The promoter of the developmentally regulated sea urchin L. variegatus U1 small nuclear RNA gene has been studied using in vitro protein binding assays. This U1 gene is active in early embryogenesis but is turned off by the gastrula stage. The promoter of this gene shares four regions of homology with the early U1 promoter of the sea urchin S. purpuratus. The L. variegatus gene can be expressed after microinjection into S. purpuratus embryos, so these four regions were investigated as possible protein binding sites using DNase I footprinting and mobility shift assays. The locations in the promoters of four Boxes tested is as follows: Box 4, $-$318 to $-$298; Box 3, $-$269 to $-$251; Box 2, $-$167 to $-$149; and Box 1, $-$35 to $-$13. The developmental regulation was addressed by performing these assays using nuclear extracts from blastula stage, when expression is maximal, and extracts from gastrula stage, when transcription is turned off. / Mobility shift assays detected complexes with proteins from blastula nuclei using each of the four boxes tested. Areas of protection from DNase I cleavage were detected using one of the boxes and with a sequence immediately adjacent to Box 2. Two of the sequences tested possibly could be involved in the developmental regulation of this gene. DNase I footprints of Box 4 using blastula nuclear extract show an area of protection from cleavage from $-$307 to $-$296 nucleotides 5$\sp\prime$ of the start of the RNA. No protection is detected using gastrula nuclear extracts. Mobility shift assays also detect a complex in blastula extracts only. The other developmentally regulated complex involves Box 2. A specific complex is detected using blastula nuclear extract, but no complex is detected using gastrula extracts. / Boxes 1 and 3 form complexes detected in mobility shift assays using both blastula and gastrula extracts, and the sequence immediately 5$\sp\prime$ of Box 2 is protected from DNase I cleavage using both blastula and gastrula extracts. This protected sequence is located between $-$181 to $-$168. These data suggest that the sequence from $-$307 to $-$296, which is required for expression of the gene, binds a critical developmentally regulated protein. / Source: Dissertation Abstracts International, Volume: 52-08, Section: B, page: 4041. / Major Professor: William F. Marzluff. / Thesis (Ph.D.)--The Florida State University, 1991.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_76475 |
| Contributors | Stevenson, Karen Anne., Florida State University |
| Source Sets | Florida State University |
| Language | English |
| Detected Language | English |
| Type | Text |
| Format | 138 p. |
| Rights | On campus use only. |
| Relation | Dissertation Abstracts International |
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