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Sample records for diverse point mutations

  1. Diverse point mutations in the human glucose-6-phosphate dehydrogenase gene cause enzyme deficiency and mild or severe hemolytic anemia

    SciTech Connect

    Vulliamy, T.J.; D'Urso, M.; Battistuzzi, G.; Estrada, M.; Foulkes, N.S.; Martini, G.; Calabro, V.; Poggi, V.; Giordano, R.; Town, M.; Luzzatto, L.; Persico, M.G. )

    1988-07-01

    Glucose-6-phosphate dehydrogenase deficiency is a common genetic abnormality affecting an estimated 400 million people worldwide. Clinical and biochemical analyses have identified many variants exhibiting a range of phenotypes, which have been well characterized from the hematological point of view. However, until now, their precise molecular basis has remained unknown. The authors have cloned and sequenced seven mutant G6PD alleles. In the nondeficient polymorphic African variant G6PD A they have found a single point mutation. The other six mutants investigated were all associated with enzyme deficiency. The mutations observed show a striking predominance of C {yields} T transitions, with CG doublets involved in four of seven cases. Thus, diverse point mutations may account largely for the phenotypic heterogeneity of G6PD deficiency.

  2. High Inter-Individual Diversity of Point Mutations, Insertions, and Deletions in Human Influenza Virus Nucleoprotein-Specific Memory B Cells

    PubMed Central

    Bussmann, Bianca M.; Horn, Susanne; Sieg, Michael; Jassoy, Christian

    2015-01-01

    The diversity of virus-specific antibodies and of B cells among different individuals is unknown. Using single-cell cloning of antibody genes, we generated recombinant human monoclonal antibodies from influenza nucleoprotein-specific memory B cells in four adult humans with and without preceding influenza vaccination. We examined the diversity of the antibody repertoires and found that NP-specific B cells used numerous immunoglobulin genes. The heavy chains (HCs) originated from 26 and the kappa light chains (LCs) from 19 different germ line genes. Matching HC and LC chains gave rise to 43 genetically distinct antibodies that bound influenza NP. The median lengths of the CDR3 of the HC, kappa and lambda LC were 14, 9 and 11 amino acids, respectively. We identified changes at 13.6% of the amino acid positions in the V gene of the antibody heavy chain, at 8.4 % in the kappa and at 10.6 % in the lambda V gene. We identified somatic insertions or deletions in 8.1% of the variable genes. We also found several small groups of clonal relatives that were highly diversified. Our findings demonstrate broadly diverse memory B cell repertoires for the influenza nucleoprotein. We found extensive variation within individuals with a high number of point mutations, insertions, and deletions, and extensive clonal diversification. Thus, structurally conserved proteins can elicit broadly diverse and highly mutated B-cell responses. PMID:26086076

  3. Decoding germline de novo point mutations.

    PubMed

    Goriely, Anne

    2016-07-27

    Analysis of a large whole-genome sequencing data set of 36,441 high-quality de novo mutations (DNMs) that arose in 816 family trios provides an unprecedented view into the landscape of DNMs in the germ line. This work both refines and challenges some of the views previously held on the nature and origin of DNMs. PMID:27463396

  4. The point mutation process in proteins

    NASA Technical Reports Server (NTRS)

    Schwartz, R. M.; Dayhoff, M. O.

    1978-01-01

    An optimized scoring matrix for residue-by-residue comparisons of distantly related protein sequences has been developed. The scoring matrix is based on observed exchanges and mutabilities of amino acids in 1572 closely related sequences derived from a cross-section of protein groups. Very few superimposed or parallel mutations are included in the data. The scoring matrix is most useful for demonstrating the relatedness of proteins between 65 and 85% different.

  5. Laser desorption mass spectrometry for point mutation detection

    SciTech Connect

    Taranenko, N.I.; Chung, C.N.; Zhu, Y.F.

    1996-12-31

    A point mutation can be associated with the pathogenesis of inherited or acquired diseases. Laser desorption mass spectrometry coupled with allele specific polymerase chain reaction (PCR) was first used for point mutation detection. G551D is one of several mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene present in 1-3% of the mutant CFTR alleles in most European populations. In this work, two different approaches were pursued to detect G551D point mutation in the cystic fibrosis gene. The strategy is to amplify the desired region of DNA template by PCR using two primers that overlap one base at the site of the point mutation and which vary in size. If the two primers based on the normal sequence match the target DNA sequence, a normal PCR product will be produced. However, if the alternately sized primers that match the mutant sequence recognize the target DNA, an abnormal PCR product will be produced. Thus, the mass spectrometer can be used to identify patients that are homozygous normal, heterozygous for a mutation or homozygous abnormal at a mutation site. Another approach to identify similar mutations is the use of sequence specific restriction enzymes which respond to changes in the DNA sequence. Mass spectrometry is used to detect the length of the restriction fragments by digestion of a PCR generated target fragment. 21 refs., 10 figs., 2 tabs.

  6. Laser desorption mass spectrometry for point mutation detection

    SciTech Connect

    Taranenko, N.I.; Chung, C.N.; Zhu, Y.F.

    1996-10-01

    A point mutation can be associated with the pathogenesis of inherited or acquired diseases. Laser desorption mass spectrometry coupled with allele specific polymerase chain reaction (PCR) was first used for point mutation detection. G551D is one of several mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene present in 1-3% of the mutant CFTR alleles in most European populations. In this work, two different approaches were pursued to detect G551D point mutation in the cystic fibrosis gene. The strategy is to amplify the desired region of DNA template by PCR using two primers that overlap one base at the site of the point mutation and which vary in size. If the two primers based on the normal sequence match the target DNA sequence, a normal PCR product will be produced. However, if the alternately sized primers that match the mutant sequence recognize the target DNA, an abnormal PCR product will be produced. Thus, the mass spectrometer can be used to identify patients that are homozygous normal, heterozygous for a mutation or homozygous abnormal at a mutation site. Another approach to identify similar mutations is the use of sequence specific restriction enzymes which respond to changes in the DNA sequence. Mass spectrometry is used to detect the length of the restriction fragments generated by digestion of a PCR generated target fragment. 21 refs., 10 figs., 2 tabs.

  7. Diverse growth hormone receptor gene mutations in Laron syndrome

    SciTech Connect

    Berg, M.A.; Francke, U. ); Gracia, R.; Rosenbloom, A.; Toledo, S.P.A. ); Chernausek, S. ); Guevara-Aguirre, J. ); Hopp, M. ); Rosenbloom, A.; Argente, J. ); Toledo, S.P.A. )

    1993-05-01

    To better understand the molecular genetic basis and genetic epidemiology of Laron syndrome (growth-hormone insensitivity syndrome), the authors analysed the growth-hormone receptor (GHR) genes of seven unrelated affected individuals from the United States, South America, Europe, and Africa. They amplified all nine GHR gene exons and splice junctions from these individuals by PCR and screened the products for mutations by using denaturing gradient gel electrophoresis (DGGE). They identified a single GHR gene fragment with abnormal DGGE results for each affected individual, sequenced this fragment, and, in each case, identified a mutation likely to cause Laron syndrome, including two nonsense mutations (R43X and R217X), two splice-junction mutations, (189-1 G to T and 71+1 G to A), and two frameshift mutations (46 del TT and 230 del TA or AT). Only one of these mutations, R43X, has been previously reported. Using haplotype analysis, they determined that this mutation, which involves a CpG dinucleotide hot spot, likely arose as a separate event in this case, relative to the two prior reports of R43X. Aside from R43X, the mutations identified are unique to patients from particular geographic regions. Ten GHR gene mutations have now been described in this disorder. The authors conclude that Laron syndrome is caused by diverse GHR gene mutations, including deletions, RNA processing defects, translational stop codons, and missense codons. All the identified mutations involve the extracellular domain of the receptor, and most are unique to particular families or geographic areas. 35 refs., 3 figs., 1 tab.

  8. Predicting folding free energy changes upon single point mutations

    PubMed Central

    Zhang, Zhe; Wang, Lin; Gao, Yang; Zhang, Jie; Zhenirovskyy, Maxim; Alexov, Emil

    2012-01-01

    Motivation: The folding free energy is an important characteristic of proteins stability and is directly related to protein's wild-type function. The changes of protein's stability due to naturally occurring mutations, missense mutations, are typically causing diseases. Single point mutations made in vitro are frequently used to assess the contribution of given amino acid to the stability of the protein. In both cases, it is desirable to predict the change of the folding free energy upon single point mutations in order to either provide insights of the molecular mechanism of the change or to design new experimental studies. Results: We report an approach that predicts the free energy change upon single point mutation by utilizing the 3D structure of the wild-type protein. It is based on variation of the molecular mechanics Generalized Born (MMGB) method, scaled with optimized parameters (sMMGB) and utilizing specific model of unfolded state. The corresponding mutations are built in silico and the predictions are tested against large dataset of 1109 mutations with experimentally measured changes of the folding free energy. Benchmarking resulted in root mean square deviation = 1.78 kcal/mol and slope of the linear regression fit between the experimental data and the calculations was 1.04. The sMMGB is compared with other leading methods of predicting folding free energy changes upon single mutations and results discussed with respect to various parameters. Availability: All the pdb files we used in this article can be downloaded from http://compbio.clemson.edu/downloadDir/mentaldisorders/sMMGB_pdb.rar Contact: ealexov@clemson.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:22238268

  9. The Y-chromosome point mutation rate in humans.

    PubMed

    Helgason, Agnar; Einarsson, Axel W; Guðmundsdóttir, Valdís B; Sigurðsson, Ásgeir; Gunnarsdóttir, Ellen D; Jagadeesan, Anuradha; Ebenesersdóttir, S Sunna; Kong, Augustine; Stefánsson, Kári

    2015-05-01

    Mutations are the fundamental source of biological variation, and their rate is a crucial parameter for evolutionary and medical studies. Here we used whole-genome sequence data from 753 Icelandic males, grouped into 274 patrilines, to estimate the point mutation rate for 21.3 Mb of male-specific Y chromosome (MSY) sequence, on the basis of 1,365 meioses (47,123 years). The combined mutation rate for 15.2 Mb of X-degenerate (XDG), X-transposed (XTR) and ampliconic excluding palindromes (rAMP) sequence was 8.71 × 10(-10) mutations per position per year (PPPY). We observed a lower rate (P = 0.04) of 7.37 × 10(-10) PPPY for 6.1 Mb of sequence from palindromes (PAL), which was not statistically different from the rate of 7.2 × 10(-10) PPPY for paternally transmitted autosomes. We postulate that the difference between PAL and the other MSY regions may provide an indication of the rate at which nascent autosomal and PAL de novo mutations are repaired as a result of gene conversion. PMID:25807285

  10. Repository of mutations from Oman: The entry point to a national mutation database

    PubMed Central

    Rajab, Anna; Hamza, Nishath; Al Harasi, Salma; Al Lawati, Fatma; Gibbons, Una; Al Alawi, Intesar; Kobus, Karoline; Hassan, Suha; Mahir, Ghariba; Al Salmi, Qasim; Mons, Barend; Robinson, Peter

    2015-01-01

    The Sultanate of Oman is a rapidly developing Muslim country with well-organized government-funded health care services, and expanding medical genetic facilities. The preservation of tribal structures within the Omani population coupled with geographical isolation has produced unique patterns of rare mutations. In order to provide diagnosticians and researchers with access to an up-to-date resource that will assist them in their daily practice we collated and analyzed all of the Mendelian disease-associated mutations identified in the Omani population. By the 1 st of August 2015, the dataset contained 300 mutations detected in over 150 different genes. More than half of the data collected reflect novel genetic variations that were first described in the Omani population, and most disorders with known mutations are inherited in an autosomal recessive fashion. A number of novel Mendelian disease genes have been discovered in Omani nationals, and the corresponding mutations are included here. The current study provides a comprehensive resource of the mutations in the Omani population published in scientific literature or reported through service provision that will be useful for genetic care in Oman and will be a starting point for variation databases as next-generation sequencing technologies are introduced into genetic medicine in Oman. PMID:26594346

  11. VACTERL with the mitochondrial np 3243 point mutation.

    PubMed

    Damian, M S; Seibel, P; Schachenmayr, W; Reichmann, H; Dorndorf, W

    1996-04-24

    The VACTERL association of vertebral, anal, cardiovascular, tracheo-esophageal, renal, and limb defects is one of the more common congenital disorders with limb deficiency arising during blastogenesis. The cause is probably heterogeneous; a molecular basis has not yet been defined. We report on a family in which a female infant with VACTERL was born in 1977 and died at age 1 month due to renal failure. Because her mother and sister later developed classical mitochondrial cytopathy associated with the A-G point mutation at nucleotide position (np) 3243 of mitochondrial (mt) DNA, we performed a molecular analysis of mt DNA in preserved kidney tissue from the VACTERL case. We discovered 100% mutant mt DNA in multicystic and 32% mutant mt DNA in normal kidney tissue. Mild deficiency of complex I respiratory chain enzyme activity was found in the mother's muscle biopsy. Other maternal relatives were healthy but had low levels of mutant mt DNA in blood. This is the first report to provide a precise molecular basis for a case of VACTERL. The differing tissue pathology depending on the percentage of mutant mt DNA suggests a causal connection between the mutation and symptoms. VACTERL, and this type of multicystic renal dysplasia, are new phenotypes for the np 3243 point mutation. The possibility of a mitochondrial disorder should be born in mind and also that VACTERL may occur as a first manifestation of a mutation that has been present for generations. This would have major implications for patient management and for genetic counselling regarding both the risk of recurrence and risk of other mitochondrial syndromes in affected families. PMID:8723071

  12. VACTERL with the mitochondrial NP 3243 point mutation

    SciTech Connect

    Damian, M.S.; Dorndorf, W.; Schachenmayr, W.; Seibel, P.; Reichmann, H.

    1996-04-24

    The VACTERL association of vertebral, anal, cardiovascular, tracheo-esophageal, renal, and limb defects is one of the more common congenital disorders with limb deficiency arising during blastogenesis. The cause is probably heterogeneous; a molecular basis has not been defined. We report on a family in which a female infant with VACTERL was born in 1977 and died at age 1 month due to renal failure. Because her mother and sister later developed classical mitochondrial cytopathy associated with the A-G point mutation at nucleotide position (np) 3243 of mitochondrial (mt) DNA, we performed a molecular analysis of mt DNA in preserved kidney tissue from the VACTERL case. We discovered 100% mutant mt DNA in multicystic and 32% mutant mt DNA in normal kidney tissue. Mild deficiency of complex I respiratory chain enzyme activity was found in the mother`s muscle biopsy. Other maternal relatives were healthy but had low levels of mutant mt DNA in blood. This is the first report to provide a precise molecular basis for a case of VACTERL. The differing tissue pathology depending on the percentage of mutant mt DNA suggests a causal connection between the mutation and symptoms. VACTERL, and this type of multicystic renal dysplasia, are new phenotypes for the np 3243 point mutation. The possibility of a mitochondrial disorder should be born in mind and also that VACTERL may occur as a first manifestation of a mutation that has been present for generations. This would have major implications for patient management and for genetic counselling regarding both the risk of recurrence and risk of other mitochondrial syndromes in affected families. 19 refs., 3 figs., 1 tab.

  13. Racial diversity of actionable mutations in non-small cell lung cancer

    PubMed Central

    Bollig-Fischer, Aliccia; Chen, Wei; Gadgeel, Shirish M.; Wenzlaff, Angela S.; Cote, Michele L.; Schwartz, Ann G.; Bepler, Gerold

    2014-01-01

    Introduction Lung cancer is the leading cause of cancer-related deaths in the United States. The reasons for higher incidence and poorer survival rates among black compared to white lung cancer patients have not been defined. We hypothesized that differential incidence of somatic cancer gene mutations may be a contributing factor. Previous genomic studies of non-small cell lung cancer (NSCLC) have not adequately represented black patients. Methods A MALDI-TOF mass-spectrometry approach was used to analyze tumor DNA for 214 coding mutations in 26 cancer genes previously identified in NSCLC. The samples included NSCLC from 335 white patients and 137 black patients. For 299 of these, normal matched DNA was available and analyzed. Results EGFR exon 19 deletions were only detected in female cases, with increased odds for black women compared to white women (odds ratio=3.914, 95% CI: 1.014–15.099, p=0.048). Beyond race, variations in mutation frequencies were seen by histology. DDR2 alterations, previously described as somatic mutations, were identified as constitutional variants. Conclusions This study is among the largest comparing somatic mutations in black and white patients. The results point to the molecular diversity of NSCLC and raise new questions as to the importance of inherited alleles. Genomic tumor testing will benefit both populations, although the mutation spectrum appears to vary by sex, race, and histology. PMID:25376516

  14. EPHA2 MUTATIONS CONTRIBUTE TO CONGENITAL CATARACT THROUGH DIVERSE MECHANISMS

    PubMed Central

    Dave, Alpana; Martin, Sarah; Kumar, Raman; Craig, Jamie E.; Burdon, Kathryn P.

    2016-01-01

    Purpose Congenital cataract is a leading cause of childhood blindness. Mutations in the EPHA2 gene are one of the causes of inherited congenital cataract. The EPHA2 gene encodes a membrane-bound tyrosine kinase receptor and is highly expressed in epithelial cells, including in the ocular lens. Signaling through the EPHA2 receptor plays a pivotal role in epithelial cell homeostasis. The aim of this study was to determine the effect of congenital cataract causing mutations in the EPHA2 gene on the encoded protein in epithelial cells. Methods The effect of five disease-causing mutations, p.P584L (c.1751C>T), p.T940I (c.2819C>T), p.D942fsXC71 (c.2826–9G>A), p.A959T (c.2875G>A), and p.V972GfsX39 (c.2915_2916delTG), on localization of the protein was examined in two in vitro epithelial cell culture systems: Madin-Darby Canine Kidney (MDCK) and human colorectal adenocarcinoma (Caco-2) epithelial cells. Myc-tagged mutant constructs were generated by polymerase chain reaction (PCR)-based mutagenesis. The Myc-tagged wild-type construct was used as a control. The Myc-tagged wild-type and mutant proteins were ectopically expressed and detected by immunofluorescence labeling. Results Two of the mutations, p.T940I and p.D942fsXC71, located within the cytoplasmic sterile-α-motif (SAM) domain of EPHA2, led to mis-localization of the protein to the perinuclear space and co-localization with the cis-golgi apparatus, indicating sub-organellar/cellular retention of the mutant proteins. The mutant proteins carrying the remaining three mutations, similar to the wild-type EPHA2, localized to the cell membrane. Conclusions Mis-localization of two of the mutant proteins in epithelial cells suggests that some disease-causing mutations in EPHA2 likely affect lens epithelial cell homeostasis and contribute to cataract. This study suggests that mutations in EPHA2 contribute to congenital cataract through diverse mechanisms. PMID:26900323

  15. Point Mutation in Essential Genes with Loss or Mutation of the Second Allele

    PubMed Central

    Beck-Engeser, Gabriele B.; Monach, Paul A.; Mumberg, Dominik; Yang, Farley; Wanderling, Sherry; Schreiber, Karin; Espinosa, Rafael; Le Beau, Michelle M.; Meredith, Stephen C.; Schreiber, Hans

    2001-01-01

    Antigens that are tumor specific yet retained by tumor cells despite tumor progression offer stable and specific targets for immunologic and possibly other therapeutic interventions. Therefore, we have studied two CD4+ T cell–recognized tumor-specific antigens that were retained during evolution of two ultraviolet-light–induced murine cancers to more aggressive growth. The antigens are ribosomal proteins altered by somatic tumor-specific point mutations, and the progressor (PRO) variants lack the corresponding normal alleles. In the first tumor, 6132A-PRO, the antigen is encoded by a point-mutated L9 ribosomal protein gene. The tumor lacks the normal L9 allele because of an interstitial deletion from chromosome 5. In the second tumor, 6139B-PRO, both alleles of the L26 gene have point mutations, and each encodes a different tumor-specific CD4+ T cell–recognized antigen. Thus, for both L9 and L26 genes, we observe “two hit” kinetics commonly observed in genes suppressing tumor growth. Indeed, reintroduction of the lost wild-type L9 allele into the 6132A-PRO variant suppressed the growth of the tumor cells in vivo. Since both L9 and L26 encode proteins essential for ribosomal biogenesis, complete loss of the tumor-specific target antigens in the absence of a normal allele would abrogate tumor growth. PMID:11489948

  16. Functional diversity and mutational analysis of Agrobacterium 6B oncoproteins.

    PubMed

    Helfer, A; Pien, S; Otten, L

    2002-07-01

    Many Agrobacterium T-DNA genes belong to a diverse family of T-DNA genes, the rolB family. These genes cause various growth abnormalities but their modes of action remain largely unknown. So far, none of the RolB-like proteins has been subjected to mutational analysis. The RolB-like oncoprotein 6B, which induces tumours on species such as Nicotiana glauca and Kalanchoe tubiflora, was chosen to investigate the role of the most conserved amino acid residues within the RolB family. We first determined which of the natural 6B variants had the strongest oncogenic activity; to this end, six 6b coding sequences (A- 6b, AB- 6b, C- 6b, CG- 6b, S- 6b and T- 6b) were placed under the control of the strong constitutive 2x35S promoter and compared for tumour induction on N. glauca, N. tabacum and K. daigremontiana. Oncogenicity increased in the order C- 6b/CG- 6b, A- 6b/AB- 6b, and S- 6b/T- 6b. The most conserved amino acid residues in the strongly oncogenic T-6B protein were mutated and shown to be required for oncogenicity and accumulation of the T-6B protein in planta but not in bacteria. Hybrids between T-6B and the weakly oncogenic A-6B protein revealed an additional oncogenic determinant required for the formation of large tumours. PMID:12172796

  17. On the origin of deletions and point mutations in Duchenne muscular dystrophy: most deletions arise in oogenesis and most point mutations result from events in spermatogenesis.

    PubMed Central

    Grimm, T; Meng, G; Liechti-Gallati, S; Bettecken, T; Müller, C R; Müller, B

    1994-01-01

    We present the results of a study of the rate and origin of mutations in Duchenne muscular dystrophy (DMD). Depending on the type of mutation (deletion/duplication or point mutation) present in the patient, there are widely varying ratios of male to female mutation rates. In deletions, the male mutation rate is only 30% of the female one. In non-deletional/non-duplicational mutations (presumably containing a high proportion of point mutations) the male mutation rate is at least 2.2 as high as the female one and probably much higher. Allowing for the presence of autosomal recessive phenocopies we find that k in non-deletional/non-duplicational mutations is 40.3. These findings mean that the vast majority of deletions arise in oogenesis, while most point mutations stem from spermatogenesis. Previous investigations have shown that in other diseases and genes, most notably haemophilia B and A, but also the ZFY and ZFX genes, the male mutation rate for point mutations tends to be higher than the female one. Our results can be seen as a confirmation of this for the special case of DMD. The influence on risk figures is considerable. As an example, the risk of the mother of an isolated case of DMD without an apparent structural anomaly of the gene of being a carrier increases from 67% to at least 76%. Given the estimate of 40.3 for k, allowing for the presence of autosomal recessive phenocopies mentioned above, it increases even further to 98%. However, as confidence intervals are still large, more data are needed to improve the estimates. Germinal mosaicism in this context is discussed. PMID:8014964

  18. Impact of Mutation Type and Amplicon Characteristics on Genetic Diversity Measures Generated Using a High-Resolution Melting Diversity Assay

    PubMed Central

    Cousins, Matthew M.; Donnell, Deborah; Eshleman, Susan H.

    2013-01-01

    We adapted high-resolution melting (HRM) technology to measure genetic diversity without sequencing. Diversity is measured as a single numeric HRM score. Herein, we determined the impact of mutation types and amplicon characteristics on HRM diversity scores. Plasmids were generated with single-base changes, insertions, and deletions. Different primer sets were used to vary the position of mutations within amplicons. Plasmids and plasmid mixtures were analyzed to determine the impact of mutation type, position, and concentration on HRM scores. The impact of amplicon length and G/C content on HRM scores was also evaluated. Different mutation types affected HRM scores to varying degrees (1-bp deletion < 1-bp change < 3-bp insertion < 9-bp insertion). The impact of mutations on HRM scores was influenced by amplicon length and the position of the mutation within the amplicon. Mutations were detected at concentrations of 5% to 95%, with the greatest impact at 50%. The G/C content altered melting temperature values of amplicons but had no impact on HRM scores. These data are relevant to the design of assays that measure genetic diversity using HRM technology. PMID:23178437

  19. Marcus model of spontaneous point mutation in DNA

    NASA Astrophysics Data System (ADS)

    Turaeva, N.; Brown-Kennerly, V.

    2015-11-01

    The theoretical model of Löwdin's mechanism of spontaneous mutation based on 2D Marcus theory of DPT has been proposed in this work. The equation for the kinetics of DPT during DNA replication has been established, and the expression for the probability of spontaneous mutation has been received. The probability of spontaneous mutation formation has been estimated for tautomeric G∗-C∗ complexes, which is in the range of experimental results. The probability of spontaneous mutation as a function of temperature, replication rate, and solvent effect has been discussed. It increases with temperature and decreases with replication rate. The solvent and pH effects on the probability of spontaneous mutation can also be discussed within the framework of the model.

  20. A computer program for the estimation of protein and nucleic acid sequence diversity in random point mutagenesis libraries

    PubMed Central

    Volles, Michael J.; Lansbury, Peter T.

    2005-01-01

    A computer program for the generation and analysis of in silico random point mutagenesis libraries is described. The program operates by mutagenizing an input nucleic acid sequence according to mutation parameters specified by the user for each sequence position and type of point mutation. The program can mimic almost any type of random mutagenesis library, including those produced via error-prone PCR (ep-PCR), mutator Escherichia coli strains, chemical mutagenesis, and doped or random oligonucleotide synthesis. The program analyzes the generated nucleic acid sequences and/or the associated protein library to produce several estimates of library diversity (number of unique sequences, point mutations, and single point mutants) and the rate of saturation of these diversities during experimental screening or selection of clones. This information allows one to select the optimal screen size for a given mutagenesis library, necessary to efficiently obtain a certain coverage of the sequence-space. The program also reports the abundance of each specific protein mutation at each sequence position, which is useful as a measure of the level and type of mutation bias in the library. Alternatively, one can use the program to evaluate the relative merits of preexisting libraries, or to examine various hypothetical mutation schemes to determine the optimal method for creating a library that serves the screen/selection of interest. Simulated libraries of at least 109 sequences are accessible by the numerical algorithm with currently available personal computers; an analytical algorithm is also available which can rapidly calculate a subset of the numerical statistics in libraries of arbitrarily large size. A multi-type double-strand stochastic model of ep-PCR is developed in an appendix to demonstrate the applicability of the algorithm to amplifying mutagenesis procedures. Estimators of DNA polymerase mutation-type-specific error rates are derived using the model. Analyses of an

  1. A point mutation and a RNA processing mutation in a carbamyl phosphate synthetase I (CPSI) deficient patient

    SciTech Connect

    Hall, L.; Summer, M.; Sierra-Rivera, E.; Freeman, M.

    1994-09-01

    Deficiency of carbamyl phosphate synthetase I (CPSID) results in a life-threatening disease due to hyperammonemia. A better understanding of the molecular basis of CPSID was achieved by studying the genetic defects in a CPSID patient. CPSI message was analyzed from hepatic tissue through Northern blot analysis, reverse transcription of liver mRNA followed by polymerase chain reaction amplification (RT-PCR), dideoxy fingerprinting, and direct DNA sequencing. Northern blot analysis of the patient revealed a diminished amount of normal sized CPSI message and multiple other bands not detected in controls. Analysis of the amplified coding region revealed a single point mutation leading to an asparagine to lysine substitution at codon 715. The patient`s cDNA was homozygous and genomic DNA heterozygous for the point mutation which was not found in ten unrelated CPSID patients. The point mutation causes a change from a highly-conserved neutral amino acid to a polar basic residue within a nucleotide/bicarbonate binding domain which points to its importance in normal CPSI function. The other allele which was absent in RT-PCR fragements presumably leads to the multi-form poly-A message detected by Northern blot analysis and allows the point mutation to become the dominant expressed allele. These mutations represent the second reported molecular defect in CPSI and the first to involve a mutation in a functional domain and in RNA processing.

  2. Clonal diversity of recurrently mutated genes in myelodysplastic syndromes

    PubMed Central

    Walter, MJ; Shen, D; Shao, J; Ding, L; White, BS; Kandoth, C; Miller, CA; Niu, B; McLellan, MD; Dees, ND; Fulton, R; Elliot, K; Heath, S; Grillot, M; Westervelt, P; Link, DC; DiPersio, JF; Mardis, E; Ley, TJ; Wilson, RK; Graubert, TA

    2013-01-01

    Recent studies suggest that most cases of myelodysplastic syndrome (MDS) are clonally heterogeneous, with a founding clone and multiple subclones. It is not known whether specific gene mutations typically occur in founding clones or subclones. We screened a panel of 94 candidate genes in a cohort of 157 patients with MDS or secondary acute myeloid leukemia (sAML). This included 150 cases with samples obtained at MDS diagnosis and 15 cases with samples obtained at sAML transformation (8 were also analyzed at the MDS stage). We performed whole-genome sequencing (WGS) to define the clonal architecture in eight sAML genomes and identified the range of variant allele frequencies (VAFs) for founding clone mutations. At least one mutation or cytogenetic abnormality was detected in 83% of the 150 MDS patients and 17 genes were significantly mutated (false discovery rate ≤0.05). Individual genes and patient samples displayed a wide range of VAFs for recurrently mutated genes, indicating that no single gene is exclusively mutated in the founding clone. The VAFs of recurrently mutated genes did not fully recapitulate the clonal architecture defined by WGS, suggesting that comprehensive sequencing may be required to accurately assess the clonal status of recurrently mutated genes in MDS. PMID:23443460

  3. Single Quantum Dot Analysis Enables Multiplexed Point Mutation Detection by Gap Ligase Chain Reaction

    PubMed Central

    Song, Yunke; Zhang, Yi; Wang, Tza-Huei

    2014-01-01

    Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and tedious assay processes. In this report, we propose an assay technology which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single molecule coincidence detection and superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification. PMID:23239594

  4. Ancient founder mutation is responsible for Imerslund-Gräsbeck Syndrome among diverse ethnicities

    PubMed Central

    2011-01-01

    Background Imerslund-Gräsbeck syndrome (IGS) was described just over 50 years ago by Olga Imerslund and Ralph Gräsbeck and colleagues. IGS is caused by specific malabsorption of cobalamin (Cbl) due to bi-allelic mutations in either the cubilin gene (CUBN) or the human amnionless homolog (AMN). Mutations in the two genes are commonly seen in founder populations or in societies with a high degree of consanguineous marriages. One particular mutation in AMN, c.208-2A>G, causing an out-of-frame loss of exon 4 in the mRNA, is responsible for some 15% of IGS cases globally. We present evidence that this founder mutation causes a substantial percentage of cases among diverse ethnicities and that the mutation is as old as human civilization. Methods Partial genotyping indicated a founder event but its presence in diverse peoples of Arabic, Turkish, Jewish, and Hispanic ancestry suggested that the mutation might be recurrent. We therefore studied the flanking sequence spanning 3.5 Mb to elucidate the origin of the haplotype and estimate the age of the mutation using a Bayesian inference method based on observed linkage disequilibrium. Results The mutation's distribution, the size of the shared haplotype, and estimates of growth rate and carrier frequency indicated that the mutation was a single prehistoric event. Dating back to the ancient Middle East around 11,600 BC, the mutation predates the advent of writing, farming, and the monotheistic religions of the region. Conclusions This mutation causes over 50% of the IGS cases among Arabic, Turkish, and Sephardic Jewish families, making it a primary target for genetic screening among diverse IGS cases originating from the Middle East. Thus, rare founder mutations may cause a substantial number of cases, even among diverse ethnicities not usually thought to be related. PMID:22078000

  5. Automated detection of point mutations using fluorescent sequence trace subtraction.

    PubMed Central

    Bonfield, J K; Rada, C; Staden, R

    1998-01-01

    The final step in the detection of mutations is to determine the sequence of the suspected mutant and to compare it with that of the wild-type, and for this fluorescence-based sequencing instruments are widely used. We describe some simple algorithms forcomparing sequence traces which, as part of our sequence assembly and analysis package, are proving useful for the discovery of mutations and which may also help to identify misplaced readings in sequence assembly projects. The mutations can be detected automatically by a new program called TRACE_DIFF and new types of trace display in our program GAP4 greatly simplify visual checking of the assigned changes. To assess the accuracy of the automatic mutation detection algorithm we analysed 214 sequence readings from hypermutating DNA comprising a total of 108 497 bases. After the readings were assembled there were 1232 base differences, including 392 Ns and 166 alignment characters. Visual inspection of the traces established that of the 1232 differences, 353 were real mutations while the rest were due to base calling errors. The TRACE_DIFF algorithm automatically identified all but 36, with 28 false positives. Further information about the software can be obtained from http://www.mrc-lmb.cam.ac.uk/pubseq/ PMID:9649626

  6. Calmodulin Point Mutations Affect Drosophila Development and Behavior

    PubMed Central

    Nelson, H. B.; Heiman, R. G.; Bolduc, C.; Kovalick, G. E.; Whitley, P.; Stern, M.; Beckingham, K.

    1997-01-01

    Calmodulin (CAM) is recognized as a major intermediary in intracellular calcium signaling, but as yet little is known of its role in developmental and behavioral processes. We have generated and studied mutations to the endogenous Cam gene of Drosophila melanogaster that change single amino acids within the protein coding region. One of these mutations produces a striking pupal lethal phenotype involving failure of head eversion. Various mutant combinations produce specific patterns of ectopic wing vein formation or melanotic scabs on the cuticle. Anaphase chromosome bridging is also seen as a maternal effect during the early embryonic nuclear divisions. In addition, specific behavioral defects such as poor climbing and flightlessness are detected among these mutants. Comparisons with other Drosophila mutant phenotypes suggests potential CAM targets that may mediate these developmental and behavioral effects, and analysis of the CAM crystal structure suggests the structural consequences of the individual mutations. PMID:9409836

  7. Phylogenetic diversity, functional trait diversity and extinction: avoiding tipping points and worst-case losses

    PubMed Central

    Faith, Daniel P.

    2015-01-01

    The phylogenetic diversity measure, (‘PD’), measures the relative feature diversity of different subsets of taxa from a phylogeny. At the level of feature diversity, PD supports the broad goal of biodiversity conservation to maintain living variation and option values. PD calculations at the level of lineages and features include those integrating probabilities of extinction, providing estimates of expected PD. This approach has known advantages over the evolutionarily distinct and globally endangered (EDGE) methods. Expected PD methods also have limitations. An alternative notion of expected diversity, expected functional trait diversity, relies on an alternative non-phylogenetic model and allows inferences of diversity at the level of functional traits. Expected PD also faces challenges in helping to address phylogenetic tipping points and worst-case PD losses. Expected PD may not choose conservation options that best avoid worst-case losses of long branches from the tree of life. We can expand the range of useful calculations based on expected PD, including methods for identifying phylogenetic key biodiversity areas. PMID:25561672

  8. Identification of and Molecular Basis for SIRT6 Loss-of-Function Point Mutations in Cancer.

    PubMed

    Kugel, Sita; Feldman, Jessica L; Klein, Mark A; Silberman, Dafne M; Sebastián, Carlos; Mermel, Craig; Dobersch, Stephanie; Clark, Abbe R; Getz, Gad; Denu, John M; Mostoslavsky, Raul

    2015-10-20

    Chromatin factors have emerged as the most frequently dysregulated family of proteins in cancer. We have previously identified the histone deacetylase SIRT6 as a key tumor suppressor, yet whether point mutations are selected for in cancer remains unclear. In this manuscript, we characterized naturally occurring patient-derived SIRT6 mutations. Strikingly, all the mutations significantly affected either stability or catalytic activity of SIRT6, indicating that these mutations were selected for in these tumors. Further, the mutant proteins failed to rescue sirt6 knockout (SIRT6 KO) cells, as measured by the levels of histone acetylation at glycolytic genes and their inability to rescue the tumorigenic potential of these cells. Notably, the main activity affected in the mutants was histone deacetylation rather than demyristoylation, pointing to the former as the main tumor-suppressive function for SIRT6. Our results identified cancer-associated point mutations in SIRT6, cementing its function as a tumor suppressor in human cancer. PMID:26456828

  9. Haplotype analysis of the 185delAG BRCA1 mutation in ethnically diverse populations

    PubMed Central

    Laitman, Yael; Feng, Bing-Jian; Zamir, Itay M; Weitzel, Jeffrey N; Duncan, Paul; Port, Danielle; Thirthagiri, Eswary; Teo, Soo-Hwang; Evans, Gareth; Latif, Ayse; Newman, William G; Gershoni-Baruch, Ruth; Zidan, Jamal; Shimon-Paluch, Shani; Goldgar, David; Friedman, Eitan

    2013-01-01

    The 185delAG* BRCA1 mutation is encountered primarily in Jewish Ashkenazi and Iraqi individuals, and sporadically in non-Jews. Previous studies estimated that this is a founder mutation in Jewish mutation carriers that arose before the dispersion of Jews in the Diaspora ∼2500 years ago. The aim of this study was to assess the haplotype in ethnically diverse 185delAG* BRCA1 mutation carriers, and to estimate the age at which the mutation arose. Ethnically diverse Jewish and non-Jewish 185delAG*BRCA1 mutation carriers and their relatives were genotyped using 15 microsatellite markers and three SNPs spanning 12.5 MB, encompassing the BRCA1 gene locus. Estimation of mutation age was based on a subset of 11 markers spanning a region of ∼5 MB, using a previously developed algorithm applying the maximum likelihood method. Overall, 188 participants (154 carriers and 34 noncarriers) from 115 families were included: Ashkenazi, Iraq, Kuchin-Indians, Syria, Turkey, Iran, Tunisia, Bulgaria, non-Jewish English, non-Jewish Malaysian, and Hispanics. Haplotype analysis indicated that the 185delAG mutation arose 750–1500 years ago. In Ashkenazim, it is a founder mutation that arose 61 generations ago, and with a small group of founder mutations was introduced into the Hispanic population (conversos) ∼650 years ago, and into the Iraqi–Jewish community ∼450 years ago. The 185delAG mutation in the non-Jewish populations in Malaysia and the UK arose at least twice independently. We conclude that the 185delAG* BRCA1 mutation resides on a common haplotype among Ashkenazi Jews, and arose about 61 generations ago and arose independently at least twice in non-Jews. PMID:22763381

  10. Haplotype analysis of the 185delAG BRCA1 mutation in ethnically diverse populations.

    PubMed

    Laitman, Yael; Feng, Bing-Jian; Zamir, Itay M; Weitzel, Jeffrey N; Duncan, Paul; Port, Danielle; Thirthagiri, Eswary; Teo, Soo-Hwang; Evans, Gareth; Latif, Ayse; Newman, William G; Gershoni-Baruch, Ruth; Zidan, Jamal; Shimon-Paluch, Shani; Goldgar, David; Friedman, Eitan

    2013-02-01

    The 185delAG* BRCA1 mutation is encountered primarily in Jewish Ashkenazi and Iraqi individuals, and sporadically in non-Jews. Previous studies estimated that this is a founder mutation in Jewish mutation carriers that arose before the dispersion of Jews in the Diaspora ~2500 years ago. The aim of this study was to assess the haplotype in ethnically diverse 185delAG* BRCA1 mutation carriers, and to estimate the age at which the mutation arose. Ethnically diverse Jewish and non-Jewish 185delAG*BRCA1 mutation carriers and their relatives were genotyped using 15 microsatellite markers and three SNPs spanning 12.5 MB, encompassing the BRCA1 gene locus. Estimation of mutation age was based on a subset of 11 markers spanning a region of ~5 MB, using a previously developed algorithm applying the maximum likelihood method. Overall, 188 participants (154 carriers and 34 noncarriers) from 115 families were included: Ashkenazi, Iraq, Kuchin-Indians, Syria, Turkey, Iran, Tunisia, Bulgaria, non-Jewish English, non-Jewish Malaysian, and Hispanics. Haplotype analysis indicated that the 185delAG mutation arose 750-1500 years ago. In Ashkenazim, it is a founder mutation that arose 61 generations ago, and with a small group of founder mutations was introduced into the Hispanic population (conversos) ~650 years ago, and into the Iraqi-Jewish community ~450 years ago. The 185delAG mutation in the non-Jewish populations in Malaysia and the UK arose at least twice independently. We conclude that the 185delAG* BRCA1 mutation resides on a common haplotype among Ashkenazi Jews, and arose about 61 generations ago and arose independently at least twice in non-Jews. PMID:22763381

  11. Point Mutations Effects on Charge Transport Properties of the Tumor-Suppressor Gene p53

    NASA Astrophysics Data System (ADS)

    Roemer, Rudolf A.; Shih, Chi-Tin; Roche, Stephan

    2008-03-01

    We report on a theoretical study of point mutations effects on charge transfer properties in the DNA sequence of the tumor-suppressor p53 gene. On the basis of effective tight-binding models which simulate hole propagation along the DNA, a statistical analysis of mutation-induced charge transfer modifications is performed. In contrast to non-cancerous mutations, mutation hotspots tend to result in significantly weaker changes of transmission properties. This suggests that charge transport could play a significant role for DNA-repairing deficiency yielding carcinogenesis.

  12. Site-specific somatic mitochondrial DNA point mutations in patients with thymidine phosphorylase deficiency

    PubMed Central

    Nishigaki, Yutaka; Martí, Ramon; Copeland, William C.; Hirano, Michio

    2003-01-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). This deficiency of TP leads to increased circulating levels of thymidine (deoxythymidine, dThd) and deoxyuridine (dUrd) and has been associated with multiple deletions and depletion of mitochondrial DNA (mtDNA). Here we describe 36 point mutations in mtDNA of tissues and cultured cells from MNGIE patients. Thirty-one mtDNA point mutations (86%) were T-to-C transitions, and of these, 25 were preceded by 5′-AA sequences. In addition, we identified a single base-pair mtDNA deletion and a TT-to-AA mutation. Next-nucleotide effects and dislocation mutagenesis may contribute to the formation of these mutations. These results provide the first demonstration that alterations of nucleoside metabolism can induce multiple sequence-specific point mutations in humans. We hypothesize that, in patients with TP deficiency, increased levels of dThd and dUrd cause mitochondrial nucleotide pool imbalances, which, in turn, lead to mtDNA abnormalities including site-specific point mutations. PMID:12813027

  13. Site-specific somatic mitochondrial DNA point mutations in patients with thymidine phosphorylase deficiency.

    PubMed

    Nishigaki, Yutaka; Martí, Ramon; Copeland, William C; Hirano, Michio

    2003-06-01

    Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by loss-of-function mutations in the gene encoding thymidine phosphorylase (TP). This deficiency of TP leads to increased circulating levels of thymidine (deoxythymidine, dThd) and deoxyuridine (dUrd) and has been associated with multiple deletions and depletion of mitochondrial DNA (mtDNA). Here we describe 36 point mutations in mtDNA of tissues and cultured cells from MNGIE patients. Thirty-one mtDNA point mutations (86%) were T-to-C transitions, and of these, 25 were preceded by 5'-AA sequences. In addition, we identified a single base-pair mtDNA deletion and a TT-to-AA mutation. Next-nucleotide effects and dislocation mutagenesis may contribute to the formation of these mutations. These results provide the first demonstration that alterations of nucleoside metabolism can induce multiple sequence-specific point mutations in humans. We hypothesize that, in patients with TP deficiency, increased levels of dThd and dUrd cause mitochondrial nucleotide pool imbalances, which, in turn, lead to mtDNA abnormalities including site-specific point mutations. PMID:12813027

  14. Structural Analysis of Single-Point Mutations Given an RNA Sequence: A Case Study with RNAMute

    NASA Astrophysics Data System (ADS)

    Churkin, Alexander; Barash, Danny

    2006-12-01

    We introduce here for the first time the RNAMute package, a pattern-recognition-based utility to perform mutational analysis and detect vulnerable spots within an RNA sequence that affect structure. Mutations in these spots may lead to a structural change that directly relates to a change in functionality. Previously, the concept was tried on RNA genetic control elements called "riboswitches" and other known RNA switches, without an organized utility that analyzes all single-point mutations and can be further expanded. The RNAMute package allows a comprehensive categorization, given an RNA sequence that has functional relevance, by exploring the patterns of all single-point mutants. For illustration, we apply the RNAMute package on an RNA transcript for which individual point mutations were shown experimentally to inactivate spectinomycin resistance in Escherichia coli. Functional analysis of mutations on this case study was performed experimentally by creating a library of point mutations using PCR and screening to locate those mutations. With the availability of RNAMute, preanalysis can be performed computationally before conducting an experiment.

