Makabe, Kazuhiro W. and Kirchhamer, Carmen V. and Britten, Roy J. and Davidson, Eric H. (1995) Cis-regulatory control of the SM50 gene, an early marker of skeletogenic lineage specification in the sea urchin embryo. Development, 121 (7). pp. 1957-1970. ISSN 0950-1991 http://resolver.caltech.edu/CaltechAUTHORS:20120215-152338740
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The SM50 gene encodes a minor matrix protein of the sea urchin embryo spicule. We carried out a detailed functional analysis of a cis-regulatory region of this gene, extending 440 bp upstream and 120 bp downstream of the transcription start site, that had been shown earlier to confer accurate skeletogenic expression of an injected expression vector. The distal portion of this fragment contains elements controlling amplitude of expression, while the region from −200 to +105 contains spatial control elements that position expression accurately in the skeletogenic lineages of the embryo. A systematic mutagenesis analysis of this region revealed four adjacent regulatory elements, viz two copies of a positively acting sequence (element D) that are positioned just upstream of the transcription start site; an indispensable spatial control element (element C) that is positioned downstream of the start site; and further downstream, a second positively acting sequence (element A). We then constructed a series of synthetic expression constructs. These contained oligonucleotides representing normal and mutated versions of elements D, C, and A, in various combinations. We also changed the promoter of the SM50 gene from a TATA-less to a canonical TATA box form, without any effect on function. Perfect spatial regulation was also produced by a final series of constructs that consisted entirely of heterologous enhancers from the CyIIIa gene, the SV40 early promoter, and synthetic D, C, and A elements. We demonstrate that element C exercises the primary spatial control function of the region we analyzed. We term this a ‘locator’ element. This differs from conventional ‘tissue-specific enhancers’ in that while it is essential for expression, it has no transcriptional activity on its own, and it requires other, separable, positive regulatory elements for activity. In the normal configuration these ancillary positive functions are mediated by elements A and D. Only positively acting control elements were observed in the SM50 regulatory domain throughout this analysis.
|Additional Information:||© 1995 The Company of Biologists Limited. Received 21 February 1995; Accepted 30 March 1995. We are grateful to Dr Ellen Rothenberg for critical review of the manuscript. This research was supported by the ONR Cell Biology Program, Grant N00014-93-1-0379. K. W. M. was supported by the Japan Society for the Promotion of Science and by ONR.|
|Subject Keywords:||Strongylocentrotus purpuratus, SM50 gene, mutagenesis analysis, gene regulation, locator element, skeletogenic lineage|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Tony Diaz|
|Deposited On:||20 Mar 2012 22:56|
|Last Modified:||26 Dec 2012 14:50|
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