High frequency of homoplasmic mitochondrial DNA mutations in human tumors can be explained without selection.

Researchers in several laboratories have reported a high frequency of homoplasmic mitochondrial DNA (mtDNA) mutations in human tumors. This observation has been interpreted to reflect a replicative advantage for mutated mtDNA copies, a growth advantage for a cell containing certain mtDNA mutations, and/or tumorigenic properties of mtDNA mutations. We consider another possibility-that the observed homoplasmy arose entirely by chance in tumor progenitor cells, without any physiological advantage or tumorigenic requirement. Through extensive computer modeling, we demonstrate that there is sufficient opportunity for a tumor progenitor cell to achieve homoplasmy through unbiased mtDNA replication and sorting during cell division. To test our model in vivo, we analyzed mtDNA homoplasmy in healthy human epithelial tissues and discovered that the model correctly predicts the considerable observed frequency of homoplasmic cells. Based on the available data on mitochondrial mutant fractions and cell division kinetics, we show that the predicted frequency of homoplasmy in tumor progenitor cells in the absence of selection is similar to the reported frequency of homoplasmic mutations in tumors. Although a role for other mechanisms is not excluded, random processes are sufficient to explain the incidence of homoplasmic mtDNA mutations in human tumors.

Bell-shaped nuclei dividing by symmetrical and asymmetrical nuclear fission have qualities of stem cells in human colonic embryogenesis and carcinogenesis.

Large cell nuclei with at least eight distinct morphologies have been discovered throughout the fetal gut (5-7 weeks), colonic adenomas, and adenocarcinomas, five of which are not present in the normal adult colon. The most remarkable nuclear forms are hollow bells, approximately 10-15 microns in height and about 7-10 microns in bell mouth diameter. When encased in tubular syncytia, these bell-shaped structures divide symmetrically by an amitotic nuclear fission process resembling the separation of two paper cups. Seven other nuclear morphotypes emerge from the bell-shaped nuclei within the syncytia by asymmetrical amitotic nuclear fission. Cells containing these differentiated nuclear forms subsequently divide extra-syncytially by mitoses that form clonal populations of cells with identical nuclear morphotypes in embryos, adenomas, adenocarcinomas, and metastases. Cells with bell-shaped nuclei thus appear to be responsible for both net growth and differentiation in the embryonic gut, adenomas, and adenocarcinomas, and fulfill the requirements for post-embryonic stem cells in colon organogenesis and carcinogenesis.

A sensitive scanning technology for low frequency nuclear point mutations in human genomic DNA.

Knowledge of the kinds and numbers of nuclear point mutations in human tissues is essential to the understanding of the mutation mechanisms underlying genetic diseases. However, nuclear point mutant fractions in normal humans are so low that few methods exist to measure them. We have now developed a means to scan for point mutations in 100 bp nuclear single copy sequences at mutant fractions as low as 10(-6). Beginning with about 10(8) human cells we first enrich for the desired nuclear sequence 10,000-fold from the genomic DNA by sequence-specific hybridization coupled with a biotin-streptavidin capture system. We next enrich for rare mutant sequences 100-fold against the wild-type sequence by wide bore constant denaturant capillary electrophoresis (CDCE). The mutant-enriched sample is subsequently amplified by high fidelity PCR using fluorescein-labeled primers. Amplified mutant sequences are further enriched via two rounds of CDCE coupled with high fidelity PCR. Individual mutants, seen as distinct peaks on CDCE, are then isolated and sequenced. We have tested this approach by measuring N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced point mutations in a 121 bp sequence of the adenomatous polyposis coli gene (APC) in human lymphoblastoid MT1 cells. Twelve different MNNG-induced GC-->AT transitions were reproducibly observed in MNNG-treated cells at mutant fractions between 2 x 10(-6) and 9 x 10(-6). The sensitivity of this approach was limited by the fidelity of Pfu DNA polymerase, which created 14 different GC-->TA transversions at a mutant fraction equivalent to approximately 10(-6) in the original samples. The approach described herein should be general for all DNA sequences suitable for CDCE analysis. Its sensitivity and capacity would permit detection of stem cell mutations in tissue sectors consisting of approximately 10(8) cells.

