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At least 1,243 records · Page 69Linked to original sources
PubMed2005

Automated splicing mutation analysis by information theory.

Information theory-based software tools have been useful in interpreting noncoding sequence variation within functional sequence elements such as splice sites. Individual information analysis detects activated cryptic splice sites and associated splicing regulatory sites and is capable of distinguishing null from partially functional alleles. We present a server (https://splice.cmh.edu) designed to analyze splicing mutations in binding sites in either human genes, genome-mapped mRNAs, user-defined sequences, or dbSNP entries. Standard HUGO-approved gene symbols and HGVS-approved systematic mutation nomenclature (or dbSNP format) are entered via a web portal. After verifying the accuracy of input variant(s), the surrounding interval is retrieved from the human genome or user-supplied reference sequence. The server then computes the information contents (Ri) of all potential constitutive and/or regulatory splice sites in both the reference and variant sequences. Changes in information content are color-coded, tabulated, and visualized as sequence walkers, which display the binding sites with the reference sequence. The software was validated by analyzing approximately 1,300 mutations from Human Mutation as well as eight mapped SNPs from dbSNP designated as splice site variants. All of the splicing mutations and variants affected splice site strength or activated cryptic splice sites. The server also detected several missense mutations that were unexpectedly predicted to have concomitant effects on splicing or appeared to activate cryptic splicing.

Automation↗
PubMed1995

Mutation analysis in two newly identified rat p53 pseudogenes.

We have analysed the genomic organization of the rat p53 gene in normal intestinal rat cells. Exons 5-8 of the p53 gene were amplified by PCR from rat genomic DNA using primers complementary to stretches of nucleotides identical in rat and human cDNAs. Two amplification products of 727 and 339 bp were obtained from the rat DNA. The PCR products were subcloned into M13mp18 and sequenced. By comparison with the rat cDNA sequence the 727 bp band was identified as the functional p53 gene containing exons 5, 6 7 and 8 and introns 5 and 7. The sequences corresponding to human intron 6 are absent from the rat p53. The second amplification product was a mixture of two different 'processed' pseudogenes, in which 42 mutations accumulated in a sequence corresponding to the cDNA between exons 5 and 8. Analysis of these mutations shows that in both pseudogenes the vast majority is constituted by base substitutions, with transitions being more frequent than transversions. The most prominent mutational class is formed by G-->A transitions which are predominantly located at CpG sites. Since a high level of homology is present between the rat and the human cDNA, the type and the positions where mutations occur in the two rat pseudogenes is discussed in relation to the possible origin of these mutations in human tumors.

Amino Acid Sequence↗
PubMed1996

Linkage, association and mutational analysis of the dopamine D3 receptor gene in schizophrenia.

This study follows the observation of an association between homozygosity of an Mscl polymorphism in exon 1 and schizophrenia, which gives rise to a glycine to serine substitution and may alter the functional properties of the receptor. Alternatively the polymorphism may not itself be of functional significance but may be in linkage disequilibrium with another genetic variant in the coding or regulatory regions. To examine the second possibility we have screened all six exons of DRD3 by single-stranded conformational polymorphism analysis (SSCP) in 36 cases and 36 controls. Our findings suggest that the gene is highly conserved since we found no other mutations which alter protein structure. However we did detect a 5-bp deletion in the 3' intronic sequence flanking exon 5 which occurred in 7-8% of subjects within both case and control samples. A single bp substitution (g to a) in exon 3, which does not alter an amino acid was found in one affected individual. In addition we carried out a linkage study of 24 families multiply affected with schizophrenia and a non-parametric linkage study of 90 affected sibling pairs. These studies give no support for either major or moderate gene effects on schizophrenia susceptibility. Finally we have extended our association sample and observe a non-significant excess of homozygotes for the Mscl polymorphism in the sample overall (chi 2 = 2.09, 1 d.f., P = 0.15). The excess of homozygotes is specific to males (chi 2 = 4.617, 1 d.f., P = 0.032) and not females (chi 2 = 0.243, 1 d.f., NS). When these data are added to our previous published data a highly significant excess of homozygotes is observed in males (chi 2 = 13.766, 1 d.f., P = 0.00021) but not females (chi 2 = 0.606, 1 d.f., NS). In conclusion the accumulated data suggest strongly that genetic variation at the DRD3 locus increases susceptibility of schizophrenia, at least in males. At present the Mscl polymorphism in exon 1 of the gene remains a candidate for bringing about functional change in the receptor but this has not been formally tested. Other coding region polymorphisms have not been detected but it remains possible that variation within the promoter may alter receptor function.

Base Sequence↗
PubMed1997

A mutational analysis of conjugation in Tetrahymena thermophila. 1. Phenotypes affecting early development: meiosis to nuclear selection.

