Molecular basis of spontaneous mutation at the aprt locus of hamster cells

Mutations occurring spontaneously at the hamster aprt locus were examined at the base-pair level by amplifying target sequences using the polymerase chain reaction and then directly sequencing the double-stranded products. In a collection of 89 sequenced genes, all types of mutations were found, with transitions (mostly G.C to A.T) constituting the largest class (35%), transversions accounting for 27%, and small deletions/duplications for 25%. Simple base substitutions were distributed throughout the aprt structural gene with few sites having recurring mutations and G.C base-pairs being the predominant substitution target. Small deletions, on the other hand, were not distributed so evenly, being concentrated in a region of aprt rich in short direct and inverted repeat sequences. The base substitutions were predominantly missense, while about 10% produced nonsense codons. Splice junctions, and start and stop codons were also significant targets for mutation. No alterations were detected in three aprt-deficient strains after sequencing all exons and substantial upstream and downstream regions.     

Biochemical analysis of spontaneous fepA mutants of Escherichia coli

The fepA gene of Escherichia coli encodes the outer-membrane receptor protein for ferrienterobactin. Previous genetic studies indicated that fepA mutations occur frequently and suggested that most of the mutations were deletions. In this work seven spontaneous fepA mutations were analyzed by enzyme assay (enterobactin synthase and enterobactin esterase) and by DNA hybridization studies. In two strains, UT500 and UT700, the mutations were confined to the fepA gene. In the remaining mutants, the mutations were large deletions; in several cases, 27 kb or more of DNA had been lost. The deletions, all of which eliminated approximately the left half of the enterobactin gene cluster, extended from the vicinity of the fepC gene counterclockwise into the chromosome. A minimum of three clockwise endpoints were identified and at least two counterclockwise endpoints were detected. The variation in endpoints among the deletions argues against the involvement of a normal transposon in their formation. Also, unexpected homology was found between enterobactin gene cluster DNA and lacPOZ and pSC101.     

Genetical tests for the frequency of small deletions among ems-induced point mutations in Drosophila

In order to estimate the proportion of small deletions among EMS-induced point mutations we scored visible mutations at 6 sex-linked loci either by a specific-locus test, in which both deletions and intragenic changes survive, or in sons of attached-X females, in which deletions do not survive. About twice as many visibles were detected in the specific-locus as in the attached-X test, and between 50 and 90% of the former were lethal to males. From this we have concluded that at least 60% of EMS-induced point mutations are small deletions. The high ratio of lethal to viable visible mutations was in agreement with this conclusion. These results are compared with data from the literature, most of which report very low deletion frequencies among EMS-induced mutations. The alternative possibility, namely that EMS tends to produce clusters of linked mutations, has also been considered and finds some support in literature. Whatever the cause of the high frequency of lethals associated with specific visible mutations, our data suggest that genetic hazards from EMS may be considerable; this is indeed true for its effect of the viability of heterozygotes.     

Mutation analysis of the dystrophin gene in Southern French DMD or BMD families: from Southern blot to protein truncation test

Taq I) were defined in a sample of normal, DMD, and BMD Xchromosomes from Southern France. The determination of the grandparental origin of either deletions or point mutations revealed differences depending on the type of the mutation, with most of the deletions occurring in oogenesis and most of the point mutations occurring in spermatogenesis. Received: 31 May 1997 / Accepted: 10 December 1997     

Illegitimate recombination induced by DNA double-strand breaks in a mammalian chromosome.

We examined DNA double-strand-break-induced mutations in the endogenous adenine phosphoribosyl-transferase (APRT) gene in cultured Chinese hamster ovary cells after exposure to restriction endonucleases. PvuII, EcoRV, and StuI, all of which produce blunt-end DNA double-strand breaks, were electroporated into CHO-AT3-2 cells hemizygous at the APRT locus. Colonies of viable cells containing mutations at APRT were expanded, and the mutations that occurred during break repair were analyzed at the DNA sequence level. Restriction enzyme-induced mutations consisted of small deletions of 1 to 36 bp, insertions, and combinations of insertions and deletions at the cleavage sites. Most of the small deletions involved overlaps of one to four complementary bases at the recombination junctions. Southern blot analysis revealed more complex mutations, suggesting translocation, inversion, or insertion of larger chromosomal fragments. These results indicate that blunt-end DNA double-strand breaks can induce illegitimate (nonhomologous) recombination in mammalian chromosomes and that they play an important role in mutagenesis.     

