Genetic transformation of somatic cells. VI. Transfer of the trait of the rate of loss of transformant phenotype by means of DNA from cells containing the thymidine kinase gene of the herpes simplex virus

Using dot-hybridization with thymidine kinase gene (tk gene) of Herpes simplex virus type 1 (HSV 1) of DNA preparations obtained from isolated metaphase chromosomes and lysate fractions of metaphase cells, which presumably contain smaller particles compared to metaphase chromosomes, it has been shown that the tk gene of HSV 1 is localized in chromosomes of cells of transformant clones unstable in TK+-phenotype. The DNA isolated from the metaphase chromosomes from cells of transformant clones is 1.5- or 2-fold more efficient in transforming TK-Chinese hamster cells than is the total high molecular weight DNA from the same cells. Upon transformation of TK- cells by the high molecular weight DNA from the tk gene of HSV 1-containing clones, varying in the rate of the loss of TK+-phenotypes, the character "rate of the loss of transformant phenotype" is transferred together with the tk gene of HSV 1 in 22% of cases. Cells of rerevertant clones, produced from TK- subclones of transformant clones, display the rate of the loss of transformant phenotype characteristic of cells of parental TK+-clones. A comparison of the results allows a conclusion that DNA sequences, determining the character "rate of the loss of transformant phenotype", are linked tightly with the transforming DNA proper containing the tk gene of HSV 1, but are not localized inside such a DNA.     

Genetic transformation of somatic cells. IX. The loss of the transformant phenotype is accompanied by rearrangements in the plasmid DNA containing the thymidine kinase gene of the herpes virus

As demonstrated by dot-hybridization, the cells of HT-subclones isolated from the cells of transformant clones cultured on a non-selective medium differ significantly in the number of copies of thymidine kinase gene (tk-gene) of Herpes simplex virus (HSV1). Since the cells of transformant clones lose thymidine kinase-positive (TK+) phenotype during cultivation, this data are indicative of high frequence rearrangements in the region of transforming DNA as responsible for the transformant phenotype nonstability. These rearrangements, among other things, induce alterations in the number of copies of tk gene of HSV1. The analysis of cells of subclones isolated on a medium containing 5-bromodeoxyuridine (BrdU) shows that the number of copies of tk gene of HSV1 decreases as compared to the cells of parental clones. The decrease in the number of copies of tk gene of HSV1 in a row of BrdU-resistant subclones is accompanied by simultaneous increase in the number of sequences of pBR325 plasmide DNA to which tk gene of HSV1 is linked covalently in the pST826 plasmide introduced into cells of transformant clones. This evidence implies a most complex nature of transforming DNA rearrangements reducing the number of copies of tk gene of HSV1 due possibly to a genetic correction. The analysis of results permits a hypothesis that instability of cells in transformant phenotype may be determined by the genetic instability of insertion type. The rate of the loss of transformant phenotype depends on the frequency of rearrangements in the transforming DNA locus.     

Genetic transformation of somatic cells. VIII. The effect of the DNA methylation inhibitor 5-azacytidine on the stability of thymidine kinase-negative and -positive phenotypes of transformant clone cells

A study was made of the effect of an DNA methylation inhibitor 5-azacytidine (azaC) on the frequency of reversion to a thymidine kinase-positive (TK+) phenotype in 5-bromodeoxy-uridine (BrdU)-resistant subclones obtained from clones of Chinese hamster cells transformed by thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1). It is shown that in 8 of 15 BrdU-resistant subclones azaC increases 2-1000-fold the frequency of reversion to TK+ phenotype. Variations in the inducibility of reversions to TK+ phenotype indicate that the DNA methylation associated with TK- phenotype affects but differently tk gene of HSV1. Cultivation of TK+ cells of transformant clones in the presence of azaC may lead to stabilization (or decrease in the rate of the loss) of TK+ phenotype, or may not influence the stability of transformant phenotype. The reaction of TK+ cells of transformant clones depends both on genetically determined rate of the loss of TK+ phenotype, and on the structure of transforming DNA introduced to cells. A conclusion is drawn that the TK- phenotype of transformant clone cells arises due to processes which are not associated with methylation of tk gene of HSV1 in spite of the fact that such a methylation may later stabilize significantly the TK- phenotype.     

