Tracking insertion mutants within libraries by deep sequencing and a genome-wide screen for Haemophilus genes required in the lung.

Rapid genome-wide identification of genes required for infection would expedite studies of bacterial pathogens. We developed genome-scale "negative selection" technology that combines high-density transposon mutagenesis and massively parallel sequencing of transposon/chromosome junctions in a mutant library to identify mutants lost from the library after exposure to a selective condition of interest. This approach was applied to comprehensively identify Haemophilus influenzae genes required to delay bacterial clearance in a murine pulmonary model. Mutations in 136 genes resulted in defects in vivo, and quantitative estimates of fitness generated by this technique were in agreement with independent validation experiments using individual mutant strains. Genes required in the lung included those with characterized functions in other models of H. influenzae pathogenesis and genes not previously implicated in infection. Genes implicated in vivo have reported or potential roles in survival during nutrient limitation, oxidative stress, and exposure to antimicrobial membrane perturbations, suggesting that these conditions are encountered by H. influenzae during pulmonary infection. The results demonstrate an efficient means to identify genes required for bacterial survival in experimental models of pathogenesis, and this approach should function similarly well in selections conducted in vitro and in vivo with any organism amenable to insertional mutagenesis.

ArcA-regulated glycosyltransferase lic2B promotes complement evasion and pathogenesis of nontypeable Haemophilus influenzae.

Signaling mechanisms used by Haemophilus influenzae to adapt to conditions it encounters during stages of infection and pathogenesis are not well understood. The ArcAB two-component signal transduction system controls gene expression in response to respiratory conditions of growth and contributes to resistance to bactericidal effects of serum and to bloodstream infection by H. influenzae. We show that ArcA of nontypeable H. influenzae (NTHI) activates expression of a glycosyltransferase gene, lic2B. Structural comparison of the lipooligosaccharide (LOS) of a lic2B mutant to that of the wild-type strain NT127 revealed that lic2B is required for addition of a galactose residue to the LOS outer core. The lic2B gene was crucial for optimal survival of NTHI in a mouse model of bacteremia and for evasion of serum complement. The results demonstrate that ArcA, which controls cellular metabolism in response to environmental reduction and oxidation (redox) conditions, also coordinately controls genes that are critical for immune evasion, providing evidence that NTHI integrates redox signals to regulate specific countermeasures against host defense.

Resistance of Haemophilus influenzae to reactive nitrogen donors and gamma interferon-stimulated macrophages requires the formate-dependent nitrite reductase regulator-activated ytfE gene.

Haemophilus influenzae efficiently colonizes and persists at the human nasopharyngeal mucosa, causing disease when it spreads to other sites. Nitric oxide (NO) represents a major antimicrobial defense deployed by host cells in locations colonized by H. influenzae during pathogenesis that are likely to vary in oxygen levels. Formate-dependent nitrite reductase regulator (FNR) is an oxygen-sensitive regulator in several bacterial pathogens. We report that fnr of H. influenzae is required for anaerobic defense against exposure to NO donors and to resist NO-dependent effects of gamma interferon (IFN-gamma)-activated murine bone marrow-derived macrophages. To understand the mechanism of resistance, we investigated the role of FNR-regulated genes in defense against NO sources. Expression analysis revealed FNR-dependent activation of nrfA, dmsA, napA, and ytfE. Nonpolar deletion mutants of nrfA and ytfE exhibited sensitivity to NO donors, and the ytfE gene was more critical for survival. Compared to the wild-type strain, the ytfE mutant exhibited decreased survival when exposed to macrophages, a defect that was more pronounced after prior stimulation of macrophages with IFN-gamma or lipopolysaccharide. Complementation restored survival of the mutant to the level in the parental strain. Increased sensitivity of the ytfE mutant relative to that of the parent was abrogated by treatment of macrophages with a NO synthase inhibitor, implicating YtfE in resistance to a NO-dependent pathway. These results identify a requirement for FNR in positive control of ytfE and indicate a critical role for ytfE in resistance of H. influenzae to reactive nitrogen species and the antibacterial effects of macrophages.

The periplasmic disulfide oxidoreductase DsbA contributes to Haemophilus influenzae pathogenesis.

