Chicken ovalbumin gene fused to a herpes simplex virus alpha promoter and linked to a thymidine kinase gene is regulated like a viral gene.

Abstract

We are describing a system for the introduction, selection, and expression of eucaryotic genes in higher eucaryotic cells. The carrier consisted of the herpes simplex virus 1 (HSV-1) tk gene covalently linked to an HSV-1 alpha promoter directed away from the tk gene. In this study we fused to the alpha promoter the 5' transcribed noncoding sequences and the coding sequences of the chicken oviduct ovalbumin gene. Cells converted to the TK+ phenotype with this chimeric fragment produced an ovalbumin precursor which was processed and secreted into the extracellular fluid. The ovalbumin gene utilized the HSV-1 alpha promoter and was regulated as a viral gene inasmuch as inversion of the genomic DNA relative to the alpha promoter resulted in no ovalbumin synthesis, and production of ovalbumin was enhanced after superinfection with HSV-1. Synthesis of ovalbumin was not detected when cDNA was linked to the HSV-1 alpha promoter. The carrier system described in this study is suitable for introduction, selection, and expression of eucaryotic genes whose natural promoter is either weak or requires the presence of regulatory elements which may be absent from undifferentiated cells in culture.

Glycosylation of ovalbumin in a heterologous cell: analysis of oligosaccharide chains of the cloned glycoprotein in mouse L cells.

“…Although the exact distribution of high-mannose and hybrid chains is uncertain, the sum total of hybrid oligosaccharide chains is believed to be approximately equal to that of the high-mannose oligosaccharides (6). As (2) have shown that these cells synthesize an ovalbumin precursor that is processed and secreted into the extracellular medium after superinfection of the cells with HSV-1. These cells contain a plasmid (pRB353) with ovalbumin genomic DNA located downstream from and in the same transcriptional orientation as the a-regulated promoter of a protein 4 from HSV-1.…”

“…Because we are interested in determining the influence of the protein structure and cell type on proper-site glycosylation and oligosaccharide chain processing, we have begun to study the expression of cloned glycoproteins in heterologous cells. In this report, we examine the glycosylation of a model glycoprotein, chicken ovalbumin, in a mouse L-cell/herpes simplex virus type 1 (HSV-1) system (2).…”

“…Radiolabeled ovalbumin was prepared essentially as described (2). HSV-1 ts502A305 carries a temperature-sensitive mutation such that at the nonpermissive temperature (39°C), infected cells produce significant amounts of a-gene products.…”

Expression of hepatitis B virus S gene by herpes simplex virus type 1 vectors carrying alpha- and beta-regulated gene chimeras.

“…Available data based on chemical enucleation with actinomycin D or physical enucleation with the aid of cytochalasin B suggest that the inhibition is at least in part at the translational level (6,7,9). Although HSV-1 was shown previously to induce the expression of some host genes (10) and particularly foreign genes (e.g., ovalbumin) placed under control of viral regulatory regions and introduced into cells by transfection (11,12), the induction is selective and transient. A central question is whether the inhibitory machinery of wild-type virus permits sustained expression of a foreign gene introduced into the HSV genome.…”

“…Specifically, chimeric genes constructed by fusion of promoter-regulatory domains of a gene [e.g., the gene specifying infected cell protein (ICP) no. 0, 4, 27, or 22] to the 5'-transcribed noncoding and coding sequences of other genes are regulated as a or 8 genes, respectively (11,(13)(14)(15)(16)19). In these studies we placed the HBV S gene under the control of the a promoter of ICP4 and the , promoter of the viral thymidine kinase (TK) genes.…”