Herpes simplex virus thymidine kinase activity of thymidine kinase ...

Proc. NatL Acad. Sci. USA

Vol. 78, No. 1, pp. 582-586, January 1981 Medical Sciences

Herpes simplex virus thymidine kinase activity of thymidine kinase-deficient Escherichia coli K-12 mutant transformed by hybrid plasmids (thymidine kinase-deficient mouse L cells/biochemical transformation/anti-herpes simplex virus type 1 IgG)

SAUL KIT, HARUKI OTSUKA, HAMIDA QAVI, AND MARION HAZEN Division ofBiochemical Virology, Baylor College of Medicine, Houston, Texas 77030

Communicated by Sidney Weinhouse, August 20, 1980

by plasmids which contain the wild-type E. coli tk gene (15). Therefore, E. coli K-12 strain KY895 was transformed by derivatives ofplasmid pBR322 containing the HSV-1 tk gene, and the acquisition ofdThd phosphorylating activity was investigated.

ABSTRACT A hybrid plasmid (pAGO) that contains the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in the form of a 2-kldobase-pair (kbp) Pvu H fragment inserted at the Pvu H site of plasmid pBR322 was used to transform TK- Escherichia coli K-12 strain KY895. pAGO-transformed KY895 cells exhibited partially restored abiity to incorporate [3H]dThd into DNA and an HSV-1-specific TK activity. Bacteria cured of plasmid pAGO (or transformed by plasmid pBR322) did not show enhanced incorporation of [3H]dThd into DNA or HSV-1 TK activity. Plasmid pMH1A was derived from pAGO by deletion of 2067 bp of DNA sequence from pBR322 and 105 bp from the HSV-1 TK gene. E. coli K-12 strain KY895 cells transformed by pMH1A did not show enhanced incorporation of [3H]dThd into bacterial DNA, although pMH1A DNA isolated from transformed KY895 cells, like pAGO DNA, did transform TK- mouse fibroblast [LM(TK-)] cells to the TKV phenotype. The expression of HSV-1 TK activity by E. coli K-12 suggests that intervening sequences may be absent from the coding region of HSV-1 tk or that the coding region ofthe gene possesses short intervening sequences which do not disrupt the translational reading frame.

MATERIALS AND METHODS Cells and DNA. Mouse LM(TK-) cells (16, 17) were grown in Eagle's minimal essential medium supplemented with 10% calf serum and BrdUrd at 25 jig/ml. E. coli K-12 strain 1106 (803 rk mk supE supF), which contains plasmid pAGO, was grown in ML broth with ampicillin (Amp) at 25 ug/ml and tetracycline (Tet) at 10 ,g/ml. E. coli K-12 strain RR1 (F- pro leu thi lacY Strr rk Ink-) (18) was grown in ML broth or in complete M9 medium (supplemented with 1.5% Casamino acids, 0.5% glucose, and 0.2% glycerol) and thiamin at 40 ,ug/ml (19). E. coli K-12 strain KY895 (F- tdk--1 ilv), a derivative of strain W3110, was propagated in ML broth or complete M9 medium and isoleucine and valine, each at 20 ,g/ml. Plasmid pBR322 DNA was purchased from Bethesda Research Laboratories (Rockville, MD). Hybrid plasmid pAGO, a derivative of pBR322, contains a 2-kbp Pvu II fragment of HSV1 DNA inserted at the pBR322 Pvu II site (20). Hybrid plasmid pMHLA was derived from pAGO by deletion of a 2067-bp DNA sequence of pBR322, which extends from EcoRI to Pvu II, and a 105-bp HSV-1 tk sequence extending from Pvu II to EcoRI (11). E. coli K-12 strains transformed by pBR322 and pAGO are AmpT Tetr; those transformed by pMH1A are Ampr Tets. Plasmid DNAs were isolated from transformed bacteria as described (21) and purified by sucrose gradient centrifugation. Recombinant DNA experiments were performed in a P2 facility as recommended in the National Institutes of Health

