Xanthine-guanine Phosphoribosyltransferase Research Articles

Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

A genetic assay for aneuploidy: quantitation of chromosome loss using a mouse/human monochromosomal hydrid cell line

A genetic assay is described in which a mouse/human hybrid cell line R3-5 containing a single human chromosome (a monochromosomal hybrid) is used to detect chemically induced aneuploidy. In this assay the frequency of chromosome loss determined by the cloning efficiency of the cells in a selection medium is used as an index for the potential of a chemical to induce aneuploidy. The hybrid cells are deficient in hypoxanthine guanine phosphoribosyltransferase (HGPRT) and contain human chromosome 2, marked with Ecogpt, an E. coli gene for xanthine guanine phosphoribosyltransferase. These cells with a genotype of hgprt −/Ecogpt + can grow in medium containing mycophenolic acid and xanthine (MX medium) but not in medium containing 6-thioguanine (6-TG). The loss of the human chromosome from R3-5 cells as a result of chemical treatment produces cells with a genotype of hgprt −/Ecogpt − which are capable of growth in the medium containing 6-TG. Thus, the cloning efficiency of cells treated with a test chemical in 6-TG provides a method to determine the frequency of cells that have lost the human chromosome. Two chemicals, colcemid and nocodazole, previously known to induce aneuploidy in mammalian cells were used for a preliminary evaluation of this test system. Both of these compounds at concentrations ranging from 0.002 to 0.032 μg/ml showed a concentration-related positive response in this assay.

The role of the ras oncogene in the formation of tumours.

A c-Ha-ras 1 oncogene, cloned from the EJ human bladder carcinoma cell line, was inserted into a shuttle vector carrying the selectable marker gene gpt that encodes the enzyme xanthine-guanine phosphoribosyl transferase. This construct, pSV2gptEJ, was transfected into NIH 3T3 cells by the calcium phosphate precipitation method and cells that had incorporated the plasmid were selected by growth in the presence of mycophenolic acid to which gpt confers resistance. A number of transfectant clones were tested for tumorigenicity by inoculation into nude mice. The take incidence was variable and the tumours arose only after a prolonged latent period. Many inocula produced no tumours. These results were consistent with the view that the tumours arose by selective overgrowth of minority cell populations. Cell lines were derived by explantation of these tumours and were back-selected in 2-thioxanthine, a cytotoxic analogue of the xanthine-guanine phosphoribosyl transferase substrate. Five clones were obtained that did not express detectable levels of the c-Ha-ras 1 oncogene product, p21ras. All of them showed a less-transformed morphology than the transfected NIH 3T3 cells from which they originated. Nonetheless three of these clones were found to be tumorigenic at all sites tested. This finding demonstrates that once tumorigenic variants have been selected from the ras-transformed cells, continued production of the p21ras protein is not necessary for the maintenance of tumorigenicity. Cytogenetic analysis revealed that the transfection procedure itself introduced major and stable perturbations of the genome of the transfected cells and confirmed that tumours were produced by selective overgrowth of variants with a chromosome constitution palpably different from that of the majority of the cells injected. In the light of the complex background of genomic changes produced in NIH 3T3 cells by transfection with the c-Ha-ras 1 oncogene, no conclusion can be drawn in genetic terms concerning its dominance.

Construction of recombinant fowlpox viruses as vectors for poultry vaccines

Plasmid vectors have been constructed which allow the construction of infectious fowlpox virus (FPV) recombinants expressing foreign genes. The foreign genes were inserted within the thymidine kinase (TK) gene of FPV contained in these vectors. To facilitate the selection of recombinants the Escherichia coli xanthine guanine phosphoribosyl transferase (Ecogpt) gene was developed as a dominant selectable marker. This marker operates in a wide variety of cell types and obviates the need for TK − cell lines for selection of TK − recombinants when foreign genes have been inserted within the TK gene of FPV. The general approach adopted was to construct plasmid vectors in which the FPV TK was interrupted by the Ecogpt gene under the control of a poxvirus promoter in tandem with a gene of interest under the control of another poxvirus promoter. Selection of viruses expressing the Ecogpt gene simultaneously selects for recombinants carrying both the Ecogpt gene and the gene of interest. Using this approach a series of plasmid vectors was constructed in which the FPV TK gene was interrupted by the Ecogpt gene under the control of the P7.5 vaccinia virus promoter in tandem with the A/PR/8/34 haemagglutinin gene under the control of the PL11 vaccinia virus promoter. A recombinant FPV constructed using these plasmids had the expected genome arrangement, expressed influenza haemagglutinin, and induced haemagglutination-inhibiting antibodies when inoculated into chickens. These techniques should allow the construction of a variety of recombinant FPVs expressing poultry vaccine antigens. Such recombinants should be a very cost-effective means of delivering vaccines to poultry.

