The incidence of herpetic keratitis following in‐tranasal or direct ocular infection with thymidine kinase‐negative (TK − ) strains of herpes simplex virus (HSV)‐2 has not been well studied, and the role of the TK gene in the establishment of latency and virus reactivation is controversial. To determine whether a TK − strain of HSV‐2 could establish trigeminal ganglionic latency and be reactivated in vivo to produce recurrent keratitis or nervous system infection, an animal model of acute and recurrent infection was utilized. Rabbits were infected by the intranasal or ocular routes, and latency was reactivated by immuno‐suppression. Virus shedding in nasal and ocular secretions was monitored, and the eyes were examined for the presence of corneal epithelial lesions during acute and reactivated infections. Central nervous system (CNS) and trigeminal ganglionic tissues were assayed by histologic, virologic, and in situ hybridization techniques. All rabbits intranasally infected shed virus in both ocular and nasal secretions, whereas only 30% of rabbits infected in the eyes shed virus in nasal secretions. Virus was recovered from co‐cultivation cultures, but not from cell‐free ho‐mogenates, of trigeminal ganglionic and CNS tissues from animals inoculated by both routes. The incidence of keratitis was much greater after direct ocular inoculation, although both routes of inoculation produced CNS and ganglionic inflammatory lesions. Keratitis healed in 92% of the animals infected by the ocular route by 26 days post infection. Of rabbits initially infected in the eyes and then subjected to drug‐induced reactivation, only 30% shed virus, which was limited to a 24 hour period; there was no reappearance of epithelial keratitis, no animal became blind, and none died. In contrast, latently infected control rabbits uniformly reactivated. These studies show that this TK − HSV‐2 strain (i) replicates in the eye, (ii) is neuroinvasive but non‐neurovirulent following intranasal and direct ocular infection; (iii) sheds in the eye more frequently and for longer periods after ocular than after intranasal inoculation; (iv) induces epithelial keratitis that usually heals spontaneously; (v) establishes latency in trigeminal ganglionic neurons, but no other ganglionic cells; and, (vi) reactivates in a small proportion of animals, but does not produce recurrent ocular lesions following drug‐induced immunosuppres‐sion. Thus, the TK gene appears directly involved in HSV latency and reactivation in vivo. © 1994 Wiley‐Liss, Inc.

More than 110,000 Americans have died and more than one million are believed to be infected with the human immunodeficiency virus (HIV-1). This chapter discusses the human immunodeficiency virus type I that has been identified in retinal tissue of the AIDS patients. Eye infection, a common complication of AIDS, strikes as many as 98% of all people infected with HIV-1. Approximately 50–80% of individuals with HIV-1 infection experience significant visual loss prior to death, further complicating an already devastating disease process. Thousands of the affected are young adults and teenagers; perhaps some of the saddest cases are seen in infants and very young children who are victims of this deadly disease. It was also reported that the isolation of HIV-1 and HHV-6 from corneas of asymptomatic are from HIV-1-positive donors, suggesting that corneal tissue might be capable of supporting HIV-1 and HHV-6 infection.

Calves were vaccinated intranasally (IN) or intravenously (IV) with a thymidine kinase-negative (tk−) BHV-1 mutant. Vaccinated calves developed neutralizing antibodies but did not show clinical signs of infectious bovine rhinotracheitis (IBR). Vaccination also prevented clinical signs of IBR disease following IN challenge exposure of the calves to parental Los Angeles (LA) and USDA Cooper strains of tk+ BHV-1. Nasal swabs were collected for 10 days after the vaccination and the challenge exposures to monitor BHV-1 multiplication. At both 91 and 121 days post vaccination (PV), calves were also stressed for 5 days with dexamethasone (DEX) to induce reactivation of BHV-1 and nasal swabs were obtained. tk− BHV-1 multiplied in the nasal mucosa of IN vaccinated calves and was also recovered after DEX treatment. Likewise, tk− BHV-1 was isolated from the buffy coat fraction of IV vaccinated calves, but not from nasal swabs. tk− BHV-1 vaccination reduced the multiplication of tk+ BHV-1 in the nasal mucosa, but did not completely prevent development of a persistent infection by the challenge virus. The phenotypes of viruses isolated from the nasal swabs and buffy coats were analyzed by enzyme assays of extracts from virus-infected cells and by plaque autoradiography. These assays showed that tk− BHV-1 can persist for at least 3 months in vaccinated calves and may also be transmitted from vaccinated to control calves without revertingin vivo to tk+. The results demonstrate that the tk− BHV mutant is attenuated and efficacious as a vaccine.