Haemophilus influenzae is an obligate human pathogen that persistently colonizes the nasopharynx and causes disease when it invades the bloodstream, lungs, or middle ear. Proteins that mediate critical interactions with the host during invasive disease are likely to be secreted. Many secreted proteins require addition of disulfide bonds by the DsbA disulfide oxidoreductase for activity or stability. In this study, we evaluated the role in H. influenzae pathogenesis of DsbA, as well as HbpA, a substrate of DsbA. Mutants of H. influenzae Rd and type b strain Eagan having nonpolar deletions of dsbA were attenuated for bacteremia in animal models, and complemented strains exhibited virulence equivalent to that of the parental strains. Comparison of predicted secreted proteins in H. influenzae to known DsbA substrates in other species revealed several proteins that could contribute to the role of dsbA in virulence. One candidate, the heme transport protein, HbpA, was examined because of the importance of exogenous heme for aerobic growth of H. influenzae. The presence of a dsbA-dependent disulfide bond in HbpA was verified by an alkylation protection assay, and HbpA was less abundant in a dsbA mutant. The hbpA mutant exhibited reduced bacteremia in the mouse model, and complementation restored its in vivo phenotype to that of the parental strain. These results indicate that dsbA is required in vivo and that HbpA and additional DsbA-dependent factors are likely to participate in H. influenzae pathogenesis.

Identification and analysis of essential genes in Haemophilus influenzae.

The human respiratory pathogen Haemophilus influenzae, a Gram-negative bacterium, is the first free-living organism to have its complete genome sequenced, providing the opportunity to apply genomic-scale approaches to study gene function. This chapter provides an overview of a highly efficient, in vitro mariner transposon-based method that exploits the natural transformation feature of this organism for the identification of essential genes. In addition, we describe strategies for conditional expression systems that would facilitate further analysis of this class of genes. Finally, we outline a method based on the approach used in H. influenzae for identifying essential genes that can be applied to other bacteria that are not naturally transformable.

Maaqwi cascadensis: A large, marine diving bird (Avialae: Ornithurae) from the Upper Cretaceous of British Columbia, Canada.

Mesozoic bird fossils from the Pacific Coast of North America are rare, but small numbers are known from the Late Cretaceous aged sediments of Hornby Island, British Columbia. Most are unassociated fragments that offer little information, but additional preparation of a large coracoid has revealed more details of its structure, as well as three associated wing bones. Phylogenetic analysis suggests that Maaqwi cascadensis, gen. et sp. nov. represents a derived crown or near-crown member of Ornithurae, and specifically suggests affinities with Vegaviidae. M. cascadensis is characterized by large size, and regressions based on dimensions of the coracoid suggest a large bird, with an estimated body mass of approximately 1.5 kilograms. The bones are robust, with thick walls, suggesting that M. cascadensis was a bird adapted for diving, similar to modern loons and grebes. The wings are short, while the coracoid is unusually short and broad, similar to modern loons. Along with the Ichthyornithes and Hesperornithes, M. cascadensis and Vegaviidae appear to represent a third clade of bird that evolved to exploit marine habitats in the Late Cretaceous, one specialized for foot-propelled diving and rapid cruising flight over water.

Nigrostriatal monoamine oxidase A and B in aging squirrel monkeys and C57BL/6 mice.

In this study we assessed the two forms of monoamine oxidase (MAO) in the caudate, putamen, and substantia nigra of young (4-year-old), intermediate-aged (11-year-old), and aged (20-year-old) squirrel monkeys and in the striata of young (2-month-old) and older (10-month-old) C57Bl/6 mice. MAO A and B activities were determined by measuring the rate of oxidation of the specific substrates phenethylamine and serotonin. In squirrel monkey, the vast majority of MAO activity was MAO B with activity of this isoform 10 times greater than of MAO A, while in mice the activity of the two forms was approximately equivalent. Although mice demonstrated nearly twofold selective increases in striatal MAO B between 2 and 10 months of age, neither MAO B nor A showed statistically significant changes with age in squirrel monkeys. These results document the marked differences between nonhuman primates and rodents with respect to the relative activities and the effects of age on MAO A and B, and indicate that increased MAO is not an inevitable feature of aging.

CYP2D6 allelic frequencies in young-onset Parkinson's disease.

