Molecular analysis of chemically-induced mutations at the RpII215 locus of Drosophila melanogaster

A substantial fraction, perhaps 50% or more, of spontaneous mutations in Drosophila melanogaster have been shown by molecular analyses to be associated with the presence of a transposable element (TE) inserted into the affected gene. We are interested in the molecular structure of induced mutations in Drosophila, in particular whether TEs are also responsible for a significant proportion of chemically-induced mutations. We report here the molecular analysis of 58 mutations at the RpII215 locus induced with EMS or ENU. While we find evidence for moderately sized deletions at this locus (in 3/58, or 5% of the examined mutants), we failed to detect any mutations which were associated with an insertion event. It may be the case that induced mutations are qualitatively different from spontaneous mutations.     

Use of supF, an Escherichia coli tyrosine suppressor tRNA gene, as a mutagenic target in shuttle-vector plasmids

The Escherichia coli tyrosine amber suppressor tRNA gene, supF, has been utilized as a mutagenic target in several shuttle-vector plasmids. Data on mutagenic inactivation of suppressor activity was obtained from induced mutagenesis experiments with plasmids pZ189 and p3AC, and from studies on alterations of the supF gene transduced into E. coli. 162 single or tandem base-substitution mutations that reduce or eliminate suppressor activity were identified at 86 sites within 158 base pairs. The 2 transition and 4 transversion mutations possible in double-stranded DNA were all detectable. At 56 sites two different inactivating mutations were found; and at 20 sites all 3 possible base substitution mutations inactivated suppressor function. Most of the mutations were clustered within the mature tRNA region: 144 of the base-substitution mutations were found at 74 sites within the 85-bp mature tRNA region. Insertions of 1 or 2 bases at 4 sites and deletions of 1 to 3 bases at 15 sites were found to inactivate supF function. A few silent mutations which do not inactivate suppressor function were found: single base-substitutions at 4 sites, 14 pairs of silent double mutations, and a large deletion including the promoter region. The supF gene is thus an extremely sensitive target for mutagenic inactivation in shuttle-vector plasmids.     

Germ-line mutations in the neurofibromatosis 2 gene: correlations with disease severity and retinal abnormalities.

Neurofibromatosis 2 (NF2) features bilateral vestibular schwannomas, other benign neural tumors, and cataracts. Patients in some families develop many tumors at an early age and have rapid clinical progression, whereas in other families, patients may not have symptoms until much later and vestibular schwannomas may be the only tumors. The NF2 gene has been cloned from chromosome 22q; most identified germ-line mutations result in a truncated protein and severe NF2. To look for additional mutations and clinical correlations, we used SSCP analysis to screen DNA from 32 unrelated patients. We identified 20 different mutations in 21 patients (66%): 10 nonsense mutations, 2 frameshifts, 7 splice-site mutations, and 1 large in-frame deletion. Clinical information on 47 patients from the 21 families included ages at onset and at diagnosis, numbers of meningiomas, spinal and skin tumors, and presence of cataracts and retinal abnormalities. We compared clinical findings in patients with nonsense or frameshift mutations to those with splice-site mutations. When each patient was considered as an independent random event, the two groups differed (P < or = .05) for nearly every variable. Patients with nonsense or frameshift mutations were younger at onset and at diagnosis and had a higher frequency and mean number of tumors, supporting the correlation between nonsense and frameshift mutations and severe NF2. When each family was considered as an independent random event, statistically significant differences between the two groups were observed only for mean ages at onset and at diagnosis. A larger data set is needed to resolve these discrepancies. We observed retinal hamartomas and/or epiretinal membranes in nine patients from five families with four different nonsense mutations. This finding, which may represent a new genotype-phenotype correlation, merits further study.     

