The expression of the Escherichia coli uvrA gene in human cells

Cells cultured from xeroderma pigmentosum (XP) patients are defective in excision repair of damaged DNA specifically at the incision step. In Escherichia coli this step is mediated by the UvrA, UvrB and UvrC gene products. Our goal is to express each of these genes in XP cells, singly or in combination, and to determine the most suitable conditions for generating faithful E. coli Uvr protein copies in functional concentrations and properly localized for the eventual repair of damaged chromosomal DNA or DNA which is introduced exogenously. The E. coli gpt gene in pSV2gpt is used as a selection marker for uvr gene transfection into XP cells. The uvr genes were cloned into composite pBR322, SV40 and gpt vectors in which each E. coli gene is flanked by individual SV40 regulatory elements. SV40-transformed XP-A cells were transfected with pSV2uvrASV2gpt, gpt+ colonies were selected, and cell lines established. Several lines were examined in detail. Cell lines 714 and 1511 contain uvrA together with flanking SV40 regulatory elements integrated intact in genomic DNA and express UvrA protein as well as a 95,000-dalton UvrA-related protein. The expression of uvrA was found to be 50-100-fold lower than the expression of gpt. Attempts were made to assay the mammalian UvrA protein for functionality, but endogenous activities interfered with assays for each of the UvrA protein's three activities. The peptide maps derived from partial proteolysis of the "mammalian" UvrA protein are identical to the E. coli UvrA protein. The sub-cellular location of UvrA protein in uvrA+ XP cells was investigated by fractionation of cell extracts in which an indirect immunofluorescence method revealed its location as being largely extra-nuclear. Two uvrA+ cell lines were examined for their UV-resistant phenotype and not unexpectedly were found not to be reverted to a state of repair proficiency.     

Analyses of mutation in pSV2gpt-transformed CHO cells

We have developed a system to study mutations which affect expression of the E. coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) in hypoxanthine-guanine phosphoribosyl transferase-deficient (HPRT-) Chinese hamster ovary (CHO) cells that have been transformed by the plasmid pSV2gpt. Several gpt-transformed cell lines have been isolated and characterized with respect to integrated pSV2gpt sequences, expression of the gpt gene, and cytotoxic and mutagenic responses to UV light. While the gpt-transformed CHO and wild-type CHO-K1-BH4 cell lines have similar cytotoxic responses to UV light, the gpt-transformed cell lines respond differently from the parental CHO-K1-BH4 cell line in terms of mutation induction. As with CHO-K1-BH4 HPRT mutants, spontaneous or induced XPRT mutants derived from the gpt+ cell lines can be selected for 6-thioguanine resistance (TGr). Analysis of cell-free extracts from a number of these TGr clones indicates that the mutant phenotype is due to the absence of XPRT activity. One transformant, designated AS52, has previously been described in limited detail. Here we describe additional characteristics of this cell line, as well as several related transformants.     

Stable transformation of mouse teratocarcinoma stem cells with the dominant selective marker Eco.gpt and retention of their developmental potentialities

Transformation of PCC4 mouse teratocarcinoma stem cells was obtained using a dominant selective marker, the enzyme xanthine-guanine phosphoribosyltransferase (XGPRT), coded by the bacterial Eco.gpt gene placed under the control of the early SV40 genes in the vector pSV2gpt. An average of 20 colonies of transformed cells was obtained, using the calcium phosphate technique, 10 microg DNA vector, no carrier DNA and 1 x 10(6) recipient cells. Five independent Eco.gpt-transformed PCC4 cell lines were propagated in selective medium and assayed for XGPRT activity. All of them had the ability to convert [14C]xanthine to xanthine monophosphate. pSV2gpt sequences were present and associated with high mol. wt. cellular DNA. pSV2gpt sequences and XGPRT activity were both conserved in the three clones that were propagated in non-selective medium for 30 generations. The transformed PCC4 cells retained their ability to produce, in host mice, teratocarcinoma tumors composed of embryonal carcinoma and various differentiated tissues. Thus, pSV2gpt can be used as a dominant marker to select teratocarcinoma stem cells co-transformed with genes that are not selectable by themselves.     

