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CRISPR-Cas9 provides a tool to treat autosomal dominant disease by non-homologous end joining (NHEJ) gene disruption of the mutant allele. In order to discriminate between wild-type and mutant alleles, Streptococcus pyogenes Cas9 (SpCas9) must be able to detect a single nucleotide change. Allele-specific editing can be achieved by using either a guide-specific approach, in which the missense mutation is found within the guide sequence, or a protospacer-adjacent motif (PAM)-specific approach, in which the missense mutation generates a novel PAM. While both approaches have been shown to offer allele specificity in certain contexts, in cases where numerous missense mutations are associated with a particular disease, such as TGFBI (transforming growth factor β-induced) corneal dystrophies, it is neither possible nor realistic to target each mutation individually. In this study, we demonstrate allele-specific CRISPR gene editing independent of the disease-causing mutation that is capable of achieving complete allele discrimination, and we propose it as a targeting approach for autosomal dominant disease. Our approach utilizes natural variants in the target region that contain a PAM on one allele that lies in cis with the causative mutation, removing the constraints of a mutation-dependent approach. Our innovative patient-specific guide design approach takes into account the patient's individual genetic make-up, allowing on- and off-target activity to be assessed in a personalized manner., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

The relationship of mitochondrial DNA mutations to aging is still debated. Most mtDNA mutations are recessive: there are multiple copies per cell and mutation needs to clonally expand to cause respiratory deficiency. Overall mtDNA mutant loads are low, so effects of mutations are limited to critical areas where mutations locally reach high fractions. This includes respiratory chain deficient zones in muscle fibers, respiratory-deficient crypts in colon, and massive expansions of deleted mtDNA in substantia nigra neurons. mtDNA "mutator" mouse with increased rate of mtDNA mutations is a useful model, although rates and distribution of mutations may significantly deviate from what is observed in human aging. Comparison of species with different longevity reveals intriguing longevity-related traits in mtDNA sequence, although their significance is yet to be evaluated. The impact of somatic mtDNA mutations rapidly increases with age, so their importance is expected to grow as human life expectancy increases., (© 2014 Elsevier Inc. All rights reserved.)

Limb girdle muscular dystrophy type 2L or anoctaminopathy is a condition mainly characterized by adult onset proximal lower limb muscular weakness and raised CK values, due to recessive ANO5 gene mutations. An exon 5 founder mutation (c.191dupA) has been identified in most of the British and German LGMD2L patients so far reported. We aimed to further investigate the prevalence and spectrum of ANO5 gene mutations and related clinical phenotypes, by screening 205 undiagnosed patients referred to our molecular service with a clinical suspicion of anoctaminopathy. A total of 42 unrelated patients had two ANO5 mutations (21%), whereas 14 carried a single change. We identified 34 pathogenic changes, 15 of which are novel. The c.191dupA mutation represents 61% of mutated alleles and appears to be less prevalent in non-Northern European populations. Retrospective clinical analysis corroborates the prevalently proximal lower limb phenotype, the male predominance and absence of major cardiac or respiratory involvement. Identification of cases with isolated hyperCKaemia and very late symptomatic male and female subjects confirms the extension of the phenotypic spectrum of the disease. Anoctaminopathy appears to be one of the most common adult muscular dystrophies in Northern Europe, with a prevalence of about 20%-25% in unselected undiagnosed cases., (© 2013 WILEY PERIODICALS, INC.)

Mitochondrial defects within substantia nigra (SN) neurons are implicated in the pathogenesis of Parkinson's disease. SN neurons show increased mitochondrial defects, mitochondrial DNA deletion levels, and susceptibility to such dysfunction, although the role of mitochondria in neuronal degeneration remains uncertain. In this study, we addressed this important question by exploring changes within the mitochondria of SN neurons from patients with primary mitochondrial diseases to determine whether mitochondrial dysfunction leads directly to neuronal cell loss. We counted the pigmented neurons and quantified mitochondrial respiratory activity, deficiencies in mitochondrial proteins, and the percentage of pathogenic mutations in single neurons. We found evidence of defects of both complex I and complex IV of the respiratory chain in all patients. We found that marked neuronal cell loss was only observed in a few patients with mitochondrial disease and that all these patients had mutations in polymerase gamma (POLG), which leads to the formation of multiple mitochondrial DNA deletions over time, similar to aging and Parkinson's disease. Interestingly, we detected α-synuclein pathology in two mitochondrial patients with POLG mutations. Our observations highlight the complex relationship between mitochondrial dysfunction and the susceptibility of SN neurons to degeneration and α-synuclein pathology. Our finding that the loss of SN neurons was only severe in patients with POLG mutations suggests that acquired mitochondrial defects may be less well tolerated by SN neurons than by inherited ones.

