Identification of Sequences Which Regulate the Expression of Drosophila melanogaster Doc Elements

Abstract

Doc is one of 50 or more mobile DNA elements that have been identified in the fruit fly Drosophila melanogaster . Doc elements lack terminal repeats, instead terminating at the 3Ј end in runs of adenine residues flanked by polyadenylation signals. They differ in size, being variously truncated at the 5Ј end, and are flanked by target site duplications which vary in length from 10 to 14 bp 1 (Schneuwly et al., 1987;Driver et al., 1989). Complete family members are ϳ4.7 kb in length and potentially encode a putative nucleic acid binding protein and a reverse transcriptase (O'Hare et al., 1991). The structure and coding capacity of Doc is typical of LINEs, nomadic DNA sequences conserved in evolution from protozoa to man (Doolittle et al., 1989;Xiong and Eickbush, 1990). LINEs, also known as type II retrotransposons, use self-encoded proteins to reverse transcribe their own mRNA and integrate cDNA copies at new locations in the genome. This hypothesis has been experimentally supported by the analysis of transgenic flies carrying intron-marked Drosophila I factors (Pelisson et al., 1991;Jensen and Heidmann, 1991) and baby hamster kidney cells transfected with mouse LINE-1 elements (Evans and Palmiter, 1991).Mammalian genomes harbor Ͼ10 5 LINEs that belong to a single superfamily (Singer and Skowronski, 1985;Hutchison et al., 1989). By contrast, distinct LINE families, each including 50 -80 members, coexist in D. melanogaster. In addition to Doc, six other families of LINE elements have been described so far in this organism, including the I factor ), F (Di Nocera and Casari, 1987), G (Di Nocera, 1988, and jockey (Priimagi et al., 1988) elements, and type I and type II ribosomal DNA insertions (Jacubczak et al., 1990).LINEs differ markedly from other mobile DNA sequences that also propagate by the retrotranscription of RNA intermediates such as copia-like elements in D. melanogaster ) and the Ty element (Boeke et al., 1985) in Saccharomyces cerevisiae. These elements, also known as viral retrotransposons, resemble the integrated genomes of retroviruses as they carry LTRs. LINEs lack LTRs, and their expression is controlled by promoters which are located within the transcribed region Swergold, 1990;Minchiotti and Di Nocera, 1991;Minakami et al., 1992;Contursi et al., 1993;McLean et al., 1993).By means of transient transfection assays we monitored the expression of constructs in which the reporter CAT gene was under the control of various Doc DNA segments in Drosophila Schneider II (S2) cells. We show that distinct cis-acting DNA elements, clustered in a ϳ50-bp long DNA region located at the 5Ј end of unit-length Doc copies, cooperate to control RNA initiation. In addition, we found that sequences located ϳ200 bp downstream from the 5Ј end inhibit the expression of the reporter CAT gene in a position-and orientation-dependent manner. The inhibition appears to be due to reduced translation rather than to impaired synthesis of CAT mRNA. clones suN, 6N, and 11N, in which the 5Ј end regions of the elements su(f) S2 , Doc6...

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.…”

A Novel Intragenic Sequence Enhances Initiator-dependent Transcription in Human Embryonic Kidney 293 Cells

“…2). Functional interactions between DPE and Inrs located at a specific distance are widely documented (12)(13)(14)(15)(16). The finding that DE1 mimics DPE in the activation of the same Inr is novel, and adds knowledge on the range of core promoter elements interacting with the pol II transcriptional machinery.…”

“…This sequence, conserved ϳ30 bp downstream from the RNA start site in a variety of Drosophila TATA-less promoters, greatly enhances the activity of upstream Inr modules (12)(13)(14)(15)(16)(17). DPE interacts with specific components of the Drosophila TFIID complex (dTAFII40 and dTAFII60; see Ref.…”

Multiple downstream promoter modules regulate the transcription of the Drosophila melanogaster I, doc and F elements

“…Previous studies showed that $40 bp located at the 5 H ends of complete Drosophila I, F and Doc LINE elements contain all the information necessary to direct basal transcription (Minchiotti & Di Nocera, 1991;Contursi et al, 1993Contursi et al, , 1995Mc Lean et al, 1993). These DNA intervals can be viewed primarily as two regions, A and B ( Figure 1A).…”

“…LINEs are mobile DNA sequences conserved in evolution from protozoa to man (Singer & Skowronski, 1985; which use self-encoded proteins to reverse transcribe their own mRNA and integrate cDNA copies at new genomic locations (Pelisson et al, 1991). LINEs lack long terminal repeats (LTRs), and their transcription is regulated by cis-acting signals located at and downstream from the RNA start site(s) Mizrokhi & Mazo, 1990;Swergold, 1990;Minchiotti & Di Nocera, 1991;Minakami et al, 1992;Becker et al, 1993;Contursi et al, 1993Contursi et al, , 1995McLean et al, 1993). Whereas many eukaryotic species contain a single LINE family, eight distinct LINE families coexist in Drosophila melanogaster: the I factor (Fawcett et al, 1986), F (Di Nocera & Casari, 1987), G (Di Nocera, 1988 jockey (Priimagi et al, 1988), Doc (Driver et al, 1989) and BS (Udomkit et al, 1995) elements, and type I and type II ribosomal DNA insertions (Jacubczak et al, 1990).…”