DUBBS, D. R. (University of Minnesota, Minneapolis), AND W. F. SCHERER. Inapparent viral infection of cells in vitro. III. Manifestations of infection of L mouse cells by Japanese encephalitis virus. J. Bacteriol. 91:2349-2355. 1966.-Nine strains of Japanese encephalitis (JE) virus were propagated serially in cultures of L cells reaching titers of 103-5 to 106.3. Although cytopathic effects were not seen in cultures of contiguous L cells after infection with JE virus, cell growth was inhibited. Moreover, cell destruction was readily apparent in infected cultures of sparse, noncontiguous L cells. Differences in the size of cell population of infected and noninfected cultures (i) occurred despite only 0.2 to 3.5% of the cells in infected cultures being associated with infectious virus, (ii) were greater in actively growing cultures than in those kept in maintenance media, and (iii) were probably in part related to an interferon produced in infected cultures. Overt disease usually does not result from infection of man and animals with Japanese encephalitis (JE) virus unless the central nervous system is invaded and encephalitis develops (20, 23, 28, 30). Viremia probably results from viral replication in non-neural tissues, yet the absence of disease suggests that non-neural cells escape injury. In an attempt to understand the relationship between non-neural cells and JE virus in vivo, studies were undertaken with strain L mouse fibroblasts in vitro as a model cell derived originally from a susceptible host and easily cultivated in vitro (5, 7). This report describes two methods of detecting JE virus in L-cell cultures which ordinarily show no cytopathology: (i) by measurement of virus-induced inhibition of cell growth in cultures of contiguous L cells, and (ii) by infection of sparse populations ofwidely separated L cells which, in contrast to contiguous cells, are destroyed by JE virus. MATERIALS AND METHODS Viruses. Eight strains of JE virus from Japan were employed after three to seven mouse brain passages I Presented at the Annual Meeting of the American Association of Immunologists, 1960 (Federation Proc. 19:386, 1960). 2Present address: Division ofBiochemical Virology, Baylor University College of Medicine, Houston, Tex. 3 Present address: Department of Microbiology, Cornell University Medical College, New York, N.Y. from the following: (i) Culex tritaeniorhynchus mosquitoes [strains M5/596, M5/572, and M5/560b (24, 26)]; (ii) blood of Black-crowned Night Heron nestlings [strain 281776, 281777, 281801 (26)]; and (iii) brains of fatal human cases of encephalitis [strains 55-1735 and 55-1456 (26)]. The ninth strain, Nakayama, was isolated in 1935 from an encephalitis patient in Japan (1, 26), and underwent more than 70 mouse passages prior to use. Virus inocula for L cell cultures were 10% (w/v) suspensions of mouse brains harvested 3 to 7 days after intracranial inoculation of either weanling (3 to 4 weeks old) or suckling (1 to 5 days old) Swiss albino mice. Brains were triturated in a mixture of rabbit serum, 2 parts, and Hank's (10) balanced salt solution (BSS), 3 parts, and centrifuged at 175 X g for 10 min; supernatant fluid was stored in sealed glass ampoules on Dry Ice. A strain of western encephalitis (WE) virus adapted to destroy L cells (6) was used for interferon assays; the virus was in fluid from the 40th passage in L-cell cultures. Cell cultures. L strain mouse fibroblasts were grown in a mixture of 200 parts horse serum (HS), 1 part yeast extract (YE; Difco), and 799 parts Hanks' BSS (HS-20, YE-0.1, BSS-79.9). Tube cultures were prepared by inoculating approximately 105 cells in 1 ml of growth medium per test tube (16 by 125 mm), and incubating at 37 C for 3 to 7 days. Just prior to inoculation with virus, medium was replaced with 0.9 ml of one of three mixtures: (i) 200 parts HS, 1 part YE, and 799 parts BSS (HS-20, YE-0.1, BSS79.9); (ii) 10 parts chicken serum and 90 parts maintenance solution (21) modified to omit purines and pyrimidines (CS-10, MS-90); and (iii) 5 parts HS and 2349 on O cber 6, 2017 by gest http/jb.asm .rg/ D ow nladed fom

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.

