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PubMed1999

Action of repeat-induced point mutation on both strands of a duplex and on tandem duplications of various sizes in Neurospora.

In Neurospora crassa, DNA sequence duplications are detected and altered efficiently during the sexual cycle by a process known as RIP (repeat-induced point mutation). Affected sequences are subjected to multiple GC-to-AT mutations. To explore the pattern in which base changes are laid down by RIP we examined two sets of strains. First, we examined the products of a presumptive spontaneous RIP event at the mtr locus. Results of sequencing suggested that a single RIP event produces two distinct patterns of change, descended from the two strands of an affected DNA duplex. Equivalent results were obtained using an exceptional tetrad from a cross with a known duplication flanking the zeta-eta (zeta-eta) locus. The mtr sequence data were also used to further examine the basis for the differential severity of C-to-T mutations on the coding and noncoding strands in genes. The known bias of RIP toward CpA/TpG sites in conjunction with the sequence bias of Neurospora accounts for the differential effect. Finally, we used a collection of tandem repeats (from 16 to 935 bp in length) within the mtr gene to examine the length requirement for RIP. No evidence of RIP was found with duplications shorter than 400 bp while all longer tandem duplications were frequently affected. A comparison of these results with vegetative reversion data for the same duplications is consistent with the idea that reversion of long tandem duplications and RIP share a common step.

Base Pairing↗
PubMed2003

Effects of point mutations in the major capsid protein of beet western yellows virus on capsid formation, virus accumulation, and aphid transmission.

Point mutations were introduced into the major capsid protein (P3) of cloned infectious cDNA of the polerovirus beet western yellows virus (BWYV) by manipulation of cloned infectious cDNA. Seven mutations targeted sites on the S domain predicted to lie on the capsid surface. An eighth mutation eliminated two arginine residues in the R domain, which is thought to extend into the capsid interior. The effects of the mutations on virus capsid formation, virus accumulation in protoplasts and plants, and aphid transmission were tested. All of the mutants replicated in protoplasts. The S-domain mutant W166R failed to protect viral RNA from RNase attack, suggesting that this particular mutation interfered with stable capsid formation. The R-domain mutant R7A/R8A protected approximately 90% of the viral RNA strand from RNase, suggesting that lower positive-charge density in the mutant capsid interior interfered with stable packaging of the complete strand into virions. Neither of these mutants systemically infected plants. The six remaining mutants properly packaged viral RNA and could invade Nicotiana clevelandii systemically following agroinfection. Mutant Q121E/N122D was poorly transmitted by aphids, implicating one or both targeted residues in virus-vector interactions. Successful transmission of mutant D172N was accompanied either by reversion to the wild type or by appearance of a second-site mutation, N137D. This finding indicates that D172 is also important for transmission but that the D172N transmission defect can be compensated for by a "reverse" substitution at another site. The results have been used to evaluate possible structural models for the BWYV capsid.

Amino Acid Sequence↗
PubMed1995

Skipping of exon 12 as a consequence of a point mutation (1898 + 5G-->T) in the cystic fibrosis transmembrane conductance regulator gene found in a consanguineous Chinese family.

A point mutation (1898 + 5G-->T) located five base pairs downstream from the donor splice site in intron 12 of the CFTR gene has been identified in a consanguineous CF patient of Chinese origin. To determine if this nucleotide substitution could affect mRNA splicing, PCR analysis was performed with RNA isolated from the lymphoblastoid cell line of the mother of the deceased patient. While exon 12-minus transcript was detected in this sample, it was also found in individuals without 1898 + 5G-->T, albeit in a smaller proportion. Using a sequence polymorphism associated with each of the two alleles in the mother, however, we showed that mutant transcript was almost exclusively produced by the 1898 + 5G-->T allele. Skipping of exon 12 would result in the deletion of 29 amino acids from the first nucleotide binding domain of CFTR, rendering the protein non-functional. The possibility of a low level (< or = 2.5%) of normal transcript from the mutant allele cannot be excluded and it may explain the pancreatic sufficient phenotype of the patient. The 1898 + 5G-->T mutation was found in two other CF patients of Chinese origin, but it was not detected in 192 CF chromosomes of Caucasian origin and 30 other chromosomes from Chinese individuals without a family history of CF.

