Molecular nature of spontaneous mutations at the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in chinese hamster ovary cells

The hypoxanthine-guanine phosphoribosyltransferase (hprt) locus has been widely used as a selectable genetic marker for studies of mammalian cell mutagenesis. We report here the spontaneous mutation spectrum at the hprt locus in 64 independently isolated mutants of Chinese hamster ovary (CHO) cells. All nine hprt exons were simultaneously analyzed via multiplex polymerase chain reaction (PCR) for rapid detection of gene deletions or insertions. Structural point mutations were identified by direct sequence analysis of the PCR amplified cDNA. The molecular nature of RNA splicing errors and insertions was analyzed by solid-phase direct exon sequencing. Single base substitutions were found in 24 mutants (38%), of which 21 were missense and 3 were nonsense mutations. Transversions were about twice as frequent as transitions. Fifteen mutants (23%) had deletions involving either intragenic small fragments (2), single exons (9), or multiple exons (4). The majority of deletion breakpoints (71%) were located in regions surrounding exons 4, 5, and 6. RNA splicing mutations were observed in 15 mutants (23%) and affected exons 3–8; most (6/15) resulted in the loss of exon 7. Two insertion mutants, one with a 209 bp insert in exon 4 and the other with a 88 bp insert accompanied by a 24 bp deletion in exon 6, represent novel mutations reported for the first time in spontaneous mutants of the mammalian hprt gene. © 1995 Wiley-Liss, Inc.     

Deletion and insertion in vivo somatic mutations in the hypoxanthine phosphoribosyltransferase (hprt) gene of human T-lymphocytes

Deletion and insertion mutations have been found to be a major component of the in vivo somatic mutation spectrum in the hypoxanthine phosphoribosyltransferase (hprt) gene of T-lymphocytes. In apopulation of 172 healthy people (average age, 34; mutant frequency, 10.3 × 10⁻⁶), deletion/in sertion mutations constituted 41% (89) of the 217independent mutations, the remainder being base substitutions. Mutations were identified by multiplex PCR assay of genomic DNA for exon regions, by sequencing cDNA, or sequencing genomic DNA. The deletion and insertion mutations were divided among ±1 to 2 basepair (bp) frameshifts (14%, 30), small deletions and insertions of 3–200 bps (13%, 28), large deletions of one or more exons (12%, 27), and complex events (2%, 4). Frameshift mutations were dominated by −1 bp deletions (21 of 30). Exon 3 contained five frameshift mutations in the run of 6 Gs, the only site in the coding region with multiple frameshift mutations, possibly caused by strand dislocation during replication. Both end points were sequenced for 23 of the 28 small deletions/insertions including two tandem duplication events in exon 6. More small deletions (8/28), pos sibly mediated by trinucleotide repeats, occurred in exon 2 than in the other exons. Large deletions included total gene deletions (6), exon 2 + 3 dele tions (4), and loss of multiple (9) and single exons (8) in genomic DNA. The diverse mutation spectrum indicates that multiple mechanisms operated at many different sequences and provides a resource for examination of deletion mutation. Environ. Mol. Mutagen. 30:371–384, 1997 © 1997 Wiley-Liss, Inc.     

Characterization of mutants involving partial exon duplications in thehprt gene of Chinese hamster V79 cells

Sequencing ofhprt cDNA revealed that three spontaneous mutants in V79 Chinese hamster cells exhibit tandem duplications of exon(s), i.e., either exons 2 and 3 or exon 7. Sequences of different sizes (4.5—8 Kb) were found to be duplicated and inserted in tandem into thehprt gene. These mutants demonstrated spontaneous reversion frequencies which were about 40-fold higher than those observed with other types of spontaneous mutants, but on the same order of magnitude as spontaneous reversions in Sp5, a mutant with a duplication insertion involving exon 2 in this gene. These data suggest that all of the duplications found have the same genetic instability, regardless of the type, size or position of the duplictaed fragment. The coding sequence of thehprt cDNA and the restriction pattern of the revertants were virtually identical to the wild-type, indicating restoration of a functionalhprt gene by precise deletion of the duplicated fragment.     

