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At least 37 records · Page 2Linked to original sources
PubMed1989

Mutational analysis of the mitochondrial Rieske iron-sulfur protein of Saccharomyces cerevisiae. II. Biochemical characterization of temperature-sensitive RIP1- mutations.

Although the function of the Rieske iron-sulfur protein is generally understood, little is known of how the structure of this protein supports its mechanistic role in electron transfer in the cytochrome bc1 complex. To better understand the structural basis of iron-sulfur protein function, we have undertaken a mutational analysis of the gene encoding this protein and initially isolated five temperature-sensitive iron-sulfur protein mutants (Beckmann, J. D., Ljungdahl, P. O., and Trumpower, B. L. (1989) J. Biol. Chem. 264, 3713-3722). Each of the five ts-rip1- mutants exhibited pleiotropic effects. Although the mutant iron-sulfur proteins manifest several in vitro phenotypes in common, each exhibited unique characteristics. All of the ts-rip1- mutations resulted in membranes with decreased ubiquinol-cytochrome c oxidoreductase activities and decreased thermostability compared to membranes containing wild type iron-sulfur protein. All of the mutations conferred slight but significant resistance to the respiratory inhibitor myxothiazol, and one mutant was hypersensitive to inhibition by UHDBT, a structural analog of ubiquinone. In addition, one of the mutations completely blocks post-translational processing of the iron-sulfur protein, leading to accumulation of pre-iron-sulfur protein in mitochondrial membranes at nonpermissive temperatures. Finally, a mutation 12-amino acid residues away from the carboxyl terminus (203S) results in an extremely unstable protein. This region of the protein may be essential in blocking degradation of pre-iron-sulfur protein by cytoplasmic proteases as the protein is imported into the mitochondria, or may be a "degradation signal," which tags the iron-sulfur protein for turnover.

Antimycin A↗
PubMed1992

Mutation analysis of the cystic fibrosis transmembrane regulator gene in Native American populations of the southwest.

We report DNA and clinical analyses of cystic fibrosis (CF) in two previously unstudied, genetically isolated populations: Pueblo and Navajo Native Americans. Direct mutation analysis of six mutations of the CFTR gene--namely, delta F508, G542X, G551D, R553X, N1303K, and W1282X--was performed on PCR-amplified genomic DNA extracted from blood samples. Haplotype analyses with marker/enzyme pairs XV2c/TaqI and KM19/PstI were performed as well. Of the 12 affected individuals studied, no delta F508 mutation was detected; only one G542X mutation was found. None of the other mutations was detected. All affected individuals have either an AA, AC, or CC haplotype, except for the one carrying the G542X mutation, who has the haplotype AB. Clinically, six of the affected individuals examined exhibit growth deficiency, and five (all from the Zuni Pueblo) have a severe CF phenotype. Four of the six Zunis with CF are also microcephalic, a finding not previously noted in CF patients. Our DNA data have serious implications for risk assessment of CF carrier status for these people.

Adolescent↗
PubMed1992

Mutational analysis of SRY: nonsense and missense mutations in XY sex reversal.

XY females (n = 17) were analysed for mutations in SRY (sex-determining region Y gene), a gene that has recently been equated with the testis determining factor (TDF). SRY sequences were amplified by the polymerase chain reaction (PCR) and analysed by both the single strand conformational polymorphism assay (SSCP) and DNA sequencing. The DNA from two individuals gave altered SSCP patterns; only these two individuals showed any DNA sequence variation. In both cases, a single base change was found, one altering a tryptophan codon to a stop codon, the other causing a glycine to arginine amino acid substitution. These substitutions lie in the high mobility group (HMG)-related box of the SRY protein, a potential DNA-binding domain. The corresponding regions of DNA from the father of one individual and the paternal uncle of the other, were sequenced and found to be normal. Thus, in both cases, sex reversal is associated with de novo mutations in SRY. Combining this data with two previously published reports, a total of 40 XY females have now been analysed for mutations in SRY. The number of de novo mutations in SRY is now doubled to four, adding further strength to the argument that SRY is TDF.

Base Sequence↗
PubMed1995

Mutation analysis of Western Australian families affected by cystic fibrosis.

