Comparison of several promoters and polyadenylation signals for use in heterologous gene expression in culturedDrosophilacells

Abstract

We have directly compared the ability of four promoters and three polyadenylation (poly(A)) signals to direct heterologous gene expression in stably transfected Drosophila melanogaster S2 cells. We compared two constitutive Drosophila promoters, the actin 5C distal promoter and the alpha 1-tubulin promoter, with the tightly regulated Drosophila metallothionein (Mtn) promoter and the Bombyx mori fibroin promoter. We find that the actin 5C and induced Mtn promoters generate comparable high levels of RNA and protein in this system. The alpha 1-tubulin promoter generates about four-fold lower levels, and the fibroin promoter shows no detectable activity in S2 cells. Interestingly, genes expressed from the constitutive actin 5C and alpha 1-tubulin promoters are consistently present at three- to four-fold lower copy numbers than genes expressed from the inducible Mtn promoter or the inactive fibroin promoter. Poly(A) signals of both mammalian (SV40) and Drosophila (Mtn) origin efficiently directed stable RNA synthesis in S2 cells, and, as in mammalian cells, the SV40 late poly(A) signal was more efficient than the SV40 early poly(A) signal. Thus the process of polyadenylation appears to be conserved between mammalian and Drosophila cells.

Thermosensitive Splicing of a Clock Gene and Seasonal Adaptation

“…Expression of the luc-dper/3´UTR hybrid gene was placed under the control of the constitutive actin 5C promoter (pAct) ( Fig. 2A) (Angelichio et al 1991). To enable the simple introduction of different intron and flanking exon sequences, this parent wild-type vector (termed dmpi8) also includes two engineered Xho1 and Kpn1 restriction sites placed 10 bp upstream and downstream from the dmpi8 5´and 3´splice sites (ss), respectively.…”

The Fused Protein Kinase Regulates Hedgehog-stimulated Transcriptional Activation in Drosophila Schneider 2 Cells

“…To construct expression plasmids for epitope-tagged proteins, a DNA fragment encoding the Flag (MDYKDDDDKAGVD), HA (MVYPYDVP-DYASLVD), or Myc (MEQKLISEEDLGDPAG) sequence was ligated to the 5Ј end of the coding region of Ci, Fu, Su(fu), or Cos2 cDNA, and inserted into the expression vector pAct5C0, which carries the Drosophila actin 5C promoter (40,41). These plasmids were designated as pDA-Flag-Ci, pDA-Flag-Fu, pDA-HA-Su(fu), and pDA-Myc-Cos2.…”

Cloning, in Vitro Expression, and Functional Characterization of a Novel Human CC Chemokine of the Monocyte Chemotactic Protein (MCP) Family (MCP-4) That Binds and Signals through the CC Chemokine Receptor 2B

“…MCP-4 was expressed in a Drosophila cell culture system (17,18). The MCP-4 coding region was amplified by polymerase chain reaction (PCR) using the cDNA clone as substrate and the following pair of primers: 5Ј-TCCCCGCGGCCACCATGAAAGTTTCTGCAGTGCTT-3Ј (SacII site and initiator methionine codon underlined) and 5Ј-GCTCTA-GACTATTAAGTCTTCAGGGTGTGAGCT-3Ј (XbaI site underlined).…”

“…Expression and Purification of Recombinant MCP-4 -To allow functional studies with this novel chemokine, MCP-4 was expressed in Drosophila S2 cells, a system previously shown to correctly recognize and remove human cytokine signal sequences (18,23). Growth medium from a stably transfected Drosophila S2 cell line carrying the expression vector for MCP-4 was analyzed by immunoblot, using an antiserum raised against a synthetic MCP-4 peptide.…”