Parkinson's disease (PD) is thought to develop as a result of interactions between genetic susceptibility factors and environmental exposures. One candidate gene is CYP2D6, which codes for the debrisoquine 4-hydroxylase cytochrome P450. Impairment of debrisoquine 4-hydroxylase activity has been associated with an increased risk of PD in patients with younger age at disease onset. Genotyping studies in patients with an older age at onset have reported modest increases in risk associated with the CYP2D6 B and A alleles; however, the risk for young-onset PD has not been adequately evaluated. We designed a case-control study to investigate the role of nonfunctional CYP2D6 allelic risk factors for young-onset PD in a sizable patient population and compared the distributions of CYP2D6 genotypes between young-onset ( < or = 51 years) PD patients (n = 108) and controls (n = 236). In contrast with the results from genotyping studies conducted among patients with an older age at onset, there were no significant differences in CYP2D6 allelic frequencies between young-onset PD cases and controls. The frequency of the B allele was slightly lower in the young-onset PD cases than in the controls (0.14 versus 0.20) (X2 = 2.66, p = 0.10). The presence of one or more B alleles was not associated with an increased risk of young-onset PD (odds ratio 0.58; 95% CI 0.33 to 1.00), nor was the presence of one or more nonfunctional alleles (i.e., A, B, D, and D2) (odds ratio 0.68; 95% CI 0.41 to 1.13). This study suggests that the young-onset PD population may differ from the older-onset population with respect to risk factors.

Role of redox cycling and lipid peroxidation in bipyridyl herbicide cytotoxicity. Studies with a compromised isolated hepatocyte model system.

The role of active oxygen species and lipid peroxidation in the toxic effects of diquat, paraquat and other bipyridyl herbicides remains controversial. In vitro studies have shown that these compounds are potent generators of active oxygen species by redox cycling and that they stimulate lipid peroxidation. In vivo studies have failed, however, to show clear evidence of lipid peroxidation resulting from toxic exposures to these compounds. We have directly compared the abilities of three bipyridyl herbicides, diquat (DQ), paraquat (PQ) and benzyl viologen (BV), to generate superoxide anion radical (O2-.) in rat liver microsomes and H2O2 in hepatocytes and correlated this with their relative toxicities to a compromised isolated hepatocyte system. DQ was the most potent generator of O2-. and H2O2, being slightly more potent than BV and much better than PQ. This ability of the bipyridyls to generate active oxygen was positively correlated with the ability to induce toxicity in hepatocytes pretreated with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) to inhibit their glutathione reductase activity, i.e. DQ greater than BV greater than PQ. DQ caused a rapid depletion of cellular GSH and a concomitant increase in GSSG in this system. Toxicity, measured as loss of plasma membrane integrity, was pronounced after only 30-60 min of incubation and was accompanied by a significant increase in lipid peroxidation. The onset of lipid peroxidation could not be separated temporally from the expression of toxicity. However, the total inhibition of lipid peroxidation by the antioxidants Trolox C, promethazine and N,N'-diphenyl-p-phenylenediamine only delayed toxicity, indicating that, even though lipid peroxidation may play some role in enhancing bipyridyl herbicide toxicity, it is not essential for the toxicity to manifest itself.

N-acetylcysteine and glutathione-dependent protective effect of PZ51 (Ebselen) against diquat-induced cytotoxicity in isolated hepatocytes.

The glutathione peroxidase (GSH-Px)-like reduction of H2O2 by the selenoorganic compound 2-phenyl-1,2-benzoisoselenazol-3(H)-one (PZ51: Ebselen) was studied using glutathione (GSH) and the therapeutic agent N-acetylcysteine (NAC) to provide reducing equivalents. In a purely chemical system containing H2O2 and in an enzymatic system of glucose/glucose oxidase-generated H2O2 Ebselen alone did not reduce H2O2. Ebselen in combination with either GSH (1 mM) or NAC (1 mM) was capable of reducing H2O2 in both systems. In these non-cellular systems GSH was a more effective source of reducing equivalents than NAC. The GSH-Px-like activity of Ebselen was further investigated in a cellular system. The redox-cycling bipyridylium compound diquat generates active oxygen species, depletes intracellular glutathione, and is cytotoxic in isolated hepatocytes pretreated with the glutathione reductase inhibitor 1,3-bis(Z-chloro-ethyl)-1-nitrosourea (BCNU). Ebselen alone did not ameliorate diquat cytotoxicity, but in combination with either GSH (1 mM) or NAC (1 mM) it produced a significant delay in diquat-induced cytotoxicity. Further additions of either GSH (0.5 mM) or NAC (0.5 mM) at 30 min intervals provided significantly more protection against diquat-induced cytotoxicity and intracellular GSH depletion than the single 1 mM addition. Thus, the combination of Ebselen and NAC may provide an effective antidote in cases of overexposure to bipyridylium herbicides, such as diquat and paraquat.