Patients with disorders from mutations in the mitochondrial genome have variable phenotypes, but common to many of these disorders are underlying changes in postmitotic cells, particularly neurons and muscle fibers. The mitochondrial dysfunction caused by these mutations has been shown to be associated with signs of apoptosis and to cause cell loss. Mutations of the mitochondrial genome have also been shown to accumulate with age and in common neurodegenerative diseases, such as Parkinson's disease. This review presents recent data to show that the information gained from studying patients with mitochondrial disorders can help our understanding of the role of mitochondrial DNA mutations in brain aging and neurodegeneration.

A decline in mitochondrial function has long been shown to exist in neurodegenerative disease. Whether this decline is a secondary consequence of other factors or whether it causes the eventual death of a cell is unknown. In this review, we will discuss some of the major evidence surrounding mitochondrial DNA mutations leading to mitochondrial dysfunction in neurodegenerative disease and discuss their possible role in neurodegeneration.

Background: In clinical practice, mitochondrial disease is seldom considered until a variable combination of seizures, alteration in tone, muscle weakness, and developmental problems is evident. However, it is not uncommon for one symptom to occur in isolation and dominate the clinical phenotype. We report six patients from two families where dystonia was the principal clinical manifestation. A mitochondrial etiology was considered in each case because of the association of dystonia with other less prominent clinical features such as epilepsy., Methods: Histochemical and biochemical analyses were undertaken in skeletal muscle biopsies from individuals in both families. Sequencing of skeletal muscle mtDNA was also performed and suspected mutations were quantified by hot last cycle PCR-RFLP or primer extension assay. Functional consequences of one of the mutations were investigated by measurement of steady state levels of mitochondrial tRNA., Results: Two distinct mitochondrial pathologies were identified: a novel, homoplasmic mitochondrial tRNA(Cys) (MTTC) mutation and the primary, m.11778G>A Leber hereditary optic neuropathy (LHON) mutation. The mild nature of both mutations has permitted very high levels of mutated mtDNA to accumulate. Patients with the mutation in the MTTC gene have no wild type mtDNA detectable and although the LHON mutation is heteroplasmic in the patients we report, it is commonly observed to be homoplasmic., Conclusions: The mitochondrial etiology identified in these patients emphasizes the pathologic potential of homoplasmic mutations and has important implications for the investigation and genetic counseling of families where dystonia is the principal clinical feature. We advocate that mitochondrial disease should be given serious consideration in patients with familial, progressive dystonia, particularly when additional neurologic features such as epilepsy are present.

The population of elderly individuals has increased significantly over the past century and is predicted to rise even more rapidly in the future. Ageing is a major risk factor for many diseases such as neurodegenerative disease, diabetes and cancer. This highlights the importance of understanding the mechanisms involved in the ageing process. One plausible mechanism for ageing is accumulation of mutations in the mitochondrial genome. In this review, we discuss some of the most convincing data surrounding age-related mtDNA mutations and the evidence that these mutations contribute to the ageing process.

Mutations in the gene coding for the catalytic subunit of the mitochondrial DNA (mtDNA) polymerase gamma (POLG1) have recently been described in patients with diverse clinical presentations, revealing a complex relationship between genotype and phenotype in patients and their families. POLG1 was sequenced in patients from different European diagnostic and research centres to define the phenotypic spectrum and advance understanding of the recurrence risks. Mutations were identified in 38 cases, with the majority being sporadic compound heterozygotes. Eighty-nine DNA sequence changes were identified, including 2 predicted to alter a splice site, 1 predicted to cause a premature stop codon and 13 predicted to cause novel amino acid substitutions. The majority of children had a mutation in the linker region, often 1399G-->A (A467T), and a mutation affecting the polymerase domain. Others had mutations throughout the gene, and 11 had 3 or more substitutions. The clinical presentation ranged from the neonatal period to late adult life, with an overlapping phenotypic spectrum from severe encephalopathy and liver failure to late-onset external ophthalmoplegia, ataxia, myopathy and isolated muscle pain or epilepsy. There was a strong gender bias in children, with evidence of an environmental interaction with sodium valproate. POLG1 mutations cause an overlapping clinical spectrum of disease with both dominant and recessive modes of inheritance. 1399G-->A (A467T) is common in children, but complete POLG1 sequencing is required to identify multiple mutations that can have complex implications for genetic counselling.