Conjugation in the freshwater ciliate Tetrahymena thermophila involves a developmental program that models meiosis, fertilization, and early developmental events characteristic of multicellular eukaryotes. We describe a gallery of five early-acting conjugation mutations. These mutants, cnj1-5, exhibit phenotypes in which specific steps in the conjugal pathway have been altered or eliminated. Specifically, cnj1 and cnj2 fail to condense their micronuclear chromatin prior to each of the three prezygotic nuclear divisions. This results in nuclear division failure, failure to replicate DNA, and failure to initiate postzygotic development. The cnj3 mutant appears to exhibit a defect in chromosome separation during anaphase of mitosis. cnj4 mutants successfully carry out meiosis I, yet are unable to execute the second meiotic division and abort all further development. cnj5 mutants are unable to initiate either meiosis I or meiosis II, yet proceed to execute all subsequent developmental events. These mutant phenotypes are used to draw inferences regarding developmental dependencies that exist within the conjugation program.

Animals↗
PubMed1988

Mutational analysis of the tRNA mimicry of brome mosaic virus RNA. Sequence and structural requirements for aminoacylation and 3'-adenylation.

The genomic RNAs of brome mosaic virus (BMV) exhibit various tRNA-like properties, including specific tyrosylation by tyrosyl-tRNA synthetases and adenylation of the 3'-CCOH derivative by tRNA nucleotidyl transferases. We have studied the effect of numerous mutations in all domains of the tRNA-like structure of BMV RNA on tyrosylation and adenylation in vitro. Surprisingly few mutations resulted in more than 50% decrease in tyrosylation rates with either wheat germ or yeast synthetases; those mutations were at the 3' terminus, the pseudoknot, and the bases of arms B and E. The results suggest an interaction of synthetase with arm A as the analog of the aminoacyl acceptor stem of tRNAs, and arm B as the analog of the anticodon arm of tRNAs, although there is no apparent interaction with the terminal loop of arm B analogous to the interaction with the anticodon in tRNAs. Mutations at several loci resulted in large losses of adenylation activity catalyzed by wheat germ and Escherichia coli nucleotidyl transferases; those loci were the pseudoknot, the bases of arms B, C and D, and at the junctions of these arms with arm A. These studies have identified mutants specifically defective in one of the tRNA-like activities, which are appropriate for investigating the role of these activities during infection in vivo.

Autoradiography↗
PubMed1998

Mutation analysis by a non-radioactive single-strand conformation polymorphism assay in nine families with X-linked severe combined immunodeficiency (SCIDX1).

X-linked severe combined immunodeficiency (SCIDX1) is an inherited disease characterized by profound abnormalities of cell-mediated and humoral immunity. Patients with SCIDX1 have defects in the common cytokine receptor gamma chain gene (IL2RG) that encodes a shared, essential component of the receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9 and IL-15. We have characterized nine SCIDX1 families by using a DNA-based, non-radioactive screening method and DNA sequencing. Nine different mutations were found, scattered from exon 1 to exon 5 of the IL2RG gene. Two of these mutations have been previously identified in other unrelated patients; the other seven are novel mutations that differ from all of the 95 already reported in the IL2RG mutation data base. In addition to describing novel mutations in the IL2RG gene, this study shows that the knowledge of the genetic defect and the use of an efficient, non-radioactive, and rapid screening approach have important implications for prenatal and postnatal diagnosis, carrier female identification, and possibly prenatal therapy.

DNA Mutational Analysis↗
PubMed2003

Mutation analysis of the zinc transporter gene SLC30A4 reveals no association with periodic catatonia on chromosome 15q15.

The zinc transporter gene SLC30A4, located on chromosome 15q15-q21, has previously been reported to show altered expression patterns in post mortem analysis of the brains of schizophrenic patients. As a positional candidate we investigated SLC30A4 in the chromosome 15q15-linked schizophrenic phenotype periodic catatonia (MIM 605419), by means of a systematic mutation screening in affected individuals from exceptionally large pedigrees with perfect co-segregation of a chromosomal segment between marker D15S1042 and D15S659 in all affected individuals. The mutation scan revealed no genetic variants within the coding and the putative promoter region of SLC30A4 and, thus, excludes a genetic association of SLC30A4 with catatonic schizophrenia.

Brain↗
PubMed2001

Aromatics at the murine nicotinic receptor agonist binding site: mutational analysis of the alphaY93 and alphaW149 residues.