  15. Direct determination of the point mutation rate of a murine retrovirus.

    PubMed Central

    Monk, R J; Malik, F G; Stokesberry, D; Evans, L H

    1992-01-01

    The point mutation rate of a murine leukemia virus (MuLV) genome (AKV) was determined under conditions in which the number of replicative cycles was carefully controlled and the point mutation rate was determined by direct examination of the RNA genomes of progeny viruses. A clonal cell line infected at a low multiplicity of infection (2 x 10(-3)) was derived to provide a source of virus with high genetic homogeneity. Virus stocks from this cell line were used to infect cells at a low multiplicity of infection, and the cells were seeded soon after infection to obtain secondary clonal cell lines. RNase T1-oligonucleotide fingerprinting analyses of virion RNAs from 93 secondary lines revealed only 3 base changes in nearly 130,000 bases analyzed. To obtain an independent assessment of the mutation rate, we directly sequenced virion RNAs by using a series of DNA oligonucleotide primers distributed across the genome. RNA sequencing detected no mutations in over 21,000 bases analyzed. The combined fingerprinting and sequencing analyses yielded a mutation rate for infectious progeny viruses of one base change per 50,000 (2 x 10(-5)) bases per replication cycle. Our results suggest that over 80% of infectious progeny MuLVs may be replicated with complete fidelity and that only a low percentage undergo more than one point mutation during a replication cycle. Previous estimates of retroviral mutation rates suggest that the majority of infectious progeny viruses have undergone one or more point mutations. Recent studies of the mutation rates of marker genes in spleen necrosis virus-based vectors estimate a base substitution rate lower than estimates for infectious avian retroviruses and nearly identical to our determinations with AKV. The differences between mutation rates observed in studies of retroviruses may reflect the imposition of different selective conditions. Images PMID:1316475

  16. Interplay between DMD point mutations and splicing signals in Dystrophinopathy phenotypes.

    PubMed

    Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

    2013-01-01

    DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

  17. Interplay between DMD Point Mutations and Splicing Signals in Dystrophinopathy Phenotypes

    PubMed Central

    Juan-Mateu, Jonàs; González-Quereda, Lidia; Rodríguez, Maria José; Verdura, Edgard; Lázaro, Kira; Jou, Cristina; Nascimento, Andrés; Jiménez-Mallebrera, Cecilia; Colomer, Jaume; Monges, Soledad; Lubieniecki, Fabiana; Foncuberta, Maria Eugenia; Pascual-Pascual, Samuel Ignacio; Molano, Jesús; Baiget, Montserrat; Gallano, Pia

    2013-01-01

    DMD nonsense and frameshift mutations lead to severe Duchenne muscular dystrophy while in-frame mutations lead to milder Becker muscular dystrophy. Exceptions are found in 10% of cases and the production of alternatively spliced transcripts is considered a key modifier of disease severity. Several exonic mutations have been shown to induce exon-skipping, while splice site mutations result in exon-skipping or activation of cryptic splice sites. However, factors determining the splicing pathway are still unclear. Point mutations provide valuable information regarding the regulation of pre-mRNA splicing and elements defining exon identity in the DMD gene. Here we provide a comprehensive analysis of 98 point mutations related to clinical phenotype and their effect on muscle mRNA and dystrophin expression. Aberrant splicing was found in 27 mutations due to alteration of splice sites or splicing regulatory elements. Bioinformatics analysis was performed to test the ability of the available algorithms to predict consequences on mRNA and to investigate the major factors that determine the splicing pathway in mutations affecting splicing signals. Our findings suggest that the splicing pathway is highly dependent on the interplay between splice site strength and density of regulatory elements. PMID:23536893

  18. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS).

    PubMed Central

    Newton, C R; Graham, A; Heptinstall, L E; Powell, S J; Summers, C; Kalsheker, N; Smith, J C; Markham, A F

    1989-01-01

    We have improved the "polymerase chain reaction" (PCR) to permit rapid analysis of any known mutation in genomic DNA. We demonstrate a system, ARMS (Amplification Refractory Mutation System), that allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis. The system is simple, reliable and non-isotopic. It will clearly distinguish heterozygotes at a locus from homozygotes for either allele. The system requires neither restriction enzyme digestion, allele-specific oligonucleotides as conventionally applied, nor the sequence analysis of PCR products. The basis of the invention is that unexpectedly, oligonucleotides with a mismatched 3'-residue will not function as primers in the PCR under appropriate conditions. We have analysed DNA from patients with alpha 1-antitrypsin (AAT) deficiency, from carriers of the disease and from normal individuals. Our findings are in complete agreement with allele assignments derived by direct sequencing of PCR products. Images PMID:2785681

  19. Repair of Single-Point Mutations by Polypurine Reverse Hoogsteen Hairpins

    PubMed Central

    Solé, Anna; Villalobos, Xenia; Noé, Véronique

    2014-01-01

    Abstract Polypurine reverse Hoogsteen hairpins (PPRHs) are formed by two intramolecularly bound antiparallel homopurine domains linked by a five-thymidine loop. One of the homopurine strands binds with antiparallel orientation by Watson–Crick bonds to the polypyrimidine target sequence, forming a triplex. We had previously reported the ability of PPRHs to effectively bind dsDNA displacing the fourth strand away from the newly formed triplex. The main goal of this work was to explore the possibility of repairing a point mutation in mammalian cells using PPRHs as tools. These repair-PPRHs contain different combinations of extended sequences of DNA with the corrected nucleotide to repair the point mutation. As a model we used the dihydrofolate reductase gene. On the one hand, we demonstrate in vitro that PPRHs bind specifically to their polypyrimidine target sequence, opening the two strands of the dsDNA, and allowing the binding of a given repair oligonucleotide to the displaced strand of the DNA. Subsequently, we show at a cellular level (Chinese ovary hamster cells) that repair-PPRHs are able to correct a single-point mutation in a dihydrofolate reductase minigene bearing a nonsense mutation, both in an extrachromosomal location and when the mutated plasmid was stably transfected into the cells. Finally, this methodology was successfully applied to repair a single-point mutation at the endogenous locus, using the DA5 cell line with a deleted nucleotide in exon six of the dhfr gene. PMID:25222154

  20. Diversity and Functional Consequences of Germline and Somatic PTPN11 Mutations in Human Disease

    PubMed Central

    Tartaglia, Marco; Martinelli, Simone; Stella, Lorenzo; Bocchinfuso, Gianfranco; Flex, Elisabetta; Cordeddu, Viviana; Zampino, Giuseppe; Burgt, Ineke van der; Palleschi, Antonio; Petrucci, Tamara C.; Sorcini, Mariella; Schoch, Claudia; Foà, Robin; Emanuel, Peter D.; Gelb, Bruce D.

    2006-01-01

    Germline mutations in PTPN11, the gene encoding the protein tyrosine phosphatase SHP-2, cause Noonan syndrome (NS) and the clinically related LEOPARD syndrome (LS), whereas somatic mutations in the same gene contribute to leukemogenesis. On the basis of our previously gathered genetic and biochemical data, we proposed a model that splits NS- and leukemia-associated PTPN11 mutations into two major classes of activating lesions with differential perturbing effects on development and hematopoiesis. To test this model, we investigated further the diversity of germline and somatic PTPN11 mutations, delineated the association of those mutations with disease, characterized biochemically a panel of mutant SHP-2 proteins recurring in NS, LS, and leukemia, and performed molecular dynamics simulations to determine the structural effects of selected mutations. Our results document a strict correlation between the identity of the lesion and disease and demonstrate that NS-causative mutations have less potency for promoting SHP-2 gain of function than do leukemia-associated ones. Furthermore, we show that the recurrent LS-causing Y279C and T468M amino acid substitutions engender loss of SHP-2 catalytic activity, identifying a previously unrecognized behavior for this class of missense PTPN11 mutations. PMID:16358218

  1. Structure Based Thermostability Prediction Models for Protein Single Point Mutations with Machine Learning Tools.

    PubMed

    Jia, Lei; Yarlagadda, Ramya; Reed, Charles C

    2015-01-01

    Thermostability issue of protein point mutations is a common occurrence in protein engineering. An application which predicts the thermostability of mutants can be helpful for guiding decision making process in protein design via mutagenesis. An in silico point mutation scanning method is frequently used to find "hot spots" in proteins for focused mutagenesis. ProTherm (http://gibk26.bio.kyutech.ac.jp/jouhou/Protherm/protherm.html) is a public database that consists of thousands of protein mutants' experimentally measured thermostability. Two data sets based on two differently measured thermostability properties of protein single point mutations, namely the unfolding free energy change (ddG) and melting temperature change (dTm) were obtained from this database. Folding free energy change calculation from Rosetta, structural information of the point mutations as well as amino acid physical properties were obtained for building thermostability prediction models with informatics modeling tools. Five supervised machine learning methods (support vector machine, random forests, artificial neural network, naïve Bayes classifier, K nearest neighbor) and partial least squares regression are used for building the prediction models. Binary and ternary classifications as well as regression models were built and evaluated. Data set redundancy and balancing, the reverse mutations technique, feature selection, and comparison to other published methods were discussed. Rosetta calculated folding free energy change ranked as the most influential features in all prediction models. Other descriptors also made significant contributions to increasing the accuracy of the prediction models. PMID:26361227

  2. Structure Based Thermostability Prediction Models for Protein Single Point Mutations with Machine Learning Tools

    PubMed Central

    Jia, Lei; Yarlagadda, Ramya; Reed, Charles C.

    2015-01-01

    Thermostability issue of protein point mutations is a common occurrence in protein engineering. An application which predicts the thermostability of mutants can be helpful for guiding decision making process in protein design via mutagenesis. An in silico point mutation scanning method is frequently used to find “hot spots” in proteins for focused mutagenesis. ProTherm (http://gibk26.bio.kyutech.ac.jp/jouhou/Protherm/protherm.html) is a public database that consists of thousands of protein mutants’ experimentally measured thermostability. Two data sets based on two differently measured thermostability properties of protein single point mutations, namely the unfolding free energy change (ddG) and melting temperature change (dTm) were obtained from this database. Folding free energy change calculation from Rosetta, structural information of the point mutations as well as amino acid physical properties were obtained for building thermostability prediction models with informatics modeling tools. Five supervised machine learning methods (support vector machine, random forests, artificial neural network, naïve Bayes classifier, K nearest neighbor) and partial least squares regression are used for building the prediction models. Binary and ternary classifications as well as regression models were built and evaluated. Data set redundancy and balancing, the reverse mutations technique, feature selection, and comparison to other published methods were discussed. Rosetta calculated folding free energy change ranked as the most influential features in all prediction models. Other descriptors also made significant contributions to increasing the accuracy of the prediction models. PMID:26361227

  3. Induced Pluripotent Stem Cell Generation-Associated Point Mutations Arise during the Initial Stages of the Conversion of These Cells

    PubMed Central

    Sugiura, Mayumi; Kasama, Yasuji; Araki, Ryoko; Hoki, Yuko; Sunayama, Misato; Uda, Masahiro; Nakamura, Miki; Ando, Shunsuke; Abe, Masumi

    2014-01-01

    Summary A large number of point mutations have been identified in induced pluripotent stem cell (iPSC) genomes to date. Whether these mutations are associated with iPSC generation is an important and controversial issue. In this study, we approached this critical issue in different ways, including an assessment of iPSCs versus embryonic stem cells (ESCs), and an investigation of variant allele frequencies and the heterogeneity of point mutations within a single iPSC clone. Through these analyses, we obtained strong evidence that iPSC-generation-associated point mutations occur frequently in a transversion-predominant manner just after the onset of cell lineage conversion. The heterogeneity of the point mutation profiles within an iPSC clone was also revealed and reflects the history of the emergence of each mutation. Further, our results suggest a possible approach for establishing iPSCs with fewer point mutations. PMID:24511470

  4. Rapid evolution of cis-regulatory sequences via local point mutations

    NASA Technical Reports Server (NTRS)

    Stone, J. R.; Wray, G. A.

    2001-01-01

    Although the evolution of protein-coding sequences within genomes is well understood, the same cannot be said of the cis-regulatory regions that control transcription. Yet, changes in gene expression are likely to constitute an important component of phenotypic evolution. We simulated the evolution of new transcription factor binding sites via local point mutations. The results indicate that new binding sites appear and become fixed within populations on microevolutionary timescales under an assumption of neutral evolution. Even combinations of two new binding sites evolve very quickly. We predict that local point mutations continually generate considerable genetic variation that is capable of altering gene expression.

  5. BRAF and NRAS mutations are uncommon in melanomas arising in diverse internal organs

    PubMed Central

    Wong, C W; Fan, Y S; Chan, T L; Chan, A S W; Ho, L C; Ma, T K F; Yuen, S T; Leung, S Y

    2005-01-01

    Background: Malignant melanoma arising from different body compartments may be associated with differing aetiological factors and clinical behaviour, and may manifest diverse molecular genetic profiles. Although many studies have focused on cutaneous melanoma, little is known of mucosal and other types of melanoma. In particular, malignant melanoma of soft parts is different from other melanomas in many respects, yet manifests a common melanocytic differentiation. Mutation of BRAF is now known to be common in cutaneous melanomas, and raises possible new therapeutic options of anti-RAF treatment for these patients. Few data are available for non-cutaneous melanomas. Aims: To study the incidence of BRAF and NRAS mutations in melanomas arising in diverse internal organs. Methods: Fifty one melanomas from various internal organs were investigated for BRAF and NRAS mutation by direct DNA sequencing. Results: BRAF and NRAS mutations were found in two and five mucosal melanomas arising from the aerodigestive and female genital tracts (n = 36). Their occurrence is mutually exclusive, giving a combined mutation incidence rate of 19.4% in mucosal melanomas. Both BRAF and NRAS mutations were absent in malignant melanoma of soft parts (n = 7). BRAF mutation was also absent in uveal melanoma (n = 6), but was seen in two of five cutaneous melanomas. The incidence of BRAF or combined BRAF/NRAS mutations in all non-cutaneous groups was significantly lower than published rates for cutaneous melanomas. Conclusion: Each melanoma subtype may have a unique oncogenetic pathway of tumour development, and only a small fraction of non-cutaneous melanomas may benefit from anti-RAF treatment. PMID:15917418

  6. Diversity, Mutation and Recombination Analysis of Cotton Leaf Curl Geminiviruses

    PubMed Central

    Saleem, Huma; Nahid, Nazia; Shakir, Sara; Ijaz, Sehrish; Murtaza, Ghulam; Khan, Asif Ali; Mubin, Muhammad; Nawaz-ul-Rehman, Muhammad Shah

    2016-01-01

    The spread of cotton leaf curl disease in China, India and Pakistan is a recent phenomenon. Analysis of available sequence data determined that there is a substantial diversity of cotton-infecting geminiviruses in Pakistan. Phylogenetic analyses indicated that recombination between two major groups of viruses, cotton leaf curl Multan virus (CLCuMuV) and cotton leaf curl Kokhran virus (CLCuKoV), led to the emergence of several new viruses. Recombination detection programs and phylogenetic analyses showed that CLCuMuV and CLCuKoV are highly recombinant viruses. Indeed, CLCuKoV appeared to be a major donor virus for the coat protein (CP) gene, while CLCuMuV donated the Rep gene in the majority of recombination events. Using recombination free nucleotide datasets the substitution rates for CP and Rep genes were determined. We inferred similar nucleotide substitution rates for the CLCuMuV-Rep gene (4.96X10-4) and CLCuKoV-CP gene (2.706X10-4), whereas relatively higher substitution rates were observed for CLCuMuV-CP and CLCuKoV-Rep genes. The combination of sequences with equal and relatively low substitution rates, seemed to result in the emergence of viral isolates that caused epidemics in Pakistan and India. Our findings also suggest that CLCuMuV is spreading at an alarming rate, which can potentially be a threat to cotton production in the Indian subcontinent. PMID:26963635

  7. Clinical Significance of a Point Mutation in DNA Polymerase Beta (POLB) Gene in Gastric Cancer

    PubMed Central

    Tan, Xiaohui; Wang, Hongyi; Luo, Guangbin; Ren, Shuyang; Li, Wenmei; Cui, Jiantao; Gill, Harindarpal S.; Fu, Sidney W.; Lu, Youyong

    2015-01-01

    Gastric cancer (GC) is a major cause of global cancer mortality. Genetic variations in DNA repair genes can modulate DNA repair capability and, consequently, have been associated with risk of developing cancer. We have previously identified a T to C point mutation at nucleotide 889 (T889C) in DNA polymerase beta (POLB) gene, a key enzyme involved in base excision repair in primary GCs. The purpose of this study was to evaluate the mutation and expression of POLB in a larger cohort and to identify possible prognostic roles of the POLB alterations in GC. Primary GC specimens and their matched normal adjacent tissues were collected at the time of surgery. DNA, RNA and protein samples were isolated from GC specimens and cell lines. Mutations were detected by PCR-RFLP/DHPLC and sequencing analysis. POLB gene expression was examined by RT-PCR, tissue microarray, Western blotting and immunofluorescence assays. The function of the mutation was evaluated by chemosensitivity, MTT, Transwell matrigel invasion and host cell reactivation assays. The T889C mutation was detected in 18 (10.17%) of 177 GC patients. And the T889C mutation was associated with POLB overexpression, lymph nodes metastases and poor tumor differentiation. In addition, patients with- the mutation had significantly shorter survival time than those without-, following postoperative chemotherapy. Furthermore, cell lines with T889C mutation in POLB gene were more resistant to the treatment of 5-fluorouracil, cisplatin and epirubicin than those with wild type POLB. Forced expression of POLB gene with T889C mutation resulted in enhanced cell proliferation, invasion and resistance to anticancer drugs, along with increased DNA repair capability. These results suggest that POLB gene with T889C mutation in surgically resected primary gastric tissues may be clinically useful for predicting responsiveness to chemotherapy in patients with GC. The POLB gene alteration may serve as a prognostic biomarker for GC. PMID

  8. Point mutations throughout the GLI3 gene cause Greig cephalopolysyndactyly syndrome.

    PubMed

    Kalff-Suske, M; Wild, A; Topp, J; Wessling, M; Jacobsen, E M; Bornholdt, D; Engel, H; Heuer, H; Aalfs, C M; Ausems, M G; Barone, R; Herzog, A; Heutink, P; Homfray, T; Gillessen-Kaesbach, G; König, R; Kunze, J; Meinecke, P; Müller, D; Rizzo, R; Strenge, S; Superti-Furga, A; Grzeschik, K H

    1999-09-01

    Greig cephalopolysyndactyly syndrome, characterized by craniofacial and limb anomalies (GCPS; MIM 175700), previously has been demonstrated to be associated with translocations as well as point mutations affecting one allele of the zinc finger gene GLI3. In addition to GCPS, Pallister-Hall syndrome (PHS; MIM 146510) and post-axial polydactyly type A (PAP-A; MIM 174200), two other disorders of human development, are caused by GLI3 mutations. In order to gain more insight into the mutational spectrum associated with a single phenotype, we report here the extension of the GLI3 mutation analysis to 24 new GCPS cases. We report the identification of 15 novel mutations present in one of the patient's GLI3 alleles. The mutations map throughout the coding gene regions. The majority are truncating mutations (nine of 15) that engender prematurely terminated protein products mostly but not exclusively N-terminally to or within the central region encoding the DNA-binding domain. Two missense and two splicing mutations mapping within the zinc finger motifs presumably also interfere with DNA binding. The five mutations identified within the protein regions C-terminal to the zinc fingers putatively affect additional functional properties of GLI3. In cell transfection experiments using fusions of the DNA-binding domain of yeast GAL4 to different segments of GLI3, transactivating capacity was assigned to two adjacent independent domains (TA(1)and TA(2)) in the C-terminal third of GLI3. Since these are the only functional domains affected by three C-terminally truncating mutations, we postulate that GCPS may be due either to haploinsufficiency resulting from the complete loss of one gene copy or to functional haploinsufficiency related to compromised properties of this transcription factor such as DNA binding and transactivation. PMID:10441342

  9. Identification of eight point mutations in protein S deficiency type I--analysis of 15 pedigrees.

    PubMed

    Gómez, E; Poort, S R; Bertina, R M; Reitsma, P H

    1995-05-01

    We described molecular genetic studies of 15 patients with protein S deficiency type I (i.e. reduced total protein S antigen). All the exons of the PROS 1 gene were analyzed both by PCR and direct sequencing in all 15 probands. This analysis led to the identification of point mutations affecting eight individuals. One of these mutations (codon-25, insertion of T) has been described previously in a Dutch pedigree. The other mutations are novel and all are located in exons that code for the protein S domain that is homologous to the steroid hormone binding globulins. They include two amino acid replacements (one individual with 340 Gly--> Val, and two individuals with 467 Val --> Gly), and four frameshift mutations due to either one bp deletions (in codon 261 deletion of T and in codon 267 deletion of G) or insertions (in codon 565 insertion T and after codon 578 insertions of C). Studies performed in six families (totalling 43 subjects) showed cosegregation of the genetic abnormality with reduced plasma protein S levels, and provided genetic evidence for a heterozygous protein S deficiency in 25 of them. The yield of mutations in this study (53%) confirms that the percentage of protein S deficient cases in which a point mutation is found remains low. PMID:7482398

  10. Stochastic Drift in Mitochondrial DNA Point Mutations: A Novel Perspective Ex Silico

    PubMed Central

    Poovathingal, Suresh Kumar; Gruber, Jan; Halliwell, Barry; Gunawan, Rudiyanto

    2009-01-01

    The mitochondrial free radical theory of aging (mFRTA) implicates Reactive Oxygen Species (ROS)-induced mutations of mitochondrial DNA (mtDNA) as a major cause of aging. However, fifty years after its inception, several of its premises are intensely debated. Much of this uncertainty is due to the large range of values in the reported experimental data, for example on oxidative damage and mutational burden in mtDNA. This is in part due to limitations with available measurement technologies. Here we show that sample preparations in some assays necessitating high dilution of DNA (single molecule level) may introduce significant statistical variability. Adding to this complexity is the intrinsically stochastic nature of cellular processes, which manifests in cells from the same tissue harboring varying mutation load. In conjunction, these random elements make the determination of the underlying mutation dynamics extremely challenging. Our in silico stochastic study reveals the effect of coupling the experimental variability and the intrinsic stochasticity of aging process in some of the reported experimental data. We also show that the stochastic nature of a de novo point mutation generated during embryonic development is a major contributor of different mutation burdens in the individuals of mouse population. Analysis of simulation results leads to several new insights on the relevance of mutation stochasticity in the context of dividing tissues and the plausibility of ROS ”vicious cycle” hypothesis. PMID:19936024

  11. A New Approach To the Diagnosis of Point Mutations in Native DNA Using Graphene Oxide.

    PubMed

    Kuznetsov, A A; Maksimova, N R; Kaimonov, V S; Alexandrov, G N; Smagulova, S A

    2016-01-01

    Development of new methods for the diagnosis of point mutations is a pressing issue. We have developed a new approach to the design of graphene oxide-based test systems for the diagnosis of point mutations in native DNA. This new approach is based on the use of graphene oxide for the adsorption and quenching of fluorescently labeled primers in a post-amplification PCR mixture followed by detection of fluorescently labeled PCR products. It is possible to detect fluorescently labelled amplicons in the presence of an excess of primers in a PCR product solution due to the different affinities of single-stranded and double-stranded DNA molecules to graphene oxide, as well as the ability of graphene oxide to act as a quencher of the fluorophores adsorbed on its surface. The new approach was tested by designing a graphene oxide-based test system for the DNA diagnosis of the point mutation associated with the development of the 3M syndrome in Yakuts. The developed approach enables one to design graphene oxide-based test systems suitable for the diagnosis of any point mutations in native DNA. PMID:27437142

  12. A New Approach To the Diagnosis of Point Mutations in Native DNA Using Graphene Oxide

    PubMed Central

    Kuznetsov, A.A.; Maksimova, N.R.; Kaimonov, V.S.; Alexandrov, G.N.; Smagulova, S.A.

    2016-01-01

    Development of new methods for the diagnosis of point mutations is a pressing issue. We have developed a new approach to the design of graphene oxide-based test systems for the diagnosis of point mutations in native DNA. This new approach is based on the use of graphene oxide for the adsorption and quenching of fluorescently labeled primers in a post-amplification PCR mixture followed by detection of fluorescently labeled PCR products. It is possible to detect fluorescently labelled amplicons in the presence of an excess of primers in a PCR product solution due to the different affinities of single-stranded and double-stranded DNA molecules to graphene oxide, as well as the ability of graphene oxide to act as a quencher of the fluorophores adsorbed on its surface. The new approach was tested by designing a graphene oxide-based test system for the DNA diagnosis of the point mutation associated with the development of the 3M syndrome in Yakuts. The developed approach enables one to design graphene oxide-based test systems suitable for the diagnosis of any point mutations in native DNA. PMID:27437142

  13. Phenotypic reversion of an IS1-mediated deletion mutation: a combined role for point mutations and deletions in transposon evolution.

    PubMed

    Lida, S; Marcoli, R; Bickle, T A

    1982-01-01

    We have physically characterised a deletion mutant of the R plasmid R100 which has lost all of the antibiotic resistances, including chloramphenicol resistance (Cmr), coded by its IS1-flanked r-determinant. The deletion was mediated by one of the flanking IS1 elements and terminates within the carboxyl terminus of the Cmr gene. DNA sequence analysis showed that the mutated gene would produce a protein 20 amino acids longer than the wild-type due to fusion with an open reading frame in the IS element. Surprisingly for a deletion mutation, rare, spontaneous Cmr revertants could be recovered. Two of the four revertants studied had frame shifts due to the insertion of a single AT base pair at the same position; the revertants would code for a protein five amino acids shorter than the wild-type. The other two revertants had acquired duplications of the 34-bp inverted terminal repeat sequences of the IS1 element and would direct the synthesis of a protein six amino acids longer than the wild-type. The reverted Cmr markers were still capable of transposition. These observations suggest a role for point mutations and small DNA rearrangements in the formation of new gene organisations produced by mobile genetic elements. PMID:6329702

  14. Adenylosuccinate lyase (ADSL) and infantile autism: Absence of previously reported point mutation

    SciTech Connect

    Fon, E.A.; Sarrazin, J.; Rouleau, G.A.

    1995-12-18

    Autism is a heterogeneous neuropsychiatric syndrome of unknown etiology. There is evidence that a deficiency in the enzyme adenylosuccinate lyase (ADSL), essential for de novo purine biosynthesis, could be involved in the pathogenesis of certain cases. A point mutation in the ADSL gene, resulting in a predicted serine-to-proline substitution and conferring structural instability to the mutant enzyme, has been reported previously in 3 affected siblings. In order to determine the prevalence of the mutation, we PCR-amplified the exon spanning the site of this mutation from the genomic DNA of patients fulfilling DSM-III-R criteria for autistic disorder. None of the 119 patients tested were found to have this mutation. Furthermore, on preliminary screening using single-strand conformation polymorphism (SSCP), no novel mutations were detected in the coding sequence of four ADSL exons, spanning approximately 50% of the cDNA. In light of these findings, it appears that mutations in the ADSL gene represent a distinctly uncommon cause of autism. 12 refs., 2 figs.

  15. Using Gel Electrophoresis To Illustrate Protein Diversity and Isoelectric Point.

    ERIC Educational Resources Information Center

    Browning, Mark; Vanable, Joseph

    2002-01-01

    Demonstrates the differences in protein structures by focusing on isoelectric point with an experiment that is observable under certain pH levels in gel electrophoresis. Explains the electrophoresis procedure and reports results of the experiments. (YDS)

  16. Sensitive detection of point mutation by electrochemiluminescence and DNA ligase-based assay

    NASA Astrophysics Data System (ADS)

    Zhou, Huijuan; Wu, Baoyan

    2008-12-01

    The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence (ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273 of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point mutation detection.

  17. Method for detecting point mutations in DNA utilizing fluorescence energy transfer

    DOEpatents

    Parkhurst, Lawrence J.; Parkhurst, Kay M.; Middendorf, Lyle

    2001-01-01

    A method for detecting point mutations in DNA using a fluorescently labeled oligomeric probe and Forster resonance energy transfer (FRET) is disclosed. The selected probe is initially labeled at each end with a fluorescence dye, which act together as a donor/acceptor pair for FRET. The fluorescence emission from the dyes changes dramatically from the duplex stage, wherein the probe is hybridized to the complementary strand of DNA, to the single strand stage, when the probe is melted to become detached from the DNA. The change in fluorescence is caused by the dyes coming into closer proximity after melting occurs and the probe becomes detached from the DNA strand. The change in fluorescence emission as a function of temperature is used to calculate the melting temperature of the complex or T.sub.m. In the case where there is a base mismatch between the probe and the DNA strand, indicating a point mutation, the T.sub.m has been found to be significantly lower than the T.sub.m for a perfectly match probelstand duplex. The present invention allows for the detection of the existence and magnitude of T.sub.m, which allows for the quick and accurate detection of a point mutation in the DNA strand and, in some applications, the determination of the approximate location of the mutation within the sequence.

  18. Fast, sensitive point of care electrochemical molecular system for point mutation and select agent detection.

    PubMed

    MacLeod, J A; Nemeth, A C; Dicke, W C; Wang, D; Manalili Wheeler, S; Hannis, J C; Collier, G B; Drader, J J

    2016-07-01

    Point of care molecular diagnostics benefits from a portable battery-operated device capable of performing a fast turnaround using reliable inexpensive cartridges. We describe a prototype device for performing a molecular diagnostics test for clinical and biodefense samples in 16 minutes using a prototype capable of an 8 minute PCR reaction, followed by hybridization and detection on an electrochemical microarray based on the i-STAT® system. We used human buccal swabs for hemochromatosis testing including in-device DNA extraction. Additional clinical and biodefense samples included influenza A and bacterial select agents Bacillus anthracis, Yersinia pestis and Francisella tularensis. PMID:27280174

  19. De novo point mutations in patients diagnosed with ataxic cerebral palsy.

    PubMed

    Parolin Schnekenberg, Ricardo; Perkins, Emma M; Miller, Jack W; Davies, Wayne I L; D'Adamo, Maria Cristina; Pessia, Mauro; Fawcett, Katherine A; Sims, David; Gillard, Elodie; Hudspith, Karl; Skehel, Paul; Williams, Jonathan; O'Regan, Mary; Jayawant, Sandeep; Jefferson, Rosalind; Hughes, Sarah; Lustenberger, Andrea; Ragoussis, Jiannis; Jackson, Mandy; Tucker, Stephen J; Németh, Andrea H

    2015-07-01

    Cerebral palsy is a sporadic disorder with multiple likely aetiologies, but frequently considered to be caused by birth asphyxia. Genetic investigations are rarely performed in patients with cerebral palsy and there is little proven evidence of genetic causes. As part of a large project investigating children with ataxia, we identified four patients in our cohort with a diagnosis of ataxic cerebral palsy. They were investigated using either targeted next generation sequencing or trio-based exome sequencing and were found to have mutations in three different genes, KCNC3, ITPR1 and SPTBN2. All the mutations were de novo and associated with increased paternal age. The mutations were shown to be pathogenic using a combination of bioinformatics analysis and in vitro model systems. This work is the first to report that the ataxic subtype of cerebral palsy can be caused by de novo dominant point mutations, which explains the sporadic nature of these cases. We conclude that at least some subtypes of cerebral palsy may be caused by de novo genetic mutations and patients with a clinical diagnosis of cerebral palsy should be genetically investigated before causation is ascribed to perinatal asphyxia or other aetiologies. PMID:25981959

  20. The fitness effects of a point mutation in Escherichia coli change with founding population density.

    PubMed

    Cao, Huansheng; Plague, Gordon R

    2016-08-01

    Although intraspecific competition plays a seminal role in organismal evolution, little is known about the fitness effects of mutations at different population densities. We identified a point mutation in the cyclic AMP receptor protein (CRP) gene in Escherichia coli that confers significantly higher fitness than the wildtype at low founding population density, but significantly lower fitness at high founding density. Because CRP is a transcription factor that regulates the expression of nearly 500 genes, we compared global gene expression profiles of the mutant and wildtype strains. This mutation (S63F) does not affect expression of crp itself, but it does significantly affect expression of 170 and 157 genes at high and low founding density, respectively. Interestingly, acid resistance genes, some of which are known to exhibit density-dependent effects in E. coli, were consistently differentially expressed at high but not low density. As such, these genes may play a key role in reducing the crp mutant's fitness at high density, although other differentially expressed genes almost certainly also contribute to the fluctuating fitness differences we observed. Whatever the causes, we suspect that many mutations may exhibit density-dependent fitness effects in natural populations, so the fate of new mutations may frequently depend on the effective population size when they originate. PMID:27344657

  1. De novo point mutations in patients diagnosed with ataxic cerebral palsy

    PubMed Central

    Parolin Schnekenberg, Ricardo; Perkins, Emma M.; Miller, Jack W.; Davies, Wayne I. L.; D’Adamo, Maria Cristina; Pessia, Mauro; Fawcett, Katherine A.; Sims, David; Gillard, Elodie; Hudspith, Karl; Skehel, Paul; Williams, Jonathan; O’Regan, Mary; Jayawant, Sandeep; Jefferson, Rosalind; Hughes, Sarah; Lustenberger, Andrea; Ragoussis, Jiannis

    2015-01-01

    Cerebral palsy is a sporadic disorder with multiple likely aetiologies, but frequently considered to be caused by birth asphyxia. Genetic investigations are rarely performed in patients with cerebral palsy and there is little proven evidence of genetic causes. As part of a large project investigating children with ataxia, we identified four patients in our cohort with a diagnosis of ataxic cerebral palsy. They were investigated using either targeted next generation sequencing or trio-based exome sequencing and were found to have mutations in three different genes, KCNC3, ITPR1 and SPTBN2. All the mutations were de novo and associated with increased paternal age. The mutations were shown to be pathogenic using a combination of bioinformatics analysis and in vitro model systems. This work is the first to report that the ataxic subtype of cerebral palsy can be caused by de novo dominant point mutations, which explains the sporadic nature of these cases. We conclude that at least some subtypes of cerebral palsy may be caused by de novo genetic mutations and patients with a clinical diagnosis of cerebral palsy should be genetically investigated before causation is ascribed to perinatal asphyxia or other aetiologies. PMID:25981959

  2. Point Mutations in c-Myc Uncouple Neoplastic Transformation from Multiple Other Phenotypes in Rat Fibroblasts

    PubMed Central

    Graves, J. Anthony; Rothermund, Kristi; Wang, Tao; Qian, Wei; Van Houten, Bennett; Prochownik, Edward V.

    2010-01-01

    Deregulation of c-Myc (Myc) occurs in many cancers. In addition to transforming various cell types, Myc also influences additional transformation-associated cellular phenotypes including proliferation, survival, genomic instability, reactive oxygen species production, and metabolism. Although Myc is wild type in most cancers (wtMyc), it occasionally acquires point mutations in certain lymphomas. Some of these mutations confer a survival advantage despite partially attenuating proliferation and transformation. Here, we have evaluated four naturally-occurring or synthetic point mutations of Myc for their ability to affect these phenotypes, as well as to promote genomic instability, to generate reactive oxygen species and to up-regulate aerobic glycolysis and oxidative phosphorylation. Our findings indicate that many of these phenotypes are genetically and functionally independent of one another and are not necessary for transformation. Specifically, the higher rate of glucose metabolism known to be associated with wtMyc deregulation was found to be independent of transformation. One mutation (Q131R) was greatly impaired for nearly all of the studied Myc phenotypes, yet was able to retain some ability to transform. These findings indicate that, while the Myc phenotypes examined here make additive contributions to transformation, none, with the possible exception of increased reliance on extracellular glutamine for survival, are necessary for achieving this state. PMID:21060841

  3. Registered report: Diverse somatic mutation patterns and pathway alterations in human cancers

    PubMed Central

    Sharma, Vidhu; Young, Lisa; Allison, Anne B; Owen, Kate

    2016-01-01

    The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "Diverse somatic mutation patterns and pathway alterations in human cancers" by Kan and colleagues published in Nature in 2010 (Kan et al., 2010). The experiments to be replicated are those reported in Figures 3D-F and 4C-F. Kan and colleagues utilized mismatch repair detection (MRD) technology to identify somatic mutations in primary human tumor samples and identified a previously uncharacterized arginine 243 to histidine (R243H) mutation in the G-protein α subunit GNAO1 in breast carcinoma tissue. In Figures 3D-F, Kan and colleagues demonstrated that stable expression of mutant GNAO1R243D conferred a significant growth advantage in human mammary epithelial cells, confirming the oncogenic potential of this mutation. Similarly, expression of variants with somatic mutations in MAP2K4, a JNK pathway kinase (shown in Figures 4C-E) resulted in a significant increase in anchorage-independent growth. Interestingly, these mutants exhibited reduced kinase activity compared to wild type MAP2K4, indicating these mutations impose a dominant-negative influence to promote growth (Figure 4F). The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife. DOI: http://dx.doi.org/10.7554/eLife.11566.001 PMID:26894955

  4. Adaptation of Stenotrophomonas maltophilia in cystic fibrosis: molecular diversity, mutation frequency and antibiotic resistance.

    PubMed

    Vidigal, P G; Dittmer, S; Steinmann, E; Buer, J; Rath, P-M; Steinmann, J

    2014-07-01

    Due to the continuous exposure to a challenging environment and repeated antibiotic treatment courses, bacterial populations in cystic fibrosis (CF) patients experience selective pressure causing the emergence of mutator phenotypes. In this study we investigated the genotypic diversity, mutation frequency and antibiotic resistance of S. maltophilia isolates chronically colonizing CF patients. S. maltophilia was isolated from a total of 90 sputum samples, collected sequentially from 19 CF patients admitted between January 2008 and March 2012 at the University Hospital Essen, Germany. DNA fingerprinting by repetitive-sequence-based PCR revealed that 68.4% (n=13) of CF patients harbored different S. maltophilia genotypes during the 4-year study course. Out of 90 S. maltophilia isolates obtained from chronically colonized CF patients, 17.8% (n=16) were hypomutators, 27.7% (n=25), normomutators, 23.3% (n=21), weak hypermutators and 31.2% (n=28) strong hypermutators. We also found that mutation rates of the most clonally related genotypes varied over time with the tendency to become less mutable. Mutator isolates were found to have no significant increase in resistance against eight different antibiotics versus nonmutators. Sequencing of the mismatch repair genes mutL, mutS and uvrD revealed alterations that resulted in amino acid changes in their corresponding proteins. Here, we could demonstrate that several different S. maltophilia genotypes are present in CF patients and as a sign of adaption their mutation status switches over time to a less mutator phenotype without increasing resistance. These results suggest that S. maltophilia attempts to sustain its biological fitness as mechanism for long-term persistence in the CF lung. PMID:24836944

  5. Dew inspired breathing-based detection of genetic point mutation visualized by naked eye

    PubMed Central

    Xie, Liping; Wang, Tongzhou; Huang, Tianqi; Hou, Wei; Huang, Guoliang; Du, Yanan

    2014-01-01

    A novel label-free method based on breathing-induced vapor condensation was developed for detection of genetic point mutation. The dew-inspired detection was realized by integration of target-induced DNA ligation with rolling circle amplification (RCA). The vapor condensation induced by breathing transduced the RCA-amplified variances in DNA contents into visible contrast. The image could be recorded by a cell phone for further or even remote analysis. This green assay offers a naked-eye-reading method potentially applied for point-of-care liver cancer diagnosis in resource-limited regions. PMID:25199907

  6. Dew inspired breathing-based detection of genetic point mutation visualized by naked eye

    NASA Astrophysics Data System (ADS)

    Xie, Liping; Wang, Tongzhou; Huang, Tianqi; Hou, Wei; Huang, Guoliang; Du, Yanan

    2014-09-01

    A novel label-free method based on breathing-induced vapor condensation was developed for detection of genetic point mutation. The dew-inspired detection was realized by integration of target-induced DNA ligation with rolling circle amplification (RCA). The vapor condensation induced by breathing transduced the RCA-amplified variances in DNA contents into visible contrast. The image could be recorded by a cell phone for further or even remote analysis. This green assay offers a naked-eye-reading method potentially applied for point-of-care liver cancer diagnosis in resource-limited regions.