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.

Simian virus 40 T antigens accumulate in the cytoplasm of simian virus 40 tsA207 transformants of primary mouse kidney or human retinoblastoma cells grown at 40 degrees C in 10% serum.

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.

Although the size of marmoset herpesvirus (MarHV) DNA, estimated by velocity sedimentation in sucrose gradients, was similar to that of herpes simplex virus type 1 (HSV-1) DNA, the restriction endonuclease sites of MarHV and HSV-1 DNAs were quite different. A specific BamHI restriction fragment (6.2 x 10(6) daltons) of MarHV DNA biochemically transformed LM(TK-) mouse fibroblasts to the thymidine kinase(TK)-positive phenotype. Rabbit antisera, prepared against MarHV TK, inhibited MarHV-induced TK, but not HSV-1, HSV-2, or cellular TKs. Disc PAGE analyses and enzyme neutralization experiments with the anti-MarHV TK sera demonstrated that the TK expressed in MarHV transformants was MarHV-specific.

MKSA207 cells, a BALB/c mouse kidney line transformed by a tsA mutant of SV40, are temperature-dependent for the expression of the 'standard transformed phenotype'. At the permissive temperature (33.5 degrees C), the mKSA207 cells resembled wild-type (wt) SV40 transformants; they contained the intranuclear SV40 T antigen, grew to high saturation density in monolayer culture in either 10% or 0.5% serum, and also in methylcellulose suspension culture and became multinucleate in cytochalasin B. At the nonpermissive temperature (39.8 degrees C), the mKSA207 cells lost some of their transformed properties; they grew only to low density in 10% serum, hardly grew at all in 0.5% serum or in methylcellulose suspension culture, and remained mono- or binucleate in cytochalasin B. At 40 degrees C in low serum, mKSA207 cells lost the intranuclear T antigen and when fed 10% serum at 39.8 degrees C, accumulated large amounts of T antigen in the cytoplasm. Derivatives of mKSA207 have been selected at 39.8 degrees C in liquid medium and methylcellulose suspension culture. The heat adapted lines, like wt SV40 transformants, exhibited the standard transformed phenotype at both 33.5 and 39.8 degrees C. It is unlikely that acquisition of temperature-independence for the transformed phenotype was due to reversion of the tsA gene to wild-type because the heat-adapted cell lines displayed the cytoplasmic T antigen at 39.8 degrees C, characteristic of the parental mKSA207 cells and SV40 rescued from one of the heat-adapted lines was temperature sensitive for growth. The T antigen levels (complement fixation units per 10(6) cells) of heat-adapted lines grown at 39.8 degrees C were comparable to those of mKSA207 cells grown at 33.5 or 39.8 degrees C.

To investigate the size of herpes simplex virus (HSV) thymidine kinase (TK) polypeptides, procedures have been devised to purify the enzyme from infected cells labeled with 35S-methionine by (i) affinity chromatography on Sepharose-5'-amino-5'-deoxythymidine; (ii) preparative isoelectric focusing or preparative PAGE; and (iii) glycerol gradient centrifugation. Portions of enzyme fractions, at each purification step, were also treated with an immunoadsorbent, Sepharose-anti-HSV-1 TK immunoglobulin (IgG). Labeled polypeptides eluted from the immunoadsorbent were analyzed by electrophoresis in SDS slab gels and autoradiography. The results demonstrate that the molecular weights of HSV TK polypeptides are about 40,000. TK-negative HSV-1 mutant B2006 failed to induce the 40 K dalton polypeptide.