The effects of weak selection on neutral diversity at linked sites

The effects of selection on variability at linked sites have an important influence on levels and patterns of within-population variation across the genome. Most theoretical models of these effects have assumed that selection is sufficiently strong that allele frequency changes at the loci concerned are largely deterministic. These models have led to the conclusion that directional selection for selectively favorable mutations, or against recurrent deleterious mutations, reduces nucleotide site diversity at linked neutral sites. Recent work has shown, however, that fixations of weakly selected mutations, accompanied by significant stochastic changes in allele frequencies, can sometimes cause higher diversity at linked sites when compared with the effects of fixations of neutral mutations. This study extends this work by deriving approximate expressions for the mean conditional times to fixation and loss of mutations subject to selection, and analyzing the conditions under which selection increases rather than reduces these times. Simulations are used to examine the relations between diversity at a neutral site and the fixation and loss times of mutations at a linked site that is subject to selection. It is shown that the long-term level of neutral diversity can be increased over the purely neutral value by recurrent fixations and losses of linked, weakly selected dominant or partially dominant favorable mutations, or linked recessive or partially recessive deleterious mutations. The results are used to examine the conditions under which associative overdominance, as opposed to background selection, is likely to operate.     

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa, IX. Mutational spectra as a function of X-ray dose.

In previous studies, X-ray-induced specific-locus mutations in the adenine-3 (ad-3) region of a two-component heterokaryon (H-12) of Neurospora crassa were combined with a series of tester strains carrying markers in the ad-3 and immediately adjacent regions to map mutants that were presumed multilocus deletions (de Serres, 1989c, 1990a). Two new classes of X-ray-induced mutations were recovered: multiple-locus mutations consisting of gene/point mutations at the ad-3A or ad-3B locus with a closely linked recessive lethal mutation, or multilocus deletions covering the ad-3A, ad-3B and/or nic-2 loci with a closely linked recessive lethal mutation (designated ad-3R + RLCL and [ad-3]IR + RLCL, respectively). Thus, the ad-3 specific-locus assay can detect damage occurring at the ad-3A and the ad-3B loci, as well as at a minimum of 19 other loci in the immediately adjacent regions. The original overall spectrum of ad-3 mutations can be resolved, by genetic analysis, into a series of 30 subclasses. In the present paper, the data from the genetic analysis of 832 X-ray-induced mutants recovered from a series of 4 experiments (Webber and de Serres, 1965) have been presented in terms of Mutational Spectra organized as a function of X-ray dose. Comparison of these Spectra demonstrates the shift from high percentages of gene/point mutations (with a high percentage of mutants at the ad-3B locus showing allelic complementation) at low doses, to low percentages of gene/point mutations (with a low percentage of ad-3B mutants showing allelic complementation) and high percentages of multilocus deletion mutations and multiple-locus mutations (of genotype ad-3R + RLCL or [ad-3]IR + RLCL) at high doses. These Mutational Spectra demonstrate the marked dose-dependence of X-ray-induced specific-locus mutations in a eukaryotic organism.     

Radiation-induced forward and reverse specific locus mutations and dominant cataract mutations in treated strain BALB/c and DBA/2 male mice

Strain BALB/c and DBA/2 mice were chosen to investigate the effects of genetic background on the radiation-induced mutation rate since they exhibit differences in their radiation sensitivity. Males were exposed to 3 + 3-Gy X-irradiation and mated to untreated specific locus Test-stock females. Offspring resulting from treated spermatogonia were screened for induced specific locus forward and reverse mutations and dominant cataract mutations. Since BALB/c mice are homozygous brown and albino, specific locus forward mutations could be screened at 5 of the 7 specific loci (a, d, se, p, s), while reverse mutations could be screened at the b and c loci. Strain DBA/2 is homozygous non-agouti, brown and dilute. Therefore, specific locus forward mutations could be screened at 4 loci (c, se, p, s) and reverse mutations were screened at the a, b and d loci. Results indicate no effect of genetic background on the sensitivity to mutation induction of specific locus forward mutations, while for the dominant cataract alleles strain DBA/2 exhibited a higher mutation rate than either strain BALB/c or similarly treated (101/El X C3H/El)F1 mice. If, by confirmation, these differences should be demonstrated to be real, it is interesting that strain DBA/2 should exhibit a greater sensitivity to radiation-induced dominant mutations. First, strain DBA/2 was chosen as radiation resistant or repair competent. The observation that DBA/2 exhibited a higher sensitivity to radiation-induced mutation may indicate a role for repair, albeit misrepair, in the mutation process. Second, that the effect of genotype was only observed for the mutation rate to dominant cataract alleles may reflect a difference in the spectrum of DNA alterations which result in dominant or recessive alleles. A dominant allele is more likely misinformation, such that as heterozygote it interferes with the wild-type allele. By comparison, a recessive allele may result from any DNA alteration leading to the loss of a functional gene product. One reverse mutation at each of the a and d loci was recovered in the present experiments. The similarities of the present results for radiation-induced reverse mutations with the extensive data on the spontaneous reverse mutation rates are interesting. Reverse mutations were recovered only at the a and d loci. Further, the reverse mutations recovered at the a locus were to alternate alleles (at, Aw or Asy) while true reverse mutations were apparently recovered at the d locus.     