  7. Four novel point mutations in the PMP22 gene with phenotypes of HNPP and Dejerine-Sottas neuropathy.

    PubMed

    Brožková, Dana; Mazanec, Radim; Rychlý, Zdeněk; Haberlová, Jana; Böhm, Jiří; Staněk, Jan; Plevová, Pavlína; Lisoňová, Jana; Sabová, Jana; Sakmaryová, Iva; Seeman, Pavel

    2011-11-01

    We report four novel point mutations in the PMP22 gene with two different phenotypes: mutation p.Ser79Thr arose de novo in a patient with the Dejerine-Sottas neuropathy (DSN) phenotype; and mutations c.78+5 G>A, c.320-1 G>C, and p.Trp140Stop segregated with HNPP in 5 families.Our findings show that point mutations in PMP22 may be more likely in HNPP patients than in CMT1 patients after exclusion of CMT1A/HNPP. PMID:22006697

  8. Clinical Sensitivity of Cystic Fibrosis Mutation Panels in a Diverse Population.

    PubMed

    Hughes, Erin E; Stevens, Colleen F; Saavedra-Matiz, Carlos A; Tavakoli, Norma P; Krein, Lea M; Parker, April; Zhang, Zhen; Maloney, Breanne; Vogel, Beth; DeCelie-Germana, Joan; Kier, Catherine; Anbar, Ran D; Berdella, Maria N; Comber, Paul G; Dozor, Allen J; Goetz, Danielle M; Guida, Louis; Kattan, Meyer; Ting, Andrew; Voter, Karen Z; van Roey, Patrick; Caggana, Michele; Kay, Denise M

    2016-02-01

    Infants are screened for cystic fibrosis (CF) in New York State (NYS) using an IRT-DNA algorithm. The purpose of this study was to validate and assess clinical validity of the US FDA-cleared Illumina MiSeqDx CF 139-Variant Assay (139-VA) in the diverse NYS CF population. The study included 439 infants with CF identified via newborn screening (NBS) from 2002 to 2012. All had been screened using the Abbott Molecular CF Genotyping Assay or the Hologic InPlex CF Molecular Test. All with CF and zero or one mutation were tested using the 139-VA. DNA extracted from dried blood spots was reliably and accurately genotyped using the 139-VA. Sixty-three additional mutations were identified. Clinical sensitivity of three panels ranged from 76.2% (23 mutations recommended for screening by ACMG/ACOG) to 79.7% (current NYS 39-mutation InPlex panel), up to 86.0% for the 139-VA. For all, sensitivity was highest in Whites and lowest in the Black population. Although the sample size was small, there was a nearly 20% increase in sensitivity for the Black CF population using the 139-VA (68.2%) over the ACMG/ACOG and InPlex panels (both 50.0%). Overall, the 139-VA is more sensitive than other commercially available panels, and could be considered for NBS, clinical, or research laboratories conducting CF screening. PMID:26538069

  9. Patterns of repeat-induced point mutation in transposable elements of basidiomycete fungi.

    PubMed

    Horns, Felix; Petit, Elsa; Yockteng, Roxana; Hood, Michael E

    2012-01-01

    Transposable elements (TEs) are ubiquitous genomic parasites that have prompted the evolution of genome defense systems that restrict their activity. Repeat-induced point mutation (RIP) is a homology-dependent genome defense that introduces C-to-T transition mutations in duplicated DNA sequences and is thought to control the proliferation of selfish repetitive DNA. Here, we determine the taxonomic distribution of hypermutation patterns indicative of RIP among basidiomycetes. We quantify C-to-T transition mutations in particular di- and trinucleotide target sites for TE-like sequences from nine fungal genomes. We find evidence of RIP-like patterns of hypermutation at TpCpG trinucleotide sites in repetitive sequences from all species of the Pucciniomycotina subphylum of the Basidiomycota, Microbotryum lychnidis-dioicae, Puccinia graminis, Melampsora laricis-populina, and Rhodotorula graminis. In contrast, we do not find evidence for RIP-like hypermutation in four species of the Agaricomycotina and Ustilaginomycotina subphyla of the Basidiomycota. Our results suggest that a RIP-like process and the specific nucleotide context for mutations are conserved within the Pucciniomycotina subphylum. These findings imply that coevolutionary interactions between TEs and a hypermutating genome defense are stable over long evolutionary timescales. PMID:22250128

  10. Point mutations in the tyrosine aminotransferase gene in tyrosinemia type II.

    PubMed Central

    Natt, E; Kida, K; Odievre, M; Di Rocco, M; Scherer, G

    1992-01-01

    Tyrosinemia type II (Richner-Hanhart syndrome, RHS) is a disease of autosomal recessive inheritance characterized by keratitis, palmoplantar hyperkeratosis, mental retardation, and elevated blood tyrosine levels. The disease results from deficiency in hepatic tyrosine aminotransferase (TAT; L-tyrosine:2-oxoglutarate aminotransferase, EC 2.6.1.5), a 454-amino acid protein encoded by a gene with 12 exons. To identify the causative mutations in five TAT alleles cloned from three RHS patients, chimeric genes constructed from normal and mutant TAT alleles were tested in directing TAT activity in a transient expression assay. DNA sequence analysis of the regions identified as nonfunctional revealed six different point mutations. Three RHS alleles have nonsense mutations at codons 57, 223, and 417, respectively. One "complex" RHS allele carries a GT----GG splice donor mutation in intron 8 together with a Gly----Val substitution at amino acid 362. A new splice acceptor site in intron 2 of the fifth RHS allele leads to a shift in reading frame. Images PMID:1357662

  11. Does landscape diversity reduce the risk of catastrophic tipping points?

    NASA Astrophysics Data System (ADS)

    Temme, Arnaud; Baartman, Jantiene; Saco, Patricia; Nijp, Jelmer; Langston, Abigail

    2016-04-01

    Most studies about tipping points are based on computer simulations. These simulations, based on first principles of vegetation growth and competition, are not only able to explain a surprising number of vegetation patterns occurring in natural ecosystems, but they also predict shifts between multiple stable states that may be catastrophic. Initially, such studies were performed on simplistic 'non-landscapes' - flats or straight slopes. Recently, we have been able to resolve geomorphic redistribution processes more accurately, so that vegetation patterning can be simulated in more complex landscapes. Here, we present a first look into how such 'real landscapes' affect the risk of catastrophic shifts. We test the hypothesis that increasing complexity and organisation in a landscape reduce the risk of catastrophic shifts by effectively creating mini-refugia where vegetation persists over a wider range of boundary conditions such as precipitation. Depending on the extent of a study area, large complexity could even change the system from one with multiple stable states into one with only one stable state.

  12. Mcl-1-Bim complexes accommodate surprising point mutations via minor structural changes

    SciTech Connect

    Fire, Emiko; Gullá, Stefano V.; Grant, Robert A.; Keating, Amy E.

    2010-06-25

    Mcl-1 is an antiapoptotic Bcl-2-family protein that protects cells against death. Structures of Mcl-1, and of other anti-apoptotic Bcl-2 proteins, reveal a surface groove into which the {alpha}-helical BH3 regions of certain proapoptotic proteins can bind. Despite high overall structural conservation, differences in this groove afford binding specificity that is important for the mechanism of Bcl-2 family function. We report the crystal structure of human Mcl-1 bound to a BH3 peptide derived from human Bim and the structures for three complexes that accommodate large physicochemical changes at conserved Bim sites. The mutations had surprisingly modest effects on complex stability, and the structures show that Mcl-1 can undergo small changes to accommodate the mutant ligands. For example, a shift in a leucine side chain fills a hole left by an isoleucine-to-alanine mutation at the first hydrophobic buried position of Bim BH3. Larger changes are also observed, with shifting of helix {alpha}3 accommodating an isoleucine-to-tyrosine mutation at this same position. We surveyed the variation in available Mcl-1 and Bcl-x{sub L} structures and observed moderate flexibility that is likely critical for facilitating interactions of diverse BH3-only proteins with Mcl-1. With the antiapoptotic Bcl-2 family members attracting significant attention as therapeutic targets, these structures contribute to our growing understanding of how specificity is achieved and can help to guide the design of novel inhibitors that target Mcl-1.

  13. Toward point-of-care testing for JAK2 V617F mutation on a microchip.

    PubMed

    Wang, Hua; Liu, Weiwei; Zhang, Xinju; Xu, Xiao; Kang, Zhihua; Li, Shibao; Wu, Zhiyuan; Yang, Zhiliu; Yao, Bo; Guan, Ming

    2015-09-01

    Molecular genetics now plays a crucial role in diagnosis, the identification of prognostic markers, and monitoring of hematological malignancies. Demonstration of acquired changes such as the JAK2 V617F mutation within myeloproliferative neoplasms (MPN) has quickly moved from a research setting to the diagnostic laboratory. Microfluidics-based assays can reduce the assay time and sample/reagent consumption and enhance the reaction efficiency; however, no current assay has integrated isothermal amplification for point-of-care MPN JAK2 V617F mutation testing with a microchip. In this report, an integrated microchip that performs the whole human blood genomic DNA extraction, loop-mediated isothermal nucleic acid amplification (LAMP) and visual detection for point-of-care genetic mutation testing is demonstrated. This method was validated on DNA from cell lines as well as on whole blood from patients with MPN. The results were compared with those obtained by unlabeled probe melting curve analysis. This chip enjoys a high accuracy, operability, and cost/time efficiency within 1h. All these benefits provide the chip with a potency toward a point-of-care genetic analysis. All samples identified as positive by unlabeled probe melting curve analysis (n=27) proved positive when tested by microchip assay. None of the 30 negative controls gave false positive results. In addition, a patient with polycythemia vera diagnosed as being JAK2 V617F-negative by unlabeled probe melting curve analysis was found to be positive by the microchip. This microchip would possibly be very attractive in developing a point-of-care platform for quick preliminary diagnosis of MPN or other severe illness in resource-limited settings. PMID:26235214

  14. Retinitis pigmentosa: impact of different Pde6a point mutations on the disease phenotype.

    PubMed

    Sothilingam, Vithiyanjali; Garcia Garrido, Marina; Jiao, Kangwei; Buena-Atienza, Elena; Sahaboglu, Ayse; Trifunović, Dragana; Balendran, Sukirthini; Koepfli, Tanja; Mühlfriedel, Regine; Schön, Christian; Biel, Martin; Heckmann, Angelique; Beck, Susanne C; Michalakis, Stylianos; Wissinger, Bernd; Seeliger, Mathias W; Paquet-Durand, François

    2015-10-01

    Mutations in the PDE6A gene can cause rod photoreceptors degeneration and the blinding disease retinitis pigmentosa (RP). While a number of pathogenic PDE6A mutations have been described, little is known about their impact on compound heterozygous situations and potential interactions of different disease-causing alleles. Here, we used a novel mouse model for the Pde6a R562W mutation in combination with an existing line carrying the V685M mutation to generate compound heterozygous Pde6a V685M/R562W animals, exactly homologous to a case of human RP. We compared the progression of photoreceptor degeneration in these compound heterozygous mice with the homozygous V685M and R562W mutants, and additionally with the D670G line that is known for a relatively mild phenotype. We investigated PDE6A expression, cyclic guanosine mono-phosphate accumulation, calpain and caspase activity, in vivo retinal function and morphology, as well as photoreceptor cell death and survival. This analysis confirms the severity of different Pde6a mutations and indicates that compound heterozygous mutants behave like intermediates of the respective homozygous situations. Specifically, the severity of the four different Pde6a situations may be categorized by the pace of photoreceptor degeneration: V685M (fastest) > V685M/R562W > R562W > D670G (slowest). While calpain activity was strongly increased in all four mutants, caspase activity was not. This points to the execution of non-apoptotic cell death and may lead to the identification of new targets for therapeutic interventions. For individual RP patients, our study may help to predict time-courses for Pde6a-related retinal degeneration and thereby facilitate the definition of a window-of-opportunity for clinical interventions. PMID:26188004

  15. Genetic diversity within the R408W phenylketonuria mutation lineages in Europe.

    PubMed

    Tighe, Orna; Dunican, Donncha; O'Neill, Charles; Bertorelle, Giorgio; Beattie, Diane; Graham, Colin; Zschocke, Johannes; Cali, Francesco; Romano, Valentino; Hrabincova, Eva; Kozak, Libor; Nechyporenko, Marina; Livshits, Ludmilla; Guldberg, Per; Jurkowska, Monika; Zekanowski, Cezary; Perez, Belen; Desviat, Lourdes Ruiz; Ugarte, Magdalena; Kucinskas, Vaidutis; Knappskog, Per; Treacy, Eileen; Naughten, Eileen; Tyfield, Linda; Byck, Susan; Scriver, Charles R; Mayne, Philip D; Croke, David T

    2003-04-01

    The R408W phenylketonuria mutation in Europe has arisen by recurrent mutation in the human phenylalanine hydroxylase (PAH) locus and is associated with two major PAH haplotypes. R408W-2.3 exhibits a west-to-east cline of relative frequency reaching its maximum in the Balto-Slavic region, while R408W-1.8 exhibits an east-to-west cline peaking in Connacht, the most westerly province of Ireland. Spatial autocorrelation analysis has demonstrated that the R408W-2.3 cline, like that of R408W-1.8, is consistent with a pattern likely to have been established by human dispersal. Genetic diversity within wild-type and R408W chromosomes in Europe was assessed through variable number tandem repeat (VNTR) nucleotide sequence variation and tetranucleotide short tandem repeat (STR) allelic associations. Wild-type VNTR-8 chromosomes exhibited two major cassette sequence organizations: (a1)5-b3-b2-c1 and (a1)5-b5-b2-c1. R408W-1.8 was predominantly associated with (a1)5-B5-B2-C1. Both wild-type vntr-3 and r408w-2.3 chromosomes exhibited a single invariant cassette sequence organization, a2-b2-c1. STR allele distributions associated with the cassette variants were consistent with greater diversity in the wild-type VNTR-8 lineage and were suggestive of different levels of diversity between R408W-1.8 and R408W-2.3. The finding of greater genetic diversity within the wild-type VNTR-8 lineage compared to VNTR-3 suggests that VNTR-8 may be older within the European population. However, in the absence of a more extensive STR data-set, no such conclusions are possible for the respective R408W mutant lineages. PMID:12655548

  16. A Point Mutation within the Replicase Gene Differentially Affects Coronavirus Genome versus Minigenome Replication

    PubMed Central

    Galán, Carmen; Enjuanes, Luis; Almazán, Fernando

    2005-01-01

    During the construction of the transmissible gastroenteritis virus (TGEV) full-length cDNA clone, a point mutation at position 637 that was present in the defective minigenome DI-C was maintained as a genetic marker. Sequence analysis of the recovered viruses showed a reversion at this position to the original virus sequence. The effect of point mutations at nucleotide 637 was analyzed by reverse genetics using a TGEV full-length cDNA clone and cDNAs from TGEV-derived minigenomes. The replacement of nucleotide 637 of TGEV genome by a T, as in the DI-C sequence, or an A severely affected virus recovery from the cDNA, yielding mutant viruses with low titers and small plaques compared to those of the wild type. In contrast, T or A at position 637 was required for minigenome rescue in trans by the helper virus. No relationship between these observations and RNA secondary-structure predictions was found, indicating that mutations at nucleotide 637 most likely had an effect at the protein level. Nucleotide 637 occupies the second codon position at amino acid 108 of the pp1a polyprotein. This position is predicted to map in the N-terminal polyprotein papain-like proteinase (PLP-1) cleavage site at the p9/p87 junction. Replacement of G-637 by A, which causes a drastic amino acid change (Gly to Asp) at position 108, affected PLP-1-mediated cleavage in vitro. A correlation was found between predicted cleaving and noncleaving mutations and efficient virus rescue from cDNA and minigenome amplification, respectively. PMID:16306572

  17. 2-Amino-N6-hydroxyadenine induces gene/point mutations and multiple-locus mutations, but not multilocus deletion mutations, in the ad-3 region of a two-component heterokaryon of Neurospora crassa.

    PubMed

    de Serres, F J; Brockman, H E; Overton, L K

    1991-08-01

    The mutagenicity of 2-amino-N6-hydroxyadenine (AHA) has been studied in Neurospora crassa by treating a two-component heterokaryon (H-12) and recovering specific-locus mutations induced in the ad-3 region. This assay system permits the identification of ad-3A and/or ad-3B mutants resulting from gene/point mutations, multilocus deletion mutations, and multiple-locus mutations of various genotypes, involving one or both loci. Genetic characterization of the ad-3 mutants recovered from experiments with AHA in H-12 shows that 98.9% (270/273) of the ad-3 mutants are gene/point mutations (ad-3R), 1.1% (3/270) are unknowns, and none is a multilocus deletion mutation (ad-3IR). Among the gene/point mutations, 3.3% (9/273) are multiple-locus mutations (gene/point mutations with a closely-linked recessive lethal mutation [ad-3R + RLCL]). Another 25.3% (69/273) are multiple-locus mutations with a recessive lethal mutation located elsewhere in the genome [ad-3R + RL]. Heterokaryon tests for allelic complementation among the ad-3BR mutants showed that 90.8% (139/153) of the mutants were complementing, and 20.3% (31/153) were leaky. In addition, 32.5% (38/117) of the ad-3AR mutants were leaky. These data are consistent with the hypothesis that AHA produces specific-locus mutations in the ad-3 region of N. crassa by base-pair substitution. The data from the present experiments are compared with the data for 2-aminopurine (2AP)-induced ad-3 mutants in H-12 (de Serres and Brockman, 1991). Whereas, 2AP is a weak mutagen in H-12, AHA is extremely potent (Brockman et al., 1987). In contrast with 2AP, AHA induces ad-3 mutants exclusively by gene/point mutation in H-12. We conclude that whereas AHA induces ad-3 mutants predominantly by AT to GC base-pair transitions, 2AP induces ad-3 mutants by a wide variety of mechanisms including: (1) AT to GC and GC to AT base-pair transitions, (2) frameshift mutations, (3) other, as yet unidentified, intragenic alterations, (4) small multilocus

  18. Random mutagenesis strategies for construction of large and diverse clone libraries of mutated DNA fragments.

    PubMed

    Chusacultanachai, Sudsanguan; Yuthavong, Yongyuth

    2004-01-01

    The first important step toward a successful preparation of large and diverse DNA libraries with desired complexity is to select a suitable mutagenesis strategy. This chapter describes three different methods for random mutagenesis, the use of XL1-red cells, error-prone polymerase chain reaction (PCR), and degenerate oligonucleotides-Pfu (DOP). These mutagenesis strategies possess different benefits and pitfalls; thus, they are differentially useful for production of DNA libraries with different density and complexity. The use of XL1-red, an engineered Escherichia coli with DNA repair deficiency, is one of the simplest mutagenesis and requires no subcloning step. After plasmid encoding DNA of inter-est is transformed into the cells, the mutations are simply generated during each round of DNA replication. The mutation frequency of this method is reported to be 1 base change per 2000 nucleotides; however, it can be slightly increased by extending the culture period to allow the accumulation of more mutations. This strategy is suitable for generation of random mutations with low frequency in a large target DNA. Error-prone PCR is one of the most widely used random mutagenesis. During DNA amplification, misincorporation of nucleotides can be promoted by altering the nucleotide ratio and the concentration of divalent cations in the reaction. We discovered that, by adjusting template concentration, frequency of mutation could be controlled easily and a library with desired mutation rate could be obtained. Additionally, efficiency of subsequent cloning steps to insert the PCR product into plasmid DNA is also a key factor determining size and complexity of the libraries. DOP mutagenesis is a rapid and effective method for random mutagenesis of small DNA and peptides. This strategy uses two chemically synthesized degenerate oligonucleotides as primers. By controlling the positions and ratios of degenerate nucleotides used during oligonucleotide synthesis, it is possible to

  19. Heteroplasmy levels of mtDNA1555A>G mutation is positively associated with diverse phenotypes and mutation transmission in a Chinese family.

    PubMed

    Shen, Shan-Shan; Liu, Chang; Xu, Zhi-Yong; Hu, Yu-Hua; Gao, Guo-Feng; Wang, Sha-Yan

    2012-04-20

    The mtDNA 1555A>G mutation was considered to be one of the most common causes of aminoglycoside-induced and non-syndromic hearing loss. However, this mutation was always found in homoplasmy with high phenotypic heterogeneity. Recently this mutation in heteroplasmy has been reported in several studies. In the present study, we have collected a large Chinese family harboring heteroplasmic mtDNA 1555A>G mutation with diverse clinical phenotypes. To investigate the relationship between the mutation load and the severity of hearing loss under Eastern Asian background, we performed clinical, molecular, genetic and phylogenic analysis. This pedigree was characterized by coexistence of eight subjects with homoplasmic mutation and ten subjects with various degrees of heteroplasmy, and the results suggested that there was a strong correlation between the mutation load and the severity/age-onset of hearing loss (r=0.758, p<0.001). We noticed that the mutation level of offspring was associated with their mothers' in this pedigree, which indicated that maybe exist a regular pattern during the process of the heteroplasmic transmission. In addition, analysis of the complete mtDNA genome of this family revealed that it belonged to Eastern Asian haplogroup B4C1. In addition, a rare homoplasmic mtDNA 9128T>C variant was identified, it located at a strictly conserved site of mtDNA ATP6 gene. PMID:22475488

  20. Specific point mutations in Lactobacillus casei ATCC 27139 cause a phenotype switch from Lac- to Lac+.

    PubMed

    Tsai, Yu-Kuo; Chen, Hung-Wen; Lo, Ta-Chun; Lin, Thy-Hou

    2009-03-01

    Lactose metabolism is a changeable phenotype in strains of Lactobacillus casei. In this study, we found that L. casei ATCC 27139 was unable to utilize lactose. However, when exposed to lactose as the sole carbon source, spontaneous Lac(+) clones could be obtained. A gene cluster (lacTEGF-galKETRM) involved in the metabolism of lactose and galactose in L. casei ATCC 27139 (Lac(-)) and its Lac(+) revertant (designated strain R1) was sequenced and characterized. We found that only one nucleotide, located in the lacTEGF promoter (lacTp), of the two lac-gal gene clusters was different. The protein sequence identity between the lac-gal gene cluster and those reported previously for some L. casei (Lac(+)) strains was high; namely, 96-100 % identity was found and no premature stop codon was identified. A single point mutation located within the lacTp promoter region was also detected for each of the 41 other independently isolated Lac(+) revertants of L. casei ATCC 27139. The revertants could be divided into six classes based on the positions of the point mutations detected. Primer extension experiments conducted on transcription from lacTp revealed that the lacTp promoter of these six classes of Lac(+) revertants was functional, while that of L. casei ATCC 27139 was not. Northern blotting experiments further confirmed that the lacTEGF operon of strain R1 was induced by lactose but suppressed by glucose, whereas no blotting signal was ever detected for L. casei ATCC 27139. These results suggest that a single point mutation in the lacTp promoter was able to restore the transcription of a fully functional lacTEGF operon and cause a phenotype switch from Lac(-) to Lac(+) for L. casei ATCC 27139. PMID:19246746

  1. A Mismatch EndoNuclease Array-Based Methodology (MENA) for Identifying Known SNPs or Novel Point Mutations.

    PubMed

    Comeron, Josep M; Reed, Jordan; Christie, Matthew; Jacobs, Julia S; Dierdorff, Jason; Eberl, Daniel F; Manak, J Robert

    2016-01-01

    Accurate and rapid identification or confirmation of single nucleotide polymorphisms (SNPs), point mutations and other human genomic variation facilitates understanding the genetic basis of disease. We have developed a new methodology (called MENA (Mismatch EndoNuclease Array)) pairing DNA mismatch endonuclease enzymology with tiling microarray hybridization in order to genotype both known point mutations (such as SNPs) as well as identify previously undiscovered point mutations and small indels. We show that our assay can rapidly genotype known SNPs in a human genomic DNA sample with 99% accuracy, in addition to identifying novel point mutations and small indels with a false discovery rate as low as 10%. Our technology provides a platform for a variety of applications, including: (1) genotyping known SNPs as well as confirming newly discovered SNPs from whole genome sequencing analyses; (2) identifying novel point mutations and indels in any genomic region from any organism for which genome sequence information is available; and (3) screening panels of genes associated with particular diseases and disorders in patient samples to identify causative mutations. As a proof of principle for using MENA to discover novel mutations, we report identification of a novel allele of the beethoven (btv) gene in Drosophila, which encodes a ciliary cytoplasmic dynein motor protein important for auditory mechanosensation. PMID:27600073

  2. A Mismatch EndoNuclease Array-Based Methodology (MENA) for Identifying Known SNPs or Novel Point Mutations

    PubMed Central

    Comeron, Josep M.; Reed, Jordan; Christie, Matthew; Jacobs, Julia S.; Dierdorff, Jason; Eberl, Daniel F.; Manak, J. Robert

    2016-01-01

    Accurate and rapid identification or confirmation of single nucleotide polymorphisms (SNPs), point mutations and other human genomic variation facilitates understanding the genetic basis of disease. We have developed a new methodology (called MENA (Mismatch EndoNuclease Array)) pairing DNA mismatch endonuclease enzymology with tiling microarray hybridization in order to genotype both known point mutations (such as SNPs) as well as identify previously undiscovered point mutations and small indels. We show that our assay can rapidly genotype known SNPs in a human genomic DNA sample with 99% accuracy, in addition to identifying novel point mutations and small indels with a false discovery rate as low as 10%. Our technology provides a platform for a variety of applications, including: (1) genotyping known SNPs as well as confirming newly discovered SNPs from whole genome sequencing analyses; (2) identifying novel point mutations and indels in any genomic region from any organism for which genome sequence information is available; and (3) screening panels of genes associated with particular diseases and disorders in patient samples to identify causative mutations. As a proof of principle for using MENA to discover novel mutations, we report identification of a novel allele of the beethoven (btv) gene in Drosophila, which encodes a ciliary cytoplasmic dynein motor protein important for auditory mechanosensation. PMID:27600073

  3. Point mutations in the proximal Cys-His box of Rous sarcoma virus nucleocapsid protein.

    PubMed Central

    Dupraz, P; Oertle, S; Meric, C; Damay, P; Spahr, P F

    1990-01-01

    To extend our previous studies of the function of the Cys-His box of Rous sarcoma virus NC protein, we have constructed a series of point mutations of the conserved or nonconserved amino acids of the proximal Cys-His box and a one-amino-acid deletion. All mutants were characterized for production of viral proteins and particles, for packaging and maturation of viral RNA, for reverse transcriptase activity, and for infectivity. Our results indicated the following. (i) Mutations affecting the strictly conserved amino acids cysteine 21, cysteine 24, and histidine 29 were lethal; only the mutant His-29----Pro was still able to package viral RNA, most of it in an immature form. (ii) Mutation of the highly conserved glycine 28 to valine reduced viral RNA packaging by 90% and infectivity 30-fold, whereas mutant Gly-28----Ala was fully infectious. This suggests a steric hindrance limit at this position. (iii) Shortening the distance between cysteine 24 and histidine 29 by deleting one amino acid abolished the maturation of viral RNA and yielded noninfectious particles. (iv) Substitution of tyrosine 22 by serine lowered viral RNA packaging efficiency and yielded particles that were 400-fold less infectious; double mutant Tyr-22Thr-23----SerSer had the same infectivity as Tyr-22----Ser, whereas mutant Thr-23----Ser was fully infectious. (v) Changing glutamine 33 to a charged glutamate residue did not affect virus infectivity. Similarities and differences between our avian mutants and those in murine retroviruses are discussed. Images PMID:2168981

  4. Prediction of change in protein unfolding rates upon point mutations in two state proteins.

    PubMed

    Chaudhary, Priyashree; Naganathan, Athi N; Gromiha, M Michael

    2016-09-01

    Studies on protein unfolding rates are limited and challenging due to the complexity of unfolding mechanism and the larger dynamic range of the experimental data. Though attempts have been made to predict unfolding rates using protein sequence-structure information there is no available method for predicting the unfolding rates of proteins upon specific point mutations. In this work, we have systematically analyzed a set of 790 single mutants and developed a robust method for predicting protein unfolding rates upon mutations (Δlnku) in two-state proteins by combining amino acid properties and knowledge-based classification of mutants with multiple linear regression technique. We obtain a mean absolute error (MAE) of 0.79/s and a Pearson correlation coefficient (PCC) of 0.71 between predicted unfolding rates and experimental observations using jack-knife test. We have developed a web server for predicting protein unfolding rates upon mutation and it is freely available at https://www.iitm.ac.in/bioinfo/proteinunfolding/unfoldingrace.html. Prominent features that determine unfolding kinetics as well as plausible reasons for the observed outliers are also discussed. PMID:27264959

  5. Point-Mutation Effects on Charge-Transport Properties of the Tumor-Suppressor Gene p53

    NASA Astrophysics Data System (ADS)

    Shih, Chi-Tin; Roche, Stephan; Römer, Rudolf A.

    2008-01-01

    We report on a theoretical study of point mutations effects on charge transfer properties in the DNA sequence of the tumor-suppressor p53 gene. On the basis of effective tight-binding models which simulate hole propagation along the DNA, a statistical analysis of mutation-induced charge transfer modifications is performed. In contrast to noncancerous mutations, mutation hot spots tend to result in significantly weaker changes of transmission properties. This suggests that charge transport could play a significant role for DNA-repairing deficiency yielding carcinogenesis.

  6. Effect of point mutations on Herbaspirillum seropedicae NifA activity

    PubMed Central

    Aquino, B.; Stefanello, A.A.; Oliveira, M.A.S.; Pedrosa, F.O.; Souza, E.M.; Monteiro, R.A.; Chubatsu, L.S.

    2015-01-01

    NifA is the transcriptional activator of the nif genes in Proteobacteria. It is usually regulated by nitrogen and oxygen, allowing biological nitrogen fixation to occur under appropriate conditions. NifA proteins have a typical three-domain structure, including a regulatory N-terminal GAF domain, which is involved in control by fixed nitrogen and not strictly required for activity, a catalytic AAA+ central domain, which catalyzes open complex formation, and a C-terminal domain involved in DNA-binding. In Herbaspirillum seropedicae, a β-proteobacterium capable of colonizing Graminae of agricultural importance, NifA regulation by ammonium involves its N-terminal GAF domain and the signal transduction protein GlnK. When the GAF domain is removed, the protein can still activate nif genes transcription; however, ammonium regulation is lost. In this work, we generated eight constructs resulting in point mutations in H. seropedicae NifA and analyzed their effect on nifH transcription in Escherichia coli and H. seropedicae. Mutations K22V, T160E, M161V, L172R, and A215D resulted in inactive proteins. Mutations Q216I and S220I produced partially active proteins with activity control similar to wild-type NifA. However, mutation G25E, located in the GAF domain, resulted in an active protein that did not require GlnK for activity and was partially sensitive to ammonium. This suggested that G25E may affect the negative interaction between the N-terminal GAF domain and the catalytic central domain under high ammonium concentrations, thus rendering the protein constitutively active, or that G25E could lead to a conformational change comparable with that when GlnK interacts with the GAF domain. PMID:26176311

  7. Somatic mutations contribute to genotypic diversity in sterile and fertile populations of the threatened shrub, Grevillea rhizomatosa (Proteaceae)

    PubMed Central

    Gross, C. L.; Nelson, Penelope A.; Haddadchi, Azadeh; Fatemi, Mohammad

    2012-01-01

    Background and Aims Grevillea rhizomatosa is a spreading shrub which exhibits multiple breeding strategies within a narrow area in the fire-prone heathlands of eastern Australia. Reproductive strategies include self-compatibility, self-incompatibility and clonality (with and without sterility). The close proximity of contrasting breeding systems provides an opportunity to explore the evolution of sterility and to compare and contrast the origins of genotypic diversity (recombinant or somatic) against degrees of sexual expression. Methods ISSR markers for 120 band positions (putative loci) were used to compare genetic diversity among five populations at a macro-scale of 5 m between samples (n = 244 shrubs), and at a micro-scale of nearest neighbours for all plants in five 25-m2 quadrats with contrasting fertilities (n = 162 shrubs). Nearest-neighbour sampling included several clusters of connected ramets. Matrix incompatibility (MIC) analyses were used to evaluate the relative contribution of recombination and somatic mutation to genotype diversity. Key Results High levels of genotypic diversity were found in all populations regardless of fertilities (fertile populations, G/N ≥ 0·94; sterile populations, G/N ≥ 0·97) and most sterile populations had a unique genetic profile. Somatic mutations were detected along connected ramets in ten out of 42 ramet clusters. MIC analyses showed that somatic mutations have contributed to diversity in all populations and particularly so in sterile populations. Conclusions Somatic mutations contribute significantly to gene diversity in sterile populations of Grevillea rhizomatosa, the accumulation of which is the likely cause of male and female sterility. High levels of genetic diversity therefore may not always be synonymous with sexual fitness and genetic health. We hypothesize that frequent fires drive selection for clonal reproduction, at the cost of flowering such that sexual functions are not maintained through selection

  8. How Do Single Point Mutations Impact Protein Folding in Parkinson's Disease

    NASA Astrophysics Data System (ADS)

    Wise-Scira, Olivia; Roque, Andrew; Xu, Liang; Coskuner, Orkid

    2010-10-01

    Although the structures of the wild type (WT) and mutants (A53T, A30P, E46K) of α-synuclein (α-syn) proteins related to Parkinson's disease have been studied extensively using both experimental and theoretical tools, the relationships between the structural properties and thermodynamic preferences at a molecular level with dynamics are unknown. Such an understanding is required for accessing detailed knowledge regarding to the ``early aggregation and monomer'' hypothesis in Parkinson's disease. We investigated the impact of these single point mutations on the structures and conformational preferences of α-syn monomers in aqueous solution as well as the impact of the aqueous solution environment on the proteins. Obtained qualitative and quantitative results provide new insights into the structure-function relationships of these proteins and help us to understand the molecular mechanism hidden behind the ``early aggregation and monomer'' hypothesis. Our results show that the tertiary structure of the α-syn proteins varies significantly with dynamics, however, this variability is not easily reflected in the changes of the relative amounts of the secondary structural components. The obtained structures also demonstrate that a single point mutation can have a significant effect on protein folding. The structures of each of the WT, A53T, A30P, and E46K α-syn monomers differ from each other throughout and the presence of aqueous solution significantly impacts the α-syn protein structures.

  9. Point Mutations in Membrane Proteins Reshape Energy Landscape and Populate Different Unfolding Pathways

    PubMed Central

    Sapra, K. Tanuj; Balasubramanian, G. Prakash; Labudde, Dirk; Bowie, James U.; Muller, Daniel J.

    2009-01-01

    Using single-molecule force spectroscopy, we investigated the effect of single point mutations on the energy landscape and unfolding pathways of the transmembrane protein bacteriorhodopsin. We show that the unfolding energy barriers in the energy landscape of the membrane protein followed a simple two-state behavior and represent a manifestation of many converging unfolding pathways. Although the unfolding pathways of wild-type and mutant bacteriorhodopsin did not change, indicating the presence of same ensemble of structural unfolding intermediates, the free energies of the rate-limiting transition states of the bacteriorhodopsin mutants decreased as the distance of those transition states to the folded intermediate states decreased. Thus, all mutants exhibited Hammond behavior and a change in the free energies of the intermediates along the unfolding reaction coordinate and, consequently, their relative occupancies. This is the first experimental proof showing that point mutations can reshape the free energy landscape of a membrane protein and force single proteins to populate certain unfolding pathways over others. PMID:18191146

  10. Investigating the Impact of Asp181 Point Mutations on Interactions between PTP1B and Phosphotyrosine Substrate

    NASA Astrophysics Data System (ADS)

    Liu, Mengyuan; Wang, Lushan; Sun, Xun; Zhao, Xian

    2014-05-01

    Protein tyrosine phosphatase 1B (PTP1B) is a key negative regulator of insulin and leptin signaling, which suggests that it is an attractive therapeutic target in type II diabetes and obesity. The aim of this research is to explore residues which interact with phosphotyrosine substrate can be affected by D181 point mutations and lead to increased substrate binding. To achieve this goal, molecular dynamics simulations were performed on wild type (WT) and two mutated PTP1B/substrate complexes. The cross-correlation and principal component analyses show that point mutations can affect the motions of some residues in the active site of PTP1B. Moreover, the hydrogen bond and energy decomposition analyses indicate that apart from residue 181, point mutations have influence on the interactions of substrate with several residues in the active site of PTP1B.

  11. Subjects harboring presenilin familial Alzheimer’s disease mutations exhibit diverse white matter biochemistry alterations

    PubMed Central

    Roher, Alex E; Maarouf, Chera L; Malek-Ahmadi, Michael; Wilson, Jeffrey; Kokjohn, Tyler A; Daugs, Ian D; Whiteside, Charisse M; Kalback, Walter M; Macias, MiMi P; Jacobson, Sandra A; Sabbagh, Marwan N; Ghetti, Bernardino; Beach, Thomas G

    2013-01-01

    Alzheimer’s disease (AD) dementia impacts all facets of higher order cognitive function and is characterized by the presence of distinctive pathological lesions in the gray matter (GM). The profound alterations in GM structure and function have fostered the view that AD impacts are primarily a consequence of GM damage. However, the white matter (WM) represents about 50% of the cerebrum and this area of the brain is substantially atrophied and profoundly abnormal in both sporadic AD (SAD) and familial AD (FAD). We examined the WM biochemistry by ELISA and Western blot analyses of key proteins in 10 FAD cases harboring mutations in the presenilin genes PSEN1 and PSEN2 as well as in 4 non-demented control (NDC) individuals and 4 subjects with SAD. The molecules examined were direct substrates of PSEN1 such as Notch-1 and amyloid precursor protein (APP). In addition, apolipoproteins, axonal transport molecules, cytoskeletal and structural proteins, neurotrophic factors and synaptic proteins were examined. PSEN-FAD subjects had, on average, higher amounts of WM amyloid-beta (Aβ) peptides compared to SAD, which may play a role in the devastating dysfunction of the brain. However, the PSEN-FAD mutations we examined did not produce uniform increases in the relative proportions of Aβ42 and exhibited substantial variability in total Aβ levels. These observations suggest that neurodegeneration and dementia do not depend solely on enhanced Aβ42 levels. Our data revealed additional complexities in PSEN-FAD individuals. Some direct substrates of γ-secretase, such as Notch, N-cadherin, Erb-B4 and APP, deviated substantially from the NDC group baseline for some, but not all, mutation types. Proteins that were not direct γ-secretase substrates, but play key structural and functional roles in the WM, likewise exhibited varied concentrations in the distinct PSEN mutation backgrounds. Detailing the diverse biochemical pathology spectrum of PSEN mutations may offer valuable

  12. Diverse Renal Phenotypes Observed in a Single Family with a Genetic Mutation in Paired Box Protein 2

    PubMed Central

    Iwafuchi, Yoichi; Morioka, Tetsuo; Morita, Takashi; Yanagihara, Toshio; Oyama, Yuko; Morisada, Naoya; Iijima, Kazumoto; Narita, Ichiei

    2016-01-01

    A common renal phenotype of paired box protein 2 (PAX2) mutations is renal coloboma syndrome. We report a single family with diverse renal phenotypes associated with PAX2 mutation. The proband presented steroid-resistant focal segmental glomerulosclerosis with optic coloboma, whereas his two sons showed severe renal hypoplasia with end-stage renal disease, with or without optic coloboma. In all three cases, a heterozygous PAX2 genetic mutation was identified (exon 2; NM_003987.3:c.76dupG, p.Val26Glyfs*28). Based on histopathological findings of the proband, we hypothesized that autophagic dysfunction was associated with the pathophysiology of the focal segmental glomerulosclerosis with PAX2 mutation. Detailed funduscopic examination – including the optic disc – might be useful for the diagnosis of renal anomalies associated with PAX2 mutation. PMID:27226968

  13. Diverse Renal Phenotypes Observed in a Single Family with a Genetic Mutation in Paired Box Protein 2.

    PubMed

    Iwafuchi, Yoichi; Morioka, Tetsuo; Morita, Takashi; Yanagihara, Toshio; Oyama, Yuko; Morisada, Naoya; Iijima, Kazumoto; Narita, Ichiei

    2016-01-01

    A common renal phenotype of paired box protein 2 (PAX2) mutations is renal coloboma syndrome. We report a single family with diverse renal phenotypes associated with PAX2 mutation. The proband presented steroid-resistant focal segmental glomerulosclerosis with optic coloboma, whereas his two sons showed severe renal hypoplasia with end-stage renal disease, with or without optic coloboma. In all three cases, a heterozygous PAX2 genetic mutation was identified (exon 2; NM_003987.3:c.76dupG, p.Val26Glyfs*28). Based on histopathological findings of the proband, we hypothesized that autophagic dysfunction was associated with the pathophysiology of the focal segmental glomerulosclerosis with PAX2 mutation. Detailed funduscopic examination - including the optic disc - might be useful for the diagnosis of renal anomalies associated with PAX2 mutation. PMID:27226968

  14. Kinase inhibitor-responsive genotypes in EGFR mutated lung adenocarcinomas: moving past common point mutations or indels into uncommon kinase domain duplications and rearrangements.

    PubMed

    Costa, Daniel B

    2016-06-01

    The most frequent epidermal growth factor receptor (EGFR) mutations found by traditional or comprehensive molecular profiling of lung adenocarcinomas include indels of exon 19 (the exon 19 deletion delE746_A750 being the most common) and the exon 21 L858R point mutation. The current approval labels for first line palliative gefitinib 250 mg/day, erlotinib 150 mg/day and afatinib 40 mg/day for advanced lung cancers require the presence of the aforementioned classical/sensitizing EGFR mutations. Other gefitinib, erlotinib and afatinib sensitizing mutations include exon 18 indels, G719X, exon 19 insertions, A763_Y764insFQEA, S768I and L861Q; for which off-label EGFR kinase inhibitor use is generally agreed upon by thoracic oncologists. The main biological mechanism of resistance to approved first line EGFR inhibitors is the selection/acquisition of EGFR-T790M that in itself can be inhibited by osimertinib 80 mg/day, a 3(rd) generation EGFR inhibitor that is bypassed by EGFR-C797X mutations. Another class of de novo inhibitor insensitive mutation includes EGFR exon 20 insertions. More recently, the dichotomy of only point mutations or indels explaining aberrant kinase activation of EGFR plus inhibitor response has been shattered by the discovery of uncommon (<0.5% of all EGFR mutations) genomic events involving exon 18-25 kinase domain duplications (KDD) and rearrangements (EGFR-RAD51 or EGFR-PURB). The latter lead to oncogene addiction, enhanced sensitivity to kinase inhibitors in vitro and clinical responses to approved EGFR inhibitors. The enhanced landscape of EGFR inhibitor-responsive genotypes highlights that comprehensive molecular profiling may be necessary to maximize the identification of all cases that can benefit from precision oncology. PMID:27413714

  15. Kinase inhibitor-responsive genotypes in EGFR mutated lung adenocarcinomas: moving past common point mutations or indels into uncommon kinase domain duplications and rearrangements

    PubMed Central

    2016-01-01

    The most frequent epidermal growth factor receptor (EGFR) mutations found by traditional or comprehensive molecular profiling of lung adenocarcinomas include indels of exon 19 (the exon 19 deletion delE746_A750 being the most common) and the exon 21 L858R point mutation. The current approval labels for first line palliative gefitinib 250 mg/day, erlotinib 150 mg/day and afatinib 40 mg/day for advanced lung cancers require the presence of the aforementioned classical/sensitizing EGFR mutations. Other gefitinib, erlotinib and afatinib sensitizing mutations include exon 18 indels, G719X, exon 19 insertions, A763_Y764insFQEA, S768I and L861Q; for which off-label EGFR kinase inhibitor use is generally agreed upon by thoracic oncologists. The main biological mechanism of resistance to approved first line EGFR inhibitors is the selection/acquisition of EGFR-T790M that in itself can be inhibited by osimertinib 80 mg/day, a 3rd generation EGFR inhibitor that is bypassed by EGFR-C797X mutations. Another class of de novo inhibitor insensitive mutation includes EGFR exon 20 insertions. More recently, the dichotomy of only point mutations or indels explaining aberrant kinase activation of EGFR plus inhibitor response has been shattered by the discovery of uncommon (<0.5% of all EGFR mutations) genomic events involving exon 18–25 kinase domain duplications (KDD) and rearrangements (EGFR-RAD51 or EGFR-PURB). The latter lead to oncogene addiction, enhanced sensitivity to kinase inhibitors in vitro and clinical responses to approved EGFR inhibitors. The enhanced landscape of EGFR inhibitor-responsive genotypes highlights that comprehensive molecular profiling may be necessary to maximize the identification of all cases that can benefit from precision oncology. PMID:27413714

  16. HIV type 1 pol gene diversity and archived nevirapine resistance mutation in pregnant women in Rwanda.

    PubMed

    Servais, Jean; Lambert, Christine; Karita, Etienne; Vanhove, Dirk; Fischer, Aurelie; Baurith, Therese; Schmit, Jean-Claude; Schneider, François; Hemmer, Robert; Arendt, Vic

    2004-03-01

    This study aimed to find out whether genetic polymorphisms were present in positions potentially affecting susceptibility to antiretrovirals in non-B subtypes from HIV-1-infected patients in Rwanda. Viral pol gene diversity was investigated by direct sequencing in 43 treatment-naive women. In addition, 10 DNA sequences from uncultured peripheral blood mononuclear cells were analyzed 6 weeks after a single dose of nevirapine (prevention of mother-to-child transmission program). Phylogenetic analyses have shown 34 subtype A1, 6 subtype C, and 2 subtype D strains. In addition, an A/C recombinant between the protease (PR) (subtype A1) and the reverse transcriptase (RT) (subtype C) was identified. In the PR coding region, high numbers of polymorphisms were found, including substitutions in secondary PR resistance sites. PR 35D, 36I, and 37N were always present within subtype A as were PR 93L in subtype C strains. PR 10I/V, 20R, 33F, and 77V were found in subtype A whereas PR 36I was highly prevalent in subtype C strains. The A/C recombinant displayed substitutions related to resistance (PR 10, 33, 36 and RT 118). One nevirapine resistance mutation (RT 181Y/C) was found in proviral DNA after 6 weeks. In conclusion, subtypes A and C are predominant in this cohort in Rwanda. Substitutions similar to secondary protease inhibitor resistance mutations are common before treatment whereas major resistance mutation may be archived after a single dose of nevirapine. Accordingly, the hypothesis of a genetic background effect in non-B strains has to be further addressed in programs of introduction of antivirals in Africa. PMID:15117451

  17. Diversity of Pol IV Function Is Defined by Mutations at the Maize rmr7 Locus

    PubMed Central

    Erhard, Karl F.; Hollick, Jay B.

