Cloning of the active thymidine kinase gene of herpes simplex virus type 1 in Escherichia coli K-12.

Colbère-Garapin, Florence; Chousterman, S.; Horodniceanu, F; Kourilsky, Philippe; Garapin, Axel-Claude

doi:10.1073/pnas.76.8.3755

Cited by 223 publications

(117 citation statements)

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“…This EcoRI site may be within a sequence modulating transcription (Dierks et al, 1981). Digestion of HSV-1 B a m H I p with EcoRI has been reported to abolish the TKtransforming activity of the fragment (Wigler et al, 1977) although in more recent studies a low level of transforming activity has been reported following EcoRI digestion (Colbere- Garapin et al, 1979). Deletion mutants retaining this site are therefore of obvious value in determining the role of sequences upstream of the TK mRNA-coding region.…”

Section: Superinfection Of Lmtk-cells Biochemically Transformed With mentioning

confidence: 99%

“…The direction of transcription of the HSV-1 gene has been determined . Recently, that portion of the HSV-1 genome coding for the TK gene has been cloned (Wilkie et al, 1979 b;Colbere-Garapin et al, 1979;Enquist et al, 1979) and sequenced (McKnight, 1980;Wagner et al, 1981 ;Preston & McGeoch, 1981). The cloned gene retains biological activity (McKnight & Gavis, 1980;Cordingley & Preston, 1981;Garapin et al, 1981) and is becoming widely used in genetic manipulation of the HSV genome (Mocarski et In this paper we extend our previous report on the production of TK-deletion mutants of…”

Section: Introductionmentioning

confidence: 99%

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Thymidine Kinase Deletion Mutants of Herpes Simplex Virus Type 1

Sanders

Wilkie

Davison

1982

Journal of General Virology

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277 SUMMARYDeletions in the cloned thymidine kinase (TK) gene of herpes simplex virus type 1 (HSV-I), strain 17 syn ÷, were produced by two methods. Removal of a 506 base pair fragment from between the unique SstI and BglII restriction endonuclease sites of pTK1 (HSV-1 BamHI p cloned in pAT153) and subsequent transformation of Escherichia coli resulted in the isolation of 50 deleted plasmids. Sequential digestion of pTK1 with BgllI and nuclease BAL 31 followed by ligation and recleavage with BglII resulted in the isolation of 31 deleted plasmids. Three clones, pTK2, pTK3 and pTK4, obtained following BglII and SstI treatment of pTK 1 were recombined with wild-type (wt) HSV-1 (17) syn ÷ DNA in baby hamster kidney (BHK) cells to produce TKdeletion mutants HSV-1 (17)TK 1301, HSV-1 (17)TK 1302 and HSV-1 (17)TK 1303 respectively. 5-Bromo-2'-deoxyuridine, 5-bromo-2'-deoxycytidine and 9-(2-hydroxyethoxymethyl)guanine were used to reduce the background of TK + virus in heterogeneous recombinant stocks analysed for the presence of TK-recombinants. All recombinant clones isolated produced a small syncytial plaque morphology in BHK cells. The mutants HSV-1 (17) TK 1301 and HSV-I (17) TK 1302 were TK-, failed to produce polypeptides of molecular weights 43 000 and 19 000 found in wt-infected cells and demonstrated one-step growth curves different from wt virus and the TK-mutant HSV-I (17)dPyk -7. Superinfection studies with HSV-1 (17)TK 1301, HSV-1 (17)TK 1302, HSV-I (MDK) and HSV-1 (17) dPyk -7 indicated that all TK-mutants except dPyK -7 produce a tram-acting gene product which can switch on the transforming HSV-1 TK gene.

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Section: Superinfection Of Lmtk-cells Biochemically Transformed With mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Thymidine Kinase Deletion Mutants of Herpes Simplex Virus Type 1

Sanders

Wilkie

Davison

1982

Journal of General Virology

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“…The coding sequence of the HSVtk gene was excised from the pAG0 (pBR322 plasmid carrying the HSVtk gene with its promoter) 45 by double digestion with PvuII and BglII, followed by treatment with Klenow fragment to create blunt ends. The 1.8 kb HSVtk gene fragment was then ligated into pAFP-Luc that had been double digested with HindIII-XbaI to remove the luciferase gene and treated with Klenow fragment to create blunt ends.…”

