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. 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.     

Genetic transformation of somatic cells. VII. The loss of the transformant phenotype is accompanied by both stable and unstable changes in DNA

From 6 clones of Chinese hamster cells varying in the rate of the loss of transformant phenotype and containing a thymidine kinase gene (tk-gene) of Herpes simplex virus type 1 (HSV1), 25 subclones negative in thymidine kinase (TK-) were isolated on a medium with 50 micrograms/ml 5-bromodeoxyuridine (BrdU). A study was made of the frequency of spontaneous reversions to the TK+ phenotype in cell populations of BrdU-resistant subclones, and of the transforming activity (upon transformation of TK- cells of A238 clone to the TK+ phenotype) of DNA preparations from a row BrdU-resistant subclones. In 7 of 11 BrdU-resistant subclones the TK- phenotype is associated with changes reducing significantly the transforming activity of DNA. Some of these alterations are stable and undergo no spontaneous reversion, while the other ones are unstable, being reversed or suppressed at a high frequency. BrdU-resistant subclones produced from clones more stable in transformant phenotype are on the whole more stable in the TK- phenotype than BrdU-resistant subclones from the clones with the high rate of the loss of the TK+ phenotype.     

Genetic transformation of somatic cells. V. Inheritability of the rate of loss of the trait and the stabilization of the transformant phenotype

Subclones were isolated both on selective and nonselective medium from the Chinese hamster cells transformed by thymidine kinase gene (TK-gene) of Herpes simplex virus (HSV-1) and varying in the rate of the loss of transformant phenotype. The study of the stability of thymidine kinase-positive (TK+) phenotype in cell populations the subclones shows that the nonstability and the rate of the loss of transformant phenotype are the characters that are inherited in the cell generations. Durable cultivation on a HAT-selective medium may lead to a complete or partial (expressed as a reduced rate of the loss of the character) stabilization of TK+-phenotype of the cells of transformant clones. The rate of stabilization of TK+-phenotype may differ depending on the structure of transforming DNA introduced into cells of transformant clones.     

Genetic transformation of somatic cells. IV. The rate of loss of transformant phenotype depends on the structure of the transforming DNA and changes when the cells are treated with the tumor promotor 12-o-tetradecanoyl-phorbol-13-acetate

Analysis was made of the phenotype stability of some clones of thymidine kinase deficient (TK-) Chinese hamster cells transformed by thymidine kinase gene (TK-gene) of Herpes simplex virus type (HSV 1). The presence of a fragment of human satellite DNA III in the plasmid DNA carrying the TK-gene of HSV 1 reduced notably the rate of the loss of TK+-phenotype, and the treatment of the cells with a tumour promoter--12-o-tetradecanoyl-phorbol-13-acetate--immediately after transformation destabilizes TK+-phenotype of transformant clones. Removal of the eukaryotic carrier DNA for the plasmid DNA without the TK-gene of HSV 1 destabilizes the clone transformant phenotype. Changes in the structure of the plasmid DNA containing no TK-gene of HSV 1 and introduced into cells simultaneously with TK-gene containing plasmids affects the rate of the loss of TK+-phenotype transformed cells.     

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.     

Genetic transformation of somatic cells. II. An analysis of the status of the plasmid nucleotide sequences in chromosomal DNA and the thymidine kinase activity in transformant clone cells

Chinese hamster A238 TK- -cells were transformed with plasmids (derivatives of pBR325) containing thymidine kinase (TK) gene of Herpes simplex virus type 1 (HSV1). The results of dot- and blot-hybridization indicate the presence of pBR325 sequences in the chromosomal fractions of DNA in the transformant clones. These sequences are probably tandemly arranged, and each cluster contains 25--50 copies. SV40 sequences cloned in pBR325 were introduced into the Chinese hamster cells by co-transformation with TK-gene of HSV1-containing plasmid DNA, and all the co-transformant clones selected for TK+-phenotype were shown by hydridization to contain 3V40 DNA fragments. Isoelectrofocusing in polyacrylamide gel shows that thymidine kinase from TK+-transformant clones is of viral type (isoelectric point 7), in contrast to the cellular enzyme (coded by chromosomal gene) having alkaline isoelectric point (pH 9). The results suggest that the true TK+-transformant cells are selected by the procedure used in this study.     