PCR-directed DNA sequencing of “nondeletion” HPRT− mutants induced by bleomycin in CHO K1-BH4 cells

Bleomycin is one of the radiomimetic antibiotics which induces DNA double-strand breaks by highly specific free radical attack on deoxyribose moieties in DNA. Earlier, we have shown that bleomycin induces a high proportion of large deletions involving one or more exons in the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in a Chinese hamster ovary (CHO) cell line CHO K1-BH4, in which no spontaneously occurring large deletions were detected by a polymerase chain reaction (PCR)-based deletion screening assay. Here we report the molecular nature of another class of mutants in which we did not observe any abnormal exon pattern. We refer to these mutants as the “nondeletion” type. Since bleomycin is a reactive oxygen species (ROS)-generating agent, we also studied whether the change of intracellular levels of ROS may affect the bleomycin-induced mutation spectra. We therefore also investigated the hprt mutation spectra induced by bleomycin with pretreatment by TRIEN (triethylenetetramine), a superoxide dismutase (SOD) inhibitor, and TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl), a SOD mimic. Analysis of these three bleomycin-induced “nondeletion” mutation spectra revealed that 5′-GT C-3′ or 5′-GC C-3′ sequences were the hot spots for single basepair deletions. Other types of mutation include abnormal cDNA or no cDNA amplification on the hprt locus. Due to the small sample size, we are unable to draw a definitive conclusion about the effects of TRIEN and TEMPOL on bleomycin-induced spectrum of “nondeletion” type hprt mutations. Environ. Mol. Mutagen. 32:244–250, 1998 © 1998 Wiley-Liss, Inc.     

Molecular analysis of T-lymphocyte HPRT- mutations in individuals exposed to ionizing radiation in Goiânia,Brazil

We have characterized 54 HPRT^- point mutations in T-lymphocytes from 17 individuals exposed to ionizing radiation of ¹³⁷Cs in Goiânia, Brazil and compared this spectrum to that of 30 HPRT^- mutants from 9 unexposed Brazilian controls. The average internal exposure of the exposed group was 205 mCi, and the average external exposure was 1.7 Gy. The average HPRT^- mutant frequency for the exposed group was 13.3 × 10^-5, approximately a 10-fold increase over the mutant frequency of the unexposed controls, which was 1.56 × 10^-5. The types of point mutations characterized included base substitutions, small deletions, frameshifts, in-sertions, complex mutations, and losses of exon sequences from the mRNA. The relative frequency of the different mutation types was similar in the two studied groups. However, in our study the distribution of events within the hprt coding sequence seemed to cluster at the same regions of the gene. These observations imply that the hprt gene does not present a homogeneous target to radiation mutagenesis, and perhaps this class of information may be used to detect radiation exposure in human populations. Environ. Mol. Mutagen. 29:107-116, 1997. © 1997 Wiley-Liss, Inc.     

Mutational spectrum of ICR-191 at the hprt locus in human lymphoblastoid cells

Human TK6 lymphoblasts were treated with the acridine derivative ICR-191, and mutants at the hprt locus were isolated. Mutant hprt cDNA was reverse-transcribed from mRNA, amplified by polymerase chain reaction (PCR), and sequenced. Additions of single G:C base pairs (+1 frameshift mutations) in repetitive G:C sequences were found in 82% (32/39) of the mutants. Sixteen of the +1 frameshifts analyzed were located in a single sequence of six consecutive guanine bases in exon 3. The remaining +1 frameshifts occurred at six different GGG sequences (14 mutants) and a single GGGG sequence (2 mutants) in other hprt exons. The repetitive guanine sequences that underwent frameshift mutagenesis were located in both the transcribed and nontranscribed strands of hprt. No single base deletions (-1 frameshift mutations) were observed. Base substitutions were observed in 13% (5/39) of the clones analyzed and occurred at both G:C and A:T bases. Loss of exon 4 from the cDNA was also observed in 5% (2/39) of the mutants. Hprt mutants containing seven consecutive guanines (produced from a +1 frameshift in a GGGGGG sequence) were treated with ICR-191 and wild-type revertants selected in CHAT medium. Revertants were recovered at a frequency of approximately 10^?7 and contained the wild-type sequence (GGGGGG) in all clones analyzed. The observed frequency of ICR-191-induced -1 frameshift reversion in the GGGGGGG sequence was ～500-fold lower than the estimated frequency of +1 frameshifts observed in the wild-type GGGGGG sequence following the same ICR-191 treatment. These results suggest that ICR-191 produces predominantly +1 frameshift mutations at the hprt locus in human cells. © 1994 Wiley-Liss, Inc.     