Involvement of RecB-mediated (but not RecF-mediated) repair of DNA double-strand breaks in the gamma-radiation production of long deletions in Escherichia coli.

Experiments were designed to determine the association between the repair of gamma-radiation-induced DNA double-strand breaks (DSB) and the induction of 700-1000 bp long deletions (Lac(-)----Lac+), base substitutions (leuB19----Leu+), and frameshifts (trpE9777----Trp+) in Escherichia coli K-12. Over the range of 2.5-20 krad, deletions were induced with linear kinetics, as has been shown for the induction of DSB, while the induction kinetics of base substitutions and frameshifts were curvilinear. Like the repair of DSB, deletion induction showed an absolute requirement for an intact recB gene as well as a dependency on the type of preirradiation growth medium; these requirements were not seen for base substitutions or frameshifts. In addition, about 80% of the spontaneous deletions were absent in the recB21 strain. A recC1001 mutation, which confers a 'hyper-Rec' phenotype, increased the rate of gamma-radiation-induced deletions as well as the low-dose production of base substitutions and frameshifts. A recF143 mutation increased the yield of gamma-radiation-induced deletions without increasing base substitutions or frameshifts. A mutS mutation markedly enhanced the gamma-radiation induction of frameshifts, and had a slight effect on base substitutions, but did not affect the induction of deletions. Resistance to gamma-irradiation and the capacity to repair DSB (albeit at about half the normal rate) were restored to the radiosensitive recB21 strain by the addition of the sbcB21 and sbcC201 mutations. However, the radioresistant recB sbcBC strain, which is recombination proficient via the RecF pathway, was still grossly deficient in the ability to produce deletions. A model for deletion induction as a by-product of the recB-dependent (Chi-dependent) repair of gamma-radiation-induced DSB is discussed, as is the inability to detect deletions in cells that use only the recF-dependent (Chi-independent) mechanism to repair DSB.     

DNA sequence analysis of spontaneous tonB deletion mutations in a polA1 strain of Escherichia coli K12.

By DNA sequence analysis, we have determined a spectrum of 61 spontaneous mutations occurring in the endogenous tonB gene in the polA1 strain of Escherichia coli. The overall mutation frequency was approximately 2.4-fold higher in the polA1 strain and this was attributable to enhanced rates of deletion and frameshift mutations. Among 39 deletions, a hot spot (17 mutations) was detected: a 13-bp deletion presumably directed by a 3-bp repeated sequence at its end points. The remaining 22 were distributed among 19 different mutations either flanked (16/19) or not flanked (3/19) by repeated sequences. Single-base frameshifts accounted for 8 mutations of either repeated (3/8) or nonrepeated (5/8) bases among which 6 were minus one frameshift. In contrast to previous reports, we did not frequently observe a 5'-GTGG-3' sequence in the vicinity of the deletions and frameshifts. The results presented here indicated an anti-deletion and anti-frameshift role for DNA polymerase I.     

A macaque's-eye view of human insertions and deletions: differences in mechanisms

Insertions and deletions (indels) cause numerous genetic diseases and lead to pronounced evolutionary differences among genomes. The macaque sequences provide an opportunity to gain insights into the mechanisms generating these mutations on a genome-wide scale by establishing the polarity of indels occurring in the human lineage since its divergence from the chimpanzee. Here we apply novel regression techniques and multiscale analyses to demonstrate an extensive regional indel rate variation stemming from local fluctuations in divergence, GC content, male and female recombination rates, proximity to telomeres, and other genomic factors. We find that both replication and, surprisingly, recombination are significantly associated with the occurrence of small indels. Intriguingly, the relative inputs of replication versus recombination differ between insertions and deletions, thus the two types of mutations are likely guided in part by distinct mechanisms. Namely, insertions are more strongly associated with factors linked to recombination, while deletions are mostly associated with replication-related features. Indel as a term misleadingly groups the two types of mutations together by their effect on a sequence alignment. However, here we establish that the correct identification of a small gap as an insertion or a deletion (by use of an outgroup) is crucial to determining its mechanism of origin. In addition to providing novel insights into insertion and deletion mutagenesis, these results will assist in gap penalty modeling and eventually lead to more reliable genomic alignments.     