Base Sequence↗
PubMed1994

Prenatal diagnosis and carrier detection in mucopolysaccharidosis type II by mutation analysis. A 47,XXY male heterozygous for a missense point mutation.

Identification of iduronate-2-sulphatase (IDS) gene mutations in patients with mucopolysaccharidosis type II (MPS II, Hunter syndrome) allows fast and reliable carrier detection and prenatal diagnosis. We describe here three cases of prenatal diagnosis by direct detection of the gene mutation. In addition to two affected male fetuses from two different families, a 47,XXY fetus carrying both the normal and the mutant allele was diagnosed in a third family. The latter pregnancy was carried to term and the child is obviously not affected by MPS II.

Alleles↗
PubMed1991

Primary hyperoxaluria type I due to a point mutation of T to C in the coding region of the serine:pyruvate aminotransferase gene.

cDNA clones for serine:pyruvate aminotransferase (SPT, alternative name: alanine:glyoxylate aminotransferase) were obtained from a cDNA library constructed from the liver of a primary hyperoxaluria type I (PH1) case in which the SPT activity was approximately one-hundredth that in control liver. Six clones were isolated from 100,000 transformants and all of them contained an approximately 1.5 kbp insert which included the whole coding region for human SPT. Nucleotide sequence analysis revealed a point mutation of T to C at position 634 (relative to the 5'-end of the cDNA) encoding a Ser to Pro substitution at residue 205. The T to C conversion created a new SmaI site, which enabled us to demonstrate that the point mutation had occurred in the patient's SPT gene. SmaI digestion of genomic DNA may be useful for the diagnostic gene analysis of this type of PH1.

Adult↗
PubMedDate not supplied

Plasmodium falciparum: in vitro activity of sulfadoxine and dapsone in field isolates from Kenya: point mutations in dihydropteroate synthase may not be the only determinants in sulfa resistance.

We have determined the relationship between point mutations in the gene that encodes the sulfa target, dihydropteroate synthase (DHPS) and the chemosensitivity profile to sulfadoxine and dapsone in 67 isolates from Kilifi, Kenya. We assessed the presence of mutations at codons 436, 437, 540, 581, and 613 of dhps. The results showed that the dhps genotype had a strong influence on the sensitivity to sulfadoxine and dapsone, but that the correlation was far from perfect. Eleven isolates carried a wild-type dhps allele, but were resistant to sulfadoxine (IC(50) values >10 microg/ml), and 4/28 isolates were classed as sensitive to sulfadoxine (IC(50) values <10 microg/ml), but carried a triple mutant (436/437/613) allele of dhps. These data show that in low folate medium in vitro, the dhps genotype alone did not account completely for sulfadoxine or dapsone resistance; other factors such as the utilisation of exogenous folate must also be considered.

Alleles↗
PubMed2005

Collateral damage: spread of repeat-induced point mutation from a duplicated DNA sequence into an adjoining single-copy gene in Neurospora crassa.

Repeat-induced point mutation (RIP) is an unusual genome defense mechanism that was discovered in Neurospora crassa. RIP occurs during a sexual cross and induces numerous G : C to A : T mutations in duplicated DNA sequences and also methylates many of the remaining cytosine residues. We measured the susceptibility of the erg-3 gene, present in single copy, to the spread of RIP from duplications of adjoining sequences. Genomic segments of defined length (1, 1.5 or 2 kb) and located at defined distances (0, 0.5, 1 or 2 kb) upstream or downstream of the erg-3 open reading frame (ORF) were amplified by polymerase chain reaction (PCR), and the duplications were created by transformation of the amplified DNA. Crosses were made with the duplication strains and the frequency of erg-3 mutant progeny provided a measure of the spread of RIP from the duplicated segments into the erg-3 gene. Our results suggest that ordinarily RIP-spread does not occur. However, occasionally the mechanism that confines RIP to the duplicated segment seems to fail (frequency 0.1-0.8%) and then RIP can spread across as much as 1 kb of unduplicated DNA. Additionally, the bacterial hph gene appeared to be very susceptible to the spread of RIP-associated cytosine methylation.