The authors describe a 47-year-old man who presented with proximal muscle weakness, myalgia, elevated creatine kinase, and features of a pure myopathic syndrome in whom they have identified a novel mutation in the mitochondrial tRNA(Ala) gene. This 5591G>A transition is heteroplasmic, segregates with cytochrome c oxidase deficiency in single muscle fibers, and fulfills recognized criteria for pathogenicity. This case exemplifies the wide-ranging clinical spectrum of mitochondrial disease presentations.

The authors describe a patient who presented with headache, seizures, and severe cerebral edema in whom they identified a novel mutation in the mitochondrial (mt-) tRNA(His) gene. This G12147A transition is heteroplasmic, predicted to disrupt a highly conserved base pair, and segregates with the cytochrome c oxidase deficiency in single muscle fibers.

We have extended our previous analysis of the pedigree rate of control-region divergence in the human mitochondrial genome. One new germline mutation in the mitochondrial DNA (mtDNA) control region was detected among 185 transmission events (generations) from five Leber hereditary optic neuropathy (LHON) pedigrees. Pooling the LHON pedigree analyses yields a control-region divergence rate of 1.0 mutation/bp/10(6) years (Myr). When the results from eight published studies that used a similar approach were pooled with the LHON pedigree studies, totaling >2,600 transmission events, a pedigree divergence rate of 0.95 mutations/bp/Myr for the control region was obtained with a 99.5% confidence interval of 0.53-1.57. Taken together, the cumulative results support the original conclusion that the pedigree divergence rate for the control region is approximately 10-fold higher than that obtained with phylogenetic analyses. There is no evidence that any one factor explains this discrepancy, and the possible roles of mutational hotspots (rate heterogeneity), selection, and random genetic drift and the limitations of phylogenetic approaches to deal with high levels of homoplasy are discussed. In addition, we have extended our pedigree analysis of divergence in the mtDNA coding region. Finally, divergence of complete mtDNA sequences was analyzed in two tissues, white blood cells and skeletal muscle, from each of 17 individuals. In three of these individuals, there were four instances in which an mtDNA mutation was found in one tissue but not in the other. These results are discussed in terms of the occurrence of somatic mtDNA mutations.

Aims/hypothesis: To examine whether there is a high content of mutated mitochondrial DNA in individual pancreatic beta cells from a patient with the A3243G mitochondrial DNA mutation., Methods: Tissues were available from a patient with diabetes and the A3243G mutation including pancreatic tissue. We quantified the amount of mutated mitochondrial DNA in tissue homogenates and single pancreatic beta cells using hot last cycle PCR., Results: The percentage ratio of mutated to wild-type mtDNA was high in tissues such as muscle and brain (>60%), but surprisingly low in both pancreatic islets and in individual beta cells from these islets. The islets were smaller in the patient than in control subjects in keeping with a decreased beta-cell mass., Conclusions/interpretation: These observations suggest that either the beta cells show increased sensitivity to the effects mtDNA mutations on respiratory chain function, and/or cells with a high mutant load are preferentially removed leading to a progressive decrease in the islet beta-cell mass.

Purpose: To evaluate somatic mitochondrial (mt)DNA mutations in the macula during ageing., Methods: Ten 30-microm cryostat sections from the macula (foveal and perifoveal regions) and peripheral retina of 14 donors (aged 14-94 years) were cut for cytochrome c oxidase cytochemistry. The photoreceptor layer was microdissected and DNA extracted for 4977-bp mtDNA (mtDNA(4977)) quantification using PCR. Dual cytochemistry for cytochrome c oxidase and succinate dehydrogenase allowed the detection of cytochrome c oxidase-deficient cones., Results: Findings showed a progressive accumulation of mtDNA(4977) from ages 14 to 94 years. From ages 14 to 60 years there was an increase from 0.006% to 0.25%, and from ages 60 to 94 years there was a steeper increase from 0.25% to 5.39%. Counts of cones in the dual-reacted preparations showed more cytochrome c oxidase-deficient cones in the foveal region than elsewhere., Conclusions: The results show that mitochondrial DNA deletions and cytochrome c oxidase-deficient cones accumulate in the ageing retina, particularly in the foveal region. These defects may contribute to the changes in macular function observed in ageing and age-related maculopathy.