Expression of adenovirus E1a and E1b gene products and the Escherichia coli XGPRT gene in KB cells

The recombinant plasmid pSV2-gpt, which contains the Escherichia coli XGPRT gene under the control of a simian virus 40 early promoter, was modified to contain the type 2 adenovirus (Ad2) XhoI-C (0 to 15.5 map units) restriction endonuclease fragment. Plasmid (pLB206) DNA was introduced into human KB cells by Ca2+-mediated DNA transfection, and transformants were selected in medium containing xanthine, aminopterin, and mycophenolic acid, as a consequence of expression of the dominant, selectable XGPRT gene. A series of 13 gpt+ cell lines were isolated and tested for their ability to complement Ad5 deletion mutants in E1a (H5dl312) and E1b (H5dl315). Four classes of gpt+ KB cell lines were identified, including clones constitutively expressing both E1a and E1b, only E1a, or only E1b or not expressing either E1a or E1b. DNA and RNA filter transfer hybridization analysis substantiated the conclusions that those cell lines capable of complementing viral host range mutants contained the appropriate viral DNA sequences and cytoplasmic polyadenylated RNA species. DNA filter transfer hybridization studies also revealed that the transfected vector DNA was stably integrated into chromosomal DNA in the KB transformants and the number of integrated sites ranged from 1 to 3. The gpt+ KB cell line that only expressed E1b gene functions only contained viral E1b gene sequences; those cell lines that expressed neither E1a nor E1b gene function contained only small or no regions of Ad2 DNA. When weaned off the selective medium, transformed KB cell lines stably maintained their inserted DNA in the absence of selective pressure and could easily be adapted to growth in suspension culture.     

The genomic instability associated with integrated simian virus 40 DNA is dependent on the origin of replication and early control region.

DNA rearrangements in the form of deletions and duplications are found within and near integrated simian virus 40 (SV40) DNA in nonpermissive cell lines. We have found that rearrangements also occur frequently with integrated pSV2neo plasmid DNA. pSV2neo contains the entire SV40 control region, including the origin of replication, both promoters, and the enhancer sequences. Linearized plasmid DNA was electroporated into X1, an SV40-transformed mouse cell line that expresses SV40 large T antigen (T Ag) and shows very frequent rearrangements at the SV40 locus, and into LMtk-, a spontaneously transformed mouse cell line that contains no SV40 DNA. Stability was analyzed by subcloning G-418-resistant clones and examining specific DNA fragments for alterations in size. Five independent X1 clones containing pSV2neo DNA were unstable at both the neo locus and the T Ag locus. By contrast, four X1 clones containing mutants of pSV2neo with small deletions in the SV40 core origin and three X1 clones containing a different neo plasmid lacking SV40 sequences were stable at the neo locus, although they were still unstable at the T Ag locus. Surprisingly, five independent LMtk- clones containing pSV2neo DNA were unstable at the neo locus. LMtk- clones containing origin deletion mutants were more stable but were not as stable as the X1 clones containing the same plasmid DNA. We conclude that the SV40 origin of replication and early control region are sufficient viral components for the genomic instability at sites of SV40 integration and that SV40 T Ag is not required.     

Quantification of expression of linked cloned genes in a simian virus 40-transformed xeroderma pigmentosum cell line.