Studies on the mechanism of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine cytotoxicity in isolated hepatocytes.

Oxidative stress and covalent binding have been proposed as possible mechanisms involved in the cytotoxic effects of the parkinsonism-causing compound 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). However, the toxicity induced by MPTP in isolated rat hepatocytes seems to be relatively independent of oxygen radical-induced oxidative stress. Here we demonstrate that MPTP cytotoxicity is not potentiated by pretreatment with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of glutathione reductase, nor prevented by the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) or the iron-chelating agent desferrioxamine. Moreover, preincubation of hepatocytes with diethylmaleate to lower the level of intracellular reduced glutathione (to 20% of the initial value) did not affect either the rate or extent of MPTP cytotoxicity. Thus, nucleophilic soluble thiols do not seem to play a protective role against MPTP-induced cell damage, in contrast to what one would have expected if covalent protein binding and oxidative stress were involved as toxic mechanisms. On the other hand, MPTP cytotoxicity was potentiated by pretreatment of hepatocytes with cytochrome P-450 inhibitors (e.g., SKF 525A and metyrapone) and a more rapid depletion of ATP was observed in these experimental conditions. We conclude that mitochondrial damage and subsequent ATP depletion are likely to play a critical role in the toxicity of MPTP to isolated hepatocytes and that the metabolism of MPTP via the cytochrome P-450 monooxygenase system can be considered to be a detoxifying pathway.

Determination of the intracellular protein thiol distribution of hepatocytes using monobromobimane derivatisation of intact cells and isolated subcellular fractions.

The derivatisation of intact rat hepatocytes with monobromobimane resulted in rapid labelling of accessible protein thiols in several subcellular fractions. The derivatisation procedure did not cause acute cytotoxicity, nor did it alter the buoyant densities of the fractions or their gross protein compositions. Quantitation of the fluorescence irreversibly associated with the fractions demonstrated considerable intracellular heterogeneity in this pool of thiols. Values were highest in cytosol (ca. 90 nmol/mg protein), intermediate in microsomes (ca. 65 nmol/mg protein) and mitochondria (ca. 45 nmol/mg protein) and lowest in a crude fraction containing both nuclei and plasma membrane (ca. 35 nmol/mg protein). Similar values were obtained from microsomes and cytosol derivatised after fractionation but there were significant increases of ca. 100% in corresponding values from isolated mitochondria and the nuclear/plasma membrane fraction. These results are discussed in terms of the dynamic fluxes in monobromobimane protein thiols during fractionation and the applicability of this noninvasive method to studies of the mechanism(s) of toxicity of reactive xenobiotics and the role(s) of protein thiols in normal cellular function.

Discriminative ability and predictive validity of the timed up and go test in identifying older people who fall: systematic review and meta-analysis.

OBJECTIVES: To investigate the discriminative ability and diagnostic accuracy of the Timed Up and Go Test (TUG) as a clinical screening instrument for identifying older people at risk of falling. DESIGN: Systematic literature review and meta-analysis. SETTING AND PARTICIPANTS: People aged 60 and older living independently or in institutional settings. MEASUREMENTS: Studies were identified with searches of the PubMed, EMBASE, CINAHL, and Cochrane CENTRAL data bases. Retrospective and prospective cohort studies comparing times to complete any version of the TUG of fallers and non-fallers were included. RESULTS: Fifty-three studies with 12,832 participants met the inclusion criteria. The pooled mean difference between fallers and non-fallers depended on the functional status of the cohort investigated: 0.63 seconds (95% confidence (CI) = 0.14-1.12 seconds) for high-functioning to 3.59 seconds (95% CI = 2.18-4.99 seconds) for those in institutional settings. The majority of studies did not retain TUG scores in multivariate analysis. Derived cut-points varied greatly between studies, and with the exception of a few small studies, diagnostic accuracy was poor to moderate. CONCLUSION: The findings suggest that the TUG is not useful for discriminating fallers from non-fallers in healthy, high-functioning older people but is of more value in less-healthy, lower-functioning older people. Overall, the predictive ability and diagnostic accuracy of the TUG are at best moderate. No cut-point can be recommended. Quick, multifactorial fall risk screens should be considered to provide additional information for identifying older people at risk of falls.