Assessing in vivo mutation frequencies and creating a high-resolution genome-wide map of fitness costs of Hepatitis C virus

Like many viruses, Hepatitis C Virus (HCV) has a high mutation rate, which helps the virus adapt quickly, but mutations come with fitness costs. Fitness costs can be studied by different approaches, such as experimental or frequency-based approaches. The frequency-based approach is particularly useful to estimate in vivo fitness costs, but this approach works best with deep sequencing data from many hosts are. In this study, we applied the frequency-based approach to a large dataset of 195 patients and estimated the fitness costs of mutations at 7957 sites along the HCV genome. We used beta regression and random forest models to better understand how different factors influenced fitness costs. Our results revealed that costs of nonsynonymous mutations were three times higher than those of synonymous mutations, and mutations at nucleotides A or T had higher costs than those at C or G. Genome location had a modest effect, with lower costs for mutations in HVR1 and higher costs for mutations in Core and NS5B. Resistance mutations were, on average, costlier than other mutations. Our results show that in vivo fitness costs of mutations can be site and virus specific, reinforcing the utility of constructing in vivo fitness cost maps of viral genomes.     

Atypical forms of incontinentia pigmenti in male individuals result from mutations of a cytosine tract in exon 10 of NEMO (IKK-gamma)

Familial incontinentia pigmenti (IP [MIM 308310]), or Bloch-Sulzberger syndrome, is an X-linked dominant and male-lethal disorder. We recently demonstrated that mutations in NEMO (IKK-gamma), which encodes a critical component of the NF-kappaB signaling pathway, were responsible for IP. Virtually all mutations eliminate the production of NEMO, causing the typical skewing of X inactivation in female individuals and lethality in male individuals, possibly through enhanced sensitivity to apoptosis. Most mutations also give rise to classic signs of IP, but, in this report, we describe two mutations in families with atypical phenotypes. Remarkably, each family included a male individual with unusual signs, including postnatal survival and either immune dysfunction or hematopoietic disturbance. We found two duplication mutations in these families, at a cytosine tract in exon 10 of NEMO, both of which remove the zinc (Zn) finger at the C-terminus of the protein. Two deletion mutations were also identified in the same tract in additional families. However, only the duplication mutations allowed male individuals to survive, and affected female individuals with duplication mutations demonstrated random or slight skewing of X inactivation. Similarly, NF-kappaB activation was diminished in the presence of duplication mutations and was completely absent in cells with deletion mutations. These results strongly indicate that male individuals can also suffer from IP caused by NEMO mutations, and we therefore urge a reevaluation of the diagnostic criteria.     

Formamide Sensitivity: A Novel Conditional Phenotype in Yeast

Yeast mutants unable to grow in the presence of 3% formamide have been isolated in parallel with mutants sensitive to either 37° or 6% ethanol. The number of formamide-sensitive mutations that affect different genes that can be identified from yeast cells is at least as large as the number of thermosensitive or ethanol-sensitive mutations. These mutations are of two types: those that are sensitive to formamide, temperature and/or ethanol simultaneously; and those that are specific for formamide sensitivity and show no temperature or ethanol sensitivity phenotype. Those genes susceptible to giving rise to formamide-sensitive alleles include the structural gene for DNA ligase, CDC9, and the structural gene for arginine permease, CAN1. The results indicate that formamide sensitivity can be used as a novel conditional phenotype for mutations on both essential and nonessential genes. This work also confirms that ethanol-sensitivity can be used as a conditional phenotype to identify mutations in at least as many genes as those susceptible to temperature or formamide sensitive mutations.     