    2009-01-01

    Mutations affecting the heritable maintenance of epigenetic states in maize identify multiple small RNA biogenesis factors including NRPD1, the largest subunit of the presumed maize Pol IV holoenzyme. Here we show that mutations defining the required to maintain repression7 locus identify a second RNA polymerase subunit related to Arabidopsis NRPD2a, the sole second largest subunit shared between Arabidopsis Pol IV and Pol V. A phylogenetic analysis shows that, in contrast to representative eudicots, grasses have retained duplicate loci capable of producing functional NRPD2-like proteins, which is indicative of increased RNA polymerase diversity in grasses relative to eudicots. Together with comparisons of rmr7 mutant plant phenotypes and their effects on the maintenance of epigenetic states with parallel analyses of NRPD1 defects, our results imply that maize utilizes multiple functional NRPD2-like proteins. Despite the observation that RMR7/NRPD2, like NRPD1, is required for the accumulation of most siRNAs, our data indicate that different Pol IV isoforms play distinct roles in the maintenance of meiotically-heritable epigenetic information in the grasses. PMID:19936246

  18. A maternally inherited autosomal point mutation in human phospholipase C zeta (PLCζ) leads to male infertility

    PubMed Central

    Kashir, Junaid; Konstantinidis, Michalis; Jones, Celine; Lemmon, Bernadette; Chang Lee, Hoi; Hamer, Rebecca; Heindryckx, Bjorn; Deane, Charlotte M.; De Sutter, Petra; Fissore, Rafael A.; Parrington, John; Wells, Dagan; Coward, Kevin

    2012-01-01

    BACKGROUND Male factor and idiopathic infertility contribute significantly to global infertility, with abnormal testicular gene expression considered to be a major cause. Certain types of male infertility are caused by failure of the sperm to activate the oocyte, a process normally regulated by calcium oscillations, thought to be induced by a sperm-specific phospholipase C, PLCzeta (PLCζ). Previously, we identified a point mutation in an infertile male resulting in the substitution of histidine for proline at position 398 of the protein sequence (PLCζH398P), leading to abnormal PLCζ function and infertility. METHODS AND RESULTS Here, using a combination of direct-sequencing and mini-sequencing of the PLCζ gene from the patient and his family, we report the identification of a second PLCζ mutation in the same patient resulting in a histidine to leucine substitution at position 233 (PLCζH233L), which is predicted to disrupt local protein interactions in a manner similar to PLCζH398P and was shown to exhibit abnormal calcium oscillatory ability following predictive 3D modelling and cRNA injection in mouse oocytes respectively. We show that PLCζH233L and PLCζH398P exist on distinct parental chromosomes, the former inherited from the patient's mother and the latter from his father. Neither mutation was detected utilizing custom-made single-nucleotide polymorphism assays in 100 fertile males and females, or 8 infertile males with characterized oocyte activation deficiency. CONCLUSIONS Collectively, our findings provide further evidence regarding the importance of PLCζ at oocyte activation and forms of male infertility where this is deficient. Additionally, we show that the inheritance patterns underlying male infertility are more complex than previously thought and may involve maternal mechanisms. PMID:22095789

  19. Evolution of Salmonella enterica Virulence via Point Mutations in the Fimbrial Adhesin

    PubMed Central

    Kisiela, Dagmara I.; Chattopadhyay, Sujay; Libby, Stephen J.; Karlinsey, Joyce E.; Fang, Ferric C.; Tchesnokova, Veronika; Kramer, Jeremy J.; Beskhlebnaya, Viktoriya; Samadpour, Mansour; Grzymajlo, Krzysztof; Ugorski, Maciej; Lankau, Emily W.; Mackie, Roderick I.; Clegg, Steven; Sokurenko, Evgeni V.

    2012-01-01

    Whereas the majority of pathogenic Salmonella serovars are capable of infecting many different animal species, typically producing a self-limited gastroenteritis, serovars with narrow host-specificity exhibit increased virulence and their infections frequently result in fatal systemic diseases. In our study, a genetic and functional analysis of the mannose-specific type 1 fimbrial adhesin FimH from a variety of serovars of Salmonella enterica revealed that specific mutant variants of FimH are common in host-adapted (systemically invasive) serovars. We have found that while the low-binding shear-dependent phenotype of the adhesin is preserved in broad host-range (usually systemically non-invasive) Salmonella, the majority of host-adapted serovars express FimH variants with one of two alternative phenotypes: a significantly increased binding to mannose (as in S. Typhi, S. Paratyphi C, S. Dublin and some isolates of S. Choleraesuis), or complete loss of the mannose-binding activity (as in S. Paratyphi B, S. Choleraesuis and S. Gallinarum). The functional diversification of FimH in host-adapted Salmonella results from recently acquired structural mutations. Many of the mutations are of a convergent nature indicative of strong positive selection. The high-binding phenotype of FimH that leads to increased bacterial adhesiveness to and invasiveness of epithelial cells and macrophages usually precedes acquisition of the non-binding phenotype. Collectively these observations suggest that activation or inactivation of mannose-specific adhesive properties in different systemically invasive serovars of Salmonella reflects their dynamic trajectories of adaptation to a life style in specific hosts. In conclusion, our study demonstrates that point mutations are the target of positive selection and, in addition to horizontal gene transfer and genome degradation events, can contribute to the differential pathoadaptive evolution of Salmonella. PMID:22685400

  20. Screening of point mutations by multiple SSCP analysis in the dystrophin gene

    SciTech Connect

    Lasa, A.; Baiget, M.; Gallano, P.

    1994-09-01

    Duchenne muscular dystrophy (DMD) is a lethal, X-linked neuromuscular disorder. The population frequency of DMD is one in approximately 3500 boys, of which one third is thought to be a new mutant. The DMD gene is the largest known to date, spanning over 2,3 Mb in band Xp21.2; 79 exons are transcribed into a 14 Kb mRNA coding for a protein of 427 kD which has been named dystrophin. It has been shown that about 65% of affected boys have a gene deletion with a wide variation in localization and size. The remaining affected individuals who have no detectable deletions or duplications would probably carry more subtle mutations that are difficult to detect. These mutations occur in several different exons and seem to be unique to single patients. Their identification represents a formidable goal because of the large size and complexity of the dystrophin gene. SSCP is a very efficient method for the detection of point mutations if the parameters that affect the separation of the strands are optimized for a particular DNA fragment. The multiple SSCP allows the simultaneous study of several exons, and implies the use of different conditions because no single set of conditions will be optimal for all fragments. Seventy-eight DMD patients with no deletion or duplication in the dystrophin gene were selected for the multiple SSCP analysis. Genomic DNA from these patients was amplified using the primers described for the diagnosis procedure (muscle promoter and exons 3, 8, 12, 16, 17, 19, 32, 45, 48 and 51). We have observed different mobility shifts in bands corresponding to exons 8, 12, 43 and 51. In exons 17 and 45, altered electrophoretic patterns were found in different samples identifying polymorphisms already described.

  1. Insights into enzyme point mutation effect by molecular simulation: phenylethylamine oxidation catalyzed by monoamine oxidase A.

    PubMed

    Oanca, Gabriel; Purg, Miha; Mavri, Janez; Shih, Jean C; Stare, Jernej

    2016-05-21

    The I335Y point mutation effect on the kinetics of phenylethylamine decomposition catalyzed by monoamine oxidase A was elucidated by means of molecular simulation. The established empirical valence bond methodology was used in conjunction with the free energy perturbation sampling technique and a classical force field representing the state of reactants and products. The methodology allows for the simulation of chemical reactions, in the present case the breaking of the α-C-H bond in a phenylethylamine substrate and the subsequent hydrogen transfer to the flavin cofactor, resulting in the formation of the N-H bond on flavin. The empirical parameters were calibrated against the experimental data for the simulated reaction in a wild type protein and then used for the calculation of the reaction free energy profile in the I335Y mutant. In very good agreement with the measured kinetic data, mutation increases the free energy barrier for the rate limiting step by slightly more than 1 kcal mol(-1) and consequently decreases the rate constant by about an order of magnitude. The magnitude of the computed effect slightly varies with simulation settings, but always remains in reasonable agreement with the experiment. Analysis of trajectories reveals a major change in the interaction between phenyl rings of the substrate and the neighboring Phe352 residue upon the I335Y mutation due to the increased local polarity, leading to an attenuated quadrupole interaction between the rings and destabilization of the transition state. Additionally, the increased local polarity in the mutant allows for a larger number of water molecules to be present near the active site, effectively shielding the catalytic effect of the enzyme and contributing to the increased barrier. PMID:27121693

  2. Point mutation impairs centromeric CENH3 loading and induces haploid plants.

    PubMed

    Karimi-Ashtiyani, Raheleh; Ishii, Takayoshi; Niessen, Markus; Stein, Nils; Heckmann, Stefan; Gurushidze, Maia; Banaei-Moghaddam, Ali Mohammad; Fuchs, Jörg; Schubert, Veit; Koch, Kerstin; Weiss, Oda; Demidov, Dmitri; Schmidt, Klaus; Kumlehn, Jochen; Houben, Andreas

    2015-09-01

    The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called "CENP-A") is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923-937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest. PMID:26294252

  3. Repeat-Induced Point Mutation and the Population Structure of Transposable Elements in Microbotryum violaceum

    PubMed Central

    Hood, Michael E.; Katawczik, Melanie; Giraud, Tatiana

    2005-01-01

    Repeat-induced point mutation (RIP) is a genome defense in fungi that hypermutates repetitive DNA and is suggested to limit the accumulation of transposable elements. The genome of Microbotryum violaceum has a high density of transposable elements compared to other fungi, but there is also evidence of RIP activity. This is the first report of RIP in a basidiomycete and was obtained by sequencing multiple copies of the integrase gene of a copia-type transposable element and the helicase gene of a Helitron-type element. In M. violaceum, the targets for RIP mutations are the cytosine residues of TCG trinucleotide combinations. Although RIP is a linkage-dependent process that tends to increase the variation among repetitive sequences, a chromosome-specific substructuring was observed in the transposable element population. The observed chromosome-specific patterns are not consistent with RIP, but rather suggest an effect of gene conversion, which is also a linkage-dependent process but results in a homogenization of repeated sequences. Particular sequences were found more widely distributed within the genome than expected by chance and may reflect the recently active variants. Therefore, sequence variation of transposable elements in M. violaceum appears to be driven by selection for transposition ability in combination with the context-specific forces of the RIP and gene conversion.

  4. Identification of Oncogenic Point Mutations and Hyperphosphorylation of Anaplastic Lymphoma Kinase in Lung Cancer12

    PubMed Central

    Wang, Yi-Wei; Tu, Pang-Hsien; Lin, Kuen-Tyng; Lin, Shu-Chen; Ko, Jenq-Yuh; Jou, Yuh-Shan

    2011-01-01

    The oncogenic property of anaplastic lymphoma kinase (ALK) plays an essential role in the pathogenesis of various cancers and serves as an important therapeutic target. In this study, we identified frequent intragenic loss of heterozygosity and six novel driver mutations within ALK in lung adenocarcinomas. Overexpression of H694R or E1384K mutant ALK leads to hyperphosphorylation of ALK, and activation of its downstream mediators STAT3, AKT, and ERK resulted in enhanced cell proliferation, colony formation, cell migration, and tumor growth in xenograft models. Furthermore, the activated phospho-Y1604 ALK was increasingly detected in 13 human lung cancer cell lines and 263 lung cancer specimens regardless of tumor stages and types. Treatment of two different ALK inhibitors, WHI-P154 and NVP-TAE684, resulted in the down-regulation of aberrant ALK signaling, shrinkage of tumor, and suppression of metastasis and significantly improved survival of ALK mutant-bearing mice. Together, we identified that novel ALK point mutations possessed tumorigenic effects mainly through hyperphosphorylation of Y1604 and activation of downstream oncogenic signaling. The upregulated phospho-Y1604 ALK could serve as a diagnostic biomarker for lung cancer. Furthermore, targeting oncogenic mutant ALKs with inhibitors could be a promising strategy to improve the therapeutic efficacy of fatal lung cancers. PMID:21847362

  5. Point mutation impairs centromeric CENH3 loading and induces haploid plants

    PubMed Central

    Karimi-Ashtiyani, Raheleh; Ishii, Takayoshi; Niessen, Markus; Stein, Nils; Heckmann, Stefan; Gurushidze, Maia; Banaei-Moghaddam, Ali Mohammad; Fuchs, Jörg; Schubert, Veit; Koch, Kerstin; Weiss, Oda; Demidov, Dmitri; Schmidt, Klaus; Kumlehn, Jochen; Houben, Andreas

    2015-01-01

    The chromosomal position of the centromere-specific histone H3 variant CENH3 (also called “CENP-A”) is the assembly site for the kinetochore complex of active centromeres. Any error in transcription, translation, modification, or incorporation can affect the ability to assemble intact CENH3 chromatin and can cause centromere inactivation [Allshire RC, Karpen GH (2008) Nat Rev Genet 9 (12):923–937]. Here we show that a single-point amino acid exchange in the centromere-targeting domain of CENH3 leads to reduced centromere loading of CENH3 in barley, sugar beet, and Arabidopsis thaliana. Haploids were obtained after cenh3 L130F-complemented cenh3-null mutant plants were crossed with wild-type A. thaliana. In contrast, in a noncompeting situation (i.e., centromeres possessing only mutated or only wild-type CENH3), no uniparental chromosome elimination occurs during early embryogenesis. The high degree of evolutionary conservation of the identified mutation site offers promising opportunities for application in a wide range of crop species in which haploid technology is of interest. PMID:26294252

  6. Abnormal behavior associated with a point mutation in the structural gene for monoamine oxidase A

    SciTech Connect

    Brunner, H.G. ); Nelen, M.; Ropers, H.H.; van Oost, B.A. )

    1993-10-22

    Genetic and metabolic studies have been done on a large kindred in which several males are affected by a syndrome of borderline mental retardation and abnormal behavior. The types of behavior that occurred include impulsive aggression, arson, attempted rape, and exhibitionism. Analysis of 24-hour urine samples indicated markedly disturbed monoamine metabolism. This syndrome was associated with a complete and selective deficiency of enzymatic activity of monoamine oxidase A (MAOA). In each of five affected males, a point mutation was identified in the eighth exon of the MAOA structural gene, which changes a glutamine to a termination codon. Thus, isolated complete MAOA deficiency in this family is associated with a recognizable behavioral phenotype that includes disturbed regulation of impulsive aggression.

  7. Identification of two different point mutations associated with the fluoride-resistant phenotype for human butyrylcholinesterase

    SciTech Connect

    Nogueira, C.P.; McGuire, M.C.; Adkins, S.; Van Der Spek, A.F.L.; La Du, B.N. ); Bartels, C.F.; Lockridge, O. Eppley Institute, Univ. of Nebraska Medical Center, Omaha, NE ); Lubrano, T.; Rubinstein, H.M. Loyola Univ. Stritch School of Medicine, Maywood, IL ); Lightstone, H. )

    1992-10-01

    The fluoride variant of human butyrylcholinesterase owes its name to the observation that it is resistant to inhibition by 0.050 mM sodium fluoride in the in vitro assay. Individuals who are heterozygous for the fluoride and atypical alleles experience about 30 min of apnea, rather than the usual 3-5 min, after receiving succinyldicholine. Earlier the authors reported that the atypical variant has a nucleotide substitution which changes Asp 70 to Gly. In the present work they have identified two different point mutations associated with the fluoride-resistant phentotype. Fluoride-1 has a nucleotide substitution which changes Thr 243 to Met (ACG to ATG). Fluoride-2 has a substitution which changes Gly 390 to Val (GGT to GTT). These results were obtained by DNA sequence analysis of the butyrylcholinesterase gene after amplification by PCR. The subjects for these analyses were 4 patients and 21 family members. 36 refs., 8 figs.

  8. Exome sequencing of desmoplastic melanoma identifies recurrent NFKBIE promoter mutations and diverse activating mutations in the MAPK pathway.

    PubMed

    Shain, A Hunter; Garrido, Maria; Botton, Thomas; Talevich, Eric; Yeh, Iwei; Sanborn, J Zachary; Chung, Jongsuk; Wang, Nicholas J; Kakavand, Hojabr; Mann, Graham J; Thompson, John F; Wiesner, Thomas; Roy, Ritu; Olshen, Adam B; Gagnon, Alexander; Gray, Joe W; Huh, Nam; Hur, Joe S; Busam, Klaus J; Scolyer, Richard A; Cho, Raymond J; Murali, Rajmohan; Bastian, Boris C

    2015-10-01

    Desmoplastic melanoma is an uncommon variant of melanoma with sarcomatous histology, distinct clinical behavior and unknown pathogenesis. We performed low-coverage genome and high-coverage exome sequencing of 20 desmoplastic melanomas, followed by targeted sequencing of 293 genes in a validation cohort of 42 cases. A high mutation burden (median of 62 mutations/Mb) ranked desmoplastic melanoma among the most highly mutated cancers. Mutation patterns strongly implicate ultraviolet radiation as the dominant mutagen, indicating a superficially located cell of origin. Newly identified alterations included recurrent promoter mutations of NFKBIE, encoding NF-κB inhibitor ɛ (IκBɛ), in 14.5% of samples. Common oncogenic mutations in melanomas, in particular in BRAF (encoding p.Val600Glu) and NRAS (encoding p.Gln61Lys or p.Gln61Arg), were absent. Instead, other genetic alterations known to activate the MAPK and PI3K signaling cascades were identified in 73% of samples, affecting NF1, CBL, ERBB2, MAP2K1, MAP3K1, BRAF, EGFR, PTPN11, MET, RAC1, SOS2, NRAS and PIK3CA, some of which are candidates for targeted therapies. PMID:26343386

  9. Reconstruction of thermotolerant yeast by one-point mutation identified through whole-genome analyses of adaptively-evolved strains

    PubMed Central

    Satomura, Atsushi; Miura, Natsuko; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2016-01-01

    Saccharomyces cerevisiae is used as a host strain in bioproduction, because of its rapid growth, ease of genetic manipulation, and high reducing capacity. However, the heat produced during the fermentation processes inhibits the biological activities and growth of the yeast cells. We performed whole-genome sequencing of 19 intermediate strains previously obtained during adaptation experiments under heat stress; 49 mutations were found in the adaptation steps. Phylogenetic tree revealed at least five events in which these strains had acquired mutations in the CDC25 gene. Reconstructed CDC25 point mutants based on a parental strain had acquired thermotolerance without any growth defects. These mutations led to the downregulation of the cAMP-dependent protein kinase (PKA) signaling pathway, which controls a variety of processes such as cell-cycle progression and stress tolerance. The one-point mutations in CDC25 were involved in the global transcriptional regulation through the cAMP/PKA pathway. Additionally, the mutations enabled efficient ethanol fermentation at 39 °C, suggesting that the one-point mutations in CDC25 may contribute to bioproduction. PMID:26984760

  10. Global Rebalancing of Cellular Resources by Pleiotropic Point Mutations Illustrates a Multi-scale Mechanism of Adaptive Evolution.

    PubMed

    Utrilla, Jose; O'Brien, Edward J; Chen, Ke; McCloskey, Douglas; Cheung, Jacky; Wang, Harris; Armenta-Medina, Dagoberto; Feist, Adam M; Palsson, Bernhard O

    2016-04-27

    Pleiotropic regulatory mutations affect diverse cellular processes, posing a challenge to our understanding of genotype-phenotype relationships across multiple biological scales. Adaptive laboratory evolution (ALE) allows for such mutations to be found and characterized in the context of clear selection pressures. Here, several ALE-selected single-mutation variants in RNA polymerase (RNAP) of Escherichia coli are detailed using an integrated multi-scale experimental and computational approach. While these mutations increase cellular growth rates in steady environments, they reduce tolerance to stress and environmental fluctuations. We detail structural changes in the RNAP that rewire the transcriptional machinery to rebalance proteome and energy allocation toward growth and away from several hedging and stress functions. We find that while these mutations occur in diverse locations in the RNAP, they share a common adaptive mechanism. In turn, these findings highlight the resource allocation trade-offs organisms face and suggest how the structure of the regulatory network enhances evolvability. PMID:27135538

  11. Conversion of a dodecahedral protein capsid into pentamers via minimal point mutations.

    PubMed

    Chen, Hsiao-Nung; Woycechowsky, Kenneth J

    2012-06-12

    Protein self-assembly relies upon the formation of stabilizing noncovalent interactions across subunit interfaces. Identifying the determinants of self-assembly is crucial for understanding structure-function relationships in symmetric protein complexes and for engineering responsive nanoscale architectures for applications in medicine and biotechnology. Lumazine synthases (LS's) comprise a protein family that forms diverse quaternary structures, including pentamers and 60-subunit dodecahedral capsids. To improve our understanding of the basis for this difference in assembly, we attempted to convert the capsid-forming LS from Aquifex aeolicus (AaLS) into pentamers through a small number of rationally designed amino acid substitutions. Our mutations targeted side chains at ionic (R40), hydrogen bonding (H41), and hydrophobic (L121 and I125) interaction sites along the interfaces between pentamers. We found that substitutions at two or three of these positions could reliably generate pentameric variants of AaLS. Biophysical characterization indicates that this quaternary structure change is not accompanied by substantial changes in secondary or tertiary structure. Interestingly, previous homology-based studies of the assembly determinants in LS's had identified only one of these four positions. The ability to control assembly state in protein capsids such as AaLS could aid efforts in the development of new systems for drug delivery, biocatalysis, or materials synthesis. PMID:22606973

  12. Exome sequencing for bipolar disorder points to roles of de novo loss-of-function and protein-altering mutations.

    PubMed

    Kataoka, M; Matoba, N; Sawada, T; Kazuno, A-A; Ishiwata, M; Fujii, K; Matsuo, K; Takata, A; Kato, T

    2016-07-01

    Although numerous genetic studies have been conducted for bipolar disorder (BD), its genetic architecture remains elusive. Here we perform, to the best of our knowledge, the first trio-based exome sequencing study for BD to investigate potential roles of de novo mutations in the disease etiology. We identified 71 de novo point mutations and one de novo copy-number mutation in 79 BD probands. Among the genes hit by de novo loss-of-function (LOF; nonsense, splice site or frameshift) or protein-altering (LOF, missense and inframe indel) mutations, we found significant enrichment of genes highly intolerant (first percentile of intolerant genes assessed by Residual Variation Intolerance Score) to protein-altering variants in general population, an observation that is also reported in autism and schizophrenia. When we performed a joint analysis using the data of schizoaffective disorder in published studies, we found global enrichment of de novo LOF and protein-altering mutations in the combined group of bipolar I and schizoaffective disorders. Considering relationship between de novo mutations and clinical phenotypes, we observed significantly earlier disease onset among the BD probands with de novo protein-altering mutations when compared with non-carriers. Gene ontology enrichment analysis of genes hit by de novo protein-altering mutations in bipolar I and schizoaffective disorders did not identify any significant enrichment. These results of exploratory analyses collectively point to the roles of de novo LOF and protein-altering mutations in the etiology of bipolar disorder and warrant further large-scale studies. PMID:27217147

  13. Neutron radiation can activate K-ras via a point mutation in codon 146 and induces a different spectrum of ras mutations than does gamma radiation.

    PubMed Central

    Sloan, S R; Newcomb, E W; Pellicer, A

    1990-01-01

    Neutron radiation is known to produce tumors in animals and cause cell transformation. We have developed a protocol to efficiently induce thymic lymphomas in RF/J mice by a single acute dose of neutron irradiation. Activated ras genes were detected in 17% (4 of 24) of the tumors analyzed. One of the tumors contained a K-ras gene activated by a point mutation in codon 146. Activating ras mutations at position 146 have not been previously detected in any known human or animal tumors. The spectrum of ras mutations detected in neutron radiation-induced thymic lymphomas was different from that seen in thymic lymphomas induced by gamma radiation in the same strain of mice. These results may have important implications for the mechanisms by which different types of radiation damage DNA. Images PMID:2403644

  14. A Noncoding Point Mutation of Zeb1 Causes Multiple Developmental Malformations and Obesity in Twirler Mice

    PubMed Central

    Kurima, Kiyoto; Hertzano, Ronna; Gavrilova, Oksana; Monahan, Kelly; Shpargel, Karl B.; Nadaraja, Garani; Kawashima, Yoshiyuki; Lee, Kyu Yup; Ito, Taku; Higashi, Yujiro; Eisenman, David J.; Strome, Scott E.; Griffith, Andrew J.

    2011-01-01

    Heterozygous Twirler (Tw) mice develop obesity and circling behavior associated with malformations of the inner ear, whereas homozygous Tw mice have cleft palate and die shortly after birth. Zeb1 is a zinc finger protein that contributes to mesenchymal cell fate by repression of genes whose expression defines epithelial cell identity. This developmental pathway is disrupted in inner ears of Tw/Tw mice. The purpose of our study was to comprehensively characterize the Twirler phenotype and to identify the causative mutation. The Tw/+ inner ear phenotype includes irregularities of the semicircular canals, abnormal utricular otoconia, a shortened cochlear duct, and hearing loss, whereas Tw/Tw ears are severely malformed with barely recognizable anatomy. Tw/+ mice have obesity associated with insulin-resistance and have lymphoid organ hypoplasia. We identified a noncoding nucleotide substitution, c.58+181G>A, in the first intron of the Tw allele of Zeb1 (Zeb1Tw). A knockin mouse model of c.58+181G>A recapitulated the Tw phenotype, whereas a wild-type knockin control did not, confirming the mutation as pathogenic. c.58+181G>A does not affect splicing but disrupts a predicted site for Myb protein binding, which we confirmed in vitro. In comparison, homozygosity for a targeted deletion of exon 1 of mouse Zeb1, Zeb1ΔEx1, is associated with a subtle abnormality of the lateral semicircular canal that is different than those in Tw mice. Expression analyses of E13.5 Twirler and Zeb1ΔEx1 ears confirm that Zeb1ΔEx1 is a null allele, whereas Zeb1Tw RNA is expressed at increased levels in comparison to wild-type Zeb1. We conclude that a noncoding point mutation of Zeb1 acts via a gain-of-function to disrupt regulation of Zeb1Tw expression, epithelial-mesenchymal cell fate or interactions, and structural development of the inner ear in Twirler mice. This is a novel mechanism underlying disorders of hearing or balance. PMID:21980308

  15. Diverse mutations in patients with Menkes disease often lead to exon skipping

    SciTech Connect

    Das, S.; Levinson, Levinson, B.; Whitney, S.; Vulpe, C.; Packman, S.; Gitschier, J.

    1994-11-01

    Fibroblast cultures from 12 unrelated patients with classical Menkes disease were analyzed for mutations in the MNK gene, by reverse transcription-PCR (RT-PCR) and chemical cleavage mismatch detection. Mutations were observed in 10 patients, and in each case a different mutation was present. All of the mutations would be predicted to have adverse effects on protein expression. Mutations that resulted in splicing abnormalities, detected by RT-PCR alone, were observed in six patients and included two splice-site changes, a nonsense mutation, a missense mutation, a small duplication, and a small deletion. Chemical cleavage analysis of the remaining six patients revealed the presence of one missense mutation. A valine/leucine polymorphism was also observed. These findings, combined with the prior observation of deletions in 15%-20% of Menkes patients, suggest that Southern blot hybridization and RT-PCR will identify mutations in the majority of patients. 26 refs., 3 figs., 2 tabs.

  16. Point mutations in the murine fumarylacetoacetate hydrolase gene: Animalmodels for the human genetic disorder hereditary tyrosinemia type 1

    SciTech Connect

    Aponte, Jennifer; Sega, Gary A; Hauser, Loren John; Dhar, Madhu; Withrow, Catherine; Carpenter, D A; Rinchik, Eugene M.; Culiat, Cymbeline T; Johnson, Dabney K

    2001-01-01

    Hereditary tyrosinemia type 1 (HT1) is a severe autosomal recessive metabolic disease associated with point mutations in the human fumarylacetoacetate hydrolase (FAH) gene that disrupt tyrosine catabolism. An acute form of HT1 results in death during the first months of life because of hepatic failure, whereas a chronic form leads to gradual development of liver disease often accompanied by renal dysfunction, childhood rickets, neurological crisis, and hepatocellular carcinoma. Mice homozygous for certain chromosome 7 deletions of the albino Tyr; c locus that also include Fah die perinatally as a result of liver dysfunction and exhibit a complex syndrome characterized by structural abnormalities and alterations in gene expression in the liver and kidney. Here we report that two independent, postnatally lethal mutations induced by N-ethyl-N-nitrosourea and mapped near Tyr are alleles of Fah. The Fah6287SB allele is a missense mutation in exon 6, and Fah5961SB is a splice mutation causing loss of exon 7, a subsequent frameshift in the resulting mRNA, and a severe reduction of Fah mRNA levels. Increased levels of the diagnostic metabolite succinylacetone in the urine of the Fah6287SB and Fah5961SB mutants indicate that these mutations cause a decrease in Fah enzymatic activity. Thus, the neonatal phenotype present in both mutants is due to a deficiency in Fah caused by a point mutation, and we propose Fah5961SB and Fah6287SB as mouse models for acute and chronic forms of human HT1, respectively.

  17. Modeling of antigenomic therapy of mitochondrial diseases by mitochondrially addressed RNA targeting a pathogenic point mutation in mitochondrial DNA.

    PubMed

    Tonin, Yann; Heckel, Anne-Marie; Vysokikh, Mikhail; Dovydenko, Ilya; Meschaninova, Mariya; Rötig, Agnès; Munnich, Arnold; Venyaminova, Alya; Tarassov, Ivan; Entelis, Nina

    2014-05-01

    Defects in mitochondrial genome can cause a wide range of clinical disorders, mainly neuromuscular diseases. Presently, no efficient therapeutic treatment has been developed against this class of pathologies. Because most of deleterious mitochondrial mutations are heteroplasmic, meaning that wild type and mutated forms of mitochondrial DNA (mtDNA) coexist in the same cell, the shift in proportion between mutant and wild type molecules could restore mitochondrial functions. Recently, we developed mitochondrial RNA vectors that can be used to address anti-replicative oligoribonucleotides into human mitochondria and thus impact heteroplasmy level in cells bearing a large deletion in mtDNA. Here, we show that this strategy can be also applied to point mutations in mtDNA. We demonstrate that specifically designed RNA molecules containing structural determinants for mitochondrial import and 20-nucleotide sequence corresponding to the mutated region of mtDNA, are able to anneal selectively to the mutated mitochondrial genomes. After being imported into mitochondria of living human cells in culture, these RNA induced a decrease of the proportion of mtDNA molecules bearing a pathogenic point mutation in the mtDNA ND5 gene. PMID:24692550

  18. Point mutations in dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum isolates from Venezuela.

    PubMed

    Urdaneta, L; Plowe, C; Goldman, I; Lal, A A

    1999-09-01

    The present study was designed to characterize mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum in the Bolivar region of Venezuela, where high levels of clinical resistance to sulfadoxine-pyrimethamine (SP, Fansidar; F. Hoffman-La Roche, Basel, Switzerland) has been documented. We used a nested mutation-specific polymerase chain reaction and restriction digestion methods to measure 1) the prevalence of DHFR mutations at 16, 50, 51, 59, 108, and 164 codon positions, and 2) the prevalence of mutations in the 436, 437, 581, and 613 codon sites in DHPS gene. In the case of the DHFR gene, of the 54 parasite isolates analyzed, we detected the presence of Asn-108 and Ile-51 in 96% of the isolates and Arg-50 mutation in 64% of the isolates. Each of these mutations has been associated with high level of resistance to pyrimethamine. Only 2 samples (4%) showed the wild type Ser-108 mutation and none showed Thr-108 and Val-16 mutations that are specific for resistance to cycloguanil. In the case of DHPS gene, we found a mutation at position 437 (Gly) in 100% of the isolates and Gly-581 in 96% of the isolates. The simultaneous presence of mutations Asn-108 and Ile-51 in the DHFR gene and Gly-437 and Gly-581 in the DHPS gene in 96% of the samples tested suggested that a cumulative effect of mutations could be the major mechanism conferring high SP resistance in this area. PMID:10497990

  19. Practicability of detecting somatic point mutation from RNA high throughput sequencing data.

    PubMed

    Sheng, Quanhu; Zhao, Shilin; Li, Chung-I; Shyr, Yu; Guo, Yan

    2016-05-01

    Traditionally, somatic mutations are detected by examining DNA sequence. The maturity of sequencing technology has allowed researchers to screen for somatic mutations in the whole genome. Increasingly, researchers have become interested in identifying somatic mutations through RNAseq data. With this motivation, we evaluated the practicability of detecting somatic mutations from RNAseq data. Current somatic mutation calling tools were designed for DNA sequencing data. To increase performance on RNAseq data, we developed a somatic mutation caller GLMVC based on bias reduced generalized linear model for both DNA and RNA sequencing data. Through comparison with MuTect and Varscan we showed that GLMVC performed better for somatic mutation detection using exome sequencing or RNAseq data. GLMVC is freely available for download at the following website: https://github.com/shengqh/GLMVC/wiki. PMID:27046520

  20. Novel point mutations in the German cockroach para sodium channel gene are associated with knockdown resistance (kdr) to pyrethroid insecticides.

    PubMed

    Liu, Z; Valles, S M; Dong, K

    2000-10-01

    Knockdown resistance (kdr) to pyrethroid insecticides has been attributed to point mutations in the para sodium channel gene in more than a half dozen insect pest species. In this study, we identified two novel para mutations in five highly resistant kdr-type German cockroach strains. The two mutations, from glutamic acid (E434) to lysine (K434) and from cysteine (C764) to arginine (R764), respectively, are located in the first intracellular linker connecting domains I and II. E434K is located near the beginning of the linker (closest to domain I), whereas C764R is found toward the end of the linker (closest to domain II). Two additional mutations from aspartic acid (D58) to glycine (G58), and from proline (P1880) to leucine (L1888), respectively, were found in one of the resistant strains. The four mutations coexist with the previously identified leucine to phenylalanine (L993F) kdr mutation in IIS6, and are present only in the highly resistant individuals of a given strain. These findings suggest that these mutations might be responsible for high levels of knockdown resistance toward pyrethroid insecticides in the German cockroach. PMID:10899465

  1. Point mutations in the 3' minor domain of 16S rRNA of E.coli.

    PubMed Central

    Jemiolo, D K; Zwieb, C; Dahlberg, A E

    1985-01-01

    Point mutations were produced near the 3' end of E. coli 16S rRNA by bisulfite mutagenesis in a 121 base loop-out (1385 to 1505) in a heteroduplex of wild type (pKK3535) and deletion mutant plasmids. Two highly conserved, single stranded regions flank an irregular helix (1409-1491) in the area studied. Only a single mutation was isolated in the flanking regions, a transition at C1402, (normally methylated on the base and ribose in rRNA). Mutations occurred throughout the irregular helix. All mutant rRNAs were processed and assembled into 30S subunits capable of interacting with 50S subunits. Growth rates ranged from faster to significantly slower than cells with the wild type transcript. In particular, mutations at C1467 or C1469 cause slow growth. These two transitions (in a bulge region within the helix) reduced the bulge by additional base pairing. PMID:3909106

  2. HIV-1 Drug Resistance Mutations: Potential Applications for Point-of-Care Genotypic Resistance Testing.

    PubMed

    Rhee, Soo-Yon; Jordan, Michael R; Raizes, Elliot; Chua, Arlene; Parkin, Neil; Kantor, Rami; Van Zyl, Gert U; Mukui, Irene; Hosseinipour, Mina C; Frenkel, Lisa M; Ndembi, Nicaise; Hamers, Raph L; Rinke de Wit, Tobias F; Wallis, Carole L; Gupta, Ravindra K; Fokam, Joseph; Zeh, Clement; Schapiro, Jonathan M; Carmona, Sergio; Katzenstein, David; Tang, Michele; Aghokeng, Avelin F; De Oliveira, Tulio; Wensing, Annemarie M J; Gallant, Joel E; Wainberg, Mark A; Richman, Douglas D; Fitzgibbon, Joseph E; Schito, Marco; Bertagnolio, Silvia; Yang, Chunfu; Shafer, Robert W

    2015-01-01

    The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART) in the low- and middle-income countries (LMICs) hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in areas with rising transmitted drug resistance (TDR) and enable care-providers to determine which individuals with virological failure (VF) on a first- or second-line ART regimen require a change in treatment. An inexpensive near point-of-care (POC) genotypic resistance test would be useful in settings where the resources, capacity, and infrastructure to perform standard genotypic drug resistance testing are limited. Such a test would be particularly useful in conjunction with the POC HIV-1 viral load tests that are currently being introduced in LMICs. A POC genotypic resistance test is likely to involve the use of allele-specific point mutation assays for detecting drug-resistance mutations (DRMs). This study proposes that two major nucleoside reverse transcriptase inhibitor (NRTI)-associated DRMs (M184V and K65R) and four major NNRTI-associated DRMs (K103N, Y181C, G190A, and V106M) would be the most useful for POC genotypic resistance testing in LMIC settings. One or more of these six DRMs was present in 61.2% of analyzed virus sequences from ART-naïve individuals with intermediate or high-level TDR and 98.8% of analyzed virus sequences from individuals on a first-line NRTI/NNRTI-containing regimen with intermediate or high-level acquired drug resistance. The detection of one or more of these DRMs in an ART-naïve individual or in a individual with VF on a first-line NRTI/NNRTI-containing regimen may be considered an indication for a protease inhibitor (PI)-containing regimen or closer virological monitoring based on cost-effectiveness or country policy. PMID:26717411

  3. HIV-1 Drug Resistance Mutations: Potential Applications for Point-of-Care Genotypic Resistance Testing

    PubMed Central

    Rhee, Soo-Yon; Jordan, Michael R.; Raizes, Elliot; Chua, Arlene; Parkin, Neil; Kantor, Rami; Van Zyl, Gert U.; Mukui, Irene; Hosseinipour, Mina C.; Frenkel, Lisa M.; Ndembi, Nicaise; Hamers, Raph L.; Rinke de Wit, Tobias F.; Wallis, Carole L.; Gupta, Ravindra K.; Fokam, Joseph; Zeh, Clement; Schapiro, Jonathan M.; Carmona, Sergio; Katzenstein, David; Tang, Michele; Aghokeng, Avelin F.; De Oliveira, Tulio; Wensing, Annemarie M. J.; Gallant, Joel E.; Wainberg, Mark A.; Richman, Douglas D.; Fitzgibbon, Joseph E.; Schito, Marco; Bertagnolio, Silvia; Yang, Chunfu; Shafer, Robert W.

    2015-01-01

    The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART) in the low- and middle-income countries (LMICs) hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in areas with rising transmitted drug resistance (TDR) and enable care-providers to determine which individuals with virological failure (VF) on a first- or second-line ART regimen require a change in treatment. An inexpensive near point-of-care (POC) genotypic resistance test would be useful in settings where the resources, capacity, and infrastructure to perform standard genotypic drug resistance testing are limited. Such a test would be particularly useful in conjunction with the POC HIV-1 viral load tests that are currently being introduced in LMICs. A POC genotypic resistance test is likely to involve the use of allele-specific point mutation assays for detecting drug-resistance mutations (DRMs). This study proposes that two major nucleoside reverse transcriptase inhibitor (NRTI)-associated DRMs (M184V and K65R) and four major NNRTI-associated DRMs (K103N, Y181C, G190A, and V106M) would be the most useful for POC genotypic resistance testing in LMIC settings. One or more of these six DRMs was present in 61.2% of analyzed virus sequences from ART-naïve individuals with intermediate or high-level TDR and 98.8% of analyzed virus sequences from individuals on a first-line NRTI/NNRTI-containing regimen with intermediate or high-level acquired drug resistance. The detection of one or more of these DRMs in an ART-naïve individual or in a individual with VF on a first-line NRTI/NNRTI-containing regimen may be considered an indication for a protease inhibitor (PI)-containing regimen or closer virological monitoring based on cost-effectiveness or country policy. PMID:26717411

  4. High rate of A2142G point mutation associated with clarithromycin resistance among Iranian Helicobacter pylori clinical isolates.

    PubMed

    Khashei, Reza; Dara, Mahintaj; Bazargani, Abdollah; Bagheri Lankarani, Kamran; Taghavi, Alireza; Moeini, Maryam; Dehghani, Behzad; Sohrabi, Maryam

    2016-09-01

    This study aimed to investigate the clarithromycin resistance and its associated molecular mechanisms among Helicobacter pylori isolates from dyspeptic patients in Shiraz, Iran. From January to May 2014, 100 H. pylori strains were isolated from patients with gastroduodenal disorders. The resistance to clarithromycin was quantitatively evaluated, using Epsilometer (E-test) method. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed on all the isolates to detect A2143G and A2142G mutations in 23S rRNA gene. The H. pylori isolation rate was found to be 31.4%. E-test showed that 20% of isolates were resistant to clarithromycin (MIC ≥ 1 mg/L). MIC of clarithromycin ranged between 0.016 and 24 mg/L. Findings of PCR-RFLP showed that the A2142G was the most (90%) frequently point mutation, followed by the A2143G (10%). No statistically significant difference was found between H. pylori clarithromycin resistance point mutations and patients' gender or age. To the best of our knowledge, this is the first report of high frequency of A2142G point mutation in Iran and probably in other regions of the world. Considering the increasing trend of H. pylori resistance to clarithromycin due to these mutations, it is crucial to investigate the new therapeutic approaches against H. pylori infection. PMID:27357065

  5. Sdt97: A Point Mutation in the 5′ Untranslated Region Confers Semidwarfism in Rice

    PubMed Central

    Tong, Jiping; Han, Zhengshu; Han, Aonan; Liu, Xuejun; Zhang, Shiyong; Fu, Binying; Hu, Jun; Su, Jingping; Li, Shaoqing; Wang, Shengjun; Zhu, Yingguo

    2016-01-01

    Semidwarfism is an important agronomic trait in rice breeding programs. The semidwarf mutant gene Sdt97 was previously described. However, the molecular mechanism underlying the mutant is yet to be elucidated. In this study, we identified the mutant gene by a map-based cloning method. Using a residual heterozygous line (RHL) population, Sdt97 was mapped to the long arm of chromosome 6 in the interval of nearly 60 kb between STS marker N6 and SNP marker N16 within the PAC clone P0453H04. Sequencing of the candidate genes in the target region revealed that a base transversion from G to C occurred in the 5′ untranslated region of Sdt97. qRT-PCR results confirmed that the transversion induced an obvious change in the expression pattern of Sdt97 at different growth and developmental stages. Plants transgenic for Sdt97 resulted in the restoration of semidwarfism of the mutant phenotype, or displayed a greater dwarf phenotype than the mutant. Our results indicate that a point mutation in the 5′ untranslated region of Sdt97 confers semidwarfism in rice. Functional analysis of Sdt97 will open a new field of study for rice semidwarfism, and also expand our knowledge of the molecular mechanism of semidwarfism in rice. PMID:27172200

  6. Sdt97: A Point Mutation in the 5' Untranslated Region Confers Semidwarfism in Rice.

    PubMed

    Tong, Jiping; Han, Zhengshu; Han, Aonan; Liu, Xuejun; Zhang, Shiyong; Fu, Binying; Hu, Jun; Su, Jingping; Li, Shaoqing; Wang, Shengjun; Zhu, Yingguo

    2016-01-01

    Semidwarfism is an important agronomic trait in rice breeding programs. The semidwarf mutant gene Sdt97 was previously described. However, the molecular mechanism underlying the mutant is yet to be elucidated. In this study, we identified the mutant gene by a map-based cloning method. Using a residual heterozygous line (RHL) population, Sdt97 was mapped to the long arm of chromosome 6 in the interval of nearly 60 kb between STS marker N6 and SNP marker N16 within the PAC clone P0453H04. Sequencing of the candidate genes in the target region revealed that a base transversion from G to C occurred in the 5' untranslated region of Sdt97 qRT-PCR results confirmed that the transversion induced an obvious change in the expression pattern of Sdt97 at different growth and developmental stages. Plants transgenic for Sdt97 resulted in the restoration of semidwarfism of the mutant phenotype, or displayed a greater dwarf phenotype than the mutant. Our results indicate that a point mutation in the 5' untranslated region of Sdt97 confers semidwarfism in rice. Functional analysis of Sdt97 will open a new field of study for rice semidwarfism, and also expand our knowledge of the molecular mechanism of semidwarfism in rice. PMID:27172200

  7. Heteroduplex analysis of the dystrophin gene: Application to point mutation and carrier detection

    SciTech Connect

    Prior, T.W.; Papp, A.C.; Snyder, P.J.; Sedra, M.S.; Western, L.M.; Bartolo, C.; Mendell, J.R.; Moxley, R.T.