The spontaneous expression ofchromosomal genes from higher eukaryotes in prokaryotic cells may be limited by incompatibilities at the levels of RNA and protein synthesis and by the existence within eukaryotic genes of intervening sequences which are copied into primary transcripts and subsequently eliminated by processes not yet found in prokaryotes (1-3). The biological activity of genes from lower eukaryotes has been demonstrated in Escherichia coli by using phenotypic selection for functions that complement mutationally inactivated homologous bacterial genes (4-8). In addition, the phenotypic expression in E. coli of a cDNA sequence coding for mouse dihydrofolate reductase has been demonstrated (9). However, the expression of enzyme-encoding genes from higher eukaryotes or their viruses has not been demonstrated. We have recently found that the coding region of the HSV-1 thymidine kinase (TK) gene (tk) is contained within a 1. 1- to 1. 2kilobase-pair (kbp) DNA sequence (10, 11). This was accomplished through the use ofcloned hybrid plasmids containing an active herpes simplex virus type 1 (HSV-1) tk gene. Because HSV-1 TK has a molecular weight of about 80,000 and consists of two subunits of molecular weight 40,000 (12), the findings suggested that the HSV-1 tk gene had either small intervening sequences or none at all. These considerations prompted us to investigate the possible expression ofthe HSV-1 tk gene in aTKdeficient mutant of Escherichia coli K-12 (strain KY895) (13, 14). This strain lacks the ability to incorporate exogenous dThd into its DNA but acquires this capability and TK activity after infection with bacteriophages T2 and T4 (13) or after transformation

guidelines. Transformation Experiments. E. coli K-12 strains RR1 and KY895 were prepared for transformation by CaCl2 treatment (22) and transformed with plasmid DNA (5 ng to 1 ,g) (11). Plasmid DNA 100 IL (5; ng to 1 ,ug) in 10 mM Tris-HCl, pH 7.6/10 mM CaCl2 was added to 200 ,ul of CaCl2-treated cells. The mixture was kept at 4°C for 30 min; then the temperature was raised to 37°C for 5 min, 0.3 ml of ML medium was added, and incubation was continued for 45 min at 37°C with gentle shaking. Samples were plated on trypticase soy agar (TSA) plates supplemented with appropriate antibiotics. LM(TK-) mouse fibroAbbreviations: TK, thymidine kinase; tk, TK gene; HSV-1, herpes simplex virus type 1; bp, base pair(s); kbp, kilobase pair(s); Amp, ampicillin; Tet, tetracycline; AO, acridine orange; TSA, trypticase soy agar; EtBr, ethidium bromide; EACA, e-aminocaproic acid; Cap, chloramphenicol; RM, relative electrophoretic mobility; HATG, 0.1 mM hypoxanthine/ 1 ,IM aminopterin/40 ,uM thymidine/L10 ,uM glycine.

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Medical Sciences: Kit et aL

Proc. NatL Acad. Sci. USA 78 (1981)

were deproteinized with 1% sodium dodecyl sulfate at neutral pH. The DNA was then analyzed by CsCl/EtBr density gradient centrifugation (24). Heavy density plasmid and light density bacterial DNAs were recovered and their radioactivities were determined. Preparation ofExtracts and TK Assays. Cytosol extracts containing HSV-1 TK activity were prepared from LM(TK-) cells infected for 6 hr at 37°C with HSV-1 at an input multiplicity of5-10 plaque-forming units per cell (12). Extracts from E. coli K-12 strains were prepared essentially as described by Godson and Sinsheimer (25), except that deoxycholate and DNase were omitted from lysing mixtures, or by the method ofInuzuka and Helinski (26). The following constituents were added to bacterial lysates at the indicated final concentrations: KCl (0.1 M); ATP (2.5 mM); MgCl2 (1.25 mM); dThd (0.2 mM); e-aminocaproic acid (EACA) (0.05 M); and glycerol (10%, vol/vol). Bacterial lysates were centrifuged for 1 hr at 4°C at 105,000 x g, supernatant fluids were collected, and 10% Polymin P (Fluka AG, Switzerland) solution was added to a final concentration of 0. 6% to precipitate nucleic acids. Solid ammonium sulfate was added to the final supernatants to 40% saturation. After a 30-min mixing at 4°C, precipitates were collected by centrifugation and dissolved in enzyme buffer (0.15 M KCl/10 mM Tris-HCl, pH 8/ 3 mM 2-mercaptoethanol, supplemented with dThd, ATP, Mg2+, EACA, and glycerol at the concentrations indicated previously), and portions were assayed'for TK activity (12). Polyacrylamide disc gel electrophoresis and serological studies on the capacity of anti-HSV-1 IgG (no. 46) to inhibit the TK activities of HSV-1-infected LM(TK-) cells and pAGO-transformed E. coli K-12 KY895 cells were carried out as described (10, 12).