Splicing Defects in the COL3A1 Gene: Marked Preference for 5′ (Donor) Splice-Site Mutations in Patients with Exon-Skipping Mutations and Ehlers-Danlos Syndrome Type IV

Ehlers-Danlos syndrome (EDS) type IV results from mutations in the COL3A1 gene, which encodes the constituent chains of type III procollagen. We have identified, in 33 unrelated individuals or families with EDS type IV, mutations that affect splicing, of which 30 are point mutations at splice junctions and 3 are small deletions that remove splice-junction sequences and partial exon sequences. Except for one point mutation at a donor site, which leads to partial intron inclusion, and a single base-pair substitution at an acceptor site, which gives rise to inclusion of the complete upstream intron into the mature mRNA, all mutations result in deletion of a single exon as the only splice alteration. Of the exon-skipping mutations that are due to single base substitutions, which we have identified in 28 separate individuals, only two affect the splice-acceptor site. The underrepresentation of splice acceptor-site mutations suggests that the favored consequence of 3' mutations is the use of an alternative acceptor site that creates a null allele with a premature-termination codon. The phenotypes of those mutations may differ, with respect to either their severity or their symptomatic range, from the usual presentation of EDS type IV and thus have been excluded from analysis.     

The helical domains of the stem region of dengue virus envelope protein are involved in both virus assembly and entry

The envelope (E) of dengue virus (DENV) is a determinant of tropism and virulence. At the C terminus of E protein, there is a stem region containing two amphipathic α-helical domains (EH1 and EH2) and a stretch of conserved sequences in between. The crystal structure of E protein at the postfusion state suggested the involvement of the stem during the fusion; however, the critical domains or residues involved remain unknown. Site-directed mutagenesis was carried out to replace each of the stem residues at the hydrophobic face with an alanine or proline in a DENV serotype 4 (DENV4) precursor membrane (prM)/E expression construct. Most of the 15 proline mutations at either EH1 or EH2 severely affected the assembly of virus-like particles (VLPs). Radioimmunoprecipitation and membrane flotation assays revealed that EH1 mutations primarily affect prM-E heterodimerization and EH2 mutations affect the membrane binding of the stem. Introducing four proline mutations at either EH1 or EH2 into a DENV2 replicon packaging system greatly affects assembly and entry. Moreover, introducing these mutations into a DENV2 infectious clone confirmed the impairment in assembly and infectivity. Sequencing analysis of adaptive mutations in passage 5 viruses revealed a change to a leucine or wild-type residue at the original site, suggesting the importance of maintaining the helical structure. Collectively, these findings suggest that the EH1 and EH2 domains are involved in both assembly and entry steps of the DENV replication cycle; this feature, together with the high degree of sequence conservation, suggests that the stem region is a potential target of antiviral strategies.     

Mutagenic repair in Escherichia coli. VIII. Effect of gyrB mutations on ultraviolet light mutagenesis

Introduction into Escherichia coli WP2 bacteria of a mutation in the gyrB locus previously shown to reduce the degree of chromosomal superhelicity caused a small decrease in the frequency of UV-induced mutations to streptomycin resistance (but not significantly) and to tryptophan independence (mostly ochre suppressors) in excision repair-proficient bacteria. It did not influence the 'broth effect' or the rate or extent of 'mutation frequency decline' of suppressor mutations. In an excision-deficient (uvrA 155) background the yield of UV-induced streptomycin-resistant mutations was lower in gyrB bacteria at all doses; the yield of tryptophan-independent mutations was slightly lower at low doses and slightly higher at high doses. In both excision-proficient and -deficient bacteria the yield of UV-induced mutations to rifampicin resistance was apparently lower in gyrB mutants but this could be due at least in part to a hypersensitivity of some Rifr gyrB bacteria to UV. The number of spontaneous tryptophan-independent mutations was lower in gyrB bacteria but this was almost certainly due to their poorer viability on tryptophan-limiting plates and not to a lower spontaneous mutation rate. In a temperature-sensitive presumed gyrase-deficient strain a small decrease in mutant yield at low doses was observed following incubation at restrictive temperature before UV. This was ascribed to an enhancement of excision repair. Our failure to find any significant effect of gyrB mutations does not support the hypothesis that hairpin formation (which should be dependent on a high degree of superhelicity) is involved in determining the 'broth effect', 'mutation frequency decline' or the probability that a mutation will occur spontaneously. Dramatic effects of superhelicity on UV mutagenesis also seem to be unlikely.     

