We have utilized a recombinant vector system designed to study gene control elements by fusing them to the Escherichia coli galactokinase gene (galK) to examine the effects of gene expression that result from introducing changes into the 5' noncoding leader region of the galK transcription unit. We varied the length of the 5' leader region and found no effect on galK expression, provided that the galK initiation codon remained the first AUG in the transcription unit. Using synthetic linkers, we then inserted specific sequences, each containing a single AUG codon at a defined position within the leader region. We found that the AUG inserts had widely different effects on galK expression and that the sequences surrounding the inserted AUG codons determined the magnitude of these effects. In addition, we placed these upstream inserted AUG codons into each of the three possible translation reading frames so that translation occurring in these frames terminates prior to, within, or downstream of the galK initiation codon, respectively. Single-nucleotide frameshift mutations were also introduced into one of these constructs to shift upstream translation into the other two possible reading frames. Depending upon where upstream translation stopped relative to the galK initiation codon, we observed consistently different effects on galK expression. Our results show that an upstream AUG that interferes with downstream translation initiation exerts its greatest effect when it translates out-of-frame through the downstream initiator into the gene. If translation is stopped upstream of or within the initiator, an unexpectedly high level of expression from the downstream AUG is maintained.

The Drosophila metallothionein promoter (Mtn) was used to obtain efficient, regulated expression of foreign gene products inserted in high copy numbers into Drosophila melanogaster Schneider 2 cells. An expression unit comprised of a reporter gene [Escherichia coli galactokinase (ga/K)] fused to the Mtn promoter was stably introduced into Schneider 2 cells in up to several hundred copies per cell in a single transfection-selection event. This system contrasts dramatically with other eukaryotic systems that permit only a few copies of a gene to be stably inserted in a single transfection-selection event. The transfected Drosophila $2 cell lines expressed high levels of both ga/K mRNA and protein in response to metal induction. Most important, and in contrast to mammalian cells, expression remained fully regulated even at high copy number, with low basal expression maintained in the absence of inducer. This regulated system was used to obtain efficient expression in Drosophila cells of an otherwise lethal or growth-inhibitory gene product, the human H-ras oncogene. The ability to obtain regulated high-level expression of potentially lethal foreign proteins is unique to the Drosophila cell system.

We have established a stable, continuous culture Drosophila Schneider 2 cell line that efficiently expresses a secreted, truncated form of the HIV envelope gp120 protein in a regulated manner. The Drosophila produced recombinant gp120 protein is highly glycosylated, is recognized by gp120-specific monoclonal antibodies, binds to the CD4 receptor and has the ability to inhibit syncytia formation between uninfected CD4+ cells and HIV infected cells. We conclude that this recombinant Drosophila envelope protein is an appropriate mimic of the authentic viral envelope protein. Thus, the Drosophila cell provides a continuous, stable culture system for the efficient expression of secreted forms of complex surface glycoproteins in quantities sufficient for detailed analyses.

A series of plasmids was constructed to study the effect of two enhancers, the simian virus 40 72-base-pair repeat and the Harvey sarcoma virus 73-base-pair repeat, on the mouse ma'J-globin promoter. These plasmids contain the mouse Pm"-globin promoter linked to the Escherichia coli galK gene, thus allowing galactokinase enzyme activity to be used as a measure of promoter function. In CV-1 (primate) cells, it was found that an enhancer is required for optimal promoter activity and that the simian virus 40 (primate) enhancer increases galactokinase fourfold more than the Harvey sarcoma virus (mouse) enhancer. In L (mouse) cells, however, the Harvey sarcoma virus enhancer is 1.3-fold stronger than the simian virus 40 enhancer. These data support the hypothesis that enhancer activity can be species specific. Furthermore, when both enhancers are present on the same plasmid, their effect is additive on the ,-globin promoter whether the plasmid is in CV-1 cells or L cells.The mouse ,B-globin gene serves as a valuable model for the study of gene expression in mammals. We wished to develop expression vectors which would allow us to optimize the expression of cloned mouse 3-globin genes that are transferred into mouse tissue culture cells or into intact animals. Important elements that have been shown to stimulate the expression of certain mammalian genes have recently been discovered. These are cis DNA sequences called enhancers (1,2,(4)(5)(6)(7)(11)(12)(13)(14). As a first step toward obtaining regulated globin gene expression in mice, we examined the effect of two different enhancing sequences, the monkey simian virus 40 (SV40) 72-base-pair (bp) repeat and the mouse Harvey sarcoma virus (HaSV) 73-bp repeat, on the expression of a cloned mouse P'ai-globin promoter in both monkey (CV-1) cells and mouse (L) cells.Specifically, we asked several questions. (i) Studies have been performed with the SV40 enhancer with its natural promoter, the SV40 early promoter (2,4,6,11,13). Does the SV40 enhancer have a similar effect on the mouse pglobin promoter? (ii) The SV40 and the murine Moloney sarcoma enhancer act differently on the SV40 early promoter (11), and the SV40 and polyoma enhancers show cell-specific stimulation of the SV40 and rabbit P-globin promoters (4). Is this cell (species)-specific effect a general one? (iii) Do two different enhancers have an additive effect on promoter activity?To answer these questions, we constructed a series of plasmids containing the mouse jmajglobin promoter linked to the Escherichia coli galactokinase gene (galK). This allows us to measure galactokinase (Gal K) levels as a function of the activity of the P-globin promoter.Some constructs also contain a second assayable E. coli gene, gpt, which is driven by the SV40 early promoter, giving an internal control for transformation frequency. These constructions are based on the system described by Schumperli et al. (16). The SV40 72-bp repeat and the HaSV 73-bp repeat were added singly and in combination to these constructs. The Gal K and gpt...

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.