Concentration-dependent mutation by alkylating agents in human lymphoblasts and Salmonella typhimurium: N-methyl-N-nitrosourethane and beta-propiolactone.

The toxic and mutagenic effects of the alkylating agents N-methyl-N-nitrosourethane (MNUT) and beta-propiolactone (BPL) were quantitatively measured in human lymphoblasts and Salmonella typhimurium. Forward mutation to 6-thioguanine resistance was measured in the human lymphoblasts, and forward mutation to 8-azaguanine resistance was measured in the bacterial cells after equigenerational (1.5 doubling times) exposures. In both systems, the induced mutant fraction rose linearly as a function of concentration for BPL and was biphasic for MNUT. The responses of the two assay systems to eight alkylating agents were compared. The exposure of the cells to each alkylating agent was calculated as exposure concentration multiplied by the time of exposure, and allowance was made for the decomposition of the alkylating agents during exposure (integral exposure). Human cells were 2.5--13 times more sensitive than was S. typhimurium to the alkylating agents methyl methanesulfonate, ethyl methanesulfonate, propyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, methylnitrosourea, and MNUT. S. typhimurium cells were three times more sensitive to butyl methanesulfonate and 25 times more sensitive to BPL than were human cells.

Mutation of human cells by kerosene soot.

The polycyclic aromatic hydrocarbon fraction of a kerosene soot induced forward mutation in human diploid lymphoblasts when coincubated with Sprague-Dawley rat liver postmitochondrial supernatant. Two components of the kerosene soot extract, benzo[a]pyrene (BP) and cyclopenta[cd]pyrene (CP), were also tested. TP was not mutagenic at the concentration found in the soot extract, although it was active at higher concentrations. The amount of CP present could account for approximately 8% of the total mutation observed with the soot. The results were compared to data obtained previously in a similar mutation assay in Salmonella typhimurium. The protocol described permits the facile assay of mutation at the hgprt locus in human lymphoblasts; such mutation is induced by compounds of complex mixtures requiring mixed-function oxygenase activity for metabolism to genetically active derivatives.

Pyruvate utilization, phosphocholine and adenosine triphosphate (ATP) are markers of human breast tumor progression: a 31P- and 13C-nuclear magnetic resonance (NMR) spectroscopy study.

We have used 31P- and 13C-nuclear magnetic resonance spectroscopy to measure key metabolite levels and fluxes through enzymes regulating phospholipid and mitochondrial metabolism in normal human mammary epithelial cells. We have compared these values to those found in a progression series of breast cancer cell lines of varying metastatic potential established from a single patient. We find a 16-19-fold increase in phosphocholine content in two primary breast cancer cell lines (21PT and 21NT) and a 27-fold increase in phosphocholine content in the metastatic breast cancer cell line (21MT-2) compared with the normal breast epithelial cell strain 76N. Thus, phosphocholine may serve as a metabolic marker for the human breast cell progression state. A 30% decrease in ATP levels, a 83% decrease in phosphocreatine levels, along with a 2-fold increase in NAD(+) + NADH levels in 21PT, 21NT, and 21MT-2 cells compared to the normal breast cells further suggests impaired mitochondrial metabolism in the breast carcinoma cell lines. Consistent with this suggestion is our finding that the primary breast cancer cell lines (21PT and 21NT) and the metastatic breast cell line (21MT-2) showed a 50 and 89% relative reduction, respectively, in the flux of pyruvate utilized for mitochondrial energy generation compared to pyruvate utilized to replenish tricarboxylic acid cycle intermediates. These results demonstrate that diminished mitochondrial energy generation may be quantitatively related to the progression state of human breast cells.

Quantitative differential effects of rhodamine 123 on normal cells and human colon cancer cells by magnetic resonance spectroscopy.