Escherichia coli gpt gene provides dominant selection for vaccinia virus open reading frame expression vectors.

Mycophenolic acid, an inhibitor of purine metabolism, was shown to block the replication of vaccinia virus in normal cell lines. This observation led to the development of a dominant one-step plaque selection system, based on expression of the Escherichia coli gpt gene, for the isolation of recombinant vaccinia viruses. Synthesis of xanthine-guanine phosphoribosyltransferase enabled only the recombinant viruses to form large plaques in a selective medium containing mycophenolic acid, xanthine, and hypoxanthine. To utilize the selection system efficiently, we constructed a series of plasmids that contain the E. coli gpt gene and allow insertion of foreign genes into multiple unique restriction endonuclease sites in all three reading frames between the translation initiation codon of a strong late promoter and synthetic translation termination sequences. The selection-expression cassette is flanked by vaccinia virus DNA that directs homologous recombination into the virus genome. The new vectors allow high-level expression of complete or partial open reading frames and rapid construction of recombinant viruses by facilitating the cloning steps and by simplifying their isolation. The system was tested by cloning the E. coli beta-galactosidase gene; in 24 h, this enzyme accounted for approximately 3.5% of the total infected-cell protein.

A dominant selectable marker for the construction of recombinant poxviruses

Mycophenolic acid has been shown to be a potent inhibitor of vaccinia virus growth. By inserting the Escherichia coli xanthine-guanine phosphoribosyl transferase gene ( gpt) into the vaccinia virus genome under control of the P-7.5 promoter this inhibition was overcome. When coupled in tandem with another gene of interest, recombinant vaccinia viruses can be positively selected carrying both genes. Since the gpt gene operates as a selectable marker in most mammalian cells it will be useful as a dominant selectable marker for the construction of recombinant viruses based on other host-specific poxviruses.

Characterisation of two differently processed forms of human recombinant factor IX synthesised in CHO cells transformed with a polycistronic vector.

A stable transformed cell line constitutively expressing human factor IX has been established. Wild-type Chinese hamster ovary cells (CHO cells) were transformed using a polycistronic expression vector carrying a previously isolated factor IX cDNA and a selection gene encoding the Escherichia coli xanthine-guanine phosphoribosyl transferase. One clone, CHO 622.4, contains a high number of genomically integrated plasmids and secretes 1-3 mg factor IX l-1 day-1 into the culture medium with a biological activity ranging from 25% to 40%. The recombinant molecule was purified either by conventional chromatography or by immunoaffinity chromatography using antibodies specific to a calcium-induced factor IX conformer. The purified recombinant protein migrates as a single band with the same mobility as that of natural factor IX on SDS/polyacrylamide gels. N-terminal sequencing shows tow differently processed forms of recombinant factor IX: whereas the majority of the zymogen is correctly processed, approximately 20% of the purified recombinant molecule contains an 18-amino-acid NH2-extension corresponding to the precursor form of factor IX. Analysis of the 4-carboxyglutamic acid content indicates a high but incomplete carboxylation (70%) of the recombinant molecule as compared to natural factor IX. The carbohydrate composition of both the natural and recombinant molecules has been determined. Both molecules have a N-glycan structure of similar complexity, indicating that factor IX contains all the information to direct the same glycosylation pattern in human liver cells and in an unrelated cell line such as CHO-K1.

Molecular analysis of formaldehyde-induced mutations in human lymphoblasts and E. coli.

The molecular nature of formaldehyde (HCHO)-induced mutations was studied in both human lymphoblasts and E. coli. Thirty HPRT- human lymphoblast colonies induced by eight repetitive 150 microM HCHO treatments were characterized by Southern blot analysis. Fourteen of these mutants (47%) had visible deletions of some or all of the X-linked HPRT bands, indicating that HCHO can induce large losses of DNA in human lymphoblasts. In E. coli, DNA alterations induced by HCHO were characterized with use of the xanthine guanine phosphoribosyl transferase (gpt) gene as the genetic target. Exposure of E. coli to 4 mM HCHO for 1 hr induced large insertions (41%), large deletions (18%), and point mutations (41%). Dideoxy DNA sequencing revealed that most of the point mutations were transversions at GC base pairs. In contrast, exposure of E. coli to 40 mM HCHO for 1 hr produced 92% point mutations, 62% of which were transitions at a single AT base pair in the gene. Therefore, HCHO is capable of producing different genetic alterations in E. coli at different concentrations, suggesting fundamental differences in the mutagenic mechanisms operating at the two concentrations used. Naked pSV2gpt plasmid DNA was exposed to 3.3 or 10 mM HCHO and transformed into E. coli. Most of the resulting mutations were frameshifts, again suggesting a different mutagenic mechanism.

