Molecular analysis of DNA adducts and hprt mutations produced by 6-nitrosochrysene in Chinese hamster ovary cells

We have characterized the mutational spectrum of 6-nitrosochrysenein the hprt gene of Chinese hamster ovary (CHO-K1) cells andalso examined the adducts formed by this compound in CHO-K1cells by quantitative ³²P-postlabeling analysis. Seventy percentof the identified mutations were simple basepair substitutions,and they occurred more often at A:T (14/17) than at G:C. Furthermore,13 of the basepair substitutions at A:T had the mutated dA,the probable adducted residue, on the non-transcribed DNA strand.The preference for mutation at A:T contrasted sharply with thedistribution of adducts formed by 6-nitrosochrysene: 80% ofthe identified adducts were with dG, while only 20% were probablyformed through binding with dA. Analyses conducted with excision-repair-defectiveCHO-UV5 cells revealed both a preference for basepair substitutionat A:T and an adduct profile that were similar to those foundfor repair-proficient CHO-K1 cells. However, basepair substitutionsfrom CHO-UV5 cell mutants had the mutated dAs distributed randomlybetween the non-transcribed and transcribed DNA stands. Themutational spectra found for solvent control CHO-K1 and CHO-UV5cells differed from those of the 6-nitrosochrysene-treated cultures.These findings suggest that 6-nitrosochrysene-treated cultures.These findings suggest that 6-nitrosochrysene-induced mutationsare targeted to DNA damage, but that 6-nitrosochrysene-deriveddA adducts are much more effective at producing mutations than6-nitrosochrysene-derived dG adducts. The extreme strand biasfor mutated dAs in the CHO-K1 mutational spectrum appears toresult from preferential removal of 6-nitrosochrysene-inducedDNA lesions from the transcribed DNA strand.     

Trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene is metabolized to form a major adduct with deoxyguanosine and produces mutations in the hprt gene of Chinese hamster ovary cells at G:C basepairs

6-Nitrochrysene can be activated to genotoxic derivatives bytwo major metabolic pathways: nitroreduction to N-hydroxy-6-aminochrysene,and a combination of ring-oxidation and nitroreduction thatinvolves the intermediate formation of trans-1,2-dihydro-1,2-dihydroxy-6-aminochrysene(6-AC-1,2-dihydrodiol). The DNA adduct formed from this latterpathway was evaluated by reacting individual deoxynucleoside5'-monophosphates with 6-AC-1,2-dihydrodiol in the presenceof liver microsomal enzymes from 3-methylcholanthrene-pretreatedrats. Binding was greatest to deoxyguanosine monophosphate andthe major deoxyguanosine (dG) adduct co-chromatographed withthe single major adduct formed from the microsome-catalyzedreaction of 6-AC-1,2-dihydrodiol with DNA. In order to characterizethe mutational changes associated with the 6-AC-1,2-dihydrodiolpathway, we analyzed the mutational spectrum produced by 6-AC-1,2-dihydrodiolin the hypoxanthine-guanine phosphoribosyl-transferase (hprt)gene of CHO-K1 cells. cDNA was synthesized from the RNA of 286-thioguanine-resistant mutants, the hprt coding region amplifiedby the polymerase chain reaction, and the DNA products directlysequenced. Twenty independent primary mutations were found:12 G:C T:A transversions, three G:C C:G transversions, oneG:C A:T transition, one A:T T:A transversion, two –1frameshift mutations in sequences containing consecutive guanines,and one 11 bp deletion. All G:C basepair substitutions had themutated dG on the non-transcribed strand and 86% of the G:Cbasepair substitutions had one purine 3' to the mutated dG.The pattern of 6-AC-1,2-dihydrodiol-induced basepair substitutionswas distinct from the pattern observed in solvent control mutants.These results are consistent with the formation of a promutagenicdG adduct from a metabolite of 6-AC-1,2-dihydrodiol.     

