:Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo. F J Calzone, N Thézé, P Thiebaud, R L Hill, R J Britten, and E H Davidson Genes Dev. September 1988 2: 1074-1088; doi:10.1101/gad.2.9.1074
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:67166
Deposited By: Ruth Sustaita
Deposited On:18 May 2016 18:33
Last Modified:11 Nov 2021 00:28

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