Base Sequence↗
PubMed1991

Three distinct point mutations in the factor IX gene of three Japanese CRM+ hemophilia B patients (factor IX BMNagoya 2, factor IX Nagoya 3 and 4).

Enzymatic DNA amplification and complete sequence analysis were used to investigate human factor IX coding sequences in three CRM+ hemophilia B patients. In a patient with severe hemophilia B and a markedly prolonged ox-brain prothrombin time, a C to T transition in exon VI changed the codon for Arg180 to Trp (factor IX BMNagoya 2). This mutation would impair the cleavage by factor XIa required for activation of the zymogen. In a patient with mild hemophilia B, a G to A transition in exon VI changed the codon for Arg145 to His(factor IX Nagoya 3). This substitution also would be predicted to preclude the cleavage of factor IX by factor XIa at this peptide bond (Arg145-Ala146). Furthermore, this point mutation creates a new NlaIII restriction site which provides a quick and reliable method for carrier detection in the affected family members. A patient with severe hemophilia B (factor IX Nagoya 4) had a G to A transition in exon II changing the codon for Glu21 to Lys. This novel point mutation is assumed to impair the function of factor IX by disrupting the calcium binding of factor IX.

Amino Acid Sequence↗
PubMed1988

A point mutation in the tobacco mosaic virus capsid protein gene induces hypersensitivity in Nicotiana sylvestris.

In Nicotiana sylvestris, the N' gene confers hypersensitive resistance to some strains of tobacco mosaic virus (TMV) but not to the common strain. TMV sequences responsible for inducing local lesion formation in this host were identified by using cDNA clones to construct genomic recombinants between the common strain genome and a local-lesion-inducing mutant. To assay for sequences conferring the mutant phenotype, in vitro transcripts of recombinants were inoculated onto leaves of N. sylvestris and observed for the formation of either local lesions or a systemic infection. Sequences from the mutant that converted the hybrid genome to the mutant phenotype were located between nucleotides 5972 and 6206. Sequence analysis of this region revealed point mutations in the mutant at nucleotides 6157 (cytosine to uracil) and 6199 (adenine to guanine). The mutation at 6157 changes the capsid protein gene to specify phenylalanine rather than serine at position 148; nucleotide 6199 occurs in the 3' nontranslated region. When each point mutation was individually substituted into the wild-type background, transcripts containing only the alteration at 6157 produced local lesions on N. sylvestris, whereas transcripts containing only the alteration at 6199 produced systemic mosaic symptoms. The frequency of mutation was examined by partially sequencing virion RNA from six additional independent local-lesion mutants. Five mutants had the same alteration at 6157 as the original mutant and none had the alteration at 6199. This work demonstrates that the capsid protein gene of TMV is multifunctional, both encoding the virion structural protein and mediating the outcome of infection in N. sylvestris.

Journal Article↗
PubMed2006

On the effect of a point mutation on the reactivity of CuZn superoxide dismutase: a theoretical study.

In this paper, we investigate the effects of a point mutation on the enzymatic activity of copper-zinc superoxide dismutase, which we recently studied in detail by means of a theoretical-computational procedure. Comparison of the reactivity of the initial catalytic steps in this mutant (G93A mutation far from the active site) with our previous data, reveals the beautiful mechanical-dynamical architecture of the enzyme, altered by such an apparently irrelevant mutation. Finally, our results suggest a possible atomic-molecular-based explanation for the mutant-pathology correlation, in line with the most recent experimental data.

Electromagnetic Fields↗
PubMed1997

Detection of p53 point mutations by double-gradient, denaturing gradient gel electrophoresis.

