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PubMed1995

Identification of six mutations (R31L, 441delA, 681delC, 1461ins4, W1089R, E1104X) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Six new mutations have been identified in the CFTR gene. These mutations, representing three different categories--missense (R31L, W1098R), nonsense (E1104X), and frameshift (441delA, 681delC, 1461ins4)--are located in exons 2, 4, 5, 9, and 17b of the gene and presumed to cause cystic fibrosis (CF) in patients. All these mutations are probably rare in the population, as no additional examples were found for any of them in a cohort of 545 CF patients. Our study also revealed a benign sequence variation (3499 + 45T-->C) in intron 17b.

Adult↗
PubMed1992

F-SSCP: fluorescence-based polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis.

A fluorescence-based method for polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis, F-SSCP, was developed in which the target sequence is amplified by the PCR using fluorescent primers. The amplified products are then heat-denatured and applied to a water-jacket controlled gel in an automated DNA sequencer. The separated strands are detected as laser-excited fluorescence at the bottom of the gel, and mutations are detected as shifts in the position of the peaks in the fluorogram. The system does not involve radioactivity, and the conditions of electrophoresis are more strictly controlled than in the previous system, which relied on ambient air-cooling to maintain the gel at a constant temperature. The nature of the output data allows direct quantitative interpretation, and so the relative abundance of each allele in a mixture of two or more alleles can easily be estimated. The application of F-SSCP for detection of mutations and loss of heterozygosities of p53 in tumor tissues is reported.

Base Sequence↗
PubMed1992

Absence of p53 germ-line mutations in bilateral breast cancer patients.

The cause of Li-Fraumeni syndrome, a rare group syndrome of familial cancers, has recently been identified. Patients with this inherited condition are highly susceptible to specific neoplasms, including early-onset breast cancers. The available evidence links Li-Fraumeni syndrome to inherited mutations of the tumor suppressor gene p53. Moreover, somatically acquired p53 mutations and gene deletions are common feature in breast cancer of sporadic origin. These findings suggest that germline p53 mutations are important in familial and, possibly sporadic, breast tumors. We have therefore screened lymphocyte DNA from 19 unrelated bilateral cancer patients for germline p53 mutations in exons 5, 6, 7 and 8. We have however detected no germline mutations by means of the single-strand confirmation polymorphism technique in any of the lymphocyte DNAs examined and conclude that p53 mutations are not generally involved in bilateral breast cancer.

Adult↗
PubMed1992

ras mutations in endocrine tumors: mutation detection by polymerase chain reaction-single strand conformation polymorphism.

To elucidate the molecular basis for endocrine tumorigenesis, ras mutations in human endocrine tumors were analyzed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Mutations of the H-, K-, N-ras genes were examined in genomic DNAs from 169 successfully amplified primary endocrine tumors out of 189 samples. Four out of 24 thyroid follicular adenomas analyzed contained mutated N-ras codon 61, and one contained the mutated H-ras codon 61. One of the 19 pheochromocytomas revealed mutation of the H-ras codon 13. No mutations of the ras gene were detected in pituitary adenomas, parathyroid tumors, thyroid cancers, endocrine pancreatic tumors, and adrenocortical tumors. Based on these findings we conclude that activation of the ras gene may play a role in the tumorigenesis of a limited number of thyroid follicular adenomas and pheochromocytomas, and that mutation of the ras gene is not frequent in other human endocrine tumors.

Adult↗
PubMed1992

Mutation analysis of genetic diseases by asymmetric-PCR SSCP and ethidium bromide staining: application to neurofibromatosis and cystic fibrosis.

The Single Strand Conformation Polymorphism (SSCP) technique is widely used in mutation analysis. We have introduced several modifications to the SSCP method, which overcome the problem of incomplete denaturation or reannealing of DNA during electrophoresis. The modifications consist of asymmetrical PCR amplification of the sequence of interest, electrophoresis with a higher concentration of acrylamide, and the analysis of the DNA fragments under u.v. light. We have applied this method to the analysis of two specific diseases: neurofibromatosis type 1 (NF1) and cystic fibrosis (CF) from PCR amplified exons. Two single nucleotide changes were observed with this method.

Base Sequence↗
PubMed1992

Use of single strand conformation polymorphism analysis to detect point mutations in human mitochondrial DNA.

