Mutagenicity of 2-amino-N6-hydroxyadenine (AHA) at three loci in L5178Y/tk+/- mouse lymphoma cells: molecular and preliminary cytogenetic characterizations of AHA-induced tk-/- mutants.

2-Amino-N6-hydroxyadenine (AHA) is a remarkably efficient and specific inducer of point mutations in Neurospora, with few or no larger scale events being detected (de Serres et al., 1985). In the present studies, AHA is shown to be a potent point mutagen at the tk +/-, hprt+ and Na+/K+ ATPase loci in L5178Y/tk (+/-)-3.7.2C mouse lymphoma cells. Both large and small colony tk-/- mutants were analyzed at the molecular level and a preliminary assessment was made of small colony mutant karyotypes (230 bands/haploid metaphase cell; large colony mutants typically have normal karyotypes and were not analyzed). AHA induced greatly delayed (7-9 cell doublings) cytotoxicity, suggestive of a mutational mechanism (e.g., base-pair substitution) requiring DNA replication prior to its phenotypic expression. Approximately one-third of the tk -/- mutants formed small colonies, a phenotype which is typically associated with alterations to chromosome 11b, the site of the functional tkb allele in the parental cells. However, banded karyotypes have provided convincing evidence for alterations chromosome 11b in only 2 of the 7 small colony mutants analyzed. Southern blot analysis showed that 78% (21/27) of these small colony mutants have retained the Nco-1 6.3-kb band, which is diagnostic of the tkb allele. This makes AHA unique among the mutagens examined so far in inducing small colony mutants without inducing large losses of tkb DNA. Although a dose-dependent increase in the proportion of small colony mutants was noted, no significant dose-dependent differences were seen at the molecular level in the relatively few mutants analyzed. The majority of AHA-induced tk -/- mutants formed large colonies. Southern blot analysis showed that 86% (25/29) of these had retained the Nco-1 6.3-kb band which is diagnostic of the tkb allele. It is concluded that AHA induces primarily micromutations (less than 100 base pairs), probably through a base-pair substitution mechanism, at the tk, hprt and Na+/K+ ATPase loci in this system, with some larger scale damage (kilobases of DNA at the molecular level; chromosome 11b damage at the cytogenetic level) also occurring.     

Unexpected loss of genomic DNA from agarose gel plugs

Intact chromosomal DNAs are routinely prepared by embedding cells in agarose plugs before lysis. The large sizes of the genomic DNAs cause their retention while other macromolecules diffuse into and out of the gel matrix during lysis, washing and restriction cleavage incubations. However, in an analysis of agarose-embedded chromosomal DNAs cleaved with restriction enzymes, fragments larger than 30 kilobases were found to have eluted from the gel plugs. Since loss of fragments from gel plugs may affect qualitative and quantitative interpretations of electrophoretic patterns, an analysis of the diffusion of DNA segments from agarose plugs was performed. The two variables monitored were the time dependence and the DNA fragment size dependence of the diffusion process. The results indicate that small fragments (less than or equal to 2 kilobases) are quickly lost from 1% agarose gel plugs; moreover, significant amounts of large DNA segments (i.e., the 48.5-kilobase lambda phage chromosome) are also lost. In addition to urging caution in the analysis of restriction cleavage data, these observations suggest that intact small organelle genomes and extrachromosomal DNAs also may be lost from genomic DNAs prepared in agarose gel plugs.     

Mutagenicity of 2-amino-N6-hydroxyadenine to TK6 human lymphoblast cells

TK6 human lymphoblast cells (tk +/-; hprt+) were treated with various concentrations of 2-amino-N6-hydroxyadenine (AHA) for 24 h. AHA was quite toxic to TK6 cells in the dose range 0-0.05 micrograms/ml, but additional toxicity was not observed between 0.05 and 0.10 micrograms/ml. AHA induced mutations at 2 distinct genetic loci: the autosomal thymidine kinase (tk) and the X-linked hypoxanthine-guanine phosphoribosyl transferase (hprt). Significant levels of both tk-NG mutants (normal growth rate of 16-18 h, colonies visible after 10-11 days incubation) and tk-SG mutants (slow growth rate of greater than 24 h, colonies visible after 18 days incubation) were induced. 15 hprt- mutants were isolated and analyzed by Southern blot. 8 of these had normal restriction fragment patterns after digestion with PstI, EcoRI, and HindIII, and were defined as 'point' mutations; the remaining 7 had partial deletions of the hprt gene. 32 tk- mutants were also isolated. 3 of 22 normal growth mutants and 6 of 10 slow growth mutants had lost the active tk allele. These data suggest that both point mutations and larger-scale alterations are induced by AHA.     