blast cells were biochemically transformed with pAGO and pMHLA DNAs as described (10, 11). Elimination of Plasmids from Transformed Bacteria. Acridine orange (AO) was used for selective inhibition of the replication of plasmid pAGO (23). Cultures treated with AO at 100 ,ug/ml were plated on TSA plates to isolate single colonies which were tested for sensitivity to Amp and Tet. Colonies that were sensitive to both antibiotics were then tested for the ability to incorporate [3H]dThd into DNA. Analyses of Plasmid DNAs. Plasmid DNAs were cleaved with restriction nucleases and the fragments were separated by electrophoresis in 1% agarose gels, stained with ethidium bromide (EtBr), visualized over a long-wave UV illuminator, and photographed (10, 11). Incorporation of [3H]dThd into Bacterial DNAs. Incorporation of [3H]dThd into HClO4-insoluble material was routinely used as a measure of bacterial DNA synthesis. Overnight cultures (5 or 10 ml) were initiated from bacterial slants or from two or three drops from overnight cultures in complete M9 medium with appropriate amino acid, vitamin, and antibiotic supplements. Fresh cultures (5 ml) in supplemented M9 medium were then started by inoculating bacteria at a concentration giving OD of 0.025-0.06 (600 nm). Cultures were incubated at 370C with and without FdUrd (25 gg/ml) [and dAdo (10 Ag/ml), dCyd (10 Ag/ml), and Urd (25 ,ug/ml)]. At 2 and 4 hr after the addition of 0. l ml of [3H]dThd (New England Nuclear), 1-ml samples of bacterial suspensions were withdrawn, transferred to centrifuge tubes, and precipitated at 40C with 0.5 M HC104 containing 10 ,g of nonlabeled dThd per ml. The bacterial suspensions were transferred to Whatman GF/C filters and washed at 4°C with an-excess of 0.5 M HCIO4 plus dThd and with ethanol, dried, and transferred to counting vials; 10 ml of OCS (Amersham/Searle, Arlington Heights, IL) was added to the vials, and the radioactivity was determined. To verify that the [3H]dThd was incorporated into DNA, in some experiments labeled DNA was extracted from bacteria as described by Birnboim and Doly (21), except that the extracts o

5831

RESULTS Transformation of E. coli K-12 Strains by Plasmid DNAs. DNAs from plasmids pBR322, pAGO, and pMH1A previously have been used to transform E. coli K-12 strain RR1 (11, 18) (Fig. 1). The plasmid DNAs isolated from transformed RR1

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FIG. 1. Derivation of plasmid pAGO by insertion of HSV-1 tk [2025base-pair (bp) Pvu II-Pvu II fragment] at the unique Pvu II site of plasmid pBR322 and derivation of plasmid pMH1 from EcoRI-cleaved pAGO by elimination of a 2172-bp fragment. Restriction maps of the HSV-1 tk and of the plasmids are shown. pBR322 and pAGO are Ampr and Tetr; pMH1 is Ampr but Tet8. The unique pBR322 EcoRI site was chosen as 0 in the maps of pAGO and pMH1. Restriction sites are indicated with their approximate distance from position 0 clockwise in base pairs.