Five hybrid plasmids were constructed, each containing a portion of the vaccinia virus DNA HindIII-J fragment. These plasmid DNAs were used in marker rescue experiments to map the mutations in the thymidine kinase (TK) gene of three TK− vaccinia virus mutants. The TK gene of each of the three mutants was rescued by DNA from plasmid pPJ701, which contained about one-half of the HindIII-J fragment. Two mutants, 1004B and 1017-1, but not the third, 1016-1, were rescued by DNA of two plasmids, pPJ702 and pPJ703, which contained 16 and 18%, respectively, of one end of the J fragment. Mutant 1016-1 could be rescued by plasmid pPJ705 containing a 1.69-kb fragment of the HindIII-J fragment. The J fragment DNA in plasmid pPJ705 is located adjacent to that and separated by an EcoRI site from pPJ703 in the vaccinia virus genome. These results indicate that the mutation site in the TK gene of 1016-1 differs from that in 1004B or 1017-1 and suggests that the structural gene for the vaccinia virus TK lies near one end of the HindIII-J fragment and spans the EcoRI site.

To analyze the boundaries of the functional coding region of the HSV-2(333) thymidine kinase gene (TK gene), deletion mutants of hybrid plasmid pMAR401 H2G, which contains the 17.5 kbp BglII-G fragment of HSV-2 DNA, were prepared and tested for capacity to transform LM(TK−) cells to the thymidine kinase-positive phenotype. These studies showed that hybrid plasmids containing 2.2–2.4 kbp subfragments of HSV-2 BglII-G DNA transformed LM(TK−) cells to the thymidine kinase-positive phenotype and suggested that the region critical for transformation might be less than 2 kbp. That the activity expressed in the transformants was HSV-2 thymidine kinase was shown by experiments with type-specific enzyme-inhibiting rabbit antisera and by disc-polyacrylamide gel electrophoresis analyses. DNA fragments of the HSV-2 TK gene were subcloned in phage M13mp9 and M13mp8. A sequence of 1656 bp containing the entire coding region of the TK gene and the flanking sequences was determined by the dideoxynucleotide chain termination method. Comparisons with the HSV-1(Cl 101) TK gene revealed that PstI, PvuII, and EcoRI cleavage sites had homologous locations as did promoter, translational start and stop, and polyadenylation signals. Extensive homology was observed in the nucleotide sequence preceding the ATG translational start signal and in portions of the coding region of the genes. Comparisons of the predicted amino acid sequences of the HSV-1 and HSV-2 thymidine kinase polypeptides revealed that both were enriched in alanine, arginine, glycine, leucine, and proline residues and that clear, but interrupted homology existed within several regions of the polypeptide chains. Stretches of 15–30 amino acid residues were identical in conserved regions. The possibility is suggested that domains containing some of the conserved amino acid sequences might have a role in substrate binding and as major antigenic determinants.