Previous studies have shown that thymidine kinase (TK)-negative mutants isolated from HSV-1 clone 101, HSV-2 (333), marmoset herpesvirus, and pseudorabies virus enhanced the resident TK activity of biochemically transformed mouse fibroblast [LM(TK−)/HSV-1 Cl 7] cells. The present study shows that 10 additional TK-negative HSV-1 mutants isolated from HSV-1 strains HF and KOS also stimulated the resident TK activity of LM(TK−)/HSV-1 Cl 7 cells. To identify possible HSV-1 gene(s) regulating the formation of the resident HSV-1 TK, the LM(TK−)/HSV-1 Cl 7 cells were superinfected at permissive (34.5°) and nonpermissive (39°) temperatures with TK-negative mutants that were also temperature-sensitive (ts) for virus replication. TK-negative, HSV-1 ts mutants from 12 complementation groups (tsA1, B2, C4, E6, F18, G3, J12, K13, M19, N20, O22, and ts-24) were studied. All of these TK-negative HSV-1 ts mutants enhanced the resident TK activity of LM(TK−)/HSV-1 Cl 7 cells at the permissive temperature, and all mutants, except HSV-1 ts B2, did so when infections were carried out at the nonpermissive temperature. The resident TK activity of LM(TK−)/HSV-1 Cl 7 cells was either unaffected, or it was depressed after infection at 39° by mutant ts B2 at multiplicities of infection of 3–25 PFU/cell. The experiments indicate that the product of HSV-1 gene B is one of the regulatory proteins required for TK enhancement in biochemically transformed cells.

Association of herpes simplex virus (HSV)-related antigens with chromosomes was demonstrated in human and mouse cells biochemically transformed by HSV that had been irradiated with ultraviolet light. This was accomplished by using peroxidase-anti-peroxidase immunological staining with rabbit antisera that had high neutralizing titers against both HSV-specific thymidine kinase activity and virus infectivity. Antisera-against HSV did not react with chromosomes of uninfected cells nor did normal sera react with any of the constitutents of biochemically transformed cells. Methanol/acetic acid treatment of biochemically transformed cells eliminated their nuclear staining for HSV-related antigens. In vitro binding of HSV-related antigens to chromosomes was demonstrated by incubating soluble antigens from high salt extracts of HSV-infected cells with methanol/acetic acid-fixed chromosomes of biochemically transformed or uninfected cells, followed by exposure to antiserum against HSV and peroxidase-anti-peroxidase staining. There was no staining when soluble extracts from uninfected cells were substituted for those from HSV-infected cells. The results show that cells biochemically transformed and lytically infected by HSV, respectively, contain antigens, which like the Epstein-Barr virus-associated nuclear antigen (EBNA), bind to chromosomes in vivo and in vitro.

The role of SV40 gene A product in initiation of cellular DNA synthesis was investigated, using a mouse kidney line [mKSA207] transformed by SV40ts A207. mKSA207 cells were temperature sensitive for growth, lost SV40 T antigen (Tag) when incubated in low serum at 40°C, and accumulated Tag in the cytoplasm when fed 10% serum and incubated at the nonpermissive temperature (39.7°C). Following serum addition, the percentage of mKSA207 cells synthesizing DNA was essentially the same at nonpermissive (39.7°C) and permissive temperatures (33.5°C). The cells entered S phase asynchronously at both temperatures, but most cells entered S within 16 h, and before Tag accumulated. mKSA207 synchronized by a double thymidine block also synthesized DNA at 39.7°C and entered a second S phase. Tag-depleted or Tag-synchronized mKSA207, when fused with chick erythrocytes (CE), activated CE DNA synthesis. At nonpermissive temperatures (39.7°C), 40% of CE nuclei in heterokaryons with Tag-depleted mKSA207 displayed3H-thymidine-labeled nuclei 28–40 h after fusion, when only 12% of CE nuclei were Tag+. The experiments indicate that SV40 gene A product probably does not have a direct role as initiator of cellular DNA synthesis.