584


Proc. NatL Acad. Sci. USA 78 (1981)

cells, designated pBR322-RR1, pAGO RR1, and pMHlA RR1, respectively, have now been used to transform E. coli K-12 strain KY895. Plasmid DNAs isolated from transformed colonies of E. coli K-12 KY895 were designated pBR322KY895, pAGO KY895, and pMH1A-KY895. Antibiotic-resistant colonies were obtained with an efficiency of 105-107 per ,g of DNA in all cases. Biochemical. Transformation of LM(TK-) Cells by Plasmid DNAs. To learn whether plasmid DNAs isolated from E. coli strain KY895 retained an active tk, biochemical transformation experiments were carried out (10, 11). Superhelical pAGO RR1 and pAGO KY895 DNAs had equivalent activities in transforming mouse LM(TK-) cells to the TK+ phenotype, as did superhelical pMHlA'RRl and pMHLA'KY895 DNAs (Table 1). However, both pMH1A DNAs, which lack a 105-bp sequence extending from the Pvu II to the EcoRI recognition site of HSV1 tk (11, 20), were less efficient than pAGO DNAs in transforming LM(TK-) cells. Thus, the plasmids propagated in strain KY895 retained the active coding region for HSV-1 tk. Agarose Gel Electrophoresis of Restriction Nuclease-Cleaved Plasmid DNAs. To learn whether plasmid DNAs were altered when propagated in strain KY895 cells, pAGO (6386 bp) and pMH1A (4214 bp) DNAs were isolated from transformed E. coli K-12 strain RR1 and KY895 cells and cleaved with several restriction nucleases, and the fragments were analyzed by agarose gel electrophoresis. The results confirmed previous studies on the restriction patterns of pAGO and pMH1A DNAs (11, 20) and may be summarized as follows. (i) BamHI, which cleaves pBR322 and pAGO DNAs at a site in the Tetr gene (20, 27), and Sac I, which has one recognition site in the HSV-1 tk of pAGO (20), each converted superhelical pAGO DNA to unit length linear DNA. (ii) Superhelical pMH1A DNA was converted to its linear form by Sac I but not by BamHI. (iii) pAGO DNA was converted to two fragments by Pvu II (2 and 4.4 kbp) and to three fragments by HincII (1. 1, 1. 6, and 3.7 kbp) and Pst I (0. 88, 2.5, and3.0kbp). (iv) SuperhelicalpMHlA.DNAwas converted to linear DNA by Pvu II and it was cleaved by HincIl to two DNA fragments (0.55 and 3.7 kbp) (Fig. 1). These experiments demonstrated that the plasmid DNAs isolated from E. coli K-12 strain RR1 and KY895 cells had the same nuclease patterns. Incorporation of [3HldThd into DNA of Plasmid-Transformed E. coli K-12 Strains. E coli K-12 strain KY895 is deficient in TK activity (13-15). Hence, it utilizes exogenous dThd poorly for DNA synthesis. To learn whether plasmids containing active HSV-1 tk could restore the ability of strain KY895 to incorporate exogenous dThd into DNA, the experiments depicted in Table 2 and Fig. 2 were carried out. Strain KY895 cells transformed by plasmid pAGO were more than 10 times as efTable 1. Biochemical transformation ofLM(TK-) cells by superhelical plasmid DNAs isolated from E. coli K-12 strains Plasmid DNA DNA ng/dish Colonies, no./dish*

pAGO-RR1

505

239 133

pAGO-KY895

50

268

pMHlA-RR1

50 5 50

430 23

pMH1A*KY895 *

Three dishes per group; mean-is shown. The transforming activity of DNA preparations was tested as described (10, 11). TK+ transformants were isolated in medium containing 0.1 mM hypoxanthine, 1 uM aminopterin, 40 pM thymidine, and 10 tpM glycine (HATG medium).

Table 2. Incorporation of (3H]dThd into DNA of

plasmid-transformedE. coli K-12 strains

Exp. 1

2

3

Bacterial strain RR1 (no plasmid) KY895 (no plasmid) KY895 (pAGO-RR1) KY895 (pMHlA-RR1)

cpm x 10-3/ml ofbacterial suspension labeled for: 2hr 4hr No With No With FdUrd FdUrd FdUrd FdUrd 270.0 314.0 1.7 1.7 36.2 27.4 1.9 2.5

RR1 (no plasmid) KY895 (pBR322 RR1) colony 1 KY895 (pBR322 RR1) colony 2 KY895 (801) (cured of pAGO) KY895 (802) (cured of pAGO) KY895 (pAGO-RR1) colony 3 KY895 (pAGO-RR1) colony 4