Marmoset herpesvirus (MarHV) deletion mutants in the thymidine kinase (TK) gene were isolated after infection of OMK cells with TK+ MarHV DNA and the hybrid plasmid, pMAR401, which lacks a 2.6‐kb KpnI‐M fragment in the coding region of the MarHV TK gene. After plaque purification in TK− HeLa(BU25) cells, the DNA's from five recombinant araT‐resistant MarHV clones were analyzed with restriction nucleases to verify that the 2.6‐kb KpnI‐M fragment (and a 0.9‐kb Bg1II‐Q fragment) were deleted from the viral DNA's. Molecular hybridization experiments using 32P‐labeled pMAR4 probes and viral DNA fragments also showed that the recombinant viral DNA's lacked the KpnI‐M and Bg1II‐Q fragments, that BamHI‐I of parental virus was shortened, and that three new HindIII fragments replaced the parental virus HindIII‐G fragment. The recombinants did not induce TK activity in LM (TK−) cells. To study the relative virulence of the recombinants, 3‐week‐old Swiss mice were injected intracerebrally (Ic) or subcutaneously (Sc) in the sacro‐lumbar region with either parental or recombinant viruses. The LD50 for the parental virus was 3 p.f.u. (Ic) and 7,600 p.f.u. (Sc). The recombinant viruses were significantly less virulent than TK+ MarHV after Ic inoculation (LD50 of 62,000 and 32,000 p.f.u., respectively, for viruses 5D‐6B and 5D‐4B) and gave no fatalities after Sc inoculation. Mice surviving TK− MarHV infections were protected from challenge with TK+ parental MarHV. Recombinant TK− MarHV's may be useful as vectors for the expression of foreign genes in animal cells and as the starting material for the design of vaccines.

Virus isolated from a woman presenting with the first symptomatic episode of genital herpes was identified as herpes simplex virus type 1 (HSV-1) by restriction nuclease fingerprinting. Testing for IgM antibody to HSV indicated that the patient had recently contracted a new HSV infection. Virus microneutralization and the micro-solid phase radioimmunometric test for IgG, however, showed that the patient had had prior infection with herpes simplex virus type 2 (HSV-2); thus the HSV-1 infection was acquired despite the presence of antibody to HSV-2. Genital herpes recurred about four, seven, and nine months after the HSV-1 infection. Isolates from the latter three episodes all were of an identical strain of HSV-2 and were not recombinants or a mixture of the viruses. The data show that two distinctly different herpes simplex viruses can initiate genital infections in one individual and suggest that HSV-2 is more likely to recur than HSV-1.

Cytosol thymidine kinase (TK) activity is enhanced at 6 hr after bovine embryo tracheal (EBTr) and rabbit skin fibroblast (RAB-9) cells are infected with the Los Angeles and Cooper strains of bovine herpesvirus type 1 (BHV-1). To learn whether this enhancement resulted from the induction of a virus-specific TK activity, biochemical and genetic studies were carried out. The biochemical experiments demonstrated that: (i) the BHV-1-induced TK activity had a relative disc PAGE mobility (Rm) characteristic of other herpesvirus-encoded TKs and distinctly different from the Rm value of the cytosol TK of host cells; and (ii) the BHV-1-induced TK was significantly more sensitive to competitive inhibition by arabinosylthymine (araT) than the cytosol TKs of EBTr and RAB-9 cells. The genetic experiments entailed the isolation of a bromodeoxyuridine (BrdUrd)-resistant rabbit cell line [RAB(BU)] deficient in cytosol TK activity and of BrdUrd- and araT-resistant BHV-1 mutants. RAB(BU) cells acquired TK activity after they were infected by wild-type, TK+ BHV-1, but not drug-resistant BHV-1 mutants. The experiments strongly suggest that wild-type BHV-1 induces a virus-specific TK activity.