Glucocerebrosidase mutations in Gaucher disease.

BACKGROUND: Thirty-six mutations that cause Gaucher disease, the most common glycolipid storage disorder, are known. Although both alleles of most patients with the disease contain one of these mutations, in a few patients one or both disease-producing alleles have remained unidentified. Identification of mutations in these patients is useful for genetic counseling. MATERIALS AND METHODS: The DNA from 23 Gaucher disease patients in whom at least one glucocerebrosidase allele did not contain any of the 36 previously described mutations has been examined by single strand conformation polymorphism (SSCP) analysis, followed by sequencing of regions in which abnormalities were detected. RESULTS: Eight previously undescribed mutations were detected. In exon 3, a deletion of a cytosine at cDNA nt 203 was found. In exon 6, three missense mutations were identified: a C-->A transversion at cDNA nt 644 (Ala176-->Asp), a C-->A transversion at cDNA nt 661 that resulted in a (Pro182-->Thr), and a G-->A transition at cDNA nt 721 (Gly202-->Arg). Two missense mutations were found in exon 7: a G-->A transition at cDNA nt 887 (Arg257-->Gln) and a C-->T at cDNA nt 970 (Arg285-->Cys). Two missense mutations were found in exon 9: a T-->G at cDNA nt 1249 (Trp378-->Gly) and a G-->A at cDNA nt 1255 (Asp380-->Asn). In addition to these disease-producing mutations, a silent C-->G transversion at cDNA nt 1431, occurring in a gene that already contained the 1226G mutation, was found in one family. CONCLUSIONS: The mutations described here and previously known can be classified as mild, severe, or lethal, on the basis of their effect on enzyme production and on clinical phenotype, and as polymorphic or sporadic, on the basis of the haplotype in which they are found. Rare mutations such as the new ones described here are sporadic in nature.     

Recessive mutations conferring resistance to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae

A total of 37 recessive mutations showing enhanced resistance to the glucose repression of galactokinase synthesis have been isolated by a selection procedure with a GAL81 gal7 double mutant. These mutations were grouped into three different complementation classes. One class, reg1, contains mutants arising from mutations at a site close to, but complementing, the gal3 locus. The reg1 mutant also showed resistance to the glucose repression of invertase synthesis but not to that of alpha-D-glucosidase. The two other classes were identified as arising from recessive mutations at the GAL82 locus and the GAL83 locus, respectively, at which various dominant mutations were isolated previously. When in a constitutive background due to the GAL81 or gal80 mutation, the GAL82 and GAL83 mutations did not show a mutually additive effect on the resistance to glucose repression of galactokinase synthesis, while the reg1 and GAL82 (or GAL83) mutations did. Based upon the specific behavior of cells with various genotypes for the above genes in response to the concentration of galactose and glucose in the medium, we propose a model involving three independent circuits for glucose signals in the regulation of the structural genes for the galactose pathway enzymes.     

s-RT-MELT for rapid mutation scanning using enzymatic selection and real time DNA-melting: new potential for multiplex genetic analysis

The rapidly growing understanding of human genetic pathways, including those that mediate cancer biology and drug response, leads to an increasing need for extensive and reliable mutation screening on a population or on a single patient basis. Here we describe s-RT-MELT, a novel technology that enables highly expanded enzymatic mutation scanning in human samples for germline or low-level somatic mutations, or for SNP discovery. GC-clamp-containing PCR products from interrogated and wild-type samples are hybridized to generate mismatches at the positions of mutations over one or multiple sequences in-parallel. Mismatches are converted to double-strand breaks using a DNA endonuclease (Surveyor™) and oligonucleotide tails are enzymatically attached at the position of mutations. A novel application of PCR enables selective amplification of mutation-containing DNA fragments. Subsequently, melting curve analysis, on conventional or nano-technology real-time PCR platforms, detects the samples that contain mutations in a high-throughput and closed-tube manner. We apply s-RT-MELT in the screening of p53 and EGFR mutations in cell lines and clinical samples and demonstrate its advantages for rapid, multiplexed mutation scanning in cancer and for genetic variation screening in biology and medicine.     