A new genetic approach for studying hormonal regulation of urokinase-type plasminogen activator gene expression in LLC-PK1 cells.

In LLC-PK1 cells, a cyclic AMP (cAMP)-elevating peptide hormone, calcitonin, induces urokinase-type plasminogen activator (uPA) gene transcription without concomitant protein synthesis. To understand the molecular mechanism of the uPA gene regulation by cAMP, we developed a system which allows us to obtain mutant cells with modified regulatory proteins. A uPA-gpt hybrid gene was constructed, in which the regulatory region of the uPA gene was linked to a bacterial xanthine-guanine phosphoribosyltransferase gene (gpt), and it was transfected into LLC-PK1 cells. A stably transformed cell line, which expressed gpt only in the presence of calcitonin, was obtained, and then these cells were treated with a chemical mutagen, ethyl methanesulfonate. Cells were screened for constitutive gpt expression and, as mutations in regulatory proteins should affect the two genes at the same time, cells were further screened for an increased basal uPA mRNA level. Several such clones were obtained and none of them had modified cAMP-dependent protein kinase activity, suggesting that mutations were in the post-protein kinase step in the pathway of hormone action. Five clones were fused with the parent LLC-PK1 cells, and all of the fusion cells showed reduced basal uPA mRNA levels, indicating that they were recessive mutants. One clone was analyzed further for sensitivity to calcitonin in the induction of uPA mRNA, and it showed a significantly different dose-response pattern compared with parent cells. These results suggest that the uPA gene is regulated, at least partly, by a negatively regulating factor and that the action of cAMP is linked to this factor.

A New Genetic Approach for Studying Hormonal Regulation of Urokinase-Type Plasminogen Activator Gene Expression in LLC-PK1 Cells

In LLC-PK1 cells, a cyclic AMP (cAMP)-elevating peptide hormone, calcitonin, induces urokinase-type plasminogen activator (uPA) gene transcription without concomitant protein synthesis. To understand the molecular mechanism of the uPA gene regulation by cAMP, we developed a system which allows us to obtain mutant cells with modified regulatory proteins. A uPA-gpt hybrid gene was constructed, in which the regulatory region of the uPA gene was linked to a bacterial xanthine-guanine phosphoribosyltransferase gene (gpt), and it was transfected into LLC-PK1 cells. A stably transformed cell line, which expressed gpt only in the presence of calcitonin, was obtained, and then these cells were treated with a chemical mutagen, ethyl methanesulfonate. Cells were screened for constitutive gpt expression and, as mutations in regulatory proteins should affect the two genes at the same time, cells were further screened for an increased basal uPA mRNA level. Several such clones were obtained and none of them had modified cAMP-dependent protein kinase activity, suggesting that mutations were in the post-protein kinase step in the pathway of hormone action. Five clones were fused with the parent LLC-PK1 cells, and all of the fusion cells showed reduced basal uPA mRNA levels, indicating that they were recessive mutants. One clone was analyzed further for sensitivity to calcitonin in the induction of uPA mRNA, and it showed a significantly different dose-response pattern compared with parent cells. These results suggest that the uPA gene is regulated, at least partly, by a negatively regulating factor and that the action of cAMP is linked to this factor.

Transfection and stable expression of a dominant selective marker Ecogpt in a cultured cell line of the fish, Carassius auratus

ABSTRACT The synthetic plasmid, pSV2-gpt, was transfected into the cultured fish cells (RBCF-1 line) using polycation and dimethyl sulphoxide. The maximum transfection frequency estimated by colony number in the selection medium was 585 trans-fectants/5× 10s treated cells per 50 ng plasmid DNA. The isolated transfectants expressed the xanthine guanine phosphoribosyltransferase (XGPRT) activity encoded by the plasmid DNA associated with the promoter of simian virus 40 (SV40). The resistance to mycophenolic acid and the XGPRT activity of every transfectant examined were stable, indicating the possibility that pSV2-gpt was integrated into the genomic DNA of the host fish cells. Our results, in addition to those already reported for mammalian and avian cell lines, indicate that the promoter in the early region of SV40 can function in cultured fish cells. This success in obtaining cultured fish cells with a dominant selective marker will provide a useful clue for somatic cell genetic studies of fish in the future.