To learn whether interferon could prevent the biochemical transformations induced by cloned herpesvirus thymidine kinase (TK) genes, LM(TK−) mouse fibroblast cultures were pretreated for 24 h with 2.4–40 international units (I.U.)/ml mouse α + β interferon, and subsequently transformed to the TK+ phenotype with recombinant plasmids containing the herpes simplex virus type 1 (HSV-1) TK gene (pAGO and pMH110) and the marmoset herpesvirus (MarHV) TK gene (pMAR035). Mouse α + β interferon inhibuted transformation and the inhibition was interferon dose-dependent. Transformation was also inhibited when LM(TK−) cells were pretreated for 2 – 5 h with 40 I.U./ml interferon. Maximal inhibitions of TK+ colony formation were observed following a 9–20 h pretreatment period with interferon. In contrast, 40 I.U./ml interferon treatment for 20 h did not reduce the rate or extent of LM(TK−) cell growth. Experiments in which cultures were first treated with plasmid pAGO and only afterwards treated with interferon also showed that, as the interferon concentration used, interferon did not inhibit the outgrowth of transformated colonies. Enzyme assays showed that pretreatment with interferon inhibited the induction of TK activity in cells that had been transfected with pAGO DNA.

A hybrid plasmid (pAGO) that contains the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in the form of a 2-kilobase-pair (kbp) Pvu II fragment inserted at the Pvu II site of plasmid pBR322 was used to transform TK- Escherichia coli K-12 strain KY895. pAGO-transformed KY895 cells exhibited partially restored ability 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 TK+ 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 of the gene possesses short intervening sequences which do not disrupt the translational reading frame.

To analyze the site of integration of the herpes simplex virus type 1 (HSV‐1) thymidine kinase (TK) gene in biochemically transformed human cells, TK‐HeLa‐(BU25) cells were transformed to the TK + phenotype by a cloned, 2 kbp Pvull fragment of HSV‐1 DNA. The transformed cells [HeLa(BU25)/TF pAGO PP3] were fused with mouse LM(TK − ) cells, and human‐mouse somatic cell hybrid clones (LH PP3 clones 1,2,3,5 and 6) were isolated in HATG‐ouabain selective medium. The HeLa(BU25)/TF pAGO PP3 cells and the LH PP3 hybrid clones expressed HSV‐1‐specific TK activity and a her‐pesvirus‐associated nuclear antigen, and contained herpesvirus nucleotide sequences. Molecular hybridization experiments were carried out to map the HSV‐1 and flanking cellular nucleotide sequences in the biochemically transformed cells. These experiments demonstrated that the HSV‐1 nucleotide sequences were integrated at a single site, and that the same cellular nucleotide sequences flanked the viral DNA in transformed HeLa(BU25)/TF pAGO PP3 and LH PP3 clone 5 cells. TK − revertant subclones isolated by growing the LH PP3 clone 5 cells in BrdUrd (and diphtheria toxin) failed to form colonies in HATG medium, but retained HSV‐1 nucleotide sequences. Isozyme analyses on 21 gene‐enzyme systems representing 21 human chromosomes revealed that all of the LH PP3 clonal lines expressed human hexosaminidase B, which has been assigned to chromosome 5, and all were sensitive to diphtheria toxin, which is also a marker for chromosome 5. Chromosome analyses showed that chromosome 5 was the only human chromosome present in mitoses of LH PP3 clone 5 cells and that human chromosome 5 was present in most of the mitoses of LH PP3 clone 1,2,3, and 6 cells. The latter clones also contained 1 or 2 additional human chromosomes in some of the cells. As expected from the molecular hybridization analyses, TK − revertants of LH PP3 clone 5 cells retained portions of chromosome 5 and expressed human hexosaminidase B. The results indicate that HSV‐1 nucleotide sequences were stably integrated in the biochemically transformed cells, most likely in human chromosome 5.