Human tissues acquire somatic mitochondrial DNA (mtDNA) mutations with age. Very high levels of specific mtDNA mutations accumulate within individual cells, causing a defect of mitochondrial oxidative metabolism. This is a fundamental property of nondividing tissues, but it is not known how it comes about. To explore this problem, we developed a model of mtDNA replication within single human cells. Using this model, we show that relaxed replication of mtDNA alone can lead, through random genetic drift, to the clonal expansion of single mutant events during human life. Significant expansions primarily develop from mutations acquired during a critical period in childhood or early adult life.

Leber's hereditary optic neuropathy (LHON) is a common cause of bilateral optic nerve disease. The majority of LHON patients harbour one of three point mutations of the mitochondrial DNA (mtDNA) complex I, or NADH:ubiquinone oxidoreductase (ND) genes (G11778A in ND4, G3460A in ND1, T14484C in ND6). As a consequence, screening for these mutations has become part of the routine clinical investigation of young adults who present with bilateral optic neuropathy, and the absence of these mutations is interpreted as indicating there is a low likelihood that an optic neuropathy is LHON. However, there are many individuals who develop the clinical features of LHON but who do not harbour one of these primary LHON mutations. We describe two LHON pedigrees that harbour the same novel point mutation within the mtDNA ND6 gene (A14495G). This mutation was heteroplasmic in both families, and sequencing of the mitochondrial genome confirmed that the mutation arose on two independent occasions. This is the seventh mutation in the ND6 gene that causes optic neuropathy, indicating that this gene is a hot spot for LHON mutations. Protein modelling studies indicate that all of these pathogenic mutations lie within close proximity to one another in a hydrophobic cleft or pocket. This is the first evidence for a relationship between a specific disease phenotype and a specific structural domain within a mitochondrial respiratory chain subunit. These findings suggest that the mtDNA ND6 gene should be sequenced in all patients with LHON who do not harbour one of the three common LHON mutations.

The chronological accumulation of mitochondrial DNA mutations has been proposed as a potential mechanism in the physiological processes of ageing and age-related disease. We discuss the evidence behind this theory and relate some of the ageing mitochondrial changes to mitochondrial DNA disorders. In particular, we describe the aggregation of cytochrome c oxidase-deficient cells in both skeletal muscle and the CNS in normal ageing as seen in the mitochondrial DNA disorders. These mitochondrial enzyme-deficient cells have been shown to occur in significant quantities in both muscle and CNS in patients with mitochondrial DNA disorders. In both ageing and mtDNA disorder muscle these cytochrome c-deficient fibres contain high levels of a single mutant strain of mitochondrial DNA. Whether these mutations are a primary or secondary event in the physiology of ageing remains to be determined.

The coding sequence for the human mitochondrial genome (mtDNA) was published in 1981. Within a decade, the first pathogenic mtDNA mutations were described in humans with sporadic and maternally inherited disease. The last ten years has seen a profusion of reports describing new pathogenic mutations associated with a diverse range of clinical phenotypes. Although we have seen great advances in our understanding of the molecular mechanisms involved in the pathogenesis of mtDNA disease, we are only just beginning to tackle some of the more difficult questions. In this review we describe recent advances in our understanding of mtDNA disease and highlight ways that this knowledge might lead to novel therapies in the future.

Genetic defects affecting the mitochondrial respiratory chain are an important cause of neurological disease. Previously, we identified a family with complex II deficiency and late-onset neurodegenerative disease with progressive optic atrophy, ataxia, and myopathy. The affected family members are now shown to carry a C-to-T transition in one allele of the nuclear gene encoding the flavoprotein subunit of complex II. Mutation of the equivalent base in Escherichia coli generates an inactive enzyme unable to bind flavin adenine dinucleotide covalently. Compatible with these findings, our patients have an approximate 50% decrease in complex II and succinate dehydrogenase activity. These results suggest that genetic defects of nuclear-encoded subunits of the mitochondrial respiratory chain can result in late-onset neurodegenerative disease.

