The role of nucleotide excision repair in protecting embryonic stem cells from genotoxic effects of UV-induced DNA damage.

In this study the role of nucleotide excision repair (NER) in protecting mouse embryonic stem (ES) cells against the genotoxic effects of UV-photolesions was analysed. Repair of cyclobutane pyrimidine dimers (CPD) in transcribed genes could not be detected whereas the removal of (6-4) photoproducts (6-4PP) was incomplete, already reaching its maximum (30%) 4 h after irradiation. Measurements of repair replication revealed a saturation of NER activity at UV doses >5 J/m2 while at a lower dose (2.5 J/m2) the repair kinetics were similar to those in murine embryonic fibroblasts (MEFs). Cytotoxic and mutagenic effects of photolesions were determined in ES cells differing in NER activity. ERCC1-deficient ES cells were hypermutable (10-fold) compared to wild-type cells, indicating that at physiologically relevant doses ES cells efficiently remove photolesions. The effect of the NER deficiency on cytoxicity was only 2-fold. Exposure to high UV doses (10 J/m2) resulted in a rapid and massive induction of apoptosis. Possibly, to avoid the accumulation of mutated cells, ES cells rely on the induction of a strong apoptotic response with a simultaneous shutting down of NER activity.

Nucleotide excision repair modulates the cytotoxic and mutagenic effects of N-n-butyl-N-nitrosourea in cultured mammalian cells as well as in mouse splenocytes in vivo.

The butylating agent N-n-butyl-N-nitrosourea (BNU) was employed to study the role of nucleotide excision repair (NER) in protecting mammalian cells against the genotoxic effects of monofunctional alkylating agents. The direct acting agent BNU was found to be mutagenic in normal and XPA mouse splenocytes after a single i.p. treatment in vivo. After 25 and 35 mg/kg BNU, but not after 75 mg/ kg, 2- to 3-fold more hprt mutants were detected in splenocytes from XPA mice than from normal mice. Using O6-alkylguanine-DNA alkyltransferase (AGT)-deficient hamster cells, it was found that NER-deficient CHO UV5 cells carrying a mutation in the ERCC-2 gene were 40% more mutable towards lesions induced by BNU when compared with parental NER-proficient CHO AA8 cells. UV5 cells were 1.4-fold more sensitive to the cytotoxic effects of BNU compared with AA8 cells. To investigate whether this increased sensitivity of NER-deficient cells is modulated by AGT activity, cell survival studies were performed in human and mouse primary fibroblasts as well. BNU was 2.7-fold more toxic for mouse XPA fibroblasts compared with normal mouse fibroblasts. Comparable results were found for human fibroblasts. Taken together these data indicate that the role of NER in protecting rodent cells against the mutagenic and cytotoxic effects of the alkylating agent BNU depends on AGT.

Modulation of the toxic and mutagenic effects induced by methyl methanesulfonate in Chinese hamster ovary cells by overexpression of the rat N-alkylpurine-DNA glycosylase.

Exposure of mammalian cells to alkylating agents causes transfer of alkyl groups to N- as well as O-atoms of DNA bases. Especially the O-alkylated G and T bases have strong mutagenic properties, since they are capable of mispairing during replication. The mutagenic potential of N-alkylbases is less clear although specific base excision repair (BER) pathways exist which remove those lesions from the DNA. We investigated the relative contribution of N-alkylations to mutation induction at the Hprt gene in cultured Chinese hamster ovary cells (CHO). To this end BER activity in CHO cells was modulated by introduction of an expression vector carrying the rat N-alkylpurine-DNA glycosylase (APDG) gene, which codes for a glycosylase that is able to remove 3-methyladenine and 7-methylguanine from DNA thereby generating apurinic sites. Upon selection of a CHO clone which 10 times overproduced APDG compared to control CHO cells, mutation induction, the mutational spectrum, and cell survival were determined in both cell lines following treatment with methyl methanesulfonate (MMS). The results show that over-expression of APDG renders CHO cells more sensitive for mutation induction as well as cytotoxicity induced by MMS. The involvement of apurinic sites in induction of base pair changes at positions where 3-methyladenine was induced is inferred from the observation that the mutational spectrum of MMS-induced mutations in APDG-CHO cells showed twice as much base pair changes at AT base pairs (33.3%) compared to the spectrum of MMS-induced mutations in CHO-control cells (15.8%).

Preliminary study on the effect of miniaturisation and use of volatile mobile phases in LC for the on-line LC-MS analysis of basic pharmaceuticals.