We wished to determine whether simian virus 40 (SV40)-transformed xeroderma pigmentosum cells, despite their defective DNA repair, were suitable for DNA-mediated gene transfer experiments with linked genes. Expression of a nonselectable gene (cat, coding for chloramphenicol acetyltransferase [CAT]) linked to a selectable gene (gpt, coding for xanthine-guanine phosphoribosyltransferase [XPRT]) in the plasmid pSV2catSVgpt was quantified after transfection of SV40-transformed xeroderma pigmentosum [XP20s(SV40)] and normal human [GM0637(SV40)] fibroblast cell lines. A novel autoradiographic assay with [3H]xanthine incorporation showed 0.5 to 0.7% phenotypic expression of XPRT in both cell lines. Without selection, transient CAT activity was 20 times greater in the GM0637(SV40) than in the XP20s(SV40) cells, and transient XPRT activity was 5 times greater. Both of these transient activities were increased and equalized in both cell lines by transfection with pRSVcat or pRSVgpt. Genotypic transformation to gpt+ occurred at a frequency of 2 X 10(-4) to 4 X 10(-4) in both cell lines with pSV2catSVgpt. After 2 to 3 months in selective medium, stable expression of the (nonselected) cat gene was found in 11 (92%) of 12 gpt-containing clones derived from GM0637(SV40) cells and in 13 (81%) of 16 gpt-containing clones from XP20s(SV40) cells. However, the levels of CAT activity did not correlate with those of XPRT activity, and both of these activities varied more than 100-fold among different clones. Copies (1 to 4) of the gpt gene were integrated in four clones of the GM0637(SV40) cells having an XPRT activity of 1 to 5 nmol/min per mg, but 5 to 80 copies were integrated in four XP20s(SV40) clones with an XPRT activity of 0.8 to 1.8 nmol/min per mg. This study shows that XP20s(SV40) is as suitable for gene transfer experiments as the normal human line GM0637(SV40).     

Persistence of freely replicating SV40 recombinant molecules carrying a selectable marker in permissive simian cells

We have demonstrated that a SV40-pBR322 recombinant vector (pSV2-gpt) carrying a bacterial gene of selectable phenotype (Eco-gpt) may persist extrachromosomally in COS1 cells, a simian cell line that endogenously produces SV40 large T antigen. The amount of circular (supercoiled) recombinant DNA was estimated to be between 5 and 2000 copies per cell among several pSV2-transformed COS1 clonal lines examined. Complete pSV2 molecules were found in the majority of the transformants, although some of the pSV2 DNAs recovered were shown to have deletions in the pBR322 region. Our results indicate that removal of the pBR322 "inhibitory sequence" in pSV2 is not necessary for stable maintenance of these recombinant molecules in COS1 cells. In addition, large amounts of pSV2-related high molecular weight DNAs, probably concatemers of pSV2, were detected in the transformed lines.     

Phenotypic changes and gene expression in human colon mucosal epithelial cells upon transfection of a SV40 DNA-GPT recombinant

Summary Phenotypic changes (increased longevity, decreased growth factor requirements, altered cell surface features, growth in semisolid agarose, and SV40 T antigen expression) suggesting in vitro transformation were displayed by human normal colon mucosal epithelial cells transfected with pSV3gpt, a pBR322 recombinant containing the SV40 “early” T antigen coding region and the dominant selectable marker bacterial gene, xanthine-guanine phosphoribosyltransferase. In contrast, control cultures which received neither DNA nor the recombinatn pSV2gpt (which is identical to pSV3gpt but lacks the SV40 T antigen region) were not phenotypically altered.     

High spontaneous mutation frequency in shuttle vector sequences recovered from mammalian cellular DNA.

The recombinant shuttle vector pSV2gpt was introduced into V79 Chinese hamster cells, and stable transformants expressing the Escherichia coli gpt gene were selected. Two transformants carrying tandem duplications of the plasmid at a single site were identified and fused to simian COS-1 cells. Plasmid DNA recovered from the heterokaryons was used to transform a Gpt- derivative of E. coli HB101, and the relative frequency of plasmids carrying a mutation in the gpt gene was determined. The high frequency of Gpt- plasmids (ca. 1%) was similar to that observed when plasmid was recovered from COS-1 cells which had been transfected with pSV2gpt. Most of the mutant plasmids had rearrangements in the region containing the gpt gene.     

Inactivation of a transfected gene in human fibroblasts can occur by deletion, amplification, phenotypic switching, or methylation.