The transfer and stable integration of the HSV thymidine kinase gene into mouse cells.

Treatment of mutant mouse cells (Ltk-) deficient in thymidine kinase with Bam I restriction endonuclease-cleaved HSV-1 DNA results in the appearance of numerous surviving colonies which stably express thte tk+ phenotype. Through a series of electrophoretic fractionations in concert with transfection assays, we isolated a 3.4 kb fragment which contains the thymidine kinase gene and which alone is competent in the biochemical transformation of Ltk- cells. In this report, we have examined the distribution of tk sequences in the DNA of several transformed clones following stable gene transfer. A series of complementary experiments involving reassociation kinetics in solution and annealings with tk DNA to restriction-cleaved cellular DNA following electrophoresis and transfer to filters allow us to make the following general conclusions concerning the fate of the tk gene in all clones examined: the tk gene is present in all cells at a frequency of one copy per chromosomal complement; the tk gene is stably integrated in the DNA of all transformants; and integration is not site-specific and occurs at different loci in the DNA of all transformants examined. The existence of a single active tk gene in tk+ transformants now facilitates an analysis of the sequence organization of tk- mutant cells and provides a useful model system for studies on the transfer of cellular genes.     

Biochemical transfer of single-copy eucaryotic genes using total cellular DNA as donor.

Previous studies from our laboratories have demonstrated the feasibility of transferring the thymidine kinase (tk) gene from restriction endonuclease-generated fragments of herpes simplex virus (HSV) DNA to cultured mammalian cells. In this study, high molecular weight DNA from cells containing only one copy of the HSV gene coding for tk was successfully used to transform L+K-cells to the tk+ phenotype. The acquired phenotype was demonstrated to be donor-derived by analysis of the electrophoretic mobility of the tk activity, and the presence of HSV DNA sequences in the recipient cells was demonstrated. In companion experiments, we used high molecular weight DNA derived from tissues and cultured cells of a variety of species to transfer tk activity. The tk+ mouse cells transformed with human DNA were shown to express human type tk activity as determined by isoelectric focusing.     

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.     

Chromosomal recombination and breakage associated with instability in mouse centrometric satellite DNA

A mouse L cell line containing the centromeric insertion of herpes thymidine kinase genes (tk) was previously shown to undergo a high frequency of DNA rearrangement at the site of tk insertion. Analysis of TK- revertants had demonstrated that DNA rearrangements were usually associated with DNA deletion and were always mediated by intrachromosomal recombinations. In this study, we further analyzed several TK+ subclones to examine the mode of DNA rearrangements in the absence of negative selection pressure. In two clones, LC2-3F and LC2-3E17, rearrangements were accompanied by DNA amplification and were mediated by intrachromosomal recombination. In subclone LC2-3E17-19, we further detected perturbations in the pattern of centromeric heterochromatization. This was associated with chromosome instability, as evidenced by chromosome breakage at the centromere. The analysis of three other sibling clones, LC2-3, LC2-6 and LC2-15, further suggests that reciprocal recombination events may play a role in such centromeric rearrangements. These results suggest that DNA rearrangements in the centromere may be mediated by a number of different mechanisms, and generally do not affect chromosome stability except when accompanied by changes in the pattern of heterochromatization.     