Spectrum of small mutations in the dystrophin coding region.

Duchenne and Becker muscular dystrophies (DMD and BMD) are caused by defects in the dystrophin gene. About two-thirds of the affected patients have large deletions or duplications, which occur in the 5' and central portion of the gene. The nondeletion/duplication cases are most likely the result of smaller mutations that cannot be identified by current diagnostic screening strategies. We screened approximately 80% of the dystrophin coding sequence for small mutations in 158 patients without deletions or duplications and identified 29 mutations. The study indicates that many of the DMD and the majority of the BMD small mutations lie in noncoding regions of the gene. All of the mutations identified were unique to single patients, and most of the mutations resulted in protein truncation. We did not find a clustering of small mutations similar to the deletion distribution but found > 40% of the small mutations 3' of exon 55. The extent of protein truncation caused by the 3' mutations did not determine the phenotype, since even the exon 76 nonsense mutation resulted in the severe DMD phenotype. Our study confirms that the dystrophin gene is subject to a high rate of mutation in CpG sequences. As a consequence of not finding any hotspots or prevalent small mutations, we conclude that it is presently not possible to perform direct carrier and prenatal diagnostics for many families without deletions or duplications.     

Hereditary Angioedema Due to C1 Inhibitor Deficiency in Serbia: Two Novel Mutations and Evidence of Genotype-Phenotype Association

Hereditary angioedema due to C1 inhibitor deficiency (C1-INH-HAE) is a rare autosomal dominant disease characterized by recurrent life-threatening oedemas and/or abdominal pain and caused by mutations affecting the C1 inhibitor gene, SERPING1. We sought to investigate the spectrum of SERPING1 mutations in Serbia and the possible genotype-phenotype association. C1-INH-HAE was diagnosed on the basis of clinical and laboratory criteria in 40 patients from 27 families; four were asymptomatic. Mutational analysis of the SERPING1 gene was performed by sequencing and multiplex ligation-dependent probe amplification. Disease-causing mutations in SERPING1 were identified in all patients. In C1-INH-HAE type I, we identified 19 different mutations, including 6 missense mutations, 6 nonsense mutations, 2 small deletions, 1 small insertion, 2 splicing defects and 2 large deletions. Two of the mutations (c.300C>T and c.1184_1185insTA) are reported here for the first time. All C1-INH-HAE type II patients from three families harboured the same substitution (c.1396C>T). Based on the type of mutation identified in the SERPING1 gene, patients were divided into two groups: group 1 (nonsense, frameshift, large deletions/insertions, splicing defect, and mutations at Arg444) or group 2 (missense, excluding mutations at Arg444). Significant differences were found in the clinical severity score (P = 0.005), prevalence of laryngeal (P = 0.040) and facial (P = 0.013) oedema, and long-term prophylaxis (P = 0.023) between the groups with different types of mutations. Because our population consisted of related subjects, differences in the severity score between mutation groups were further confirmed using the generalized estimating equation (P = 0.038). Our study identified 20 different disease-causing mutations, including two novel mutations, in all C1-INH-HAE patients, highlighting the heterogeneity of mutations in the SERPING1 gene. Furthermore, it appears that mutations with a clear effect on C1-INH function might be responsible for a more severe disease phenotype.     

The molecular analysis ofbrown eye color mutations isolated from geographically discrete populations ofDrosophila melanogaster

A large proportion of spontaneous mutations inDrosophila melanogaster strains of laboratory origin are associated with insertions of mobile DNA elements. As a first step toward determining whether spontaneous laboratory mutations are predictive for mutational events occurring in the wild, recessivebrown (bw) eye color mutants were isolated. By inbreeding the progeny of wild-caughtDrosophila melanogaster females,bw mutations were isolated from seven separate geographic sites distributed among Japan, California, Siberia and Hungary. Among a total of 14 mutations studied, no case of transposon mutagenesis was found. At least 4 mutations are associated with small deletions in thebw gene. The remainder are inseparable from wild-typebw by Southern analysis and are presumed to be basepair changes or very small indels. Although only two spontaneousbw mutants of laboratory origin have been analyzed molecularly, one is a mobile element insertion.     