Plasmids containing the bacterial gpt gene under control of the simian virus 40 promoter were transfected into a simian virus 40-transformed human fibroblast line. Two transfectants, E2 and C10, which contain stably integrated single copies of the gpt gene, were isolated. These two lines produce Gpt- variants spontaneously with a frequency of about 10(-4). We carried out a detailed molecular analysis of the spectrum of alterations which gave rise to the Gpt- phenotype in these variants. DNA from 14 of 19 Gpt- derivatives of one of the cell lines (E2) contains deletions or rearrangements of gpt-containing sequences. In four of the remaining five lines, the Gpt- phenotype was correlated with reduced levels of expression rather than with changes in the gross structure of the gpt gene, and it was possible to reactivate the gpt gene. In one Gpt- line, gpt mRNA was present at normal levels, but no active enzyme was produced. Spontaneous Gpt- derivatives of the other cell line (C10) produced a completely different spectrum of alterations. Very few deletions were found, but several derivatives contained additional extrachromosomal gpt sequences, and, remarkably, in two other Gpt- lines, gpt-containing sequences were amplified more than 100-fold. The phenotypes of the majority of the Gpt- derivatives of C10 could be attributed to alterations in gene expression caused by methylation.     

Characteristics of an SV40-plasmid recombinant and its movement into and out of the genome of a murine cell

A bacterial plasmid carrying the early region of SV40 (pOT) has been stably established in high molecular weight (hmw) DNA of mouse L cells by selection for the herpes virus thymidine kinase (tk) gene. DNA blotting has demonstrated that most cell lines contain multiple discrete copies of pOT, generally with an intact SV40 early region. No free copies of pOT have been detected. Both pOT and tk sequences may be amplified up to 20–200 copies of the SV40 early region. In contrast to the uniform staining pattern normally observed in SV40-transformed lines, indirect immunofluorescence using antiserum to the SV40 T antigen has demonstrated that the expression of the early region is heterogeneous in these cell lines. This fraction expressing T is characteristic of a given cell line, and varies from 0 to 99% positive. Several pOT cell lines have been fused to simian cells, and replicating low molecular weight DNAs were isolated from the heterokaryons. Transformation of E. coli with this DNA demonstrates that pOT can be rescued from hmw DNA in L cells and reestablished as a plasmid in E. coli. Excision is generally precise when pOT is introduced to the murine cells as a supercoiled molecule, and imprecise when pOT is introduced in linear form.     

Establishment and characterization of a permanent pSV ori--transformed ataxia-telangiectasia cell line

A permanent ataxia-telangiectasia (A-T) cell line has been established from the fibroblast strain AT2SF after transfection with the bacterial plasmid pSV ori-, which contains replication origin-defective SV40 sequences. The original transfection frequency, as measured by transformed foci, was markedly reduced in two A-T strains when compared with either normal or xeroderma pigmentosum fibroblasts. As with SV40 virion-transformed fibroblasts, pSV ori--transformed cells entered a crisis phase, from which about one-fourth of the original clones from A-T and normal fibroblasts recovered. Both the pSV ori--transformed TAT2SF cell line and an SV40 virion-transformed AT5BI (GM5489) cell line retained their characteristic sensitivity to the lethal effects of ionizing radiation, as well as their X ray-resistant DNA synthesis. Southern blot analysis of cellular SV40 sequences demonstrated a single major integration site of pSV ori- in the AT2SF cells. In contrast, AT5BI cells transformed with SV40 virions demonstrated a high degree of heterogeneity of integrated viral sequences. Neither the TAT2SF nor the GM5489 transformed cell line contains any detectable freely replicating SV40 viral sequences, which are seen in many other semipermissive SV40-transformed cells.     

Expression of a gene encoding a rabbit sperm membrane protein in mammalian cells.