Molecular characterization of mutation and comparison of mutation profiles in the hprt gene of Chinese hamster ovary cells treated with benzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide, 1-nitrobenzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide,and 3-nitrobenzo[a]pyrene trans-7,8-diol-anti-9,10-epoxide

Both 1- and 3-nitrobenzo[a]pyrene (nitro-8aP) are environmental contaminants, potent mutagens in Salmonella, and moderate mutagens in Chinese hamster ovary (CHO) cells. The mutagenicity of their oxidized metabolites, trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-1-nitrobenzo[a]pyrene (1-nitro-Bap-DE) and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydro-3-nitrobenzo[a]pyrene (3-nitro-Bap-DE), together with trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP-DE), was determined in CHO-K1 cells, and the resulting mutations at the hprt locus were characterized by polymerase chain reaction (PCR) amplification of reverse-transcribed hprt mRNA, followed by DNA sequence analysis. The mutant frequencies, in mutants/10⁶ clonable cells, at 30 and 100 ng/ml, were BaP-DE, 248 and 456; 1-nitro-BaP-DE, 68 and 260; 3-nitro-BaP-DE, 81 and 232, respectively. In general, the three diolepoxides exhibited similar mutational spectra: 1) 64% (23/36 sequenced mutants) of BaP-DE, 53% (19/36) of 1-nitro-BaP-DE, and 64% (23/36) of 3-nitro-BaP-DE mutants resulted from simple base pair substitution, with the predominant mutation being G→T transversion; 2) 90%, 100%, and 100% of mutations at G:C had the mutated dG on the nontranscribed DNA strand; and 3) about one quarter of the mutants produced by each mutagen had one or more PCR products with partial or complete exon deletions. The mutagens induced few frameshifts or complex mutations. Among the differences in mutational specificity for the three diolepoxides, the proportion of substituted dGs with 3′ purines was significant (P < 0.05) for BaP-DE (16/19, 84%) and 3-nitro-BaP-DE (17/20, 85%), but not significant for 1-nitro-BaP-DE-induced mutants (11/17, 65%, P > 0.05). Also, high proportions of BaP-DE and 3-nitro-BaP-DE base pair substitutions at G:C occurred in DNA sequence contexts of 5′-GG-3′, 5′-GGA-3′, and 5′-TGGA-3′, while the proportions of 1-nitro-BaP-DE mutants in these contexts were often lower. The results indicate that nitro substitution at C1 or C3 of BaP-DE reduces mutational potency in CHO cells and appears to have only subtle effects upon the mutational pattern in the hprt gene. © 1996 Wiley-Liss, Inc.     

A new T-lymphocyte cloning assay for detection of in vivo mutations in the human hypoxanthine-guanine phosphoribosyltransferase gene

The X-linked hypoxonthine-guanine phosphoribosyltransferase (hprt) gene is a target of analyses of in vivo mutation frequencies in circulating T-lymphocytes. We established a novel, accessory cell-free cloning method of T-lymphocytes with a hprt mutation by a combined use of recombinant interleukin-2, conditioned medium from activating T-lymphocytes and culture plates coated with anti-CD3 monoclonal antibody. Using the method, we examined mutation frequencies of the hprt gene in T-lymphocytes from six healthy individuals, nine patients with colon cancer including two patients from different families with hereditary nonpolyposis colon cancer and six cancer-free relatives of the patients. In six healthy individuals, the mean cloning efficiency and mutation frequency (MF) of the hprt gene in T-lymphocytes were 0.51 ± 0.28 and 9.4 ± 7.5 × 10⁻⁶, respectively. These data were similar to the reported values. The mean MFs in the nine colon cancer patients (10.6 ± 7.3 × 10⁻⁶) were not significantly different from those of the 12 cancer-free individuals (11.6 ± 9.4 × 10⁻⁶). The correlation between mutation frequencies and age of the individuals was significant regardless of the presence or absence of cancers. The single-strand conformation polymorphism analyses of nested RT-PCR products of hprt mRNA were done in 33 mutant clones from five members of a family of which MF values were high. All the analyzed mutant clones show a genetic aberration in the coding region of the hprt gene. At least 28 of 33 mutants were independent. Our method provides a new versatile tool for in vivo analysis for mutations of the hprt gene. Environ. Mol. Mutagen. 30:31–39, 1997 © 1997 Wiley-Liss, Inc.     

