TK Activity Research Articles

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.

High efficiency transformation by direct microinjection of DNA into cultured mammalian cells

Direct microinjection of DNA by glass micropipettes was used to introduced the Herpes simplex virus thymidine kinase gene into cultured mammalian cells. When DNA was delivered directly into the nuclei of LMTK −, a mouse cell line deficient in thymidine kinase activity, 50–100% of the cells expressed TK enzymatic activity. In contrast, no TK activity could be detected when the DNA was injected into the cytoplasm. The number of injected LMTK − cells capable of indefinite growth in a TK + selective medium (that is, transformants) depended on the nature of the plasmid DNA into which the HSV-TK gene was inserted. One cell in 500–1000 cells which received nuclear injections with pBR322/TK DNA gave rise to a viable colony when grown in HAT medium (that is, a TK + selective medium). The transformation frequency increased to one in five injected cells when specific SV40 DNA sequences were also introduced into the HSV-TK plasmid. With the microinjection procedure transformation frequency was relatively insensitive to DNA concentration and did not depend on co-injecting with a carrier DNA. Most of the transformants were stable in nonselective medium as soon as they could be tested.

Mutagenicity testing in mammalian cells: II. Validation of multiple drug-resistance markers having practical application for screening potential mutagens

Chinese hamster ovary (CHO) cell lines heterozygous at both the adenine phosphoribosyltransferase ( aprt) and thymidine kinase ( tk) loci were used for single-step selection of spontaneous and induced mutants resistant to 8-azaadenine (AA r), 6-thioguanine (TG r), ouabain (OUA r), or 5-fluorodeoxyuridine (FUdR r). Mutation data are reported for direct mutagens (EMS, ethyl methane-sulfonate; MNNG, N-methyl- N′-nitro- N-nitrosoguanidine; NQO, 4-nitroquinoline 1-oxide) and promutagens (DMN, dimethylnitrosamine; BP, benzo[ a]-pyrene) activated by rat-liver homogenates. Optimal plating densities were established for AA r, TG r, OUA r and FUdR r. The induced mutant frequencies as a function of relative cell survival after treatment with EMS, DMN or BP were 2–4 d for AA r, 6–8 d for TG r, 1–3 d for FUdR r. The induced mutant frequencies as a function of relative cell survival after treatment with EMS, DMN or BP showed locus-specific differences in sensitivity. Of 61 clonal isolates resistant to AA and assayed for APRT activity, 87% had ⩽ 5% wild-type activity; of 30 TG r clones assayed, 83% had ⩽ 5% wild-type HGPRT activity. Of 42 FUdR r clones assayed, 98% had ⩽ 1% wild-type TK activity. 50 clones selected in medium containing FUdR displayed cross-resistance to 5-bromodeoxyuridine (BUdR) and trifluorothymidine (TFT) and all were sensitive to HAT (hypoxanthine-amethopterin-thymidine) medium. The tk locus showed the largest mutational response as a function of cell survival after mutagen treatment. The rapid expression kinetics for FUdR r and the possibility that the locus detects a broader spectrum of genetic lesions than the other drug-resistance markers are discussed in terms of a sensitive screening assay for detecting potential mutagens.

Effects of arabinofuranosylthymine on Epstein-Barr virus replication

The effect of 1-β- d-arabinofuranosylthymine (AraT) on Epstein-Barr virus (EBV) replication was studied using the producer lymphoblastoid cell line P3HR-1. Virus-specific early and late capsid antigens were induced with a tumor promoter, 12- O-tetradecanoyl-phorbol-13-acetate (TPA) alone, or in combination with sodium butyrate. The expression of VCA was completely inhibited by the analog after chemical induction of these cells, whereas no effect on early antigen production or on the increase of an EBV-specific DNA polymerase activity was observed. The activation of P3HR-1 TK − cells by TPA-sodium butyrate induced high stimulation of [ 3H]thymidine incorporation into the viral DNA, which was entirely inhibited by the analog, as demonstrated by CsCl gradient analysis. The work reported here strongly suggests that AraT is an efficient inhibitor of EBV replication, acting during the period of initiation of viral DNA replication.

Introduction of a viral thymidine kinase gene and the human beta-globin gene into developmentally multipotential mouse teratocarcinoma cells.