1. Two aromatic residues of the muscle nicotinic receptor putative agonist binding site, a tyrosine in position alpha93 and a tryptophan in position alpha149, were mutated to phenylalanine and the effects of the mutations on receptor properties were investigated using single-channel patch clamp. 2. The alphaY93F mutation reduced the receptor affinity by approximately 4-fold and the channel opening rate constant by 48-fold. The alphaW149F mutation reduced the receptor affinity by approximately 12-fold and the channel opening rate constant by 93-fold. 3. The kinetic properties of hybrid receptors that contained one wild-type and one mutated alpha subunit were also examined. Only one type of hybrid receptor activity was detected. The hybrid receptors had a channel opening rate constant intermediate to those of the wild-type and mutant receptors. It was concluded that the ligand binding sites in the mutated muscle nicotinic receptor contributed equally to channel gating. In the case of the alphaW149F mutation, the presence of the mutation in one of the binding sites had no effect on the binding properties of the other, non-mutated, site. 4. The mutant channel opening and closing rate constants were also estimated in the presence of tetramethylammonium. The data suggested significant interaction between the acetyl group of acetylcholine and the alphaY93 residue.

Acetylcholine↗
PubMed2007

C-kit Asp-816-Val mutation analysis in patients with mastocytosis.

BACKGROUND: Mastocytosis is a heterogeneous group of disorders characterized by abnormal accumulation of mast cells. OBJECTIVE: Skin biopsies from 24 patients (23 with proven mastocytosis) were screened for the presence of the c-kit Asp-816-Val mutation. METHODS: In frozen biopsies, RNA was isolated, cDNA synthesis and PCR, the expected PCR product of 346 bp was obtained from 23 patients. RESULTS: In patients with urticaria pigmentosa, the mutation was detected in 38% of the adults and 25% of the children. With regard to the clinical presentation of the disease, no difference was found between adult patients with and without the mutation, as detected with our assay. One out of the 2 children with the mutation had an atypical presentation of the disease. CONCLUSION: the mutation could not be detected in all the patients, probably due to lack of sensitivity of the methods.

Adolescent↗
PubMed2000

Mutation analysis of the growth differentiation factor-9 and -9B genes in patients with premature ovarian failure and polycystic ovary syndrome.

OBJECTIVES: To investigate whether growth differentiation factor (GDF)-9 or GDF-9B/bone morphogenetic protein (BMP)-15 mutation is present in Japanese women with premature ovarian failure (POF) and polycystic ovary syndrome (PCOS). DESIGN: Clinical and molecular studies. SETTING: Outpatient clinic in a university hospital. PATIENT(S): Fifteen women with POF, 38 women with PCOS, and 3 normal fertile controls. INTERVENTION(S): Extraction of DNA from blood samples for subsequent polymerase chain reaction (PCR). MAIN OUTCOME MEASURE(S): Amplified DNA was analyzed by denaturing gradient gel electrophoresis (DGGE) and/or by direct sequencing. RESULT(S): No missense mutation was found in any exons of the GDF-9 gene and the GDF-9B/BMP-15 gene in patients with POF and PCOS. CONCLUSION(S): The missense mutation in the GDF-9 gene or the GDF-9B/BMP-15 gene is uncommon in anovulatory Japanese women with POF and PCOS.

Adult↗
PubMed1998

Optimization of PCR and electrophoresis conditions enhances mutation analysis of the BRCA1 gene.

Single-strand confirmation polymorphism (SSCP) analysis on MDE gels has been extensively used to detect BRCA1 gene mutations. In screening a large cohort of ovarian cancer patients, our group was not completely satisfied with the standard published techniques. Although conventional primer sets usually amplified well, we were able to enhance band spread and resolution by varying the PCR and electrophoresis conditions for many of the individual exons. These alterations enhanced our ability to detect polymorphisms and mutations. When utilizing SSCP screening of a gene as large as BRCA1, no one set of conditions or even method may be optimal for all of the exons. Rather, a variety of different methods and conditions should be studied for each set of amplimers under analysis.

Buffers↗
PubMed2006

Duplication of the mmoX gene in Methylosinus sporium: cloning, sequencing and mutational analysis.

The soluble methane monooxygenase (sMMO) is a key enzyme for methane oxidation, and is found in only some methanotrophs, including Methylosinus sporium 5. sMMO expression is regulated at the level of transcription from a sigma(54) promoter by a copper-switch, and is only expressed when the copper-to-biomass ratio during growth is low. Extensive phylogenetic and genetic analyses of sMMOs and other soluble di-iron monooxygenases reveal that these enzymes have only been acquired relatively recently through horizontal gene transfer. In this study, further evidence of horizontal gene transfer was obtained, through cloning and sequencing of the genes encoding the sMMO enzyme complex plus the regulatory genes mmoG and mmoR, and identification of a duplicate copy of the mmoX gene in Ms. sporium. mmoX encodes the alpha subunit of the hydroxylase of the sMMO enzyme, which constitutes the active site (Prior & Dalton, 1985). The mmoX genes were characterized at the molecular and biochemical levels. Although both copies were transcribed, only mmoX copy 1 was essential for sMMO activity. Construction of an sMMO(-) mutant by marker-exchange mutagenesis gave some possible insights into the role of the water-soluble pigment in siderophore-mediated iron acquisition. Finally, the amenability of Ms. sporium to genetic manipulation was demonstrated by complementing the sMMO(-) mutant by heterologous expression of sMMO genes from Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), and it was shown that Ms. sporium could be used as an alternative model organism for molecular analysis of MMO regulation.