Nonsynchronized and hydroxyurea (HU)-synchronized SV40-transformed human cells (W98VaD) were fused with chick embryo erythrocytes (CE). The uptake of T antigen by CE nuclei was compared with initiation of chick nuclear DNA synthesis. Uptake of T antigen by CE nuclei occurred at about the same time after fusion with asynchronous as with HU-synchronized cells. CE nuclei rapidly became T antigen-positive between 16 h and 28 h after fusion and usually almost all CE nuclei were T antigen-positive by 48 h after fusion. In contrast, initiation of chick nuclear DNA synthesis occurred as a function of time after reversal of the HU block, when the host cell nuclei were also synthesizing DNA. Chick nuclear DNA synthesis occurred in many heterokaryons before the CE nuclei became T antigen-positive by immunofluorescence.

Thymidine kinase (TK) activity of herpes simplex virus-transformed cells [LM(TK − )/HSV-1 and LM(TK-)/HSV-2] was stimulated 6 to 9 hr after the cells were infected with TK-negative mutants of either type 1 or type 2 herpes simplex virus (HSV-1 TK-, HSV-2 TK − ) or with TK-negative mutants of marmoset herpesvirus (MarHV TK − ) and pseudorabies virus (PRV TK − ) Inhibitors of DNA synthesis did not prevent this stimulation, but inhibitors of RNA and protein synthesis did. In contrast, equine herpesvirus (EHV-1) and TK-negative mutants of vaccinia virus did not enhance the TK activity of the transformed cells, showing that stimulation was not a consequence merely of nonspecific virus infection. HSV-1 132015, a mutant deficient in inducing TK activity in productively infected LM(TK − ) cells at 37°, stimulated HSV-transformed cell TK at this temperature, suggesting that the gene which controls TK turn-on probably functions normally in mutant 132015. These experiments suggest that enhancement by TK-negative herpesviruses can occur regardless of whether the superinfecting herpesvirus produces an inactive TK polypeptide or none at all. Autoradiographic and enzyme studies on a subline of LM(TK-)/HSV-1 cells grown in nonselective medium without HAT (hypoxanthine, aminopterin, and thymidine) demonstrated that essentially all of the cells contained significant TK activity. The TK activity of this subline and of clonal sublines isolated in nonselective medium or in counterselective medium with bromodeoxyuridine (BrUdR) was enhanced by HSV-1 TK − infection, supporting the suggestion by Davidson and co-workers that the HSV TK gene was retained in most transformed cells grown in nonselective medium. Immunological studies, using type-specific immunoglobulin (IgG) fractions prepared from sera of rabbits that had been immunized with HSV-1 TK or HSV-2 TK, demonstrated that the enhanced TK activity of HSV-1-transformed cells was inhibited by anti-HSV-1 IgG and that of HSV-2-transformed cells was inhibited by anti-HSV-2 IgG, regardless of whether the transformed cells were superinfected with HSV-1 TK − , HSV-2 TK − or MarHV TK − . Neither antiserum inhibited the TK activity induced by wild-type MarHV in LM(TK − ) cells, indicating that the TK activity stimulated by TK-negative herpesvirus infections of HSV-transformed cells had the antigenic specificity of the resident virus TK.

Herpes simplex virus (HSV)‐related antigens have been demonstrated in the nuclei and cytoplasm of human and mouse cells biochemically transformed by ultraviolet light‐irradiated HSV. This was accomplished by using peroxidase/anti‐peroxidase immunological staining and indirect immunofluorescence with rabbit antisera that had high neutralizing titers against the HSV‐specific thymidine kinase activity and virus infectivity. HSV‐1 antisera reacted with antigens in cells biochemically transformed by type 1 HSV, but not with those of cells biochemically transformed by type 2 HSV. Similarly, HSV‐2 antisera reacted with antigens in cells biochemically transformed by HSV‐2, but not with those in cells biochemically transformed by HSV‐1. In contrast, herpes virus‐related antigens were detected in cells cytolytically infected with HSV‐1 and with HSV‐2 by either type 1 or type 2 HSV antisera. These observations suggest that the antigens detected in the biochemically transformed cells were a type‐specific subset of the HSV‐related antigens synthesized in cells undergoing productive infection by HSV‐1 and HSV‐2.