Teratocarcinoma (TCC) stem cells provide unique prospects for the introduction of specific genes into mice, by virtue of their dual capacity for propagation in vitro and for normal differentiation in embryos. In this study, we have demonstrated that foreign genes amenable to selection in culture can be transferred into the stem cells and expressed. These cells maintain expression of the gene for long periods during differentiation in tumors in vivo in the absence of selective pressure. The cells also integrate an unlinked nonselectable gene at high frequency. Addition of the cloned herpes simplex virus (HSV) thymidine kinase (tk; ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21) gene to cultures of tk(-)TCC cells yielded tk(+) colonies at a frequency of one colony per 4 mug of plasmid DNA. This transformation efficiency, although appreciably lower than for mouse L tk(-) cells, permits the isolation of many transformants. The HSV provenance of the transformed phenotype was verified by the characteristic electrophoretic mobility of the tk protein and by neutralization of the tk activity with specific antiserum. Moreover, blot hybridization tests revealed at least one intact copy of the viral tk gene integrated into the DNA of transformed cells. When injected into syngeneic mice, the cells formed solid tumors with various differentiating tissues. From blot hybridization comparisons with their cell lines of origin, seven of nine tumors examined had maintained the HSV tk gene without significant loss or rearrangement. Viral tk enzyme activity could also be demonstrated in at least some of the tumors. Cotransfer of the cloned human beta-globin gene along with the unlinked HSV tk gene was successful in 2 of 10 tk(+) transformants. Thus, defined genes can be stably introduced into TCC cells in culture and maintained in vivo in a form in which they are transcribed and translated to produce a functional protein.

Thymidine kinase-deficient mutants of [formula omitted]; relationships between enzyme activity levels and ploidy

Thymidine (TK) and deoxycytidine (dCK) kinase levels, chromosome counts and whole culture protein:DNA ratios have been determined in the TK −plasmodial strains of P. polycephalum —TU84 and TU63—and on several wild type ( wt ) plasmodial and amoebal strains from Wis 1 and Colonia backgrounds. It was found that (a) dCK levels and protein:DNA ratios were similar in all plasmodia, regardless of ploidy or genetic background; (b) TU84 and 63 were haploid; (c) TK specific activity was similar in all wt plasmodia but lower in amoebae; (d) TK activities of mutants and wt were approximately additive in heterokaryons. It was concluded that TK deficiency is due to a defect in a structural gene and that wt strains are TK +/TK + and mutants hemizygous TK −.

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.

Reversible inactivation of autosomal alleles in Chinese hamster cells.

Evidence is presented which suggests that a CHO-derived thymidine kinase (Tk) heterozygote, ts201, can reversibly inactivate the wild type Tk allele, and that this event may result in simultaneous inactivation of a linked allele, galactokinase (Glk). The clone ts201 was isolated as a revertant of a stable Tk- 5-bromodeoxyuridine (BrdU)-resistant mutant of CHO. The reacquired Tk activity differed from that of the wild type with respect to Km and heat resistance, supporting the contention that ts201 was genetically heterozygous (Tk+/-). At frequencies varying from 0.02-0.5, segregants of ts201 could be isolated by cloning in BrdU at 39 degrees. These derivatives were phenotypically Tk- (by enzymology and by autoradiography of 3H-dT labeled cells), but after removal of BrdU, reacquired the Tk+ phenotype at frequencies varying randomly from clone to clone. From mutagenized populations of ts201 two variants were isolated, 71t and 72c, by selection at 39 degrees in 2-deoxygalactose (2-dgal). Resistance to 2-dgal has been correlated with a mutation in the gene for Glk, which is syntenic with that for Tk and evidence is presented suggesting that 71t and 72c are Glk+/-. Cloning efficiencies in doubly selective media and autoradiographic data showed that at 39 degrees a coordination existed between the levels of Tk and Glk, which in 71t was inverse, and in 72c, direct. These data led to the hypothesis that pairs of linked alleles can be inactivated or re-expressed simultaneously. The inactivating effect was temperature-sensitive, since at 33 degrees, the cloning efficiency in BrdU and in 2-dgal was very low.

Characterization of pyrimidine deoxyribonucleoside kinase (thymidine kinase) and thymidylate kinase as a multifunctional enzyme in cells transformed by herpes simplex virus type 1 and in cells infected with mutant strains of herpes simplex virus

Pyrimidine deoxyribonucleoside kinase (thymidine kinase [TK]) was purified from two herpes simplex virus type 1 (HVS-1)-transformed TK-deficient mouse (LMTK-) cell lines and from LMTK- cells infected with HSV-1 mutant viruses coding for variant TK enzymes. These preparations exhibited normal or variant virus-induced thymidylate kinase activities correlating with their relative TK activities. Neither virus-induced activity was detected in LMTK- cells infected with an HSV-1 TK-deficient mutant. These results suggest that HSV-1 thymidylate kinase activity and TK activity are mediated by the same protein.