De novo methylation as major event in the inactivation of transfected herpesvirus thymidine kinase genes in human cells

Spontaneous inactivation of integrated thymidine kinase genes was studied in three human cell lines, one with multiple copies and two with a single copy of a transfected shuttle plasmid containing two selectable genes: the HSV tk gene and the Eco gpt gene. Selection for gpt expression prevented the isolation of TK- mutants which are the result of plasmid loss. Under these conditions TK- clones were isolated with a frequency of 5.10(-6) both with the cell line containing 5 or 6 copies of the tk gene and with one of the two cell lines containing one copy of this gene. This inactivity of the tk gene was associated with de novo methylation as the number of HAT-resistant (TK+) clones strongly increased after growth of the TK- derivatives in the presence of the demethylating agent, 5-azacytidine. Digestion with methylation-sensitive restriction enzymes revealed two different patterns of DNA methylation in the genomic DNA of TK- variants. In the TK- derivatives of the cell line containing multiple copies of the tk gene many HpaII restriction sites in the gene copies were insensitive to digestion. These HpaII sites were, however, not methylated in TK- variants of the cell line containing one copy of the plasmid, and methylated CpGs could be detected only with EcoRI which recognizes the cGAATTCg sequence in the tk promoter region. With the other of the two single-copy TK+ cell lines no TK- mutants were obtained, suggesting that the position of a gene in the genome is an important factor in determining the frequency and the extent of de novo methylation. Additionally, we observed that remethylation is an even more efficient process of gene inactivation as TK+ clones reactivated with 5-azacytidine can become TK- again at a 100-fold higher rate than the original TK+ cell line.     

Mutant of herpes simplex virus type 1 conditionally able to transform thymidine kinaseless L cells to a tk+ phenotype.

After nitrous acid mutagenesis of herpes simplex virus type 1 (HSV-1), a mutant, 1093, was isolated which, during productive infection, induced very low levels of thymidine kinase (tk). The mutant virus was found, after UV irradiation, to be unable to transform L cells lacking tk (Ltk-) to a tk+ phenotype as chararcterized by growth of the cells in a modified HAT-selective medium containing 1.6 X 10(-5) M thymidine. Cells transformed by wild-type virus grew vigorously under the same conditions. The mutant was able to transform Ltk- cells if the medium contained 10(-3) M thymidine. These transformed cells maintained their conditional character and would not grow in low concentrations of thymidine in selective medium. Therefore, this mutant is conditional on the thymidine concentration in the selection medium in its ability to transform Ltk- cells to a tk+ phenotype. The conditionally transformed cells could be supertransformed with wild-type UV-irradiated HSV-1 to a phenotype which would grow in low-thymidine selective medium. The frequency of supertransformation closely approximated the frequency of transformation of Ltk- cells by wild-type virus. Supertransformation at high frequency could not be effected by mutant 1093 or the tk- mutant B2006. These results indicate that the presence of HSV-1 genetic information in HSV-1-transformed cells does not preclude the acquisition by these cells of at least one additional HSV-1 gene, that for tk.     

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.     

Transfer of genes to Chinese hamster ovary cells by DNA-mediated transformation

We have transferred DNa to Chinese hamster ovary (CHO) cells by DNA-mediated transformation. CHO tk- cells were transformed with the clones gene for herpes simplex virus thymidine kinase (HSV-tk) and were found to have a 50-fold lower frequency of transformation than mouse Ltk- cells at the same DNA dosage. By altering the amount of tk gene and carrier DNA present, frequencies of up to 5 x 10(-5) were obtained. CHO HSV-tk+ transformants were very stable, and in several clones the HSV-tk gene copies integrated in higher-molecular-weight DNA. These cells also exhibited cotransformation for unselected markers. CHO lines were also transformed at a frequency of 10(-4) with the bacterial gene Ecogpt in a SV40-pBR322 vector. CHO tk-cells could be transformed at a frequency of 10(-7) with cellular DNA isolated from CHO tk+ cells. CHO cells offer a well-defined genetic system within which to transfer either cloned or whole cellular DNAs.     