Measuring herpes simplex virus thymidine kinase reporter gene expression in vitro

The herpes simplex 1 virus thymidine kinase (HSV1-tk) positron emission tomography (PET) reporter gene (PRG) or its mutant HSV1-sr39tk are used to investigate intracellular molecular events in cultured cells and for imaging intracellular molecular events and cell trafficking in living subjects. Two in vitro methods are available to assay gene expression of HSV1-tk or HSV1-sr39tk in cells or tissues. One method determines the level of HSV1-TK or HSV1-sr39TK enzyme activity in cell or tissue lysates by measuring the amount of the radiolabeled substrates that have been phosphorylated by these enzymes in a fixed amount of cell lysate protein after a fixed incubation time. The other method, called the 'cell-uptake assay', takes into account the natural uptake and efflux characteristics of the radiolabeled substrate by specific cells, in addition to the level of HSV1-TK or HSV1-sr39TK activity. Both of these assays can be used to validate molecular models in cultured cells, prior to studying them in living research subjects. Each of these assays can be completed in one day.     

Factors Governing Expression of the Herpes Simplex Virus Gene for Thymidine Kinase in Clonal Derivatives of Transformed Mouse L Cells

The cells used in this study are sublines of a transformed mouse L cell line (designated H2) that carries the herpes simplex virus (HSV) gene for thymidine kinase (tk) as well as other viral genetic information acquired after exposure of the parental Ltk(-) cells to UV-irradiated HSV type 1. These sublines of the H2 cell line were isolated by cloning under nonselective conditions and were shown to express widely different levels of viral tk. Selective media were used to isolate phenotypically tk(-) and tk(+) variants in sequence from one of the clonal derivatives. As previously reported, superinfection of the tk(+) cell lines with tk(-) HSV type 1 resulted in enhancement of tk activity. A new finding was that viral tk activity could be induced by superinfection in at least 30% of cells from the phenotypically tk(-) sublines, indicating that a functional viral tk gene was retained in a significant proportion of the cells. Experiments were designed to test for the presence of regulatory factors that could influence tk expression in the nonsuperinfected sublines of H2. Absence of freely diffusible regulatory factors was indicated by the finding that the fusion of phenotypically tk(-) and tk(+) cells and untransformed cells in appropriate combinations did not affect the levels of tk detected. Moreover, there was no evidence for the presence in phenotypically tk(+) transformed cells of HSV-specific regulatory factors that could influence expression of tk from a superinfecting viral genome. Phenotypically tk(+) sublines of H2 were found to differ from the phenotypically tk(-) sublines and from untransformed cells in that the tk(+) cells synthesized viral proteins earlier and produced greater yields of infectious HSV progeny after superinfection with wild-type tk(+) virus. We can conclude that the absence of tk expression in the tk(-) H2 sublines cannot be accounted for by rearrangements or loss of DNA sequences encoding the enzyme itself or of sequences necessary for induction of the gene by superinfecting HSV. Moreover, it appears that the expression of tk in the tk(+) H2 sublines correlates with the presence of some factor that can enhance (or the absence of some factor that can depress) HSV replication and gene expression.     

Mapping of the herpes simplex virus DNA sequences present in herpes simplex virus type-1 thymidine kinase-transformed cells

Analyses of the herpes simplex virus (HSV) DNA sequences which are present in three HSV thymidine kinase-transformed (HSVtk+) mouse cell lines have revealed that these cells contain relatively large and variable portions of the viral genome. Two of these cell lines do not contain the viral DNA sequences known to encode the early viral genes normally responsible for regulating tk gene expression during lytic HSV infections. This finding suggests that cell-associated viral tk gene expression may be regulated by cellular rather than viral control mechanisms. In addition, we have compared the viral DNA sequences present in one unstable HSVtk+ cell line to those present in tk- revertant and tk+ rerevertant cell lines sequentially derived from it. Our results have shown that within the limits of sensitivity of our mapping approach, these three related cell lines contain the same set of viral DNA sequences. Thus, gross changes in viral DNA content do not appear to be responsible for the different tk phenotypes of these cells.     

Development and characterization of a mouse cell line expressing the human V2 vasopressin receptor gene

Human genomic DNA and the HSV tk gene were cotransfected into mouse Ltk- cells and assayed for the acquisition of a Gs-coupled receptor to obtain cell lines expressing human receptors that are so far unavailable. The transfected cells were distributed into 96-well microtitration plates at a density such that after HAT (100 microM hypoxanthine, 1 microM aminopterin, and 10 microM thymidine) selection each well contained, on the average, two to three tk+ cell clones. After replication, half of them were tested for expression of a new phenotype: an adenylyl cyclase stimulatory receptor not normally expressed in the Ltk- recipient cell. The screen yielded a positive result on testing cells arising from the third transfection, the newly expressed receptor is that for arginine vasopressin, commonly referred to as type 2 or V2. DNA from primary transformants (HTB-1 cells) served to obtain secondary transformants by the same technique (HTB-2 cells). Pharmacological properties confirmed that this new receptor, which stimulates adenylyl cyclase activity 7- to 10-fold, is the human V2 receptor and not the activated homologous murine gene. The new cell line provides a permanent accessible source to study the human receptor, by-passing the need for human kidneys. The V2 receptor was susceptible to homologous down-regulation in the HTB-2 cell, but no down-regulation of the cell authentic prostaglandin E1 receptor was observed. The vasopressin receptor did not modify phospholipase-C activity in these cells as expected from V2 receptors. Thus, we successfully applied genomic DNA-mediated gene transfer and were able to develop a cell line expressing a Gs-coupled human receptor of low abundance and poor accessibility.     

