The plasmid vector used as a vehicle to test the cis-regulatory function of randomly cloned genomic DNA fragments was modified from a previous construct, EpGFP (Arnone et al., 1997; Arnone et al., 1998). The grand-parent of this plasmid was pCMV (Clontech); it has ampicillin resistance. EpGFP contains the region around the start of transcription of the endo16 gene (from -117 to +20).The activity of this element has been described in detail elsewhere (Yuh and Davidson, 1996; Yuh et al., 1996,1998, 2001). In addition to a TATA-containing basal promoter, it contains auxiliary sites for a ubiquitous sea urchin transcription factor which promotes DNA looping and has weak transcriptional enhancement activity(GCF1; Zeller et al., 1995). The vector also includes a sea urchin ribosome binding site from the cyIIa gene (Arnone et al., 1998), positioned at the start of the coding sequence of the GFP reporter. The original EpGFP construct was further modified by the addition of a double-stranded oligonucleotide polylinker inserted between the MluI and XmaI sites in the Multiple Cloning Site (MCS), so that the MCS contains the following restriction enzyme sites: 5'-KpnI-SacI-MluI-EcoRI-SpeI-BglII-SmaI/XmaI-3', situated just upstream of the endo16 basal promoter.
Yuh, C. H., Moore, J. G. and Davidson, E. H. Quantitative functional interrelations within thecis-regulatory system of the S. purpuratus Endo16 gene. Development 122, 4045-4056, 1996.
Yuh, C.-H. and Davidson, E. H. Modular cis-regulatory organization of Endo16, a gut-specific gene of the sea urchin embryo. Development 122, 1069-1082, 1996.
Yuh, C.-H., Bolouri, H. and Davidson, E. H. Genomic cis-regulatory logic: Functional analysis and computational model of a sea urchin gene control system. Science 279, 1896-1902, 1998.
Yuh, C.-H., Bolouri, H. and Davidson, E. H. cis-Regulatory logic in the endo16 gene: Switching from a specification to a differentiation mode of control. Development 128, 617-628, 2001.
Zeller, R. W., Coffman, J. A., Harrington, M. G., Britten, R. J. and Davidson, E. H. SpGCF1, a sea urchin embryo transcription factor, exists as five nested variants encoded by a single mRNA. Dev. Biol. 169, 713-727, 1995.
Arnone, M. and Davidson, E. H. The hardwiring of development: Organization and function of genomic regulatory systems. Development 124, 1851-1864, 1997.
Arnone, M. I., Martin, E. L. and Davidson. E. H. Cis-regulation downstream of cell type specification: A single compact element controls the complex expression of the CyIIa gene in sea urchin embryos. Development 125, 1381-1395, 1998.