gpt-gus fusion gene for selection and marker in recombinant poxviruses.

Poxviruses have become a useful eukaryotic expression vector system and have been used to express a variety of viral (especially poxviral), eukaryotic and bacterial proteins and as recombinant vaccines (11). During construction, the identification of recombinant poxviruses is greatly facilitated by co-expression of either a dominant selection gene or a color screening marker. Examples of the former are E. coli guanine phosphoribosyl transferase gene (gpt) (4) and the neomycinresistance gene derived from Tn5 (5), and the E. coli genes lacZ (encoding βgalactosidase) (2,10) and gusA (encoding β-glucuronidase) (1) have been used for color screening. Although the dominant selection offers the advantage of selective amplification of the recombinant virus, the phenotype of recombinant virus plaques is usually similar to the parental wild-type virus. Consequently, a second screening is required. Color screening markers make recombinant viruses produce easily identifiable plaques. However, they do not offer a selective advantage, and the subsequent isolation and purification can be troublesome. In this communication, we report construction of a fusion gene comprised of E. coli gpt and gusA genes, which can offer both dominant selection and color screening. Under the control of a single promoter, the complete cassette can be easily transferred into other vectors by blunt-end ligation. E. coli GPT, which can initiate a salvage pathway for purine synthesis inhibited by mycophenolic acid (MPA), has been widely used for dominant selection of recombinant poxviruses (4,9). The E. coli gusA gene, which had been used effectively as a color reporter in plants (7), invertebrates (8) and mammalian cells (6), has recently been introduced as a color screening marker for isolation of the recombinant vaccinia virus (1). The gusA gene is significantly smaller than lacZ and lacks several of the restriction sites present in the latter, which have often complicated cloning strategies. A further advantage of gusA is that it may be used as a color marker in viruses that already express β-galactosidase (1). The strategy used to construct the gpt-gusA fusion gene is illustrated in Figure 1 with primer sequences shown in Figure 2. The fusion gene (Figure 2C) encodes an intact GPT, an extra amino acid (alanine) as a linker and GUS with the alteration of only the second amino acid (leucine → valine). Initially, the construct was tested by transient assays, which demonstrated GUS activity in the transfected poxvirus-infected cells. Since gusA is the C-terminal half of the fusion gene, this suggested that the whole fusion protein was produced. Therefore, this cassette, containing the gpt-gusA fusion gene regulated by a synthetic early/late promoter (E/L) (1), was isolated by digestion with ScaI and cloned into the ectromelia virus (EV) homolog of the vaccinia virus F3L gene. A recombinant EV was produced