Genetic instability is a typical feature of tumor cells. This evidence has stimulated the development of rapid methods for detection of gene mutations. A new, improved protocol for denaturing gradient gel electrophoresis (DGGE), to screen for point mutations in genomic DNA, is reported: double gradient (DG) DGGE. In this technique, to the primary, denaturing gradient (typically 30-80% or 40-80% urea/formamide) a secondary gradient, colinear with the first, is superimposed: a porosity gradient (typically 6.5-12% polyacrylamide). The secondary gradient acts by recompacting smeared and diffuse bands of heteroduplexes, which are often indistinguishable from background fluorescence, and by augmenting the resolution between closely spaced homoduplex zones. This allows proper densitometric quantitation of the ratio of the two homoduplex bands. The reliability of this technique has been documented by detection of a number of mutations in exons 6 and 8 of the p53 gene which had escaped revelation by single-strand conformational polymorphism (SSCP) analysis. Additionally, the precise assessment of ratio of the doublet of homoduplex bands has allowed quantitation of the extent of p53 mutation in a mixed cell population extracted from a tumor specimen.

Electrophoresis, Polyacrylamide Gel↗
PubMed1992

Detection of point mutations in human DNA by analysis of RNA conformation polymorphism(s).

RNA molecules were found to separate into numerous metastable conformational forms upon non-denaturing gel electrophoresis. The equilibration of the conformations was accelerated by heating or mild denaturing conditions. Single-base substitutions in the sequence of the RNAs caused changes in the conformational patterns, including mobility shifts of major and minor conformations, appearance of new conformations and loss of other conformations. This sequence-dependent RNA conformational polymorphism was used to detect point mutations in p53 and, dihydrofolate reductase genes. Sense and anti-sense RNA strands corresponding to the same segment of the p53 gene gave entirely different conformational patterns. To generate the RNA, short regions of the target genes (up to about 250 bp) were amplified by the polymerase chain reaction and the resulting DNA segments transcribed to RNA by T7 RNA polymerase. The method is rapid, simple, amenable to non-radioactive visualization and was successful in several cases when DNA single-strand conformational polymorphism analysis (Orita et al. (1989) Genomics 5, 874-879) failed to detect the point mutation.

Base Sequence↗
PubMed1994

Point mutations in mitochondrial tRNA genes: sequence analysis of chronic progressive external ophthalmoplegia (CPEO)

We have sequenced all mitochondrial tRNA genes from 9 Japanese patients with chronic progressive external ophthalmoplegia (CPEO) who had no detectable large mtDNA deletions nor mutations previously reported, and identified 6 different base substitutions in 6 patients. Since 5 of the 6 substitutions were homoplasmic in distribution and recognizable in some normal controls, they were thought to be polymorphisms in normal individuals. One mutation at nucleotide (nt) 12311 in the tRNA(Leu(CUN)) gene was not present in 90 normal controls nor in 103 patients with other mitochondrial myopathies. This mutation was in a heteroplasmic state, and the mutated site was conserved among other species during evolution, suggesting a disease-related mutation. However, the significance of this mutation has to be studied further. In Japanese CPEO patients without large deletions, a point mutation in the mitochondrial tRNA gene is not likely to be a frequent cause.

Adult↗
PubMed2001

Seven novel and four recurrent point mutations in the factor VIII (F8C) gene.

Haemophilia A is a X-linked bleeding disorder, caused by deficiency in the activity of coagulation factor VIII due to mutations in the corresponding gene. The most common defect in patients is an inversion of the factor VIII gene that accounts for nearly 45% of individuals with severe hemophilia A. Point mutations and small deletions/insertions are responsible for the majority of cases with moderate to mild clinical course and for half of the severe hemophilia A occurrences. The majority of these mutations are "private", because of the high mutation rate for this particular gene. We report on eleven pathological changes in the factor VIII sequence detected in male patients with haemophilia A or in female obligate carriers. Seven of these mutations are novel [E204N, E265X, M320T, F436C, S535C, N2129M and R2307P] and four have been previously identified [V162M, R527W, R1966X, and R2159C]. Genotype-phenotype correlations and computer prediction analysis on the effect of missense mutations on the secondary structure of the factor VIII protein are performed and the relationships evaluated.

