Expression of the avian gag-myc oncogene in Saccharomyces cerevisiae

Abstract

The gag-myc oncogenic sequence of the avian retrovirus MC29 was first inserted in a multicopy expression vector allowing its expression in Saccharomyces cerevisiae. The oncogene transcripts were detected in yeast by Northern blot hybridization and gag-myc proteins were revealed by immunoprecipitation. On solid medium, the average size of gag-myc transformant colonies was smaller than control. In liquid cultures, the gag-myc strains had a doubling time of 4.7 h compared with 3.1 h in the controls. In one of the recipient strains, and after an initial transient period of 5 days, the gag-myc transformants became physiologically indistinguishable from control. In another recipient strain, the slow-growth phenotype is permanent. Plasmid instability is increased in gag-myc transformants. When a single copy of the gag-myc gene was inserted in a yeast chromosome, no phenotype was observed, showing that slow growth is the consequence of plasmid loss.

Cloning of the Multicopy Suppressor Gene SUR7: Evidence for a Functional Relationship between the Yeast Actin-binding Protein Rvs167 and a Putative Membranous Protein

“…In the plasmid pTGORF1, the ORF1 is inserted in the multicopy plasmid pTG887 (generous gift from Transgene, France). It is then under the control of the PGK promoter, which allows its overexpression in S. cerevisiae (Durrens et al, 1990). The 1040 bp fragment, containing ORF1, was obtained from a PCR-amplified fragment of pSP35 using orf1-2 and dis1 as primers (Table 2), and digestion with BamHI and EcoRI enzymes.…”

Characterization of the ORF YBR264c in Saccharomyces cerevisiae, which encodes a new yeast Ypt that is degraded by a proteasome-dependent mechanism

“…The multicopy pMA3a carries the marker leu2-d and is maintained in up to 200 copies per cell (Spalding and Tuite 1989). The multicopy plasmid pTG887 allows gene expression in yeast under control of the PGK promoter (Durrens et al 1990). The 1446-bp DNA fragment containing the 657-bp YBR264c ORF anked by 297 bp at the 5¢ end and 492 bp at the 3¢ end, was ampli®ed by PCR with appropriate primers from the X2180 genome, and inserted into the EcoRV site of the plasmid pKS+ (Stratagene).…”

Functional expression in Saccharomyces cerevisiae of the Lactococcus lactis mleS gene encoding the malolactic enzyme

“…The 1.6 kb PCR fragment was ligated to the multicopy yeast plasmid pTG887 (Transgene S.A. and [9]). This procedure allowed insertion of the 1.6 kb fragment in the proper orientation for transcription of the mleS gene from the PGK promoter of pTG887.…”