Acquired B Antigen, Tk Activation and A(1) Destroying Enzyme Activity in a Patient with Septicaemia

During the course of septicaemia, a group A(1) patient developed several RBC antigenic modifications. Acquired B antigen, Tk transformation and a partial disappearance of A(1) receptors associated with an increase of H reactivity were observed.

Characterization of Human TK- Cell Lines Transformed to a TK+ Phenotype by Herpes Simplex Virus Type 2 DNA

Human TK- cells carrying the HSV-2 TK gene as a result of transformation with virus DNA express a TK activity of virus origin and maintain the TK+ phenotype when grown in HAT medium. Under non-selective conditions, however, reversion to a TK- phenotype occurs with a significant frequency characteristic of each transformed line. Once reversion has occurred the TK- phenotype appears to be stable, since only very rare instances of TK- to TK+ reversion have been observed. TK- revertants were susceptible to re-transformation by virus DNA, but no reactivation of a silent virus TK gene could be obtained by superinfecting them with a TK- virus mutant. The data presented are consistent with the hypothesis that acquisition of the TK- phenotype is brought about by loss of the virus sequences coding for TK.

Biological properties of equine herpesvirus type 1 DNA: transfectivity and transforming capacity

DNA extracted from purified virions of equine herpesvirus type 1 (EHV-1) was examined for its transfectivity and transforming ability. The infectivity of the herpesvirus DNA was demonstrated by addition of calcium phosphate-DNA coprecipitates to monolayers of permissive horse cells, with resultant plaque formation. The efficiency of transfection (50 to 100 plaque-forming units/microgram of DNA) was reduced by treatment of the viral DNA with deoxyribonuclease or sonication but not with Pronase or antivirus neutralizing serum. When nonpermissive mouse 3T3 Cells lacking the enzyme thymidine kinase (TK-) were transfected with intact EHV-1 DNA, clones of cells transformed to the TK+ phenotype were isolated in selective HAT medium (hypoxanthine, aminopterin, thymidine), which prevents growth of the TK- parental phenotype. The efficiency of transformation ranged from one to five transformants per microgram of EHV-1 DNA. The TK activity of the biochemically transformed cells was characterized by biochemical, electrophoretic, and immunological techniques. By these criteria, the TK activity was identical to the EHV-1 TK and different from the host wild-type enzyme. In contrast to the parental TK+ 3T3 cells, the EHV-1-transformed TK+ cells were unable to grow in the presence of arabinosylthymine, a drug selectively phosphorylated by herpesvirus TKs. These results indicate that stable transfer of EHV-1 genes into nonpermissive cells can be achieved with purified viral DNA.

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 Ltk − 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.

Novel phenotypes arising from selection of hamster melanoma cells for resistance to BUdR

Pigmented clones of the Syrian hamster melanoma line RPMI 3460 were selected for resistance to high levels of the thymidine analogue 5-bromodeoxyuridine (BUdR). The resistant lines obtained were characterized as to the percentage of thymidine residues in nuclear DNA replaced by BUdR (BUdR substitution), the level of in vitro thymidine kinase (EC 2.7.1.75) (TK) activity the ability to utilize exogenous thymidine for growth, and pigmentation. In contrast to the BUdR resistant cells generally reported in the literature, most of the lines isolated in this study have large amounts of BUdR in their nuclear DNA and/or high levels of TK activity. The ability of the cells to utilize exogenous thymidine was found to correlate well with the level of BUdR substitution. However, there was a definite lack of correlation between the level of in vitro TK activity and the level of BUdR-substitution. Further, the phenotypes of the cells selected in 10 −4 M BUdR appear to be to some extent clonally determined.

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.

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 Hind 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.

Herpes simplex virus gene expression in transformed cells. I. Regulation of the viral thymidine kinase gene in transformed L cells by products of superinfecting virus