DNA↗
PubMed2002

Seven novel and four recurrent point mutations in the factor VIII (F8C) gene.

Haemophilia A is a X-linked bleeding disorder, caused by deficiency in the activity of coagulation factor VIII due to mutations in the corresponding gene. The most common defect in patients is an inversion of the factor VIII gene that accounts for nearly 45% of individuals with severe hemophilia A. Point mutations and small deletions/insertions are responsible for the majority of cases with moderate to mild clinical course and for half of the severe hemophilia A occurrences. The majority of these mutations are "private", because of the high mutation rate for this particular gene. We report on eleven pathological changes in the factor VIII sequence detected in male patients with haemophilia A or in female obligate carriers. Seven of these mutations are novel [E204N, E265X, M320T, F436C, S535C, N2129M and R2307P] and four have been previously identified [V162M, R527W, R1966X, and R2159C]. Genotype-phenotype correlations and computer prediction analysis on the effect of missense mutations on the secondary structure of the factor VIII protein are performed and the relationships evaluated.

Adult↗
PubMed1992

Point mutation of an FGF receptor abolishes phosphatidylinositol turnover and Ca2+ flux but not mitogenesis.

Stimulation of certain receptor tyrosine kinases results in the tyrosine phosphorylation and activation of phospholipase C gamma (PLC gamma), an enzyme that catalyses the hydrolysis of phosphatidylinositol (PtdIns). This hydrolysis generates diacylglycerol and free inositol phosphate, which in turn activate protein kinase C and increase intracellular Ca2+, respectively. PLC gamma physically associates with activated receptor tyrosine kinases, suggesting that it is a substrate for direct phosphorylation by these kinases. Here we report that a fibroblast growth factor (FGF) receptor with a single point mutation at residue 766 replacing tyrosine with phenylalanine fails to associate with PLC gamma in response to FGF. This mutant receptor also failed to mediate PtdIns hydrolysis and Ca2+ mobilization after FGF stimulation. However, the mutant receptor phosphorylated itself and several other cellular proteins, and it mediated mitogenesis in response to FGF. These findings show that a point mutation in the FGF receptor selectively eliminates activation of PLC gamma and that neither Ca2+ mobilization nor PtdIns hydrolysis are required for FGF-induced mitogenesis.

Amino Acid Sequence↗
PubMed1994

Episodic ataxia/myokymia syndrome is associated with point mutations in the human potassium channel gene, KCNA1.

Episodic ataxia (EA) is a rare, familial disorder producing attacks of generalized ataxia, with normal or near-normal neurological function between attacks. One type of EA is characterized by brief episodes of ataxia with myokymia (rippling of muscles) evident between attacks. Linkage studies in four such families suggested localization of an EA/myokymia gene near the voltage gated K+ channel gene, KCNA1 (Kv1.1), on chromosome 12p. Mutation analysis of the KCNA1 coding region in these families identified four different missense point mutations present in the heterozygous state, indicating that EA/myokymia can result from mutations in this gene.

Amino Acid Sequence↗
PubMed1991

Specific diagnosis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in dried blood spots by a polymerase chain reaction (PCR) assay detecting a point-mutation (G985) in the MCAD gene.

The discovery of a point-mutation, adenine-to-guanine, at position 985 in the gene coding for MCAD (G985), gave the basis for an easy and specific polymerase chain reaction test. We tested the specificity of such a PCR based assay and detected correctly G985 and A985 in sequence verified cDNA clones. We showed that the G985 mutation is present in genomic DNA from 48 of 50 patients with confirmed MCAD deficiency, originating from various European countries, Australia and the USA. On the basis of this high frequency of the G985 mutation among patients, we improved and optimized the assay with respect to reliability and convenience for routine diagnostic and screening purposes. As little as 2 microliters blood from filter-paper blood-spots (Guthrie spots) is sufficient for the test.

Acyl-CoA Dehydrogenase↗