Rhodamine 123 is a lipophilic cationic compound that is selectively taken up by cancer cell mitochondria. This compound is toxic to epithelial cancer cells in vitro and displays significant anticancer activity in vivo. However, the mechanism of action of rhodamine 123 in intact, actively metabolizing cell preparations is unknown. We have used 31P- and 13C-nuclear magnetic resonance spectroscopy to quantitatively characterize how rhodamine 123 affects the energetics of human colon cancer cells (HCT-116) and spontaneously immortalized normal epithelial cells (CV-1). Rhodamine 123 differentially altered the phosphorus and glucose metabolism of HCT-116 and CV-1 cells. 31P-nuclear magnetic resonance detected mitochondrial poisoning in the HCT-116 human colon cancer cell line in its early stages after selective uptake of rhodamine 123. When we compared administration of rhodamine 123 and [1-C13]glucose to administration of [1-C13]glucose alone in the HCT-116 cells, we noted a marked decrease in intracellular pH to 6.7 +/- 0.06 (mean +/- SD) units, a 2.2-fold increase in lactate production, and a 1.8-fold increase in glucose consumption after 10 h. In addition, we found a 2-fold rise in intracellular free magnesium 12 h after rhodamine 123 administration. These results suggest that when rhodamine 123 inhibits mitochondrial ATP production, it initially stimulates cytoplasmic glycolysis in an attempt to maintain cellular energy demands. The marked fall in intracellular pH and rise in intracellular free magnesium after administration of rhodamine 123 may inhibit activity of several glycolytic enzymes: this effect would inhibit cytoplasmic ATP generation and interfere with multiple cell enzymatic processes, leading to cell death. The CV-1 cells showed no change in intracellular pH, intracellular free magnesium, or magnesium-bound ATP levels over the 24-h period following rhodamine 123 administration. Rhodamine 123 also failed to alter glucose utilization and lactate production levels significantly in the CV-1 cells. These results prove the usefulness of 31P- and 13C-nuclear magnetic resonance spectroscopy for quantifying differing effects of rhodamine 123 on the high energy phosphate metabolism and glucose metabolism of HCT-116 and CV-1 cells.

Alkyl methane sulfonate mutation of diploid human lymphoblasts and Salmonella typhimurium.

Concentration dependence of mutation in equigenerational exposures to methyl, ethyl, propyl, and butyl methanesulfonates has been determined in diploid human lymphoblasts and Salmonella typhimurium. Forward mutation was measured at the hypoxanthine guanine phosphoribosyltransferase locus in human lymphoblasts and at the putative guanine phosphoribosyltransferase locus in S. typhimurium. Reverse mutation at the his G46 locus was also measured in S. typhimurium. This analysis and previous reports support the conclusion that S. typhimurium and mammalian cells are essentially equisensitive to the mutagenic effects of ethyl methanesulfonate when concentration and exposure time are taken into account. Comparison of forward and reverse mutation assays in S. typhimurium reveals no important differences in sensitivities for the four compounds studied.

Cell density dependence of focus formation in the C3H/10T1/2 transformation assay.

Some of the dynamics of neoplastic transformation in vitro have been studied with the use of benzo(a)pyrene as the carcinogen in the C3H/10T1/2 morphological transformation assay. Experiments that involved the dsipersion of cells into new culture dishes at various times after carcinogen treatment have shown that no change in the fraction of potentially transformed cells occurs while cultures grow to form a confluent monolayer, that little or no change in the fraction of potentially transformed cells occurs for approximately 3 weeks after confluence is attained, and that this fraction increases rapidly some 7 weeks after BP treatment. When confluent benzo(a)pyrene-treated cultures are dispersed in new culture dishes prior to the onset of growth toward focus formation, the formation of transformed foci is suppresssed at high cell densities of seeding. This phenomenon is independent of the total number of divisions undergone by cells after treatment. We suggest that phenotypic expression of morphological transformation is dependent on colony interactions in the C3H/10T1/2 system, which we do not yet understand, but which are independent of time posttreatment either in cell generations or absolute time.

Killing and mutation of human lymphoblast cells by aflatoxin B1: evidence for an inducible repair response.

