Transfer of the gene for thymidine kinase to thymidine kinase-deficient human cells by purified herpes simplex viral DNA.

Abstract

Transformation of human cells from a thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75)negative to a thymidine kinase-positive phenotype has been achieved by using pd DNA from herpes simplex virus type 2. The specific activityo tLieDNA was in the range 0.5 to 2.0 transformants per jig and the efficiency of gene transfer was up to 1 transformant per 105 recipient cells. Several transformed lines able to grow continuously in medium selective for thymidine kinase-positive cells have been established. All of these lines express a thymidine kinase activity of viral origin but they differ from each other in the stability of enzyme expression. Subclones derived from a given transformed line inherited the degree of stability of the parental line. The ability to use pure DNA to transfer genes between cultured mammalian cells would be of considerable importance to studies on the genetics of higher organisms. Although the transfer of viral genes that induce oncogenic transformation of cultured cells can be achieved routinely with purified viral DNA (1) and is likely to involve events similar to those expected to occur in the transfer of other types of genes, neither the viral gene products responsible for oncogenic transformation nor their functions are well defined. Consequently, oncogenic transformation is not a suitable model system for studying the characteristics of gene transfer in general. However, it is well established that herpes simplex viruses (HSV) code for a thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.75) (TK) (2) and that this enzyme can be transferred to thymidine kinase-deficient (TK-) cells by transformation with UV-inactivated HSV-1 or HSV-2 (3) or with temperaturesensitive mutants of these viruses (4). The viral TK is well characterized and can be easily distinguished from most mammalian cell TKs (2). Moreover, a number of TK-mammalian cell lines are available as recipients for the viral enzyme. Thus, the transfer of TK would appear to be an excellent model system for studying gene transfer between mammalian cells.Our aim is to learn how viral genes are processed by recipient mammalian cells-i.e., whether and how they become integrated into the recipient cell genome, how their expression is controlled, and what determines the efficiency of these processes. Studies such as these hopefully may lead to DNA-mediated transfer of genes between mammalian cells as well as to a better understanding of some of the processes involved in oncogenic transformation. In this article we describe the transfer of the HSV-2 TK to human TK-cells by infection with purified HSV-2 DNA and the preliminary characterization of the resulting transformed lines. Asg/ml (a-BrdUrd)-selective for TK-cells; in this case, the lines were first either extensively washed free of HAT medium or were cultured for at least one passage in a-MEM. All lines were routinely subcultured twice weekly with a split ratio of 1:6 to 1:8. Periodic checks of cultures for mycoplasma contamination (8) were negative...

Viral oncoapoptosis of human tumor cells

“…28 Human 143B cells are an osteogenic murine sarcoma virustransformed line 29 that was selected in bromodeoxyuridine for the loss of thymidine kinase. 30 Infection of HeLa, HEp-2, and 143B cells with rd D27 led to apoptosis, since PARP and procaspase-3 processing was detected, while wt HSV or mock infection did not ( Figure 1a). As an additional control, rd D3 infection of HEp-2 cells was also performed.…”

Molecularly Cloned Bovine Papillomavirus DNA Transforms Mouse Fibroblasts in Vitro

“…Mouse NIH 3T3 fibroblasts (Jainchill et al, 1969) were maintained in SF12 medium (Flow Laboratories) supplemented with 15% foetal calf serum (FCS; Flow Laboratories). DNAmediated cell transformation was accomplished by a modification (Spandidos & Paul, 1982) of previously published methods (Graham & van der Eb, 1973;Bacchetti & Graham, 1977). Cells were treated with 0-01, 0-1, 1 or 10 ktg of each recombinant DNA.…”

Evidence for the Presence of 5S rRNA in Mammalian Mitochondria

“…To construct plasmid pT7.5S.Dra, we amplified human DNA encoding the 121-nucleotide (nt) 5S rRNA from 143B.TK Ϫ cells (Bacchetti and Graham, 1977) with primers 5S-F (5Ј-GTCTACGGCCATAC-CACCCTG-3Ј) and 5S-R (5Ј-AAAGCCTACAGCACCCGGTAT-3Ј), by the use of Pwo DNA polymerase (Boehringer Mannheim, Indianapolis, IN), and cloned this DNA into SmaI-digested pUC19 (plasmid p5S). By the use of primers T7.5S-F (5Ј-taatacgactcactataGTC-TACGGCCATACCACCC-3Ј [T7 promoter in lowercase and 5S RNA sequence in uppercase]) and 5S.Dra-R (5Ј-tttAAAGCCTA-CAGCACCCGG-3Ј [DraI half-site in lowercase with the DraI site underlined]) and with p5S as template, a 141-bp fragment was amplified and cloned into EcoRV-digested pZErO-2.1 (Invitrogen, San Diego, CA).…”