In this paper we show that the expression of the herpes simplex virus type 1 (HSV-1) gene for thymidine kinase (tk) in HSV-transformed cells is subject to regulation by two viral products synthesized during productive infection of these cells with a tk- mutant of HSV-1. The cell line used in this study is a derivative of tk-deficient mouse L cells that, after exposure to UV-inactivated HSV-1, had acquired the HSV-1 gene for tk (which we term a resident viral gene) and consequently expressed the tk+ phenotype (LVtk+ cells). Productive infection of these cells with HSV-1(tk-) at appropriate multiplicities caused significant enhancement of the viral tk activity. The results of several experiments allow us to conclude that this enhancement was due to increased synthesis of tk specified by the HSV-1 gene resident in the LVtk+ cells and that a specific protein made early after infection with HSV-1(tk-) mediated the enhancement, probably by increasing the production of mRNA from the viral tk gene resident in the LVtk+ cells. Our data also indicate that another HSV-1(tk-) product acted to turn off tk synthesis. The finding that tk activity continued to increase for a longer time after infection of the LVtk+ cells at 2 PFU/cell than after infection at higher multiplicities suggested the synthesis of a product which inhibited tk synthesis and whose concentration reached critical levels earlier at higher multiplicities of infection. Inhibition of DNA synthesis after infection, a treatment that depresses the synthesis of late viral proteins, prolonged the synthesis of tk in LVtk+ cells infected at either 2 or 5 PFU/cell. Infection of the LVtk+ cells with HSV-2(tk-) resulted in only small increases in tk activity, indicating some type specificity in recognition of viral products that can modify the expression of the HSV-1 tk gene resident in these cells.

The Influence of Changing Crypt Cell-Kinetics on Functional Differentiation in the Small Intestine of the Rat: Nucleotide and Protein Synthesis

In the intestinal epithelium, cell production and cell differentiation take place in the crypts and require both the synthesis of (deoxy)nucleotides for the assembly of DNA + RNA polymers and the synthesis of structural proteins and enzymes. These processes were studied in isolated cell compartments in normal animals and in animals recovering from radiation damage in which the proliferation zone becomes extended to the base of the villus. The correlation of DNA + RNA synthesis and enzymes involved in nucleotide synthesis was investigated. Hypoxanthine phosphoribosyl transferase was measured as an enzyme which is involved in the conversion of nucleotides. The activity of HPRT was found to be twice as high in the villus than in the crypt cells. Thymidine-kinase, in contrast, which is assumed to be specific for proliferating tissues showed a much higher activity in crypt than in villus cells. TK activity increases when cell production increases during regeneration of the epithelium after X-irradiation. However, 5′-nucleoti-dase activity which is found to be localised ultrastructurally in the same place as alkaline phosphatase, interferes with the assay of thymidine kinase. Protein synthesis was investigated by intraperitoneal and intraluminal administration of 3H-la-belled amino acids. The incorporation in crypt and villus cells was analysed by autoradiography. All three of the 3H-labelled amino acids, i.e., L-leucine, L-methionine and L-proline were intensively incorporated in the crypt and the lower half of the villus after intraperitoneal injection. The cells of the upper 20 cell positions beneath the tip of the villus did not show grains. Normal and irradiated animals showed a similar pattern of incorporation. When the labelled amino acids were administered intraluminally, grains could also be observed at the tip of the villi, implying protein synthesis in cells immediately before their extrusion into the lumen. This suggests that protein synthesis at the tip is only possible with amino acids absorbed from the intestinal lumen.

Pyrimidinyl Nicotinic Acid and Cerebrocortical Necrosis

Pyrimidinyl nicotinic acid (PNA) exhibits great structural similarities to amprolium, which is a known thiamine antagonist. Experimental cerebrocortical necrosis (CCN) has been induced by oral administration of amprolium. PNA has been demonstrated in ruminai fluid from animals suffering from CCN. The aim of this investigation was to study whether PNA can act as thiamine antagonist and whether it can give rise to CCN. For this purpose PNA was synthesized and was daily given intravenously to a calf. The dose corresponded to roughly 10 times the content of thiamine in the blood. After three weeks the dose was doubled. During the entire experimental period comprising nine weeks no clinical sign of thiamine deficiency or CCN was noticeable. The values for all recorded blood chemical parameters, with the exception of occasional GOT and PK values, were within the normal limits of variation. Rats were used in a similar experiment with the same aim. PNA was homogeneously added to their feed in quantities equivalent to five and 10 times the thiamine content. The rats were clinically healthy throughout the experimental period comprising eight weeks. No significant difference in TK activity and TPP effect was observed between the experimental groups and the control group.

Thymidine-, uridine- and choline-kinase in rabbit kidney cells infected with herpesvirus hominis, type I and II.

Data are presented about the activity of the thymidine-, uridine- and choline-kinase after infection with 21 strains ofherpesvirus hominis of serotype I or II in rabbit kidney cells. Type I strains increase the activity of the thymidine-kinase 15–20 fold over the controls, whereas the type II strains demonstrate a moderate activity, the level of the enzyme is increased 2–5 fold. One giant cell forming strain exhibits unusual properties, the TK activity decreases in correlation to the controls. The uridine- and choline-kinases induce the respective enzymes in different manner. The strains tested are divided into 5 groups depending upon the activity of the enzymatic activity. The implications of the results are discussed.