Diploid human lymphoblast cells exhibit apparent saturation of mutation induced by exposure to aflatoxin B1, despite a linear increase in the amount and proportion of the aflatoxin-DNA adducts formed. The saturation is neither a cell cycle phenomenon nor a result of a genetically heterozygous population. Examination of the biphasic nature of aflatoxin-DNA adduct loss in vivo shows initial, rapid removal of all adduct species, followed by a slow loss of the aflatoxin-N7-guanine adduct alone. We hypothesize that these data reveal two modes of adduct loss in these cells. The first is an inducible, error-free system that is short-lived, turning off as adduct levels fall below the induction threshold of some 1000 total adducts/cell. The second loss is slower and results from spontaneous depurination of remaining aflatoxin-N7-guanines. Our data are in agreement with the possibility that apurinic sites thus generated are responsible for the mutation observed. A major paradox arises from the fact that aflatoxin-related premutagenic depurinations are estimated to be only 10% of the number of spontaneous depurinations estimated by others to occur in human cells in a 1-h period.

Mutagenicity of hydroxamic acids and the probable involvement of carbamoylation.

A series of hydroxamic acids (aceto-, propiono-, benzo-, and p-nitrobenzo-) and seven derivatives of these were examined for biological activity using Salmonella typhimurium. Acylation to yield O-acetyl and O-benzoyl derivatives markedly enhanced toxic properties and was necessary for mutagenic activity for all but p-nitrobenzohydroxamic acid. The dose necessary to produce a minimum significant mutagenic response varied from 21 microM for O-benzoyl benzohydroxamate to 430 microM for O-acetyl acetohydroxamate. These two compounds were also tested with human lymphoblasts to which they were toxic at 100 microM but not mutagenic. O-Acetyl benzohydroxamate, a mutagen, was prepared wih a 14C label in the carbonyl carbon atom of the benzoyl group and was shown to form an adduct in vitro with DNA and polyguanylic acid. The level of binding was 1 mol of 14C per 5 X 10(4) mol of DNA phosphate and 1 mol of 14C per 10(5) mol of polyguanylic acid phosphate.

Mutational spectra of a 100-base pair mitochondrial DNA target sequence in bronchial epithelial cells: a comparison of smoking and nonsmoking twins.

Seventeen separate mitochondrial hot spot mutations in a 100-bp target sequence (mitochondrial bp 10,030-10,130) were detected and measured in bronchial epithelial cell samples isolated from smokers and nonsmokers. Among the individuals sampled were three pairs of monozygotic twins in which one twin had never smoked and had a nonsmoking spouse, and the other had a smoking history of >10 pack-years. Individual point mutations present at frequencies as low as 10(-6) were detected. Partially denaturing electrophoresis was used to separate mutant from nonmutant sequences on the basis of their melting temperatures, and the target sequence was subsequently amplified via high-fidelity PCR with Pfu DNA polymerase. Tests were performed to determine whether mismatch intermediates or DNA adducts present in the cellular DNA were converted to mutants during PCR. Hot spot mutations were clearly observed in bronchial epithelial cells, and the same hot spots were observed consistently in different samples. Significant numerical variability in the mutant fractions for individual mutants was observed among samples and are ascribed to unequal mitochondrial segregation in stem and transition cells. The mutational spectra in smokers' samples did not differ significantly from the mutational spectra in nonsmokers' samples for this 100 bp of mitochondrial DNA. No smoking-specific hot spots were detected. The overall mutant fractions in smokers' samples were not elevated compared to those of nonsmokers. As much variability was observed between two samples from the same individual's lung as between a sample from a smoker and a sample from a nonsmoker. These findings demonstrate that inhaled tobacco smoke does not induce prominent point mutations in this 100-bp target mitochondrial sequence in smokers' bronchial epithelial cells. Endogenous factors (e.g., DNA replication errors or DNA damage by endogenous reactive chemicals) are suggested to be more likely to represent the most important contributors to mitochondrial mutagenesis.