To investigate the chromosomal site(s) of integration of the herpes simplex virus type 2 (HSV‐2) thymidine kinase (TK) gene in biochemically transformed human cells, transformed human cells[HeLa (BU25)/HSV‐2‐6 Cl 4] were fused with TK‐deficient mouse fibroblast [LM(TK − )] cells and seven lines of human—mouse somatic cell hybrids [HL/1… HL/7] were isolated. Enzyme assays demonstrated that the HL somatic cell hybrid lines expressed significant TK activity. That the TK activity was HSV‐2 specific was verified by disc PAGE analyses, by superinfection experiments with TK‐negative HSV mutants, and by enzyme neutralization experiments with anti‐HSV‐2 TK immunoglobulins. The seven HL somatic cell hybrid lines contained a complement of mouse chromosomes and one or more human chromosomes. The sensitivities of the HL hybrid lines to poliovirus types 1 and 2 and to diphtheria toxin were also studied. The poliovirus challenge experiments showed that hybrid lines HL/1, HL/3, HL/4 and HL/5 were completely resistant to poliovirus and that less than 25% of the cells in line HL/2 were destroyed, indicating that with the exception of HL/2, human chromosome 19 was not retained in the human—mouse hybrid cells. Hybrid lines HL/1‐1, HL/2‐1, HL/3, HL/4, HL/5, HL/6 and HL/7 were insensitive to diphtheria toxin, indicating that human chromosome 5 was not retained in these human—mouse hybrid cells. Karyotype analyses showed that the only human chromosome found in hybrid lines HL/3, HL/4, HL/5 and HL/6 was marker chromosome M13; this chromosome was present in the majority of HL/3 cells and in all HL/4, HL/5, and HL/6 hybrid cells. The same chromosome was present in all cells of hybrid HL/7, in 17/19 analyzed mitosis of HL/2, and in all cells of HSV‐2 TK‐positive HL/2 subclones but, in addition, several other human chromosomes were found in the HL/7 and HL/2 hybrid lines. After counterselection with bromodeoxyuridine (Brd Urd), chromosome M13 was absent from all analyzed cells of HSV‐2 TK‐negative, Brd Urdcounterselected HL/2 subclones, but some of the other human chromosomes were still present. Morphology and G banding analyses suggest that M13 is an isochromosome formed from the short arm of human chromosome X. The only human chromosome present in cells of somatic hybrid HL/1 and four of its HSV‐2 TK‐positive subclones was chromosome 17 with a translocation on the short arm. This chromosome was present in all cells of hybrid HL/1 and its subclones and was absent in all cells of four analyzed HSV‐2 TK‐negative, Brd Urdcounterselected subclones. The preceding experiments demonstrate that the HSV‐2 TK gene is associated with human marker chromosome M13 and modified human chromosome 17 in HSV‐2‐transformed human line, HeLa(BU25)/HSV‐2‐6 Cl 4.

Chinese hamster cell line K12 is temperature-sensitive for the initiation of DNA synthesis. K12 cells synchronized by serum deprivation were collected in early G1(G0).Heterokaryons were formed by fusing chick erythrocytes with serum-starved K12 cells through the use of UV-irradiated Sendai virus. At the permissive temperature (36.5‡ C), erythrocyte nuclei in heterokaryons enlarged, the chromatin dispersed, and erythrocyte nuclei synthesized DNA at about the same time as the K12 nuclei. At the restrictive temperature (41‡ C), erythrocyte nuclei enlarged, but neither erythrocyte nor K12 nuclei initiated DNA synthesis. When erythrocyte nuclei were fused with Wg-1A cells, the wild-type parent for ts K12 cells, both kinds of nuclei synthesized DNA at 36.5‡ C and 41‡ C. Activation of erythrocyte nuclei was inefficient in heterokaryons incubated in low-serum medium. The results indicate that serum factors and a cellular function defined by the K12 mutation are required for activation of chick erythrocyte nuclear DNA synthesis.