Host-specificities of papillomavirus, Moloney murine sarcoma virus and simian virus 40 enhancer sequences.

The bovine papillomavirus (BPV-1), Moloney murine sarcoma virus (MoMuSV) and simian virus 40(SV40) genomes have been shown to contain sequences termed 'enhancers' which activate the expression of linked genes. DNA fragments containing these three enhancers have been inserted into recombinant plasmids upstream from the herpes simplex virus thymidine kinase (tk) gene, and their effect on tk expression monitored. Two types of assay have been used. Firstly, the ability of recombinant plasmids to transform TK- recipient cells to a TK+ phenotype was measured. Secondly, the amount of tk-specific RNA and TK enzyme activity transiently expressed after DNA transfection was determined. Both types of assay gave similar results. The enhancers increased tk gene expression by regulating the amount of full length tk mRNA present shortly after transfection independent of gene copy number. Furthermore, marked species specificity in the relative efficiencies of different enhancers was observed, including that of the BPV-1 enhancer for the first time. The MoMuSV enhancer showed preference for murine fibroblasts, while the papillomavirus enhancer showed a marked preference for bovine cells. In contrast, the SV40 enhancer gave the same relative increase in tk gene expression in the murine, rat, bovine and human cells tested.     

Expression of the herpes thymidine kinase gene in Xenopus laevis oocytes: an assay for the study of deletion mutants constructed in vitro.

When Xenopus laevis oocyte nuclei are injected with a recombinant plasmid containing the Herpes Simplex Virus (HSV) thymidine kinase (tk) gene, a 100-fold increase in tk enzymatic activity is observed. Three lines of evidence show that this increase in tk activity is a result of the expression of the HSV tk gene. First, the enzymatic activity is selectively inactivated by the IgG fraction of antiserum raised against HSV tk protein. Second, a polypeptide that comigrates with authentic HSV tk on polyacrylamide gels is synthesized uniquely by oocytes injected with the HSV tk gene. Third, the induced tk activity found in injected oocytes is capable of phosphorylating deoxycytidine, a substrate that is utilized by HSV tk but not by cellular tk. We have used these observations to establish an assay for examining the activity of mutated variants of the HSV tk gene. Two sets of deletion mutants of the tk gene were constructed in vitro. In one set varying amounts of 5' flanking and intragenic sequences are deleted. The other set is deleted at the 3' end of the gene. By testing the activity of each mutant in the oocyte injection assay we have delimited functional boundaries corresponding to the 5' and 3' termini of the HSV tk gene.     

Identification of the herpes simplex virus DNA sequences present in six herpes simplex virus thymidine kinase-transformed mouse cell lines

We have used a novel filter hybridization approach to detect and map the herpes simplex virus (HSV) DNA sequences which are present in four HSV thymidine kinase (HSVtk+)-transformed cell lines which were derived by exposure of thymidine kinase negative (tk-) mouse cells to UV light-irradiated HSV type 2 (HSV-2). In addition, we have mapped the HSV-1 DNA sequences which are present in two HSV-1tk+-transformed cell lines produced by transfection of tk- mouse cells with sheared HSV-1 DNA. The results of these studies can be summarized as follows. (i) The only HSV DNA sequences which were common to all HSVtk+-transformed cells were those located between map coordinates 0.28 and 0.32. Thus, this region contains all of the viral DNA sequences which are necessary for the expression of HSV-mediated tk transformation. (ii) Many of the cell lines also contained variable amounts of non-tk gene viral DNA sequences located between map coordinates 0.11 to 0.57 and 0.82 to 1.00, suggesting that incorporation of the viral DNA sequences located between these map coordinates is a relatively random event. (iii) The viral DNA sequences located between map coordinates 0 to 0.11 and 0.57 to 0.82 were uniformly absent from all of the HSVtk+ cell lines tested, suggesting that there is a strong negative selective pressure against incorporation of these viral DNA sequences.     

Carrier DNA affects the integration of HSV-1 TK gene in the genome of L929TK- cells.

