Temporal regulation in development: negative and positive cis regulators dictate the precise timing of expression of a Drosophila chorion gene.

Abstract

We have used germ-line transformation to dissect the cis regulatory elements responsible for the transcriptional control of an internally marked Drosophila chorion gene (s15-P) during development. A 73-base-pair segment of the proximal 5'-flanking DNA contains sequences essential for the tissue-specific expression and the precise "late" temporal regulation of that gene. A substitute s36-1 segment of similar location can provide the tissue-specific function and imparts an early temporal regulation characteristic of gene s36-1. Within the regulatory DNA of sl5-P, at least three adjacent elements are recognizable: an essential operationally positive element (TCACGT) that is shared by s36-1 and other chorion genes, irrespective of temporal specificity; a second positive element that is required for the normal late expression of slS-P; and, farthest upstream, a negative element that represses precocious expression during the early choriogenic stages.In Drosophila melanogaster, the proteinaceous eggshell (chorion), which is a multilayered protective egg covering, is constructed during the final stages of oogenesis (1-3). Crucial to its successful morphogenesis and function (4) is the programmed, sequential synthesis of the various chorion proteins during the 5-to 6-hr choriogenic period (oogenic stages 11-14). The genes encoding these proteins are regulated, being expressed exclusively in the "1000 follicle cells that surround each maturing oocyte during overlapping "early" or "late" periods that are characteristic for each gene (5, 6). For example, transcripts of the very late gene known as sJS-J are detectable in stage 13 and are particularly abundant in stage 14 follicles; although low levels of transient and nontranslated transcripts are seen occasionally during prechoriogenic stages, they are totally absent from stages 11 and 12 (5). The sJS-1 gene is found at chromosomal locus 66D12-15, clustered with three additional chorion genes that also have late but nonidentical temporal specificities (5, 7).We have been investigating the cis-acting elements responsible for the detailed developmental regulation of sl5-1 expression by using in vitro mutagenesis of the 5'-flanking region of the gene, followed by P-element-mediated transformation (8, 9) and assay of in vivo gene expression (10). With the exception of some quantitative regulators, it appears that the cis regulatory elements dictating developmental specificity are found upstream and closely associated with the gene (11). In the present report, we analyze by in vitro mutagenesis the cis regulation of an s5-1 gene, marked with a unique DNA insertion and transformed in isolation of the other three chorion genes that normally flank it. The results identify a 6-base-pair (bp) element as essential for chorion gene expression and in addition reveal that the precise temporal specificity of this gene is under the control of closely linked positive and negative regulatory elements in the proximal 5'-flanking DNA. MATERIALS AND METHODSMarking of the siS-1 Gene an...

Proteins that bind to Drosophila chorion cis-regulatory elements: a new C2H2 zinc finger protein and a C2C2 steroid receptor-like component.

“…However, the effect of this mutation, coupled with the observation that A-189/-69 suppresses late expression ( Fig. 1; Mariani et al 1988) and that A-189/ -69 hinders CF2 binding and complex II formation but not the binding of CF1 or complex I formation (data not shown), all argue that CF2 may act as a late activator . If CF2 binds in vivo to two secondary sites, as it does in the presence of added Zn 2+ in vitro, the phenotype of mutation d may grossly underestimate the importance of CF2.…”

“…For in vivo analysis of s15 promoter mutations, the s15 gene was marked by an insertion of silkmoth DNA and used as a reporter (s15-P; Mariani et al 1988). Ovaries from conditioned transformed females were dissected, and stage 14 follicles were harvested and total RNA extracted, and gel-blot hybridization was performed as in Mariani et al (1988).…”

“…Ovaries from conditioned transformed females were dissected, and stage 14 follicles were harvested and total RNA extracted, and gel-blot hybridization was performed as in Mariani et al (1988). For dot-blot hybridizations, 5~g of total RNA (for detection of both endogenous s15-1 transcripts and transformant-specific s15-P transcripts) from the above samples was incubated in 200 ~1 of a solution of 5 mM Tris-HC1 (pH 8.0), 0.5 mM EDTA (pH 8.0), 3 x SSC, and 7% (wt/vol) formaldehyde for 15 min at 65~ prior to dot-blotting to Biotrans (ICN) nylon membranes with a suction manifold.…”

Genetic and molecular analysis of maternal information in region 32 of Drosophila melanogaster

“…A peculiar feature of the VMP gene family was discovered: a conserved hydrophobic domain, constituted of about 30 amino acids, is present in all the genes (Popodi et al, 1988;Scherer et al, 1988;Gigliotti et al, 1989). Synthesis of the proteinaceous eggshell in D. melanogaster is a system particularly well suited for cis-regulatory signals controlling gene expression in eukaryotes (Komitopoulou et al, 1983;Mariani et al, 1987;Mitsialis et al, 1987;Parks and Spradling, 1987;Orr et al? 1989;Tolias and Kafatos, 1990).…”

Building up the Drosophila eggshell: First of all the eggshell genes must be transcribed

“…The relevant regulatory elements controlling s15 chorion gene expression have been identified in a 73-bp segment (Ϫ118/Ϫ46) of the proximal 5Ј-flanking DNA. Within this region, at least three regulatory elements were found: an essential positive element (TCACGT) shared by s36 chorion gene and other chorion genes, irrespective of temporal specificity; a second positive element required for the normal late expression of s15 chorion gene; a negative element repressing precocious expression during early choriogenic stages (Mariani et al, 1988). Furthermore, a more detailed mutational analysis of the Ϫ189/Ϫ39 s15 promoter region has shown that it encompasses many positive and negative, at least partially degenerate, cisregulatory elements, which are involved in specifying the highly precise expression pattern of the s15 gene during development (Mariani et al, 1996).…”