Biochemical transformation of LM(TK-) cells by hybrid plasmids containing the coding region of the herpes simplex virus type 1 thymidine kinase gene

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.     

Replication of pSV2-gpt in COS-1 cells: Stability of plasmid DNA in the presence and absence of biochemical selection

We have previously demonstrated that COS-1 cell lines transformed by pSV2-gpt and maintained under biochemical selection replicate multiple copies of extrachromosomal plasmid DNA (1). We have now examined the replication and stability of this DNA in a representative cell line. In situ hybridization analyses revealed that intense replication of pSV2-gpt occurs in only a small subpopulation of cells and results from bursts of plasmid replication that occur periodically and spontaneously in the cell population. This suggests that COS-1 cells are only semipermissive for pSV2-gpt replication. No correlation was observed between levels of pSV2-gpt replication and the presence or absence of biochemical selection for the Gptmarker. However, growth of cells under nonselective conditions led to a rapid and progressive loss of pSV2-gpt DNA. This loss correlated with segregation of Gpt ^– revertants that lacked detectable plasmid sequences. Hence, maintenance of pSV2-gpt in the cell line was dependent on continuous biochemical selection. Stable replication of pSV2-gpt could be observed as late as four months after transfection, suggesting that this system might be useful for propagation of cloned DNA in COS-1 cells for extended periods of time. However, by nine months, extensive rearrangements of pSV2-gpt sequences were detected, indicating ultimate instability of the plasmid in the host cells.     

Mitotic stability of transforming DNA is determined by its chromosomal configuration in the fungus Cochliobolus heterostrophus

Summary Cochliobolus heterostrophus was transformed with a plasmid (pH1S) containing a bacterial gene (hygB), which confers resistance to the antibiotic hygromycin B when under control of an 838-bp fragment of promoter 1 from C. heterostrophus. The plasmid integrated at either homologous (52% single copy, 33% tandemly repeated copies) or ectopic (4% single copy, 11% tandemly repeated copies) sites on different chromosomes, resulting in four distinct configurations of integrated DNA. All four configurations were highly stable during mitotic growth; virtually no loss of integrated DNA was detected after five subcultures on nonselective medium or after seven cycles of pathogenesis on maize, the normal host of this fungus. However, deletion of integrated DNA was detected after eight or more disease cycles. The frequency of deletion depended on the configuration of the recombinant chromosome. A single copy of pH1S integrated at an homologous site was flanked by direct repeats of the target sequence and was least stable; up to 50% of the population lacked integrated DNA after 12 disease cycles. A single copy integrated at an ectopic site had no repeated DNA directly associated with it and was the most stable; no deletions were detected after 12 disease cycles. Tandemly repeated copies of pH1S integrated at either homologous or ectopic sites appeared to have intermediate stability; 2–18% of each population lost at least one copy after 12 disease cycles, although in no case were all copies deleted. Cytosine residues of integrated DNA were methylated during mitotic growth, but this had no apparent effect on the expression of hygB.     

Transfer of the Herpes simplex thymidine kinase gene from human cells to mouse cells by means of metaphase chromosomes

Thymidine kinase (TK)-deficient human cells were infected with ultraviolet light-inactivated Herpes simplex virus type 1, and transformed cells that expressed Herpes TK activity were isolated. Purified metaphase chromosomes were isolated from the transformed human line and incubated with TK-deficient mouse cells. TK ⁺ cells were selected, and it was shown that these cells were gene transferents which expressed Herpes TK activity, identical to that found in the transformed human cells. The gene transferents contained no intact human chromosomes. When removed from selective pressure, the gene transferents rapidly lost the TK ⁺ phenotype. However, upon continued growth in nonselective medium, a subpopulation in which the TK ⁺ phenotype had become more stabilized appeared. These results suggest that the Herpes gene for thymidine kinase has integrated into the genome of the HSV-transformed human cells and that it can be transferred to other cells by means of purified metaphase chromosomes.     