To enhance to compatibility of the on-line coupling of liquid chromatography (LC) with mass spectrometry (MS) for the analysis of basic pharmaceuticals, the use of volatile mobile phase systems in combination with miniaturised LC was investigated. Multifactor analysis of variance (MANOVA) was used to evaluate the data obtained for the various variables (modifier, stationary phase, buffer, buffer pH and buffer concentration) on the resolution, peak symmetry and retention of four basic compounds analysed using LC columns with internal diameters (I.D.) of 0.3, 1.0 and 4.6 mm (conventional). Preliminary results obtained with the investigated micro and conventional columns showed similar behaviour with respect to ruggedness. The various investigated variables showed that miniaturisation by simply downscaling dimensions can result in varying selectivity and peak shapes for basic compounds. When comparing volatile mobile phases (containing ammonium acetate or ammonium citrate) and a conventional non-volatile mobile phase (containing sodium phosphate) under pH 3 conditions, similar separation performances were observed. In the present study, ammonium citrate as the buffering salt, a high buffer concentration and methanol as the modifier showed the best peak symmetry.

Alkylpurine-DNA-N-glycosylase knockout mice show increased susceptibility to induction of mutations by methyl methanesulfonate.

Alkylpurine-DNA-N-glycosylase (APNG) null mice have been generated by homologous recombination in embryonic stem cells. The null status of the animals was confirmed at the mRNA level by reverse transcription-PCR and by the inability of cell extracts of tissues from the knockout (ko) animals to release 3-methyladenine (3-meA) or 7-methylguanine (7-meG) from 3H-methylated calf thymus DNA in vitro. Following treatment with DNA-methylating agents, increased persistence of 7-meG was found in liver sections of APNG ko mice in comparison with wild-type (wt) mice, demonstrating an in vivo phenotype for the APNG null animals. Unlike other null mutants of the base excision repair pathway, the APNG ko mice exhibit a very mild phenotype, show no outward abnormalities, are fertile, and have an apparently normal life span. Neither a difference in the number of leukocytes in peripheral blood nor a difference in the number of bone marrow polychromatic erythrocytes was found when ko and wt mice were exposed to methylating or chloroethylating agents. These agents also showed similar growth-inhibitory effects in primary embryonic fibroblasts isolated from ko and wt mice. However, treatment with methyl methanesulfonate resulted in three- to fourfold more hprt mutations in splenic T lymphocytes from APNG ko mice than in those from wt mice. These mutations were predominantly single-base-pair changes; in the ko mice, they consisted primarily of AT-->TA and GC-->TA transversions, which most likely are caused by 3-meA and 3- or 7-meG, respectively. These results clearly show an important role for APNG in attenuating the mutagenic effects of N-alkylpurines in vivo.

Elevated frequencies of benzo(a)pyrene-induced Hprt mutations in internal tissue of XPA-deficient mice.

Xeroderma pigmentosum (XP) patients are hypersensitive to sunlight and have a high predisposition to developing cancer. At the cellular level, XP patients are defective in nucleotide excision repair (NER). Recently, mice have been generated via gene targeting that are deficient in the expression of the XPA gene [A. de Vries et al., Nature (Lond.), 377: 169-173, 1995]. We have assessed the consequences of defective NER for mutagenesis in normal and XPA mice exposed to benzo(a)pyrene and 2-acetylaminofluorene. To study mutagenesis, mature T lymphocytes were isolated from the spleen and stimulated to proliferate in vitro to select for mutants at the endogenous Hprt locus. Background mutant frequencies in normal and XPA mice were very similar and not influenced by age. Single doses of benzo(a)pyrene administered i.p. resulted in a dose-dependent increase of the Hprt mutant frequency in normal mice. In addition, after chronic exposure to benzo(a)pyrene, Hprt mutants were readily detectable in XPA mice at an early onset of treatment but only at a later stage in normal mice. In contrast, chronic treatment of either normal or XPA mice with 2-acetylaminofluorene did not increase Hprt mutant frequency above the background frequency. This absence of significant induction of Hprt mutants can be entirely attributed to the low frequency of 2-acetylaminofluorene-induced DNA adducts in lymphoid tissue. These results provide the first direct evidence in mammals that deficient NER leads to enhanced mutagenesis in endogenous genes in internal tissue after exposure to relevant environmental mutagens, such as benzo(a)pyrene.

Effect of nucleotide excision repair on hprt gene mutations in rodent cells exposed to DNA ethylating agents.

