Expression of a Bacterial Gene in Mammalian Cells

Abstract

Transfection of cultured monkey kidney cells with recombinant DNA constructed with a cloned Escherichia coli gene that codes for xanthine-guanine phosphoribosyltransferase and several different SV40 DNA-based vectors, results in the synthesis of readily measurable quantities of the bacterial enzyme. Moreover, the physiological defect in purine nucleotide synthesis characteristic of human Lesch-Nyhan cells can be overcome by the introduction of the bacterial gene into these cells.

Dissociation of p53-mediated suppression of homologous recombination from G1/S cell cycle checkpoint control

“…An EcoRI linker was inserted into the ClaI site of pBABEpuro (Morgenstern and Land, 1990), the puromycin resistance gene under the control of an early SV40 promoter was taken out as an EcoRI fragment and inserted into pD2 which contains a copy of the XGPRT gene inactivated by a 5' prime deletion mutation of 460 bp (Hamilton and Thacker, 1987). Another XGPRT copy was constructed by amplifying a 930-bp fragment from pSV2gpt (Mulligan and Berg, 1980) via a standard PCR approach. Primers 5'-CGC GGG ATC CCA GCT GTG GAA TGT G-3' and 5'-CGC GGG ATC CAT CAA CAA CAT AGT C-3' annealed to the PvuII site at the beginning of the SV40 promoter and to the EcoRV site within the XGPRT gene, respectively (new BamHI sites are underlined).…”

Activation of a translocated human c-myc gene by an enhancer in the immunoglobulin heavy-chain locus

“…For this purpose, the 2.2 kilobase (kb) XbaI fragment (see Figure 1) containing most of the sequence between the J H cluster and S μ , plus about 330 bp derived from the 5' flanking region of the translocated, truncated c-myc gene, was inserted into the EcoRI site of plasmid pSER 2 . This plasmid was derived from pSV2.gpt 29 (which contains the mycophenolic acid-resistance conferring gene Ecogpt) by deletion from the latter of the SV40 enhancer sequences. As a result, pSER transforms cells to mycophenolic acid resistance (gpt + phenotype) at a much lower frequency than does pSV2.gpt (see Table 1).…”

Sorting and endocytosis of viral glycoproteins in transfected polarized epithelial cells.

“…The ends were then made blunt with Klenow polymerase, and Hindlll linkers were added. The modified cDNA was removed from pUC8PAHA together with the polyadenylation signal by Hindlil-BamHl digestion and ligated to the large Hind llI-BamHl fragment of the expression vector pSV2gpt (24), placing it downstream from the SV40 early promoter. The extent of the deletions in each clone was first assessed by restriction mapping using the Psi 1 site downstream from the initiation codon, and then by sequencing using the dideoxy method (38) after subcloning the relevant fragments into M I3 vectors (46).…”