Drosophila retrotransposon promoter includes an essential sequence at the initiation site and requires a downstream sequence for full activity.

Abstract

We describe a 98-base-pair region (-38 to +60) in the long terminal repeat of the Drosophila gypsy retrotransposon that is sufficient for accurate normal-level transcription. We find that, unlike most RNA polymerase H (pot H) promoters, the gypsy promoter includes downstam sequences that are required for full activity. Also unlike most pol I promoters, the gypsy promoter, which lacks a TATA motif, was found to have an essential sequence at the transcription initiation site, mutation of which abolishes transcription. These three uncommon features of the gypsy promoter may be characteristic of a subset of pol H promoters, exemplified by certain retrotransposons and developmental genes of Drosophila and by Tdt, the mouse terminal deoxynucleotidyltransferase (TdT) gene.Mobile genetic elements are a common feature of eukaryotic genomes. Examples are the gypsy and copia elements of Drosophila, the Ty elements of yeast, and the integrated murine leukemia virus (1-3). Relatively little is known regarding the detailed structure of retrotransposon promoters and enhancers and the nature, effects, and genetic specification of trans-acting factors that control retrotransposon expression. We report here that the gypsy promoter has three unusual features, at least some of which are found in certain other retrotransposons and developmental genes. MATERIALS AND METHODSPlasmids and Constructions. The full-length gypsychloramphenicol acetyltransferase (CAT) construct consists of the first 970 base pairs (bp) of the b94e gypsy ligated to the CAT gene, followed by hsp7O DNA that provides a cleavage and polyadenylylation signal. The CAT-hsp70 segment was derived from a hsp7O-CAT construct described elsewhere (4). The 5'-and 3'-deletion derivatives were made by BAL 31 nuclease digestion of the full-length construct. The A35 construct is identical to the full-length construct, except that bp +14 to +48 were replaced by 16 bp of vector sequence.The mutated initiator construct is identical to the -38 to +60 deletion derivative of the full-length construct, except that the TCAGTT sequence at the start site has been changed to GGATCC by site-directed mutagenesis (5).Transfection. Drosophila Schneider 2 cells (-5 x 107) were transfected in 10 ml with 50 ,ug of gypsy-CAT DNA and 5 tg of heat shock protein hsp82-lacZ DNA (6) by the calcium phosphate method (7). Cells were harvested after 48 hr and extracted for CAT assays with '4C-labeled chloramphenicol (12) report that deletion of the leader region from a gypsy-CAT construct (equivalent to our +249 truncation) decreased transcription 5-fold.The leader in their construct contained a 110-bp segment at +537 not present in the gypsy we used. Whether the apparent difference can be ascribed to this or to some other factor is not clear. (b) CAT assays comparing expression of the -38 to +60, A35, and mutated initiator constructs. ated chloramphenicol was determined by scintillation counting of acetylated and nonacetylated chloramphenicol recovered from TLC plates. Each value was normalized...

Identification and characterization of an early gene in the Lymantria dispar multinucleocapsid nuclear polyhedrosis virus

“…The early transcriptional start sites are at residues 231 and 232, as determined by primer extension mapping (see below). These start sites are within a region (GTCAGTCT) that shows homology to the transcriptional start sites (with the consensus sequence NTCAGTYN) in Drosophila melanogaster retrotransposon and developmental genes and in the developmentally controlled mouse terminal deoxynucleotidyltransferase (Tdt) gene (Table 1; Jarrell & Meselson, 1991). These promoters represent a subset of RNA polymerase II promoters that lack TATA motifs.…”

“…T (Bischoff & Slavicek, 1994), AcMNPV lef-6 (Passarelli & Miller, 1994), mouse Tdt (Smale & Baltimore, 1989), Drosophila gypsy retrotransposon mdg4 (Jarrell & Meselson, 1991), Drosophila developmental genes Antennapedia (Perkins et al, 1988) and engrailed (Soeller et aL, 1988), and with the Drosophila consensus start site sequence (Jarrell & Meselson, 1991).…”

“…This is the first early gene identified in LdMNPV that is expressed at high levels in the presence of cycloheximide. The G22 gene promoter contains a sequence that shows homology to a subset of RNA polymerase II promoters in Drosophila (Jarrell & Meselson, 1991) positioned upstream of certain retrotransposon and developmental genes which lack TATA motifs.…”

The downstream core promoter element, DPE, is conserved fromDrosophila to humans and is recognized by TAFII60 of Drosophila

“…Such downstream promoter sequences have been found in TATA-containing promoters (see, e.g., Lewis and Manley 1985;Nakatani et al 1990; Lee et al 1992;Emanuel and Gilmour 1993;Purnell and Gilmour 1993), as well as in TATA-less promoters (see, e.g., Biggin and Tjian 1988;Perkins et al 1988;Soeller et al 1988;Smale and Baltimore 1989;Jarrell and Meselson 1991;Contursi et al 1995;Minchiotti et al 1997). It appears that many of these downstream promoter sequences are involved in basal transcription, but it is also important to consider that some downstream promoter sequences might be binding sites for sequence-specific transcriptional activators.…”

Both internal and external regulatory elements control expression of the pea Fed-1 gene in transgenic tobacco seedlings.

“…Although there is as yet no direct evidence, this difference suggests that the interna1 cis elements in the two systems may exert their effects by different mechanisms. The 4CL-7 element may b e analogous to the downstream portions of certain animal and vira1 promoters that require specific sequences on both sides of the initiation start site (e.g., Jarrell and Meselson, 1991), whereas the Fed-7 interna1 element(s) might act by affecting processes such as transcriptional elongation or mRNA stability, or perhaps by a mechanism similar to that of the TAR element in the human immunodeficiencyvirus (e.g., Braddock et al, 1991;Gatignol et al, 1991).…”