The role of the nucleotide excision repair (NER) pathway in removal of DNA ethylation damage was investigated by means of hprt mutational spectra analysis in the NER-deficient Chinese hamster ovary cell line UV5, which lacks ERCC2/XPD, and in its parental cell line AA8. Both cell lines were exposed to ethyl methanesulfonate (EMS) or N-ethyl-N-nitrosourea (ENU). EMS gave a similar dose-dependent increase in hprt mutants in UV5 compared with AA8. In both cell lines EMS-induced mutations in the hprt coding region consisted almost exclusively of GC-->AT transitions, probably due to the direct miscoding lesion O6-ethylguanine. ENU, an agent that in addition to O6-ethylguanine also induces other O-alkylation products, was significantly more mutagenic in UV5 than in AA8. Mutational spectra analysis showed that the proportions of ENU-induced GC-->AT, AT-->TA and AT-->GC base pair changes were similar for both cell lines. ENU-induced DNA lesions that may be involved in GC-->AT transitions are O6-ethylguanine and O2-ethylcytosine, the latter being a chemically stable DNA lesion of which the miscoding properties and repair characteristics are largely unknown. ENU-induced AT-->TA transversions are probably caused by O2-ethylthymine, which mispairs with thymine. In AA8 thymines in ENU-induced AT-->TA transversions were exclusively located in the non-transcribed strand of the hprt gene, whereas in UV5 30% of these thymines were found in the transcribed strand. Together, these results indicate that O6-ethylguanine is a poor substrate for NER in rodent cells and that O2-ethylpyrimidines are preferentially removed from the transcribed strand of the hprt gene by NER.

Induction of hprt gene mutations in splenic T-lymphocytes from the rat exposed in vivo to DNA methylating agents is correlated with formation of O6-methylguanine in bone marrow and not in the spleen.

The suitability of splenic T-lymphocytes as a substitute tissue for detection of genotoxic effects induced in vivo by chemical agents that are organ-specifically activated was tested in rats exposed to single doses at the potent lung-carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), acetoxymethylmethylnitrosamine (AMMN) or N-methyl-N-nitrosourea (MNU). NNK, AMMN and MNU methylate DNA most likely via the formation of a methanediazohydroxide ion that decomposes to a methyl diazonium ion. For all three agents, an increase in the levels of 06-methylguanine and 7-methylguanine in DNA of rat liver and lung was detected by reverse phase HPLC and electrochemical detection. Treatment with NNK did not result in the formation of O6-methylguanine and 7-methylguanine in DNA of bone marrow and spleen, corresponding with the absence of metabolic activation pathways for this compound in these tissues. For AMMN formation of both 06-methylguanine and 7-methylguanine was detectable in DNA of the spleen, whereas in DNA of bone marrow only very low frequencies of 7-methylguanine were found at a toxic dose. MNU induced O6-methylguanine and 7-methylguanine in both spleen and bone marrow. Using splenic T-lymphocytes from the rat no increase above control levels of the hprt mutant frequencies was found for NNK and AMMN for all exposure levels tested, 32 days after chemical exposure. For MNU a dose-dependent increase in hprt mutant frequency was found at exposure levels of 0.097 mmol/kg up to 0.582 mmol/kg. DNA sequence analysis was performed on PCR products of hprt cDNA from 39 MNU-induced 6-thioguanine-resistant T-lymphocyte clones. Single base pair substitutions were found in 25 of these mutants (64%), GC-->AT transitions being the predominant type of mutation (19 of 25; 76%). These mutations are probably caused by mispairing of 06-methylguanine with thymine during DNA replication. The results indicate that formation of mutagenic lesions in the spleen is not correlated with an enhanced frequency of 6-thioguanine-resistant splenic T-lymphocyte clones from rats, 32 days after exposure in vivo to DNA damaging agents. This suggests that mutation-fixation in T-lymphocytes does not occur in the spleen but at other sites in the body such as bone marrow, after which these mutated cells migrate to the spleen.

Identification and genetic analysis of a common molecular variant of histidine-rich glycoprotein with a difference of 2kD in apparent molecular weight.