Myoclonus epilepsy with ragged-red fibers (MERRF) has been shown to be associated with a specific point mutation at the nucleotide 8344 in the tRNA(Lys) gene of mitochondrial DNA (mtDNA). We screened 6 patients with clinically diagnosed MERRF and 1 patient with ocular myopathy for point mutations in the tRNA(Lys) gene, using single strand conformation polymorphism (SSCP) analysis, which can detect even a 1-basepair difference between 2 DNA sequences. Using SSCP and consequent DNA sequencing, we identified the known MERRF mutation in 4 out of 6 MERRF patients, as well as in 1 patient with a new clinical phenotype associated with this mutation: progressive external ophthalmoplegia, muscle weakness and a lipoma, but no myoclonus or epilepsy. Two of the patients with clinical MERRF had neither the MERRF-mutation nor any other mutations in the tRNA(Lys) gene. Using SSCP analysis, we also detected a new polymorphism in 1 patient. Thus, SSCP analysis can be applied to search effectively and rapidly for point mutations or polymorphisms in mitochondrial DNA.

Adolescent↗
PubMed1992

Detection of p53 gene mutations in human ovarian and endometrial cancers by polymerase chain reaction-single strand conformation polymorphism analysis.

The presence of mutations in the p53 gene was examined in ovarian cancers by a polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. The primers were designed to amplify exons 5 through 9 that contain phylogenetically conserved domains of the p53 gene. Mutations were detected in 5 out of 10 cases, one of which contained a deletion in the second allele. A single base substitution was detected in 4 cases at codons 162, 175, 205 and 273 and a single base insertion in one case within codon 315. A high frequency of p53 mutations in ovarian cancers and lack of mutation in 6 benign ovarian tumors and 2 normal ovaries suggested that the mutation of the p53 gene was associated with the genesis and/or progression of ovarian cancer. In 1 of 7 endometrial cancers, two mutations at codons 239 and 254 were detected.

Alleles↗
PubMed1986

Nucleotide sequence of an heterochromatic segment recognized by the antibodies to Z-DNA in fixed metaphase chromosomes.

The purpose of this work was to analyse at the molecular level the DNA recognized by the antibodies to Z-DNA in in situ experiments. Antibodies to Z-DNA interact strongly with R-band positive heterochromatic segments of fixed metaphase chromosomes of Cebus (Viegas-Pequignot et al., 1983). These segments are constituted of a satellite DNA the repeat unit of which is about 1520 base pairs long. The base sequence of the repeat unit has been determined. It contains a (AC)n rich region which, in vitro, adopts the Z conformation under topological constraints. Experiments with nuclei suggest that this sequence is not predominantly in the Z conformation in vivo. The polymorphic structure of the (AC)n rich region argues for an active recombination sequence.

Animals↗
PubMed1992

Characteristics of polymorphism at a VNTR locus 3' to the apolipoprotein B gene in five human populations.

We have analyzed the allele frequency distribution at the hypervariable locus 3' to the apolipoprotein B gene (ApoB 3' VNTR) in five well-defined human populations (Kacharis of northeast India, New Guinea Highlanders of Papua New Guinea, Dogrib Indians of Canada, Pehuenche Indians of Chile, and a relatively homogeneous Caucasian population of northern German extraction) by using the PCR technique. A total of 12 segregating alleles were detected in the pooled sample of 319 individuals. A fairly consistent bimodal pattern of allele frequency distribution, apparent in most of these geographically and genetically diverse populations, suggests that the ApoB 3' VNTR polymorphism predates the geographic dispersal of ancestral human populations. In spite of the observed high degree of polymorphism at this locus (expected heterozygosity levels 55%-78%), the genotype distributions in all populations (irrespective of their tribal or cosmopolitan nature) conform to their respective Hardy-Weinberg predictions. Furthermore, analysis of the congruence between expected heterozygosity and the observed number of alleles reveals that, in general, the allele frequency distributions at this locus are in agreement with the predictions of the classical mutation-drift models. The data also show that alleles that are shared by all populations have the highest average frequency within populations. These findings demonstrate the potential utility of highly informative hypervariable loci such as the ApoB 3' VNTR locus in population genetic research, as well as in forensic medicine and determination of biological relatedness of individuals.

Alleles↗
PubMed1992

A new mutant transthyretin (Arg 10) associated with familial amyloid polyneuropathy.

We report a new kindred with systemic amyloidosis presenting as peripheral neuropathy in the sixth and seventh decades of life. Polymorphism in exon 2 of the transthyretin (TTR) gene was suggested by single strand conformation polymorphism analysis and determined by direct DNA sequencing. We also developed restriction fragment length polymorphism analysis by polymerase chain reaction using a primer with an induced mutation. The point mutation (cytosine for thymine at position 1038 of the TTR gene) is responsible for substitution of arginine for cysteine at position 10 of the TTR molecule. It is hypothesised that the TTR molecules which have no cysteine have a unique structure in heterozygous TTR polymers and are responsible for amyloid fibril formation.

Aged↗
PubMed1989

DNA duplex with the potential to change handedness after every half a turn.

