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. 1988 Oct;85(20):7607–7611. doi: 10.1073/pnas.85.20.7607

An in vivo titration of regulatory factors required for expression of a fusion gene in transgenic sea urchin embryos.

D L Livant 1, A E Cutting 1, R J Britten 1, E H Davidson 1
PMCID: PMC282241  PMID: 3174654

Abstract

We report that endogenous regulatory factors mediating expression of a lineage-specific sea urchin embryo gene can be titrated in vivo by introduction of a sufficient molar excess of DNA-binding sites. Thus we obtain an estimate of the quantity of limiting factor(s) required for developmental activation and transcriptional expression, which can be compared with estimates of factor prevalence obtained by measurements in vitro carried out under equilibrium conditions. A fusion construct in which the bacterial gene for chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol O3-acetyltransferase, EC 2.3.1.28) is controlled by cis-regulatory elements of the CyIIIa cytoskeletal actin gene (CyIIIa-CAT) was introduced in varying numbers of copies into sea urchin eggs. The activity of the CyIIIa-CAT fusion gene in 24-hr blastula-stage embryos was shown to saturate as the number of exogenous genes was increased. The mean number of CyIIIa-CAT fusion genes per nucleus at which half saturation was obtained was 105 +/- 40 (mean +/- SD). This result suggests that equilibrium parameters measured earlier in vitro may apply, at least approximately, within the embryo nuclei.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akhurst R. J., Calzone F. J., Lee J. J., Britten R. J., Davidson E. H. Structure and organization of the CyIII actin gene subfamily of the sea urchin, Strongylocentrotus purpuratus. J Mol Biol. 1987 Mar 20;194(2):193–203. doi: 10.1016/0022-2836(87)90368-8. [DOI] [PubMed] [Google Scholar]
  2. Angerer R. C., Davidson E. H. Molecular indices of cell lineage specification in sea urchin embryos. Science. 1984 Dec 7;226(4679):1153–1160. doi: 10.1126/science.6594757. [DOI] [PubMed] [Google Scholar]
  3. Baker R. E., Gabrielsen O., Hall B. D. Effects of tRNATyr point mutations on the binding of yeast RNA polymerase III transcription factor C. J Biol Chem. 1986 Apr 25;261(12):5275–5282. [PubMed] [Google Scholar]
  4. Cameron R. A., Hough-Evans B. R., Britten R. J., Davidson E. H. Lineage and fate of each blastomere of the eight-cell sea urchin embryo. Genes Dev. 1987 Mar;1(1):75–85. doi: 10.1101/gad.1.1.75. [DOI] [PubMed] [Google Scholar]
  5. Cox K. H., Angerer L. M., Lee J. J., Davidson E. H., Angerer R. C. Cell lineage-specific programs of expression of multiple actin genes during sea urchin embryogenesis. J Mol Biol. 1986 Mar 20;188(2):159–172. doi: 10.1016/0022-2836(86)90301-3. [DOI] [PubMed] [Google Scholar]
  6. Emerson B. M., Lewis C. D., Felsenfeld G. Interaction of specific nuclear factors with the nuclease-hypersensitive region of the chicken adult beta-globin gene: nature of the binding domain. Cell. 1985 May;41(1):21–30. doi: 10.1016/0092-8674(85)90057-1. [DOI] [PubMed] [Google Scholar]
  7. Flytzanis C. N., Britten R. J., Davidson E. H. Ontogenic activation of a fusion gene introduced into sea urchin eggs. Proc Natl Acad Sci U S A. 1987 Jan;84(1):151–155. doi: 10.1073/pnas.84.1.151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Franks R. R., Hough-Evans B. R., Britten R. J., Davidson E. H. Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA. Development. 1988 Feb;102(2):287–299. doi: 10.1242/dev.102.2.287. [DOI] [PubMed] [Google Scholar]
  9. Hough-Evans B. R., Franks R. R., Cameron R. A., Britten R. J., Davidson E. H. Correct cell-type-specific expression of a fusion gene injected into sea urchin eggs. Dev Biol. 1987 Jun;121(2):576–579. doi: 10.1016/0012-1606(87)90193-x. [DOI] [PubMed] [Google Scholar]
  10. Lee J. J., Calzone F. J., Britten R. J., Angerer R. C., Davidson E. H. Activation of sea urchin actin genes during embryogenesis. Measurement of transcript accumulation from five different genes in Strongylocentrotus purpuratus. J Mol Biol. 1986 Mar 20;188(2):173–183. doi: 10.1016/0022-2836(86)90302-5. [DOI] [PubMed] [Google Scholar]
  11. Lee J. J., Shott R. J., Rose S. J., 3rd, Thomas T. L., Britten R. J., Davidson E. H. Sea urchin actin gene subtypes. Gene number, linkage and evolution. J Mol Biol. 1984 Jan 15;172(2):149–176. doi: 10.1016/s0022-2836(84)80035-2. [DOI] [PubMed] [Google Scholar]
  12. McMahon A. P., Flytzanis C. N., Hough-Evans B. R., Katula K. S., Britten R. J., Davidson E. H. Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis. Dev Biol. 1985 Apr;108(2):420–430. doi: 10.1016/0012-1606(85)90045-4. [DOI] [PubMed] [Google Scholar]
  13. McMahon A. P., Flytzanis C. N., Hough-Evans B. R., Katula K. S., Britten R. J., Davidson E. H. Introduction of cloned DNA into sea urchin egg cytoplasm: replication and persistence during embryogenesis. Dev Biol. 1985 Apr;108(2):420–430. doi: 10.1016/0012-1606(85)90045-4. [DOI] [PubMed] [Google Scholar]
  14. Shott R. J., Lee J. J., Britten R. J., Davidson E. H. Differential expression of the actin gene family of Strongylocentrotus purpuratus. Dev Biol. 1984 Feb;101(2):295–306. doi: 10.1016/0012-1606(84)90143-x. [DOI] [PubMed] [Google Scholar]

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