184.0

550.0

250.0

567.0

1.6

4.7

2.3

4.0

1.7

4.4

2.1

4.5

1.4

4.9

1.4

4.8

1.4

4.5

1.4

5.0

17.9

80.0

23.1

101.0

12.5

51.2

20.0

67.3

RR1 (no plasmid) KY895 (801) (cured of pAGO) KY895 (pBR322-RRI) colony 1 KY895 (no plasmid) KY895 (pMHlA RR1) KY895 (pAGORR1) colony 4

432.0

416.0

1.8

1.4

1.9 1.5 2.2

2.0 1.7 2.2

15.9

25.9

503.0 578.0 RR1 (no plasmid) 3.4 3.0 KY895 (no plasmid) KY895 (801) (cured of 3.6 3.3 pAGO) KY895 (pAGO RR1) 37.1 58.3 colony 4 Cultures (5 ml) in M9 medium with appropriate supplements were started by inoculating bacteria at a concentration of 0.025-0.06 OD (600 nm). In Exp. 2, cultures were preincubated (with or without FdUrd, dAdo, dCyd, and Urd) for 1 hr at 370C prior to the addition of 13H]dThd. For Exps. 3 and 4, cultures were preincubated for 90 and 60 min, respectively, prior to [3H]dThd addition. To each culture, 100 ,uCi of [3H]dThd and 10 ug of unlabeled dThd were added (except that in Exp. 4 the label was increased to 200 ,Ci of [3H]dThd and 20 pg of unlabeled dThd), and incubation was continued for 2 and 4 hr, at which times 1 ml ofbacterial suspension was harvested and analyzed. The OD (600 nm) of strain RR1 cultures was about 2.5 at the conclusion of the 4-hr labeling period; that ofthe other cultures (without FdUrd) varied from about L.2 to 1.7.

4

fective as untransformed KY895 cells and about 1/10th as effective as wild-type TK+ strain RR1 cells in incorporating [3H]dThd into DNA (Table 2). Furthermore, the utilization of exogenous. [3H]dThd for DNA synthesis was increased by treating strain RR1- or pAGO-transformed cells. with FdUrd which inhibits de novo dTMP synthesis and, hence, enhances the utilization of exogenous dThd. Partial restoration ofthe ability to incorporate [3H]dThd into DNA did not occur in strain KY895 cells that were transformed by plasmid pBR322, which does not contain a tk, nor in KY895 cells that had been cured of plasmid pAGO by AO treatment. It is interesting that KY895 cells transformed by plasmid pMH1A, which lacks the 2067-bp pBR322 DNA se-

Proc. Natl. Acad. Sci. USA 78 (1981)


0 -

G)

04

U4

3

7

11 15 19 23 27

3

7

11 15 19 23 27 Fraction

from EcoRI to Pvu II and the 105-bp HSV-1 tk DNA sefrom Pvu II to EcoRI (11, 20) (Fig. 1), likewise failed to show enhanced [3H]dThd utilization. On the other hand, KY895(801) cells that had been cured of the resident plasmid by AO treatment did exhibit increased [3H]dThd incorporation into DNA after they were retransformed by pAGO (data not shown). In the preceding experiments, the incorporation of [3H]dThd into HCl04-insoluble material was taken as a measure of DNA synthesis. To verify that [3H]dThd was being incorporated into DNA, bacterial lysates were prepared and DNA was purified by centrifugation in CsCl/EtBr density gradients. In some experiments, the incorporation of [3H]dThd into DNA was also studied in bacteria that had first been treated for 1 hr with chloramphenicol (Cap) to permit the completion of a round of bacterial DNA synthesis and then had been incubated with Cap and [3H]dThd for 4 hr. Although Cap inhibits the synthesis of bacterial chromosomal DNA, it does not inhibit the synthesis of certain plasmid DNAs, and it may be used to amplify plasmid DNAs preferentially. Fig. 2A shows that E. coli K-12 strain RR1 DNA (no plasmid) had a light density in CsCl/EtBr gradients and that Cap decreased the incorporation of [3H]dThd into DNA to about 6% of the control value. TK-negative pBR322-transformed E. coli K-