Polymorphic forms of the DNA duplex with long stretches of structural monotony are known. Several alternating purine-pyrimidine sequences have been shown to adopt left-handed Z-conformation. We report a DNA sequence d(CGCGCGATCGAT)n exhibiting alternating right-handed B and left-handed Z helical conformation after every half a turn. Further, this unusual conformation with change in handedness after every six base pairs was induced at physiological superhelical density.

Base Sequence↗
PubMed1992

Detection of a Ha-ras point mutation by polymerase chain reaction-single strand conformation polymorphism analysis in 2-amino-3,4-dimethylimidazo[4,5-f]quinoline-induced mouse forestomach tumors.

Ha-ras activation in forestomach squamous cell carcinomas of CDF1 mice induced by 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), one of the heterocyclic amines isolated from broiled sardine was analyzed. Mutations were detected in two of three primary original carcinomas and two of four cell lines derived from other independent carcinomas by the polymerase chain reaction followed by analysis of single strand conformation polymorphism and direct sequencing. All the mutations detected were G----T transversions at the second letter of codon 13 resulting in an amino acid change from Gly to Val. This finding together with the previous reports on squamous cell carcinomas of the rat Zymbal gland suggest that MeIQ induces a specific type of mutation at a specific site of the Ha-ras gene during squamous cell carcinogenesis, in a species-independent manner.

Animals↗
PubMed1992

Lack of allelic preference in amplification and loss of the c-myc oncogene in methylcholanthrene-induced mouse sarcomas.

Sarcomas were induced in F1 mice between C57BL/6N and C3H/He strains by subcutaneous injection of methylcholanthrene. The c-myc oncogene was found to be amplified in 16 cases among 43 sarcomas of C57BL/6N x C3H/He mice and 1 case among 5 sarcomas of the reciprocal cross. The origin of the amplified allele was determined by the polymerase chain reaction single strand conformation polymorphism analysis. Among the 17 sarcomas, only one had both of the alleles amplified. The rest of the tumors carried the amplified c-myc allele coming either from C57BL/6N (9 cases) or from C3H/He (8 cases). These results indicate that the c-myc allele is amplified randomly in methylcholanthrene-induced mouse sarcomas irrespective of its origin, such as paternal or maternal allele and C57BL/6N or C3H/He allele. In addition to these changes, the unamplified c-myc oncogene was found to be lost in 12 cases out of the 17 sarcomas with the amplification.

Alleles↗
PubMed1990

Mercury-induced DNA polymorphism: probing the conformation of Hg(II)-DNA via staphylococcal nuclease digestion and circular dichroism measurements.

Exposing native calf thymus DNA to increasing concentrations of Hg(ClO4)2 not only produces dramatic changes in its circular dichroism (CD) but results also in the decrease, and ultimate cessation, of endonucleolytic DNA cleavage by staphylococcal nuclease. Let r = [moles of added Hg(II)]/[mole of DNA base]: the conservative CD spectrum of the DNA B-form becomes nonconservative in appearance at 0.01 less than r less than 0.12 (resembling DNA in C-form geometry) and assumes the spectral characteristics of a left-handed DNA double helix at 0.12 less than r less than or equal to 1.0. DNA cleavage proceeds at or near the rates exhibited by untreated DNA at 0 less than r less than 0.08. At Hg(II) levels of 0.08 less than r less than 0.5, the rate of DNA hydrolysis decreases monotonically with increasing Hg(II) concentrations, and at r greater than 0.4, DNA cleavage ceases. Both the CD changes and the changes in the rate of DNA digestion are totally reversible upon the removal of Hg(II), at least up to r = 1.0, demonstrating that Hg(II) keeps all base pairs in register. For comparison purposes, native calf thymus DNA was also treated with methylmercury [CH3Hg(II)], an agent known to disrupt the secondary structure of DNA. The treatment yielded single-stranded methylmercurated DNA with preserved right-handed helix screwness. In addition, this DNA is digested by staphylococcal nuclease much more rapidly than double-stranded control DNA. Lastly, neither the CD nor the cleavage rate changes are reversible upon the removal of methylmercury.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Intrahelical pseudoknots and interhelical associations mediated by mispaired human minisatellite DNA sequences in vitro.