In order to delimit the approximate boundaries of the marmoset herpesvirus (MarHV) thymidine kinase (TK) gene, HindIII and BamHI digests of MarHV DNA were cloned in plasmid pBR322. Several recombinant plasmids which transformed E. coli K12 strain RR1 to ampicillin resistance were isolated. The MarHV DNA inserts in these plasmids accounted for about half of the MarHV genome. One of the plasmids, pMAR4, contained a 9.1-kbp fragment of MarHV DNA (HindIII-G), transformed LM(TK−) cells to TK+, and hybridized to the BamHI-I fragment of MarHV DNA, which had previously been shown to have TK-transforming activity. pMAR4 DNA had little or no homology to the 2-kbp PuvII fragment of HSV-1 DNA, which contains the HSV-1 TK gene. Cleavage with PvuII, SacI, SmaI, and KpnI inactivated the TK-transforming activity of pMAR4, but cleavage with HindIII, PstI, EcoRI, XhoI, XbaI, and BamHI did not. Deletion mutants pMAR401 and pMAR420, which lacked the 2.6-kbp KpnI and the 2.75-kbp EcoRI fragments, respectively, of pMAR4, lost transforming activity, whereas pMAR410, which lacked a 2.9kbp XhoI fragment of pMAR4 did not. Recombinant plasmid pMAR430, which contained a 3-kbp PstI fragment of pMAR4, also transformed LM(TK−) cells to TK+. The results strongly suggest that the coding region of the MarHV TK gene was within a 2.4-kbp pMAR4 sequence extending from the PstI (0.33 kbp) to the EcoRI (2.7 kbp) cleavage sites.

The recombinant plasmid pAGO contains the Herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene and consists of a 2-kb PvuII fragment of HSV-1 DNA inserted into the PvuII site of pBR322. A deletion mutant of pAGO, designated pMH110, has been isolated which removes the normal HSV-1 TK gene promoter but places the promoter of the pBR322 tetracycline-resistance (tetr) gene only about 400 bp from the translational start codon of the HSV-1 TK polypeptide. In contrast to pAGO, which transforms mouse LM(TK-) cells to TK+ but is only weakly expressed in TK- bacteria, pMH110 not only efficiently transforms LM(TK-) cells to TK+ but also enables TK- Escherichia coli K-12 cells to form colonies on selective plates containing 5-fluorodeoxyuridine (FdUrd) plus thymidine (dThd) and to exhibit fully restored ability to incorporate [3H]dThd into DNA. The levels of TK activity expressed by bacteria harboring pMH110 were about as high as those expressed by bacteria harboring plasmid pTK3, which contains the wild-type E. coli TK gene. The TK activity expressed in bacteria harboring pMH110 was partially purified and shown to be HSV-1-specific by serological and disc PAGE analyses and by experiments demonstrating that this enzyme phosphorylated [125I]deoxycytidine.

To study the expression of SV40 tsA genomes that had been non-selectively introduced into mouse cells, SV40 tsA207 DNA was cleaved with BamH I and ligated to BamH I-cleaved plasmid pAGO DNA, which contains a functional HSV-1 thymidine kinase (TK) gene in the form of 2 kbp Pvu II fragment inserted at the Pvu II site of pBR322. Recombinant plasmids (11-12 kbp) were isolated and amplified in E. coli K12 strain RRI. Restriction nuclease analyses demonstrated that recombinant plasmids pSB15 and pSB10 contained intact SV40 genomes with the polarity of transcription oriented in the same direction (clockwise) or the opposite direction (counterclockwise), respectively, in relation to that of the HSV-1 TK gene. Cla I-cleaved pSB10 and pSB15 DNAs were used to transform LM(TK-) cells to TK+. Serological and disc PAGE analyses showed that clonal lines transformed by these plasmids all expressed the selected marker, HSV-1 TK. Molecular hybridization experiments showed that transformed clonal lines TF pSB10 C7 and TF pSB15 C10 had integrated intact SV40 genomes at one integration site, TF pSB10 C3 had integrated an SV40 genome with a small deletion near the BamH I site, but TF pSB15 Cl had integrated a plasmid from which most of the SV40 nucleotide sequences had been deleted. IF assays with hamster anti-SV40 tumor sera showed that TF pSB10 C7 and TF pSB15 C10 strongly expressed SV40 T antigens in over 90% of the cells, TF pSB10 C3 expressed SV40 T antigens in a minority of the cells, and TF pSB15 C1 did not express SV40 T antigens at all. [35S]-methionine labelling and immunoprecipitation experiments showed that, at 36.5 degrees C: (1) TF pSB10 C7 and TF pSB15 C10 expressed 92K and 20K mol. wt. species of SV40 T antigens and 50-55K cellular protein; (2) expression of all three was reduced in TF pSB10 C3 cells; and (3) TF pSB15 C1 expressed none of the SV40 T antigens, nor did parental LM(TK-) or TF 8-2 transformed cells (which contained the HSV-1 TK gene but not SV40 DNA). At 40 degrees C, labelling of the 50-55K cellular protein was markedly reduced in TF pSB10 C7 and pSB15 C10 cells. The results suggest that SV40 large T antigen (92K) induces and/or stabilizes the 50-55K cellular protein in these mouse cells.