L929TK- cells were cotransfected with DNA mixtures containing tk gene of HSV-1, plasmids carrying LTR of MoMLV or RSV and carrier DNA of salmon sperm or chromosomal DNA of recipient cells. Selection of TK+ transformants was conducted in DMEM supplemented with HAT. Plasmids carrying LTR sequences of MoMLV or RSV retroviruses showed enhancing effect on the frequency of TK+ transformation. Southern blot analysis of chromosomal DNA of TK+ transformants demonstrated in clones deriving from cotransfections of tk gene and carrier DNA of L929TK- cells multiple copies of tk gene integrated into several genomic sites of host. Single copies of tk gene integrated into different sites of host genome occurred in chromosomal DNA of TK+ clones deriving from cotransfections of tk gene and carrier DNA of salmon sperm. Cells cotransfected with tk gene and plasmids carrying LTR sequences of MoMLV or RSV formed three dimensional colonies in semisolid agar medium. No effect of carrier DNA on the morphology of TK+ transformant clones was noticed.     

Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells.

Treatment of Ltk^?, mouse L cells deficient in thymidine kinase (tk), with Bam I restriction endonuclease cleaved DNA from herpes simplex virus-1 (HSV-1) produced tk⁺ clones with a frequency of 10^?6/2 μg of HSV-1 DNA. Untreated cells or cells treated with Eco RI restriction endonuclease fragments produced no tk+ clones under the same conditions. The thymidine kinase activities of four independently derived clones were characterized by biochemical and serological techniques. By these criteria, the tk activities were found to be identical to HSV-1 tk and different from host wildtype tk. The tk⁺ phenotype was stable over several hundred cell generations, although the rate of reversion to the tk^? phenotype, as judged by cloning efficiency in the presence of bromodeoxyuridine, was high (1–5 × 10^?3). HSV-1 DNA Bam restriction fragments were separated by gel electrophoresis, and virtually all activity, as assayed by transfection, was found to reside in a 3.4 kb fragment. Transformation efficiency with the isolated fragment is 20 fold higher per gene equivalent than with the unfractionated total Bam digest. These results prove the usefulness of transfection assays as a means for the bioassay and isolation of restriction fragments carrying specific genetic information. Cells expressing HSV-1 tk may also provide a useful model system for the detailed analysis of eucaryotic and viral gene regulation.     

Studying DNA mutations in human cells with the use of an integrated HSV thymidine kinase target gene

A shuttle vector carrying the origin of SV40 replication, the thymidine kinase (tk) gene of herpes simplex virus and the E. coli xanthine guanine phosphoribosyl transferase (gpt) gene has been introduced into human TK- cells. A transformed cell line containing only one stably integrated copy of the shuttle vector was used to study mutations in the introduced tk gene at the molecular level. Without selection for gpt expression, spontaneous TK- mutants arose at a frequency of approximately 10(-4)/generation, and were caused by deletion of plasmid sequences. However, when selection for expression of the gpt gene was applied, the background level of mutations at the tk gene was below 4.10(-6). From this cell line, TK- mutants were obtained after treatment with N-ethyl-N-nitrosourea (ENU). COS fusion appeared to be an efficient method for rescue and amplification of the integrated shuttle vector from the human chromosome. After further amplification and analysis in E. coli, rescued tk genes were easily identified and were shown to be physically unaltered by the rescue procedure. In contrast to rescued tk genes from TK+ cells, those obtained from the ENU-induced TK- mutants were unable to complement thymidine kinase-negative E. coli cells. Two such tk mutations were mapped in E. coli by marker rescue analysis. A GC----AT transition was the cause of both mutations. We show here that plasmid rescue by COS fusion is a reliable system for studying gene mutations in human cells, since no sequence changes occurred in rescued DNA except for the 2 ENU-induced sequence changes.     

Biochemical transformation of mouse cells by fragments of herpes simplex virus DNA.

Mouse L cells lacking the enzyme thymidine kinase (LMTK-) have been converted to a TK+ phenotype by infection with fragmented HSV2 strain 333 DNA. The DNA fragments used were either unique, produced by cleavage with the restriction endonucleases Eco RI and Hild III, or randomly produced by mechanical shearing. Survival in HAT medium was used initially to establish the TK+ phenotype; clones possessing the ability to grow in selective medium were picked on the basis of differing morphology and growth rates. Cytosol extracts of these clones possessed virus-specified TK activity identical to that present in cells lytically infected with HSV2, as indicated by thermolability and mobility on polyacrylamide gel electrophoresis. The transformed cells also exhibit HSV-specific immunofluorescence. Based on these transformation studies, it is possible to assign a map location to the TK gene on the HSV genome.