The segregation and transmission of mitochondrial genomes in humans are complicated processes, but are particularly important for understanding the inheritance and clinical abnormalities of mitochondrial disorders. This review describes three aspects of mitochondrial genetics. First, that the segregation and transmission of mitochondrial (mt)DNA molecules are likely to be determined by their physical association within the organelles and by the dynamics of mitochondrial structure and subcellular organization. Second, that the transmission of heteroplasmic mtDNA sequence changes from one generation to the next often involves rapid shifts in allele frequency. For >20 years, the standard explanation has been that there is a developmental bottleneck in which, at some stage of oogenesis, there is a reduction in the effective number of mitochondrial units of inheritance. The third aspect is that ongoing analyses of the segregation and transmission of pathogenic mtDNA mutations indicate the operation of multiple genetic processes. Thus, the segregation and transmission of mtDNA mutations occurs predominantly, but not exclusively, under conditions of random genetic drift. However, there is also evidence for bias due to incomplete ascertainment of pedigrees and for negative selection of pathogenic mutations in rapidly dividing somatic tissues such as the white blood cell population.

Since Harman in 1972 first proposed a role in the process of aging for the mitochondrial genome, a wealth of evidence has been accumulated to support this theory. We discuss the hereditary mitochondrial DNA disorders, which we believe may give insight into both normal aging and neurodegenerative conditions. We then review the evidence for the role of mitochondrial DNA mutations in both aging and age-related disorders and also discuss new approaches for investigating the mitochondrial genome at a single cell level, by observing the activity of the mitochondrial enzyme cytochrome c oxidase.

Heteroplasmic mitochondrial DNA (mtDNA) defects are an important cause of neurological disease. Although hearing impairment is common in patients with mtDNA defects, the spectrum and pathophysiology of the hearing loss is not well characterized. We therefore studied the relationship between cochlear and brainstem auditory function in 23 patients harbouring a range of different mtDNA mutations. Based upon the pure tone audiogram, patients fell into three distinct groups: (i) normal hearing, (ii) mild to moderate predominantly high frequency hearing loss, and (iii) severe or profound hearing loss at all frequencies. Within this study group only certain genetic defects were associated with hearing loss, and for individuals harbouring the A3243G point mutation, the severity of the hearing loss correlated with the percentage level of mutated mtDNA (mutation load) in skeletal muscle. The 10 patients who had a moderate hearing loss or less had normal brainstem auditory evoked responses and MRI, but it was not possible to interpret the brainstem auditory evoked responses in 13 patients with severe hearing loss. Otoacoustic emissions were absent in patients with a moderate or more severe hearing loss. These findings are consistent with a predominantly cochlear origin for the hearing deficit, which is determined by the precise genetic defect and the percentage mutation load.

Heteroplasmic mitochondrial DNA (mtDNA) defects are an important cause of inherited human disease. On a cellular level, the percentage of mutant mtDNA is the principal factor behind the expression of the genetic defect. Marked variation in the level of mutant mtDNA among tissues is thought to be responsible for the diverse clinical phenotypes associated with the same pathogenic mtDNA mutation. This study was designed to determine whether the percentage level of a pathogenic mtDNA molecule is determined by a purely random process. The tissue distribution of the A3243G MELAS point mutation was analyzed in five individuals who were members of a family with maternally inherited diabetes and deafness. The level of mutant mtDNA was measured in four tissues in three individuals and three tissues in two individuals. The highest level of mutant mtDNA occurred in skeletal muscle, followed by hair follicles, and then buccal mucosa, with the lowest levels in blood (leucocyte/platelet fraction). The probability of observing any strict hierarchy in family is 4.82 x 10(-5). These results indicate that the distribution of the A3243G mutation is not solely determined by random processes., (Copyright 1999 Wiley-Liss, Inc.)

Defects of the mitochondrial genome are important causes of disease. Despite major advances in our investigation of patients, there is no effective therapy. Progress in this area is limited by the absence of any animal models in which we can evaluate treatment. To develop such a model we have injected human myoblasts into the tibialis anterior of SCID mice after inducing necrosis. After injection of normal human myoblasts, regenerating fibers expressed human beta-spectrin, confirming they were derived from fusion of human myoblasts. The stability of the muscle fibers was inferred by demonstrating the formation of motor end plates on the regenerating fibers. In addition, we show the presence of human cytochrome c oxidase subunit II, which is encoded by the mitochondrial genome, in the regenerated fibers. After injection of human myoblasts containing either the A8344G or the T8993C heteroplasmic mitochondrial DNA mutations, human beta-spectrin positive fibers were found to contain the mutation at a similar level to the injected myoblasts. These studies highlight the potential value of this model for the study of mitochondrial DNA defects.