Recombinant plasmid pAGO codes for herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) and consists of a 2-kbp HSV-1 DNA fragment inserted at the unique PvuII cleavage site of plasmid pBR322. A hybrid plasmid, designated pMH110, has been derived from plasmid pAGO by deleting the 1689-bp pBR322 nucleotide sequence of pAGO, which extends from the BamHI to the PvuII cleavage site, and the 250-bp HSV-1 nucleotide sequence of pAGO, which extends from the PvuII to the BglII cleavage site. Plasmid pMH110 biochemically transformed LM(TK−)cells to the TK+ phenotype. The biochemically transformed cell lines had the following properties: (i) they were resistant to the growth-inhibiting effects of 1 mM thymidine; and (ii) they expressed an HSV-1-specific TK activity. This HSV-1 TK activity was purified after labeling biochemically transformed cell lines [LM(TK−)/TF pMH110 E2 and LM(TK−)/TF pMH110 Hc2] with [35S]methionine. Immunoprecipitation experiments revealed that the TK polypeptides made in the biochemically transformed cells had molecular weights of about 39,000 to 40,000, which are about the same as the molecular weights of the TK polypeptides previously purified from HSV-1-infected LM(TK−) cells and other biochemically transformed cell lines. The experiments support the hypothesis that the functional coding region of the HSV-1 TK gene is 3′ to the BglII cleavage site, and they also suggest that the HSV-1 TK messenger RNA may have been initiated in cells transformed by HincII- and EcoRI-cleaved pMH110 DNA at a site in cellular (or plasmid) DNA upstream from the HSV-1 DNA BglII cleavage site.

To investigate the chromosomal sites of integration of the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in HSV-1-transformed human HeLa(BU25)/KOS 8-1 cells, the biochemically transformed cells were fused with TK-negative mouse LM(TK-) cells, and human-mouse somatic cell hybrid lines (LH81) were isolated using a HATG-ouabain selection system. The presence of HSV-1 TK activity in the hybrid lines was verified by disc polyacrylamide gel electrophoresis (PAGE) and by enzyme neutralization with type-specific rabbit anti-HSV-1 TK immunoglobulin. Karyotype analyses of several somatic cell hybrid clones using G-banding, Hoechst 33258 staining, and combined G-banding and Hoechst staining demonstrated that they retained only a few human chromosomes. A marker chromosome, M7, consisting of a chromosome 17 translocated to the short arm of 3, occurred in 25 of the 28 metaphases examined. Also chromosomes 8 and X were found in a minority of metaphases. Isozyme analyses showed that all 19 hybrid clones analyzed expressed human aminoacylase-1 (ACY1) and esterase D (ESD), markers for 3 and 13, respectively. Back-selection of somatic cell hybrid clones with 5-bromodeoxyuridine resulted in the isolation of several subclones lacking HSV-1 TK activity, human ACY1, human ESD, and the human chromosomes. These experiments suggest that the HSV-1 TK gene is associated with either M7 or a segment of 13, or both, in biochemically transformed HeLa(BU25)/KOS 8-1 cells. These experiments also permit localization of the ACY1 structural gene to the pter leads to p12 region of 3.