Construction of a mammalian transducing vector from the genome of Moloney murine leukemia virus.

A 0.9-kilobase DNA fragment from the genome of Moloney murine leukemia virus, including the viral long terminal repeat, was covalently linked to the herpes simplex virus I thymidine kinase (tk) gene whose promoter was previously removed. The hybrid DNA structure was introduced into the chromosome of tk- mouse cells at single copy numbers, via transfection procedures. Cells expressing the newly introduced tk gene were identified by the HAT selection procedure and analyzed for tk- and moloney murine leukemia virus-specific DNA and RNA sequences by blot hybridization procedures. Expression of the tk gene is dependent on function(s) provided in cis by the viral DNA fragment. Vectors derived from this region are termed rGag (rG) vectors.     

Cloning of type 8 capsule genes and analysis of gene clusters for the production of different capsular polysaccharides in Staphylococcus aureus.

Eleven serotypes of capsular polysaccharide from Staphylococcus aureus have been reported. We have previously cloned a cluster of type 1 capsule (cap1) genes responsible for type 1 capsular polysaccharide biosynthesis in S. aureus M. To clone the type 8 capsule (cap8) genes, a plasmid library of type 8 strain Becker was screened with a labelled DNA fragment containing the cap1 genes under low-stringency conditions. One recombinant plasmid containing a 14-kb insert was chosen for further study and found to complement 14 of the 18 type 8 capsule-negative (Cap8-) mutants used in the study. Additional library screening, subcloning, and complementation experiments showed that all of the 18 Cap8- mutants were complemented by DNA fragments derived from a 20.5-kb contiguous region of the Becker chromosome. The mutants were mapped into six complementation groups, indicating that the cap8 genes are clustered. By Southern hybridization analyses under high-stringency conditions, we found that DNA fragments containing the cap8 gene cluster show extensive homology with all 17 strains tested, including type 1 strains. By further Southern analyses and cloning of the cap8-related homolog from strain M, we show that strain M carries an additional capsule gene cluster different from the cap1 gene cluster. In addition, by using DNA fragments containing different regions of the cap8 gene cluster as probes to hybridize DNA from different strains, we found that the central region of the cap8 gene cluster hybridizes only to DNAs from certain strains tested whereas the flanking regions hybridize to DNAs of all strains tested. Thus, the cap8 gene clusters and its closely related homologs are likely to have organizations similar to those of the encapsulation genes of other bacterial systems.     

Preliminary molecular analysis of the TK locus in L5178Y large- and small-colony mouse lymphoma cell mutants

Mouse lymphoma cells of the L5178Y TK+/- -3.7.2C line were exposed to sidestream and mainstream cigarette smoke condensates (CSC). Cells which survived the trifluorothymidine (TFT) challenge fell in 2 classes: large- and small-colony formers. Southern blot analysis of NcoI-digested DNA from mutant colonies yielded 2 distinct restriction fragment banding patterns when probed with the thymidine kinase (TK) cDNA clone pMtk4. One such pattern was composed of 4 bands at 6.4, 5.5, 4.7 and 2.9 kilobase pairs (kb) and was identical to that of TK+/- controls. A second pattern differed from the first only in the absence of the 6.4-kb band. The majority (83/95) of both large and small colonies derived from cells exposed to CSC exhibited restriction fragment banding patterns lacking the 6.4-kb band. The data from the present study suggest that there is no association between mutant colony size and the presence of the 6.4-kb NcoI restriction fragment at the TK locus in the mouse lymphoma mutants analyzed.     

A rapid method for determining the relationship between cDNA clones

We describe a technique for rapidly screening the inserts of plasmids for homology to each other by using DNA fragments isolated in agarose gels to probe Southern blots of DNA prepared by the "miniprep" alkaline lysis method. The procedure includes a technique for labeling DNA fragments in agarose gel slices without further purification. The protocol results in a significant savings in time and expense and a considerable increase in fragment yield over methods involving fragment purification from polyacrylamide or agarose gels.     