Herpesvirus-associated nuclear antigen(s) in cells biochemically transformed by fragments of herpesvirus DNA and in somatic cell hybrids

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.     

Role of the TK+ phenotype in the stability of Pigeonpox virus recombinant

Summary Insertion of foreign DNA containing the E. coli gpt marker by homologous recombination in the pigeonpox virus (PPV) thymidine kinase (TK) gene and selection for the presence of this DNA in the viral genome produced unstable recombinants after 3 plaque purifications. We highlight the persistence of duplicated TK DNA sequences arising from single crossing over, due to the growth advantage of TK⁺ virus. Restoration of the TK function by coinsertion of the vaccinia virus TK gene led to stable TK⁺ recombinants arising from double crossing over.     

A transforming plasmid from HSV-2 transformed cells contains rat DNA homologous to the HSV-1 and HSV-2 genomes

Morphologically transformed rat (3Y1) cell lines were established following transfection with HSV-2 mtrII DNA sequences (0.585 to 0.601 map units). The mtrII sequences were cloned in plasmids containing the neor gene. Cells resistant to the antibiotic G-418 were passed into soft agarose, and clonal lines were established from individual colonies. The DNAs from two cell lines examined by Southern blot hybridization were shown to contain the original transfected viral DNA sequences in a fashion consistent with a multiple and complex pattern of integration. From one cell line, an approximately 20-kbp plasmid was isolated after transformation of bacteria with Hirt supernatant DNA. This plasmid was capable of rapidly transforming rat cells at a greater than 1000-fold higher frequency than the mtrII DNA. This plasmid consists mainly of unique sequence rat DNA with two copies of the HSV-2 mtrII region DNA (HSV-2 genomic map unit location of ca. 0.595) present at sites distant from each other. The rat DNA in the rescued plasmid is homologous to the putative focus-forming sequences present in the HSV-2 mtrIII (0.53 to 0.58 map units) and the colinear HSV-1 DNA. The genomic copy of these rat sequences in four HSV-2 mtrII transformed cell lines appears to have undergone rearrangement. These data provide evidence that the HSV-2 mtrII sequences are involved in transformation, and that the HSV-2 mtrII region may affect transformation by rearranging the cellular sequences that are homologous to mtrIII.     

Ultraviolet-induced reactivation,amplification, and hypomethylation of a herpes simplex virus thymidine kinase gene

A line of mouse cells containing a methylated and inactive herpes simplex virus thymidine kinase(TK) gene was irradiated with ultraviolet (UV) light in an attempt to induce expression of the inactiveTK gene. UV irradiation was shown to be capable of inducing expression of the viralTK gene in a dose-dependent manner. Analysis of the methylation pattern of the viralTK gene indicated that the activeTK gene in three UV-induced TK⁺ cell lines was methylated to a lesser extent than was the inactive viralTK gene in the parental cells. Analysis of the copy number of the viralTK gene in parental and UV-induced cell lines also revealed that the viralTK gene was amplified 3- to 20-fold in three of four UV-induced TK⁺ cell lines tested.     

Cloning of the marmoset herpesvirus thymidine kinase gene and analyses of the boundaries of the coding region

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.     

Transformation of rat cells by the hybrid virus Ad2(2+) HEY.

A set of four isogenic rat cell lines transformed by Ad22+ HEY have been studied. While all of the cell lines synthesize SV40 T antigen, only one expresses adenovirus 2 T antigen: none expresses SV40 V antigen or adenovirus 2 fibre antigen. Three cell lines contain 1 to 2 virus equivalents of SV40 and adenoviral sequences per diploid quantity of rate cell DNA and the fourth line contains five copies of SV40 and 20 copies of the adenovirus genome. At least three of the cell lines contain DNA sequences from the helper adenovirus 2 in addition to sequences from the Ad2+ HEY genome. The patterns of integrated virus sequences are complex suggesting multiple insertions of both adenovirus and SV40 DNA sequences. SV40 can be rescued from three cell lines by fusion with permissive cells.     