Two forms of histidine-rich glycoprotein (HRG) were detected on SDS-PAGE by silver staining and immunoblotting after isolation of the protein from pooled plasma using immuno-affinity chromatography followed by chromatography with heparin-Sepharose. Both forms were single-chain molecules and the apparent molecular weights of form 1 and form 2 were 77 kD and 75 kD respectively. Mendelian inheritance of both HRG forms was observed in four families with 24 informative meioses, strongly suggesting that the two forms are encoded by different alleles. The frequency of form 1 and form 2 in a group of 36 individuals was 0.35 and 0.65 respectively. The difference between the two molecular variants was studied by direct sequence analysis of amplified exons of the HRG gene from 6 individuals who were homozygous either for form 1 or form 2. Five amino acid polymorphisms in three different exons were observed: Ile/Thr in exon4; Pro/Ser in exon 5; His/Arg, Arg/Cys and Asn/Ile in exon 7. Analysis of these polymorphisms in 20 volunteers showed that only the Pro/Ser polymorphism at position 186 in exon 5 was coupled to the form of the HRG protein. Ser was found in form 1 and Pro in form 2. The presence of Ser at position 186 introduces a consensus sequence for a N-glycosylation site (Asn-X-Ser/Thr). By removing N-linked sugars with N-glycanase, it could be demonstrated that the difference between the two forms of HRG is caused by an extra carbohydrate group at Asn 184 in form 1.

Detection of point mutations in T lymphocytes.

We exposed experimental animals to a series of alkylating agents that induced mutations at the X-linked hprt gene of T lymphocytes. We then isolated the mutant cells and analyzed the molecular nature of the mutations by amplification of hprt cDNA sequences with the use of reverse transcriptase PCR followed by DNA sequence analysis, and then correlated the mutational spectra obtained to the spectra of DNA adducts caused by the alkylating agents used. The nature of the base-pair changes causing the mutations was characteristic for the reaction pattern of the genotoxic agent with DNA. However, we also found a clear influence of DNA repair processes; i.e., in those cells that were able to remove certain types of DNA damage, the class of mutations expected from that type of damage was reduced.

Marked differences in the role of O6-alkylguanine in hprt mutagenesis in T-lymphocytes of rats exposed in vivo to ethylmethanesulfonate, N-(2-hydroxyethyl)-N-nitrosourea, or N-ethyl-N-nitrosourea.

The role of DNA alkylation at the O6 position of guanine in the induction of gene mutations in vivo was studied in the hprt gene of rat T-lymphocytes from spleen exposed in vivo to the monofunctional ethylating agents ethylmethanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU), or the hydroxyethylating agent N-(2-hydroxyethyl)-N-nitrosourea (HOENU). All chemicals showed an exposure-dependent increase in hprt mutant frequency. HOENU and ENU, however, were much more mutagenic than EMS when compared at equimolar levels. DNA sequence analysis was performed on PCR products of hprt cDNA from 40 EMS-, 35 HOENU-, and 46 ENU-induced 6-thioguanine-resistant T-lymphocyte clones. Thirty EMS-induced mutants contained a single base pair substitution with GC to AT transitions being the predominant type of mutation (26 of 30) which are probably caused by mispairing of O6-ethylguanine with T during DNA replication. No strand specificity of mutated G's among GC to AT transitions was observed. Twenty-three HOENU- and 42 ENU-induced mutants contained a single base pair substitution. In contrast to EMS, GC to AT transitions were found at a low frequency, 4 of 23 for HOENU and 5 of 42 for ENU, indicating that O6-hydroxyethylguanine and O6-ethylguanine are less important in HOENU- and ENU-induced mutagenesis in vivo, respectively. Also here no strand bias for mutated G's was observed, although the number of this type of mutation was limited. The most frequently induced base pair alterations by HOENU and ENU were transversions at AT base pairs, 16 of 23 and 28 of 42, respectively, with AT to TA being the predominant type of mutation. In both ENU and HOENU mutational spectra, an extreme strand bias for mutated T's toward the nontranscribed strand was found. The results suggest that DNA damage induced in rat T-lymphocytes in vivo by HOENU and ENU is processed in similar ways.

Mechanisms and biomarkers of genotoxicity. Molecular dosimetry of chemical mutagens.

The relevance of the use of DNA adduct frequencies as a parameter for the extent of mutation induction by monofunctional alkylating agents was investigated in cultured Chinese hamster cells and in rat skin fibroblasts treated in vivo with the test chemicals. The nature of the biologically significant DNA adducts was investigated by DNA sequence analysis of mutations induced at the hypoxanthine-guanine phophoribosyltransferase (hprt) gene. The results show that under conditions where O6-alkylguanine is a persistent DNA lesion more than 50% of the mutations are GC to AT transitions indicating that the frequency of O6-alkylguanine is a good parameter for mutation induction. However, in target cells which are able to remove alkyl groups from the O6 position of guanine, alkylating agents with a low nucleophilic selectivity (e.g. N-ethyl-N-nitrosourea (ENU) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG)) exert most of their mutagenic activity most likely via the induction of O2-ethylthymine.