The molecular analysis ofbrown eye color mutations isolated from geographically discrete populations ofDrosophila melanogaster

A large proportion of spontaneous mutations inDrosophila melanogaster strains of laboratory origin are associated with insertions of mobile DNA elements. As a first step toward determining whether spontaneous laboratory mutations are predictive for mutational events occurring in the wild, recessivebrown (bw) eye color mutants were isolated. By inbreeding the progeny of wild-caughtDrosophila melanogaster females,bw mutations were isolated from seven separate geographic sites distributed among Japan, California, Siberia and Hungary. Among a total of 14 mutations studied, no case of transposon mutagenesis was found. At least 4 mutations are associated with small deletions in thebw gene. The remainder are inseparable from wild-typebw by Southern analysis and are presumed to be basepair changes or very small indels. Although only two spontaneousbw mutants of laboratory origin have been analyzed molecularly, one is a mobile element insertion.     

Genetic tests for the detection of chemically induced small deletions in Drosophila chromosomes

We have developed genetic tests for estimating the proportion of small deletions among chemically induced point mutations in Drosophila. The criteria used allow the detection of deletions that are large enough to include a viable visible mutation as well as a lethal, or a sex-linked lethal as well as a gene that is required for the development of a spermatogonium into a spermatozoon. On these criteria, we have concluded that DEB produces a high proportion of deletions among point mutations; that HA produces no deletions; and that DEN produces either no deletions or only very small ones that cannot be detected by our methods.     

A spectrum of ABCC6 mutations is responsible for pseudoxanthoma elasticum

To better understand the pathogenetics of pseudoxanthoma elasticum (PXE), we performed a mutational analysis of ATP-binding cassette subfamily C member 6 (ABCC6) in 122 unrelated patients with PXE, the largest cohort of patients yet studied. Thirty-six mutations were characterized, and, among these, 28 were novel variants (for a total of 43 PXE mutations known to date). Twenty-one alleles were missense variants, six were small insertions or deletions, five were nonsense, two were alleles likely to result in aberrant mRNA splicing, and two were large deletions involving ABCC6. Although most mutations appeared to be unique variants, two disease-causing alleles occurred frequently in apparently unrelated individuals. R1141X was found in our patient cohort at a frequency of 18.8% and was preponderant in European patients. ABCC6del23-29 occurred at a frequency of 12.9% and was prevalent in patients from the United States. These results suggested that R1141X and ABCC6del23-29 might have been derived regionally from founder alleles. Putative disease-causing mutations were identified in approximately 64% of the 244 chromosomes studied, and 85.2% of the 122 patients were found to have at least one disease-causing allele. Our results suggest that a fraction of the undetected mutant alleles could be either genomic rearrangements or mutations occurring in noncoding regions of the ABCC6 gene. The distribution pattern of ABCC6 mutations revealed a cluster of disease-causing variants within exons encoding a large C-terminal cytoplasmic loop and in the C-terminal nucleotide-binding domain (NBD2). We discuss the potential structural and functional significance of this mutation pattern within the context of the complex relationship between the PXE phenotype and the function of ABCC6.     

Implication of annexin 1 in phagocytosis: effects of n-terminal domain deletions and point mutations of the phosphorylation site Ser-27

Directed mutagenesis, in the form of deletions and point mutations, was used to investigate the regulatory importance of the N-terminal domain of annexin 1. Wild-type and mutant forms were fused to green fluorescent protein (GFP) to track their localization and introduced in to J-774A.1 cells by transfection. The fusion of annexin 1 to GFP at the N- or C-terminal end did not alter the cellular distribution or co-localization with phagosomes. The effects of mutations were determined according to these characteristics. The prominent effect resulted from S27E mutation which mimics the phosphorylated state of Ser-27. Although still retaining the granular structures in the cytoplasm, S27E annexin 1 failed to associate with the phagosomal protein complex. This suggests an essential regulatory role of the phosphorylation of residue 27 in annexin 1 function.     