Spectrum of point mutations in the coding region of the hypoxanthine- guanine phosphoribosyltransferase (hprt) gene in human T-lymphocytes in vivo

The hypoxanthine-guanine phosphoribosyl transferase (hprt) locus in 6- thioguanine (TG) resistant T-lymphocytes is a useful target for the study of somatic in vivo mutagenesis, since it provides information about a broad spectrum of mutation. Mutations in the hprt coding region were studied in 124 TG-resistant T-cell clones from 38 healthy, non- smoking male donors from a previously studied population of bus maintenance workers, fine-mechanics and laboratory personnel. Their mean age was 43 years (range 23-64) and their hprt mutant frequency was 9.3 +/- 5.2 x 10(-6) (mean +/- SD, range 1.4-22.6 x 10(-6)). Sequence analysis of hprt cDNA identified 115 unique mutations; 76% were simple base substitutions, 10% were +/-1 bp frameshifts, and 10% were small deletions within exons (3-52 bp). In addition, two tandem base substitutions and one complex mutation were observed. Simple base substitutions were observed at 55 (20%) of 281 sites known to be mutable in the hprt coding sequence. The distribution of these mutations was significantly different than would be expected based upon a Poisson distribution (P < 0.0001), suggesting the existence of 'hotspots'. All of the 87 simple base substitutions occurred at known mutable sites, but eight were substitutions of a kind that have not previously been reported at these sites. The most frequently mutated sites were cDNA positions 197 and 146, with six and five independent mutations respectively. Four mutations were observed at position 131, and three each at positions 143, 208, 508 and 617. Transitions (52%) were slightly more frequent than tranversions (48%), and mutations at GC base pairs (56%) more common than mutations at AT base pairs (44%). GC > AT was the most common type of base pair substitution (37%). The majority of the mutations at GC base pairs (78%) occurred at sites with G in the non-transcribed strand. All but one of eight mutations at CpG- sites were of the kind expected from deamination of methylated cytosine. Deletion of a single base pair (-1 frameshift) was three times more frequent than insertion of a single bp (+1 frameshift). Almost half (6/13) of the small (3-52 bp) deletions within the coding sequence clustered in the 5' end of exon 2. Short repeats and other sequence motifs that have been associated with replication error were found in the flanking regions of most of the frameshifts and small deletions. However, several differences in the local sequence context between +/-1 frameshift and deletion mutations were also noticed. The present results identify positions 197, 146 and possibly 131 as hotspots for base substitution mutations, and confirm previously reported hotspots at positions 197, 508 and 617. In addition, the earlier notion of a deletion hotspot in the 5'end of exon 2 was confirmed. The observations of these mutational cluster regions in different human populations suggest that they are due to endogeneous mechanisms of mutagenesis, or to ubiquitous environmental influences. The emerging background spectrum of somatic in vivo mutation in the human hprt gene provides a useful basis for comparisons with radiation or chemically induced mutational spectra, as well as with gene mutations in human tumors.      

Frequency and molecular analysis of hprt mutations induced by estradiol in Chinese hamster V79 cells

The natural hormone estradiol (E2) induces tumors in rodents and various types of DNA damage in vitro and in vivo, but has not been mutagenic in bacterial or mammalian assays. Recent reports of chromosomal and genetic lesions induced by E2 has led us to re-examine the mutation frequency and molecular alterations of the hypoxanthine-guanine phosphoribosyltransferase (hprt) gene in Chinese hamster V79 cells. E2 at both physiological and pharmacological concentrations (10-11, 10-10, and 10-7, 10-6 M) significantly increased the mutation frequency of the hprt gene by 2. 57-, 3.45-, 2.63-, and 8.78-fold, respectively, compared to the controls, while 10-13, 10-12, 10-9, or 10-8 M E2 induced little change (< or =0.93-fold). PCR and a molecular analysis of the hprt coding sequence identified genetic lesions in the cDNA and/or genomic DNA in 15 of the 21 picked E2-induced mutants (71%). Simple base substitutions, such as Tright curved arrow G or Tright curved arrow A transversions, were the most common mutations (8/21 or 38%) and frequently occurred at 122 bp or 407 bp of the hprt coding sequence. Deletion mutations were detected in 6 of the 21 clones (29%). An Aright curved arrow G and a Cright curved arrow T transition and a four-base insertion (TATT) were identified each in one mutant clone. A RT-PCR analysis demonstrated an abundant expression of the estrogen receptor-alpha (ERalpha). However, ICI 182,780, an antagonist of ERalpha, acted in an additive manner with E2 and increased the hprt mutation frequency. In conclusion, E2 induces a low frequency of mutations (deletions and point mutations) in V79 cells, which is consistent with the weak carcinogenic activity of this hormone. The mutagenic effects of E2 in V79 cells are not mediated by the ERalpha.     