quence quence

12 KY895 cells incorporated few cpm from [3H]dThd into light

density chromosomal DNA, and this labeling was decreased to background levels by Cap treatment (Fig. 2B). Similar results were obtained with KY895 cells that had been cured of plasmid pAGO (data not shown). In contrast, E. coli K-12 KY895 (pAGO-RR1), which contains the HSV-1 tk, incorporated significant amounts of [3H]dThd into both bacterial and plasmid DNAs (Fig. 2C). Although Cap inhibited the labeling of light density bacterial DNA, it enhanced the labeling of heavy density plasmid DNA. Detection of HSV-1 TK Activity in Bacterial Extracts of

pAGO-Transformed Cells. Extracts were prepared from wildtype E. coli K-12 strain RR1, TK- mutant KY895, and plasmid pAGO-transformed strain KY895 cells. After partial purification by ammonium sulfate precipitation, TK activity was assayed (12-15). The specific activity of TK partially purified from strain KY895 (pAGO RRl) cells was about 25% of that of TK+ E. coli K12 strain RR1 cells. To investigate whether an HSV-1-specific TK activity was expressed by pAGO-transformed KY895 cells, serological and heat inactivation studies and polyacrylamide disc gel electrophoretic analyses were performed. Previous experiments had shown that: (i) anti-HSV-1 IgG 46

3

7

11 15 19 23 27

585

FIG. 2. Analyses, by CsCl/ EtBr density gradient centrifugation, of [3 H]dTMP-labeled DNAs isolated from E. coli K-12 strain RR1 (A) andzfrom strain KY895 cells transformed by plasmids pBR322 (B) and pAGO (C). Exponentially. growing cells were incubated for 1 hr with (- -.) and without (-) Cap (200 Lg/ml) and then labeled for 4 hr at 38°C with [3H]dThd (4 ,ug; 40 gCi/ml) in the presence or absence of Cap. DNA samples were centrifuged for 44 hr at 23-C at 44,000 rpm in the Spinco L2 centrifuge and 50 Ti rotor. Twelve-drop fractions were collected on Whatman GF/A filters, washed at 4°C with 5% trichloroacetic acid and with ethanol, dried, and assayed for radioactivity (24).

inhibits the cytosol TK activity of HSV-1-infected and -transformed LM(TK-) cells but has little or no inhibitory activity for the cytosol TK activities of uninfected mammalian cells or cells infected by other herpesviruses; (ii) HSV-1 TK activity has a relative electrophoretic mobility (RM) of about 0.6-0.7, distinctly different from that of the cytosol TK activity of uninfected cells (10, 12, 28); (iii) wild-type E. coli TK has an R1M value of about 0.8-0.9 (15) and withstands heating for 5 min at 70'C (15, 29, 30); and (iv) HSV-1 TK activity is extremely thermolabile (12). Anti-HSV-1 IgG 46 completely inhibited the TK activities of HSV-1-infected LM(TK-) and pAGO-transformed E. coli K-12 strain KY895 cells but had no inhibiting activitv for the wildtype E. coli K-12 TK activity of strain RR1 cells (or the residual TK activity of pAGO-cured strain KY895 cells) (Table 3). Heating the TK activities of HSV-1-infected LM(TK-) and pAGOtransformed strain KY895 cells completely inactivated these activities, but wild-type E. coli K-12 strain RR1 TK activity was thermostable under the same conditions (Table 4). Electrophoresis at pH 8.6 in 5% polyacrylamide disc gels was carried out on bacterial extracts as described (10-12) except that Table 3. Effect ofrabbit anti-HSV-1 IgG 46 on TK activities of HSV1-infected LM(TK-) cells andE. coli K-12 strains % TK activity after treatment with Anti-HSV-1 Normal rabbit IgG 46 TK extracts from serum IgG* 4 100 (11,500) HSV-1-infected LM(TK-) E. coli K-12 strain RR1 (no 100 100 (23,200) plasmid) E. coli K-12 strain KY895 1 100 (100,200) (pAGOMRR1) E. coli K-12 strain KY895 100 100 (850) (801) (cured of pAGO) Anti-HSV-1 IgG 46 was diluted 1:4- with normal rabbit serum IgG, and 0.15 ml was mixed with 0.15 ml of enzyme extracts from HSV-1infected LM(TK-) cells or from strain RR1 cells (wild-type E. coli TK+) and incubated for 10 min at 4°C. Portions (0.05 ml) in triplicate plus 0.075 ml of TK reaction mixture containing [3H]dThd and ATP-Mg2e were then incubated for 15 min at 38°C, and the conversion of[3H]dThd to [3H]dTMP was determined (10, 12). In the case of extracts from pAGO-transformed KY895 and cured KY895 (801) cells, 0.03 ml of extract was incubated for 10 min at 4°C with 0.09 ml of anti-HSV-1 IgG 46; then, 30-ILI portions in duplicate were mixed with 0.05 ml of TK reaction mixture and incubated for 2 hr at 38°C and [3H]dTMP formation was determined. * Values in parentheses are cpm of [3H]dTMP per 20-,ul portion of reaction mixture.