    1994-03-01

    Approximately one-third of Duchenne muscular dystrophy patients have undefined mutations in the dystrophin gene. For carrier and prenatal studies in families without detectable mutations, the indirect restriction fragment length polymorphism linkage approach is used. Using a multiplex amplification and heteroduplex analysis of dystrophin exons, the authors identified nonsense mutations in two DMD patients. Although the nonsense mutations are predicted to severely truncate the dystrophin protein, both patients presented with mild clinical courses of the disease. As a result of identifying the mutation in the affected boys, direct carrier studies by heteroduplex analysis were extended to other relatives. The authors conclude that the technique is not only ideal for mutation detection but is also useful for diagnostic testing. 29 refs., 4 figs.

  8. Post-zygotic Point Mutations Are an Underrecognized Source of De Novo Genomic Variation

    PubMed Central

    Acuna-Hidalgo, Rocio; Bo, Tan; Kwint, Michael P.; van de Vorst, Maartje; Pinelli, Michele; Veltman, Joris A.; Hoischen, Alexander; Vissers, Lisenka E.L.M.; Gilissen, Christian

    2015-01-01

    De novo mutations are recognized both as an important source of genetic variation and as a prominent cause of sporadic disease in humans. Mutations identified as de novo are generally assumed to have occurred during gametogenesis and, consequently, to be present as germline events in an individual. Because Sanger sequencing does not provide the sensitivity to reliably distinguish somatic from germline mutations, the proportion of de novo mutations that occur somatically rather than in the germline remains largely unknown. To determine the contribution of post-zygotic events to de novo mutations, we analyzed a set of 107 de novo mutations in 50 parent-offspring trios. Using four different sequencing techniques, we found that 7 (6.5%) of these presumed germline de novo mutations were in fact present as mosaic mutations in the blood of the offspring and were therefore likely to have occurred post-zygotically. Furthermore, genome-wide analysis of “de novo” variants in the proband led to the identification of 4/4,081 variants that were also detectable in the blood of one of the parents, implying parental mosaicism as the origin of these variants. Thus, our results show that an important fraction of de novo mutations presumed to be germline in fact occurred either post-zygotically in the offspring or were inherited as a consequence of low-level mosaicism in one of the parents. PMID:26054435

  9. A novel von Hippel-Lindau point mutation presents as apparently sporadic pheochromocytoma.

    PubMed

    Rich, Thereasa A; Jonasch, Eric; Matin, Surena; Waguespack, Steven G; Gombos, Dan S; Santarpia, Libero; Stolle, Catherine; Jimenez, Camilo

    2008-07-01

    Von Hippel Lindau disease is a common cause of apparently sporadic pheochromocytomas. Herein, we describe a 20-year-old man with an apparently sporadic pheochromocytoma associated with a novel, relatively conservative germline Gly104Val VHL gene mutation, which is localized within exon 1 of the VHL gene corresponding to the beta -domain of the VHL protein (pVHL). The nearly asymptomatic patient's father also carries the same mutation. Similar to other mutations localized in the same codon, the Gly104Val VHL mutation seems to have an attenuated disease phenotype. PMID:18584357

  10. Disentangling the effects of mating systems and mutation rates on cytoplamic diversity in gynodioecious Silene nutans and dioecious Silene otites

    PubMed Central

    Lahiani, E; Dufaÿ, M; Castric, V; Le Cadre, S; Charlesworth, D; Van Rossum, F; Touzet, P

    2013-01-01

    Many flowering plant species exhibit a variety of distinct sexual morphs, the two most common cases being the co-occurrence of females and males (dioecy) or the co-occurrence of hermaphrodites and females (gynodioecy). In this study, we compared DNA sequence variability of the three genomes (nuclear, mitochondrial and chloroplastic) of a gynodioecious species, Silene nutans, with that of a closely related dioecious species, Silene otites. In the light of theoretical models, we expect cytoplasmic diversity to differ between the two species due to the selective dynamics that acts on cytoplasmic genomes in gynodioecious species: under an epidemic scenario, the gynodioecious species is expected to exhibit lower cytoplasmic diversity than the dioecious species, while the opposite is expected in the case of balancing selection maintaining sterility cytoplasms in the gynodioecious species. We found no difference between the species for nuclear gene diversity, but, for the cytoplasmic loci, the gynodioecious S. nutans had more haplotypes, and higher nucleotide diversity, than the dioecious relative, S. otites, even though the latter has a relatively high rate of mitochondrial synonymous substitutions, and therefore presumably a higher mutation rate. Therefore, as the mitochondrial mutation rate cannot account for the higher cytoplasmic diversity found in S. nutans, our findings support the hypothesis that gynodioecy in S. nutans has been maintained by balancing selection rather than by epidemic-like dynamics. PMID:23591518

  11. Site-directed point mutations in embryonic stem cells: a gene-targeting tag-and-exchange strategy.

    PubMed Central

    Askew, G R; Doetschman, T; Lingrel, J B

    1993-01-01

    Sequential gene targeting was used to introduce point mutations into one alpha 2 isoform Na,K-ATPase homolog in mouse embryonic stem (ES) cells. In the first round of targeted replacement, the gene was tagged with selectable markers by insertion of a Neor/HSV-tk gene cassette, and this event was selected for by gain of neomycin (G418) resistance. In the second targeted replacement event, the tagged genomic sequence was exchanged with a vector consisting of homologous genomic sequences carrying five site-directed nucleotide substitutions. Embryonic stem cell clones modified by exchange with the mutation vector were selected for loss of the HSV-tk gene by resistance to ganciclovir. Candidate clones were further screened and identified by polymerase chain reaction and Southern blot analysis. By this strategy, the endogenous alpha 2 isoform Na,K-ATPase gene was altered to encode two other amino acids so that the enzyme is resistant to inhibition by cardiac glycosides while maintaining its transmembrane ion-pumping function. Since the initial tagging event and the subsequent mutation-exchange event are independent of one another, a tagged cell line can be used to generate a variety of mutant lines by exchange with various mutation vectors at the tagged locus. This method should be useful for testing specific mutations introduced into the genomes of tissue culture cells and animals and for developing animal models encompassing the mutational variability of known genetic disorders. Images PMID:8391633

  12. Novel point mutations in the ERG11 gene in clinical isolates of azole resistant Candida species

    PubMed Central

    Silva, Danielly Beraldo dos Santos; Rodrigues, Luana Mireli Carbonera; de Almeida, Adriana Araújo; de Oliveira, Kelly Mari Pires; Grisolia/, Alexéia Barufatti

    2016-01-01

    The azoles are the class of medications most commonly used to fight infections caused by Candida sp. Typically, resistance can be attributed to mutations in ERG11 gene (CYP51) which encodes the cytochrome P450 14α-demethylase, the primary target for the activity of azoles. The objective of this study was to identify mutations in the coding region of theERG11 gene in clinical isolates of Candidaspecies known to be resistant to azoles. We identified three new synonymous mutations in the ERG11 gene in the isolates of Candida glabrata (C108G, C423T and A1581G) and two new nonsynonymous mutations in the isolates of Candida krusei - A497C (Y166S) and G1570A (G524R). The functional consequence of these nonsynonymous mutations was predicted using evolutionary conservation scores. The G524R mutation did not have effect on 14α-demethylase functionality, while the Y166S mutation was found to affect the enzyme. This observation suggests a possible link between the mutation and dose-dependent sensitivity to voriconazole in the clinical isolate of C. krusei. Although the presence of the Y166S in phenotype of reduced azole sensitivity observed in isolate C. kruseidemands investigation, it might contribute to the search of new therapeutic agents against resistant Candida isolates. PMID:26982177

  13. Novel point mutations in the ERG11 gene in clinical isolates of azole resistant Candida species.

    PubMed

    Silva, Danielly Beraldo dos Santos; Rodrigues, Luana Mireli Carbonera; Almeida, Adriana Araújo de; Oliveira, Kelly Mari Pires de; Grisolia, Alexéia Barufatti

    2016-03-01

    The azoles are the class of medications most commonly used to fight infections caused by Candida sp. Typically, resistance can be attributed to mutations in ERG11 gene (CYP51) which encodes the cytochrome P450 14α-demethylase, the primary target for the activity of azoles. The objective of this study was to identify mutations in the coding region of theERG11 gene in clinical isolates of Candida species known to be resistant to azoles. We identified three new synonymous mutations in the ERG11 gene in the isolates of Candida glabrata (C108G, C423T and A1581G) and two new nonsynonymous mutations in the isolates of Candida krusei--A497C (Y166S) and G1570A (G524R). The functional consequence of these nonsynonymous mutations was predicted using evolutionary conservation scores. The G524R mutation did not have effect on 14α-demethylase functionality, while the Y166S mutation was found to affect the enzyme. This observation suggests a possible link between the mutation and dose-dependent sensitivity to voriconazole in the clinical isolate of C. krusei. Although the presence of the Y166S in phenotype of reduced azole sensitivity observed in isolate C. krusei demands investigation, it might contribute to the search of new therapeutic agents against resistant Candida isolates. PMID:26982177

  14. A novel type II collagen gene mutation in a family with spondyloepiphyseal dysplasia and extensive intrafamilial phenotypic diversity.

    PubMed

    Nakashima, Yasuharu; Sakamoto, Yuma; Nishimura, Gen; Ikegawa, Shiro; Iwamoto, Yukihide

    2016-01-01

    The purpose of this study was to describe a family with spondyloepiphyseal dysplasia caused by a novel type II collagen gene (COL2A1) mutation and the family's phenotypic diversity. Clinical and radiographic examinations of skeletal dysplasia were conducted on seven affected family members across two generations. The entire coding region of COL2A1, including the flanking intron regions, was analyzed with PCR and direct sequencing. The stature of the subjects ranged from extremely short to within normal height range. Hip deformity and advanced osteoarthritis were noted in all the subjects, ranging from severe coxa plana to mild acetabular dysplasia. Atlantoaxial subluxation combined with a hypoplastic odontoid process was found in three of the subjects. Various degrees of platyspondyly were confirmed in all subjects. Genetically, a novel COL2A1 mutation (c.1349G>C, p.Gly450Ala) was identified in all the affected family members; however, it was not present in the one unaffected family member tested. We described a family with spondyloepiphyseal dysplasia and a novel COL2A1 mutation (c.1349G>C, p.Gly450Ala). Phenotypes were diverse even among individuals with the same mutation and within the same family. PMID:27274858

  15. A novel type II collagen gene mutation in a family with spondyloepiphyseal dysplasia and extensive intrafamilial phenotypic diversity

    PubMed Central

    Nakashima, Yasuharu; Sakamoto, Yuma; Nishimura, Gen; Ikegawa, Shiro; Iwamoto, Yukihide

    2016-01-01

    The purpose of this study was to describe a family with spondyloepiphyseal dysplasia caused by a novel type II collagen gene (COL2A1) mutation and the family’s phenotypic diversity. Clinical and radiographic examinations of skeletal dysplasia were conducted on seven affected family members across two generations. The entire coding region of COL2A1, including the flanking intron regions, was analyzed with PCR and direct sequencing. The stature of the subjects ranged from extremely short to within normal height range. Hip deformity and advanced osteoarthritis were noted in all the subjects, ranging from severe coxa plana to mild acetabular dysplasia. Atlantoaxial subluxation combined with a hypoplastic odontoid process was found in three of the subjects. Various degrees of platyspondyly were confirmed in all subjects. Genetically, a novel COL2A1 mutation (c.1349G>C, p.Gly450Ala) was identified in all the affected family members; however, it was not present in the one unaffected family member tested. We described a family with spondyloepiphyseal dysplasia and a novel COL2A1 mutation (c.1349G>C, p.Gly450Ala). Phenotypes were diverse even among individuals with the same mutation and within the same family. PMID:27274858

  16. Identification of Point Mutations in Clinical Staphylococcus aureus Strains That Produce Small-Colony Variants Auxotrophic for Menadione

    PubMed Central

    Dean, Melissa A.; Olsen, Randall J.; Long, S. Wesley; Rosato, Adriana E.

    2014-01-01

    Staphylococcus aureus small-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of five S. aureus SCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice. PMID:24452687

  17. Diversity of [beta]-globin mutations in Israeli ethnic groups reflects recent historic events

    SciTech Connect

    Filon, D.; Oron, V.; Krichevski, S.; Shaag, A.; Goldfarb, A.; Aker, M.; Rachmilewitz, E.A.; Rund, D.; Oppenheim, A. )

    1994-05-01

    The authors characterized nearly 500 [beta]-thalassemia genes from the Israeli population representing a variety of ethnic subgroups. They found 28 different mutations in the [beta]-globin gene, including three mutations ([beta][sup S], [beta][sup C], and [beta][sup O-Arab]) causing hemoglobinopathies. Marked genetic heterogeneity was observed in both the Arab (20 mutations) and Jewish (17 mutations) populations. On the other hand, two ethnic isolates - Druze and Samaritans - had a single mutation each. Fifteen of the [beta]-thalassemia alleles are Mediterranean in type, 5 originated in Kurdistan, 2 are of Indian origin, and 2 sporadic alleles came from Europe. Only one mutant allele-nonsense codon 37-appears to be indigenous to Israel. While human habitation in Israel dates back to early prehistory, the present-day spectrum of [beta]-globin mutations can be largely explained by migration events that occurred in the past millennium. 26 refs., 2 figs., 3 tabs.

  18. Relics of repeat-induced point mutation direct heterochromatin formation in Neurospora crassa

    PubMed Central

    Lewis, Zachary A.; Honda, Shinji; Khlafallah, Tamir K.; Jeffress, Jennifer K.; Freitag, Michael; Mohn, Fabio; Schübeler, Dirk; Selker, Eric U.

    2009-01-01

    Both RNAi-dependent and -independent mechanisms have been implicated in the establishment of heterochromatin domains, which may be stabilized by feedback loops involving chromatin proteins and modifications of histones and DNA. Neurospora crassa sports features of heterochromatin found in higher eukaryotes, namely cytosine methylation (5mC), methylation of histone H3 lysine 9 (H3K9me), and heterochromatin protein 1 (HP1), and is a model to investigate heterochromatin establishment and maintenance. We mapped the distribution of HP1, 5mC, H3K9me3, and H3K4me2 at 100 bp resolution and explored their interplay. HP1, H3K9me3, and 5mC were extensively co-localized and defined 44 heterochromatic domains on linkage group VII, all relics of repeat-induced point mutation. Interestingly, the centromere was found in an ∼350 kb heterochromatic domain with no detectable H3K4me2. 5mC was not found in genes, in contrast to the situation in plants and animals. H3K9me3 is required for HP1 localization and DNA methylation in N. crassa. In contrast, we found that localization of H3K9me3 was independent of 5mC or HP1 at virtually all heterochromatin regions. In addition, we observed complete restoration of DNA methylation patterns after depletion and reintroduction of the H3K9 methylation machinery. These data show that A:T-rich RIP'd DNA efficiently directs methylation of H3K9, which in turn, directs methylation of associated cytosines. PMID:19092133

  19. A single-point mutation enhances dual functionality of a scorpion toxin.

    PubMed

    Wang, Xueli; Gao, Bin; Zhu, Shunyi

    2016-01-01

    Scorpion venom represents a tremendous, hitherto partially explored peptide library that has been proven to be useful not only for understanding ion channels but also for drug design. MeuTXKα3 is a functionally unknown scorpion toxin-like peptide. Here we describe new transcripts of this gene arising from alternative polyadenylation and its biological function as well as a mutant with a single-point substitution at site 30. Native-like MeuTXKα3 and its mutant were produced in Escherichia coli and their toxic function against Drosophila Shaker K(+) channel and its mammalian counterparts (rKv1.1-rKv1.3) were assayed by two-electrode voltage clamp technique. The results show that MeuTXKα3 is a weak toxin with a wide-spectrum of activity on both Drosophila and mammalian K(+) channels. The substitution of a proline at site 30 by an asparagine, an evolutionarily conserved functional residue in the scorpion α-KTx family, led to an increased activity on rKv1.2 and rKv1.3 but a decreased activity on the Shaker channel without changing the potency on rKv1.1, suggesting a key role of this site in species selectivity of scorpion toxins. MeuTXKα3 was also active on a variety of bacteria with lethal concentrations ranging from 4.66 to 52.01μM and the mutant even had stronger activity on some of these bacterial species. To the best of our knowledge, this is the first report on a bi-functional short-chain peptide in the lesser Asian scorpion venom. Further extensive mutations of MeuTXKα3 at site 30 could help improve its K(+) channel-blocking and antibacterial functions. PMID:26358403

  20. The Molecular Basis of Muscular Dystrophy in the mdx Mouse: A Point Mutation

    NASA Astrophysics Data System (ADS)

    Sicinski, Piotr; Geng, Yan; Ryder-Cook, Allan S.; Barnard, Eric A.; Darlison, Mark G.; Barnard, Pene J.

    1989-06-01

    The mdx mouse is an X-linked myopathic mutant, an animal model for human Duchenne muscular dystrophy. In both mouse and man the mutations lie within the dystrophin gene, but the phenotypic differences of the disease in the two species confer much interest on the molecular basis of the mdx mutation. The complementary DNA for mouse dystrophin has been cloned, and the sequence has been used in the polymerase chain reaction to amplify normal and mdx dystrophin transcripts in the area of the mdx mutation. Sequence analysis of the amplification products showed that the mdx mouse has a single base substitution within an exon, which causes premature termination of the polypeptide chain.

  1. Recessively inherited L-DOPA-responsive dystonia caused by a point mutation (Q381K) in the tyrosine hydroxylase gene.

    PubMed

    Knappskog, P M; Flatmark, T; Mallet, J; Lüdecke, B; Bartholomé, K

    1995-07-01

    Tyrosine hydroxylase (TH) catalyzes the conversion of L-tyrosine to L-dihydroxyphenylalanine (L-DOPA), the rate-limiting step in the biosynthesis of dopamine. Recently, we described a point mutation in hTH (Q381K) in a family of two siblings suffering from progressive L-DOPA-responsive dystonia (DRD), representing the first reported mutation in this gene. We here describe the cloning, expression and steady-state kinetic properties of the recombinant mutant enzyme. When expressed by a coupled in vitro transcription-translation system and in E. coli, the mutant enzyme represents a kinetic variant form, with a reduced affinity for L-tyrosine. The 'residual activity' of about 15% of the corresponding wild-type hTH (isoform hTH1), at substrate concentrations prevailing in vivo, is compatible with the clinical phenotype of the two Q381K homozygote patients carrying this recessively inherited mutation. PMID:8528210

  2. Genetic and phenotypic diversity of NHE6 mutations in Christianson syndrome

    PubMed Central

    Pescosolido, Matthew F.; Stein, David M.; Schmidt, Michael; Achkar, Christelle Moufawad El; Sabbagh, Mark; Rogg, Jeffrey M.; Tantravahi, Umadevi; McLean, Rebecca L.; Liu, Judy S.; Poduri, Annapurna; Morrow, Eric M.

    2015-01-01

    Objective Recently, Christianson syndrome (CS) has been determined to be caused by mutations in the X-linked Na+/H+ Exchanger 6 (NHE6). We aimed to determine the diagnostic criteria and mutational spectrum for CS. Methods Twelve independent pedigrees (14 boys, ages 4 to 19) with mutations in NHE6 were administered standardized research assessments and mutations were characterized. Results The mutational spectrum was composed of 9 single nucleotide variants (SNVs), 2 indels and 1 CNV deletion. All mutations were protein-truncating or splicing mutations. We identified two recurrent mutations (c.1498 c>t, p.R500X; and c.1710 g>a, p.W570X). Otherwise, all mutations were unique. In our study, seven of 12 mutations (58%) were de novo, in contrast to prior literature wherein mutations were largely inherited. We also report prominent neurological, medical and behavioral symptoms. All CS participants were non-verbal and had intellectual disability, epilepsy and ataxia. Many had prior diagnoses of autism and/or Angelman syndrome. Other neurologic symptoms included eye movement abnormalities (79%), postnatal microcephaly (92%) and MRI evidence of cerebellar atrophy (33%). Regression was noted in 50%, with recurrent presentations involving loss of words and/or the ability to walk. Medical symptoms, particularly gastrointestinal symptoms, were common. Height and body mass index measures were below normal ranges in most participants. Behavioral symptoms included hyperkinetic behavior (100%) and a majority exhibited high pain threshold. Interpretation This is the largest cohort of independent CS pedigrees reported. We propose diagnostic criteria for CS. CS represents a novel neurogenetic disorder with general relevance to autism, intellectual disability, Angelman syndrome, epilepsy and regression. PMID:25044251

  3. Identification of a Novel GJA8 (Cx50) Point Mutation Causes Human Dominant Congenital Cataracts

    NASA Astrophysics Data System (ADS)

    Ge, Xiang-Lian; Zhang, Yilan; Wu, Yaming; Lv, Jineng; Zhang, Wei; Jin, Zi-Bing; Qu, Jia; Gu, Feng

    2014-02-01

    Hereditary cataracts are clinically and genetically heterogeneous lens diseases that cause a significant proportion of visual impairment and blindness in children. Human cataracts have been linked with mutations in two genes, GJA3 and GJA8, respectively. To identify the causative mutation in a family with hereditary cataracts, family members were screened for mutations by PCR for both genes. Sequencing the coding regions of GJA8, coding for connexin 50, revealed a C > A transversion at nucleotide 264, which caused p.P88T mutation. To dissect the molecular consequences of this mutation, plasmids carrying wild-type and mutant mouse ORFs of Gja8 were generated and ectopically expressed in HEK293 cells and human lens epithelial cells, respectively. The recombinant proteins were assessed by confocal microscopy and Western blotting. The results demonstrate that the molecular consequences of the p.P88T mutation in GJA8 include changes in connexin 50 protein localization patterns, accumulation of mutant protein, and increased cell growth.

  4. A new point mutation in the iron-sulfur subunit of succinate dehydrogenase confers resistance to boscalid in Sclerotinia sclerotiorum.

    PubMed

    Wang, Yong; Duan, Yabing; Wang, Jianxin; Zhou, Mingguo

    2015-09-01

    Research has established that mutations in highly conserved amino acids of the succinate dehydrogenase (SDH) complex in various fungi confer SDH inhibitor (SDHI) resistance. For Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic fungus with a broad host range and a worldwide distribution, boscalid resistance has been attributed to the mutation H132R in the highly conserved SdhD subunit protein of the SDH complex. In our previous study, however, only one point mutation, A11V in SdhB (GCA to GTA change in SdhB), was detected in S. sclerotiorum boscalid-resistant (BR) mutants. In the current study, replacement of the SdhB gene in a boscalid-sensitive (BS) S. sclerotiorum strain with the mutant SdhB gene conferred resistance. Compared with wild-type strains, BR and GSM (SdhB gene in the wild-type strain replaced by the mutant SdhB gene) mutants were more sensitive to osmotic stress, lacked the ability to produce sclerotia and exhibited lower expression of the pac1 gene. Importantly, the point mutation was not located in the highly conserved sequence of the iron-sulfur subunit of SDH. These results suggest that resistance based on non-conserved vs. conserved protein domains differs in mechanism. In addition to increasing our understanding of boscalid resistance in S. sclerotiorum, the new information will be useful for the development of alternative antifungal drugs. PMID:25441450

  5. A Targeted Q-PCR-Based Method for Point Mutation Testing by Analyzing Circulating DNA for Cancer Management Care.

    PubMed

    Thierry, Alain R

    2016-01-01

    Circulating cell-free DNA (cfDNA) is a valuable source of tumor material available with a simple blood sampling enabling a noninvasive quantitative and qualitative analysis of the tumor genome. cfDNA is released by tumor cells and exhibits the genetic and epigenetic alterations of the tumor of origin. Circulating cell-free DNA (cfDNA) analysis constitutes a hopeful approach to provide a noninvasive tumor molecular test for cancer patients. Based upon basic research on the origin and structure of cfDNA, new information on circulating cell-free DNA (cfDNA) structure, and specific determination of cfDNA fragmentation and size, we revisited Q-PCR-based method and recently developed a the allele-specific-Q-PCR-based method with blocker (termed as Intplex) which is the first multiplexed test for cfDNA. This technique, named Intplex(®) and based on a refined Q-PCR method, derived from critical observations made on the specific structure and size of cfDNA. It enables the simultaneous determination of five parameters: the cfDNA total concentration, the presence of a previously known point mutation, the mutant (tumor) cfDNA concentration (ctDNA), the proportion of mutant cfDNA, and the cfDNA fragmentation index. Intplex(®) has enabled the first clinical validation of ctDNA analysis in oncology by detecting KRAS and BRAF point mutations in mCRC patients and has demonstrated that a blood test could replace tumor section analysis for the detection of KRAS and BRAF mutations. The Intplex(®) test can be adapted to all mutations, genes, or cancers and enables rapid, highly sensitive, cost-effective, and repetitive analysis. As regards to the determination of mutations on cfDNA Intplex(®) is limited to the mutational status of known hotspot mutation; it is a "targeted approach." However, it offers the opportunity in detecting quantitatively and dynamically mutation and could constitute a noninvasive attractive tool potentially allowing diagnosis, prognosis, theranostics

  6. Simultaneous DNA and RNA Mapping of Somatic Mitochondrial Mutations across Diverse Human Cancers

    PubMed Central

    Stewart, James B.; Alaei-Mahabadi, Babak; Sabarinathan, Radhakrishnan; Samuelsson, Tore; Gorodkin, Jan; Gustafsson, Claes M.; Larsson, Erik

    2015-01-01

    Somatic mutations in the nuclear genome are required for tumor formation, but the functional consequences of somatic mitochondrial DNA (mtDNA) mutations are less understood. Here we identify somatic mtDNA mutations across 527 tumors and 14 cancer types, using an approach that takes advantage of evidence from both genomic and transcriptomic sequencing. We find that there is selective pressure against deleterious coding mutations, supporting that functional mitochondria are required in tumor cells, and also observe a strong mutational strand bias, compatible with endogenous replication-coupled errors as the major source of mutations. Interestingly, while allelic ratios in general were consistent in RNA compared to DNA, some mutations in tRNAs displayed strong allelic imbalances caused by accumulation of unprocessed tRNA precursors. The effect was explained by altered secondary structure, demonstrating that correct tRNA folding is a major determinant for processing of polycistronic mitochondrial transcripts. Additionally, the data suggest that tRNA clusters are preferably processed in the 3′ to 5′ direction. Our study gives insights into mtDNA function in cancer and answers questions regarding mitochondrial tRNA biogenesis that are difficult to address in controlled experimental systems. PMID:26125550

  7. Single-point mutation-mediated local amphipathic adjustment dramatically enhances antibacterial activity of a fungal defensin.

    PubMed

    Wu, Jiajia; Gao, Bin; Zhu, Shunyi

    2016-07-01

    The emergence and rapid spread of multiresistant bacteria has lead to an urgent need for novel antimicrobials. Based on single-point substitutions, we generated a series of mutants of micasin, a dermatophytic defensin, with enhanced activities against multiple clinical isolates of Staphylococcus species, including 4 antibiotic-resistant strains. We first mapped the functional surface of micasin by alanine-scanning mutational analysis of its highly exposed residues, through which we found that substitution of site 8 (acidic Glu) dramatically enhanced bacterial killing of this peptide. Structural analysis indicates that this single point mutation could result in a functional local amphipathic architecture. Four different types of side chains (hydrophobic, cationic polar, neutral polar, and acidic polar) were introduced at site 8 to clarify the role of this local architecture in micasin function. The results show that all mutants displayed increased antibacterial activity with the exception of the acidic replacement. These mutants with enhanced activity exhibited low hemolysis and cytotoxicity and showed high serum stability, indicating their therapeutic potential. Our work represents the first example of structural fine-tuning to largely improve the antibacterial potency of a dermatophytic defensin.-Wu, J., Gao, B., Zhu, S. Single-point mutation-mediated local amphipathic adjustment dramatically enhances antibacterial activity of a fungal defensin. PMID:27084888

  8. Analbuminemia: three cases resulting from different point mutations in the albumin gene.

    PubMed Central

    Watkins, S; Madison, J; Galliano, M; Minchiotti, L; Putnam, F W

    1994-01-01

    Analbuminemia is a very rare recessive disorder in which subjects have little or no circulating albumin, although albumin is normally the most abundant plasma protein and has many functions. Analbuminemia is caused by a variety of mutations in the albumin gene and is exhibited only by subjects homozygous for the defect. Previously the mutation had been identified at the molecular level in only two human cases; in one case it resulted from an exon-splicing defect, and in the other case it was caused by a nucleotide insertion that caused a frameshift and premature stop codon. In this investigation we identified the mutations in three unrelated subjects from different countries. In each instance a single-nucleotide mutation produced a stop codon, but the mutations occurred at three different sites: (i) in an Italian male a C-->T transition at nt 2368 in the genomic sequence of albumin, (ii) a C-->T transition at nt 4446 for an American female, and (iii) a G-->A transition at nt 7708 in a Canadian male. The size of the albumin fragment that might have been produced for the three cases varied from 31- to 213-amino acid residues, but no evidence for a circulating albumin fragment was obtained. The paradox is that analbuminemia is extremely rare (frequency < 1 x 10(6)); yet the virtual absence of albumin is tolerable despite its multiple functions. Images PMID:7937781

  9. Real-time resolution of point mutations that cause phenovariance in mice

    PubMed Central

    Wang, Tao; Zhan, Xiaowei; Bu, Chun-Hui; Lyon, Stephen; Pratt, David; Hildebrand, Sara; Choi, Jin Huk; Zhang, Zhao; Zeng, Ming; Wang, Kuan-wen; Turer, Emre; Chen, Zhe; Zhang, Duanwu; Yue, Tao; Wang, Ying; Shi, Hexin; Wang, Jianhui; Sun, Lei; SoRelle, Jeff; McAlpine, William; Hutchins, Noelle; Zhan, Xiaoming; Fina, Maggy; Gobert, Rochelle; Quan, Jiexia; Kreutzer, McKensie; Arnett, Stephanie; Hawkins, Kimberly; Leach, Ashley; Tate, Christopher; Daniel, Chad; Reyna, Carlos; Prince, Lauren; Davis, Sheila; Purrington, Joel; Bearden, Rick; Weatherly, Jennifer; White, Danielle; Russell, Jamie; Sun, Qihua; Tang, Miao; Li, Xiaohong; Scott, Lindsay; Moresco, Eva Marie Y.; McInerney, Gerald M.; Karlsson Hedestam, Gunilla B.; Xie, Yang; Beutler, Bruce

    2015-01-01

    With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled “superpedigrees” are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening. PMID:25605905

  10. Real-time resolution of point mutations that cause phenovariance in mice.

    PubMed

    Wang, Tao; Zhan, Xiaowei; Bu, Chun-Hui; Lyon, Stephen; Pratt, David; Hildebrand, Sara; Choi, Jin Huk; Zhang, Zhao; Zeng, Ming; Wang, Kuan-wen; Turer, Emre; Chen, Zhe; Zhang, Duanwu; Yue, Tao; Wang, Ying; Shi, Hexin; Wang, Jianhui; Sun, Lei; SoRelle, Jeff; McAlpine, William; Hutchins, Noelle; Zhan, Xiaoming; Fina, Maggy; Gobert, Rochelle; Quan, Jiexia; Kreutzer, McKensie; Arnett, Stephanie; Hawkins, Kimberly; Leach, Ashley; Tate, Christopher; Daniel, Chad; Reyna, Carlos; Prince, Lauren; Davis, Sheila; Purrington, Joel; Bearden, Rick; Weatherly, Jennifer; White, Danielle; Russell, Jamie; Sun, Qihua; Tang, Miao; Li, Xiaohong; Scott, Lindsay; Moresco, Eva Marie Y; McInerney, Gerald M; Karlsson Hedestam, Gunilla B; Xie, Yang; Beutler, Bruce

    2015-02-01

    With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening. PMID:25605905

  11. Identification of weak points prone for mutation in ferredoxin of Trichomonas vaginalis.

    PubMed

    Wiwanitkit, V

    2008-01-01

    Trichomonas vaginalis, the causative agent for human trichomoniasis, is a problematic sexually transmitted disease mainly in women. At present, metronidazole-resistant trichomoniasis is an infrequent but challenging problem with no universally successful treatment. Genetic mutation is believed to be an important factor leading to increasing drug resistance. Understanding the mutation status will help to design accurate strategies of therapy against mutant strains of T. vaginalis. The author performed a bioinformatic analysis to determine positions that tend to comply peptide motifs in the amino acid sequence of ferredoxin of T. vaginalis. Based on this study, the weak linkages in the studied protein can be identified and can be useful information for prediction of possible new mutations that can lead to drug resistance. In addition, the results from this study can be good information for further research on the diagnosis for mutants and new effective drug development. PMID:18445954

  12. Phenotypical diversity of patients with LEOPARD syndrome carrying the worldwide recurrent p.Tyr279Cys PTPN11 mutation.

    PubMed

    Nemes, Edina; Farkas, Katalin; Kocsis-Deák, Barbara; Drubi, Andrea; Sulák, Adrienn; Tripolszki, Kornélia; Dósa, Piroska; Ferenc, Lakatos; Nagy, Nikoletta; Széll, Márta

    2015-12-01

    LEOPARD syndrome (LS, OMIM 151100) is a rare monogenic disorder. The name is an acronym of its major features such as multiple lentigines, electrocardiographic conduction defects, ocular hypertelorism, pulmonary stenosis, abnormalities of genitalia, retardation of growth and sensorineural deafness. LS develops due to mutations in the protein-tyrosine phosphatase nonreceptor-type 11, PTPN11. Here, we have investigated a 51-year-old Hungarian male patient affected by LS. Direct sequencing of the PTPN11 gene revealed a worldwide recurrent missense mutation (c.836A/G; p.Tyr279Cys), which has been previously identified in 47 LS patients. Comparison of the clinical phenotypes of our patient and the ones reported in the literature demonstrates great phenotypic diversity despite the same genotype. PMID:26377839

  13. Point mutations in an epigenetic factor lead to multiple types of bone tumors: role of H3.3 histone variant in bone development and disease

    PubMed Central

    Kato, Shigeaki; Ishii, Takeaki; Kouzmenko, Alexander

    2015-01-01

    Coordinated post-translational modifications (PTMs) of nucleosomal histones emerge as a key mechanism of gene regulation by defining chromatin configuration. Patterns of histone modifications vary in different cells and constitute core elements of cell-specific epigenomes. Recently, in addition to canonical histone proteins produced during the S phase of cell cycle, several non-canonical histone variants have been identified and shown to express in a DNA replication-independent manner. These histone variants generate diversity in nucleosomal structures and add further complexity to mechanisms of epigenetic regulation. Cell-specific functions of histone variants remain to be determined. Several recent studies reported an association between some point mutations in the non-canonical histone H3.3 and particular types of brain and bone tumors. This suggests a possibility of differential physiological effects of histone variants in different cells and tissues, including bone. In this review, we outline the roles of histone variants and their PTMs in the epigenetic regulation of chromatin structure and discuss possible mechanisms of biological effects of the non-canonical histone mutations found in bone tumors on tumorigenesis in differentiating bone stem cells. PMID:26157578

  14. Point mutations in the potato leafroll virus major capsid protein alter virion stability and aphid transmission

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The coat protein (CP) of potato leafroll virus (PLRV) is the primary component of the capsid and is a multifunctional protein known to be involved in vector transmission and virus movement within plant hosts, in addition to particle assembly. Thirteen mutations were generated in various regions of ...

  15. Gene Coexpression Analyses Differentiate Networks Associated with Diverse Cancers Harboring TP53 Missense or Null Mutations

    PubMed Central

    Oros Klein, Kathleen; Oualkacha, Karim; Lafond, Marie-Hélène; Bhatnagar, Sahir; Tonin, Patricia N.; Greenwood, Celia M. T.

    2016-01-01

    In a variety of solid cancers, missense mutations in the well-established TP53 tumor suppressor gene may lead to the presence of a partially-functioning protein molecule, whereas mutations affecting the protein encoding reading frame, often referred to as null mutations, result in the absence of p53 protein. Both types of mutations have been observed in the same cancer type. As the resulting tumor biology may be quite different between these two groups, we used RNA-sequencing data from The Cancer Genome Atlas (TCGA) from four different cancers with poor prognosis, namely ovarian, breast, lung and skin cancers, to compare the patterns of coexpression of genes in tumors grouped according to their TP53 missense or null mutation status. We used Weighted Gene Coexpression Network analysis (WGCNA) and a new test statistic built on differences between groups in the measures of gene connectivity. For each cancer, our analysis identified a set of genes showing differential coexpression patterns between the TP53 missense- and null mutation-carrying groups that was robust to the choice of the tuning parameter in WGCNA. After comparing these sets of genes across the four cancers, one gene (KIR3DL2) consistently showed differential coexpression patterns between the null and missense groups. KIR3DL2 is known to play an important role in regulating the immune response, which is consistent with our observation that this gene's strongly-correlated partners implicated many immune-related pathways. Examining mutation-type-related changes in correlations between sets of genes may provide new insight into tumor biology. PMID:27536319

  16. Development of a Magnetic Electrochemical Bar Code Array for Point Mutation Detection in the H5N1 Neuraminidase Gene

    PubMed Central

    Krejcova, Ludmila; Hynek, David; Kopel, Pavel; Merlos Rodrigo, Miguel Angel; Adam, Vojtech; Hubalek, Jaromir; Babula, Petr; Trnkova, Libuse; Kizek, Rene

    2013-01-01

    Since its first official detection in the Guangdong province of China in 1996, the highly pathogenic avian influenza virus of H5N1 subtype (HPAI H5N1) has reportedly been the cause of outbreaks in birds in more than 60 countries, 24 of which were European. The main issue is still to develop effective antiviral drugs. In this case, single point mutation in the neuraminidase gene, which causes resistance to antiviral drug and is, therefore, subjected to many studies including ours, was observed. In this study, we developed magnetic electrochemical bar code array for detection of single point mutations (mismatches in up to four nucleotides) in H5N1 neuraminidase gene. Paramagnetic particles Dynabeads® with covalently bound oligo (dT)25 were used as a tool for isolation of complementary H5N1 chains (H5N1 Zhejin, China and Aichi). For detection of H5N1 chains, oligonucleotide chains of lengths of 12 (+5 adenine) or 28 (+5 adenine) bp labeled with quantum dots (CdS, ZnS and/or PbS) were used. Individual probes hybridized to target molecules specifically with efficiency higher than 60%. The obtained signals identified mutations present in the sequence. Suggested experimental procedure allows obtaining further information from the redox signals of nucleic acids. Moreover, the used biosensor exhibits sequence specificity and low limits of detection of subnanogram quantities of target nucleic acids. PMID:23860384

  17. Diversity and Convergence of Sodium Channel Mutations Involved in Resistance to Pyrethroids

    PubMed Central

    Rinkevich, Frank D.; Du, Yuzhe; Dong, Ke

    2013-01-01

    Pyrethroid insecticides target voltage-gated sodium channels, which are critical for electrical signaling in the nervous system. The intensive use of pyrethroids in controlling arthropod pests and disease vectors has led to many instances of pyrethroid resistance around the globe. In the past two decades, studies have identified a large number of sodium channel mutations that are associated with resistance to pyrethroids. The purpose of this review is to summarize both common and unique sodium channel mutations that have been identified in arthropod pests of importance to agriculture or human health. Identification of these mutations provides valuable molecular markers for resistance monitoring in the field and helped the discovery of the elusive pyrethroid receptor site(s) on the sodium channel. PMID:24019556

  18. The identification of point mutations in Duchenne muscular dystrophy patients by using reverse-transcription PCR and the protein truncation test

    SciTech Connect

    Gardner, R.J.; Bobrow, M.; Roberts, R.G.

    1995-08-01

    The protein truncation test (PTT) is a mutation-detection method that monitors the integrity of the open reading frame (ORF). More than 60% of cases of Duchenne muscular dystrophy (DMD) result from gross frameshifting deletions in the dystrophin gene that are detectable by multiplex PCR system. It has become apparent that virtually all of the remaining DMD mutations also disrupt the translational reading frame, making the PTT a logical next step toward a comprehensive strategy for the identification of all DMD mutations. We report here a pilot study involving 22 patients and describe the mutations characterized. These constitute 12 point mutations or small insertions/deletions and 4 gross rearrangements. We also have a remaining five patients in whom there does not appear to be mutation in the ORF. We believe that reverse-transcription-PCR/PTT is an efficient method by which to screen for small mutations in DMD patients with no deletion. 29 refs., 2 figs., 3 tabs.

  19. Large Deletions and Point Mutations Involving DOCK8 in the Autosomal Recessive Form of the Hyper-IgE Syndrome

    PubMed Central

    Engelhardt, Karin R.; McGhee, Sean; Winkler, Sabine; Sassi, Atfa; Woellner, Cristina; Lopez-Herrera, Gabriela; Chen, Andrew; Kim, Hong Sook; Lloret, Maria Garcia; Schulze, Ilka; Ehl, Stephan; Thiel, Jens; Pfeifer, Dietmar; Veelken, Hendrik; Niehues, Tim; Siepermann, Kathrin; Weinspach, Sebastian; Reisli, Ismail; Keles, Sevgi; Genel, Ferah; Kütükçüler, Necil; Camcioğlu, Yildiz; Somer, Ayper; Aydiner, Elif Karakoc; Barlan, Isil; Gennery, Andrew; Metin, Ayse; Degerliyurt, Aydan; Pietrogrande, Maria C.; Yeganeh, Mehdi; Baz, Zeina; Al-Tamemi, Salem; Klein, Christoph; Puck, Jennifer M.; Holland, Steven M.; McCabe, Edward R. B.; Grimbacher, Bodo; Chatila, Talal

    2010-01-01

    Background The genetic etiologies of the hyper-IgE syndromes are diverse. Approximately 60-70% of patients with hyper-IgE syndrome have dominant mutations in STAT3, and a single patient was reported to have a homozygous TYK2 mutation. In the remaining hyper-IgE syndrome patients, the genetic etiology has not yet been identified. Methods We performed genome-wide single nucleotide polymorphism analysis for nine subjects with autosomal recessive hyper-IgE syndrome to locate copy number variations and homozygous haplotypes. Homozygosity mapping was performed with twelve subjects from seven additional families. The candidate gene was analyzed by genomic and cDNA sequencing to identify causative alleles in a total of 27 patients with autosomal recessive hyper-IgE syndrome. Findings Subtelomeric microdeletions were identified in six subjects at the terminus of chromosome 9p. In all patients the deleted interval involved DOCK8, encoding a protein implicated in the regulation of the actin cytoskeleton. Sequencing of subjects without large deletions revealed 16 patients from nine unrelated families with distinct homozygous mutations in DOCK8 causing premature termination, frameshift, splice site disruption, single exon- and micro-deletions. DOCK8 deficiency was associated with impaired activation of CD4+ and CD8+ T cells. Interpretation Autosomal recessive mutations in DOCK8 are responsible for many, though not all, cases of autosomal recessive hyper-IgE syndrome. DOCK8 disruption is associated with a phenotype of severe cellular immunodeficiency characterized by susceptibility to viral infections, atopic eczema, defective T cell activation and TH17 cell differentiation; and impaired eosinophil homeostasis and dysregulation of IgE. PMID:20004785

  20. A Human Disease-causing Point Mutation in Mitochondrial Threonyl-tRNA Synthetase Induces Both Structural and Functional Defects.