DNA base changes in benzo[a]pyrene diol epoxide-induced dihydrofolate reductase mutants of Chinese hamster ovary cells.

We previously treated Chinese hamster ovary (CHO) cells with benzo[a]pyrene diol epoxide (BPDE) and mutants at the dihydrofolate reductase (dhfr) locus were isolated. On the basis of Southern blotting and RNA heteroduplex mapping experiments, 14 of the 15 mutants were presumed to carry point mutations. Two restriction fragment length polymorphism mutants were cloned and sequenced; one carried a point mutation, the other a -1 frameshift mutation. Using polymerase chain reaction techniques and direct sequencing of amplified mutant DNA, we have now determined the induced changes in the remaining 12 cell lines. All changes occurred at guanine bases; all target guanines except one were on the non-transcribed coding strand. Most mutants (79%) contained base substitutions; the rest (3/14) carried frameshift mutations. Of the point mutations, all but one (91%) were GC----TA transversions either in the dhfr coding sequence or at splice sites. The single exception was a GC----AT transition. Of the frameshift mutations, two were deletions of a GC pair and the other was an insertion of an AT pair. Four different mutations (29%) were clustered in a 3 bp region in exon 4. Tandem guanine bases adjacent to adenine were favored sites for mutation occurring in 9/14 cases (64%). These results are consistent with data previously obtained by others in the supF shuttle vector system and the CHO aprt gene.     

Molecular analysis of hprt mutations generated in Chinese hamster ovary EM9 cells by uranyl acetate, by hydrogen peroxide, and spontaneously

Naturally occurring uranium and depleted uranium (DU) are believed to be health hazards by virtue of both their chemical and radiological properties. The mechanism(s) behind uranium's chemotoxic effects has yet to be elucidated. Previous work has shown that DU, as uranyl acetate (UA), was mutagenic at the hypoxanthine (guanine) phosphoribosyltransferase (hprt) locus in XRCC1-deficient CHO EM9 cells. The purpose of the current study was to characterize the mutations induced by UA at the hprt locus of CHO EM9 cells and compare the mutation spectrum of UA with those of hydrogen peroxide and spontaneous mutations in the same line. The hypothesis being tested was that if DU as UA is chemically genotoxic then the mutation spectrum induced by the heavy metal should be distinct from that produced spontaneously or by H2O2. A total of 59 UA-induced, 38 spontaneous, and 45 H2O2-induced mutations were identified. Base substitutions comprised 29%, 42%, and 16% of UA, spontaneous, and H2O2 mutants, respectively. The frequency of G --> T or C --> A substitutions was not significantly different in spontaneous or H2O2-induced mutants than in UA-induced mutants, suggesting a possible role for 8-oxodG damage in UA mutagenesis. However, the observation that UA produced significantly more major genomic rearrangements (multiexon insertions and deletions) than occurred spontaneously suggests the possibility that DNA strand breaks or crosslinks could also be UA-induced mutagenic lesions. The unique mutation spectrum elicited by exposure to UA suggests that UA generates mutations in ways that are different from spontaneous and free radical as well as radiological mechanisms.     

Mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline in human lymphoblastoid cells

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ), a heterocyclic aromatic amine that has been identified in cooked meats and cigarette condensates, is mutagenic in human lymphoblastoid TK6 cells at the thymidine kinase and hypoxanthine-guanine phosphoribosyl transferase (hprt) loci. Treatment of the cells with IQ following activation with either an exogenous metabolizing mixture (S9) or following photoactivation of the azido-derivative of IQ (N3-IQ) showed that the photolytic-derivative of N3-IQ was more active. This observation is consistent with other reports that indicate that the weak mutagenicity of IQ in mammalian cells is caused by the lack of enzymes required for the ultimate activation of the compound within the cells. Two DNA adducts were found by 32P-post-labelling in the cells treated with the photoactivated N3-IQ. The major adduct was identified as N- (deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline (dG-C8-IQ) and the minor adduct as 5-(deoxyguanosin-N2-yl)-2-amino-3- methylimidazo[4,5-f]quinoline (dG-N2-IQ). The ratio of the dG-C8IQ to the dG-N2-IQ adducts was approximately 3:1 and did not significantly change in cultures treated with different concentrations of the mutagen. Approximately 50% of the adducts were removed 9 h after treatment with IQ and <10% of these adducts remained after 24 h. There was no significant preferential repair of either adduct under the experimental conditions used. The identification of 15 mutations induced at the hprt locus (of the 44 mutants analysed) showed IQ to be efficient at inducing single base deletions in a run of guanines. Six single guanine deletions were observed in the run of six guanines in exon III and one deletion of a single guanine was observed in a non- repetitive sequence in exon VI. Other mutations observed were two GC-- >TA transversions, two GC-->CG transversions, one AT-->TA transversion and one GC-->AT transition. In addition, two multiple mutations were found. The majority of the identified mutations (12/15) occurred at GC base pairs and suggests either the dG-C8-IQ or the dG-N2-IQ adduct to be the pre-mutagenic lesion.      

Mutagenicity of vinyl chloride and its reactive metabolites, chloroethylene oxide and chloroacetaldehyde, in a metabolically competent human B-lymphoblastoid line

Vinyl chloride (VC), a known human and rodent carcinogen, is metabolically activated by cytochrome P450 to chloroethylene oxide (CEO), which can rearrange to chloroacetaldehyde (CAA) or undergo hydrolysis. To further understand the roles of CEO and CAA in VC mutagenesis, the types and frequencies of mutations induced at the hypoxanthine (guanine) phosphoribosyl-transferase (hprt) locus were examined in a human B-lymphoblastoid line constitutively expressing human cytochrome P450 2E1 (H2E1 cells). VC was toxic and mutagenic to H2E1 cells as a function of incubation time; exposure to 7.5% VC in air resulted in 75% survival and an hprt mutant frequency of 42 x 10(-6) after 48 h, compared to 5.7 +/- 2.7 x 10(-6) for unexposed cells. The exposure of H2E1 cells to 0.8 to 15.0% VC in air produced similar mutant frequencies without a clear dose-response relationship, suggesting saturation of metabolic activation. Both CEO and CAA exhibited dose-dependent increases in cell killing and mutant frequency in H2E1 cells. Treatment with 16 microM CEO for 24 h resulted in 75% survival and an induced mutant frequency of 23 x 10(-6), while 16 microM CAA produced 5% survival and an induced mutant frequency of 20 x 10(-6). Structural alterations at the hprt locus in independent thioguanine-resistant clones were examined by Southern blot analysis of Pst I-digested DNA with a full-length human hprt cDNA probe. Ten percent (5/50) of VC-induced and 18% (7/38) of CEO-induced mutants showed detectable deletions, compared with 45% (9/20) of CAA-induced mutants. Thus, VC and CEO displayed similar toxicity/mutation profiles and a similar frequency of large deletions, whereas CAA displayed greater toxicity and a larger frequency of deletion mutations. These results suggest that the majority of mutations induced by VC occur through its metabolite, CEO.      