AT base pairs are the main target for mutations at the hprt locus of rat skin fibroblasts exposed in vitro to the monofunctional alkylating agent N-ethyl-N-nitrosourea.

Spectra of N-ethyl-N-nitrosourea (ENU)-induced mutations differ widely among various in vitro and in vivo mutational systems. To investigate possible reasons for these differences, a mutational system is needed in which the same target gene is used for comparison in the same type of cells in vitro and in vivo. In the present study, this was achieved by analysing at the molecular level 35 hprt mutant rat fibroblast clones obtained from cell populations exposed in vitro to ENU and comparing the mutational spectrum with the previously determined spectrum of ENU-induced hprt mutants in the same target cells exposed in vivo. Twenty-eight mutants contained a single base pair alteration in the hprt coding sequence. Most of these changes were found at AT base pairs (19/28), the AT to TA transversion being the most frequent kind of mutation (12/19), which is probably caused by O2-ethylthymine. Transversions at AT base pairs showed all mutated T's to be located in the nontranscribed strand of the hprt gene, suggesting a strand specific fixation of mutations induced by O2-ethylthymine, which appears to be a general feature of ENU- and ENNG-induced hprt mutations in mammalian cells. GC to AT transitions, probably caused by O6-ethylguanine, were detected at a lower frequency (7/28). This in vitro mutational spectrum was very similar to that of the same target cells exposed in vivo to ENU. A comparison of the mutational spectra in AGT-proficient and AGT-deficient rodent cells exposed to ethylating agents showed that in contrast to the situation in AGT-proficient rat fibroblasts, GC to AT base pair changes (and not AT to TA) are the predominant mutations in AGT-deficient hamster cells.

Molecular dosimetry of 7-alkyl- and O6-alkylguanine in DNA by electrochemical detection.

A methodology is described for the quantitation of 7-alkyl- and O6-alkylguanine in DNA isolated from experimental animals exposed to alkylating agents. Following purification, the DNA is hydrolysed under acid conditions after which 7-alkyl- and O6-alkylguanine are separated from unmodified bases by HPLC using a strong cation exchange column. The fractions containing the methylated purines are subsequently analyzed by HPLC using a reverse phase column coupled to an electrochemical detector (amperometric). This method allows the detection of 10-20 fmoles 7-alkyl- and O6-alkylguanine, when pure markers are analyzed. In practice, the detection limit is 0.5 adducts per 10(6) nucleotides for the methylated and 1 adduct per 10(6) nucleotides for the ethylated form of 7-alkyl- and O6-alkylguanine using 25 micrograms DNA.

Formation and persistence of DNA adducts in pouch skin fibroblasts and liver tissue of rats exposed in vivo to the monofunctional alkylating agents N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea.

Base substitutions and frameshifts induced by genotoxic agents are considered to result mainly from incomplete repair and incorrect replication of modified nucleotides in DNA. In this study, induction and persistence of O6-alkyl- and 7-alkylguanine adducts were determined by reverse phase HPLC and electrochemical detection in DNA of pouch skin fibroblasts and liver tissue of rats exposed in vivo to the monofunctional alkylating agents N-methyl-N-nitrosourea (MNU) and N-ethyl-N-nitrosourea (ENU). Although an exposure dependent increase in the level of adducts was found for both chemicals, a much lower frequency of both O6-alkylguanine and 7-alkylguanine was detected after ENU treatment than after MNU treatment, indicating that MNU is much more reactive with DNA than ENU. The persistence of O6-alkyl- and 7-alkylguanine was studied for up to 48 h at exposure levels of 60 mg/kg for MNU and 100 mg/kg for ENU. A time-dependent decline in the levels of both adducts was observed, but w6-alkylguanine was more rapidly lost than 7-alkylguanine in both pouch skin fibroblasts and liver. Furthermore, DNA adducts were faster lost from liver than from pouch skin fibroblasts. The loss of O6-alkylguanine adducts is probably mediated by the action of O6-alkylguanine-DNA alkyltransferase (AGT) in the target tissues since AGT activity was detectable in protein extracts of pouch skin fibroblasts and liver from unexposed rats and from exposed rats, 48 h but not 1 h after MNU and ENU treatment. AGT activity recovered faster in liver tissue than in pouch skin fibroblasts, and after ENU exposure an induction of AGT activity was observed in the liver but not in pouch skin fibroblasts. The difference in the level of O6-alkylguanine in DNA of pouch skin fibroblasts introduced upon exposure to MNU and ENU may explain the molecular nature of most base pair changes observed previously in spectra of hprt mutants induced in these cells in vivo. The frequency of O6-methylguanine upon MNU exposure remains relatively high with time and these adducts most likely cause GC to AT transitions. In the case of ENU, O6-ethylguanine was detected at very low frequencies resulting in a low contribution of GC to AT transitions. Rather, the ENU spectrum is dominated by base pair changes at AT base pairs.