    PubMed

    Wang, Yong; Zhou, Xiao-Long; Ruan, Zhi-Rong; Liu, Ru-Juan; Eriani, Gilbert; Wang, En-Duo

    2016-03-18

    Mitochondria require all translational components, including aminoacyl-tRNA synthetases (aaRSs), to complete organelle protein synthesis. Some aaRS mutations cause mitochondrial disorders, including human mitochondrial threonyl-tRNA synthetase (hmtThrRS) (encoded by TARS2), the P282L mutation of which causes mitochondrial encephalomyopathies. However, its catalytic and structural consequences remain unclear. Herein, we cloned TARS2 and purified the wild-type and P282L mutant hmtThrRS. hmtThrRS misactivates non-cognate Ser and uses post-transfer editing to clear erroneously synthesized products. In vitro and in vivo analyses revealed that the mutation induces a decrease in Thr activation, aminoacylation, and proofreading activities and a change in the protein structure and/or stability, which might cause reduced catalytic efficiency. We also identified a splicing variant of TARS2 mRNA lacking exons 8 and 9, the protein product of which is targeted into mitochondria. In HEK293T cells, the variant does not dimerize and cannot complement the ThrRS knock-out strain in yeast, suggesting that the truncated protein is inactive and might have a non-canonical function, as observed for other aaRS fragments. The present study describes the aminoacylation and editing properties of hmtThrRS, clarifies the molecular consequences of the P282L mutation, and shows that the yeast ThrRS-deletion model is suitable to test pathology-associated point mutations or alternative splicing variants of mammalian aaRS mRNAs. PMID:26811336

  1. A point mutation within CD45 exon A is the cause of variant CD45RA splicing in humans.

    PubMed

    Zilch, C F; Walker, A M; Timón, M; Goff, L K; Wallace, D L; Beverley, P C

    1998-01-01

    The leukocyte common antigen (CD45) is alternatively spliced, generating various isoforms expressed on hemopoietic cells. The splicing pattern of CD45 in T cells is altered in some individuals who show abnormal expression of high molecular weight isoforms containing exon A. The variant splicing pattern was shown to be associated with heterozygosity for a silent point mutation within CD45 exon A. This C to G transition is located 77 nucleotides downstream of the splice acceptor junction of exon A (198 bp total length). Here we report that this mutation is the cause of abnormal splicing. To isolate the mutant gene, somatic cell hybrids of lymphocytes with a CD45 splicing defect and a mouse lymphoid line were produced and clones expressing different isoforms of CD45 were isolated. Expression of the high molecular weight isoform containing exon A was associated with the mutation within exon A. All hybrids expressing the low molecular weight isoforms lacking exon A contained the normal allele of CD45 only. In addition, minigenes including this mutation were constructed and transfected into various cell lines (COS-7, HeLa, CHO). Semi-quantitative reverse transcription polymerase chain reaction showed an increase of more than tenfold in splicing to CD45RA (concomitant with a decrease in splicing to CD45RO) when compared with the normal minigene. Taken together, these results demonstrate a causal relationship between the mutation in CD45 exon A and the variant splicing pattern observed. The involvement of trans-acting splicing factors that interact with this region of CD45 pre-mRNA is currently under investigation. PMID:9485182

  2. Dynamic Harmony Search with Polynomial Mutation Algorithm for Valve-Point Economic Load Dispatch

    PubMed Central

    Karthikeyan, M.; Sree Ranga Raja, T.

    2015-01-01

    Economic load dispatch (ELD) problem is an important issue in the operation and control of modern control system. The ELD problem is complex and nonlinear with equality and inequality constraints which makes it hard to be efficiently solved. This paper presents a new modification of harmony search (HS) algorithm named as dynamic harmony search with polynomial mutation (DHSPM) algorithm to solve ORPD problem. In DHSPM algorithm the key parameters of HS algorithm like harmony memory considering rate (HMCR) and pitch adjusting rate (PAR) are changed dynamically and there is no need to predefine these parameters. Additionally polynomial mutation is inserted in the updating step of HS algorithm to favor exploration and exploitation of the search space. The DHSPM algorithm is tested with three power system cases consisting of 3, 13, and 40 thermal units. The computational results show that the DHSPM algorithm is more effective in finding better solutions than other computational intelligence based methods. PMID:26491710

  3. Phenotypic Switching in Mycoplasma gallisepticum Hemadsorption Is Governed by a High-Frequency, Reversible Point Mutation

    PubMed Central

    Winner, Florian; Markovà, Ivana; Much, Peter; Lugmair, Albin; Siebert-Gulle, Karin; Vogl, Gunther; Rosengarten, Renate; Citti, Christine

    2003-01-01

    Mycoplasma gallisepticum is a flask-shaped organism that commonly induces chronic respiratory disease in chickens and infectious sinusitis in turkeys. Phenotypic switching in M. gallisepticum hemadsorption (HA) was found to correlate with phase variation of the GapA cytadhesin concurrently with that of the CrmA protein, which exhibits cytadhesin-related features and is encoded by a gene located downstream of the gapA gene as part of the same transcription unit. In clones derived from strain Rlow, detailed genetic analyses further revealed that on-off switching in GapA expression is governed by a reversible base substitution occurring at the beginning of the gapA structural gene. In HA− variants, this event generates a stop codon that results in the premature termination of GapA translation and consequently affects the expression of CrmA. Sequences flanking the mutation spot do not feature any repeated motifs that could account for error-prone mutation via DNA slippage and the exact mechanism underlying this high-frequency mutational event remains to be elucidated. An HA− mutant deficient in producing CrmA, mHAD3, was obtained by disrupting the crmA gene by using transposition mutagenesis. Despite a fully functional gapA gene, the amount of GapA detected in this mutant was considerably lower than in HA+ clonal variants, suggesting that, in absence of CrmA, GapA might be subjected to a higher turnover. PMID:12595441

  4. Point mutations in the promoter region of the CYBB gene leading to mild chronic granulomatous disease

    PubMed Central

    Weening, R S; De Boer, M; Kuijpers, T W; Neefjes, V M E; Hack, W W M; Roos, D

    2000-01-01

    Chronic granulomatous disease (CGD) is a clinical syndrome of recurrent bacterial and fungal infections caused by a rare disorder of phagocytic cells. In CGD, the phagocytes are unable to generate oxygen radicals after stimulation of these cells, due to a defect in the NADPH oxidase system. This NADPH oxidase is a multicomponent enzyme of at least four subunits, of which the β-subunit of cytochrome b558, gp91-phox, is encoded by an X-linked gene (called CYBB). We report here five patients from two families; in each family we found a different mutation in the promoter region of CYBB. Both mutations prevented the expression of gp91-phox in the patients' neutrophils and thus caused inability of these cells to generate oxygen radicals. However, the mutations left the gp91-phox expression and the function of the NADPH oxidase in the patients' eosinophils intact. The relatively mild course of the CGD in these patients can probably be attributed to the fact that the eosinophils have retained their oxidative capacity. Furthermore, our results indicate that neutrophils and eosinophils differ in their regulation of gp91-phox expression. PMID:11122248

  5. Distance from sub-Saharan Africa predicts mutational load in diverse human genomes.

    PubMed

    Henn, Brenna M; Botigué, Laura R; Peischl, Stephan; Dupanloup, Isabelle; Lipatov, Mikhail; Maples, Brian K; Martin, Alicia R; Musharoff, Shaila; Cann, Howard; Snyder, Michael P; Excoffier, Laurent; Kidd, Jeffrey M; Bustamante, Carlos D

    2016-01-26

    The Out-of-Africa (OOA) dispersal ∼ 50,000 y ago is characterized by a series of founder events as modern humans expanded into multiple continents. Population genetics theory predicts an increase of mutational load in populations undergoing serial founder effects during range expansions. To test this hypothesis, we have sequenced full genomes and high-coverage exomes from seven geographically divergent human populations from Namibia, Congo, Algeria, Pakistan, Cambodia, Siberia, and Mexico. We find that individual genomes vary modestly in the overall number of predicted deleterious alleles. We show via spatially explicit simulations that the observed distribution of deleterious allele frequencies is consistent with the OOA dispersal, particularly under a model where deleterious mutations are recessive. We conclude that there is a strong signal of purifying selection at conserved genomic positions within Africa, but that many predicted deleterious mutations have evolved as if they were neutral during the expansion out of Africa. Under a model where selection is inversely related to dominance, we show that OOA populations are likely to have a higher mutation load due to increased allele frequencies of nearly neutral variants that are recessive or partially recessive. PMID:26712023

  6. Diverse deafness mechanisms of connexin mutations revealed by studies using in vitro approaches and mouse models

    PubMed Central

    Dinh, Emilie Hoang; Ahmad, Shoeb; Chang, Qing; Tang, Wenxue; Stong, Benjamin; Lin, Xi

    2009-01-01

    Mutations in connexins (Cxs), the constitutive protein subunits of gap junction (GJ) intercellular channels, are one of the most common human genetic defects that cause severe prelingual non-syndromic hearing impairments. Many subtypes of Cxs (e.g., Cxs 26, 29, 30, 31, 43) and pannexins (Panxs) are expressed in the cochlea where they contribute to the formation of a GJ-based intercellular communication network. Cx26 and Cx30 are the predominant cochlear Cxs and they co-assemble in most GJ plaques to form hybrid GJs. The cellular localization of specific Cx subtypes provides a basis for understanding the molecular structure of GJs and hemichannels in the cochlea. Information about the interactions among the various co-assembled Cx partners is critical to appreciate the functional consequences of various types of genetic mutations. In vitro studies of reconstituted GJs in cell lines have yielded surprisingly heterogeneous mechanisms of dysfunction caused by various Cx mutations. Availability of multiple lines of Cx-mutant mouse models has provided some insight into the pathogenesis processes in the cochlea of deaf mice. Here we summarize recent advances in understanding the structure and function of cochlear GJs and give a critical review of current findings obtained from both in vitro studies and mouse models on the mechanisms of Cx mutations that lead to cell death in the cochlea and hearing loss. PMID:19230829

  7. Distance from sub-Saharan Africa predicts mutational load in diverse human genomes

    PubMed Central

    Henn, Brenna M.; Botigué, Laura R.; Peischl, Stephan; Dupanloup, Isabelle; Lipatov, Mikhail; Maples, Brian K.; Martin, Alicia R.; Musharoff, Shaila; Cann, Howard; Snyder, Michael P.; Excoffier, Laurent; Kidd, Jeffrey M.; Bustamante, Carlos D.

    2016-01-01

    The Out-of-Africa (OOA) dispersal ∼50,000 y ago is characterized by a series of founder events as modern humans expanded into multiple continents. Population genetics theory predicts an increase of mutational load in populations undergoing serial founder effects during range expansions. To test this hypothesis, we have sequenced full genomes and high-coverage exomes from seven geographically divergent human populations from Namibia, Congo, Algeria, Pakistan, Cambodia, Siberia, and Mexico. We find that individual genomes vary modestly in the overall number of predicted deleterious alleles. We show via spatially explicit simulations that the observed distribution of deleterious allele frequencies is consistent with the OOA dispersal, particularly under a model where deleterious mutations are recessive. We conclude that there is a strong signal of purifying selection at conserved genomic positions within Africa, but that many predicted deleterious mutations have evolved as if they were neutral during the expansion out of Africa. Under a model where selection is inversely related to dominance, we show that OOA populations are likely to have a higher mutation load due to increased allele frequencies of nearly neutral variants that are recessive or partially recessive. PMID:26712023

  8. Intrahost diversity of feline coronavirus: a consensus between the circulating virulent/avirulent strains and the internal mutation hypotheses?

    PubMed

    Hora, Aline S; Asano, Karen M; Guerra, Juliana M; Mesquita, Ramon G; Maiorka, Paulo; Richtzenhain, Leonardo J; Brandão, Paulo E

    2013-01-01

    To evaluate the most controversial issue concerning current feline coronavirus (FCoV) virology, the coexisting hypotheses of the intrahost and interhost origins of feline infectious peritonitis virus (FIPV) in regard to the pathogenesis of feline infectious peritonitis (FIP), this study aimed to assess the molecular diversity of the membrane gene FCoVs in 190 samples from 10 cats with signs of FIP and in 5 faecal samples from cats without signs of FIP. All samples from the non-FIP cats and 25.26% of the samples from the FIP cats were positive for the FCoV membrane (M) gene. Mutations in this gene consisted of SNP changes randomly scattered among the sequences; few mutations resulted in amino acid changes. No geographic pattern was observed. Of the cats without FIP that harboured FECoV, the amino acid sequence identities for the M gene were 100% among cats (Cats 1-3) from the same cattery, and the overall sequence identity for the M gene was ≥91%. In one cat, two different lineages of FCoV, one enteric and one systemic, were found that segregated apart in the M gene tree. In conclusion, the in vivo mutation transition hypothesis and the circulating high virulent-low virulent FCoV hypothesis have been found to be plausible according to the results obtained from sequencing the M gene. PMID:23589704

  9. Intrahost Diversity of Feline Coronavirus: A Consensus between the Circulating Virulent/Avirulent Strains and the Internal Mutation Hypotheses?

    PubMed Central

    Hora, Aline S.; Asano, Karen M.; Guerra, Juliana M.; Mesquita, Ramon G.; Maiorka, Paulo; Richtzenhain, Leonardo J.; Brandão, Paulo E.

    2013-01-01

    To evaluate the most controversial issue concerning current feline coronavirus (FCoV) virology, the coexisting hypotheses of the intrahost and interhost origins of feline infectious peritonitis virus (FIPV) in regard to the pathogenesis of feline infectious peritonitis (FIP), this study aimed to assess the molecular diversity of the membrane gene FCoVs in 190 samples from 10 cats with signs of FIP and in 5 faecal samples from cats without signs of FIP. All samples from the non-FIP cats and 25.26% of the samples from the FIP cats were positive for the FCoV membrane (M) gene. Mutations in this gene consisted of SNP changes randomly scattered among the sequences; few mutations resulted in amino acid changes. No geographic pattern was observed. Of the cats without FIP that harboured FECoV, the amino acid sequence identities for the M gene were 100% among cats (Cats 1–3) from the same cattery, and the overall sequence identity for the M gene was ≥91%. In one cat, two different lineages of FCoV, one enteric and one systemic, were found that segregated apart in the M gene tree. In conclusion, the in vivo mutation transition hypothesis and the circulating high virulent-low virulent FCoV hypothesis have been found to be plausible according to the results obtained from sequencing the M gene. PMID:23589704

  10. Three Novel Heterozygous Point Mutations of NR3C1 Causing Glucocorticoid Resistance.

    PubMed

    Vitellius, Géraldine; Fagart, Jérôme; Delemer, Brigitte; Amazit, Larbi; Ramos, Nelly; Bouligand, Jérôme; Le Billan, Florian; Castinetti, Frédéric; Guiochon-Mantel, Anne; Trabado, Séverine; Lombès, Marc

    2016-08-01

    Generalized glucocorticoid resistance is associated with glucocorticoid receptor (GR; NR3C1) mutations. Three novel heterozygous missense NR3C1 mutations (R477S, Y478C, and L672P) were identified in patients presenting with adrenal incidentalomas, glucocorticoid excess without Cushing syndrome. Dexamethasone (DXM) binding studies demonstrated that the affinity of GRR477S and GRY478C mutants, located in the DNA-binding domain (DBD) of GR, was similar to wild-type GR (Kd  = 2-3 nM). In contrast, GRL672P mutant, located in the ligand-binding domain (LBD) of GR, was unable to bind glucocorticoids and was more sensitive to protein degradation. GR subcellular distribution revealed a marked decrease in DXM-induced nuclear translocation of GRR477S and GRY478C mutants, whereas GRL672P remained exclusively cytoplasmic. Chromatin immunoprecipitation demonstrated impaired recruitment of DBD mutants onto the regulatory sequence of FKBP5. Transactivation assays disclosed the lack of transcriptional activity of GRR477S and GRL672P , whereas GRY478C had a reduced transactivation capacity. Three-dimensional modeling indicated that R477S lost two essential hydrogen bonds with DNA, Y478C resulted in altered interaction with surrounding amino-acids, destabilizing DBD, whereas L672P altered the H8 helix folding, leading to unstructured LBD. This study identifies novel NR3C1 mutations with their molecular consequences on altered GR signaling and suggests that genetic screening of NR3C1 should be conducted in patients with subclinical hypercorticism. PMID:27120390

  11. Single-Point Mutation with a Rotamer Library Toolkit: Toward Protein Engineering.

    PubMed

    Pottel, Joshua; Moitessier, Nicolas

    2015-12-28

    Protein engineers have long been hard at work to harness biocatalysts as a natural source of regio-, stereo-, and chemoselectivity in order to carry out chemistry (reactions and/or substrates) not previously achieved with these enzymes. The extreme labor demands and exponential number of mutation combinations have induced computational advances in this domain. The first step in our virtual approach is to predict the correct conformations upon mutation of residues (i.e., rebuilding side chains). For this purpose, we opted for a combination of molecular mechanics and statistical data. In this work, we have developed automated computational tools to extract protein structural information and created conformational libraries for each amino acid dependent on a variable number of parameters (e.g., resolution, flexibility, secondary structure). We have also developed the necessary tool to apply the mutation and optimize the conformation accordingly. For side-chain conformation prediction, we obtained overall average root-mean-square deviations (RMSDs) of 0.91 and 1.01 Å for the 18 flexible natural amino acids within two distinct sets of over 3000 and 1500 side-chain residues, respectively. The commonly used dihedral angle differences were also evaluated and performed worse than the state of the art. These two metrics are also compared. Furthermore, we generated a family-specific library for kinases that produced an average 2% lower RMSD upon side-chain reconstruction and a residue-specific library that yielded a 17% improvement. Ultimately, since our protein engineering outlook involves using our docking software, Fitted/Impacts, we applied our mutation protocol to a benchmarked data set for self- and cross-docking. Our side-chain reconstruction does not hinder our docking software, demonstrating differences in pose prediction accuracy of approximately 2% (RMSD cutoff metric) for a set of over 200 protein/ligand structures. Similarly, when docking to a set of over 100

  12. Conspicuous involvement of desmin tail mutations in diverse cardiac and skeletal myopathies.

    PubMed

    Bär, Harald; Goudeau, Bertrand; Wälde, Sarah; Casteras-Simon, Monique; Mücke, Norbert; Shatunov, Alexey; Goldberg, Y Paul; Clarke, Charles; Holton, Janice L; Eymard, Bruno; Katus, Hugo A; Fardeau, Michel; Goldfarb, Lev; Vicart, Patrick; Herrmann, Harald

    2007-04-01

    Myofibrillar myopathy (MFM) encompasses a genetically heterogeneous group of human diseases caused by mutations in genes coding for structural proteins of muscle. Mutations in the intermediate filament (IF) protein desmin (DES), a major cytoskeletal component of myocytes, lead to severe forms of "desminopathy," which affects cardiac, skeletal, and smooth muscle. Most mutations described reside in the central alpha-helical rod domain of desmin. Here we report three novel mutations--c.1325C>T (p.T442I), c.1360C>T (p.R454W), and c.1379G>T (p.S460I)--located in desmin's non-alpha-helical carboxy-terminal "tail" domain. We have investigated the impact of these and four--c.1237G>A (p.E413K), c.1346A>C (p.K449T), c.1353C>G (p.I451M), and c.1405G>A (p.V469M)--previously described "tail" mutations on in vitro filament formation and on the generation of ordered cytoskeletal arrays in transfected myoblasts. Although all but two mutants (p.E413K, p.R454W) assembled into IFs in vitro and all except p.E413K were incorporated into IF arrays in transfected C2C12 cells, filament properties differed significantly from wild-type desmin as revealed by viscometric assembly assays. Most notably, when coassembled with wild-type desmin, these mutants revealed a severe disturbance of filament-formation competence and filament-filament interactions, indicating an inherent incompatibility of mutant and wild-type protein to form mixed filaments. The various clinical phenotypes observed may reflect altered interactions of desmin's tail domain with different components of the myoblast cytoskeleton leading to diminished biomechanical properties and/or altered metabolism of the individual myocyte. Our in vitro assembly regimen proved to be a very sensible tool to detect if a particular desmin mutation is able to cause filament abnormalities. PMID:17221859

  13. Whole exome sequencing identifies the first STRADA point mutation in a patient with polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome (PMSE).

    PubMed

    Bi, Weimin; Glass, Ian A; Muzny, Donna M; Gibbs, Richard A; Eng, Christine M; Yang, Yaping; Sun, Angela

    2016-08-01

    Polyhydramnios, megalencephaly, and symptomatic epilepsy syndrome (PMSE) is an ultra rare neurodevelopmental disorder characterized by severe, infantile-onset intractable epilepsy, neurocognitive delay, macrocephaly, and craniofacial dysmorphism. The molecular diagnosis of this condition has thus far only been made in 16 Old Order Mennonite patients carrying a homozygous 7 kb founder deletion of exons 9-13 of STRADA. We performed clinical whole exome sequencing (WES) on a 4-year-old Indian male with global developmental delay, history of failure to thrive, infantile spasms, repetitive behaviors, hypotonia, low muscle mass, marked joint laxity, and dysmorphic facial features including tall forehead, long face, arched eyebrows, small chin, wide mouth, and tented upper lip. A homozygous single nucleotide duplication, c.842dupA (p.D281fs), in exon 10 of STRADA was identified. Sanger sequencing confirmed the mutation in the individual and identified both parents as carriers. In light of the molecular discoveries, the patient's clinical phenotype was considered to be a good fit for PMSE. We identified for the first time a homozygous point mutation in STRADA causing PMSE. Additional bi-allelic mutations related to PMSE thus far have not been observed in Baylor ∼6,000 consecutive clinical WES cases, supporting the rarity of this disorder. Our findings may have treatment implications for the patient since previous studies have shown rapamycin as a potential therapeutic agent for the seizures and cognitive problems in PMSE patients. © 2016 Wiley Periodicals, Inc. PMID:27170158

  14. Genetic diversity and mutation of avian paramyxovirus serotype 1 (Newcastle disease virus) in wild birds and evidence for intercontinental spread

    USGS Publications Warehouse

    Ramey, Andy M.; Reeves, Andrew B.; Ogawa, Haruko; Ip, Hon S.; Imai, Kunitoshi; Bui, V. N.; Yamaguchi, Emi; Silko, N. Y.; Afonso, C.L.

    2013-01-01

    Avian paramyxovirus serotype 1 (APMV-1), or Newcastle disease virus, is the causative agent of Newcastle disease, one of the most economically important diseases for poultry production worldwide and a cause of periodic epizootics in wild birds in North America. In this study, we examined the genetic diversity of APMV-1 isolated from migratory birds sampled in Alaska, Japan, and Russia and assessed the evidence for intercontinental virus spread using phylogenetic methods. Additionally, we predicted viral virulence using deduced amino acid residues for the fusion protein cleavage site and estimated mutation rates for the fusion gene of class I and class II migratory bird isolates. All 73 isolates sequenced as part of this study were most closely related to virus genotypes previously reported for wild birds; however, five class II genotype I isolates formed a monophyletic clade exhibiting previously unreported genetic diversity, which met criteria for the designation of a new sub-genotype. Phylogenetic analysis of wild-bird isolates provided evidence for intercontinental virus spread, specifically viral lineages of APMV-1 class II genotype I sub-genotypes Ib and Ic. This result supports migratory bird movement as a possible mechanism for the redistribution of APMV-1. None of the predicted deduced amino acid motifs for the fusion protein cleavage site of APMV-1 strains isolated from migratory birds in Alaska, Japan, and Russia were consistent with those of previously identified virulent viruses. These data therefore provide no support for these strains contributing to the emergence of avian pathogens. The estimated mutation rates for fusion genes of class I and class II wild-bird isolates were faster than those reported previously for non-virulent APMV-1 strains. Collectively, these findings provide new insight into the diversity, spread, and evolution of APMV-1 in wild birds.

  15. A Point Mutation in PDGFRB Causes Autosomal-Dominant Penttinen Syndrome

    PubMed Central

    Johnston, Jennifer J.; Sanchez-Contreras, Monica Y.; Keppler-Noreuil, Kim M.; Sapp, Julie; Crenshaw, Molly; Finch, NiCole A.; Cormier-Daire, Valerie; Rademakers, Rosa; Sybert, Virginia P.; Biesecker, Leslie G.

    2015-01-01

    Penttinen syndrome is a distinctive disorder characterized by a prematurely aged appearance with lipoatrophy, epidermal and dermal atrophy along with hypertrophic lesions that resemble scars, thin hair, proptosis, underdeveloped cheekbones, and marked acro-osteolysis. All individuals have been simplex cases. Exome sequencing of an affected individual identified a de novo c.1994T>C p.Val665Ala variant in PDGFRB, which encodes the platelet-derived growth factor receptor β. Three additional unrelated individuals with this condition were shown to have the identical variant in PDGFRB. Distinct mutations in PDGFRB have been shown to cause infantile myofibromatosis, idiopathic basal ganglia calcification, and an overgrowth disorder with dysmorphic facies and psychosis, none of which overlaps with the clinical findings in Penttinen syndrome. We evaluated the functional consequence of this causative variant on the PDGFRB signaling pathway by transfecting mutant and wild-type cDNA into HeLa cells, and transfection showed ligand-independent constitutive signaling through STAT3 and PLCγ. Penttinen syndrome is a clinically distinct genetic condition caused by a PDGFRB gain-of-function mutation that is associated with a specific and unusual perturbation of receptor function. PMID:26279204

  16. Identification of a point mutation impairing the binding between aquaporin-4 and neuromyelitis optica autoantibodies.

    PubMed

    Pisani, Francesco; Mola, Maria Grazia; Simone, Laura; Rosito, Stefania; Alberga, Domenico; Mangiatordi, Giuseppe Felice; Lattanzi, Gianluca; Nicolotti, Orazio; Frigeri, Antonio; Svelto, Maria; Nicchia, Grazia Paola

    2014-10-31

    Neuromyelitis optica (NMO) is characterized by the presence of pathogenic autoantibodies (NMO-IgGs) against supra-molecular assemblies of aquaporin-4 (AQP4), known as orthogonal array of particles (OAPs). NMO-IgGs have a polyclonal origin and recognize different conformational epitopes involving extracellular AQP4 loops A, C, and E. Here we hypothesize a pivotal role for AQP4 transmembrane regions (TMs) in epitope assembly. On the basis of multialignment analysis, mutagenesis, NMO-IgG binding, and cytotoxicity assay, we have disclosed the key role of aspartate 69 (Asp(69)) of TM2 for NMO-IgG epitope assembly. Mutation of Asp(69) to histidine severely impairs NMO-IgG binding for 85.7% of the NMO patient sera analyzed here. Although Blue Native-PAGE, total internal reflection fluorescence microscopy, and water transport assays indicate that the OAP Asp(69) mutant is similar in structure and function to the wild type, molecular dynamic simulations have revealed that the D(69)H mutation has the effect of altering the structural rearrangements of extracellular loop A. In conclusion, Asp(69) is crucial for the spatial control of loop A, the particular molecular conformation of which enables the assembly of NMO-IgG epitopes. These findings provide additional clues for new strategies for NMO treatment and a wealth of information to better approach NMO pathogenesis. PMID:25239624

  17. Microphthalmia and cataract in rats with a novel point mutation in connexin 50 - L7Q

    PubMed Central

    Chylíková, Blanka; Martínek, Jindřich; Křen, Vladimír

    2008-01-01

    Purpose We isolated an autosomal semi-dominant cataract from our inbred SHR/OlaIpcv rat colony. Heterozygotes express pulverulent cataract with smaller eyes; homozygotes express marked microphthalmia with hypoplastic lens. We call this mutation Dca (for dominant cataract). In this study, we focus on the identification of the responsible gene. Methods We performed linkage mapping using 93 F2(SHR-Dca x PD) hybrids and a panel of microsatellite markers. In a separate group of animals with a SHR genetic background, we examined the lenses histologically using Epon semi-thin sections and toluidine blue staining. We also assessed the weight of the eyes as an immediate measure for microphthalmia. Results We mapped the Dca gene to chromosome 2, spanning 8.6 Mbp between markers D2Rat134 and D2Rat186. By sequencing the most plausible candidate gene, Gja8 (coding for connexin 50), we found a T to A transversion at codon 7, leading to a substitution of glutamine for leucin (L7Q). L7Q lies within the NH2-terminal cytosolic domain, presumably involved in voltage gating. Histology revealed disturbances in cell to cell contacts in the lens. Conclusions L7Q is a novel mutation in connexin 50 (Gja8), causing semi-dominant pulverulent cataracts. Dca rats can serve as a model for cataract development. A study on the properties of the mutant protein may offer an insight into the connexin channel function. PMID:18470322

  18. Activation of diverse signaling pathways by oncogenic PIK3CA mutations

    PubMed Central

    Wu, Xinyan; Renuse, Santosh; Sahasrabuddhe, Nandini A.; Zahari, Muhammad Saddiq; Chaerkady, Raghothama; Kim, Min-Sik; Nirujogi, Raja S.; Mohseni, Morassa; Kumar, Praveen; Raju, Rajesh; Zhong, Jun; Yang, Jian; Neiswinger, Johnathan; Jeong, Jun-Seop; Newman, Robert; Powers, Maureen A.; Somani, Babu Lal; Gabrielson, Edward; Sukumar, Saraswati; Stearns, Vered; Qian, Jiang; Zhu, Heng; Vogelstein, Bert; Park, Ben Ho; Pandey, Akhilesh

    2014-01-01

    The PIK3CA gene is frequently mutated in human cancers. Here we carry out a SILAC-based quantitative phosphoproteomic analysis using isogenic knockin cell lines containing ‘driver’ oncogenic mutations of PIK3CA to dissect the signaling mechanisms responsible for oncogenic phenotypes induced by mutant PIK3CA. From 8,075 unique phosphopeptides identified, we observe that aberrant activation of PI3K pathway leads to increased phosphorylation of a surprisingly wide variety of kinases and downstream signaling networks. Here, by integrating phosphoproteomic data with human protein microarray-based AKT1 kinase assays, we discover and validate six novel AKT1 substrates, including cortactin. Through mutagenesis studies, we demonstrate that phosphorylation of cortactin by AKT1 is important for mutant PI3K enhanced cell migration and invasion. Our study describes a quantitative and global approach for identifying mutation-specific signaling events and for discovering novel signaling molecules as readouts of pathway activation or potential therapeutic targets. PMID:25247763

  19. From the nephrologist's point of view: diversity of causes and clinical features of acute kidney injury

    PubMed Central

    Bienholz, Anja; Wilde, Benjamin; Kribben, Andreas

    2015-01-01

    Acute kidney injury (AKI) is a clinical syndrome with multiple entities. Although AKI implies renal damage, functional impairment or both, diagnosis is solely based on the functional parameters of serum creatinine and urine output. The latest definition was provided by the Kidney Disease Improving Global Outcomes (KDIGO) working group in 2012. Independent of the underlying disease, and even in the case of full recovery, AKI is associated with an increased morbidity and mortality. Awareness of the patient's individual risk profile and the diversity of causes and clinical features of AKI is pivotal for optimization of prophylaxes, diagnosis and therapy of each form of AKI. A differentiated and individualized approach is required to improve patient mortality, morbidity, long-term kidney function and eventually the quality of life. In this review, we provide an overview of the different clinical settings in which specific forms of AKI may occur and point out possible diagnostic as well as therapeutic approaches. Secifically AKI is discussed in the context of non-kidney organ failure, organ transplantation, sepsis, malignancy and autoimmune disease. PMID:26251707

  20. Building diversity in REU programs through MIMSUP at the Shannon Point Marine Center

    NASA Astrophysics Data System (ADS)

    Bingham, B. L.; Sulkin, S.

    2011-12-01

    The road to a career in the ocean sciences can be long and challenging, particularly for students from racial/ethnic groups underrepresented in the field. For the past 21 years, faculty and staff at the Shannon Point Marine Center, Western Washington University have annually administered the NSF-funded Multicultural Initiative in the Marine Sciences: Undergraduate Participation (MIMSUP) program. The goal of MIMSUP is to increase diversity in the ocean sciences by moving students though their undergraduate programs into advanced education and leadership positions in the field. Helping students find positions in REU and other focused research programs is an important step along this path. Primary obstacles for the students include 1) a lack of knowledge about opportunities available to them, 2) a lack of experience preparing quality applications and 3) a lack of confidence in their ability to compete for positions. Focused mentoring, with an emphasis on skills development is important in helping outstanding, though inexperienced, students find and excel in REU programs.

  1. Intrachromosomal Amplification, Locus Deletion and Point Mutation in the Aquaglyceroporin AQP1 Gene in Antimony Resistant Leishmania (Viannia) guyanensis

    PubMed Central

    Monte-Neto, Rubens; Laffitte, Marie-Claude N.; Leprohon, Philippe; Reis, Priscila; Frézard, Frédéric; Ouellette, Marc

    2015-01-01

    Background Antimony resistance complicates the treatment of infections caused by the parasite Leishmania. Methodology/Principal Findings Using next generation sequencing, we sequenced the genome of four independent Leishmania guyanensis antimony-resistant (SbR) mutants and found different chromosomal alterations including aneuploidy, intrachromosomal gene amplification and gene deletion. A segment covering 30 genes on chromosome 19 was amplified intrachromosomally in three of the four mutants. The gene coding for the multidrug resistance associated protein A involved in antimony resistance was also amplified in the four mutants, most likely through chromosomal translocation. All mutants also displayed a reduced accumulation of antimony mainly due to genomic alterations at the level of the subtelomeric region of chromosome 31 harboring the gene coding for the aquaglyceroporin 1 (LgAQP1). Resistance involved the loss of LgAQP1 through subtelomeric deletions in three mutants. Interestingly, the fourth mutant harbored a single G133D point mutation in LgAQP1 whose role in resistance was functionality confirmed through drug sensitivity and antimony accumulation assays. In contrast to the Leishmania subspecies that resort to extrachromosomal amplification, the Viannia strains studied here used intrachromosomal amplification and locus deletion. Conclusions/Significance This is the first report of a naturally occurred point mutation in AQP1 in antimony resistant parasites. PMID:25679388

  2. Splitting the difference: the germline-somatic mutation debate on generating antibody diversity.

    PubMed

    Silverstein, Arthur M

    2003-09-01

    In the debate about the mechanism for the generation of immunological diversity, the initial positions of both 'somaticists' and 'germliners' were diametrically opposed. Then, as data developed favoring first one and then the other side, concessions were made, until the final solution showed that each had been at least partially correct. PMID:12942083

  3. Genetic diversity estimates point to immediate efforts for conserving the endangered Tibetan sheep of India.

    PubMed

    Sharma, Rekha; Kumar, Brijesh; Arora, Reena; Ahlawat, Sonika; Mishra, A K; Tantia, M S

    2016-06-01

    Tibetan is a valuable Himalayan sheep breed classified as endangered. Knowledge of the level and distribution of genetic diversity in Tibetan sheep is important for designing conservation strategies for their sustainable survival and to preserve their evolutionary potential. Thus, for the first time, genetic variability in the Tibetan population was accessed with twenty five inter-simple sequence repeat markers. All the microsatellites were polymorphic and a total of 148 alleles were detected across these loci. The observed number of alleles across all the loci was more than the effective number of alleles and ranged from 3 (BM6506) to 11 (BM6526) with 5.920 ± 0.387 mean number of alleles per locus. The average observed heterozygosity was less than the expected heterozygosity. The observed and expected heterozygosity values ranged from 0.150 (BM1314) to 0.9 (OarCP20) with an overall mean of 0.473 ± 0.044 and from 0.329 (BM8125) to 0.885 (BM6526) with an overall mean 0.672 ± 0.030, respectively. The lower heterozygosity pointed towards diminished genetic diversity in the population. Thirteen microsatellite loci exhibited significant (P < 0.05) departures from the Hardy-Weinberg proportions in the population. The estimate of heterozygote deficiency varied from - 0.443 (OarCP20) to 0.668 (OarFCB128) with a mean positive value of 0.302 ± 0.057. A normal 'L' shaped distribution of mode-shift test and non-significant heterozygote excess on the basis of different models suggested absence of recent bottleneck in the existing Tibetan population. In view of the declining population of Tibetan sheep (less than 250) in the breeding tract, need of the hour is immediate scientific management of the population so as to increase the population hand in hand with retaining the founder alleles to the maximum possible extent. PMID:27014586

  4. Genetic diversity estimates point to immediate efforts for conserving the endangered Tibetan sheep of India

    PubMed Central

    Sharma, Rekha; Kumar, Brijesh; Arora, Reena; Ahlawat, Sonika; Mishra, A.K.; Tantia, M.S.

    2016-01-01

    Tibetan is a valuable Himalayan sheep breed classified as endangered. Knowledge of the level and distribution of genetic diversity in Tibetan sheep is important for designing conservation strategies for their sustainable survival and to preserve their evolutionary potential. Thus, for the first time, genetic variability in the Tibetan population was accessed with twenty five inter-simple sequence repeat markers. All the microsatellites were polymorphic and a total of 148 alleles were detected across these loci. The observed number of alleles across all the loci was more than the effective number of alleles and ranged from 3 (BM6506) to 11 (BM6526) with 5.920 ± 0.387 mean number of alleles per locus. The average observed heterozygosity was less than the expected heterozygosity. The observed and expected heterozygosity values ranged from 0.150 (BM1314) to 0.9 (OarCP20) with an overall mean of 0.473 ± 0.044 and from 0.329 (BM8125) to 0.885 (BM6526) with an overall mean 0.672 ± 0.030, respectively. The lower heterozygosity pointed towards diminished genetic diversity in the population. Thirteen microsatellite loci exhibited significant (P < 0.05) departures from the Hardy–Weinberg proportions in the population. The estimate of heterozygote deficiency varied from − 0.443 (OarCP20) to 0.668 (OarFCB128) with a mean positive value of 0.302 ± 0.057. A normal ‘L’ shaped distribution of mode-shift test and non-significant heterozygote excess on the basis of different models suggested absence of recent bottleneck in the existing Tibetan population. In view of the declining population of Tibetan sheep (less than 250) in the breeding tract, need of the hour is immediate scientific management of the population so as to increase the population hand in hand with retaining the founder alleles to the maximum possible extent. PMID:27014586

  5. A vertically-stacked, polymer, microfluidic point mutation analyzer: Rapid, high accuracy detection of low-abundance K-ras mutations

    PubMed Central

    Han, Kyudong; Lee, Tae Yoon; Nikitopoulos, Dimitris E.; Soper, Steven A.; Murphy, Michael C.

    2011-01-01

    Recognition of point mutations in the K-ras gene can be used for the clinical management of several types of cancers. Unfortunately, several assay and hardware concerns must be addressed to allow users not well-trained in performing molecular analyses the opportunity to undertake these measurements. To provide for a larger user-base for these types of molecular assays, a vertically-stacked microfluidic analyzer with a modular architecture and process automation was developed. The analyzer employed a primary PCR coupled to an allele-specific ligase detection reaction (LDR). Each functional device, including continuous flow thermal reactors for the PCR and LDR, passive micromixers and ExoSAP-IT® purification, was designed and tested. Individual devices were fabricated in polycarbonate using hot embossing and assembled using adhesive bonding for system assembly. The system produced LDR products from a DNA sample in ~1 h, an 80% reduction in time compared to conventional bench-top instrumentation. Purifying the post-PCR products with the ExoSAP-IT® enzyme led to optimized LDR performance minimizing false positive signals and producing reliable results. Mutant alleles in genomic DNA were quantified to the level of 0.25 ng of mutant DNA in 50 ng of wild-type DNA for a 25 μL sample, equivalent to DNA from 42 mutant cells. PMID:21771577

  6. A point mutation in the microtubule binding region of the Ncd motor protein reduces motor velocity.

    PubMed Central

    Moore, J D; Song, H; Endow, S A

    1996-01-01

    Non-claret disjunctional (Ncd) is a kinesin-related microtubule motor protein in Drosophila that functions in meiotic spindle assembly in oocytes and spindle pole maintenance in early embryos. The partial loss-of-function mutant ncdD retains mitotic, but not meiotic, function. The predicted NcdD mutant protein contains a V556-->F mutation in the putative microtubule binding region of the Ncd motor domain. Here we report an analysis of the properties of recombinant Ncd and NcdD proteins. A GST-NcdD fusion protein translocated microtubules approximately 10-fold more slowly than the corresponding wild-type protein in gliding assays. The maximum microtubule-stimulated ATPase activity of an NcdD motor domain protein was reduced approximately 3-fold and an approximately 3-fold greater concentration of microtubules was required for half-maximal stimulation of ATPase activity, compared with the corresponding wild-type protein. The Km for ATP and basal rate of ATP turnover were, in contrast, similar for the NcdD mutant and wild-type Ncd motor domain proteins. Pelleting assays demonstrated that the binding of the mutant NcdD motor protein to microtubules was reduced in the absence of nucleotide, relative to wild-type. The reduced velocity of NcdD translocation on microtubules is therefore correlated with reductions in microtubule-stimulated ATPase activity and affinity of the mutant motor for microtubules. The characteristics of the NcdD motor explain its meiotic loss of function, and are consistent with partial motor activity of Ncd being sufficient for its mitotic, but not its meiotic, role. Images PMID:8670831

  7. Extremely high genetic diversity in a single tumor points to prevalence of non-Darwinian cell evolution

    PubMed Central

    Ling, Shaoping; Hu, Zheng; Yang, Zuyu; Yang, Fang; Li, Yawei; Lin, Pei; Chen, Ke; Dong, Lili; Cao, Lihua; Tao, Yong; Hao, Lingtong; Chen, Qingjian; Gong, Qiang; Wu, Dafei; Li, Wenjie; Zhao, Wenming; Tian, Xiuyun; Hao, Chunyi; Hungate, Eric A.; Catenacci, Daniel V. T.; Hudson, Richard R.; Li, Wen-Hsiung; Lu, Xuemei; Wu, Chung-I

    2015-01-01

    The prevailing view that the evolution of cells in a tumor is driven by Darwinian selection has never been rigorously tested. Because selection greatly affects the level of intratumor genetic diversity, it is important to assess whether intratumor evolution follows the Darwinian or the non-Darwinian mode of evolution. To provide the statistical power, many regions in a single tumor need to be sampled and analyzed much more extensively than has been attempted in previous intratumor studies. Here, from a hepatocellular carcinoma (HCC) tumor, we evaluated multiregional samples from the tumor, using either whole-exome sequencing (WES) (n = 23 samples) or genotyping (n = 286) under both the infinite-site and infinite-allele models of population genetics. In addition to the many single-nucleotide variations (SNVs) present in all samples, there were 35 “polymorphic” SNVs among samples. High genetic diversity was evident as the 23 WES samples defined 20 unique cell clones. With all 286 samples genotyped, clonal diversity agreed well with the non-Darwinian model with no evidence of positive Darwinian selection. Under the non-Darwinian model, MALL (the number of coding region mutations in the entire tumor) was estimated to be greater than 100 million in this tumor. DNA sequences reveal local diversities in small patches of cells and validate the estimation. In contrast, the genetic diversity under a Darwinian model would generally be orders of magnitude smaller. Because the level of genetic diversity will have implications on therapeutic resistance, non-Darwinian evolution should be heeded in cancer treatments even for microscopic tumors. PMID:26561581

  8. Extremely high genetic diversity in a single tumor points to prevalence of non-Darwinian cell evolution.

    PubMed

    Ling, Shaoping; Hu, Zheng; Yang, Zuyu; Yang, Fang; Li, Yawei; Lin, Pei; Chen, Ke; Dong, Lili; Cao, Lihua; Tao, Yong; Hao, Lingtong; Chen, Qingjian; Gong, Qiang; Wu, Dafei; Li, Wenjie; Zhao, Wenming; Tian, Xiuyun; Hao, Chunyi; Hungate, Eric A; Catenacci, Daniel V T; Hudson, Richard R; Li, Wen-Hsiung; Lu, Xuemei; Wu, Chung-I

    2015-11-24

    The prevailing view that the evolution of cells in a tumor is driven by Darwinian selection has never been rigorously tested. Because selection greatly affects the level of intratumor genetic diversity, it is important to assess whether intratumor evolution follows the Darwinian or the non-Darwinian mode of evolution. To provide the statistical power, many regions in a single tumor need to be sampled and analyzed much more extensively than has been attempted in previous intratumor studies. Here, from a hepatocellular carcinoma (HCC) tumor, we evaluated multiregional samples from the tumor, using either whole-exome sequencing (WES) (n = 23 samples) or genotyping (n = 286) under both the infinite-site and infinite-allele models of population genetics. In addition to the many single-nucleotide variations (SNVs) present in all samples, there were 35 "polymorphic" SNVs among samples. High genetic diversity was evident as the 23 WES samples defined 20 unique cell clones. With all 286 samples genotyped, clonal diversity agreed well with the non-Darwinian model with no evidence of positive Darwinian selection. Under the non-Darwinian model, MALL (the number of coding region mutations in the entire tumor) was estimated to be greater than 100 million in this tumor. DNA sequences reveal local diversities in small patches of cells and validate the estimation. In contrast, the genetic diversity under a Darwinian model would generally be orders of magnitude smaller. Because the level of genetic diversity will have implications on therapeutic resistance, non-Darwinian evolution should be heeded in cancer treatments even for microscopic tumors. PMID:26561581

  9. Spectrum of BRCA1/2 point mutations and genomic rearrangements in high-risk breast/ovarian cancer Chilean families.

    PubMed

    Gonzalez-Hormazabal, Patricio; Gutierrez-Enriquez, Sara; Gaete, Daniel; Reyes, Jose M; Peralta, Octavio; Waugh, Enrique; Gomez, Fernando; Margarit, Sonia; Bravo, Teresa; Blanco, Rafael; Diez, Orland; Jara, Lilian

    2011-04-01

    The distribution of BRCA1/2 germline mutations in breast/ovarian cancer (BC/OC) families varies among different populations. In the Chilean population, there are only two reports of mutation analysis of BRCA1/2, and these included a low number of BC and/or OC patients. Moreover, the prevalence of BRCA1/2 genomic rearrangements in Chilean and in other South American populations is unknown. In this article, we present the mutation-detection data corresponding to a set of 326 high-risk families analyzed by conformation-sensitive gel electrophoresis and heteroduplex analysis. To determine the contribution of BRCA1/2 LGRs in Chilean BC patients, we analyzed 56 high-risk subjects with no pathogenic BRCA1/2 point mutations. Germline BRCA1/2 point mutations were found in 23 (7.1%) of the 326 Chilean families. Families which had at least three BC and/or OC cases showed the highest frequency of mutations (15.9%). We identified 14 point pathogenic mutations. Three recurrent mutations in BRCA1 (c.187_188delAG, c.2605_2606delTT, and c.3450_3453delCAAG) and three in BRCA2 (c.4969_4970insTG, c.5374_5377delTATG, and c.6503_6504delTT) contributed to 63.6 and 66.7% of all the deleterious mutations of each gene, which may reflect the presence of region-specific founder effects. Taken together BRCA1/2 recurrent point mutations account for 65.2% (15/23) of the BRCA1/2 (+) families. No large deletions or duplications involving BRCA1/2 were identified in a subgroup of 56 index cases negative for BRCA1/2 point mutations. Our study, which is the largest conducted to date in a South American population, provides a comprehensive analysis on the type and distribution of BRCA1/2 mutations and allelic variants. PMID:20859677

  10. Predicting protein thermal stability changes upon point mutations using statistical potentials: Introducing HoTMuSiC

    PubMed Central

    Pucci, Fabrizio; Bourgeas, Raphaël; Rooman, Marianne

    2016-01-01

    The accurate prediction of the impact of an amino acid substitution on the thermal stability of a protein is a central issue in protein science, and is of key relevance for the rational optimization of various bioprocesses that use enzymes in unusual conditions. Here we present one of the first computational tools to predict the change in melting temperature ΔTm upon point mutations, given the protein structure and, when available, the melting temperature Tm of the wild-type protein. The key ingredients of our model structure are standard and temperature-dependent statistical potentials, which are combined with the help of an artificial neural network. The model structure was chosen on the basis of a detailed thermodynamic analysis of the system. The parameters of the model were identified on a set of more than 1,600 mutations with experimentally measured ΔTm. The performance of our method was tested using a strict 5-fold cross-validation procedure, and was found to be significantly superior to that of competing methods. We obtained a root mean square deviation between predicted and experimental ΔTm values of 4.2 °C that reduces to 2.9 °C when ten percent outliers are removed. A webserver-based tool is freely available for non-commercial use at soft.dezyme.com. PMID:26988870

  11. Understanding the lid movements of LolA in Escherichia coli using molecular dynamics simulation and in silico point mutation.