Characterization of mutants of herpes simplex viruses, types 1 and 2, that produce fragments of the thymidine kinase polypeptide

Seven tk- mutants of herpes simplex virus, type 2 (HSV-2), and three tk- mutants of herpes simplex virus, type 1 (HSV-1), were isolated which did not produce the thymidine kinase (TK) polypeptides but formed smaller polypeptides not seen in wild-type infected cells. Positive TK mRNA selection by hybridization to the cloned tk genes followed by in vitro translation identified the TK polypeptides. Comparisons of the products of partial proteolysis of the polypeptides of four HSV-2 and two HSV-1 tk- mutants to those of the parental TK polypeptides indicated that, in each case, the novel polypeptide was a fragment of the TK polypeptide, showing that these mutants have defects in the structural gene for tk. HSV-2 mutants of this sort have not been previously described. They and the HSV-1 mutants are similar to HSV-1 mutants reported previously. In addition, it was found that TK mRNA was present early in infection but was absent late in infection, suggesting that the shutoff of TK synthesis is due to message degradation. Also, HSV-2 TK mRNA did not hybridize to the cloned HSV-1 tk gene indicating that these genes have extensively diverged.     

Restriction fragment length polymorphism of the rat albumin gene in Sprague-Dawley rats and its application in genetic study of analbuminemia.

Restriction fragment length polymorphism of the rat albumin gene was discovered in a stock of Sprague-Dawley rats by Southern blots of rat liver DNAs using cloned albumin cDNA, prAlb-1 (1), as a probe. The polymorphic DNA fragments were observed when rat DNAs were digested with either Hind III or Pst 1 and the difference in length of the DNA fragments in Hind III or Pst 1 digests was estimated as 1.4 kbp. When DNAs were digested with EcoR I, restriction fragment length polymorphism was not observed. Therefore, this polymorphic DNA was concluded to be located in the flanking sequence. Structural analysis of the cloned albumin gene showed that the polymorphism was located in the 3'-flanking sequence. With this polymorphism as a marker of the albumin structural gene, the phenotype of analbuminemia, which is an autosomally recessive trait, was found to be linked to the structural gene of albumin.     

Expression of unselected adenovirus genes in human cells co-transformed with the HSV-1 tk gene and adenovirus 2 DNA

We have introduced adenovirus 2 genes into high molecular weight DNA of permissive human cells by co-transformation of tk- human 143 cells with Ad2 restriction enzyme fragments and a cloned Bam HI fragment that carries the HSV-1 thymidine kinase gene. Tk+ cells were isolated after selection and maintenance in HAT medium. Several co-transformed lines are able to complement the growth of Ad5 dl312 (delta 1.2--3.7) and Ad5 dl434 (delta 2.6--8.7), deletion mutants that lack sequences from the left end of the viral genome. The amount and arrangement of viral sequences in the co-transformed cell lines have been analyzed by restriction endonuclease digestion and filter hybridization. Most of the cell lines contain a single insertion of the HSV-1 tk fragment and a single insert of adenoviral DNA. However, one line (B1) contains at least four different insertions, two of which are present in multiple copies. The adenoviral DNA in all cell lines is composed of sequences from the left end of the genome and extends for varying lengths in different lines. Two cell lines that complement deletion mutants efficiently synthesize both early region 1a and 1b mRNAs. The B1 line synthesizes low levels of 1a mRNA, higher levels of 1b mRNA and a unique mRNA that maps to the right of the 1b gene family. When grown continuously in HAT medium, some cell lines are quite stable while others are fairly unstable. Some tk+ subclones support the growth of viral mutants as well as the parental line while others give reduced levels of complementation. For all tk+ subclones examined, the alteration or reduction in viral gene expression is independent of changes in the pattern of integration of viral DNA.     

Genetics of Bacillus subtilis chemotaxis: isolation and mapping of mutations and cloning of chemotaxis genes