The effect of monofunctional or difunctional platinum adducts and of various other associated DNA damage on the expression of transfected DNA in mammalian cell lines sensitive or resistant to difunctional agents

The effects of introducing various DNA damage into pSV2gpt DNA on the subsequent expression of xanthine guanine phosphoribosyltransferase (XGPRT), after its transfection into two Walker 256 cell lines, one which is inherently sensitive only to difunctional agents while the other shows a normal sensitivity, have been examined. Both the sensitive (WS) and the relatively resistant (WR) cell lines were shown to be equally capable of both ligation of DNA double-strand breaks (although the efficiency varied with the actual site of the break) introduced into pSV2gpt and homologous recombination of pSV2gpt fragments (recombination events are thought to be important in the repair of DNA-DNA interstrand crosslinks). Reacting the plasmid with either the difunctional platinum compound, Cisplatin, or the monofunctional reacting Pt(Dien) caused a dose-dependent decrease in the subsequent expression of XGPRT. This decrease was about the same with either agent in either cell line when expressed as a function of dose of drug. However, when the actual binding of platinum to DNA by these compounds was measured, a large difference (due to the higher specific binding of Pt(Dien) to DNA) in the effects of the difunctional, as opposed to the monofunctional agent, was apparent and this was a reflection of the relative cytotoxicities of these compounds towards mammalian cells. Although at doses of Cisplatin equitoxic to WS and WR cells 20-fold less Pt is bound to the DNA of WS cells, no significant difference was seen on the expression of pSV2gpt, reacted with this agent, between WS or WR cells. Based upon a knowledge of the proportions of adducts formed in DNA reacted with Cisplatin, the lesion that inactivates expression of XGPRT was probably the intrastrand crosslink and it was calculated that due to the size of the plasmid, the interstrand crosslink was unlikely to be present at these inactivating doses. It is suggested that the inherent sensitivity of WS cells only to difunctional agents is due to their response to such relatively rare lesions such as a DNA-DNA interstrand crosslink.

Expression of human class II major histocompatibility complex antigens using retrovirus vectors.

Retrovirus vectors [direct orientation (DO) vectors] that permit the simultaneous expression of an inserted protein-coding sequence and a dominant-acting selectable marker have been constructed. In these vectors, an internal simian virus 40 or human metallothionein promoter sequence serves to drive the expression of the bacterial neomycin phosphotransferase or guanine-xanthine phosphoribosyltransferase genes, whereas the viral long terminal repeat sequences are utilized to promote expression of inserted sequences. In some of the vectors, the viral 5' splice site, normally used in the biogenesis of the subgenomic env-encoding mRNA, has been eliminated. These vectors yield high transient and stable titers of virus after transfection of viral packaging cell lines, show little or no depression of virus titer with a variety of inserts, and faithfully transmit recombinant proviral sequences to recipient cells. To characterize the expression potential of these vectors, a variety of inserts encoding the alpha and beta subunits of the human major histocompatibility complex class II antigen HLA-DR have been introduced into these vectors. NIH 3T3 cells infected by viruses containing HLA-DR alpha or beta cDNAs express these proteins as shown by immunoprecipitation of metabolically labeled extracts. In addition, through the sequential infection of cells with retrovirus constructions expressing two different selectable markers, both subunits of the class II antigen have been introduced into NIH 3T3 cells. Such infected cells express HLA-DR molecules at the cell surface.

Repression of insulin gene expression by adenovirus type 5 E1a proteins.

Insulin gene transcription relies on enhancer and promoter elements which are active in pancreatic beta cells. We showed that adenovirus type 5 infection of HIT T-15 cells, a transformed hamster beta cell line, represses insulin gene transcription and mRNA levels. Using expression plasmids transiently introduced into HIT T-15 cells, we showed that adenovirus type 5 E1a transcription regulatory proteins repress insulin enhancer-promoter element activity as assayed with a surrogate xanthine-guanine phosphoribosyltransferase gene. We relate E1a repression of the insulin gene to other examples of repression of enhancer-dependent genes by E1a and discuss the possible relationship of this repression to insulin gene regulation.