Many patients with inherited mitochondrial encephalopathies have one of two pathogenic mutations of mitochondrial DNA (mtDNA): A3243G or A8344G. Individuals who harbour these mutations carry both mutant and wild-type alleles within each cell (heteroplasmy). Despite clear evidence of a direct relationship between the level of mutation and mitochondrial respiratory chain function in vitro, it has been more difficult to demonstrate a clear correlation between clinical phenotype and the level of mutant mtDNA in vivo. To address this issue, we identified 245 individuals who carry either the A3243G or A8344G mutations, and studied the relationship between the incidence of specific clinical features and the level of mutant mtDNA in blood (for A3243G, n = 73; for A8344G, n = 25) and/or skeletal muscle (for A3234G, n = 111; for A8344G, n = 55). Within this study group, the frequency of key clinical features was significantly different for individuals harbouring the A3243G and A8344G mutations. For both mutations, there was a correlation between the frequency of the more common clinical features and the level of mutant mtDNA in muscle. In contrast, we did not observe a correlation between the frequency of clinical features and the level of mutant mtDNA in blood. Therefore, measurement of the level of the A3243G and A8344G mutations in muscle will allow the identification of individuals who are at risk of developing specific complications, thus improving the prognostic advice that can be given to patients and family members who carry these mutations.

Biochemical and molecular genetic evidence is presented that in six independent pedigrees the development of Leber hereditary optic neuropathy (LHON) is due to the same primary mutation in the mitochondrial ND1 gene. A LHON family from the Newcastle area of Great Britain was analyzed in depth to determine the mitochondrial genetic etiology of their disease. Biochemical assays of mitochondrial electron transport in organelles isolated from the platelet/white-blood-cell fraction have established that the members of this family have a substantial and specific lowering of flux through complex I (NADH-ubiquinone oxidoreductase). To determine the site of the primary mitochondrial gene mutation in this pedigree, all seven mitochondrial complex I genes were sequenced, in their entirety, from two family members. The primary mutation was identified as a homoplasmic transition at nucleotide 3460, which results in the substitution of threonine for alanine at position 52 of the ND1 protein. This residue occurs within a very highly conserved hydrophilic loop, is invariantly alanine or glycine in all ND1 proteins, and is adjacent to an invariant aspartic acid residue. This is only the second instance in which both a biochemical abnormality and a mitochondrial gene mutation have been identified in an LHON pedigree. The sequence analysis of the ND81 gene was extended to a further 11, unrelated LHON pedigrees that had been screened previously and found not to carry the mitochondrial ND4/R340H mutation. The ND1/A52T mutation at nucleotide 3460 was found in five of these 11 pedigrees. In contrast, this sequence change was not found in any of the 47 non-LHON controls. The possible role of secondary complex I mutations in the etiology of LHON is also addressed in these studies.

The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts by ultraviolet light (UV) were compared under conditions of maximum expression of the 6-thioguanine resistance (TGr) phenotype. Selection was with 20 micrograms TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions of the UV dose but the slope of the line for normal cells extrapolated to zero induced mutants at 3 J/m2. The postreplication repair-defective XP4BE cells showed a higher frequency of TGr colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TGr colonies was not a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than about 50%. The UV doses and induced mutation frequencies corresponding to 37% survival of cloning abilities were 6.7 J/m2 and 6.2 X 10(-5), respectively, for normal cells and 3.75 J/m2 and 17.3 X 10(-5) for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.

The forward mutation selection system based on resistance to 8-azaguanine has been widely used with cells cultured from a diversity of species and with a variety of mutagens. Ouabain resistance is an alternative selective system. Both systems show a substantial influence of expression time on the number of resistant variants observed after addition of the selective agent such that the frequency reaches a maximum which is dose dependent, and then declines rapidly. Metabolic cooperation has been propsed as the mechanism responsible for this decline with the 8-azaguanine system, but it is less likely to account for the loss of ouabain-resistant variants where it is necessary to invoke generalised effects on the viability of variants due to overcrowding on the plates. A comparison of the two selective systems showed that, with the exception of gamma-irradiation, which was apparently non-mutagenic in the ouabain system, there was broad agreement between the two systems for each mutagen tested. Ethyl methanesulphonate was the most efficient mutagen by a substantial factor. Ouabain resistance permitted greater discrimination particularly between weak mutagens because of the low frequency of spontaneous variants (4 x 10(-7) and also produced data with less intrinsic variability. The absence of gamma ray induced mutation in the ouabain system shows that it may fail to detect certain types of mutagens. Thus the two systems should be used to complement each other. Mutation by the fungicide captan was evaluated using both systems and the positive results indicate that it may pose a hazard to man.