The Genetic Analysis of Distributive Segregation in Drosophila Melanogaster. II. Further Genetic Analysis of the Nod Locus

In Drosophila melanogster females the segregation of nonexchange chromosomes is ensured by the distributive segregation system. The mutation noda specifically impairs distributive disjunction and induces nonexchange chromosomes to undergo nondisjunction, as well as both meiotic and mitotic chromosome loss. We report here the isolation of seven recessive X-linked mutations that are allelic to noda. As homozygotes, all of these mutations exhibit a phenotype that is similar to that exhibited by noda homozygotes. We have also used these mutations to demonstrate that nod mutations induce nonexchange chromosomes to nondisjoin at meiosis II. Our data demonstrate that the effects of noda on meiotic chromosome behavior are a general property of mutations at the nod locus. Several of these mutations exhibit identical phenotypes as homozygotes and as heterozygotes with a deficiency for the nod locus; these likely correspond to complete loss-of-function or null alleles. None of these mutations causes lethality, decreases the frequency of exchange, or impairs the disjunction of exchange chromosomes in females. Thus, either the nod locus defines a function that is specific to distributive segregation or exchange can fully compensate for the absence of the nod+ function.     

Influence of uvrB and pKM101 on the spectrum of spontaneous, UV- and gamma-ray-induced base substitutions that revert hisG46 in Salmonella typhimurium

Oligonucleotide probes were used to identify base substitutions in 1089 revertants of hisG46 in Salmonella typhimurium that arose spontaneously or following irradiation with UV- or gamma-rays. The hisG46 allele, carrying a mutant CCC codon (Pro) in place of the wild-type codon CTC (Leu69) reverted via 6 distinguishable mutational events--C to T transitions at codon sites 1 or 2, C to A or C to G transversions at codon site 1, C to A at codon site 2, and an extragenic suppressor mutation. The distribution of hisG46 revertants differed among treatments and was influenced by the DNA-repair capacity of the bacteria. Plasmid pKM101 enhanced the frequencies of both spontaneous and induced mutations; transversion events were enhanced more efficiently by pKM101 than were transition events. Compared to Uvr+ bacteria, Uvr- bacteria had higher frequencies of spontaneous and induced mutations; transition mutations were enhanced more efficiently than were transversion mutations. The influence of DNA-repair activities on the mutational spectra provides some insights on the origins of spontaneous and UV-induced mutations.     

Isolation and characterization of dominant mutations resistant to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae.

Seven dominant mutations showing greatly enhanced resistance to the glucose repression of galactokinase synthesis have been isolated from GAL81 mutants, which have the constitutive phenotype but are still strongly repressible by glucose for the synthesis of the Leloir enzymes. These glucose-resistant mutants were due to semidominant mutations at either of two loci, GAL82 and GAL83. Both loci are unlinked to the GAL81- gal4, gal80, or gal7 X gal10 X gal1 locus or to each other. The GAL83 locus was mapped on chromosome V at a site between arg9 and cho1. The GAL82 and GAL83 mutations produced partial resistance of galactokinase to glucose repression only when one or both of these mutations were combined with a GAL81 or a gal80 mutation. The GAL82 and GAL83 mutations are probably specific for expression of the Leloir pathway and related enzymes, because they do not affect the synthesis of alpha-D-glucosidase, invertase, or isocitrate lyase.     

Dominant Maternal-Effect Mutations of DROSOPHILA MELANOGASTER Causing the Production of Double-Abdomen Embryos

Dominant mutations at two loci, BicaudalC (BicC ) and BicaudalD (BicD), cause heterozygous females to produce double-abdomen embryos. These mutations cause the production of embryos with a range of defects extending from the anterior end of the differentiated embryo. The same array of defective embryos is caused by mutations at either locus and is similar to that produced by the original mutation at bicaudal (bic). The array of defective embryos suggests that these mutations cause the loss of positional values from the anterior end of the embryo, associated with a duplication of the posterior end if too few positional values remain. BicaudalD mutations appear to be antimorphic, gain-of-function mutations, whereas BicaudalC mutations are likely to be hypomorphic or amorphic mutations. Mutations at all these loci (bic, BicC and BicD) act as mutual enhancers of each other, and a number of other maternal-effect mutations also act to either enhance or suppress the expression of these dominant bicaudal mutations.