    PubMed

    Murahari, Priyadarshini; Anishetty, Sharmila; Pennathur, Gautam

    2013-12-01

    The Lol system in Escherichia coli is involved in localization of lipoproteins and hence is essential for growth of the organism. LolA is a periplasmic chaperone that binds to outer-membrane specific lipoproteins and transports them from inner membrane to outer membrane through LolB. The hydrophobic lipid-binding cavity of LolA consists of α-helices which act as a lid in regulating the transfer of lipoproteins from LolA to LolB. The current study aims to investigate the structural changes observed in LolA during the transition from open to closed conformation in the absence of lipoprotein. Molecular dynamics (MD) simulations were carried out for two LolA crystal structures; LolA(R43L), and in silico mutated MsL43R for a simulation time of 50 ns in water environment. We have performed an in silico point mutation of leucine to arginine in MsL43R to evaluate the importance of arginine to induce structural changes and impact the stability of protein structure. A complete dynamic analysis of open to closed conformation reveals the existence of two distinct levels; closing of lid and closing of entrance of hydrophobic cavity. Our analysis reveals that the structural flexibility of LolA is an important factor for its role as a periplasmic chaperone. PMID:23962984

  12. Identification of point mutations and large intragenic deletions in Fanconi anemia using next-generation sequencing technology.

    PubMed

    Nicchia, Elena; Greco, Chiara; De Rocco, Daniela; Pecile, Vanna; D'Eustacchio, Angela; Cappelli, Enrico; Corti, Paola; Marra, Nicoletta; Ramenghi, Ugo; Pillon, Marta; Farruggia, Piero; Dufour, Carlo; Pallavicini, Alberto; Torelli, Lucio; Savoia, Anna

    2015-11-01

    Fanconi anemia (FA) is a rare bone marrow failure disorder characterized by clinical and genetic heterogeneity with at least 17 genes involved, which make molecular diagnosis complex and time-consuming. Since next-generation sequencing technologies could greatly improve the genetic testing in FA, we sequenced DNA samples with known and unknown mutant alleles using the Ion PGM (™) system (IPGM). The molecular target of 74.2 kb in size covered 96% of the FA-coding exons and their flanking regions. Quality control testing revealed high coverage. Comparing the IPGM and Sanger sequencing output of FANCA,FANCC, and FANCG we found no false-positive and a few false-negative variants, which led to high sensitivity (95.58%) and specificity (100%) at least for these two most frequently mutated genes. The analysis also identified novel mutant alleles, including those in rare complementation groups FANCF and FANCL. Moreover, quantitative evaluation allowed us to characterize large intragenic deletions of FANCA and FANCD2, suggesting that IPGM is suitable for identification of not only point mutations but also copy number variations. PMID:26740942

  13. A unique point mutation in the fibroblast growth factor receptor 3 gene (FGFR3) defines a new craniosynostosis syndrome

    SciTech Connect

    Muenke, M.; Gripp, K.W.; McDonald-McGinn, D.M.

    1997-03-01

    The underlying basis of many forms of syndromic craniosynostosis has been defined on a molecular level. However, many patients with familial or sporadic craniosynostosis do not have the classical findings of those craniosynostosis syndromes. Here we present 61 individuals from 20 unrelated families where coronal synostosis is due to an amino acid substitution (Pro250Arg) that results from a single point mutation in the fibroblast growth factor receptor 3 gene on chromosome 4p. In this instance, a new clinical syndrome is being defined on the basis of the molecular finding. In addition to the skull findings, some patients had abnormalities on radiographs of hands and feet, including thimble-like middle phalanges, coned epiphyses, and carpal and tarsal fusions. Brachydactyly was seen in some cases; none had clinically significant syndactyly or deviation of the great toe. Sensorineural hearing loss was present in some, and developmental delay was seen in a minority. While the radiological findings of hands and feet can be very helpful in diagnosing this syndrome, it is not in all cases clearly distinguishable on a clinical basis from other craniosynostosis syndromes. Therefore, this mutation should be tested for in patients with coronal synostosis. 54 refs., 4 figs., 2 tabs.

  14. Modulating non-native aggregation and electrostatic protein-protein interactions with computationally designed single-point mutations.

    PubMed

    O'Brien, C J; Blanco, M A; Costanzo, J A; Enterline, M; Fernandez, E J; Robinson, A S; Roberts, C J

    2016-06-01

    Non-native protein aggregation is a ubiquitous challenge in the production, storage and administration of protein-based biotherapeutics. This study focuses on altering electrostatic protein-protein interactions as a strategy to modulate aggregation propensity in terms of temperature-dependent aggregation rates, using single-charge variants of human γ-D crystallin. Molecular models were combined to predict amino acid substitutions that would modulate protein-protein interactions with minimal effects on conformational stability. Experimental protein-protein interactions were quantified by the Kirkwood-Buff integrals (G22) from laser scattering, and G22 showed semi-quantitative agreement with model predictions. Experimental initial-rates for aggregation showed that increased (decreased) repulsive interactions led to significantly increased (decreased) aggregation resistance, even based solely on single-point mutations. However, in the case of a particular amino acid (E17), the aggregation mechanism was altered by substitution with R or K, and this greatly mitigated improvements in aggregation resistance. The results illustrate that predictions based on native protein-protein interactions can provide a useful design target for engineering aggregation resistance; however, this approach needs to be balanced with consideration of how mutations can impact aggregation mechanisms. PMID:27160179

  15. Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus.

    PubMed Central

    Krempl, C; Schultze, B; Laude, H; Herrler, G

    1997-01-01

    Enteropathogenic transmissible gastroenteritis virus (TGEV), a porcine coronavirus, is able to agglutinate erythrocytes because of sialic acid binding activity. Competitive inhibitors that may mask the sialic acid binding activity can be inactivated by sialidase treatment of virions. Here, we show that TGEV virions with efficient hemagglutinating activity were also obtained when cells were treated with sialidase prior to infection. This method was used to analyze TGEV mutants for hemagglutinating activity. Recently, mutants with strongly reduced enteropathogenicity that have point mutations or a deletion of four amino acids within residues 145 to 155 of the S protein have been described. Here, we show that in addition to their reduced pathogenicity, these mutants also have lost hemagglutinating activity. These results connect sialic acid binding activity with the enteropathogenicity of TGEV. PMID:9060696

  16. Functional analysis of linker insertions and point mutations in the alpha-Amy2/54 GA-regulated promoter.

    PubMed

    Tregear, J W; Primavesi, L F; Huttly, A K

    1995-11-01

    Functional analysis of a gibberellin-regulated wheat alpha-amylase promoter, alpha-Amy2/54, has indicated that three regions were essential for expression. By studying the ability of mutant promoters, containing a randomly inserted 22 bp excision linker, to direct expression in oat aleurone protoplasts we have refined the positions and extents of these three cis elements and also demonstrated the presence of two additional elements. By converting the linker insertions to either single base point mutations or deletions using the class IIS restriction endonuclease BsmI we have shown that nucleotides -119 and -109 within the GARE -121GTAACAGAGTCTGG-108 and nucleotide -152 within the proposed element -156GATTGACTTGACC-144 are essential for high level expression from this promoter. PMID:8541501

  17. Point Mutations in Exon 1B of APC Reveal Gastric Adenocarcinoma and Proximal Polyposis of the Stomach as a Familial Adenomatous Polyposis Variant.

    PubMed

    Li, Jun; Woods, Susan L; Healey, Sue; Beesley, Jonathan; Chen, Xiaoqing; Lee, Jason S; Sivakumaran, Haran; Wayte, Nicci; Nones, Katia; Waterfall, Joshua J; Pearson, John; Patch, Anne-Marie; Senz, Janine; Ferreira, Manuel A; Kaurah, Pardeep; Mackenzie, Robertson; Heravi-Moussavi, Alireza; Hansford, Samantha; Lannagan, Tamsin R M; Spurdle, Amanda B; Simpson, Peter T; da Silva, Leonard; Lakhani, Sunil R; Clouston, Andrew D; Bettington, Mark; Grimpen, Florian; Busuttil, Rita A; Di Costanzo, Natasha; Boussioutas, Alex; Jeanjean, Marie; Chong, George; Fabre, Aurélie; Olschwang, Sylviane; Faulkner, Geoffrey J; Bellos, Evangelos; Coin, Lachlan; Rioux, Kevin; Bathe, Oliver F; Wen, Xiaogang; Martin, Hilary C; Neklason, Deborah W; Davis, Sean R; Walker, Robert L; Calzone, Kathleen A; Avital, Itzhak; Heller, Theo; Koh, Christopher; Pineda, Marbin; Rudloff, Udo; Quezado, Martha; Pichurin, Pavel N; Hulick, Peter J; Weissman, Scott M; Newlin, Anna; Rubinstein, Wendy S; Sampson, Jone E; Hamman, Kelly; Goldgar, David; Poplawski, Nicola; Phillips, Kerry; Schofield, Lyn; Armstrong, Jacqueline; Kiraly-Borri, Cathy; Suthers, Graeme K; Huntsman, David G; Foulkes, William D; Carneiro, Fatima; Lindor, Noralane M; Edwards, Stacey L; French, Juliet D; Waddell, Nicola; Meltzer, Paul S; Worthley, Daniel L; Schrader, Kasmintan A; Chenevix-Trench, Georgia

    2016-05-01

    Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present. PMID:27087319

  18. Mutations in cardiac T-box factor gene TBX20 are associated with diverse cardiac pathologies, including defects of septation and valvulogenesis and cardiomyopathy.

    PubMed

    Kirk, Edwin P; Sunde, Margaret; Costa, Mauro W; Rankin, Scott A; Wolstein, Orit; Castro, M Leticia; Butler, Tanya L; Hyun, Changbaig; Guo, Guanglan; Otway, Robyn; Mackay, Joel P; Waddell, Leigh B; Cole, Andrew D; Hayward, Christopher; Keogh, Anne; Macdonald, Peter; Griffiths, Lyn; Fatkin, Diane; Sholler, Gary F; Zorn, Aaron M; Feneley, Michael P; Winlaw, David S; Harvey, Richard P

    2007-08-01

    The T-box family transcription factor gene TBX20 acts in a conserved regulatory network, guiding heart formation and patterning in diverse species. Mouse Tbx20 is expressed in cardiac progenitor cells, differentiating cardiomyocytes, and developing valvular tissue, and its deletion or RNA interference-mediated knockdown is catastrophic for heart development. TBX20 interacts physically, functionally, and genetically with other cardiac transcription factors, including NKX2-5, GATA4, and TBX5, mutations of which cause congenital heart disease (CHD). Here, we report nonsense (Q195X) and missense (I152M) germline mutations within the T-box DNA-binding domain of human TBX20 that were associated with a family history of CHD and a complex spectrum of developmental anomalies, including defects in septation, chamber growth, and valvulogenesis. Biophysical characterization of wild-type and mutant proteins indicated how the missense mutation disrupts the structure and function of the TBX20 T-box. Dilated cardiomyopathy was a feature of the TBX20 mutant phenotype in humans and mice, suggesting that mutations in developmental transcription factors can provide a sensitized template for adult-onset heart disease. Our findings are the first to link TBX20 mutations to human pathology. They provide insights into how mutation of different genes in an interactive regulatory circuit lead to diverse clinical phenotypes, with implications for diagnosis, genetic screening, and patient follow-up. PMID:17668378

  19. Locus control region HS2 point mutations are generally not responsible for elevated fetal hemoglobin expression of sickle cell patients

    SciTech Connect

    Gilman, J.G.

    1994-09-01

    The locus control region (LCR), composed of four hypersensitive sites (HS1-4) 5{prime} of the {epsilon} globin gene, confers strong, copy-number dependent expression on globin genes in transgenic mice. Several {beta}-globin gene cluster haplotypes carry the sickle cell gene, and show variable levels of fetal hemoglobin (Hb F) expression in association with DNA sequence differences in HS2, {gamma} and {beta} globin promoters, and {gamma}IVSII: The Senegal (SEN or No. 3) haplotype generally has high (>10%) Hb F, Benin (BEN or No. 19) has intermediate Hb F (but some low and some high), and Banu (BAN or No. 20) generally has low Hb F. Huisman and colleagues have proposed that `factors produced under conditions of hematopoietic stress, together with genetic determinants on the haplotype-3 like LCR sequences, allow for high level expression of {gamma} globin genes`. We have now used slot blot to screen high Hb F (>9.5%) and low Hb F cases for two of the three HS2 point mutations described by Oener et al. Comparing eight high Hb F BEN/BEN with two low Hb F BEN/BEN, all ten had the BEN mutations considered by Oener et al. to be associated with low Hb F. Comparing three high Hb F BEN/BAN with two low Hb F BEN/BAN, all five were heterozygous at three positions; this is consistent with BEN having G and T and BAN having A at both positions. DNA sequencing of HS2 for BAN, which is generally associated with low HB F, showed that the point mutations at all three positions were those seen in SEN (generally high Hb F); only the AT repeat region showed major differences, confirming results of Huisman and colleagues. Hence, if there is any effect of HS2 of the Senegal sickle cell haplotype in causing elevated Hb F under hematopoietic stress, it must be due to specific variation in the AT repeat region, which Oener et al. have suggested may bind a silencer.

  20. Mediterranean Founder Mutation Database (MFMD): Taking Advantage from Founder Mutations in Genetics Diagnosis, Genetic Diversity and Migration History of the Mediterranean Population.

    PubMed

    Charoute, Hicham; Bakhchane, Amina; Benrahma, Houda; Romdhane, Lilia; Gabi, Khalid; Rouba, Hassan; Fakiri, Malika; Abdelhak, Sonia; Lenaers, Guy; Barakat, Abdelhamid

    2015-11-01

    The Mediterranean basin has been the theater of migration crossroads followed by settlement of several societies and cultures in prehistoric and historical times, with important consequences on genetic and genomic determinisms. Here, we present the Mediterranean Founder Mutation Database (MFMD), established to offer web-based access to founder mutation information in the Mediterranean population. Mutation data were collected from the literature and other online resources and systematically reviewed and assembled into this database. The information provided for each founder mutation includes DNA change, amino-acid change, mutation type and mutation effect, as well as mutation frequency and coalescence time when available. Currently, the database contains 383 founder mutations found in 210 genes related to 219 diseases. We believe that MFMD will help scientists and physicians to design more rapid and less expensive genetic diagnostic tests. Moreover, the coalescence time of founder mutations gives an overview about the migration history of the Mediterranean population. MFMD can be publicly accessed from http://mfmd.pasteur.ma. PMID:26173767

  1. Structural analysis of point mutations at the Vaccinia virus A20/D4 interface.

    PubMed

    Contesto-Richefeu, Céline; Tarbouriech, Nicolas; Brazzolotto, Xavier; Burmeister, Wim P; Peyrefitte, Christophe N; Iseni, Frédéric

    2016-09-01

    The Vaccinia virus polymerase holoenzyme is composed of three subunits: E9, the catalytic DNA polymerase subunit; D4, a uracil-DNA glycosylase; and A20, a protein with no known enzymatic activity. The D4/A20 heterodimer is the DNA polymerase cofactor, the function of which is essential for processive DNA synthesis. The recent crystal structure of D4 bound to the first 50 amino acids of A20 (D4/A201-50) revealed the importance of three residues, forming a cation-π interaction at the dimerization interface, for complex formation. These are Arg167 and Pro173 of D4 and Trp43 of A20. Here, the crystal structures of the three mutants D4-R167A/A201-50, D4-P173G/A201-50 and D4/A201-50-W43A are presented. The D4/A20 interface of the three structures has been analysed for atomic solvation parameters and cation-π interactions. This study confirms previous biochemical data and also points out the importance for stability of the restrained conformational space of Pro173. Moreover, these new structures will be useful for the design and rational improvement of known molecules targeting the D4/A20 interface. PMID:27599859

  2. A Point Mutation in Myh10 Causes Major Defects in Heart Development and Body Wall Closure

    PubMed Central

    Ma, Xuefei; Adelstein, Robert S.

    2014-01-01

    Background The three isoforms of nonmuscle myosin II (NMII-A, NMII-B and NMII-C) play various roles during mouse embryonic development. Previous work, using knockout and hypomorphic mice, showed that MYH10 encoding myosin heavy chain II-B is critical for cardiac and brain development. Ablating or decreasing NMII-B by 80% results in cardiac (ventricular septal defect, double outlet of the right ventricle) and brain defects but not midline fusion defects. Neither NMII-A nor II-C appear to play roles in early myocardial development. Methods and Results We had previously generated point mutant knock-in mice and now report novel findings due to expressing motor deficient NMII-B at wild-type levels. Homozygous mice die at E14.5 in cardiac failure exhibiting abnormalities not seen in NMII-B null and hypomorphic mice: a failure in midline fusion resulting in a cleft palate, ectopia cordis, and a large omphalocele. Fusion of the sternum and endocardial cushions is impaired in the mutant mice associated with a failure in apoptosis of the mesenchyme cells. Failure to disassemble myocyte cell-cell adhesions during cardiac outflow tract development contributes to impaired outflow tract myocardialization and displacement of the aorta to the right ventricle. Conclusions Expression of motor impaired NMII-B disrupts normal ventral body wall closure, due to a dominant negative effect. This is not due to the loss of NMII-B function but rather to a gain-of-function resulting from prolonged crosslinking of NMII-B to actin-filaments thereby interfering with the dynamics of actomyosin cytoskeletal structure. Moreover impaired NMII-B motor activity inhibits outflow tract myocardialization leading to mis-localization of the aorta. PMID:24825879

  3. Spliceosomal gene mutations are frequent events in the diverse mutational spectrum of chronic myelomonocytic leukemia but largely absent in juvenile myelomonocytic leukemia

    PubMed Central

    Kar, Sarah Abu; Jankowska, Anna; Makishima, Hideki; Visconte, Valeria; Jerez, Andres; Sugimoto, Yuka; Muramatsu, Hideki; Traina, Fabiola; Afable, Manuel; Guinta, Kathryn; Tiu, Ramon V.; Przychodzen, Bartlomiej; Sakaguchi, Hirotoshi; Kojima, Seiji; Sekeres, Mikkael A.; List, Alan F.; McDevitt, Michael A.; Maciejewski, Jaroslaw P.

    2013-01-01

    Chronic myelomonocytic leukemia is a heterogeneous disease with multifactorial molecular pathogenesis. Various recurrent somatic mutations have been detected alone or in combination in chronic myelomonocytic leukemia. Recently, recurrent mutations in spliceosomal genes have been discovered. We investigated the contribution of U2AF1, SRSF2 and SF3B1 mutations in the pathogenesis of chronic myelomonocytic leukemia and closely related diseases. We genotyped a cohort of patients with chronic myelomonocytic leukemia, secondary acute myeloid leukemia derived from chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia for somatic mutations in U2AF1, SRSF2, SF3B1 and in the other 12 most frequently affected genes in these conditions. Chromosomal abnormalities were assessed by nucleotide polymorphism array-based karyotyping. The presence of molecular lesions was correlated with clinical endpoints. Mutations in SRSF2, U2AF1 and SF3B1 were found in 32%, 13% and 6% of cases of chronic myelomonocytic leukemia, secondary acute myeloid leukemia derived from chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia, respectively. Spliceosomal genes were affected in various combinations with other mutations, including TET2, ASXL1, CBL, EZH2, RAS, IDH1/2, DNMT3A, TP53, UTX and RUNX1. Worse overall survival was associated with mutations in U2AF1 (P=0.047) and DNMT3A (P=0.015). RAS mutations had an impact on overall survival in secondary acute myeloid leukemia (P=0.0456). By comparison, our screening of juvenile myelomonocytic leukemia cases showed mutations in ASXL1 (4%), CBL (10%), and RAS (6%) but not in IDH1/2, TET2, EZH2, DNMT3A or the three spliceosomal genes. SRSF2 and U2AF1 along with TET2 (48%) and ASXL1 (38%) are frequently affected by somatic mutations in chronic myelomonocytic leukemia, quite distinctly from the profile seen in juvenile myelomonocytic leukemia. Our data also suggest that spliceosomal mutations are of ancestral origin. PMID:22773603

  4. Myeloma Ig heavy chain V region sequences reveal prior antigenic selection and marked somatic mutation but no intraclonal diversity

    SciTech Connect

    Vescio, R.A.; Cao, J.; Hong, C.H.

    1995-09-01

    The IgV{sub H} region sequence in 48 patients with multiple myeloma (MM) was analyzed to characterize the malignant cell of origin. The sequences were obtained after amplification of bone marrow cDNA by using V{sub H} family-specific and C{sub H} primers, then compared with either directly sequenced patient germ-line or published V{sub H} gene sequences to assay for somatic mutation. Because somatic hypermutation of the V{sub H} gene occurs late in B cell development, its presence has been helpful in determining the cell of origin in other B cell malignancies. Overall, a median of 8.2% of the nucleotides had evidence of substitution within each V{sub H} gene sequence (range = 2.7% to 16.5%), which is more prevalent than in any other reported tumor type. Strong evidence of prior antigenic selection pressure was also evident. The ratio of nucleotide substitutions that resulted in amino acid replacement was significantly higher in the complementarity-determining region than in the framework region (3.25 vs. 1.56, respectively; p < 0.00005). No V{sub H} gene intraclonal diversity was noted, despite sequencing multiple clones (3-16) from each patient, nor was there evidence of further V{sub H} gene somatic mutation over the course of three patients` disease. These findings strongly imply that the malignant clone in MM evolves from a cell late in B cell development. 63 refs., 4 figs., 2 tabs.

  5. Assessing the Metabolic Diversity of Streptococcus from a Protein Domain Point of View

    PubMed Central

    Koehorst, Jasper J.; Martins dos Santos, Vitor A. P.; Schaap, Peter J.

    2015-01-01

    Understanding the diversity and robustness of the metabolism of bacteria is fundamental for understanding how bacteria evolve and adapt to different environments. In this study, we characterised 121 Streptococcus strains and studied metabolic diversity from a protein domain perspective. Metabolic pathways were described in terms of the promiscuity of domains participating in metabolic pathways that were inferred to be functional. Promiscuity was defined by adapting existing measures based on domain abundance and versatility. The approach proved to be successful in capturing bacterial metabolic flexibility and species diversity, indicating that it can be described in terms of reuse and sharing functional domains in different proteins involved in metabolic activity. Additionally, we showed striking differences among metabolic organisation of the pathogenic serotype 2 Streptococcus suis and other strains. PMID:26366735

  6. Discovery of Point Mutations in the Voltage-Gated Sodium Channel from African Aedes aegypti Populations: Potential Phylogenetic Reasons for Gene Introgression

    PubMed Central

    Muranami, Yuto; Kawashima, Emiko; Osei, Joseph H. N.; Sakyi, Kojo Yirenkyi; Dadzie, Samuel; de Souza, Dziedzom K.; Appawu, Maxwell; Ohta, Nobuo; Minakawa, Noboru

    2016-01-01

    Background Yellow fever is endemic in some countries in Africa, and Aedes aegpyti is one of the most important vectors implicated in the outbreak. The mapping of the nation-wide distribution and the detection of insecticide resistance of vector mosquitoes will provide the beneficial information for forecasting of dengue and yellow fever outbreaks and effective control measures. Methodology/Principal Findings High resistance to DDT was observed in all mosquito colonies collected in Ghana. The resistance and the possible existence of resistance or tolerance to permethrin were suspected in some colonies. High frequencies of point mutations at the voltage-gated sodium channel (F1534C) and one heterozygote of the other mutation (V1016I) were detected, and this is the first detection on the African continent. The frequency of F1534C allele and the ratio of F1534C homozygotes in Ae. aegypti aegypti (Aaa) were significantly higher than those in Ae. aegypti formosus (Aaf). We could detect the two types of introns between exon 20 and 21, and the F1534C mutations were strongly linked with one type of intron, which was commonly found in South East Asian and South and Central American countries, suggesting the possibility that this mutation was introduced from other continents or convergently selected after the introgression of Aaa genes from the above area. Conclusions/Significance The worldwide eradication programs in 1940s and 1950s might have caused high selection pressure on the mosquito populations and expanded the distribution of insecticide-resistant Ae. aegypti populations. Selection of the F1534C point mutation could be hypothesized to have taken place during this period. The selection of the resistant population of Ae. aegypti with the point mutation of F1534C, and the worldwide transportation of vector mosquitoes in accordance with human activity such as trading of used tires, might result in the widespread distribution of F1534C point mutation in tropical countries

  7. Activation of the proto-oncogene p60c-src by point mutations in the SH2 domain.

    PubMed Central

    O'Brien, M C; Fukui, Y; Hanafusa, H

    1990-01-01

    To investigate the importance of a conserved region spanning residues 137 to 241 in the noncatalytic domain of p60c-src (SH2 region), we used oligonucleotide-directed mutagenesis to change residues that are highly conserved in this region. Chicken embryo fibroblasts infected with a p60c-src variant containing arginine instead of tryptophan at residue 148 (W148R) appeared more rounded than cells overexpressing a normal c-src gene, and they formed colonies in soft agar. p60c-src variants containing serine instead of arginine at residue 155 (R155S) or isoleucine instead of glycine at residue 170 (G170I) also appeared transformed and were anchorage independent, but to a lesser extent than W148R. Mutation of residue 201 from histidine to leucine (H201L) had no observable effect. The in vitro kinase activity of cells infected with W148R or G170I was elevated twofold. Expression of p60W148R (or, to a lesser extent, of p60G170I) increased the number of proteins phosphorylated on tyrosine in infected cells. All of the mutants were phosphorylated in vivo on Tyr-527, instead of Tyr-416 as observed for p60v-src. Immunoprecipitated p60W148R and p60G170I were found to be associated with a phosphatidylinositol kinase activity, a factor which appears to be necessary for transformation by tyrosine-specific protein kinases. These results show that a single point mutation in the SH2 region of the cellular src gene can activate its transforming potential. This type of activation is in a new category of alterations at the amino terminus that activate but do not cause a shift in phosphorylation at the carboxy terminus. Images PMID:2111444

  8. Anti-GD(2) with an FC point mutation reduces complement fixation and decreases antibody-induced allodynia.

    PubMed

    Sorkin, Linda S; Otto, Mario; Baldwin, William M; Vail, Emily; Gillies, Stephen D; Handgretinger, Rupert; Barfield, Raymond C; Ming Yu, Hui; Yu, Alice L

    2010-04-01

    Monoclonal antibodies against GD(2) ganglioside, such as ch14.18, the human-mouse chimeric antibody, have been shown to be effective for the treatment of neuroblastoma. However, treatment is associated with generalized, relatively opiate-resistant pain. We investigated if a point mutation in ch14.18 antibody (hu14.18K332A) to limit complement-dependent cytotoxicity (CDC) would ameliorate the pain behavior, while preserving antibody-dependent cellular cytotoxicity (ADCC). In vitro, CDC and ADCC were measured using europium-TDA assay. In vivo, allodynia was evaluated by measuring thresholds to von Frey filaments applied to the hindpaws after injection of either ch14.18 or hu14.18K332 into wild type rats or rats with deficient complement factor 6. Other rats were pretreated with complement factor C5a receptor antagonist and tested following ch14.18 injection. The mutation reduces the antibody's ability to activate complement, while maintaining its ADCC capabilities. Injection of hu14.18K322 (1 or 3mg/kg) produced faster resolving allodynia than that engendered by ch14.18 (1mg/kg). Injection of ch14.18 (1mg/kg) into rats with C6 complement deficiency further reduced antibody-induced allodynia, while pre-treatment with complement factor C5a receptor antagonist completely abolished ch14.18-induced allodynia. These findings showed that mutant hu14.18 K322 elicited less allodynia than ch14.18 and that ch14.18-elicited allodynia is due to activation of the complement cascade: in part, to formation of membrane attack complex, but more importantly to release of complement factor C5a. Development of immunotherapeutic agents with decreased complement-dependent lysis while maintaining cellular cytotoxicity may offer treatment options with reduced adverse side effects, thereby allowing dose escalation of therapeutic antibodies. PMID:20171010

  9. A genomic point mutation in the extracellular domain of the thyrotropin receptor in patients with Graves` ophthalmopathy

    SciTech Connect

    Bahn, R.S.; Dutton, C.M.; Heufelder, A.E.; Sarkar, G. |

    1994-02-01

    Orbital and pretibial fibroblasts are targets of autoimmune attack in Graves` ophthalmopathy (GO) and pretibial dermopathy (PTD). The fibroblast autoantigen involved in these peripheral manifestations of Graves` disease and the reason for the association of GO and PTD with hyperthyroidism are unknown. RNA encoding the full-length extracellular domain of the TSH receptor has been demonstrated in orbital and dermal fibroblasts from patients with GO and normal subjects, suggesting a possible antigenic link between fibroblasts and thyrocytes. RNA was isolated from cultured orbital, pretibial, and abdominal fibroblasts obtained from patients with severe GO (n = 22) and normal subjects (n = 5). RNA was reverse transcribed, and the resulting cDNA was amplified by the polymerase chain reaction, using primers spanning overlapping regions of the entire extracellular domain of the TSH receptor. Nucleotide sequence analysis showed an A for C substitution in the first position of codon 52 in 2 of the patients, both of whom had GO, PTD, and acropachy. Genomic DNA isolated from the 2 affected patients, and not from an additional 12 normal subjects, revealed the codon 52 mutation by direct sequencing and AciI restriction enzyme digestions. In conclusion, the authors have demonstrated the presence of a genomic point mutation, leading to a threonine for proline amino acid shift in the predicted peptide, in the extracellular domain of the TSH receptor in two patients with severe GO, PTD, acropachy, and high thyroid-stimulating immunoglobulin levels. RNA encoding this mutant product was demonstrated in the fibroblasts of these patients. They suggest that the TSH receptor may be an important fibroblast autoantigen in GO and PTD, and that this mutant form of the receptor may have unique immunogenic properties. 28 refs., 3 figs., 2 tabs.

  10. A point mutation in Euglene gracilis chloroplast tRNA{sup Glu} uncouples protein and chlorophyll biosynthesis

    SciTech Connect

    Stange-Thomann, N.; Thomann, H.U.; Lloyd, A.J.; Soell, D.; Lyman, H.

    1994-08-16

    The universal precursor of tetrapyrrole pigments (e.g., chlorophylls and hemes) is 5-aminolevulinic acid (ALA), which in Euglena gracilis chloroplasts is derived via the two-step C{sub 5} pathway from glutamate charged to tRNA{sup Glu}. The first enzyme in this pathway, Glu-tRNA reductase (GluTR) catalyzes the reduction of glutamyl-tRNA{sup Glu} (Glu-tRNA) to glutamate 1-semialdehyde (GSA) with the release of the uncharged tRNA{sup Glu}. The second enzyme, GSA-2, 1-aminomutase, converts GSA to ALA. tRNA{sup Glu} is a specific cofactor for the NADPH-dependent reduction by GluTR, an enzyme that recognizes the tRNA in a sequence-specific manner. This RNA is the normal tRNA{sup Glu}, a dual-function molecule participating both in protein and in ALA and, hence, chlorophyll biosynthesis. A chlorophyll-deficient mutant of E. gracilis (Y{sub 9}ZNaIL) does not synthesize ALA from glutamate, although it contains GluTR and GSA-2,1-aminomutase activity. The tRNA{sup Glu} isolated from the mutant can still be acrylated with glutamate in vitro and in vitro. Furthermore, it supports chloroplast protein synthesis; however, it is a poor substrate for GluTR. Sequence analysis of the tRNA and of its gene revealed a C56 {yields} U mutation in the resulting gene product. C56 is therefore an important identity element for GluTR. Thus, a point mutation in the T loop of tRNA uncouples protein from chlorophyll biosynthesis.

  11. Characterization of point mutations in the cdtA gene of the cytolethal distending toxin of Actinobacillus actinomycetemcomitans

    PubMed Central

    Cao, Linsen; Volgina, Alla; Huang, Chuang-ming; Korostoff, Jonathan; DiRienzo, Joseph M.

    2006-01-01

    Summary The Cdt is a family of gram-negative bacterial toxins that typically arrest eukaryotic cells in the G0/G1 or G2/M phase of the cell cycle. The toxin is a heterotrimer composed of the cdtA, cdtB and cdtC gene products. Although it has been shown that the CdtA protein subunit binds to cells in culture and in an enzyme-linked immunosorbent assay (CELISA) the precise mechanisms by which CdtA interacts with CdtB and CdtC has not yet been clarified. In this study we employed a random mutagenesis strategy to construct a library of point mutations in cdtA to assess the contribution of individual amino acids to binding activity and to the ability of the subunit to form biologically active holotoxin. Single unique amino acid substitutions in seven CdtA mutants resulted in reduced binding of the purified recombinant protein to Chinese hamster ovary cells and loss of binding to the fucose-containing glycoprotein, thyroglobulin. These mutations clustered at the 5′- and 3′-ends of the cdtA gene resulting in amino acid substitutions that resided outside of the aromatic patch region and a conserved region in CdtA homologues. Three of the amino acid substitutions, at positions S165N (mutA81), T41A (mutA121) and C178W (mutA221) resulted in gene products that formed holotoxin complexes that exhibited a 60% reduction (mutA81) or loss (mutA121, mutA221) of proliferation inhibition. A similar pattern was observed when these mutant holotoxins were tested for their ability to induce cell cycle arrest and to convert supercoiled DNA to relaxed and linear forms in vitro. The mutations in mutA81 and mutA221 disrupted holotoxin formation. The positions of the amino acid substitutions were mapped in the Haemophilus ducreyi Cdt crystal structure providing some insight into structure and function. PMID:16313618

  12. Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations

    PubMed Central

    Gady, Antoine LF; Hermans, Freddy WK; Van de Wal, Marion HBJ; van Loo, Eibertus N; Visser, Richard GF; Bachem, Christian WB

    2009-01-01

    Background The establishment of mutant populations together with the strategies for targeted mutation detection has been applied successfully to a large number of organisms including many species in the plant kingdom. Considerable efforts have been invested into research on tomato as a model for berry-fruit plants. With the progress of the tomato sequencing project, reverse genetics becomes an obvious and achievable goal. Results Here we describe the treatment of Solanum lycopersicum seeds with 1% EMS and the development of a new mutated tomato population. To increase targeted mutant detection throughput an automated seed DNA extraction has been combined with novel mutation detection platforms for TILLING in plants. We have adapted two techniques used in human genetic diagnostics: Conformation Sensitive Capillary Electrophoresis (CSCE) and High Resolution DNA Melting Analysis (HRM) to mutation screening in DNA pools. Classical TILLING involves critical and time consuming steps such as endonuclease digestion reactions and gel electrophoresis runs. Using CSCE or HRM, the only step required is a simple PCR before either capillary electrophoresis or DNA melting curve analysis. Here we describe the development of a mutant tomato population, the setting up of two polymorphism detection platforms for plants and the results of the first screens as mutation density in the populations and estimation of the false-positives rate when using HRM to screen DNA pools. Conclusion These results demonstrate that CSCE and HRM are fast, affordable and sensitive techniques for mutation detection in DNA pools and therefore allow the rapid identification of new allelic variants in a mutant population. Results from the first screens indicate that the mutagen treatment has been effective with an average mutation detection rate per diploid genome of 1.36 mutation/kb/1000 lines. PMID:19811648

  13. From Whole Gene Deletion to Point Mutations of EP300-Positive Rubinstein-Taybi Patients: New Insights into the Mutational Spectrum and Peculiar Clinical Hallmarks.

    PubMed

    Negri, Gloria; Magini, Pamela; Milani, Donatella; Colapietro, Patrizia; Rusconi, Daniela; Scarano, Emanuela; Bonati, Maria Teresa; Priolo, Manuela; Crippa, Milena; Mazzanti, Laura; Wischmeijer, Anita; Tamburrino, Federica; Pippucci, Tommaso; Finelli, Palma; Larizza, Lidia; Gervasini, Cristina

    2016-02-01

    Rubinstein-Taybi syndrome (RSTS) is a rare congenital neurodevelopmental disorder characterized by growth deficiency, skeletal abnormalities, dysmorphic features, and intellectual disability. Causative mutations in CREBBP and EP300 genes have been identified in ∼55% and ∼8% of affected individuals. To date, only 28 EP300 alterations in 29 RSTS clinically described patients have been reported. EP300 analysis of 22 CREBBP-negative RSTS patients from our cohort led us to identify six novel mutations: a 376-kb deletion depleting EP300 gene; an exons 17-19 deletion (c.(3141+1_3142-1)_(3590+1_3591-1)del/p.(Ile1047Serfs*30)); two stop mutations, (c.3829A>T/p.(Lys1277*) and c.4585C>T/p.(Arg1529*)); a splicing mutation (c.1878-12A>G/p.(Ala627Glnfs*11)), and a duplication (c.4640dupA/p.(Asn1547Lysfs*3)). All EP300-mutated individuals show a mild RSTS phenotype and peculiar findings including maternal gestosis, skin manifestation, especially nevi or keloids, back malformations, and a behavior predisposing to anxiety. Furthermore, the patient carrying the complete EP300 deletion does not show a markedly severe clinical picture, even if a more composite phenotype was noticed. By characterizing six novel EP300-mutated patients, this study provides further insights into the EP300-specific clinical presentation and expands the mutational repertoire including the first case of a whole gene deletion. These new data will enhance EP300-mutated cases identification highlighting distinctive features and will improve the clinical practice allowing a better genotype-phenotype correlation. PMID:26486927

  14. Improvement of the Mutation-Discrimination Threshold for Rare Point Mutations by a Separation-Free Ligase Detection Reaction Assay Based on Fluorescence Resonance Energy Transfer.

    PubMed

    Hagihara, Kenta; Tsukagoshi, Kazuhiko; Nakajima, Chinami; Esaki, Shinsuke; Hashimoto, Masahiko

    2016-01-01

    We previously developed a separation-free ligase detection reaction assay based on fluorescence resonance energy transfer from a donor quantum dot to an acceptor fluorescent dye. This assay could successfully detect one cancer mutation among 10 wild-type templates. In the current study, the mutation-discrimination threshold was improved by one order of magnitude by replacing the original acceptor dye (Alexa Fluor 647) with another fluorescent dye (Cyanine 5) that was spectrally similar but more fluorescent. PMID:26960620

  15. Interface design challenges for single point access to diverse and dispersed science databases

    NASA Technical Reports Server (NTRS)

    Harberts, R. L.; Pfister, R. G.; Dobinson, E. R.

    1992-01-01

    Efforts to relate the diversity of terminology in science data bases in a logical way for information system interfaces are discussed. Attention is given to the NASA development of the Information Management System (V. 0 IMS), a prototypic common interface for accessing dispersed earth science data.