Mutational specificity of the carcinogen 3-amino-1-methyl-5H-pyrido[4,3-b]-indole in mammalian cells

We have used the pZipHprtNeo shuttle vector to determine the types of DNA sequence alterations induced by a potent carcinogen 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P2). The shuttle vector contains a human cDNA hprt as the target gene and is stably integrated into a chromosome of the mouse cell line VH12. After Trp-P2 treatment, 59 independent HPRT- mutant clones of VH12 were isolated and altered sequences of the mutant hprt- cDNA genes were determined. Mutations induced by Trp-P2 comprised a variety of events; base substitutions, frameshifts, deletions and complex. Frameshifts were the most frequent mutational events (51%), and base substitutions were the next most frequent (30%) followed by deletions (14%). Examination of the DNA sequence context in the mutant genes revealed that approximately 70% of mutations induced by Trp-P2 occurred at G:C sites and thymine residues were the suggested target for the remainder of mutations. The results seem consistent with the previously reported finding that in vivo, metabolically activated Trp-P2 specifically binds to the C8 position of guanine residues in DNA to form C8G-Trp-P2 adducts (Hashimoto et al., Mutat, Res., 105, 9-13, 1982). As for molecular mechanisms, we showed that slippage and slippage misalignment could predict the generation of a large portion of Trp-P2-induced mutations found in the cDNA gene.     

Mutation of p53 gene codon 63 in saliva as a molecular marker for oral squamous cell carcinomas

The inactivation of tumor suppressor gene (TSG) is important during multistage carcinogenesis. The p53 TSG is frequently mutated in oral squamous cell carcinomas. These mutations can serve as very specific markers for the presence of tumor cells in a background of normal cells. In this study, 10 oral squamous cell carcinoma patients and 27 normal dental students were collected from Chung Shan Medical and Dental College Hospital, Taichung, Taiwan. Extractions of DNA from saliva were obtained. Exon 4 and intron 6 within the p53 gene were amplified with polymerase chain reactions (PCRs) followed by DNA sequence analysis. DNA sequence analysis of PCR products revealed that five of eight (62.5%) tumor saliva samples and five of 27 (18. 52%) healthy saliva samples contained p53 exon 4 codon 63 mutations. These results were significantly different by using Chi-square test (P<0.05). The majority of the base substitutions were C deletions. Probable hot spots for the mutation were identified at exon 4 codon 63, which has not been observed before in head and neck cancers. Our study indicated that mutation of p53 codon 63 in saliva might be a molecular marker for oral squamous cell carcinomas. In addition, the amount of DNA recovered from saliva in most cases is sufficiently large and its quality suitable to enable PCR amplification which could be used in the search for mutations. The protocol described is rapid, cheap, and easy to perform, and may be useful for epidemiological studies for oral carcinogenesis.     

Kinds of mutations induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX) in the hprt gene of Chinese hamster ovary cells

The kinds of mutations induced by 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone(MX) in the protein coding region of the hprt gene of Chinesehamster ovary (CHO) cells were determined by direct sequencingof polymerase chain reaction (PCR)-amplified cDNA. Primary mutationswere found in 15 of 19 of the mutants: 11 were G:CT:A transversions,two were A:TT:A transversions and two were deletions of singleG:C base pairs (-1 frameshifts). The remaining four mutantshad large alterations in the cDNA that were explained by mRNAsplicing errors. A group of control mutants had more diversehprt cDNA alterations than MX-induced mutants. Transversionsyielding an A:T base pair were the predominant type of MX-inducedmutations, in agreement with previous findings in bacteria.This specificity may be explained by the ‘A rule’,that DNA polymerases preferentially insert, adenine nucleotidesopposite non-instructional lesions.     

p53 mutations, protein expression and cell proliferation in squamous cell carcinomas of the head and neck.