A general mammalian expression vector designated pSV2-EP was reconstructed by inserting an oligonucleotide fragment into pSV2-dhfr. This vector allowed insertion of cDNAs with EcoRI cohesive ends. The pSV2-EP contains a simian virus 40 (SV40) early promoter, origin for DNA replication, SV40 poly-A site, splicing site, an initiator ATG downstream from the promoter and an EcoRI site for the insertion of cDNA fragment screened from lambda gt11 expression libraries. A recombinant plasmid (pS-VRS-1) was constructed by inserting RSD-1, a cDNA encoding a rabbit sperm tail protein, into the EcoRI site of the pSV2-EP vector. Chinese hamster ovarian (CHO) dhfr-negative cells were cotransformed with pSV2-dhfr and pSVRS-1 by the calcium phosphate method. In selective culture medium without thymidine and hypoxanthine, several cell lines were obtained containing mRNA and DNA that hybridized with RSD-1. One of these transformed cell lines stained intensely with anti-rSMP-B antibodies, demonstrating that the RSD-1 was expressed in the transformed CHO cells.     

Studying DNA mutations in human cells with the use of an integrated HSV thymidine kinase target gene

A shuttle vector carrying the origin of SV40 replication, the thymidine kinase (tk) gene of herpes simplex virus and the E. coli xanthine guanine phosphoribosyl transferase (gpt) gene has been introduced into human TK- cells. A transformed cell line containing only one stably integrated copy of the shuttle vector was used to study mutations in the introduced tk gene at the molecular level. Without selection for gpt expression, spontaneous TK- mutants arose at a frequency of approximately 10(-4)/generation, and were caused by deletion of plasmid sequences. However, when selection for expression of the gpt gene was applied, the background level of mutations at the tk gene was below 4.10(-6). From this cell line, TK- mutants were obtained after treatment with N-ethyl-N-nitrosourea (ENU). COS fusion appeared to be an efficient method for rescue and amplification of the integrated shuttle vector from the human chromosome. After further amplification and analysis in E. coli, rescued tk genes were easily identified and were shown to be physically unaltered by the rescue procedure. In contrast to rescued tk genes from TK+ cells, those obtained from the ENU-induced TK- mutants were unable to complement thymidine kinase-negative E. coli cells. Two such tk mutations were mapped in E. coli by marker rescue analysis. A GC----AT transition was the cause of both mutations. We show here that plasmid rescue by COS fusion is a reliable system for studying gene mutations in human cells, since no sequence changes occurred in rescued DNA except for the 2 ENU-induced sequence changes.     

Regulation of gene amplification and expression in cells that constitutively express a temperature sensitive SV40 T-antigen.

Simian cells have been transformed with SV40 origin-defective recombinant plasmids containing the tsA209 T-antigen gene. These plasmids contain deletions of either 5 or 52 nucleotides that include the BglI site at the SV40 ori, are defective for replication in COS-1 cells but retain a functional SV40 early promoter. Two cell lines transformed with these plasmids, U4 and S7, and their respective clonal derivatives E5 and F11, contain the tsA209 T-antigen gene integrated into the cell DNA and express T-antigen as detected by immunoprecipitation and immunofluorescence. These cells behave as ts-COS cells, since they complement in a temperature dependent manner the replication of an SV40 derived recombinant plasmid. When transfected with recombinant plasmids containing the chloramphenicol acetyl transferase (CAT) gene cloned into SV40 replicons, ts-COS cells were able to regulate the induction of the CAT activity by temperature. The ratios of CAT activity observed at permissive versus restrictive temperature were in the range of 20-400. Thus, these ts-COS cells are useful systems for the regulated expression of cloned genes in simian cells.     

Quantitative and molecular analyses of ethyl methanesulfonate- and ICR 191-induced mutation in AS52 cells