Mutagenicity of N-nitroso bile acid conjugates in Salmonella typhimurium and diploid human lymphoblasts.

Two N-nitroso bile acid conjugates, N-nitrosotaurocholic acid and N-nitrosoglycocholic acid, were tested for mutagenicity by forward mutation assay in Salmonella typhimurium TM 677 and in diploid human lymphoblasts, line TK6. N-Nitrosoglycocholic acid and N-nitrosotaurocholic acid showed similar concentration-response curves in the bacterial assay with statistically significant mutant fractions observed at about 0.12 mM. Nonnitrosated parent compounds were nonmutagenic. However, in the human cell assay, N-nitrosotaurocholic acid gave statistically significant mutant fractions only at 0.4 mM, but N-nitrosoglycocholic acid was mutagenic at 0.05 microM, some 9000 times more potent. Experiments with quantitative Ames' S. typhimurium reversion assays indicated mutagenesis via base substitution.

Chemically induced mutations in mitochondrial DNA of human cells: mutational spectrum of N-methyl-N'-nitro-N-nitrosoguanidine.

We have observed a reproducible mitochondrial mutational spectrum in the MT1 human lymphoblastoid line treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The MNNG spectrum was distinct from the spontaneous mutational spectrum. However, our ability to observe MNNG-induced mitochondrial mutations above the high level of accumulated spontaneous mutations was dependent on the MT1 phenotype. MT1 cells are markedly resistant to the cytotoxicity but not the mutagenicity of MNNG, presumably as a result of inactivation of both copies of the hMSH6 (GTBP) mismatch repair gene. Thus, we were able to use conditions of treatment that yielded induced mitochondrial mutant fractions beyond the practical limits for human cell experiments in mismatch-proficient human cell lines. In contradistinction, when MT1 cells were treated repeatedly with maximum tolerated concentrations of (+/-) anti-benzo(a)pyrene diol-epoxide, no induced mitochondrial mutations above the spontaneous background were observed. A single dose of 4 microM MNNG (survival, 0.85) induced a mutant fraction of 8 x 10(-3) in the nuclear hypoxanthine-guanine phosphoribosyltrans. ferase gene, and a clear and reproducible pattern of seven MNNG-induced hotspot mutations was observed within the mitochondrial DNA target sequence studied (mitochondrial bp 10,030-10,130). All of the MNNG-induced hotspot mutations were G:C to A:T transitions present at frequencies between 6 x 10(-5) and 30 x 10(-5). Additional experiments supported the conclusion that MNNG-induced hotspot mutations observed were generated in living cells as a result of MNNG treatment and not from mismatch intermediates or DNA adducts converted into mutations during the PCR process.

Normal bronchial epithelial cell expression of glutathione transferase P1, glutathione transferase M3, and glutathione peroxidase is low in subjects with bronchogenic carcinoma.