OBJECTIVE: To document the results of mutation analysis on 160 individuals with cystic fibrosis and 31 obligate carriers of the cystic fibrosis gene in 191 Western Australian families to facilitate accurate genetic counselling. METHODS: We tested for 17 mutations of the cystic fibrosis gene by either a variation of the polymerase chain reaction amplification refractory mutation system (PCR-ARMS) or with a series of restriction enzyme cuts and dot blots using chemiluminescent probes. RESULTS: At least one of the two intragenic mutations causing cystic fibrosis was identified in 98% of affected individuals and both were detected in 68%. The delta F508 deletion occurred in 89.8% of patients: 51% were homozygous for this defect. In carriers, 85% of the mutations were detected with a panel of 16 probes, identifying 17 intragenic defects: the delta F508 deletion occurred in 72.4%. Both cystic fibrosis mutations were detected in 68% of cystic fibrosis families. CONCLUSIONS: By analysis with 16 intragenic cystic fibrosis genomic probes, we have documented the frequencies of various mutations in the Western Australian population. These data will be useful in accurate genetic counselling for affected families and carrier screening for the general population.

Adult↗
PubMed1991

Transposon Tn5supF-based reverse genetic method for mutational analysis of Escherichia coli with DNAs cloned in lambda phage.

An efficient method for systematic mutational analysis of the Escherichia coli genome was developed. It entails Tn5supF transposition to lambda-E. coli hybrid phage clones (Kohara library) and then transduction of recipient cells to Sup+. Essential and nonessential genes are distinguished by the ability of insertion mutant phage to form haploid versus only heterozygous partial diploid bacterial recombinants.

Bacteriophage lambda↗
PubMed1994

Mutation analysis in families with discordant phenotypes of phenylalanine hydroxylase deficiency. Inheritance and expression of the hyperphenylalaninaemias.

Neonatal hyperphenylalaninaemia caused by mutations in the gene encoding phenylalanine hydroxylase (PAH) represents a wide spectrum of metabolic phenotypes, ranging from classical phenylketonuria (PKU) to mild hyperphenylalaninaemia (MHP). The marked interindividual heterogeneity is due to the expression of multiple PAH mutations in genetic compounds. We have investigated four unusual families in which both PKU and MHP were present. In each family three different mutations in the PAH gene were identified, including two associated with PKU and one associated with MHP. The unexpected outcome of discordant phenotypes within the families described is explained by previously unrecognized parental MHP. By mutation analysis we have also predicted the phenotypical outcome in a hyperphenylalaninaemic infant born to a mother who before pregnancy had been diagnosed as having MHP. Our results demonstrate the utility of nucleic acid analysis in follow-up in PKU screening programmes.

Amino Acid Metabolism, Inborn Errors↗
PubMed1992

[Prenatal diagnosis of cystic fibrosis by mutational analysis].

The authors give a review about the latest method of the prenatal diagnosis of cystic fibrosis. Examples were chosen from their own cases to illustrate the possibilities of the prenatal diagnosis based on the mutation analysis of the CFTR gene. Using both mutation and haplotype analysis, 10 prenatal diagnosis were performed from chorionic villus samples taken in the early stage of the pregnancy (10-12 weeks). There were 5 healthy and 5 affected fetuses found. The advantage of this method, that in certain cases, diagnosis is available for families having no live affected child.

Child↗
PubMed1991

Mutational analysis of the Drosophila miniature-dusky (m-dy) locus: effects on cell size and circadian rhythms.

A mutational analysis has been performed to explore the function of the Drosophila melanogaster miniature-dusky (m-dy) locus. Mutations at this locus affect wing development, fertility and behavior. The genetic characterization of 13 different mutations suggests that m and dy variants are alleles of a single complex gene. All of these mutations alter wing size, apparently by reducing the volume of individual epidermal cells of the developing wing. In m mutants, epidermal cell boundaries persist in the mature wing, whereas they normally degenerate 1-2 hr after eclosion in wild-type or dy flies. This has permitted the direct visualization of cell size differences among several m mutants. Mutations at the m-dy locus also affect behavioral processes. Three out of nine dy alleles (dyn1, dyn3 and dyn4) lengthen the circadian period of the activity and eclosion rhythms by approximately 1.5 hr. In contrast, m mutants have normal circadian periods, but an abnormally large percentage of individuals express aperiodic bouts of activity. These behavior genetic studies also indicate that an existing "rhythm" mutation known as Andante is an allele of the m-dy locus. The differential effects of certain m-dy mutations on wing and behavioral phenotypes suggest that separable domains of function exist within this locus.

Alleles↗
PubMed1993

Mutational analysis using denaturing gradient gel electrophoresis and PCR.

Denaturing gradient gel electrophoresis (DGGE) separates (DNA) molecules based on their sequence. Using the proper conditions, all base-pair substitutions can be resolved from the wild-type sequence using DGGE. Polymerase chain reaction (PCR) permits rapid amplification of a given region of the genome. In this paper, we demonstrate the utility of DGGE combined with PCR for mutation analysis by presenting different examples: (i) analysis of mouse p53 cDNA for mutations, (ii) simultaneous analysis of thousands of 4NQO-induced mutants for mutations in HPRT exon 3, (iii) examination of the fidelity of the thermostable DNA polymerase isolated from Pyrococcus furiosus (Pfu), (iv) purification of mutant DNA from contaminating wild-type DNA from mouse spleenic T-cell clones.