A pSV2gpt-transformed Chinese hamster ovary (CHO) cell line has been used to study mutation at the molecular level. This cell line, designated AS52, was constructed from a hypoxanthine-guanine phosphoribosyl transferase (HPRT)-deficient CHO cell line, and has been previously shown to contain a single, functional copy of the E. coli xanthine-guanine phosphoribosyl transferase (XPRT) gene (gpt) stably integrated into the Chinese hamster genome. In this study, conditions for its use in the study of mammalian cell mutagenesis have been stringently defined. The spontaneous mutation rate (2 X 10(-6)/cell division) and phenotypic expression time (7 days) of the gpt locus compare favorably with those of the hprt locus in wild-type CHO-K1-BH4 cells. While both cell lines exhibit similar cytotoxic responses to ethyl methanesulfonate (EMSO and ICR 191, significant differences in mutation induction were observed. Ratios of XPRT to HPRT mutants induced per unit dose of EMS and ICR 191 are 0.70 and 1.6, respectively. Southern blot hybridization analyses revealed that most XPRT mutant cell lines which arose following treatment with EMS (20/22) or ICR 191 (20/24) exhibited no alterations of the gpt locus detectable by this technique. Similar observations were made for the hprt locus in EMS-(21/21) and ICR 191-induced (22/22) HPRT mutants. In contrast, most spontaneous gpt mutants (14/23) contained deletions, while most spontaneous hprt mutants (18/23) exhibited no detectable alterations. Results of this study indicate that the AS52 cell line promises to be useful for future study of mutation in mammalian cells at the DNA sequence level.     

Failure to detect a DNA repair-related defect in the transfection of ataxia-telangiectasia cells by enzymatically restricted plasmid

We have transfected two SV40-transformed human fibroblast cell lines with plasmids in which double-strand breaks have been introduced by restriction enzymes, within or near the selected gene. Restriction of pSV2gpt with KpnI, as previously shown by Cox et al. (1986), reduced the frequency of transfection more in the ionizing radiation-sensitive ataxia-telangiectasia line AT5BIVA than in the resistant line MRC5V1. When the related plasmid pSV2neo was restricted with SmaI, the reduction in transfection was less in the ataxia-telangiectasia than in the normal cell line. Under our conditions, the apparent defect in transfection of AT5BIVA by pSV2gpt appears to be a result of the unusual sensitivity of the repair-deficient recipient to the selective agent. Loss of potential transfectants is exacerbated when transient gene expression is reduced by restriction of the plasmid. We suggest that a reduction in yield of transfectants with restricted plasmid in ataxia-telangiectasia cells cannot readily be used as evidence of a defect in DNA repair. Our results are also relevant to standard transfection experiments, since they emphasize the importance of optimizing selection when transient expression may be reduced, to ensure that potential transfectants are not killed by the selection regime.     

Transformation of DNA repair-deficient human diploid fibroblasts with a simian virus 40 plasmid

Fibroblasts from patients with xeroderma pigmentosum (XP) complementation groups A, C, D, E, and G, as well as Bloom syndrome (BS) and Fanconi anemia (FA) have been transfected with a plasmid, pSV7, containing the early region of Simian virus 40 (SV40). All of the cultures exhibited cytologic changes characteristic of transformed cells and expressed T-antigen. They also contained integrated copies of DNA derived from the vector, and in several cases, extrachromosomally replicated DNA. Not all of the transfected cultures became immortalized. The transformed xeroderma pigmentosum (XP) cultures retained their UV-sensitive phenotype in all but one case. The BS and FA cell lines retained their characteristic phenotype. All of the cultures, except the BS cells, can be readily transfected with the plasmids, pSV2neo and pSV2gpt.     

Detection of deletion mutations in pSV2gpt-transformed cells.

We have developed a system to study mutations that affect xanthine-guanine phosphoribosyltransferase gene (gpt) expression in hypoxanthine-guanine phosphoribosyltransferase-deficient CHO cells that have been transformed by the plasmid vector pSV2gpt. One isolated transformant, designated AS52, carries a single copy of the Escherichia coli gpt gene stably integrated into the high-molecular-weight DNA and expresses the bacterial gene for the enzyme xanthine-guanine phosphoribosyltransferase. Mutants deficient in this enzyme can be induced in the AS52 cell line by a variety of mutagens, and spontaneous or induced mutants can be selected for resistance to 6-thioguanine (Tgr). Two Tgr clones derived from the AS52 line were analyzed by Southern blot hybridization and were found to contain deletions involving at least a portion of the gpt gene. Because of the small size and stability of the integrated pSV2gpt plasmid, and the well-defined selection protocol for mutant isolation, the AS52 line offers promise as a system suitable for the study of mutation at the molecular level in CHO cells.