Normal bronchial epithelial cells (NBECs) are at risk for damage from inhaled and endogenous oxidative species and from epoxide metabolites of inhaled polycyclic aromatic hydrocarbons. Epidemiological and in vitro data suggest that interindividual variation in this risk may result from variation in NBEC expression of enzymes that inactivate reactive species by conjugating them to glutathione. Quantitative competitive reverse transcription-PCR was used to measure mRNA levels of glutathione transferases (GSTs) and glutathione peroxidases (GSHPxs) in primary NBECs from subjects with or without bronchogenic carcinoma. Mean expression levels (mRNA/10(3) beta-actin mRNA) in NBECs from 23 subjects without bronchogenic carcinoma compared to those from 11 subjects with bronchogenic carcinoma respectively (in parentheses) were: mGST (26.0, 6.11), GSTM3 (0.29, 0.09), combined GSTM1,2,4,5 (0.98, 0.60), GSTT1 (0.84, 0.76), GSTP1 (287, 110), GSHPx (140, 62.1), and GSHPxA (0.43, 0.34). Levels of GSTP1, GSTM3, and GSHPx were significantly (P < 0.05) lower in NBECs from subjects with bronchogenic carcinoma. Further, the gene expression index formed by multiplying the values for mGST x GSTM3 x GSHPx x GSHPxA x GSTP1 had a sensitivity (90%) and specificity (76%) for detecting NBECs from bronchogenic carcinoma subjects that was better than any individual gene. In cultured NBECs derived from eight individuals without bronchogenic carcinoma and incubated under identical conditions such that environmental effects were minimized, the mean level of expression and degree of interindividual variation for each gene evaluated was less than that observed in primary NBECs. Data from these studies support the hypotheses that (a) interindividual variation in risk for bronchogenic carcinoma results in part from interindividual variation in NBEC expression of antioxidant genes; (b) gene expression indices will better identify individuals at risk for bronchogenic carcinoma than individual gene expression values; and (c) both hereditary and environmental exposures contribute to the level of and interindividual variation in gene expression observed in primary NBECs. Many epidemiological studies have been designed to evaluate risk associated with polymorphisms or gene expression levels of putative susceptibility genes based on measurements in surrogate tissues, such as peripheral blood lymphocytes. Based on data presented here, it will be important to include the assessment of NBECs in future studies. Measurement of antioxidant gene expression in NBECs may identify the 5-10% of individuals at risk for bronchogenic carcinoma. Bronchoscopic sampling of NBECs from smokers and ex-smokers then will allow susceptible individuals to be entered into surveillance and/or chemoprevention studies.

Mutational spectra in human B-cells. Spontaneous, oxygen and hydrogen peroxide-induced mutations at the hprt gene.

To test the hypothesis that reactive species in the oxygen cascade are responsible for spontaneous mutation, we examined the spectra of oxygen and hydrogen peroxide-induced mutations at the hprt locus in a human B-lymphoblastoid cell line. We compared these spectra with the spontaneous mutational spectrum. Large gene alterations were studied by Southern analysis of individual TGR clones. A combination of high fidelity polymerase chain reaction, denaturing gradient gel electrophoresis and direct DNA sequencing were used to detect and identify point mutations in exon 3 of hprt. With regard to spontaneous mutations, a previous study showed that 39% of the spontaneous TGR clones had large gene alterations. In the present study, the analysis of spontaneous point mutations within exon 3 revealed two hotspots. A one base-pair deletion (-A) at base-pair 256 or 257 and a two base-pair deletion (-GG) at base-pair 237 and 238, were detected in triplicate cultures. Each of the hotspots comprised about 1% of the TGR mutants. The analysis of individual oxygen-induced TGR clones (48 h, 910 microM-O2) showed 43% had large gene alterations similar to the spontaneous TGR clones. However, none of the spontaneous point mutation hotspots was found among triplicate oxygen-treated cultures. Two point mutations in common with H2O2-treated cultures were found in one of the three oxygen-treated cultures. Hydrogen peroxide-induced mutations (1 h, 20 microM) also differed from spontaneous mutations. Only 24% of the hydrogen peroxide-induced TGR clones had large gene alterations. The analysis of point mutations showed three hotspots within exon 3 of hprt. An AT to TA transversion at base-pair 259 had an average frequency of 3% of all TGR mutants (present in all of 3 H2O2-treated cultures). Two GC to CG transversions at base-pairs 243 and 202 were present at a frequency of 0.6% and 0.4%, respectively. A five base-pair deletion (base-pair 274 to 278) was present at an average frequency of 0.3%. The latter three mutations were detected in two of three H2O2-treated cultures. Thus, the point mutation spectra of both oxygen and hydrogen peroxide were significantly different from the spontaneous spectrum. The oxygen and hydrogen peroxide-induced spectra shared some features, suggesting that oxygen and hydrogen peroxide share some but not all pathways for induction of mutations within the DNA sequence studied here.(ABSTRACT TRUNCATED AT 400 WORDS)

Evolutionary trail of the mitochondrial genome as based on human 16S rDNA pseudogenes.