  16. Haplotypes in the Dystrophin DNA Segment Point to a Mosaic Origin of Modern Human Diversity

    PubMed Central

    Ziętkiewicz, Ewa; Yotova, Vania; Gehl, Dominik; Wambach, Tina; Arrieta, Isabel; Batzer, Mark; Cole, David E. C.; Hechtman, Peter; Kaplan, Feige; Modiano, David; Moisan, Jean-Paul; Michalski, Roman; Labuda, Damian

    2003-01-01

    Although Africa has played a central role in human evolutionary history, certain studies have suggested that not all contemporary human genetic diversity is of recent African origin. We investigated 35 simple polymorphic sites and one Tn microsatellite in an 8-kb segment of the dystrophin gene. We found 86 haplotypes in 1,343 chromosomes from around the world. Although a classical out-of-Africa topology was observed in trees based on the variant frequencies, the tree of haplotype sequences reveals three lineages accounting for present-day diversity. The proportion of new recombinants and the diversity of the Tn microsatellite were used to estimate the age of haplotype lineages and the time of colonization events. The lineage that underwent the great expansion originated in Africa prior to the Upper Paleolithic (27,000–56,000 years ago). A second group, of structurally distinct haplotypes that occupy a central position on the tree, has never left Africa. The third lineage is represented by the haplotype that lies closest to the root, is virtually absent in Africa, and appears older than the recent out-of-Africa expansion. We propose that this lineage could have left Africa before the expansion (as early as 160,000 years ago) and admixed, outside of Africa, with the expanding lineage. Contemporary human diversity, although dominated by the recently expanded African lineage, thus represents a mosaic of different contributions. PMID:14513410

  17. One Hundred Twenty-One Dystrophin Point Mutations Detected from Stored DNA Samples by Combinatorial Denaturing High-Performance Liquid Chromatography

    PubMed Central

    Torella, Annalaura; Trimarco, Amelia; Del Vecchio Blanco, Francesca; Cuomo, Anna; Aurino, Stefania; Piluso, Giulio; Minetti, Carlo; Politano, Luisa; Nigro, Vincenzo

    2010-01-01

    Duchenne and Becker muscular dystrophies are caused by a large number of different mutations in the dystrophin gene. Outside of the deletion/duplication “hot spots,” small mutations occur at unpredictable positions. These account for about 15 to 20% of cases, with the major group being premature stop codons. When the affected male is deceased, carrier testing for family members and prenatal diagnosis become difficult and expensive. We tailored a cost-effective and reliable strategy to discover point mutations from stored DNA samples in the absence of a muscle biopsy. Samples were amplified in combinatorial pools and tested by denaturing high-performance liquid chromatography analysis. An anomalous elution profile belonging to two different pools univocally addressed the allelic variation to an unambiguous sample. Mutations were then detected by sequencing. We identified 121 mutations of 99 different types. Fifty-six patients show stop codons that represent the 46.3% of all cases. Three non-obvious single amino acid mutations were considered as causative. Our data support combinatorial denaturing high-performance liquid chromatography analysis as a clear-cut strategy for time and cost-effective identification of small mutations when only DNA is available. PMID:19959795

  18. Synergistic and compensatory effects of two point mutations conferring target-site resistance to fipronil in the insect GABA receptor RDL.

    PubMed

    Zhang, Yixi; Meng, Xiangkun; Yang, Yuanxue; Li, Hong; Wang, Xin; Yang, Baojun; Zhang, Jianhua; Li, Chunrui; Millar, Neil S; Liu, Zewen

    2016-01-01

    Insecticide resistance can arise from a variety of mechanisms, including changes to the target site, but is often associated with substantial fitness costs to insects. Here we describe two resistance-associated target-site mutations that have synergistic and compensatory effects that combine to produce high and persistent levels of resistance to fipronil, an insecticide targeting on γ-aminobytyric acid (GABA) receptors. In Nilaparvata lugens, a major pest of rice crops in many parts of Asia, we have identified a single point mutation (A302S) in the GABA receptor RDL that has been identified previously in other species and which confers low levels of resistance to fipronil (23-fold) in N. lugans. In addition, we have identified a second resistance-associated RDL mutation (R300Q) that, in combination with A302S, is associated with much higher levels of resistance (237-fold). The R300Q mutation has not been detected in the absence of A302S in either laboratory-selected or field populations, presumably due to the high fitness cost associated with this mutation. Significantly, it appears that the A302S mutation is able to compensate for deleterious effects of R300Q mutation on fitness cost. These findings identify a novel resistance mechanism and may have important implications for the spread of insecticide resistance. PMID:27557781

  19. Synergistic and compensatory effects of two point mutations conferring target-site resistance to fipronil in the insect GABA receptor RDL

    PubMed Central

    Zhang, Yixi; Meng, Xiangkun; Yang, Yuanxue; Li, Hong; Wang, Xin; Yang, Baojun; Zhang, Jianhua; Li, Chunrui; Millar, Neil S.; Liu, Zewen

    2016-01-01

    Insecticide resistance can arise from a variety of mechanisms, including changes to the target site, but is often associated with substantial fitness costs to insects. Here we describe two resistance-associated target-site mutations that have synergistic and compensatory effects that combine to produce high and persistent levels of resistance to fipronil, an insecticide targeting on γ-aminobytyric acid (GABA) receptors. In Nilaparvata lugens, a major pest of rice crops in many parts of Asia, we have identified a single point mutation (A302S) in the GABA receptor RDL that has been identified previously in other species and which confers low levels of resistance to fipronil (23-fold) in N. lugans. In addition, we have identified a second resistance-associated RDL mutation (R300Q) that, in combination with A302S, is associated with much higher levels of resistance (237-fold). The R300Q mutation has not been detected in the absence of A302S in either laboratory-selected or field populations, presumably due to the high fitness cost associated with this mutation. Significantly, it appears that the A302S mutation is able to compensate for deleterious effects of R300Q mutation on fitness cost. These findings identify a novel resistance mechanism and may have important implications for the spread of insecticide resistance. PMID:27557781

  20. Discriminative detection of low-abundance point mutations using a PCR/ligase detection reaction/capillary gel electrophoresis method and fluorescence dual-channel monitoring.

    PubMed

    Hamada, Mariko; Shimase, Koji; Tsukagoshi, Kazuhiko; Hashimoto, Masahiko

    2014-04-01

    We applied a facile LIF dual-channel monitoring system recently developed and reported by our group to the polymerase chain reaction/ligase detection reaction/CGE method for detecting low-abundance point mutations present in a wild-type sequence-dominated population. Mutation discrimination limits and signaling fidelity of the analytical system were evaluated using three mutant variations in codon 12 of the K-ras oncogene that have high diagnostic value for colorectal cancer. We demonstrated the high sensitivity of the present method by detecting rare mutations present among an excess of wild-type alleles (one mutation among ~100 normal sequences). This method also simultaneously interrogated the allelic compositions of the test samples with high specificity through spectral discrimination of the dye-tagged ligase detection reaction products using the dual-channel monitoring system. PMID:24510795

  1. Novel Point Mutations and A8027G Polymorphism in Mitochondrial-DNA-Encoded Cytochrome c Oxidase II Gene in Mexican Patients with Probable Alzheimer Disease

    PubMed Central

    Loera-Castañeda, Verónica; Sandoval-Ramírez, Lucila; Pacheco Moisés, Fermín Paul; Macías-Islas, Miguel Ángel; Alatorre Jiménez, Moisés Alejandro; González-Renovato, Erika Daniela; Cortés-Enríquez, Fernando; Célis de la Rosa, Alfredo; Velázquez-Brizuela, Irma E.

    2014-01-01

    Mitochondrial dysfunction has been thought to contribute to Alzheimer disease (AD) pathogenesis through the accumulation of mitochondrial DNA mutations and net production of reactive oxygen species (ROS). Mitochondrial cytochrome c-oxidase plays a key role in the regulation of aerobic production of energy and is composed of 13 subunits. The 3 largest subunits (I, II, and III) forming the catalytic core are encoded by mitochondrial DNA. The aim of this work was to look for mutations in mitochondrial cytochrome c-oxidase gene II (MTCO II) in blood samples from probable AD Mexican patients. MTCO II gene was sequenced in 33 patients with diagnosis of probable AD. Four patients (12%) harbored the A8027G polymorphism and three of them were early onset (EO) AD cases with familial history of the disease. In addition, other four patients with EOAD had only one of the following point mutations: A8003C, T8082C, C8201T, or G7603A. Neither of the point mutations found in this work has been described previously for AD patients, and the A8027G polymorphism has been described previously; however, it hasn't been related to AD. We will need further investigation to demonstrate the role of the point mutations of mitochondrial DNA in the pathogenesis of AD. PMID:24701363

  2. The Exon 13 Duplication in the BRCA1 Gene Is a Founder Mutation Present in Geographically Diverse Populations

    PubMed Central

    2000-01-01

    Recently, a 6-kb duplication of exon 13, which creates a frameshift in the coding sequence of the BRCA1 gene, has been described in three unrelated U.S. families of European ancestry and in one Portuguese family. Here, our goal was to estimate the frequency and geographic diversity of carriers of this duplication. To do this, a collaborative screening study was set up that involved 39 institutions from 19 countries and included 3,580 unrelated individuals with a family history of the disease and 934 early-onset breast and/or ovarian cancer cases. A total of 11 additional families carrying this mutation were identified in Australia (1), Belgium (1), Canada (1), Great Britain (6), and the United States (2). Haplotyping showed that they are likely to derive from a common ancestor, possibly of northern British origin. Our results demonstrate that it is strongly advisable, for laboratories carrying out screening either in English-speaking countries or in countries with historical links with Britain, to include within their BRCA1 screening protocols the polymerase chain reaction–based assay described in this report. PMID:10827109

  3. Point mutations in the tumor suppressor Smad4/DPC4 enhance its phosphorylation by GSK3 and reversibly inactivate TGF-β signaling

    PubMed Central

    Demagny, Hadrien; De Robertis, Edward M

    2016-01-01

    The tumor suppressor Smad4/DPC4 is an essential transcription factor in the TGF-β pathway and is frequently mutated or deleted in prostate, colorectal, and pancreatic carcinomas. We recently discovered that Smad4 activity and stability are regulated by the FGF/EGF and Wnt signaling pathways through a series of MAPK and GSK3 phosphorylation sites located in its linker region. In the present study, we report that loss-of-function associated with 2 point mutations commonly found in colorectal and pancreatic cancers results from enhanced Smad4 phosphorylation by GSK3, generating a phosphodegron that leads to subsequent β-TrCP–mediated polyubiquitination and proteasomal degradation. Using chemical GSK3 inhibitors, we show that Smad4 point mutant proteins can be stabilized and TGF-β signaling restored in cancer cells harboring such mutations. PMID:27308538

  4. Correction of point mutations at the endogenous locus of the dihydrofolate reductase gene using repair-PolyPurine Reverse Hoogsteen hairpins in mammalian cells.

    PubMed

    Solé, Anna; Ciudad, Carlos J; Chasin, Lawrence A; Noé, Véronique

    2016-06-15

    Correction of point mutations that lead to aberrant transcripts, often with pathological consequences, has been the focus of considerable research. In this work, repair-PPRHs are shown to be a new powerful tool for gene correction. A repair-PPRH consists of a PolyPurine Reverse Hoogsteen hairpin core bearing an extension sequence at one end, homologous to the DNA strand to be repaired but containing the wild type nucleotide instead of the mutation. Previously, we had corrected a single-point mutation with repair-PPRHs using a mutated version of a dihydrofolate reductase (dhfr) minigene. To further evaluate the utility of these molecules, different repair-PPRHs were designed to correct insertions, deletions, substitutions and a double substitution present in a collection of mutants at the endogenous locus of the dhfr gene, the product of which is the target of the chemotherapeutic agent methotrexate. We also describe an approach to use when the point mutation is far away from the homopyrimidine target domain. This strategy consists in designing Long-Distance- and Short-Distance-Repair-PPRHs where the PPRH core is bound to the repair tail by a five-thymidine linker. Surviving colonies in a DHFR selective medium, lacking glycine and sources of purines and thymidine, were analyzed by DNA sequencing, and by mRNA, protein and enzymatic measurements, confirming that all the dhfr mutants had been corrected. These results show that repair-PPRHs can be effective tools to accomplish a permanent correction of point mutations in the DNA sequence of mutant mammalian cells. PMID:27063945

  5. A methylated Neurospora 5S rRNA pseudogene contains a transposable element inactivated by repeat-induced point mutation.

    PubMed Central

    Margolin, B S; Garrett-Engele, P W; Stevens, J N; Fritz, D Y; Garrett-Engele, C; Metzenberg, R L; Selker, E U

    1998-01-01

    In an analysis of 22 of the roughly 100 dispersed 5S rRNA genes in Neurospora crassa, a methylated 5S rRNA pseudogene, Psi63, was identified. We characterized the Psi63 region to better understand the control and function of DNA methylation. The 120-bp 5S rRNA-like region of Psi63 is interrupted by a 1.9-kb insertion that has characteristics of sequences that have been modified by repeat-induced point mutation (RIP). We found sequences related to this insertion in wild-type strains of N. crassa and other Neurospora species. Most showed evidence of RIP; but one, isolated from the N. crassa host of Psi63, showed no evidence of RIP. A deletion from near the center of this sequence apparently rendered it incapable of participating in RIP with the related full-length copies. The Psi63 insertion and the related sequences have features of transposons and are related to the Fot1 class of fungal transposable elements. Apparently Psi63 was generated by insertion of a previously unrecognized Neurospora transposable element into a 5S rRNA gene, followed by RIP. We name the resulting inactivated Neurospora transposon PuntRIP1 and the related sequence showing no evidence of RIP, but harboring a deletion that presumably rendered it defective for transposition, dPunt. PMID:9691037

  6. Characterization of a point mutation in the parC gene of Mycoplasma bovirhinis associated with fluoroquinolone resistance.

    PubMed

    Hirose, K; Kawasaki, Y; Kotani, K; Abiko, K; Sato, H

    2004-05-01

    Quinolone-resistant (QR) mutants of Mycoplasma bovirhinis strain PG43 (type strain) were generated by stepwise selection in increasing concentrations of enrofloxacin (ENR). An alteration was found in the quinolone resistance-determining region (QRDR) of the parC gene coding for the ParC subunit of topoisomerase IV from these mutants, but not in the gyrA, gyrB, and parE gene coding for the GyrA and GyrB subunits of DNA gyrase and the ParE subunit of topoisomerase IV. Similarly, such an alteration in QRDR of parC was found in the field isolates of M. bovirhinis, which possessed various levels of QR. The substitution of leucine (Leu) by serine (Ser) at position 80 of QRDR of ParC was observed in both QR-mutants and QR-isolates. This is the first report of QR based on a point mutation of the parC gene in M. bovirhinis. PMID:15228551

  7. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides

    PubMed Central

    Bialk, Pawel; Sansbury, Brett; Rivera-Torres, Natalia; Bloh, Kevin; Man, Dula; Kmiec, Eric B.

    2016-01-01

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9. PMID:27609304

  8. Role of single-point mutations and deletions on transition temperatures in ideal proteinogenic heteropolymer chains in the gas phase.

    PubMed

    Olivares-Quiroz, L

    2016-07-01

    A coarse-grained statistical mechanics-based model for ideal heteropolymer proteinogenic chains of non-interacting residues is presented in terms of the size K of the chain and the set of helical propensities [Formula: see text] associated with each residue j along the chain. For this model, we provide an algorithm to compute the degeneracy tensor [Formula: see text] associated with energy level [Formula: see text] where [Formula: see text] is the number of residues with a native contact in a given conformation. From these results, we calculate the equilibrium partition function [Formula: see text] and characteristic temperature [Formula: see text] at which a transition from a low to a high entropy states is observed. The formalism is applied to analyze the effect on characteristic temperatures [Formula: see text] of single-point mutations and deletions of specific amino acids [Formula: see text] along the chain. Two probe systems are considered. First, we address the case of a random heteropolymer of size K and given helical propensities [Formula: see text] on a conformational phase space. Second, we focus our attention to a particular set of neuropentapeptides, [Met-5] and [Leu-5] enkephalins whose thermodynamic stability is a key feature on their coupling to [Formula: see text] and [Formula: see text] receptors and the triggering of biochemical responses. PMID:26818963

  9. Analgesia and unwanted benzodiazepine effects in point-mutated mice expressing only one benzodiazepine-sensitive GABAA receptor subtype

    PubMed Central

    Ralvenius, William T.; Benke, Dietmar; Acuña, Mario A.; Rudolph, Uwe; Zeilhofer, Hanns Ulrich

    2015-01-01

    Agonists at the benzodiazepine-binding site of GABAA receptors (BDZs) enhance synaptic inhibition through four subtypes (α1, α2, α3 and α5) of GABAA receptors (GABAAR). When applied to the spinal cord, they alleviate pathological pain; however, insufficient efficacy after systemic administration and undesired effects preclude their use in routine pain therapy. Previous work suggested that subtype-selective drugs might allow separating desired antihyperalgesia from unwanted effects, but the lack of selective agents has hitherto prevented systematic analyses. Here we use four lines of triple GABAAR point-mutated mice, which express only one benzodiazepine-sensitive GABAAR subtype at a time, to show that targeting only α2GABAARs achieves strong antihyperalgesia and reduced side effects (that is, no sedation, motor impairment and tolerance development). Additional pharmacokinetic and pharmacodynamic analyses in these mice explain why clinically relevant antihyperalgesia cannot be achieved with nonselective BDZs. These findings should foster the development of innovative subtype-selective BDZs for novel indications such as chronic pain. PMID:25865415

  10. Point mutations in EBV gH that abrogate or differentially affect B cell and epithelial cell fusion

    SciTech Connect

    Wu Liguo; Hutt-Fletcher, Lindsey M. . E-mail: lhuttf@lsuhsc.edu

    2007-06-20

    Cell fusion mediated by Epstein-Barr virus requires three conserved glycoproteins, gB and gHgL, but activation is cell type specific. B cell fusion requires interaction between MHC class II and a fourth virus glycoprotein, gp42, which complexes non-covalently with gHgL. Epithelial cell fusion requires interaction between gHgL and a novel epithelial cell coreceptor and is blocked by excess gp42. We show here that gp42 interacts directly with gH and that point mutations in the region of gH recognized by an antibody that differentially inhibits epithelial and B cell fusion significantly impact both the core fusion machinery and cell-specific events. Substitution of alanine for glycine at residue 594 completely abrogates fusion with either B cells or epithelial cells. Substitution of alanine for glutamic acid at residue 595 reduces fusion with epithelial cells, greatly enhances fusion with B cells and allows low levels of B cell fusion even in the absence of gL.

  11. A Simple Extension to the CMASA Method for the Prediction of Catalytic Residues in the Presence of Single Point Mutations

    PubMed Central

    Flores, David I.; Sotelo-Mundo, Rogerio R.; Brizuela, Carlos A.

    2014-01-01

    The automatic identification of catalytic residues still remains an important challenge in structural bioinformatics. Sequence-based methods are good alternatives when the query shares a high percentage of identity with a well-annotated enzyme. However, when the homology is not apparent, which occurs with many structures from the structural genome initiative, structural information should be exploited. A local structural comparison is preferred to a global structural comparison when predicting functional residues. CMASA is a recently proposed method for predicting catalytic residues based on a local structure comparison. The method achieves high accuracy and a high value for the Matthews correlation coefficient. However, point substitutions or a lack of relevant data strongly affect the performance of the method. In the present study, we propose a simple extension to the CMASA method to overcome this difficulty. Extensive computational experiments are shown as proof of concept instances, as well as for a few real cases. The results show that the extension performs well when the catalytic site contains mutated residues or when some residues are missing. The proposed modification could correctly predict the catalytic residues of a mutant thymidylate synthase, 1EVF. It also successfully predicted the catalytic residues for 3HRC despite the lack of information for a relevant side chain atom in the PDB file. PMID:25268770

  12. Altering a gene involved in nuclear distribution increases the repeat-induced point mutation process in the fungus Podospora anserina.

    PubMed Central

    Bouhouche, Khaled; Zickler, Denise; Debuchy, Robert; Arnaise, Sylvie

    2004-01-01

    Repeat-induced point mutation (RIP) is a homology-dependent gene-silencing mechanism that introduces C:G-to-T:A transitions in duplicated DNA segments. Cis-duplicated sequences can also be affected by another mechanism called premeiotic recombination (PR). Both are active over the sexual cycle of some filamentous fungi, e.g., Neurospora crassa and Podospora anserina. During the sexual cycle, several developmental steps require precise nuclear movement and positioning, but connections between RIP, PR, and nuclear distributions have not yet been established. Previous work has led to the isolation of ami1, the P. anserina ortholog of the Aspergillus nidulans apsA gene, which is required for nuclear positioning. We show here that ami1 is involved in nuclear distribution during the sexual cycle and that alteration of ami1 delays the fruiting-body development. We also demonstrate that ami1 alteration affects loss of transgene functions during the sexual cycle. Genetically linked multiple copies of transgenes are affected by RIP and PR much more frequently in an ami1 mutant cross than in a wild-type cross. Our results suggest that the developmental slowdown of the ami1 mutant during the period of RIP and PR increases time exposure to the duplication detection system and thus increases the frequency of RIP and PR. PMID:15166143

  13. A simple extension to the CMASA method for the prediction of catalytic residues in the presence of single point mutations.

    PubMed

    Flores, David I; Sotelo-Mundo, Rogerio R; Brizuela, Carlos A

    2014-01-01

    The automatic identification of catalytic residues still remains an important challenge in structural bioinformatics. Sequence-based methods are good alternatives when the query shares a high percentage of identity with a well-annotated enzyme. However, when the homology is not apparent, which occurs with many structures from the structural genome initiative, structural information should be exploited. A local structural comparison is preferred to a global structural comparison when predicting functional residues. CMASA is a recently proposed method for predicting catalytic residues based on a local structure comparison. The method achieves high accuracy and a high value for the Matthews correlation coefficient. However, point substitutions or a lack of relevant data strongly affect the performance of the method. In the present study, we propose a simple extension to the CMASA method to overcome this difficulty. Extensive computational experiments are shown as proof of concept instances, as well as for a few real cases. The results show that the extension performs well when the catalytic site contains mutated residues or when some residues are missing. The proposed modification could correctly predict the catalytic residues of a mutant thymidylate synthase, 1EVF. It also successfully predicted the catalytic residues for 3HRC despite the lack of information for a relevant side chain atom in the PDB file. PMID:25268770

  14. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides.

    PubMed

    Bialk, Pawel; Sansbury, Brett; Rivera-Torres, Natalia; Bloh, Kevin; Man, Dula; Kmiec, Eric B

    2016-01-01

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRISPR/Cas9 executes double-stranded DNA cleavage efficiently, closure of the broken chromosomes is dynamic, as varying degrees of heterogeneity of the cleavage products appear to accompany the emergence of the corrected base pair. We provide a detailed analysis of allelic variance at and surrounding the target site. In one particular case, we report sequence alteration directed by a distinct member of the same gene family. Our data suggests that single-stranded DNA molecules may influence DNA junction heterogeneity created by CRISPR/Cas9. PMID:27609304

  15. Kinase Domain Point Mutations in Ph+ Acute Lymphoblastic Leukemia (ALL) Emerge Following Therapy with BCR-ABL Kinase Inhibitors

    PubMed Central

    Jones, Dan; Thomas, Deborah; Yin, C. Cameron; O'Brien, Susan; Cortes, Jorge E.; Jabbour, Elias; Breeden, Megan; Giles, Francis J.; Zhao, Weiqiang; Kantarjian, Hagop M.

    2008-01-01

    Background BCR-ABL kinase domain (KD) mutations are detected in approximately 45% of imatinib-resistant CML. Patterns of KD mutations in Philadelphia chromosome (Ph)+ acute lymphoblastic leukemia (ALL) are less well-studied. Methods We assessed KD mutations in relapsed Ph+ ALL following treatments that included one or more kinase inhibitors (n = 24) or no prior KI therapy (n = 12). Results ABL KD mutations were detected by direct sequencing in 15 of 17 (88%) relapsed Ph+ ALL with prior imatinib (n = 16) or dasatinib (n = 1) treatment, and in 6 of 7 (86%) resistant/relapsed tumors treated with 2 or more KIs, compared with 0 of 12 relapsed Ph+ ALL never treated with KI. A restricted set of mutations was seen, mostly Y253H and T315I, detected on average 13 months following KI initiation, and mutations were not detected in the initial tumor samples prior to KI therapy in 12 patients assessed. Using a more sensitive pyrosequencing method, we did not detect mutations at codons 315 and 253 in the diagnostic samples from these 12 patients or in 30 Ph+ ALL patients who never relapsed. Conclusions ABL KD mutations, especially at codons 315 and 253, emerge upon relapse in the vast majority of patients with Ph+ ALL receiving maintenance KI therapy. Ongoing KI exposure may thus alter the patterns of relapse and favor outgrowth of clones with KI-resistant mutations. PMID:18615627

  16. Somatic Point Mutations in mtDNA Control Region Are Influenced by Genetic Background and Associated with Healthy Aging: A GEHA Study

    PubMed Central

    Rose, Giuseppina; Romeo, Giuseppe; Dato, Serena; Crocco, Paolina; Bruni, Amalia C.; Hervonen, Antti; Majamaa, Kari; Sevini, Federica; Franceschi, Claudio; Passarino, Giuseppe

    2010-01-01

    Tissue specific somatic mutations occurring in the mtDNA control region have been proposed to provide a survival advantage. Data on twins and on relatives of long-lived subjects suggested that the occurrence/accumulation of these mutations may be genetically influenced. To further investigate control region somatic heteroplasmy in the elderly, we analyzed the segment surrounding the nt 150 position (previously reported as specific of Leukocytes) in various types of leukocytes obtained from 195 ultra-nonagenarians sib-pairs of Italian or Finnish origin collected in the frame of the GEHA Project. We found a significant correlation of the mtDNA control region heteroplasmy between sibs, confirming a genetic influence on this phenomenon. Furthermore, many subjects showed heteroplasmy due to mutations different from the C150T transition. In these cases heteroplasmy was correlated within sibpairs in Finnish and northern Italian samples, but not in southern Italians. This suggested that the genetic contribution to control region mutations may be population specific. Finally, we observed a possible correlation between heteroplasmy and Hand Grip strength, one of the best markers of physical performance and of mortality risk in the elderly. Our study provides new evidence on the relevance of mtDNA somatic mutations in aging and longevity and confirms that the occurrence of specific point mutations in the mtDNA control region may represent a strategy for the age-related remodelling of organismal functions. PMID:20976236

  17. Rapid mutation of Spirulina platensis by a new mutagenesis system of atmospheric and room temperature plasmas (ARTP) and generation of a mutant library with diverse phenotypes.

    PubMed

    Fang, Mingyue; Jin, Lihua; Zhang, Chong; Tan, Yinyee; Jiang, Peixia; Ge, Nan; Heping Li; Xing, Xinhui

    2013-01-01

    In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9(th) subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae. PMID:24319517

  18. Rapid Mutation of Spirulina platensis by a New Mutagenesis System of Atmospheric and Room Temperature Plasmas (ARTP) and Generation of a Mutant Library with Diverse Phenotypes

    PubMed Central

    Zhang, Chong; Tan, Yinyee; Jiang, Peixia; Ge, Nan; Heping Li; Xing, Xinhui

    2013-01-01

    In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae. PMID:24319517

  19. Beyond the Tipping Point: Issues of Racial Diversity in Magnet Schools Following Unitary Status

    ERIC Educational Resources Information Center

    Smrekar, Claire

    2009-01-01

    This article uses qualitative case study methodology to examine why the racial composition of magnet schools in Nashville, Tennessee, has shifted to predominantly African American in the aftermath of unitary status. The article compares the policy contexts and parents' reasons for choosing magnet schools at two points in time--under court order…

  20. Combined point mutation in KRAS or EGFR genes and EML4-ALK translocation in lung cancer patients.

    PubMed

    Jürgens, Jessica; Engel-Riedel, Walburga; Prickartz, Alexander; Ludwig, Corinna; Schildgen, Oliver; Tillmann, Ramona-Liza; Stoelben, Erich; Brockmann, Michael; Schildgen, Verena

    2014-03-01

    A total of three cases with novel constellations regarding mutation patterns in non-small-cell lung cancer (NSCLC) are reported. The mutation patterns that are observed are novel and unexpected. First, a combined simultaneous KRAS mutation and EML4-ALK translocation, both in the main tumor and a bone metastasis, were observed, these mutations are assumed to mutually exclude each other. A further two cases include a father and a daughter, both of whom are suffering from NSCLC with different EGFR mutation patterns. A common cause was assumed; however, could not be deduced to mutations in the KRAS, BRAF and EGFR genes. The aforementioned cases are important, as it must be taken into account that mutations previously assumed to be exclusive can occur in combination, may influence the clinical outcome and may require different therapy compared with single mutated tumors. It has to be discussed whether diagnostic algorithms need to be adapted. The cases of father and daughter show that further unknown factors can influence development of NSCLC. PMID:24754584

  1. In Vivo Analysis of Disease-Associated Point Mutations Unveils Profound Differences in mRNA Splicing of Peripherin-2 in Rod and Cone Photoreceptors.

    PubMed

    Becirovic, Elvir; Böhm, Sybille; Nguyen, Ong Nam Phuong; Riedmayr, Lisa Maria; Koch, Mirja Annika; Schulze, Elisabeth; Kohl, Susanne; Borsch, Oliver; Santos-Ferreira, Tiago; Ader, Marius; Michalakis, Stylianos; Biel, Martin

    2016-01-01

    Point mutations in peripherin-2 (PRPH2) are associated with severe retinal degenerative disorders affecting rod and/or cone photoreceptors. Various disease-causing mutations have been identified, but the exact contribution of a given mutation to the clinical phenotype remains unclear. Exonic point mutations are usually assumed to alter single amino acids, thereby influencing specific protein characteristics; however, they can also affect mRNA splicing. To examine the effects of distinct PRPH2 point mutations on mRNA splicing and protein expression in vivo, we designed PRPH2 minigenes containing the three coding exons and relevant intronic regions of human PRPH2. Minigenes carrying wild type PRPH2 or PRPH2 exon 2 mutations associated with rod or cone disorders were expressed in murine photoreceptors using recombinant adeno-associated virus (rAAV) vectors. We detect three PRPH2 splice isoforms in rods and cones: correctly spliced, intron 1 retention, and unspliced. In addition, we show that only the correctly spliced isoform results in detectable protein expression. Surprisingly, compared to rods, differential splicing leads to lower expression of correctly spliced and higher expression of unspliced PRPH2 in cones. These results were confirmed in qRT-PCR experiments from FAC-sorted murine rods and cones. Strikingly, three out of five cone disease-causing PRPH2 mutations profoundly enhanced correct splicing of PRPH2, which correlated with strong upregulation of mutant PRPH2 protein expression in cones. By contrast, four out of six PRPH2 mutants associated with rod disorders gave rise to a reduced PRPH2 protein expression via different mechanisms. These mechanisms include aberrant mRNA splicing, protein mislocalization, and protein degradation. Our data suggest that upregulation of PRPH2 levels in combination with defects in the PRPH2 function caused by the mutation might be an important mechanism leading to cone degeneration. By contrast, the pathology of rod

  2. In Vivo Analysis of Disease-Associated Point Mutations Unveils Profound Differences in mRNA Splicing of Peripherin-2 in Rod and Cone Photoreceptors

    PubMed Central

    Becirovic, Elvir; Böhm, Sybille; Nguyen, Ong Nam Phuong; Riedmayr, Lisa Maria; Koch, Mirja Annika; Schulze, Elisabeth; Kohl, Susanne; Borsch, Oliver; Santos-Ferreira, Tiago; Ader, Marius; Michalakis, Stylianos; Biel, Martin

    2016-01-01

    Point mutations in peripherin-2 (PRPH2) are associated with severe retinal degenerative disorders affecting rod and/or cone photoreceptors. Various disease-causing mutations have been identified, but the exact contribution of a given mutation to the clinical phenotype remains unclear. Exonic point mutations are usually assumed to alter single amino acids, thereby influencing specific protein characteristics; however, they can also affect mRNA splicing. To examine the effects of distinct PRPH2 point mutations on mRNA splicing and protein expression in vivo, we designed PRPH2 minigenes containing the three coding exons and relevant intronic regions of human PRPH2. Minigenes carrying wild type PRPH2 or PRPH2 exon 2 mutations associated with rod or cone disorders were expressed in murine photoreceptors using recombinant adeno-associated virus (rAAV) vectors. We detect three PRPH2 splice isoforms in rods and cones: correctly spliced, intron 1 retention, and unspliced. In addition, we show that only the correctly spliced isoform results in detectable protein expression. Surprisingly, compared to rods, differential splicing leads to lower expression of correctly spliced and higher expression of unspliced PRPH2 in cones. These results were confirmed in qRT-PCR experiments from FAC-sorted murine rods and cones. Strikingly, three out of five cone disease-causing PRPH2 mutations profoundly enhanced correct splicing of PRPH2, which correlated with strong upregulation of mutant PRPH2 protein expression in cones. By contrast, four out of six PRPH2 mutants associated with rod disorders gave rise to a reduced PRPH2 protein expression via different mechanisms. These mechanisms include aberrant mRNA splicing, protein mislocalization, and protein degradation. Our data suggest that upregulation of PRPH2 levels in combination with defects in the PRPH2 function caused by the mutation might be an important mechanism leading to cone degeneration. By contrast, the pathology of rod

  3. A protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH domain of Akt1

    NASA Astrophysics Data System (ADS)

    Deyle, Kaycie M.; Farrow, Blake; Qiao Hee, Ying; Work, Jeremy; Wong, Michelle; Lai, Bert; Umeda, Aiko; Millward, Steven W.; Nag, Arundhati; Das, Samir; Heath, James R.

    2015-05-01

    Ligands that can bind selectively to proteins with single amino-acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wild type (WT). However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical synthetic epitope-targeting strategy that we used to discover a 5-mer peptide with selectivity for the E17K-transforming point mutation in the pleckstrin homology domain of the Akt1 oncoprotein. A fragment of Akt1 that contained the E17K mutation and an I19[propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that clicked covalently onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to the WT, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 (phosphatidylinositol (3,4,5)-trisphosphate) substrate.

  4. Protein-targeting strategy used to develop a selective inhibitor of the E17K point mutation in the PH Domain of Akt1

    PubMed Central

    Deyle, Kaycie M.; Farrow, Blake; Hee, Ying Qiao; Work, Jeremy; Wong, Michelle; Lai, Bert; Umeda, Aiko; Millward, Steven W.; Nag, Arundhati; Das, Samir; Heath, James R.

    2015-01-01

    Ligands that can selectively bind to proteins with single amino acid point mutations offer the potential to detect or treat an abnormal protein in the presence of the wildtype. However, it is difficult to develop a selective ligand if the point mutation is not associated with an addressable location, such as a binding pocket. Here we report an all-chemical, synthetic epitope-targeting strategy which we used to discover a 5-mer peptide with selectivity for the E17K transforming point mutation in the Pleckstrin Homology Domain of the Akt1 oncoprotein. A fragment of Akt1 containing the E17K mutation and a I19[Propargylglycine] substitution was synthesized to form an addressable synthetic epitope. Azide-presenting peptides that covalently clicked onto this alkyne-presenting epitope were selected from a library using in situ screening. One peptide exhibits a 10:1 in vitro selectivity for the oncoprotein relative to wildtype, with a similar selectivity in cells. This 5-mer peptide was expanded into a larger ligand that selectively blocks the E17K Akt1 interaction with its PIP3 substrate. PMID:25901825

  5. A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen.

    PubMed

    Jeong, Eunjoo; Houn, Thavrak; Kuk, Yongin; Kim, Eun-Seon; Chandru, Hema Kumar; Baik, Myunggi; Back, Kyoungwhan; Guh, Ja-Ock; Han, Oksoo

    2003-10-01

    In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis protoporphyrinogen oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B. subtilis protoporphyrinogen oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant protoporphyrinogen oxidase was kinetically characterized. The mutant protoporphyrinogen oxidase showed very similar kinetic patterns to wild type protoporphyrinogen oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole. When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant protoporphyrinogen oxidase showed an increased inhibition constant about 1.5 times that of wild type protoporphyrinogen oxidase. The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B. subtilis protoporphyrinogen oxidase may not be an important determinant in the mechanism that protects protoporphyrinogen oxidase against diphenyl ether herbicides. PMID:12941291

  6. A CRISPR/Cas9 mediated point mutation in the alpha 6 subunit of the nicotinic acetylcholine receptor confers resistance to spinosad in Drosophila melanogaster.

    PubMed

    Zimmer, Christoph T; Garrood, William T; Puinean, A Mirel; Eckel-Zimmer, Manuela; Williamson, Martin S; Davies, T G Emyr; Bass, Chris

    2016-06-01

    Spinosad, a widely used and economically important insecticide, targets the nicotinic acetylcholine receptor (nAChRs) of the insect nervous system. Several studies have associated loss of function mutations in the insect nAChR α6 subunit with resistance to spinosad, and in the process identified this particular subunit as the specific target site. More recently a single non-synonymous point mutation, that does not result in loss of function, was identified in spinosad resistant strains of three insect species that results in an amino acid substitution (G275E) of the nAChR α6 subunit. The causal role of this mutation has been called into question as, to date, functional evidence proving its involvement in resistance has been limited to the study of vertebrate receptors. Here we use the CRISPR/Cas9 gene editing platform to introduce the G275E mutation into the nAChR α6 subunit of Drosophila melanogaster. Reverse transcriptase-PCR and sequencing confirmed the presence of the mutation in Dα6 transcripts of mutant flies and verified that it does not disrupt the normal splicing of the two exons in close vicinity to the mutation site. A marked decrease in sensitivity to spinosad (66-fold) was observed in flies with the mutation compared to flies of the same genetic background minus the mutation, clearly demonstrating the functional role of this amino acid substitution in resistance to spinosad. Although the resistance levels observed are 4.7-fold lower than exhibited by a fly strain with a null mutation of Dα6, they are nevertheless predicated to be sufficient to result in resistance to spinosad at recommended field rates. Reciprocal crossings with susceptible fly strains followed by spinosad bioassays revealed G275E is inherited as an incompletely recessive trait, thus resembling the mode of inheritance described for this mutation in the western flower thrips, Frankliniella occidentalis. This study both resolves a debate on the functional significance of a target

  7. Characterization of a novel large deletion and single point mutations in the BRCA1 gene in a Greek cohort of families with suspected hereditary breast cancer

    PubMed Central

    Belogianni, Ioulia; Apessos, Angela; Mihalatos, Markos; Razi, Evangelia; Labropoulos, Stefanos; Petounis, Andreas; Gaki, Vasiliki; Keramopoulos, Antonios; Pandis, Nikos; Kyriacou, Kyriacos; Hadjisavvas, Andreas; Kosmidis, Paris; Yannoukakos, Drakoulis; Nasioulas, Georgios

    2004-01-01

    those families with strong evidence of linkage to the BRCA1 locus in whom no point mutation has been identified re-examination should be carried out searching specifically for genomic rearrangements. PMID:15353005

  8. Protein Adsorption and Reorganization on Nanoparticles Probed by the Coffee-Ring Effect: Application to Single Point Mutation Detection.

    PubMed

    Devineau, Stéphanie; Anyfantakis, Manos; Marichal, Laurent; Kiger, Laurent; Morel, Mathieu; Rudiuk, Sergii; Baigl, Damien

    2016-09-14

    The coffee-ring effect denotes the accumulation of particles at the edge of an evaporating sessile drop pinned on a substrate. Because it can be detected by simple visual inspection, this ubiquitous phenomenon can be envisioned as a robust and cost-effective diagnostic tool. Toward this direction, here we systematically analyze the deposit morphology of drying drops containing polystyrene particles of different surface properties with various proteins (bovine serum albumin (BSA) and different forms of hemoglobin). We show that deposit patterns reveal information on both the adsorption of proteins onto particles and their reorganization following adsorption. By combining pattern analysis with adsorption isotherm and zeta potential measurements, we show that the suppression of the coffee-ring effect and the formation of a disk-shaped pattern is primarily associated with particle neutralization by protein adsorption. However, our findings also suggest that protein reorganization following adsorption can dramatically invert this tendency. Exposure of hydrophobic (respectively charged) residues can lead to disk (respectively ring) deposit morphologies independently of the global particle charge. Surface tension measurements and microscopic observations of the evaporating drops show that the determinant factor of the deposit morphology is the accumulation of particles at the liquid/gas interface during evaporation. This general behavior opens the possibility to probe protein adsorption and reorganization on particles by the analysis of the deposit patterns, the formation of a disk being the robust signature of particles rendered hydrophobic by protein adsorption. We show that this method is sensitive enough to detect a single point mutation in a protein, as demonstrated here by the distinct patterns formed by human native hemoglobin h-HbA and its mutant form h-HbS, which is responsible for sickle cell anemia. PMID:27562632

  9. Mechanism of Insolubilization by a Single point Mutation in αA-Crystallin Linked with Hereditary Human Cataract

    PubMed Central

    Andley, Usha P.; Hamilton, Paul D.; Ravi, Nathan

    2008-01-01

    αA-Crystallin is a small heat shock protein that functions as a molecular chaperone and a lens structural protein. The single point mutation R49C in αA-crystallin causes hereditary human cataracts. We have previously investigated the in vivo properties of this mutant in a gene knock-in mouse model. Remarkably, homozygous mice carrying the αA-R49C mutant show nearly complete lens opacity concurrent with small lenses and small eyes. Here we have investigated the 90° light scattering, viscosity, refractive index and bis-ANS fluorescence of lens proteins isolated from the αA-R49C mouse lenses and find that the concentration of total water-soluble proteins showed a pronounced decrease in αA-R49C homozygous lenses. Light scattering measurements on proteins separated by gel permeation chromatography showed a small amount of high molecular weight aggregated material in the void volume which still remains soluble in AA-R49C homozygous lens homogenates. Increased binding of β-and γ-crystallin to the α-crystallin fraction was observed in αA-R49C heterozygous and homozygous lenses but not in wild type. Quantitative analysis with the hydrophobic fluorescence probe bis-ANS showed a pronounced increase in fluorescence yield upon binding to α-crystallin in αA-R49C lenses as compared with the wild type protein. These results suggest that the decrease in solubility of the αA-R49C mutant protein was due to an increase in its hydrophobicity and supra-aggregation of αA-crystallin that leads to cataract formation. Our study further shows that analysis of mutant proteins from the knock-in mouse model is an effective way to understand the mechanism of protein insolubilization in hereditary cataracts. PMID:18700785

  10. [Radiation biology of structurally different Drosophila melanogaster genes. Report I. The vestigial gene: molecular characteristic of "point" mutations].

    PubMed

    Aleksandrov, I D; Afanas'eva, K P; Aleksandrova, M V; Lapidus, I L

    2012-01-01

    The screening of PCR-detected DNA alterations in 9 spontaneous and 59 gamma-ray-, neutron - or neutron + gamma-ray-induced Drosophila vestigial (vg) gene/"point" mutations was carried out. The detected patterns of existence or absence of either of 16 overlapping fragments into which vg gene (15.1 kb, 8 exons, 7 introns) was divided enable us to subdivide all mutants into 4 classes: (i) PCR+ (40.7%) without the detected changes; (ii) "single-site" (33.9%) with the loss of a single fragment; (iii) partial detections (15.2%) as a loss of 2-9 adjacent fragments and (iv) "cluster" mutants (10.2%) having 2-3 independent changes of(ii) and/or (iii) classes. All spontaneous mutants except one were found to be classified as (ii) whereas radiation-induced mutants are represented by all 4 classes whose interrelation is determined by the dose and radiation quality. In particular, the efficacy of neutrons was found to be nine times as large as that of gamma-rays under the "cluster" mutant induction. Essentially, the distribution of DNA changes along the gene is uneven. CSGE-assay of PCR+-exon 3 revealed DNA heteroduplexes in 5 out of 17 PCR+-mutants studied, 2 of which had small deletions (5 and 11 b) and 3 others made transitions (A --> G) as shown by the sequencing. Therefore, gamma-rays and neutrons seem to be significant environmental agents increasing the SNP risk for the population through their action on the germ cells. The results obtained are also discussed within the framework of the track structure theory and the notion of quite different chromatin organization in somatic and germ cells. PMID:22891545