We isolated a collection of chemotaxis mutants and characterized them for chemotactic phenotype and genotype. The mutations of most of these mutants mapped in the region between pyrD and thyA. However, the mutation in the gene specifying the chemotactic methyltransferase mapped very close to aroF. From a bank of phages containing Bacillus subtilis DNA we identified two lambda charon4 phages that contained genes specifying chemotactic functions. The inserted DNAs were removed by digestion with restriction endonuclease EcoRI and were found to share a 4.0-kilobase (kb) fragment. One of these DNAs also contained a 7.7-kb fragment, and the other also contained a 10.9-kb fragment. We identified mutants that were complemented by each fragment. The fragments were each ligated into plasmid pFH7 and were incorporated into lysogenic SP beta c2 or a deletion mutant of SP beta c2 in order to form transducing phages. The mutants in the collection containing mutations that mapped in the region between pyrD and thyA were tested for complementation by transducing phages containing the 4.0-kb fragment, the 7.7-kb fragment, the 4.0-kb fragment plus the 7.7-kb fragment, and the 10.9-kb fragment. A total of 24 mutants were complemented by the 4.0-kb fragment, 7 were complemented by the 7.7-kb fragment, 9 were complemented by the 4.0-kb fragment plus the 7.7-kb fragment, 15 were complemented by the 10.9-kb fragment, and 25 were complemented by none of the fragments.     

Mucopolysaccharidosis IVA: structural gene alterations identified by Southern blot analysis and identification of racial differences

Ninety-six alleles (36 alleles of Japanese and 60 of Caucasian origin) from forty-eight patients with mucopolysaccharidosis IVA were investigated for structural gene alterations using Southern blot analysis. All patients had a previously demonstrated deficiency of N-acetylgalactosamine-6-sulfate-sulfatase and exhibited a wide spectrum of clinical severity. Initially, using the fulllength cDNA as a probe, five of 36 chromosomes from the Japanese patients revealed similar rearrangements with respect to DNA digested with BamHI, SacI, and XhoI. Subsequent analysis using seven genomic fragments, covering the entire gene, enhanced the detection of aberrant fragments produced by the above restriction enzymes. Conversely, the 60 chromosomes of Caucasian origin revealed no evidence of large structural rearrangements when analyzed by these methods. There was a statistically significant difference between the two populations (P < 0.01). A severely affected Japanese patient showed structural rearrangements on both chromosomes by means of BamHI blots. An 8.0-kb fragment and a highly polymorphic 7.0-kb to 11.0-kb fragment present in normal individuals disappeared and two aberrant fragments of 11.5 kb and 12.0 kb were observed. Three other Japanese patients also showed these two aberrant fragments, in addition to the normal fragment pattern, and were thus heterozygous for this rearrangement. Interpretation of Southern blots was difficult because of the complexity of polymorphic bands resulting from variable number of tandem repeat elements. However, by utilizing these aberrant fragments or polymorphic bands, carrier detection was effective, even in families with poorly characterized mutations. Hybridization with probe MG-A (5end genomic probe in intron 1) showed a 8.4-kb fragment in BamHI blots of one Japanese and one Caucasian patient; XhoI, SacI, and EcoRI blots were normal. Since this BamHI alteration was also observed in one normal control, it appears to be a rare nonpathological polymorphism.     

Mutation at autosomal loci of Chinese hamster ovary cells: involvement of a high-frequency event silencing two linked alleles.

Two classes of cell lines heterozygous at the galactokinase (glk) locus have been isolated from Chinese hamster ovary cells. Class I, selected by plating nonmutagenized wild-type cells at low density in medium containing 2-deoxygalactose at a partially selective concentration, underwent subsequent mutation to the glk-/- genotype at a low frequency (approximately 10(-6) per cell), which was increased by mutagenesis. Class II heterozygotes, isolated by sib selection from mutagenized wild-type cells, had a higher spontaneous frequency of mutation to the homozygous state (approximately 10(-4) per cell), which was not affected by mutagenesis. About half of the glk-/- mutants derived from a class II heterozygote, but not the heterozygote itself, were functionally hemizygous at the syntenic thymidine kinase (tk) locus. Similarly, a tk+/- heterozygote with characteristics analogous to the class II glk+/- cell lines underwent high-frequency mutation to tk-/-, and most of these mutants, but not the tk+/- heterozygote, were functionally hemizygous at the glk locus. A model is proposed, similar to that for the mutational events at the adenine phosphoribosyl transferase locus (W. E. C. Bradley and D. Letovanec, Somatic Cell Genet. 8:51-66, 1982), of two different events, high and low frequency, being responsible for mutation at either of the linked loci tk and glk. The low-frequency event may be a point mutation, but the high-frequency event, in many instances, involves coordinated inactivation of a portion of a chromosome carrying the two linked alleles. Class II heterozygotes would be generated as a result of a low-frequency event at one allele, and class I heterozygotes would be generated by a high-frequency event. Supporting this model was the demonstration that all class I glk+/- lines examined were functionally hemizygous at tk.     