4-Nitroquinoline-1-oxide↗
PubMed1994

Direct mutation analysis of 495 patients for fragile X carrier status/proband diagnosis.

With the cloning of the FMR-1 gene, direct mutation analysis is possible for fragile X syndrome. We have analyzed 495 patients using the StB12.3 probe/EcoRI/EagI system of Rousseau et al. [N Engl J Med 325:1673-1681, 1991] and 167 of these also with PCR analysis according to Brown et al. [JAMA 270:1569-1575, 1993]. For 28 patients requesting carrier status due to a family history of fragile X, 10 were shown to have either premutations or full mutations; for the remainder with varied backgrounds, 1 in 182 was shown to carry a premutation. For proband diagnosis, 7 of 14 with a fragile X family history carried a full mutation; 11 of 271 with other family histories carried the full mutation.

Blotting, Southern↗
PubMed1995

Genetic counselling and gene mutation analysis in familial adenomatous polyposis in Western Australia.

OBJECTIVE: To assess the provision of accurate pre-symptomatic genetic testing with DNA analysis and appropriate counselling for individuals and families known to be at high risk of developing familial adenomatous polyposis coli (FAP). PATIENTS AND METHODS: Thirty-one families with clinically and pathologically documented FAP were ascertained from the Western Australian Polyposis Registry. DNA was collected from over 200 individuals in these families to establish their genetic risk status for FAP, either by direct mutation analysis, or by linkage analysis. Individuals undergoing DNA testing were given intensive psychosocial support and counselling. RESULTS: In 19 families DNA-based counselling could not be offered because either the adenomatous polyposis coli (APC) gene mutation could not be detected or there were insufficient family members for linkage analysis. Gene testing yielded mutations of the APC gene in 87 individuals from 12 families; by gene tracking (or linkage analysis) in three families and by mutation analysis in the remaining nine (four of which had only one affected individual). DNA results conformed with a definite clinicopathological diagnosis in 27 FAP patients and, of the remaining 60 high-risk subjects tested, 14 had inherited the mutated APC gene. CONCLUSIONS: DNA analysis allowed accurate genetic counselling for 12 of 31 families affected by FAP, thus improving the medical and personal management in asymptomatic people who would otherwise be subjected to the uncertainty of long term surveillance and repeated colonic examinations. In future a superior biomolecular approach to gene mutation analysis, such as the protein truncation test, will facilitate management for most FAP individuals and families.

Adenomatous Polyposis Coli↗
PubMed1993

Single base pair mutation analysis by PNA directed PCR clamping.

A novel method that allows direct analysis of single base mutation by the polymerase chain reaction (PCR) is described. The method utilizes the finding that PNAs (peptide nucleic acids) recognize and bind to their complementary nucleic acid sequences with higher thermal stability and specificity than the corresponding deoxyribooligonucleotides and that they cannot function as primers for DNA polymerases. We show that a PNA/DNA complex can effectively block the formation of a PCR product when the PNA is targeted against one of the PCR primer sites. Furthermore, we demonstrate that this blockage allows selective amplification/suppression of target sequences that differ by only one base pair. Finally we show that PNAs can be designed in such a way that blockage can be accomplished when the PNA target sequence is located between the PCR primers.

Alleles↗
PubMed1994

DNA mutational analysis of type 1 and type 3 Gaucher patients: how well do mutations predict phenotype?

The wide spectrum of clinical manifestations resulting from glucocerebrosidase deficiency complicates genetic counseling for Gaucher disease. The identification of mutations in the glucocerebrosidase gene has enabled studies of genotype-phenotype correlation. However, a genotypic analysis of 60 type 1 and type 3 Gaucher patients reveals that the 5 most common Gaucher mutations, N370S, L444P, R463C, 84insG, and IVS2 + 1 G-->A, can be found both in patients with and without neurologic manifestations. Moreover, although some generalizations can be made about mutations that are more frequently encountered in particular patient populations, Gaucher patients sharing identical genotypes can exhibit considerable clinical heterogeneity. Thus in considering rationale for population screening one cannot rely solely on PCR determined DNA mutation analysis to reliably predict prognosis in Gaucher disease.

Adolescent↗
PubMed1994

Mutation analysis in patients with possible but apparently sporadic Huntington's disease.