Stability of the transformants obtained by phage particle-mediated gene transfer

Recombinant lambda phage DNA, encapsulated in phage particles and coprecipitated with calcium phosphate, efficiently transforms cultured mammalian cells without a requirement for carrier DNA. The present paper analyzes the stability of the transformants obtained by the phage transfer method. lambda phage particles containing recombinant DNA that includes the thymidine kinase (TK) gene of herpes simplex virus type 1 as a selective marker were introduced into Ltk- cells deficient in TK activity, and TK+ transformants were selected in HAT medium. To test the stability of the TK+ phenotype of the transformants, seven individual transformant clones were isolated, cultured in HAT selective medium and then in non-selective medium for various lengths of time. After such culture, transformants were allowed to develop colonies in both selective and non-selective medium. For all seven transformant clones, the numbers of colonies obtained in the two types of medium were almost identical, irrespective of whether or not each transformant clone had been previously cultured for 15 to 50 days in non-selective medium. This result suggests that most transformants obtained by the phage transfer method maintain the TK+ phenotype stably, for at least 50 days, when grown in non-selective medium.     

Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome.

We have constructed a substrate to study homologous recombination between adjacent segments of chromosomal DNA. This substrate, designated lambda tk2 , consists of one completely defective and one partially defective herpes simplex virus thymidine kinase (tk) gene cloned in bacteriophage lambda DNA. The two genes have homologous 984-base-pair sequences and are separated by 3 kilobases of largely vector DNA. When lambda tk2 DNA was transferred into mouse LMtk- cells by the calcium phosphate method, rare TK+ transformants were obtained that contained many (greater than 40) copies of the unrecombined DNA. Tk- revertants, which had lost most of the copies of unrecombined DNA, were isolated from these TK+-transformed lines. Two of these Tk- lines were further studied by analysis of their reversion back to the Tk+ phenotype. They generated ca. 200 Tk+ revertants per 10(8) cells after growth in nonselecting medium for 5 days. All of these Tk+ revertants have an intact tk gene reconstructed by homologous recombination; they also retain various amounts of unrecombined lambda tk2 DNA. Southern blot analysis suggested that at least some of the recombination events involve unequal sister chromatid exchanges. We also tested three agents, mitomycin C, 12-O-tetradecanoyl-phorbol-13-acetate, and mezerein, that are thought to stimulate recombination to determine whether they affect the reversion from Tk- to Tk+. Only mitomycin C increased the number of Tk+ revertants.     

Expression of unselected adenovirus genes in human cells co-transformed with the HSV-1 tk gene and adenovirus 2 DNA

We have introduced adenovirus 2 genes into high molecular weight DNA of permissive human cells by co-transformation of tk- human 143 cells with Ad2 restriction enzyme fragments and a cloned Bam HI fragment that carries the HSV-1 thymidine kinase gene. Tk+ cells were isolated after selection and maintenance in HAT medium. Several co-transformed lines are able to complement the growth of Ad5 dl312 (delta 1.2--3.7) and Ad5 dl434 (delta 2.6--8.7), deletion mutants that lack sequences from the left end of the viral genome. The amount and arrangement of viral sequences in the co-transformed cell lines have been analyzed by restriction endonuclease digestion and filter hybridization. Most of the cell lines contain a single insertion of the HSV-1 tk fragment and a single insert of adenoviral DNA. However, one line (B1) contains at least four different insertions, two of which are present in multiple copies. The adenoviral DNA in all cell lines is composed of sequences from the left end of the genome and extends for varying lengths in different lines. Two cell lines that complement deletion mutants efficiently synthesize both early region 1a and 1b mRNAs. The B1 line synthesizes low levels of 1a mRNA, higher levels of 1b mRNA and a unique mRNA that maps to the right of the 1b gene family. When grown continuously in HAT medium, some cell lines are quite stable while others are fairly unstable. Some tk+ subclones support the growth of viral mutants as well as the parental line while others give reduced levels of complementation. For all tk+ subclones examined, the alteration or reduction in viral gene expression is independent of changes in the pattern of integration of viral DNA.