A novel plasmid shuttle vector for the detection and analysis of microsatellite instability in cell lines

Microsatellite instability is an important feature of tumors from hereditary nonpolyposis colorectal carcinoma (HNPCC) patients as well as a variety of sporadic tumors. Here, we present a novel plasmid shuttle vector for the detection of this replication error (RER⁺) phenotype in human cell lines. The episomely replicated plasmid pZCA29 harbours the bacterial β-galactosidase gene interrupted by two palindromically arranged poly-(CA)-repeat tracts. The resulting +1-frameshift leads to white colonies of Escherichia coli DH10B on X-Gal/IPTG¹ agar plates. Mutations in the repeats characteristic of the RER⁺-phenotype may result in the loss or gain of CA-repeats leading to blue bacterial colonies. We transiently transfected the colorectal cancer cell lines SW480 and HCT116 with the plasmid pZCA29, isolated replicated plasmid DNA after several days and used it to transform E. coli DH10B. We found 1.0 to 1.7% blue colonies after passage of the plasmid through the RER⁻-cell line SW480 in contrast to 3.5 to 8.1% blue colonies after transfection of the RER⁺-cell line HCT116, the mutation frequencies increasing with incubation time. Sequence analysis of mutated plasmids revealed mostly 2-bp deletions which occurred especially in one of the repeat tracts. We conclude that pZCA29 appears to be a suitable shuttle vector for the detection and analysis of a RER⁺-phenotype in cell lines.     

Expression of herpesvirus thymidine kinase gene under control of early promoter of SV40

A 0.31-kilobase fragment of SV40 DNA containing the early promoter of this virus was isolated. The pX1 plasmid containing the herpesvirus thymidine kinase gene (TK) was cleaved with BglII and SalI to remove the TK promoter. The Sv40 DNA fragment was mixed with the TK gene without its promoter and blunt-end ligated after treatment with S₁ nuclease. The newly engineered plasmid was used for the transformation of TK-deficient LtA cells to TK⁺ phenotype. Blot hybridization experiments show that the plasmid is integrated into high-molecular-weight DNA in both of the transformed colonies that we have studied. Also the mRNA in these cells is a hybrid molecule containing both TK and SV40 sequences.     

Transfer of antibiotic resistance genes between yeast and mammalian cells under conditions favoring cell fusion

Antibiotic resistance to G418 has been transferred into Chinese hamster cell lines via a plasmid vector. The same plasmid, which also contained the Leu2 gene, has been used to transform Leu2^– yeast (strain MCI6) to leucine prototrophy. Subsequent fusion between transformed yeast and untransformed hamster cells demonstrated that plasmid DNA could be transferred and its genes expressed within the mammalian cell genome. The fusion of transformed hamster cells with untransformed MC16 yeast cells demonstrated that DNA integrated within the mammalian cell genome could be transferred to correct the Leu2 deficiency and also confer G418 resistance on some yeast colonies.     

Plasmid recovery from transformants and the isolation of chromosomal DNA segments improving plasmid replication in Neurospora crassa

Summary The efficient recovery of plasmid DNA from Neurospora crassa transformants is described. Lithium acetate-treated spores were transformed with plasmid DNA and grown in mass in liquid culture. The resulting mycelial growth was harvested and plasmid DNA was extracted and used to transform E. coli to ampicillin resistance. Although at low frequency, routine recovery of plasmid pSD3 which carries the Neurospora qa-2 ⁺ gene and pBR322 sequences has been demonstrated. About 10% of the recovered plasmids carried deletions and transformed Neurospora at a higher frequency. The liquid culture procedure was also used in attempts to isolate autonomously replicating sequences (ars). In order to select for a stable vector which contains an ars sequence, a clone bank containing a selectable marker (qa-2 ⁺) and Neurospora chromosomal BamHI fragments was constructed and used to transform Neurospora. Several plasmid isolates resulting from a screening of the clone bank showed an improvement in the efficiency of recovery from Neurospora transformants. The properties of one such isolated plasmid, pJP102, suggest that it may contain an ars sequence. Some potential applications of these results for cloning in Neurospora and other filamentous fungi are discussed.     