Binding Sites↗
PubMed2006

Mutation analysis of 218 Chinese patients with Wilson disease revealed no correlation between the canine copper toxicosis gene MURR1 and Wilson disease.

Wilson disease (WD) is the most common disorder resulting in hepatic copper overload. A similar form of copper-associated cirrhosis caused by mutations of the canine copper toxicosis MURR1 gene is also observed in Bedlington terriers. Recent studies indicate that MURR1 might influence human copper metabolism and the clinical presentations of WD. However, the correlation between the MURR1 gene and the Chinese patients with WD has not been reported. In the present study, all three exons of the MURR1 gene including the intron-exon boundaries were directly sequenced in 120 unrelated healthy Chinese and 218 unrelated Chinese patients with WD. No mutations were detected in coding and splice site sequence in the human MURR1 gene. A novel polymorphism 3'+119T-->A in the 3' untranslated region (UTR) was identified in three healthy individuals and four patients with two disease-causing mutations in the ATP7B gene and a great diversity of clinical presentations. Of the ATP7B mutations reported here, Gly1268Arg is a novel one. Also, the previously described nucleotide change IVS2+63C-->G was detected in 31.66% of normal chromosomes and 26.15% of WD chromosomes. The results have indicated that there is no correlation between MURR1 and WD in the Chinese population.

3' Untranslated Regions↗
PubMed2006

Mutational analysis of beta-catenin and the RAS-RAF signalling pathway in early flat-type colorectal tumours.

Morphologically, early colorectal tumours can be divided into two groups, protruded-type and flat-type. However, little is known about genetic mechanisms of the latter. We investigated mutations of beta-catenin, KRAS, BRAF, and PIK3CA in 310 early colorectal tumours. beta-catenin mutation was detected in 7.1% of 310 tumours. beta-catenin mutation was detected in a significantly higher percentage of flat-type tumours with depressed areas (4/17, 23.5%) than in other tumours (18/293, 6.1%; p=0.0246). KRAS, BRAF, and PIK3CA mutations were detected in 21.6%, 5.4%, and 1.0% of 310 tumours, respectively. Concomitant mutations of beta-catenin and KRAS or BRAF were detected in seven tumours. Mutation of at least one gene was detected in a significantly higher percentage of flat-type tumour tissues (75/193, 38.9%) than in protruded-type tumour tissues (25/117, 21.4%; p=0.0014), and it was correlated significantly with size (p=0.0001). In conclusion, beta-catenin mutation seemed to play an important role in flat-type tumours, especially in those with depressed areas. The genetic abnormalities could arise and accumulate in the early stage of colorectal tumourigenesis, and seem to contribute to the development of flat-type tumour.

Aged↗
PubMed2004

IgV(H) and bcl6 somatic mutation analysis reveals the heterogeneity of cutaneous B-cell lymphoma, and indicates the presence of undisclosed local antigens.

Our understanding of the ontology of B-cell lymphomas (BCL) has been improved by the study of mutational status of IgV(H) and bcl6 genes, but only a few cases of cutaneous BCL have been examined for this status. We analyzed IgV(H) and bcl6 somatic mutations in 10 cutaneous BCL, classified as follicular (three primary and one secondary), primary marginal zone (two cases), and diffuse large BCL (three primary and one secondary). We observed a lower rate (<2%) of IgV(H) mutation in all marginal zone lymphomas, and a preferential usage of V(H)2-70 (one primary follicular and two primary diffuse large BCL). Fewer than expected replacement mutations in framework regions (FR) were observed in three primary follicular lymphomas (FLs) and in all diffuse large BCL, indicating a negative antigen selection pressure. Ongoing mutations were observed in eight of 10 cases. Only two primary FLs and two diffuse large BCL showed bcl6 somatic mutation. These data support the heterogeneous nature of the different cutaneous BCL, and specifically the distinction between cutaneous follicular and marginal zone lymphomas. The biased usage of V(H)2-70, the low rate of replacement mutation in the FR, and the presence of ongoing mutation imply that local antigens could modulate the growth of primary cutaneous BCL.

Adult↗