Dose-dependent changes in the spectrum of mutations induced by ionizing radiation

We examined the influence of dose on the spectrum of mutations induced at the hypoxanthine guanine phosphoribosyltransferase (Hprt) locus in Chinese hamster ovary (CHO) cells. Independent CHO-K1 cell mutants at the Hprt locus were isolated from cells exposed to 0, 0.5, 1.5, 3.0 and 6.0 Gy (137)Cs gamma rays, and the genetic changes responsible for the mutations were determined by multiplex polymerase chain reaction (PCR)-based exon deletion analysis. We observed dose-dependent changes in mutation spectra. At low doses, the principal radiation-induced mutations were point mutations. With increasing dose, multibase deletion mutations became the predominant mutation type such that by 6.0 Gy, there were almost three times more deletion mutations than point mutations. The dose response for induction of point mutations was linear while that for multibase deletions fit a linear-quadratic response. There was a biphasic distribution of deletion sizes, and different dose responses for small compared to large deletions. The frequency of large (>36 kb) total gene deletions increased exponentially, implying that they develop from the interaction between two independent events. In contrast, the dose response for deletion mutations of less than 10 kb was nearly linear, suggesting that these types of mutations develop mostly from single events and not the interactions between two independently produced lesions. The observation of dose-dependent changes in radiation-induced mutation spectra suggests that the types of alterations and therefore the risks from low-dose radiation exposure cannot be easily extrapolated from high-dose effects.     

DHPLC analysis of adenomatous polyposis coli (APC) mutations using ready-to-use APC plates: simple detection of multiple base pair deletion mutations

The adenomatous polyposis coli (APC), which is the susceptible gene for familial adenomatous polyposis (FAP) and sporadic colorectal cancer, spans 15 exons. The open reading frame of APC is 8529 bp, which encodes 2843 amino acids. Conventional genetic screening involves extensive time as well as high cost and labor. Thus, we developed a novel APC ready-to-use plate for high-throughput mutational analysis by denaturing high performance liquid chromatography (DHPLC). To prepare the ready-to-use APC plate, all 38 primer pairs and PCR mixtures were aliquoted into individual wells of a 96-well plate, and frozen at -20 degrees C until use. All 38 PCR primers were designed to be amplified at the same temperature (52 degrees C). We examined a total of 27 FAP patient samples with APC germline mutations (17 for multiple bp deletions, 1 for 1 bp deletion, 9 for nonsense mutations) and 50 APC-negative noncarriers. All 17 multiple bp deletion mutations were detected during the initial 50 degrees C running analysis and thus ruled out for further analyses. All other mutations were clearly detected under specific optimized conditions. More than 50% of the APC germline mutations were multiple base pair deletions and efficiently selected by omitting time-consuming partial denaturing conditions.     

Spectrum and frequency of jagged1 (JAG1) mutations in Alagille syndrome patients and their families.

Alagille syndrome (AGS) is a dominantly inherited disorder characterized by liver disease in combination with heart, skeletal, ocular, facial, renal, and pancreatic abnormalities. We have recently demonstrated that Jagged1 (JAG1) is the AGS gene. JAG1 encodes a ligand in the Notch intercellular signaling pathway. AGS is the first developmental disorder to be associated with this pathway and the first human disorder caused by a Notch ligand. We have screened 54 AGS probands and family members to determine the frequency of mutations in JAG1. Three patients (6%) had deletions of the entire gene. Of the remaining 51 patients, 35 (69%) had mutations within JAG1, identified by SSCP analysis. Of the 35 identified intragenic mutations, all were unique, with the exceptions of a 5-bp deletion in exon 16, seen in two unrelated patients, and a C insertion at base 1618 in exon 9, also seen in two unrelated patients. The 35 intragenic mutations included 9 nonsense mutations (26%); 2 missense mutations (6%); 11 small deletions (31%), 8 small insertions (23%), and 1 complex rearrangement (3%), all leading to frameshifts; and 4 splice-site mutations (11%). The mutations are spread across the coding sequence of the gene within the evolutionarily conserved motifs of the JAG1 protein. There is no phenotypic difference between patients with deletions of the entire JAG1 gene and those with intragenic mutations, which suggests that one mechanism involved in AGS is haploinsufficiency. The two missense mutations occur at the same amino acid residue. The mechanism by which these missense mutations lead to the disease is not yet understood; however, they suggest that mechanisms other than haploinsufficiency may result in the AGS phenotype.