Localization of Chinese hamster dihydrofolate reductase gene to band p23 of chromosome 2

It has been shown that gamma irradiation causes extensive deletions at the dihydrofolate reductase (dhfr)locus in Chinese hamster ovary (CHO) cells. We have analyzed seventeen DHFR-negative (DHFR ^–)mutants of CHO cells for cytogenetic alterations involving the dhfrlocus on chromosome 2. Five DHFR ^– mutants contained the same large deletion [del(2)(p16p23)] in the short arm of chromosome 2. This deletion comprised about 18% of the short arm and was estimated to be 41,000 kb in length. Four other DHFR ^– mutants contained smaller deletions of about 9200 kb. One of these mutants had a partial deletion of bands 2p22 and 2p23, whereas the others showed deletion of band 2p23. Inversions of chromosome 2 were seen in two other DHFR ^– mutants. An analysis of the breakpoints involved in these cytogenetic alterations indicates that the hamster dhfrgene resides in band p23 of chromosome number 2.     

Comprehensive genomic and phenotypic characterization of germline FH deletion in hereditary leiomyomatosis and renal cell carcinoma

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a familial cancer syndrome associated with the development of cutaneous and uterine leiomyomas, and an aggressive form of type 2 papillary kidney cancer. HLRCC is characterized by germline mutation of the FH gene. This study evaluated the prevalence and clinical phenotype of FH deletions in HLRCC patients. Patients with phenotypic manifestations consistent with HLRCC who lacked detectable germline FH intragenic mutations were investigated for FH deletion. A series of 28 patients from 13 families were evaluated using a combination of a comparative genomic hybridization (CGH) array and/or CLIA‐approved FH deletion/duplication analyses. Thirteen distinct germline deletions were identified in the 13 UOB families, including 11 complete FH gene deletions and 2 partial FH gene deletions. The size of eight evaluated complete FH deletions varied from ～4.74 Mb to 249 kb, with all deletions resulting in additional gene losses. Two partial FH gene deletions were identified, with one resulting in loss of exon 1 and the upstream region of the FH gene only. Kidney cancer was diagnosed in 9 (32%) of 28 patients and 7 (54%) of 13 families possessing either complete or partial FH deletions. Cutaneous and uterine leiomyomas were observed at similar rates to those in FH point mutation families. Complete or partial FH gene alterations in HLRCC families are associated with all of the canonical HLRCC manifestations, including type 2 papillary kidney cancer and should be screened for in any patient at‐risk for this disorder.     

The molecular nature of mutations induced by adriamycin at the hprt locus of V79 cells

Adriamycin (AM), a widely used chemotherapeutic drug, induceda broad spectrum of gene mutations at the hprt locus of V79cells. The frequency and distribution of AM-induced deletionswas analyzed with multiplex polymerase chain reaction in twoV79 cell lines, which differed considerably in their spontaneousdeletion frequency. Among AM-induced mutants, deletions predominatedin both cell lines. Apart from total deletions of the hprt gene,partial deletions were found which were distributed all overthe hprt gene with breakpoints in nearly all introns. Underthe same experimental conditions, chromosome aberrations wereinduced by AM which mainly represented chromatid-type aberrations.Neither the induction of gene mutations nor the induction ofchromosome aberrations was enhanced by the repair inhibitor3-aminobenzamide. These results are discussed in the contextwith our earlier findings on bleomycin-induced mutations andit is suggested that at least two mechanisms lead to the formationof gene deletions. One of them seems to be associated with amisrepair process of frank DNA double-strand breaks and relatedto chromosome aberrations while the other is not. ¹To whom correspondence should be addressed     

System Issues: Mutagenic response of the endogenous hprt gene and lacl transgene in benzo[a]pyrene-treated Big Blue™ B6C3F1 mice