Section: Plasmid Constructionmentioning

confidence: 99%

Actions of HSVtk and connexin43 gene delivery on gap junctional communication and drug sensitization in hepatocellular carcinoma

et al. 1998

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We have previously demonstrated that transfected hepatotransfected with pFTK alone. To improve these results, cellular carcinoma cells (Hepa1-6) with one copy (pAG0) stable connexin43 transduced Hepa1-6 cells were transand two copies (pYED) of the HSVtk gene, using lipofected with pFTK followed by GCV treatment. In this case, somes, induced cell death of untransfected cells in the the cell growth was markedly inhibited as compared with presence of ganciclovir (GCV). This phenomenon is called parental cells. Furthermore, we have studied the correthe 'bystander effect'. To determine whether an elevated lation between the expression of the HSVtk and the conlevel of connexin43 increases the bystander effect, we nexin43 proteins. Using flow cytometric analysis, scrape have cotransfected Hepa1-6 cells with a plasmid containloading/dye transfer and immunoblotting assay we found ing the HSVtk gene driven by the ␣-fetoprotein promoter that the cells transfected separately by pAG0, pYED, pFTK (pFTK) or pAG0 or pYED and connexin43. The results and pLTR-Cx43 showed an increase of connexin43 proshowed that, after GCV treatment, the percentage of tein. This study indicates that transfecting Hepa1-6 cells growth inhibition was higher (25-30%) in cells cotranswith both connexin43 and HSVtk genes up-regulates confected with HSVtk and connexin43 than in cells transfected nexin43 expression which enhances the bystander effect only with HSVtk gene. The IC 50 of GCV on cells transfected and subsequently tumor cell death. with pFTK/Connexin43 was 17.85-fold lower than cells

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“…1. The pLTR2 plasmid was cut with EcoRI and combined with an EcoRI fragment [excised from a pFG5 (22) plasmid] containing the entire HSV-1 structural gene for TK and the TATA box (23). Transfection of the TK gene EcoRl fragment alone into TK-cells results in a greatly decreased transfection frequency (23); ligation ofthe MTV sequences upstream from this fragment restores this frequency to that of the entire TK gene (unpublished data).…”

Section: Methodsmentioning

confidence: 99%

Glucocorticoid regulation of mouse mammary tumor virus sequences in transgenic mice

1985

Cell Differentiation

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We have introduced a chimeric plasmid, (12)(13)(14)(15)(16).As a first step to dissecting any MTV sequences involved in the mammary gland specific expression, we microinjected into murine embryos a chimeric plasmid that contains the MTV long terminal repeat (LTR) ligated to the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene with the "TATA" box and transcription initiation site and derived mice that contain germ-line copies of this plasmid.The ligation of these MTV sequences to the TK gene confers increased transcription over a basal level when cells transfected with this construct are grown in the presence of dexamethasone (this report; refs. 17 and 18). We find that the TK gene is expressed in the lactating mammary gland as well as in the ovaries of females of one line of mice. In addition, glucocorticoid-inducible transcription of the TK gene is detected in the testes of two independently derived males containing pLTR2TK in their germ line. MATERIALS AND METHODSConstruction of pLTR2TK Plasmid. The pLTR2 plasmid is subcloned from a permuted plasmid, pMTV2, which is cloned from viral DNA from an MTV-infected rat hepatoma cell line (4). For construction of the pLTR2TK plasmid see Fig. 1. The pLTR2 plasmid was cut with EcoRI and combined with an EcoRI fragment [excised from a pFG5 (22) Pronuclear Injections and DNA Transfections. Supercoiled or HindIII-linearized pLTR2TK was microinjected into one pronucleus of fertilized one-cell eggs recovered from the oviducts of either CD-1 (Charles River Breeding Laboratories) or C57BL/6 (The Jackson Laboratory) females (see Results) that had mated with males of the same strain the previous night. Approximately 5 pl of DNA or 1000-5000 copies of the plasmid were microinjected. After overnight cultivation to the two-cell stage in Whitten's medium (24), the eggs were implanted into pseudopregnant CD-1 females.Plasmid pLTR2TK was transfected into LMTK-mouse cells (25) by calcium phosphate precipitation. Twenty-four hours after the addition ofthe DNA precipitate, the cells were selected in HAT medium (0.5 mM hypoxanthine/0.4 ,M aminopterin/16 ,M thymidine), TK-positive colonies were selected (-2-3 weeks after selection) and grown. Cultures derived from single colonies were grown both in the presence and absence of 1 ,uM dexamethasone for 20-24 hr, and RNA was isolated from these.Isolation of RNA and DNA. Mice were anesthetized by intraperitoneal injection of Nembutal, and a lobe of the liver was removed through a small ventral incision. After homogenization in phosphate-buffered saline in a tissue grinder fitted with a Teflon pestle, the tissue was pelleted at 1000 rpm in an International centrifuge and lysed in 10 mM Tris, pH 7.5/100 mM NaCl/0.5% NaDodSO4/1 mM EDTA containing Abbreviations: MTV, mouse mammary tumor virus; LTR, long terminal repeat; TK, thymidine kinase; HSV-1, herpes simplex virus type 1; kb, kilobase(s); GRE, glucocorticoid regulatory element(s).

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Cloning of the active thymidine kinase gene of herpes simplex virus type 1 in Escherichia coli K-12.

Cited by 223 publications

References 24 publications

Thymidine Kinase Deletion Mutants of Herpes Simplex Virus Type 1

Thymidine Kinase Deletion Mutants of Herpes Simplex Virus Type 1

Actions of HSVtk and connexin43 gene delivery on gap junctional communication and drug sensitization in hepatocellular carcinoma

Glucocorticoid regulation of mouse mammary tumor virus sequences in transgenic mice

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