Genome-scale approaches to identify genes essential for Haemophilus influenzae pathogenesis.

Haemophilus influenzae is a Gram-negative bacterium that has no identified natural niche outside of the human host. It primarily colonizes the nasopharyngeal mucosa in an asymptomatic mode, but has the ability to disseminate to other anatomical sites to cause otitis media, upper, and lower respiratory tract infections, septicemia, and meningitis. To persist in diverse environments the bacterium must exploit and utilize the nutrients and other resources available in these sites for optimal growth/survival. Recent evidence suggests that regulatory factors that direct such adaptations also control virulence determinants required to resist and evade immune clearance mechanisms. In this review, we describe the recent application of whole-genome approaches that together provide insight into distinct survival mechanisms of H. influenzae in the context of different sites of pathogenesis.

High-throughput insertion tracking by deep sequencing for the analysis of bacterial pathogens.

Whole-genome techniques toward identification of microbial genes required for their survival and growth during infection have been useful for studies of bacterial pathogenesis. The advent of massively parallel sequencing platforms has created the opportunity to markedly accelerate such genome-scale analyses and achieve unprecedented sensitivity, resolution, and quantification. This chapter provides an overview of a genome-scale methodology that combines high-density transposon mutagenesis with a mariner transposon and deep sequencing to identify genes that are needed for survival in experimental models of pathogenesis. Application of this approach to a model pathogen, Haemophilus influenzae, has provided a comprehensive analysis of the relative role of each gene of this human respiratory pathogen in a murine pulmonary model. The method is readily adaptable to nearly any organism amenable to transposon mutagenesis.

The ArcA regulon and oxidative stress resistance in Haemophilus influenzae.

Haemophilus influenzae transits between niches within the human host that are predicted to differ in oxygen levels. The ArcAB two-component signal transduction system controls gene expression in response to respiratory conditions of growth and has been implicated in bacterial pathogenesis, yet the mechanism is not understood. We undertook a genome-scale study to identify genes of the H. influenzae ArcA regulon. Deletion of arcA resulted in increased anaerobic expression of genes of the respiratory chain and of H. influenzae's partial tricarboxylic acid cycle, and decreased anaerobic expression levels of genes of polyamine metabolism, and iron sequestration. Deletion of arcA also conferred a susceptibility to transient exposure to hydrogen peroxide that was greater following anaerobic growth than after aerobic growth. Array data revealed that the dps gene, not previously assigned to the ArcA modulon in bacteria, exhibited decreased expression in the arcA mutant. Deletion of dps resulted in hydrogen peroxide sensitivity and complementation restored resistance, providing insight into the previously uncharacterized mechanism of arcA-mediated H(2)O(2) resistance. The results indicate a role for H. influenzae arcA and dps in pre-emptive defence against transitions from growth in low oxygen environments to aerobic exposure to hydrogen peroxide, an antibacterial oxidant produced by phagocytes during infection.

Role of extracellular Ca2+ in toxic liver injury: comparative studies with the perfused rat liver and isolated hepatocytes.

The role of extracellular Ca2+ in toxic liver injury was examined in two in vitro models of the rat liver, the isolated perfused liver and isolated hepatocytes. The toxins t-butylhydroperoxide and carbon tetrachloride, as well as the Ca2+ ionophore, heptafluorodimethyloctanedione (FOD), were employed to induce cellular injury and death. Lipid peroxidation was also measured as the formation of thiobarbituric acid-reactive products. Cell death was measured by the release of lactate dehydrogenase in the perfused liver model and by the uptake of trypan blue in isolated hepatocytes. Toxin-induced cellular injury and death occurred in both in vitro models in the presence and absence of extracellular Ca2+, indicating that an influx of Ca2+ was not essential for toxic liver injury. The degree of toxicity seen in the perfused liver model was independent of increases or decreases in the total calcium concentration present in the liver tissue, providing further evidence that cell death is not dictated solely by changes in cellular calcium content. Isolated hepatocytes differed from the perfused liver, however, undergoing more lipid peroxidation and toxin-induced cell death when incubated in the absence of extracellular Ca2+ than in its presence. While suggesting that extracellular Ca2+ concentrations may have some influence on the expression of toxicity, these results demonstrate the nonessential role extracellular Ca2+ plays in the events leading to toxic liver injury.