Big Blue™ (BB) and generic B6C3F1 mice were given one to three i.p. injections of 50 mg/kg benzo[a]pyrene (B[a]P) in DMSO every other day to achieve cumulative doses of 50 to 150 mg/kg. Three weeks after treatment, the mutation frequency at the endogenous hprt gene and lacl transgene was measured in splenic T cells. Generic mice given 50, 100, and 150 mg/kg B[a]P displayed induced hprt⁻ frequencies (observed hprt⁻ frequency minus control frequency) of 5.5 ± 1.0, 11 ± 2.0, and 19 ± 2.6 × 10⁻⁶, respectively (average ± SEM). In contrast, BB mice given 50 and 150 mg/kg B[a]P displayed induced hprt⁻ frequencies of 0.9 ± 0.6 and 9.1 ± 1.5 × 10⁻⁶. ³²P postlabelling revealed that the lower hprt response in BB mice correlated with lower amounts of BP-DNA adducts in spleen, liver, and lung 24 hours offer B[a]P exposure. Western blot analysis of liver samples from B[a]P-treated mice suggests that the reduced adducts load in turn may be due to lower P450 1A1 levels in BB mice. The frequency of induced, nonsectored blue plaques (observed blue plaque frequency minus control frequency) in BB mice receiving 50 and 150 mg/kg B[a]P was 41 ± 9 and 134 ± 10 × 10⁻⁶ (15- to 40-fold higher than the induced hprt− frequency in the same treated animals). Sectored plaques were observed in both control and B[a]P groups but their frequency showed no relationship to dose (sectored frequency in all groups was approximately 20 × 10⁻⁶). To test whether persistent DNA adducts in the packaged lambda vector were contributing to the observed blue plaque frequency, purified λ-LIZ DNA was treated in vitro with B[a]P diol epoxide (BPDE), packaged, and plated on E. coli lawn cells. Treatment with BPDE did not produce significant increases in homogeneous blue plaques, suggesting that the majority of mutants obtained from B[a]P-treated BB mice occurred in vivo. These results indicate that B[a]P exposure produces many more mutations at the lacl transgene than at the endogenous hprt locus. © 1996 Wiley-Liss, Inc.     

Spontaneous mutation spectrum in the hprt gene in human lymphoblastoid TK6 cells

A spectrum of 100 mutations in the endogenous hprt gene of thehuman lymphoblastoid TK6 cell line is presented. The majorityof the mutations originates in sequences outside the codingregion of the gene. Large deletions are a major cause of inactivationof the hprt gene (57% of the mutants). Mutations in the splicesites that result in several forms of aberrantly spliced mRNAare relatively frequently recovered (16%) compared with mutantscontaining alterations in the coding region of the hprt gene(27%). The majority, but not all, of the splice mutants containan alteration in the consensus sequences of the splice sites.A spectrum of mutations in the coding region of the hprt geneenlarged to a total of 42 mutants shows that basepair substitutionspredominate (71%) and that small deletions and insertions areless frequently recovered. Basepair substitutions arise slightlymore frequently at GC basepairs than at AT basepairs. ³To whom correspondence should be addressed     

Detection of deletion mutations extending beyond the HPRT gene by multiplex PCR analysis

A multiplex PCR assay was developed for the rapid analysis of deletion size at the hypoxanthine phosphoribosyltransferase (hprt) locus. The DNA sequence of mapped DNA segments flanking thehprt gene was determined. These cloned DNAs were derived from the ends of a set of overlapping yeast artificial chromosomes (YAC) defining a contig of 8 Mb at Xq26 and includinghprt. We used bubble PCR to isolate an additional YAC end-clone. Seven primer pairs were derived from DNA sequence analysis of the clones and incorporated into a multiplex PCR assay. These primer pairs define loci located approximately 750 kb and 350 kb upstream ofhprt and 300 kb, 540 kb, 900 kb, 1260 kb, and 1400 kb downstream ofhprt. A primer pair for an unlinked and unselected gene sequence (K-ras) was also included in the multiplex reaction to serve as an internal positive control. Using this new assay,hprt mutant DNAs can be screened to determine the extent of deletion. Deletions larger than 2 Mb have been identified and show that large deletions can be tolerated at this hemizygous locus.     