Integrative transformation of the yeast Yarrowia lipolytica

Summary An EcoR1 shotgun of Yarrowia lipolytica DNA was inserted into the plasmid YIp333 which carries the LYS2 gene of S. cerevisiae. The resulting plasmid pool was transformed in both S. cerevisiae and Y. lipolytica. Whereas numerous replicating plasmids could be isolated from the S. cerevisiae Lys⁺ transformants, all transformants of Y. lipolytica so far analyzed were found to result from integrative transformation. This occurred at a frequency of 1 to 10 transformants per g of input DNA. Co-transformation occurred at high frequency and resulted in tandem integration of 2 to 10 copies of the incoming DNA. Structural and segregational stability of the transforming DNA were both high.     

Inhibition by acyclovir of cell growth and DNA synthesis of cells biochemically transformed with herpesvirus genetic information

Thymidine kinase-deficient LM cells (LMTK^?-) biochemically transformed to the TK⁺ phenotype with herpes simplex virus genetic information showed an increased uptake of and ability to phosphorylate the acyclic nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acyclovir, acycloguanosine, acyclo-Guo). In growth inhibition studies the TK⁺ transformants were much more sensitive to inhibition with acyclovir than the untransformed cells (13- to 90-fold more sensitive). The synthesis of DNA in the transformed cells was significantly reduced by acyclovir treatment, whereas acyclovir had little effect on the DNA synthesis of the untransformed cells. Alkaline sucrose gradient sedimentation analysis of cellular DNA synthesized in the presence of acyclovir showed that, in contrast to untreated untransformed cells, the DNA newly synthesized by transformed cells was considerably smaller in size. In pulse-chase experiments the small fragments of DNA synthesized in the presence of acyclo-Guo were not chased to high molecular weight DNA. Finally, acyclo-Guo was shown to be incorporated terminally at 3′-ends of growing DNA chains in replicating cells.     

Quantification of the herpes simplex virus DNA present in biochemically transformed mouse cells and their revertants.

Four cell lines biochemically transformed by u.v.-irradiated herpes simplex virus contain virus DNA fragments ranging from 3 to 22% of the HSV genome. Of five revertant clones selected for 3H-TdR or BrdUrd resistance, four had lost all detectable virus DNA while the fifth, selected for BrdUrd resistance, retained the entire virus fragment but there was a reduction of virus copies per cell from 5 to 1. Three 'supertransformed' revertant cell lines contained virus DNA fragments ranging from 12 to 28%. The number of virus DNA fragments per cell ranged from 1 to 5 and clearly indicated that a single copy of the virus thymidine kinase gene is adequate for biochemical transformation. The determination of the base composition of the transforming virus DNA fragment indicated that the transforming DNA has a base composition approximately the same as the HSV genome and does not constitute a low GC virus DNA region. Cross hybridization between HSV-1 transformed cells and HSV-2 DNA is very slight, indicating that the DNA found in clone 139 is not entirely composed of the HSV-1 and HSV-2 common sequences.     

Cell fusion-impaired variants of a mouse lymphocytic cell line obtained by single-step selection for polyethylene glycol resistance

This report describes an effective method for single-step isolation of cell line variants with reduced intercellular plasma membrane fusion ability. Variants, derived from a subline of L5178Y, were obtained by selection for growth in the presence of a toxic level of PEG 6000. These variants occurred after EMS treatment and spontaneously. Further testing revealed a pleiotropic phenotype, fusion impairment and polyethylene glycol resistance, which has been designated Pgf^R. Variant sublines, in serial subculture for more than 165 days on nonselective medium, retain a stable Pgf^R phenotype. Pgf^R variants have an approximate 7-fold reduction in intercellular fusion competence.