Identification of a Genetic Locus in Pseudomonas aureofaciens Involved in Fungal Inhibition

In iron-rich conditions, Pseudomonas aureofaciens PA147-2 produces an antibiotic-like compound that inhibits the growth of a plant fungal pathogen, Aphanomyces euteiches. To contribute to the potential use of PA147-2 as a biocontrol organism, we report the identification of a genetic locus important for antibiotic biosynthesis. Mutants defective for fungal inhibition (Af^-) were generated by Tn5 mutagenesis. Southern hybridization of total DNAs from three Af^- mutants indicated that loss of fungal inhibition was due to a single Tn5 insertion in each mutant. Restriction mapping of the mutation points showed that in two mutants the Tn5 insertions were in the same 16.0-kb EcoRI fragment and were separated by 2.1 kb. A genomic library of PA147-2 was constructed and screened by using a region of DNA flanking the Tn5 insertion in one mutant (PA109) as a probe to recover complementing cosmids. Three cosmids containing a 16.0-kb EcoRI fragment complementary to the two mutants were recovered. Allele replacement by homologous recombination with putative complementing cosmids restored one mutant to antifungal activity against A. euteiches. Southern analysis of the complemented mutants confirmed that allele replacement had occurred between cosmid DNA and Tn5. The wild-type 16.0-kb EcoRI fragment was cloned from the cosmid and complemented the two mutants to antifungal activity. An antifungal compound was isolated from PA147-2 grown on solid medium. Antifungal activity correlated to a peak on high-pressure liquid chromatography analysis. Under the same growth and extraction conditions, the antifungal activity seen in PA147-2 was absent in two Af^- mutants. Furthermore, absence of an antifungal compound in each mutant correlated to the absence of the wild-type “antifungal” peak on high-pressure liquid chromatography analysis.     

Transfer of genes to Chinese hamster ovary cells by DNA-mediated transformation

We have transferred DNa to Chinese hamster ovary (CHO) cells by DNA-mediated transformation. CHO tk- cells were transformed with the clones gene for herpes simplex virus thymidine kinase (HSV-tk) and were found to have a 50-fold lower frequency of transformation than mouse Ltk- cells at the same DNA dosage. By altering the amount of tk gene and carrier DNA present, frequencies of up to 5 x 10(-5) were obtained. CHO HSV-tk+ transformants were very stable, and in several clones the HSV-tk gene copies integrated in higher-molecular-weight DNA. These cells also exhibited cotransformation for unselected markers. CHO lines were also transformed at a frequency of 10(-4) with the bacterial gene Ecogpt in a SV40-pBR322 vector. CHO tk-cells could be transformed at a frequency of 10(-7) with cellular DNA isolated from CHO tk+ cells. CHO cells offer a well-defined genetic system within which to transfer either cloned or whole cellular DNAs.     

The transfer and stable integration of the HSV thymidine kinase gene into mouse cells.

Treatment of mutant mouse cells (Ltk-) deficient in thymidine kinase with Bam I restriction endonuclease-cleaved HSV-1 DNA results in the appearance of numerous surviving colonies which stably express thte tk+ phenotype. Through a series of electrophoretic fractionations in concert with transfection assays, we isolated a 3.4 kb fragment which contains the thymidine kinase gene and which alone is competent in the biochemical transformation of Ltk- cells. In this report, we have examined the distribution of tk sequences in the DNA of several transformed clones following stable gene transfer. A series of complementary experiments involving reassociation kinetics in solution and annealings with tk DNA to restriction-cleaved cellular DNA following electrophoresis and transfer to filters allow us to make the following general conclusions concerning the fate of the tk gene in all clones examined: the tk gene is present in all cells at a frequency of one copy per chromosomal complement; the tk gene is stably integrated in the DNA of all transformants; and integration is not site-specific and occurs at different loci in the DNA of all transformants examined. The existence of a single active tk gene in tk+ transformants now facilitates an analysis of the sequence organization of tk- mutant cells and provides a useful model system for studies on the transfer of cellular genes.     