Thirty-three patients with squamous cell carcinoma of the head and neck region were studied concerning p53 protein expression and mutations in exons 4-9 of the p53 gene using immunohistochemistry, polymerase chain reaction (PCR)-single strand conformation polymorphism analysis and DNA sequencing. Immunoreactivity was found in 64% and p53 gene mutations in 39% of the tumours. Thirty-three per cent of the immunopositive and 50% of the immunonegative tumours were mutated within exons 5-8. In one immunopositive tumour three variants of deletions were observed. Sequencing of the p53 mutated, immunonegative tumours revealed four cases with deletions, one case with a transversion resulting in a stop codon and one case with a splice site mutation which could result in omission of the following exon at splicing. All mutations in the immunonegative tumours resulted in a truncated p53 protein. No association between p53 gene status and expression of proliferating cell nuclear antigen (PCNA) or cell proliferation as judged by in vivo incorporation of the thymidine analogue iododeoxyuridine (IdUrd) was found.     

Analysis of ras gene mutations in rainbow trout liver tumors initiated by aflatoxin B1.

The suspect human hepatocarcinogen aflatoxin B1 (AFB1) is a well-known potent initiator of hepatic tumors in rainbow trout (Oncorhynchus mykiss). Both hepatocellular carcinomas and mixed hepatocellular/cholangiocellular carcinomas are induced by AFB1 in trout, with the mixed form predominating. We previously isolated two c-ras genes from trout liver cDNA, and in the present study we analyzed DNA from 14 AFB1-induced trout liver tumors for point mutations in exon 1 of both genes. Using the polymerase chain reaction (PCR) and oligonucleotide hybridization methods, a high proportion (10/14) of the AFB1-initiated tumor DNAs showed evidence of activating point mutations in the trout c-Ki-ras gene. Of the 10 mutant ras genotypes, seven were codon 12 GGA----GTA transversions, two were codon 13 GGT----GTT transversions, and one was codon 12 GGA----AGA transition. Nucleotide sequence analysis of cloned PCR products from four of these tumor DNAs provided definitive evidence for two codon 12 GGA----GTA mutations, one codon 12 GGA----AGA mutation, and one codon 13 GGT----GTT mutation, in complete agreement with the oligonucleotide hybridization results. No mutations were detected in exon 1 of a second trout ras gene also expressed in liver, nor in DNA from control livers. This is the first report of experimentally induced ras gene point mutations in a lower vertebrate fish model. The results indicate that the hepatocarcinogen AFB1 induces c-Ki-ras gene mutations in trout similar to those in rat liver tumors.     

P16 gene deletions and point mutations in patients with agnogenic myeloid metaplasia (AMM).

Studies of p16 alterations with homozygous deletions and mutation analysis were done in 32 patients with agnogenic myeloid metaplasia (AMM) including six patients in leukemic phase. No homozygous deletions were found and, one patient was found to have a shift band in exon 2C fragment by PCR-SSCP analysis. Further sequence analysis demonstrated that the mutated band was a point mutation of G to A in exon 2 codon 140 (GCG-->ACG) causing an amino acid substitution of alanine to threonine demonstrating this patient either carried an mutated gene in one allele as a polymorphism (heterozygous carrier of a mutant p16 gene) or carried a mutant p16 gene clone. This study demonstrates that p16 alterations with homozygous deletions and mutations were very rare in patients with AMM. A single patient found to have a shifted band by PCR-SSCP may be represented as a coincidence or as a polymorphism with a heterozygous carrier of mutated p16 gene, predisposable to AMM or as a mutant p16 gene which can be infrequently observed in this disease.     