586


Table 4. Effects ofpreincubation at 700C on the TK activities of extracts from HSV-1-infected LM(TK-) cells andE. coli K-12 strains* TK activityt Enzyme extracts Control preincubated TK extracts from 5 min at 70°C extracts 0 13,000 HSV-1-infectedLM(TK7) cells pAGO-transformedE. coli 6 K-12 strain KY895 1,500 E. coli K-12 strain RR1 6350 5,800 (no plasmid) E. coli K-12 strain KY895 10 4 (801) (cured of pAGO) * Control extracts were incubated for 5 min at. 40C; experimental extracts were heated for 5 min at 700C and immediately placed in ice bath. TK reaction mixture containing [3H]dThd and ATP-Mg2e was added and tubes were incubated at380. t cpm of [3H]dTMP formed per gg of protein in 15 min at 380C.

0. 1 mM (dCTP was added to the upper buffer solution and 1.75 mM dCTP was added to TK reaction mixtures to activate E. coli TK activity (15, 29, 30). The analyses demonstrated that TK+ E. coli K-12 strain RR1 cells contained a TK activity with RM =0.85, as expected, and that pAGO-transformed KY895 and HSV-1-infected LM(TK-) cells contained TK activities with RM -0.6-0.7 (data not shown). These TK activities were not detected after electrophoresis of extracts from cured.KY895 (801) cells. The experiments show that an HSV-1-specific TKactivity was expressed in E. coli K-12 strain KY895 (pAGO RR1) cells. DISCUSSION E. coli K-12 strain KY895 (TK- Amps Tets) has been transformed by a hybrid-plasmid (pAGO) which contains the HSV-1 tk. The pAGO-transformed cells exhibit a partially restored ability to incorporate [3H]dThd into bacterial and plasmid DNAs. The [3H]dThd phosphorylating activity of the pAGO-transformed cells cannot be ascribed to plasmid .transformation per se or to reversion of the E. coli tk to pseudo-wild-type but is associated with the expression of an HSV-1-specific TK activity for the following reasons: (i) KY895 cells transformed to Ampr Tetr by plasmid pBR322, from which plasmid pAGO was derived, do not exhibit partially restored [3H]dThd.phosphorylating activity; (ii) serological, thermal inactivation, and electrophoresis experiments indicate that the TK activity expressed in KY895 (pAGO.RRl) cells is HSV-1-specific; and (iii) after AO treatment, cured KY895 cells become Amps Tets, do not contain detectable HSV-l-specific TK activity, and lose the ability to incorporate [3H]dThd into DNA. The present studies do not disclose whether the HSV-1 TK activity made in pAGO transformants is derived from transcripts initiated from HSV-1 tk promoters or from an unknown pBR322 promoter. In this connection, it is interesting that: (i) plasmid pMHlA DNA, which lacks both the EcoRI to Pvu II sequence of pBR322 (27) and the 105-bp Pvu II to EcoRI sequence of HSV1 tk (11, 20) (Fig. 1), transforms mouse LM(TK-) cells less efficiently than. does pAGO DNA; and (ii) pMHlA-transformed KY895 cells do not exhibit partial restoration of [3H]dThd phosphorylating activity. The finding that pAGO-transformed KY895 cells express an HSV-1 TK activity suggests that intervening sequences may be absent from the coding region ofthe HSV-1 tk or that the coding region of the gene possesses short intervening sequences which do not disrupt the translational reading frame. The first possi. bility contrasts with findings on papovavirus and adenovirus