Repression of insulin gene expression by adenovirus type 5 E1a proteins.

Insulin gene transcription relies on enhancer and promoter elements which are active in pancreatic beta cells. We showed that adenovirus type 5 infection of HIT T-15 cells, a transformed hamster beta cell line, represses insulin gene transcription and mRNA levels. Using expression plasmids transiently introduced into HIT T-15 cells, we showed that adenovirus type 5 E1a transcription regulatory proteins repress insulin enhancer-promoter element activity as assayed with a surrogate xanthine-guanine phosphoribosyltransferase gene. We relate E1a repression of the insulin gene to other examples of repression of enhancer-dependent genes by E1a and discuss the possible relationship of this repression to insulin gene regulation.

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

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 recombinant pSV2gpt (which is identical to pSV3gpt but lacks the SV40 T antigen region) were not phenotypically altered.

DNA base changes and alkylation following in vivo exposure of Escherichia coli to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea.

Dideoxy chain-termination DNA sequencing was used to determine the specific DNA base changes induced after in vivo exposure of Escherichia coli to N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target. The resultant mutation spectra were compared with the levels of O6-alkylguanine and O4-alkylthymidine in genomic DNA immediately after exposure. All (39/39) of the MNU-induced mutations were G X C----A X T transitions. In contrast, 24/33 point mutations isolated following ENU treatment were G X C----A X T transitions, 7/33 were A X T----G X C transitions, 1/33 was a G X C----C X G transversion, and 1/33 was an A X T----C X G transversion. Three large insertions, probably of spontaneous origin, were also isolated. O4-alkylthymidine/O6-alkylguanine ratios were 0.014 for MNU and 0.28 for ENU. These data suggest that the difference in the mutation spectrum of MNU versus ENU may be attributed, in part, to the different ratio of O6-alkylguanine versus O4-alkylthymidine produced in the DNA. Of the G X C----A X T transitions, 82% of the MNU- and 71% of the ENU-induced mutations occurred at the middle guanine of the sequence 5'-GG(A or T)-3'.

The use of a selectable FokI cassette in dna replacement mutagenesis of the R388 dihydrofolate reductase gene

FokI, a class-IIS restriction endonuclease, cleaves double-stranded DNA to produce a protruding 5′ end consisting of four nucleotides, 10–13 residues 3′ from the nonpalindromic recognition sequence, GGATG. Cassettes which utilize this separation of cleavage and recognition site have been constructed for the purpose of linker mutagenesis and DNA replacement experiments. The cassettes are flanked by FokI recognition sequences oriented such that the FokI cleavage sites are several nucleotides beyond the cassette/vector fusion sites. FokI excises the cassette and several base pairs of the neighboring vector sequence. The ends produced in the vector by FokI cleavage are generally noncomplementary and suitable for the insertion of a segment of synthesized double-stranded replacement DNA. A cassette which contains a tyrosine tRNA suppressor gene ( supF) is selectable by the suppression of amber mutations in the recipient host. A vector containing a pBR322-derived origin of replication, the Escherichia coli xanthine-guanine phosphoribosyl transferase gene as a selectable marker, and no FokI sites has been constructed for use with the FokI cassettes. An experiment which utilized the FokI/supF cassette to modify the N-terminal coding region of the R388 dihydrofolate reductase gene is described.

Correction of alkylation hypersensitivity of CHO-W27-1 cells by transfection with human DNA.

Chinese hamster cells of the mutant strain W27-1 which is hypersensitive to UV and monofunctional alkylating agents were transfected with human DNA ligated to the bacterial xanthine-guanine phosphoribosyltransferase (gpt) gene. Selection was performed for resistance to mycophenolic acid and finally for survival after treatment with high doses of methyl methanesulfonate. A gpt+ transfectant was generated (T38-2-7) which acquired resistance to methyl methanesulfonate and cross-resistance to N-methyl-N'-nitro-N-nitrosoguanidine at levels comparable with the parental (wild-type) strain CHO-9. T38-2-7 cells were not more resistant, however, to UV, mitomycin C and N-hydroxyethyl-N-chloroethylnitrosourea than the mutant W27-1. The transfectant contains integrated human DNA and was shown to be deficient for the O6-methylguanine-DNA methyltransferase. The results indicate that the transfected DNA specifically complemented the defect underlying alkylation hypersensitivity of W27-1 cells or that a gene was transfected which is generally inactive in CHO cells and which causes alkylation resistance.