The human minisatellite arrays, 33.6 and 33.15, consist of tandem reiterations of a 37-nucleotide (nt) and a 16-nt repeat unit sequence, respectively, both of which contain a majority of purine bases on one strand. Knot-like tertiary structures, which mapped to the cloned arrays, were observed by electron microscopy (EM) in homoduplex molecules produced by denaturation and reannealing in vitro. They result from a primary hybridization between misaligned repeat units of the array, forming a slipped-strand structure with staggered single-stranded DNA loops, followed by a secondary hybridization between repeat units in the two loops. Depending on the relative alignment of the loops when they hybridize, a particular form of intrahelical pseudoknot is produced. Theta-shaped, figure-of-eight, and bow-shaped structures were the most common conformational isomers observed in homoduplexes flattened into two dimensions during EM preparation. At the site of a bow-shaped structure, a conformation-dependent bend of approximately 60 degrees between the flanking DNA segments is induced; the other conformations generally do not deflect the line of the main DNA axis. Paired loops, similar to the bow-shaped structure, were apically situated in some supercoiled plasmids containing the 33.6 array. Both plasmids formed intermolecular associations, consisting of two (or more) homoduplex molecules held together at or immediately adjacent to a nexus which mapped to the minisatellite sequences. These associations might arise either by interhelical hybridization between arrays or by knot-like structures interfering with branch migration of chi-form Holliday junctions.

Base Sequence↗
PubMed1992

The product of the human MUC1 gene when secreted by mouse cells transfected with the full-length cDNA lacks the cytoplasmic tail.

The polymorphic epithelial mucin (PEM) is found as a cell associated transmembrane protein with an extracellular domain made up largely of tandem repeats and also as a soluble form in some body fluids and culture supernatants. To determine whether the soluble form can arise without the mechanism of alternative splicing mouse cells have been transfected with an expression construct containing the full-length cDNA, and the supernatants of the transfectants analyzed for the presence of the mucin. The presence of mucin in the supernatants could indeed be detected in a radioimmunoassay and by immunoprecipitation using monoclonal antibodies to the tandem repeat region of the core protein, indicating that release of the soluble form can occur without alternative splicing. The soluble form was not however precipitated with a polyclonal antiserum to the cytoplasmic tail, suggesting that it was released from the membrane by the action of a protease.

Animals↗
PubMed1995

Divalent zinc cations induce the formation of two distinct homoduplexes of a d(GA)20 DNA sequence.

Homopurine DNA sequences are highly structurally polymorphic. In particular, d(GA)n DNA sequences are known to be capable of forming intramolecular foldbacks, bimolecular homoduplexes, and tetrastranded complexes. Counterions play a determinant role on the equilibria between the different structural conformers of d(GA)n sequences. In this paper, the effect of divalent zinc cations on the structure of a d(GA)20 oligonucleotide has been analyzed by CD spectroscopy and polyacrylamide gel electrophoresis. Depending on the precise experimental conditions at which zinc is added, two distinct conformations of the d(GA)20 oligonucleotide are stabilized. At neutral pH in the absence of zinc, d(GA)20 is partially organized into intramolecular foldbacks and bimolecular homoduplexes [Casasnovas et al. (1993) J. Mol. Biol. 233, 671-681]. Under these conditions, addition of zinc results in the stabilization of the bimolecular homoduplex which is nonspecific for zinc since it is also stabilized by divalent magnesium cations, increasing ionic strength, or decreasing pH. Its CD spectrum is identical to that reported earlier for parallel-stranded d(GA)n homoduplexes [Rippe et al. (1992) EMBO J. 11, 3777-3786]. On the other hand, if zinc is added under conditions where the d(GA)20 oligonucleotide is exclusively single-stranded, a different bimolecular homoduplex appears which is only observed in the presence of zinc. The zinc-specific duplex melts cooperatively, and, in contrast to the nonspecific duplex, its thermostability is high. Transition from the nonspecific to the zinc-specific duplex is observed at high zinc concentrations or at high temperatures. The transition is cooperative. These results are discussed in the context of the specific cation effects on the formation of intramolecular R.R.Y triplexes at d(GA.TC)n DNA sequences.

Animals↗
PubMed1987

Nuclease sensitivity of the mouse HPRT gene promoter region: differential sensitivity on the active and inactive X chromosomes.

We investigated the conformation of the X-linked mouse hypoxanthine-guanine phosphoribosyltransferase gene (HPRT) promoter region both in chromatin from the active and inactive X chromosomes with DNase I and in naked supercoiled DNA with S1 nuclease. A direct comparison of the chromatin structures of the active and inactive mouse HPRT promoter regions was performed by simultaneous DNase I treatment of the active and inactive X chromosomes in the nucleus of interspecies hybrid cells from Mus musculus and Mus caroli. Using a restriction fragment length polymorphism to distinguish between the active and inactive HPRT promoters, we found a small but very distinct difference in the DNase I sensitivity of active versus inactive chromatin. We also observed a single DNase I-hypersensitive site in the immediate area of the promoter which was present only on the active X chromosome. Analysis of the promoter region by S1 nuclease digestion of supercoiled plasmid DNA showed an S1-sensitive site which maps adjacent to or within the DNase I-hypersensitive site found in chromatin but upstream of the region minimally required for normal HPRT gene expression.

Animals↗