Molecular analysis of hprt gene mutations in skin fibroblasts of rats exposed in vivo to N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea.

The granuloma pouch assay in the rat is a model system in which relative frequencies of genetic and (pre-) neoplastic changes induced in vivo by carcinogenic agents can be determined within the same target tissue. The target is granuloma pouch tissue and consists of a population of (transient) proliferating fibroblasts which can be cultured in vitro. hprt gene mutations were studied in granuloma pouch tissue of rats treated with single doses of direct acting alkylating agents N-methyl-N-nitrosourea (MNU) or N-ethyl-N-nitrosourea (ENU). Both agents showed an exposure-dependent increase in the hprt mutant frequency. Thirty-seven MNU (60 mg/kg)- and 43 ENU (100 mg/kg)-induced hprt mutant cell clones were analyzed at the molecular level. Twenty-two MNU-induced and 36 ENU-induced mutants carried a single base pair change in exon sequences of the hprt gene. The predominant base pair alterations induced by MNU were GC to AT transitions (18 of 22), which are probably caused by O6-methylguanine lesions. For most of the GC to AT transitions (16 of 18), the G was located in the nontranscribed strand, suggesting a strand bias in the repair of O6-methylguanine lesions. ENU-induced mutations occurred predominantly at AT base pairs (28 of 36), being mostly AT to TA and AT to CG transversions, and are probably caused by O2-ethylthymidine. Also here, DNA repair processes seem to act with different rates/efficiencies on DNA adducts in the 2 strands of the hprt gene, since all the 24 transversions observed at AT base pairs had the thymidine residue in the nontranscribed strand. GC to AT transitions were only present at a low frequency among ENU-induced mutations, suggesting that O6-ethylguanine lesions were repaired efficiently before mutations were fixed during replication. The mutational spectra of MNU- and ENU-induced hprt mutant clones were different from spontaneously occurring hprt mutant clones. These results indicate that MNU and ENU induce different mutational spectra in vivo and that DNA repair systems remove O6-methylguanine, O2, and/or O4-ethylthymidine much faster from the transcribed strand than the nontranscribed strand of the hprt gene in these rat fibroblasts.

The gene encoding hypoxanthine-guanine phosphoribosyltransferase as target for mutational analysis: PCR cloning and sequencing of the cDNA from the rat.

In this paper, the cloning and nucleotide sequence of the cDNA of the rat gene coding for hypoxanthine-guanine phosphoribosyltransferase (hprt) is reported. Knowledge of the cDNA sequence is needed, among other reasons, for the molecular analysis of hprt mutations occurring in rat cells, such as skin fibroblasts isolated according to the granuloma pouch assay. The rat hprt cDNA was synthesized and used as a template for in vitro amplification by PCR. For this purpose, oligonucleotide primers were used, the nucleotide sequences of which were based on mouse and hamster hprt cDNA sequences. Sequence analysis of 1146 bp of the amplified rat hprt cDNA showed a single open reading frame of 654 bp, encoding a protein of 218 amino acids. In the predicted rat hprt amino acid sequence, the proposed functional domains for 5'-phosphoribosyl-1-pyrophosphate (PRPP) and nucleotide binding in phosphoribosylating enzymes as well as a region near the carboxyl terminal part were highly conserved when compared with amino acid sequences of other mammalian hprt proteins. Analysis of hprt amino acid sequences of 727 independent hprt mutants from human, mouse, hamster and rat cells bearing single amino acid substitutions revealed that a large variety of amino acid changes were located in these highly conserved regions, suggesting that all 3 domains are important for proper catalytic activity. The suitability of the hprt gene as target for mutational analysis is demonstrated by the fact that amino acid changes in at least 151 of the 218 amino acid residues of the hprt protein result in a 6-thioguanine-resistant phenotype.