Relationships between intracellular vitamin E, lipid peroxidation, and chemical toxicity in hepatocytes.

The cellular content of vitamin E was measured in isolated rat hepatocytes exposed to various types of chemical injury. Vitamin E was determined as alpha-tocopherol by HPLC with in-line uv and electrochemical detection. The cytotoxicity of diquat, a redox cycling compound, was accompanied by a decrease in cellular alpha-tocopherol and a stimulation of lipid peroxidation. Both the loss of alpha-tocopherol and the accumulation of lipid peroxidation products could be prevented by addition of either the antioxidant N,N'-diphenyl-p-phenylenediamine (DPPD) or the reducing agent dithiothreitol (DTT). DTT also prevented the oxidation of soluble and protein thiols and completely protected against cytotoxicity, while DPPD addition only delayed the onset of hepatocyte death. Cytotoxic doses of the naphthoquinone, menadione, and the pyridine compounds 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-methyl-4-phenyl-pyridinium ion did not deplete alpha-tocopherol levels, nor did they result in significant lipid peroxidation. On the other hand, a peroxidizing, but noncytotoxic dose of ADP-Fe3+ rapidly decreased cellular alpha-tocopherol levels. These data demonstrate that cellular alpha-tocopherol loss is neither a prerequisite for, nor a necessary consequence of toxicity. Moreover, a substantial depletion (ca. 50%) of alpha-tocopherol does not necessarily result in cell death. Although alpha-tocopherol protects against the oxidation of cellular lipids, the maintenance of hepatocyte alpha-tocopherol content does not prevent the oxidation of soluble and protein thiols. These other targets of oxidative damage seem to play a more critical role in hepatocyte toxicity.

Genotypic analysis of mutations in Taq I restriction recognition sites by restriction fragment length polymorphism/polymerase chain reaction.

Point mutations in somatic cells play a role in the etiology of several classes of human pathologies. Experimental procedures are required that allow the detection and quantitation of such mutations in disease-related genes in tissue biopsy samples without the need for the selection of mutated cells. We describe the genotypic analysis of single base pair mutations in the Taq I endonuclease recognition sequence TCGA, residues 2508-2511 of exon 2 of the human c-H-ras1 gene, by the restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) approach. The high thermostability of Taq I endonuclease allows the continuous removal of eventual residual wild-type sequences during the thermocycling of the PCR and reduces polymerase errors in the final RFLP/PCR product to a minimum. As few as five copies of a mutant standard containing two base pair changes in the chosen Taq I site could be rescued from 10(8) copies of wild-type DNA. Taq I RFLP/PCR holds promise for the monitoring of mutations in biochemical epidemiology.

Genotypic analysis of N-ethyl-N-nitrosourea-induced mutations by Taq I restriction fragment length polymorphism/polymerase chain reaction in the c-H-ras1 gene.

In genotypic mutation analysis DNA sequence changes are determined without the in vivo or in vitro selection of phenotypically altered cells. We have studied the induction of base-pair changes by N-ethyl-N-nitrosourea in Taq I endonuclease recognition site 2508-2511 (TCGA) of the c-H-ras1 gene in human fibroblasts by the restriction fragment length polymorphism/polymerase chain reaction (RFLP/PCR) method. This site contains the four bases, and all 12 possible single base-pair changes can be monitored. The transition of guanine to adenine at position 2510 was the major mutation detected by lambda plaque oligonucleotide hybridization and quantitative sequence analysis of the RFLP/PCR products. It involves the G residue of the CpG sequence of the coding strand. Data calibration with an internal mutant standard indicates that absolute frequencies for this transition lie in the range of 4-12 x 10(-7). The present study documents the capacity of the RFLP/PCR approach to measure mutagen-induced base-pair changes in a specific gene sequence without the selection of a phenotypically altered cell.