DHPLC检测林州市高龄组和低龄组居民食管癌p53基因外显子突变谱的比较研究

目的:研究我国食管癌高发区林州市居民高龄食管癌患者与低龄食管癌患者中抑癌基因p53全部编码外显子基因突变谱的情况,比较两组患者中p53基因突变的差异。方法:留取林州市>70岁和<40岁、低年龄组食管癌患者食管癌新鲜标本51例,提取DNA,PCR扩增p53第2～11外显子。DHPLC进行突变的筛查。突变样本进行DNA纯化测序分析。结果:51例患者中,p53至少有1个外显子基因突变的36例,突变率为70.6%。高年龄组突变率为63.3%(19/30),低年龄组突变率为80.9%(17/21),两者比较无统计学意义(P>0.05)。p53有2个外显子基因突变的10例,突变率为17.6%。高年龄组突变率为26.7%(8/30),低年龄组突变率为9.5%,两者比较无统计学意义,P>0.05。结论:p53基因突变是林州市居民食管癌发生过程中的一个发生频率很高的事件。林州市居民的p53外显子突变谱中,共有15种突变类型,共计59个突变位点,其中最多的突变类型是插入碱基C。在p53基因第2～8外显子与内含子交界的非编码区有大量的基因突变。     

The mutagenicity and DNA base sequence changes induced by 1-nitroso- and 1-nitropyrene in the cl gene of lambda prophage

The mutagenicity of 1-nitropyrene and its reduced metabolite1-nitrosopyrene was determined in the lambda cI gene of an Escherichiacoli uvr^- lysogen. 1-Nitropyrene induced a mutation frequencyof 3.8 x 10^–6, which was {small tilde} 2-fold higher thanthe background mutation frequency, whereas an equimolar doseof 1-nitrosopyrene induced a much higher mutation frequencyof 1.4 x 10^–4. Previous studies have established thatboth compounds form the same premutagenic lesion, viz. N-(deoxyguanosin-8-yl)-l-aminopyrenein bacterial DNA. In order to determine how this initial premutationallesion is converted to a stable heritable mutation, DNA sequenceswere determined for 30 mutations induced by 1-nitrosopyrenethat mapped between bp 1 and 352 in the lambda cI gene of E.colilysogens. We show here that these mutations are mainly frameshiftsinvolving the addition or deletion of a single GC or CG basepair. A small proportion of mutations were base substitutionswhich were equally divided between transitions and transversions.These also occurred primarily at GC or CG sites.     

Mechanisms of oncogenesis in patients with familial retinoblastoma.

In an analysis of mutations in the RB1 gene in three patients, selected at random, who had a positive family history of tumours, we identified mutations, in constitutional cells, involving exons 3, 13 and 17 of the RB1 gene. We used SSCP and PCR sequencing to screen affected individuals and other members of their families. In two cases the mutations were 2 bp and 1 bp deletions identified in exons 3 and 17 respectively. The third mutation was a 1 bp insertion in exon 13. All three mutations lead to the generation of downstream premature stop codons as a result of frameshift changes, although the mutation in exon 3 possibly affects the splicing mechanism. The sites within the RB1 gene where these mutations occur contain interspersed repetitive DNA sequences, direct and inverted repeat sequences and/or dyad symmetrical elements suggesting that these areas promote the appropriate local sequence environment for the generation of deletions and insertions in the RB1 gene.     

Identification of MEN1 gene mutations in families with MEN 1 and related disorders

Following identification of the MEN1 gene, we analysed patients from 12 MEN 1 families, 8 sporadic cases of MEN 1, and 13 patients with MEN 1-like symptoms (e.g. cases of familial isolated hyperparathyroidism (FIHPT), familial acromegaly, or atypical MEN 1 cases) for the presence of germline MEN1 mutations. The entire coding region of the MEN1 gene was sequenced, and mutations were detected in 11 MEN 1 families; one sporadic MEN 1 patient, one case of FIHPT and one MEN 1-like case. Constitutional DNA samples from individuals without MEN1 mutations were digested with several restriction enzymes, Southern blotted and probed with MEN1 cDNA to analyse for the presence of larger deletions of the MEN1 gene unable to be detected by PCR. One MEN 1 patient was found to carry such a deletion. This patient was heterozygous for the D418D polymorphism, however sequence analysis of RT-PCR products showed that only the variant allele was transcribed, thus confirming the result obtained by Southern analysis, which indicated loss of a region containing the initiation codon of one allele.