Human and mouse cells biochemically transformed by ultraviolet light (UV)-irradiated HSV-1 express HSV-1 thymidine kinase (TK) activity and also express type-specific herpesvirus-associated nuclear antigen(s) (HANA). To identify the HSV-1 DNA sequences coding for HANA and their location on the viral genome, studies were carried out on: (i) somatic cell hybrid clones obtained by fusing mouse [LM(TK−)] cells with UV-irradiated HSV-1-transformed human [HeLa(BU25)/KOS 8-1] cells; and (ii) LM(TK−) cells biochemically transformed with restriction endonuclease fragments of DNA which code for HSV-1 TK. Molecular hybridization experiments were also carried out and demonstrated that HSV-1 DNA sequences coding for TK were integrated in the biochemically transformed cells. The human-mouse somatic cell hybrid clones (LH81) which were HSV-1 TK+ were also HANA+, while clones counterselected in bromodeoxyuridine which had lost HSV-1 TK activity and DNA sequences likewise lost HANA. Previous studies had shown that the HSV-1 TK gene of LH81 hybrid clones was associated with a marker chromosome, designated M7, which consists of a human chromosome 17 translocated to the short arm of chromosome 3, or a modified M7 chromosome containing a translocation from a mouse chromosome. The present results indicate that at least one HANA gene was integrated in the same chromosome as the HSV-1 TK gene. LM(TK−) cells biochemically transformed by HSV-1 DNA restriction nuclease fragments of diminishing size, which map in the HpaI-I region (26.2 to 31.7) of the HSV-1 genome, were HANA+ as well as TK+. The HANA+ cells included LM(TK−)/TF pAGO PP and LM(TK−)/TF pAGO PS clones. The latter are clones of LM(TK−) cells biochemically transformed, respectively, by a PvuII fragment (1.35 × 106 daltons) and a PvuII-SmaI fragment (0.9 × 106 daltons; 30.2 to 31.1 map units) of HSV-1 DNA derived from Escherichia coli plasmid, pAGO. Since the PvuII-SmaI DNA fragment has only enough genetic information to code for a polypeptide of about 53,000 daltons and the HSV-1 TK polypeptide is about 40,000 daltons, the findings indicate that the genes for HSV-1 TK and one herpesvirus-associated nuclear antigen are either contiguous or overlapping, or HSV-1 TK and one HANA gene are identical.

The thymidine kinase (TK) gene of HSV-1 has been cloned in Escherichia coli K12 plasmids, pMH1, pMH1A, and pMH4. These plasmids contain a 1,92Obp HSV-1 TK DNA sequence, which replaces a 2,067 bp EcoR I to Pvu II sequence of plasmid pBR322 DNA. Superhelical DNAs of plasmids pMH1, pMH1A, and pMH4 as well as plasmid DNAs cleaved by EcoR I, Hinc II, Bg1 II, Sma I, and Pvu II transformed TK-deficient LM(TK-) cells to the TK+ phenotype. A 1,230bp EcoR I-Sma I fragment purified from pMH1 DNA (and from plasmid pAGO, DNA, the parent of pMH1) also transformed LM(TK-) cells. Serological and disc PAGE studies demonstrated that the TK activity expressed in biochemically transformed cells were HSV-1-specific. The experiments suggest that the HSV-1 TK coding region may be contained within a l.1kbp DNA sequence extending from about the Hinc II (or Bgl II) cleavage site to the Sma I site. 35S-methionine labeling experiments carried out on cell lines transformed by Hinc II-cleaved pMH1 DNA and by the EcoR I-Sma I fragment showed that the TKs purified from the transformed cells consisted of about 39-40,000 dalton polypeptides.