Proc. Natl. Acad. Sci. USA 78 (1981) genes (for review, see refs. 2 and 3). However, papovaviruses

and adenoviruses are highly dependent on host metabolic processes for their replication and.do not inhibit host RNA and protein synthesis until late in infection, whereas HSV-1 is- highly autonomous and shuts down. host RNA and protein synthesis early in infection. It remains to be seen whether the suggested absence of intervening sequences applies only to the. HSV-1 tk or also to other HSV-1 genes. The demonstration that HSV-1 tk can be expressed in pAGOtransformed E. coli K-12 strain KY895 cells has practical significance because: (i) TK- E. coli K-12 cells may be useful for the isolation ofadditional eukaryotic tk genes; and (ii) the HSV-1 tk can be used as a vector for the isolation and transfer ofother eukaryotic genes to and from E. coli K-12 strain KY895 and mammalian cells. We thank Mary Anne Michalak for technical assistance. E. coli K-12 strain 1106 containing plasmid pAGO and E. coli strain KY895 were obtained through the generosity of F. Colbere-Garapin (Institut Pasteur, Paris, France) and G. R. Greenberg (University of Michigan, Ann Arbor, MI), respectively. This research was aided by Grants K6-AI-235217 and CA-06656-18 from the National Institute of Allergy and Infectious Diseases and the National Cancer Institute, respectively. 1. Beggs, J. D., van den Berg, J., van Ooyen, A. & Weissmann, C. (1980) Nature (London) 283, 835-840. 2. Crick, F. (1979) Science 204, 264-271. 3. Abelson, J. (1979) Annu. Rev. Biochem. 48, 1035-1069. 4. Struhl, K., Stinchcomb, D. T. & Davis, R. W. (1980)J. Mol Biol 136, 291-307. 5. Ratzkin, B. & Carbon, J. (1977) Proc. Natl Acad. Sci. USA 74, 487-491. 6. Vapnek, D., Hautala, J. A., Jacobson, J, W., Giles, N. H. & Kushner, S. R. (1977) Proc. Nati Acad. Sci. USA 74, 3508-3512. 7. Citron, B. A., Feiss, M. & Donelson, J. E. (1979) Gene 6, 241-264. 8. Dickson, R. S. & Markin, J. S. (1978) Cell 15, 123-130. 9. Chang, A. C. Y., Nunberg, J. H., Kaufman, R. J., Erlich, H. A., Schimke, R. T. & Cohen, S. N. (1978) Nature (London) 275, 617-624. 10. Kit, S., Otsuka, H., Qavi, H., Trkula, D. & Dubbs, D. R. (1980) Virology 105, 103-122. 11. Kit, S., Otsuka, H., Qavi, H., Trkula, D., Dubbs, D. R. & Hazen, M. (1980) Nucleic Acids Res., in press. 12. Kit, S., Jorgensen, G. N., Dubbs, D. R., Trkula, D. & Zaslavsky, V. (1978) Intervirology 9,162-172. 13. Hiraga, S., Igarashi, K. & Yura, T. (1967) Biochim. Biophys. Acta 145, 41-51. 14. Igarashi, K., Hiraga, S. & Yura, T. (1967) Genetics 57, 643-654. 15. Goff, S. P. & Berg, P. (1979)J. Mol. Biol. 133,359-383. 16. Kit, S., Dubbs, D. R., Piekarski, L. J. & Hsu, T. C. (1963) Exp. Cell Res. 31, 297-312. 17. Kit, S. & Dubbs, D. R. (1963) Biochem. Biophys. Res. Commun. 11,55-59. 18. Bolivar, F., Rodriguez, R. L., Greene, P. J., Betlach, M. C., Heyneker, H. L. & Boyer, H. W. (1977) Gene 2, 95-113. 19. Cohen, S. N. & Hurwitz, J. (1968)J. Mol Biol 37, 387-406. 20. Colbere-Garapin, F., Chousterman, S., Horodniceanu, F., Kourilsky, P. & Garapin, A.-C. (1979) Proc. Natl Acad. Sci. USA 76,

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