Thymidine kinase deficient human cells have increased UV sensitivity in their capacity to support herpes simplex virus but normal UV sensitivity for colony formation

A thymidine kinase deficient (tk-) and two thymidine kinase proficient (tk+) human cell lines were compared for UV sensitivity using colony-forming ability as well as their capacity to support the plaque formation of herpes simplex type 1 (HSV-1). The tk- line (143 cells) was a derivative of one of the tk+ lines (R970-5), whereas the other tk+ line (AC4 cells) was a derivative of the 143 cells obtained by transfection with purified sheared HSV-2 DNA encoding the viral tk gene. 143, R970-5 and AC4 cells showed a similar UV sensitivity for colony-forming ability. In contrast, the capacity to support HSV-1 plaque formation immediately (within 1 h) after UV-irradiation was reduced to a greater extent in the 143 cells compared to the R970-5 and AC4 cells. Capacity curves for plaque formation of the HSV-1: KOS wild-type (tk+) strain were similar to those for the HSV-1: PTK3B mutant (tk-) strain in the 3 cell strains, indicating that the viral tk gene does not influence the ability of HSV-1 to form plaques in UV-irradiated compared to unirradiated human cells. Cellular capacity for HSV-1 plaque formation was found to recover in both tk- and tk+ cells for cultures infected 24 h after UV-irradiation. These results suggest that repair of UV-damaged DNA takes place to a similar extent in both tk- and tk+ human cells, but the kinetics of repair are initially slower in tk- compared to tk+ human cells.     

Alterations in the number of rRNA operons within the Bacillus subtilis genome

Deletions and additions of rRNA gene sets in Bacillus subtilis were observed by Southern hybridizations using cloned radiolabeled rDNA sequences. Of the ten rRNA gene sets found in B. subtilis 168M or NCTC3610, one was deleted in strains possessing the leuB1, ilvC1, argA2 and pheA1 mutations. Among EcoRI restriction fragments of genomic DNA products, a 2.9-kb 23S rRNA homolog was missing. In HindIII digest, both 5.5- and 5.1-kb hybrid bands were lost with 16S and 23S probes, respectively. Similarly, genomic DNAs digested with SmaI showed the absence of both 2.1- and 2.0-kb fragments that hybridized to 16S and 5S sequences, respectively, in wild-type genomes. In contrast, B. subtilis strain 166 and its derivatives displayed a gain of a 3.3-kb HindIII fragment homologous to 16S rRNA. Transforming the ilvC1 and leuB1 mutations into new genetic backgrounds revealed in some clones the concomitant introduction of the ribosomal defect. Transformations with the slightly heterologous donor DNA from strain W23 yielded some Leu+ and Arg+ transformants with altered hybridization patterns when probed with cloned sequences. We propose that the deletion of the rRNA operon occurred in the ilv-leu gene cluster of the B. subtilis genome as a result of unequal recombination between redundant sequences.     

Structural gene aberrations in mucopolysaccharidosis II (Hunter)

Summary A total of 14 unrelated German patients with X-linked iduronate-2-sulfatase (IDS) deficiency (Hunter syndrome, MPS II) showing variable clinical manifestations was screened for structural gene aberrations by Southern analysis. Using the IDS cDNA clone c2S15 as a probe, no Southern fragments could be detected in blots in the severely affected patient G-65 with respect to DNA digested by HindIII, PstI and TaqI, suggesting a total loss of the IDS structural gene. In this patient, the flanking loci DXS 297, DXS 296 and DXS 466 were tested. The locus DXS 466 is involved in the deletion, whereas both of the other loci are present. A normal 9.0-kb fragment disappeared and an aberrant fragment of 3.5 kb occurred in the HindIII blot of patient G-117. A normal Southern pattern was found in PstI and TaqI blots of this patient. This result can be interpreted as the generation of an additional HindIII restriction site by point mutation in an IDS gene intron.     

Role of the viral and cellular encoded thymidine kinase in the repair of UV-irradiated herpes simplex virus

A strain of herpes simplex type 1 (HSV-1:KOS) encoding a functional thymidine kinase (tk+) gene and a thymidine kinase deficient (tk-) mutant strain (HSV-1:PTK3B) were used as probes to examine the repair of UV-damaged viral DNA in one tk- (143) and two tk+ (R970-5 and AC4) human cell lines. UV survival for each HSV-1 strain was similar for infection of both tk- and tk+ cells suggesting that the repair of viral DNA was not dependent on the expression of a functional cellular tk gene. In contrast, UV survival of HSV-1:PTK3B was substantially reduced compared to HSV-1:KOS when infecting all 3 human cell lines, as well as Vero monkey kidney cells and LPM1A mouse cells. These results suggest that the repair of UV-irradiated HSV-1 in lytically infected mammalian cells depends, in part at least, on the expression of the viral encoded tk.