DNA sequence analysis of in vivo hprt mutation in human T lymphocytes

We have determined the molecular basis of hypoxanthine-guaninephosphoribosyltransferase (hprt) mutations that arose in vivoin the T lymphocytes of a normal male subject. In previous studies16% (23/141) of the mutants from this individual analyzed bySouthern blot displayed large structural alterations in hprt.Thirty-two mutants without these large hprt structural alterationsproduced sufficient hprt cDNA for polymerase chain reactionamplification and DNA sequence analysis. Base substitutionsin hprt cDNA resulting in missense mutations and one mRNA splicingaberration (inclusion of intron sequences) were observed in18/32 of these these mutants; substitutions occurred at bothAT and GC base pairs. Small deletions (3/32), a tandem changeand a single base insertion were also observed among the hprtcDNAs. Exon skipping and inclusion of hprt intron sequencesin the hprt cDNA were observed in an additional 9/32 of themutants. Analysis of T cell receptor (TCR) gene rearrangementsrevealed that six of eight mutants with an identical hprt T—Atransversion displayed the same TCR rearrangement pattern, indicatingthat they were clonally related and arose from a single in vivomutational event. ³Present address: Department of Pathology, University of NorthCarolina Chapel Hill, NC 27514, USA ⁴To whom correspondence should be addressed      

Amsacrine-induced mutations in AS52 cells

Amsacrine is an acridine-derived inhibitor of topoisomerase II that intercalates into DNA. We performed a detailed molecular analysis of 6-thioguanine (6-TG)-resistant mutant colonies arising in AS52 cells following Amsacrine treatment. AS52 cells carry a single copy of the bacterial gpt gene, functionally expressed using the SV40 early promoter and stably integrated into the Chinese hamster ovary genome. A 1-hr treatment with 0.1 to 0.5 μM Amsacrine was both cytotoxic and mutagenic, resulting in an average mutant frequency (MF) of 143 × 10⁻⁶ at 0.5 μM. Fifty independent 6-TG-resistant colonies were isolated for further study. These clones were initially characterised by PCR to estimate the relative proportion of putative point mutants and deletions or rearrangements; then a subset of mutants was further characterised by Southern blotting, Northern blotting, and DNA sequence analysis. Total deletion of the gpt gene sequences was found in 1 (2%) of the mutants, and 7 (14%) of the mutant clones had altered PCR patterns, suggesting complex deletions or rearrangements. The remaining 42 (84%) mutants had a wild-type PCR profile. Of these, 21 mutants were further analysed by Southern blotting. Interestingly, Southern blotting revealed genomic deletions/rearrangements in 12 of 21 mutants with a wild-type PCR profile. These deletions/rearrangements were further shown to affect gpt gene expression. The remaining nine mutants with a wild-type PCR profile were sequenced. Four of these mutants had mutations in the gpt structural gene. Overall, genomic deletions/rearrangements were observed in 12/21 independent mutants subjected to PCR and Southern blotting. Thus, deletions/rearrangements were the most common mutation observed following Amsacrine treatment of AS52 cells. Environ. Mol. Mutagen. 32:47–55, 1998. © 1998 Wiley-Liss, Inc.     

Quantification of hprt gene deletions mediated by illegitimate V(D)J recombination in peripheral blood cells of humans

V(D)J recombinase is normally involved in the highly regulated rearrangement of immunoglobulin and T-cell-receptor gene segments (in B and T cells, respectively) to form functional antibody genes and T-cell-receptor genes. Occasionally, this tightly controlled process acts on inappropriate places in the genome and results in deletions and translocations. Some of these illegitimate V(D)J recombinase-mediated events have been implicated in the genetic changes associated with several forms of leukemia and lymphoid malignancy. We have developed a sensitive, specific polymerase chain reaction (PCR)-based assay to quantify such events in the peripheral blood cells of humans. This assay detects a V(D)J recombinase-mediated deletion in the hprt gene, which codes for a housekeeping enzyme and is not implicated in cancer development. Alterations in this gene serve as a surrogate indicator for these illegitimate events, which may be occurring throughout the genome. The assay involves a heminested PCR with two sets of primers. Multiple replicates of genomic DNA (each representing 4 × 10⁵ cells) are amplified with specific primers under conditions in which a single copy will give a detectable PCR product. Poisson statistics are then used to estimate the deletion mutant frequency. The frequency of cells with the hprt deletion among 20 healthy young adults ranged from < 1.3 × 10⁻⁷ to 4.1 × 10⁻⁷ and was compared with the frequency of t(14; 18) previously determined in these same individuals. No correlation was found between the frequencies of these two measures of genomic rearrangement. The DNA sequences of the deletion junctions were determined and provided evidence for multiple independent mutations in some individuals. This assay may serve as a biomarker for the level of illegitimate V(D)J recombination occurring in peripheral blood cells of humans. Environ. Mol. Mutagen. 29:28–35, 1997 © l997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.