Until the advent of mutation analysis it was impossible to make a certain diagnosis of Huntington's disease (HD) in the absence of a positive family history, and sporadic cases of possible HD presented a substantial diagnostic dilemma. We have looked for the characteristic expanded trinucleotide (CAG) repeat sequence in the HD gene in 44 patients with probable or possible HD who did not have similarly affected relatives. We used two methods, the traditional widely used method, which estimates both the CAG repeat and the flanking CCG repeat and gives the CAG length by subtraction, and the more precise CAG method, which estimates the repeat length directly. With the CAG method, the HD mutation was detected in 25 (89%) of 28 patients with the typical clinical features of HD and 5 (31%) of 16 in whom the diagnosis was more doubtful. The CAG-CCG method gave results in the borderline abnormal range of repeats for 13 of the 33 patients eventually shown to have an unequivocal repeat expansion by the CAG method. Most of these patients had late onset of symptoms. There was evidence of expansion of an intermediate-length paternal allele in 1 patient and of non-paternity in another. The identification of the mutation causing HD means that it is now possible to confirm or exclude the diagnosis with confidence, even in the absence of a family history, by analysis of DNA from a blood sample. The precise method of measuring the CAG repeat, which is technically more difficult than the traditional method, may be needed to clarify results in a substantial proportion of such patients.

Adult↗
PubMed1994

Directed mutational analysis of bacteriochlorophyll a biosynthesis in Rhodobacter capsulatus.

Previous studies have established that most if not all of the genes required for synthesis of the Rhodobacter capsulatus essential photosystem are clustered on a 46 kb region of the chromosome known as the photosynthesis gene cluster. This region has recently been sequenced in its entirety by Hearst and co-workers, revealing the existence of 23 open reading frames, many of which are thought to be involved in the synthesis of bacteriochlorophyll. In this study we have undertaken a systematic directed mutational analysis of 12 open reading frames in the photosynthesis gene cluster to evaluate whether individual open reading frames have a role in photopigment biosynthesis. The results of this analysis demonstrate that mutations constructed in seven open reading frames resulted in a loss of bacteriochlorophyll biosynthesis, concomitant with the accumulation of specific intermediates in the Mg-tetrapyrrole biosynthetic pathway. One mutation was observed to result in partial disruption of bacteriochlorophyll biosynthesis, leading to the accumulation of bacteriochlorophyll as well as an intermediate in the biosynthetic pathway. We also observed that disruptions constructed in four open reading frames had no discernible effect on the synthesis of photopigments. The results of this analysis are discussed with regard to our current understanding of the role of each of these open reading frames in the synthesis of the R. capsulatus photosystem.

Bacterial Proteins↗
PubMed1992

Point mutation analysis of ras genes in spontaneous and chemically induced C57Bl/10J mouse liver tumours.

The high incidence and profile of ras gene mutations reported in spontaneous and chemically induced liver tumours of the B6C3F1 mouse provides a potential means of determining in vivo genotoxicity and its relevance to carcinogenicity. We analysed spontaneous and chemically induced [with 4-amino-biphenyl (ABP), 2-acetylaminofluorene (AAF) and diethylnitrosamine (DEN)] hepatocellular tumours of the C57Bl/10J mouse for H-ras, K-ras and N-ras gene mutations to see if mutational analysis of the ras genes could be useful for such a determination in this strain. Regions of DNA spanning codons 12, 13 and 61 of the ras genes were amplified from formalin fixed liver tumour sections using the polymerase chain reaction. Mutations were detected using allele specific oligonucleotide probing and confirmed by sequencing. We have found that there are few ras mutations in either spontaneous or chemically induced liver tumours in the C57Bl/10J mouse. Out of 25 spontaneous tumours two contained an A to T transversion and one contained an A to G transition in base 2 of H-ras codon 61 and two contained a G to A transition in base 2 of K-ras codon 13 (the K-ras mutations were only faintly detectable and may be present in a subpopulation of the tumour cells). In the case of the 18 ABP induced tumours one contained a C to A transversion in base 1 of H-ras codon 61, and one contained an A to T transversion in base 2 of H-ras codon 61 and one contained a G to C transversion in base 1 of K-ras codon 13. One C to A transversion in base 1 of H-ras codon 61 was detected out of eight AAF induced tumours. Of the 25 DEN induced tumours, one contained an A to G transition and one contained an A to C transversion in base 2 of H-ras codon 61. The data indicate that at least in hepatocellular tumours of the C57Bl/10J strain and using chronic dosing regimes the ras genes do not represent markers for in vivo genotoxic activity.

2-Acetylaminofluorene↗
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↗