In the course of studies on mutations in human mitochondrial (mt) DNA, we have uncovered and sequenced four new nuclear pseudogenes corresponding to bp 2457-2657 of the mt 16S rDNA. The four genes and their homologies with human mtDNA are E2 (62.4%), K10 (74.4%), E1 (84.6%) and LE6 (93.2%). When these five pseudogene sequences and another previously reported pseudogene sequence are compared with each other, they display what appears to be an ordered series of steps from a hypothetical common ancestor. The sequence of the hypothetical ancestor closely resembles that found in a wide variety of present-day mammalian mt genomes. The pseudogene sequences suggest an evolutionary trail of mt mutation dominated by base pair transitions punctuated by integration into the nuclear genome. Once integrated into the nuclear genome, the pseudogenes appear to follow the distinctive nuclear mutational pathway in which GC to AT transitions predominate and CpG sequences are preferentially eliminated.

Enzymatic amplification and characterization of large DNA fragments from genomic DNA.

Conditions for DNA amplification in vitro using modified T7 DNA polymerase have been devised to obtain 2000-bp DNA fragments of the HGPRT gene directly from human genomic DNA. The DNA obtained from a 1.2 x 10(5)-fold amplification has been used for direct sequencing.

Deletion mutagenesis during polymerase chain reaction: dependence on DNA polymerase.

Polymerase chain reaction (PCR) was performed with two polymerases. Thermus aquaticus DNA polymerase (Taq), and modified T7 DNA polymerase (Sequenase). Both polymerases were used to amplify the same portion of the human 18S rRNA gene. We report a PCR artifact, namely a deletion of 54 bp, when Taq polymerase was used to amplify a portion of the human 18S rRNA gene. PCR performed with Sequenase did not produce this artifact. The deletion eliminated a potentially stable hairpin loop. Our data are consistent with the following model for generation of the deletion: (i) the formation of an intrastrand hairpin, and (ii) polymerization across the base of the hairpin, thus deleting the nucleotide sequence in the hairpin. Furthermore, we show that the deletion occurs mainly during synthesis of the (-)DNA strand. Our observations suggest that similar artifacts may occur in other sequences containing stable secondary structures.

Single nucleotide polymorphism spectra in newborns and centenarians: identification of genes coding for rise of mortal disease.

Some single-nucleotide polymorphisms (SNPs) increase the risk of mortal disease. Identifying these SNPs and the genes in which they reside is an important area in human genomics. Such qualitative observations are important in themselves. However, an accurate assessment of the numerical distribution and age-dependent decline of SNPs in the population would permit calculation of the rises represented by each SNP. Such analyses have not been attempted because of a lack of an efficient and cost-effective method to detect multiple SNPs in a large number of individuals and a large number of genes. Here, we suggest the use of an analytical procedure that can scan for SNPs in 100-bp DNA sequences from as many as 10000 donors' blood cell samples, or 20000 alleles, simultaneously. Our suggestion is based on technology developed for studies of somatic mutations in human tissue DNA for point mutations at frequencies equal to or greater than 10(-6). In a simplified version of this technology, any SNP arising at frequencies at or above 5x10(-4) would be identified with useful precision. A gene would be represented by 10 or more sections of 100bp. This strategy includes splice-site mutations that represent a significant fraction of gene inactivating point mutations and would not be observed in strategies using cDNA. To illustrate the logic of the suggested approach, we use American mortality records to calculate the expected decrease in SNPs coding for premature mortality in newborns and centenarians. We consider several elementary cases: SNPs in one gene only, any of several genes, or all of several genes that create a risk of death by pancreatic cancer. The fraction of expressed polymorphisms affecting mortality should be simultaneously increased in probands and decreased in the aged relative to newborns. Silent polymorphisms in the same gene would remain unchanged in all three groups and serve as internal standards. A key point is that scanning a gene, in which loss of gene function creates the risk of mortality is expected to reveal not one, but multiple SNPs, which decline with age, as carriers die earlier in life than non-carriers. Several SNPs in a scanned gene would suggest that the decreasing SNP was genetically linked to a different polymorphism that creates the disease risk.